Dissertations / Theses on the topic 'Plants – Evolution'

Plant microRNAs have been shown to have important roles in regulating diverse processes ranging from reproductive development to stress response. In the first two chapters, I focus on miRNA diversity in Aquilegia studying both anciently evolved broadly conserved and rapidly evolving species specific miRNAs. In chapter one, I utilize Aquilegia's critical phylogenetic position between the well developed models Arabidopsis thaliana and Oryza sativa to study the evolution of ancient miRNAs across the angiosperms. In chapter two, I utilize smallRNA high-throughput sequencing to annotate Aquilegia specific miRNAs and, in the process, uncover the novel regulation of a floral homeotic gene by an Aquilegia-specific miRNA. In chapter three, I look at the tissue specific development of miRNA regulation in the bioenergetically relevant model organism Populus trichocarpa. High-throughput smallRNA sequencing from four diverse tissue sets including leaves, xylem, mechanically treated xylem, and pooled vegetative and reproductive tissues were analyzed, revealing a total of 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Expanding on my work with the petal identity pathway, I turned a broader analysis of Aquilegia petal spurs. Petal spurs are the distinguishing characteristic of Aquilegia and are argued to be a key innovation in the adaptive radiation of the genus. In the fourth chapter, I explore the cellular basis of extreme spur length diversity in the genus and find that a single parameter, cell shape, can explain this morphological range. Next, I seek to describe the cellular patterns that give rise to a spur primoridia from an initially flat laminar petal and find that spur initiation is characterized by concentrated, prolonged, and oriented cell divisions. Inspired by this quantitative analysis of growth, chapter five looks at the mechanisms of shape change in cucumber tendrils. I find that anisotropic contraction of a multi-layered gelatinous fiber ribbon explains coiling in cucumbers. Surprisingly, we discover that tendrils display twistless-overwinding when pulled and exhibit an unforeseen force-extension response as a result. These results provide the design basis for twistless springs with tunable mechanical responses and serve as a clear example of how the biological systems can inspire applied mechanical designs.

Thesis by publication.
Thesis (PhD) -- Macquarie University, School of Biological Sciences, 1990.
Bibliography: leaves 269-324.
Introduction -- Models of parental allocation -- Sex expression in homosporous pteridophytes -- The origin of heterospory -- Pollination and the origin of the seed habit -- Brood reduction in gymnosperms -- Pollination: costs and consequences -- Adaptive explanations for the rise of the angiosperms -- Parent-specific gene expression and the triploid endosperm -- New perspectives on the angiosperm female gametophyte -- Overview -- Glossary -- Kinship terms in plants -- Literature Cited.
Among vascular plants/ different life cycles are associated with characteristic ranges of propagule size. In the modern flora, isospores of homosporous pteridophytes are almost all smaller than 150 urn diameter, megaspores of heterosporous pteridophytes fall in the range 100-1000 urn diameter, gymnosperm seeds are possibly all larger than the largest megaspores, but the smallest angiosperm seeds are of comparable size to large isospores. -- Propagule size is one of the most important features of a sporophyte's reproductive strategy. Roughly speaking, larger propagules have larger food reserves, and a greater probability of successful establishment, than smaller propagules, but a sporophyte can produce more smaller propagules from the same quantity of resources. Different species have adopted very different size-versus-number compromises. The characteristic ranges of propagule size, in each of the major groups of vascular plants, suggest that some life cycles are incompatible with particular size-versus-number compromises. -- Sex expression in homosporous plants is a property of gametophytes (homosporous sporophytes are essentially asexual). Gametophytes should produce either eggs or sperm depending on which course of action gives the greatest chance of reproductive success. A maternal gametophyte must contribute much greater resources to a young sporophyte than the paternal gametophyte. Therefore, smaller gametophytes should tend to reproduce as males, and gametophytes with abundant resources should tend to reproduce as females. Consistent with these predictions, large female gametophytes release substances (antheridiogens) which induce smaller neighbouring ametophytes to produce sperm. -- The mechanism of sex determination in heterosporous species appears to be fundamentally different. Large megaspores develop into female gametophytes, and small icrospores develop into male gametophytes. Sex expression appears to be determined by the sporophyte generation. This is misleading. As argued above, the optimal sex expression of a homosporous gametophyte is influenced by its access to resources. This is determined by (1) the quantity of food reserves in its spore and (2) the quantity of resources accumulated by the gametophyte's own activities. If a sporophyte produced spores of two sizes, gametophytes developing from the larger spores' would be more likely to reproduce as females than gametophytes developing from the smaller spores, because the pre-existing mechanisms of sex determination would favor production of archegonia by larger gametophytes. Thus, the predicted mechanisms of sex determination in homosporous species could also explain the differences in sex expression of gametophytes developing from large and small spores in heterosporous species.
Megaspores of living heterosporous pteridophytes contain sufficient resources for female reproduction without photosynthesis by the gametophyte (Platyzoma excepted), whereas microspores only contain sufficient resources for male reproduction. Furthermore, many more microspores are produced than megaspores. A gametophyte's optimal sex expression is overwhelmingly determined by the amount of resources supplied in its spore by the sporophyte, and is little influenced by the particular environmental conditions where the spore lands. Gametophytes determine sex expression in heterosporous species, as well as homosporous species. A satisfactory model for the evolution of heterospory needs to explain under what circumstances sporophytes will benefit from producing spores of two distinct sizes. -- In Chapter 4, I present a model for the origin of heterospory that predicts the existence of a "heterospory threshold". For propagule sizes below the threshold, homosporous reproduction is evolutionarily stable because gametophytes must rely on their own activities to accumulate sufficient resources for successful female reproduction. Whether a gametophyte can accumulate sufficient resources before its competitors is strongly influenced by environmental conditions. Gametophytes benefit from being able to adjust their sex expression in response to these conditions. For propagule sizes above the threshold, homosporous reproduction is evolutionarily unstable, because the propagule's food reserves are more than sufficient for a "male" gametophyte to fertilize all eggs within its neighbourhood. A population of homosporous sporophytes can be invaded by sporophytes that produce a greater number of smaller spores which could land in additional locations and fertilize additional eggs. Such'spores would be male-specialists on account of their size. Therefore, both spore types would be maintained in the population because of frequency-dependent selection. -- The earliest vascular plants were homosporous. Several homosporous groups gave rise to heterosporous lineages, at least one of which was the progeniture of the seed plants. The first heterosporous species appear in the Devonian. During the Devonian, there was a gradual increase in maximum spore size, possibly associated with the evolution of trees and the appearance of the first forests. As the heterospory threshold was approached, the optimal spore size for female reproduction diverged from the optimal spore size for male reproduction. Below the threshold, a compromise spore size gave the highest fitness returns to sporophytes, but above the threshold, sporophytes could attain higher fitness by producing two types of spores. -- The evolution of heterospory had profound consequences. Once a sporophyte produced two types of spores, microspores and megaspores could become specialized for male and female function respectively. The most successful heterosporous lineage (or lineages) is that of the seed plants. The feature that distinguishes seed plants from other heterosporous lineages is pollination, the capture of microspores before, rather than after, propagule dispersal. Traditionally, pollination has been considered to be a major adaptive advance because it frees sexual reproduction from dependence on external fertilization by freeswimming sperm, but pollination has a more important advantage. In heterosporous pteridophytes, a megaspore is provisioned whether or not it will be fertilized whereas seeds are only provisioned if they are pollinated.
The total cost per seed cannot be assessed solely from the seed's energy and nutrient content. Rather, each seed also has an associated supplementary cost of adaptations for pollen capture and of resources committed to ovules that remain unpollinated. The supplementary cost per seed has important consequences for understanding reproductive strategies. First, supplementary costs are expected to be proportionally greater for smaller seeds. Thus, the benefits of decreasing seed size (in order to produce more seeds) are reduced for species with small seeds. This effect may explain minimum seed sizes. Second, supplementary costs are greater for populations at lower density. Thus, there is a minimum density below which a species cannot maintain its numbers. -- By far the most successful group of seed plants in the modern flora are the angiosperms. Two types of evidence suggest that early angiosperms had a lower supplementary cost per seed than contemporary gymnosperms. First, the minimum size of angiosperm seeds was much smaller than the minimum size of gymnosperm seeds. This suggests that angiosperms could produce small seeds more cheaply than could gymnosperms. Second, angiosperm-dominated floras were more speciose than the gymnosperm-dominated floras they replaced. This suggests that the supplementary cost per seed of angiosperms does not increase as rapidly as that of gymnosperms, as population density decreases. In consequence, angiosperms were able to displace gymnosperms from many habitats, because the angiosperms had a lower cost of rarity. -- Angiosperm embryology has a number of distinctive features that may be related to the group's success. In gymnosperms, the nutrient storage tissue of the seed is the female gametophyte. In most angiosperms, this role is taken by the endosperm. Endosperm is initiated by the fertilization of two female gametophyte nuclei by a second sperm that is genetically identical to the sperm which fertilizes the egg. Endosperm has identical genes to its associated embryo, except that there are two copies of maternal genes for every copy of a paternal gene. -- Chapter 9 presents a hypothesis to explain the unusual genetic constitution of endosperm. Paternal genes benefit from their endosperm receiving more resources than the amount which maximizes the fitness of maternal genes, and this conflict is expressed as parent-specific gene expression in endosperm. The effect of the second maternal genome is to increase maternal control of nutrient acquisition. -- Female gametophytes of angiosperms are traditionally classified as monosporic, bisporic or tetrasporic. Bisporic and tetrasporic embryo sacs contain the derivatives of more than one megaspore nucleus. Therefore, there is potential for conflict between the different nuclear types within an embryo sac, but this possibility has not been recognized by plant embryologists. In Chapter 10, I show that many previously inexplicable observations can be understood in terms of genetic conflicts within the embryo sac.
Mode of access: World Wide Web.
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This study lends strong support to the idea that members of Bromeliaceae have undergone a recent adaptive radiation, and therefore show that, at least in part, diversity in the tropics is due to a fast speciation rate and that the tropics can be a "cradle" for new diversification and exploitation of varying ecological niches through the diversification of ecophysiological traits within a lineage.

The Earth harbors around 300 000 plant species. The rich and complex environment provided by plants is considered a key factor for the extraordinary diversity of the terrestrial fauna by, for example, providing food and shelter. This thesis contributes to the understanding of these questions by investigating how the interplay of physiology, demography, and evolution gives rise to variation and diversity in fundamental plant traits. This will help us answer questions such as: How has this amazing diversity of plant species emerged? Which mechanisms maintain diversity? How are plant strategies and plant diversity influenced by variations in the environment? A plant faces multiple problems to survive and reproduce successfully. These problems can be modeled by considering traits, trade-offs and a fitness measure. For example: How to maximize growth rate, while maximizing structural stability? I will investigate four plant models in order to understand the function of plants, and mechanisms promoting diversity.  Paper I: We study how annual plants with and without growth constraints should optimize their flowering time when productivity or season length changes. With a dynamic ontogenetic growth model and optimal control theory we prove that a bang-bang reproductive control is optimal under constrained growth and constant mortality rate. We find that growth constraints can flip the direction of optimal phenological response for increasing productivity. The reason is that the growth rate of vegetative mass saturates at high productivity and therefore it is better to flower earlier and take advantage of a longer reproductive period. If season length extends equally both in the beginning and the end of the season, growth constraints control the direction of the optimal response as well. Our theory can help explaining phenological patterns along productivity gradients, and can be linked to empirical observations made on a calendar scale. Paper II: We introduce a new measure of tree crown-rise efficiency based on the loss of biomass of the tree during growth. The more mass the tree looses during growth, the less crown-rise efficient it is. Top-heavy shapes loose more biomass than bottom-heavy shapes. Light-use efficiency is defined as the mean light assimilation of the leaves in the crown times the ratio of leaf mass and total mass. We then study the trade-off between light-use efficiency to crown-rise efficiency for tree crown shapes. We assume that the total tree mass is constant, and a constant vertical light gradient represent the shading from a surrounding forest. We find large differences in crown shapes at intermediate vertical light gradient, when both self-shading and mean-field shading are important, suggesting light-use vs crown-rise efficiency as a new trade-off that can explain tree diversity. Our crown-rise efficiency measure could easily be integrated into existing forest models. Paper III: We extend an evolutionary tree crown model, where trees with different heights compete for light, with drought-induced mortality rates depending on ground-water availability and the depth of an optional taproot. The model does not include competition for ground water. Our model explains how ground-water availability can shape plant communities, when taproot and non-taproot strategies can coexist, and when only one of these strategies can persist. We investigate how emerging plant diversity varies with water table depth, soil water gradient and drought-induced mortality rate. The taproot enables plants to reach deep water, thus reducing mortality, but also carries a construction cost, thus inducing a trade-off. We find that taproots maintain plant diversity under increasing drought mortality, and that taproots evolve when groundwater is accessible at low depths. There are no viable strategies at high drought mortality and deep water table. Red Queen evolutionary dynamics appear at intermediate drought mortality in mixed communities with and without taproots, as the community never reaches a final evolutionarily stable composition. Paper IV: We extend a size-structured plant model, with self-shading and two evolving traits, crown top-heaviness and crown width-to-height ratio. The model allows us to identify salient trade-offs for the crown shape. The most important trade-off for top-heaviness is light-use efficiency vs crownrise efficiency, and the most important trade-off for width-to-height ratio is self-shading vs branch costs. We find that when the two traits coevolve; the outcome is a single common evolutionarily stable strategy (ESS), far away from the highest net primary production (NPP). When only sun angle is decreasing with increasing latitude both the crown width-to-height ratio and crown top-heaviness decrease. However, when light response in addition to the sun angle decreases with increasing latitude, the crown width-to-height ratio is nearly invariant of latitude except at low site productivity when the ratio decreases with latitude. Top-heaviness is always decreasing with increasing latitude. Finally, we find that crown top-heaviness increases with the NPP or leaf-area index (LAI) at ESS, but crown width-to-height ratio is maximal at an intermediate NPP or LAI.
Artikel I: Arters reproduktionsframgång (fitness), till exempel antal avkommor eller frön som produceras under livet, är ofta avgörande för huruvida de är evolutionärt framgångsrika eller inte. Här undersöker vi hur ettåriga växter med eller utan tillväxtbegränsningar ska optimera sin blomningstid när produktivitet eller säsongslängd ändras. Det är optimalt att gå direkt från tillväxt till blomning när tillväxten är begränsad och dödligheten är konstant. Vid ökad produktivitet sker blomningen tidigare med tillväxtbegränsningar men senare utan tillväxtbegränsningar, vilket beror på att med tillväxtbegränsningar ökar den vegetativa massan långsamt. Därför är det bättre att blomma tidigare och ta tillvara på en längre reproduktionsperiod. Vi får samma resultat om säsongslängden ökar lika mycket i början och slutet av säsongen. Vår teori kan bidra till att förutsäga blomningstider vid produktivitetsförändringar och säsongsförändringar. Artikel II: Tillväxten hos träd kan begränsas av brist på ljus, vatten, och näring, men också genom förlust av grenar. Vi introducerar ett nytt mått på tillväxteffektiviteten hos trädkronor baserat på förlust av biomassa under trädets tillväxt. Ju mer massa trädet förlorar under tillväxt, desto mindre tillväxteffektiva är de. Topptunga former förlorar mer biomassa än bottentunga former. Vi studerar avvägningar mellan ljuseffektivitet och tillväxteffektivitet för trädformer, där ljuseffektiviteten definieras som medelljusupptaget för löven i kronan. Vi antar en konstant totalmassa, och en statisk vertikal skuggning som representerar skuggningen från en omgivande skog. Vi hittar stora skillnader i kronformer vid en medelhög skuggning, då både självskuggningen och medelskuggningen har betydelse. Vårt mått för tillväxteffektivitet kan enkelt integreras i existerande skogsmodeller. Studien visar att avvägningar mellan tillväxteffektivitet och ljuseffektivitetet kan vara viktig för mångfalden av trädformer i en skog. En överraskande upptäckt är att konformade eller sfäriska trädkronor aldrig är effektiva, men däremot timglasformade kronor. Artikel III: Växter kan försvara sig på olika sätt mot torka, till exempel genom att rulla ihop bladen eller genom att reproducera tidigare och därigenom undvika uttdragen torka. Här undersöker vi fördelarna med en pålrot vid torka. En pålrot är en rot som växer nedåt för att nå djupliggande grundvatten. Vi utvidgar en evolutionär modell av trädkronor med grundvatten och en pålrot, där träd med olika höjd konkurrerar om ljus. Det finns ingen konkurrens om vatten. Vi undersöker hur mångfalden hos träden beror på vattendjup, vattengradient och dödlighet orsakad av torka. Med hjälp av pålroten kan träden nå djupt vatten och därigenom minska dödligheten, men den medför också en kostnad, så en avvägning måste göras. Vi ser att pålrötter upprätthåller mångfalden hos växterna vid ökad mortalitet, och att pålrötter uppstår när grundvattnet är grunt. Det finns inga strategier som kan överleva om grundvattnet är djupt och dödligheten är hög. Vår modell kan förklara hur grundvatten kan förändra sammansättningen på trädsamhällen, när träd med och utan pålrot kan samexistera, och under vilka förutsättningar endast en av strategierna förväntas dominera. Artikel IV: Träd som växer upp i en skog måste konkurrera med andra träd om ljus, framförallt större träd. Detta ger upphov till en asymmetrisk ljuskonkurrens, där de små träden hämmas av större träd. Små träd har därmed små chanser att överleva utom då skogen nyligen störts och det öppnas upp en glänta. Vid denna ljuskonkurrens kan man anta att trädkronans form har stor betydelse för trädets framgång. Frågan är hur de evolutionärt fördelaktiga kronformerna beror på latituden och produktiviteten. Vi antar att latituden påverkar solens genomsnittliga vinkel och ljusrespons. Vi utvidgar en storleksstrukturerad trädmodell med självskuggning där två evolverande egenskaper beskriver kronans topptyngd och bredd. Med modellen kan vi undersöka vilka strategiska avvägningar som bestämmer om kronans form blir konkurrenskraftig. En topptung krona har högt ljusupptag eftersom det finns mest ljus högt upp i grenverket. Å andra sidan har den en låg tillväxteffektivitet eftersom topptunga kronor måste tappa mycket grenar för att behålla sin form. En bred krona har en låg självskuggning eftersom bladen är utspridda. Å andra sidan har den höga kostnader för de långa grenar som krävs. Vi finner att när dessa egenskaper evolverar tillsammans så finns endast en evolutionärt stabil strategi (ESS), långt från den högsta nettoproduktionen. När endast solvinkeln minskar med ökande latitud minskar både kronans bredd och topptyngd, men när både solvinkel och ljusrespons minskar med ökande latitud så är bredden nästan oförändrad utom vid låg produktivitet då den minskar med latituden. Kronans topptyngd minskar alltid med latituden. Slutligen ser vi hur kronans topptyngd alltid ökar med nettoproduktionen vid ESS, medan kronans bredd har ett maxium för ett mellanvärde hos nettoproduktionen vid ESS.

Phyllotaxis, namely the arrangement of phylla (leaves, florets, etc.) has intrigued natural scientists for over four hundred years. Statistics show that about 90\% of the spiral patterns has their numbers of spirals belonging to two consecutive members of the regular Fibonacci sequence. (Fibonacci(-like) sequences refer to any sequences constructed with the addition rule $a_{j+2}=a_{j}+a_{j+1}$, while the regular Fibonacci sequence refers to the particular sequences 1,1,2,3,5,8,13,...) Historical research on pattern formation on plants, tracing back to as early as four hundred years ago, was mostly geometry based. Current studies focus on the activities on the cellular level and study initiation of primordia (the initial undifferentiated form of phylla) as a morphogenesis process cued by some signal. The nature of the signal and the mechanisms governing the distribution of the signal are still under investigation. The two top candidates are the biochemical hormone auxin distribution and the mechanical stresses in the plant surface (tunica). We built a model which takes into consideration the interactions between these mechanisms. In addition, this dissertation explores both analytically and numerically the conditions for the Fibonacci-like patterns to continuously evolve (i.e. as the mean radius of the generative annulus changes over time, the numbers of spirals in the pattern increase or decreases along the same Fibonacci-like sequence), as well as for different types of pattern transitions to occur. The essential condition for the Fibonacci patterns to continuously evolve is that the patterns are formed annulus by annulus on a circular domain and the pattern-forming mechanism is dominated by a quadratic nonlinearity. The predominance of the regular Fibonacci pattern is determined by the pattern transitions at early stages of meristem growth. Furthermore, Fibonacci patterns have self-similar structures across different radii, and there exists a one-to-one mapping between any two Fibonacci-like patterns. The possibility of unifying the previous theory of optimal packing on phyllotaxis and the solutions of current mechanistic partial differential equations is discussed.

Explaining the origin and maintenance of biodiversity is a central goal in ecology and evolutionary biology. Some of the most important, theoretical explanations for this diversity centre on the evolution of life histories. Comparative studies on life history evolution, have received significant attention in the zoological literature, but have lagged in plants. Recent developments, however, have emphasised the value of comparative analysis of data for many species to test existing theories of life history evolution, as well as to provide the basis for developing additional or alternative theories. The primary goal of this study was to explore existing theories of life history evolution using a dataset of demographic information in the form of matrix population models for a large number of plant species. By projecting average matrix population models for 207 plant species, life tables and fecundity schedules were obtained and, in turn, were used to estimate relevant life history parameters. These parameters were then used to explore the i) lability of life history traits in plants ii) their continuum of life history variation, iii) the evolution of senescence and iv) the significance of demographic entropy in population ecology. Elasticities and sensitivities of life history traits showed significant phylogenetic signal compared to other life history traits, although, all the values of phylogenetic signal observed were < 1 indicating that life history traits are generally labile. Eighty one percent of species in the datset had mortality curves that increased with age compared to one hundred percent of species that showed a reproductive value curve that decreases with age at the end of life. In particular, the parameters that measured pace and duration were inversely related suggesting in general, the presence of senescence in our data set. Finally, the tenets of the directionality theory based on demographic entropy were generally not confirmed. This study provides an important contribution to the life history evolution of iteroparous perennial plants and confirms existing theories on life history evolution.

In order to function properly, organisms have a complex control mechanism, in which a given gene is expressed at a particular time and place. One way to achieve this control is to regulate the initiation of transcription. This step requires the assembly of several components, i.e., a basal/general machinery common to all expressed genes, and a specific/regulatory machinery, which differs among genes and is the responsible for proper gene expression in response to environmental or developmental signals. This specific machinery is composed of transcription factors (TFs), which can be grouped into evolutionarily related gene families that possess characteristic protein domains. In this work we have exploited the presence of protein domains to create rules that serve for the identification and classification of TFs. We have modelled such rules as a bipartite graph, where families and protein domains are represented as nodes. Connections between nodes represent that a protein domain should (required rule) or should not (forbidden rule) be present in a protein to be assigned into a TF family. Following this approach we have identified putative complete sets of TFs in plant species, whose genome is completely sequenced: Cyanidioschyzon merolae (red algae), Chlamydomonas reinhardtii (green alga), Ostreococcus tauri (green alga), Physcomitrella patens (moss), Arabidopsis thaliana (thale cress), Populus trichocarpa (black cottonwood) and Oryza sativa (rice). The identification of the complete sets of TFs in the above-mentioned species, as well as additional information and reference literature are available at http://plntfdb.bio.uni-potsdam.de/. The availability of such sets allowed us performing detailed evolutionary studies at different levels, from a single family to all TF families in different organisms in a comparative genomics context. Notably, we uncovered preferential expansions in different lineages, paving the way to discover the specific biological roles of these proteins under different conditions. For the basic leucine zipper (bZIP) family of TFs we were able to infer that in the most recent common ancestor (MRCA) of all green plants there were at least four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments. Currently, following the approach described above, up to 57 TF and 11 TR families can be identified, which are among the most numerous transcription regulatory families in plants. Three families of putative TFs predate the split between rhodophyta (red algae) and chlorophyta (green algae), i.e., G2-like, PLATZ, and RWPRK, and may have been of particular importance for the evolution of eukaryotic photosynthetic organisms. Nine additional families, i.e., ABI3/VP1, AP2-EREBP, ARR-B, C2C2-CO-like, C2C2-Dof, PBF-2-like/Whirly, Pseudo ARR-B, SBP, and WRKY, predate the split between green algae and streptophytes. The identification of putative complete list of TFs has also allowed the delineation of lineage-specific regulatory families. The families SBP, bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA significantly differ in size between algae and land plants. The SBP family of TFs is significantly larger in C. reinhardtii, compared to land plants, and appears to have been lost in the prasinophyte O. tauri. The families bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA preferentially expanded with the colonisation of land, and might have played an important role in this great moment in evolution. Later, after the split of bryophytes and tracheophytes, the families MADS, AP2-EREBP, NAC, AUX/IAA, PHD and HRT have significantly larger numbers in the lineage leading to seed plants. We identified 23 families that are restricted to land plants and that might have played an important role in the colonization of this new habitat. Based on the list of TFs in different species we have started to develop high-throughput experimental platforms (in rice and C. reinhardtii) to monitor gene expression changes of TF genes under different genetic, developmental or environmental conditions. In this work we present the monitoring of Arabidopsis thaliana TFs during the onset of senescence, a process that leads to cell and tissue disintegration in order to redistribute nutrients (e.g. nitrogen) from leaves to reproductive organs. We show that the expression of 185 TF genes changes when leaves develop from half to fully expanded leaves and finally enter partial senescence. 76% of these TFs are down-regulated during senescence, the remaining are up-regulated. The identification of TFs in plants in a comparative genomics setup has proven fruitful for the understanding of evolutionary processes and contributes to the elucidation of complex developmental programs.
Organismen weisen einen komplexen Steuerungsmechanismus auf, bei dem die Aktivität eines Gens räumlich und zeitlich reguliert wird. Eine Möglichkeit der Kontrolle der Genaktivität ist Regulation der Initiation der Transkription. Eine Voraussetzung für die Transkriptionsinitiation ist die Zusammenlagerung verschiedener Komponenten: eine allgemeine Maschinerie, die für alle exprimierten Gene gleich ist und eine spezifische Maschinerie, die sich von Gen zu Gen unterscheidet und die für die korrekte Genexpression in Abhängigkeit der Entwicklung und von Umweltsignalen verantwortlich ist. Diese spezifische Maschinerie besteht aus Transkriptionsfaktoren (TFs), welche in evolutionär verwandte Genefamilien eingeteilt werden können, die charakteristische Proteindomänen aufweisen. In dieser Arbeit habe ich die Proteindomänen genutzt, um Regeln aufzustellen, die die Identifizierung und Klassifizierung von TFs erlauben. Solche Regeln wurden als Graphen modelliert, in denen die Familien und Proteindomänen als Knoten repräsentiert wurden. Verbindungen zwischen den Knoten bedeuten, dass eine Proteindomäne in einem Protein entweder vorhanden sein sollte oder nicht vorhanden sein darf, damit das Protein einer TF-Familie zugeordnet wird. Mit Hilfe dieses Ansatzes wurden vermutlich vollständige Datensätze von TFs in Pflanzenspezies generiert, deren Genom komplett sequenziert wurde: C. merolae, C. reinhardtii, O. tauri, P. patens, A. thaliana, P. trichocarpa and O. sativa. Diese kompletten TF-Sätze sowie weitergehende Informationen und Literaturhinweise wurden unter der Internetadresse http://plntfdb.bio.uni-potsdam.de/ öffentlich zugänglich gemacht. Die Datensätze erlaubten es, detailliertere evolutionäre Studien mit unterschiedlichen Schwerpunkten durchzuführen. Diese reichten von der Analyse einzelner Familien bis hin zum genomweiten Vergleich aller TF-Familien in verschiedenen Organismen. Als Resultat besonders erwähnenswert ist, dass bevorzugt einige bestimmte TF-Familien in verschiedenen Spezies expandierten. Diese Studien ebnen den Weg, um die spezifische biologische Rolle dieser Proteine unter verschiedenen Bedingungen zu ergründen. Für die wichtige TF-Familie bZIP konnte gezeigt werden, dass der letzte gemeinsame Vorfahr aller Grünpflanzen mindestens vier bZIP Gene hatte, die funktionell in die Antwort auf oxidativen Stress eingebunden waren. Aus den vier Gründergene entstand durch Genverdopplung und –differenzierung eine große Familie, die Eigenschaften hervorbrachte, die die Besiedelung neuer Lebensräume ermöglichten. Mit Hilfe des oben beschriebenen Ansatzes können derzeit aus der Vielzahl der Transkriptionsregulatorfamilien in Pflanzen bis zu 57 TF und 11 TR Familien identifiziert werden. Drei Familien mutmaßlicher TFs markieren die Trennung zwischen Rhodophyta (Rotalgen) und Chlorophyta (Grünalgen): G2-like, PLATZ und RWPRK. Diese könnten eine besondere Rolle bei der Evolution eukaryotischer photosynthetisch aktiver Organismen gespielt haben. Neun zusätzliche Familien (ABI3/VP1, AP2-EREBP, ARR-B, C2C2-CO-like, C2C2-Dof, PBF-2-like/Whirly, Pseudo ARR-B, SBP und WRKY) kennzeichnen die Trennung zwischen Grünalgen und Streptophyten. Die Identifizierung putativer kompletter Listen an TFs erlaubte auch die Identifizierung abtammungsspezifischer regulatorischer Familien. Die Familien SBP, bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP und FHA unterscheiden sich signifikant in ihrer Größe zwischen Algen und Landpflanzen. Die SBP Familie ist in C. reinhardtii signifikant größer als in Landpflanzen. In der Parasinophyte O. tauri scheint diese Familie verloren gegangen zu sein. Die Familien bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP und FHA expandierten präferenziell mit der Kolonialisation an Land. Sie könnten eine wichte Rolle während dieses einschneidenden Ereignisses der Evolution gespielt haben. Später, nach der Trennung von Bryophyten und Tracheophyten sind die Familien MADS, AP2-EREBP, NAC, AUX/IAA, PHD und HRT stärker in den Linien, die zu Samenpflanzen führten, gewachsen. 23 TF-Familien wurden identifiziert, die es nur in Landpflanzen gibt. Sie könnten eine besondere Rolle bei der Besiedelung des neuen Lebensraum gespielt haben. Aufbauend auf die Transkriptionsfaktordatensätze, die in dieser Arbeit erstellt wurden, wurde mittlerweile damit begonnen, experimentelle Hochdurchsatz-Plattformen zu entwickeln (für Reis und für C. reinhardtii), um Änderungen in der Genaktivität der TF-Gene unter verschiedenen genetischen, Entwicklungs- oder Umweltbedingungen zu untersuchen. In dieser Arbeit wird die Analyse von TFs aus A. thaliana im Verlauf der Seneszenz vorgestellt. Seneszenz ist ein Prozess, der zur Zell- und Gewebeauflösung führt, um Nährstoffe aus den Blättern für den Transport in reproduktive Organe freizusetzen. Es wird gezeigt, dass sich die Expression von 187 TF Gene verändert, wenn sich die Blätter voll entfalten und schließlich teilweise in den Prozess der Seneszenz eintreten. 76% der TFs waren runterreguliert, die übrigen waren hochreguliert.

The colonization of the arid continental surface by plants was one of the milestones in Earth's history. Morphological innovations, such as the origin of complex 3D tissues, allowed the successful colonization and radiation of plants on land. The epidermis is the outermost plant tissue that constitutes the interface between the plant and the environment. Thus, the evolution of epidermal cells was crucial for the adaptation of plants on the terrestrial arid environment. I undertook a combined approach that aims to understand the evolutionary trends that drove land plant colonization and the genetic mechanisms that underlie the development of the epidermis. This approach includes: 1) analyses of plant transcription factors (TFs) families distribution and diversification, with a particular focus on the basic Helix-Loop-Helix (bHLH) TF family, and 2) functional characterization of a putatively conserved bHLH TF subfamily involved in epidermal cell development in land plants. Here, I showed that there was a stepwise increase in the number of transcription factor (TF) families and bHLH subfamilies that predated the colonization of the terrestrial surface by plants. The subsequent increase in TF number on land was through duplication within pre-existing TF families and subfamilies. Moreover, a similar trend occurred in metazoan bHLH TF, suggesting that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans. Furthermore, I demonstrated that the function of IIIf bHLH TFs in controlling the development of the epidermal cell layer is conserved between liverworts and angiosperms. This suggests that IIIf bHLH TFs are ancient and conserved regulators of epidermal cell development since the early colonization of the land by plants. Moreover, these bHLH TFs were recruited during the evolution of land plants to control the development of seemingly unrelated morphological characters in specific lineages of extant land plants. The recruitment of ancient developmental regulators to control distinct and unrelated developmental processes in land plants might underlie the huge morphological and taxonomic radiation of plants on land.

[ES] Las proteínas DELLA son elementos centrales de la ruta de señalización por giberelinas (GAs), donde actúan como represores de las respuestas a GAs. En angiospermas, se ha observado que las DELLAs interaccionan con cientos de factores de transcripción y otros reguladores transcripcionales, modulando de este modo la expresión génica. Por lo tanto, la participación generalizada de las GAs a lo largo del ciclo vital de las plantas es una consecuencia directa de la promiscuidad de las proteínas DELLA y de su rol como reguladores transcripcionales clave. Aunque las DELLAs se encuentran en todas las plantas terrestres, solo son reguladas por GAs en traqueofitas, en las cuales se han centrado la mayoría de los estudios previos. El trabajo aquí presentado pretende descifrar en qué punto de la evolución las DELLAs adquirieron las características moleculares que las convierten en "hubs", y qué ventajas biológicas podrían estar relacionadas con la evolución de las DELLAs. En el primer capítulo, describimos análisis comparativos de redes de co-expresión génicas asociadas a DELLA en especies vasculares y no vasculares, y proponemos que las DELLAs tienen un papel crítico en la conformación de panoramas transcripcionales. Desde su aparición en el ancestro de las plantas terrestres, conectaron múltiples programas transcripcionales que serían independientes sin ellas, mejoraron la eficiencia de la transmisión de información y aumentaron el nivel de complejidad en la regulación transcripcional. También observamos que este efecto se incrementó tras su integración en la señalización por GAs. En el segundo capítulo, proporcionamos pruebas experimentales más sólidas que extienden esta conclusión. Usando una combinación de rastreos de doble híbrido en levadura dirigidos, con DELLAs de diferentes posiciones en el linaje vegetal, y complementación heteróloga en plantas de Arabidopsis y Marchantia, mostramos que la promiscuidad es una característica conservada en todas las proteínas DELLA examinadas; lo cual sugiere que esta propiedad puede haber estado codificada en la DELLA ancestral, y después se mantuvo a lo largo de la evolución, con episodios de co-evolución entre las DELLAs y sus interactores. Finalmente, la comparación de dianas transcripcionales de las DELLAs en diferentes especies muestra la llamativa conservación de un pequeño conjunto de funciones reguladas por DELLAs en plantas vasculares y no vasculares -incluyendo la respuesta a factores de estrés-, mientras que análisis comparativos de promotores indican que las dianas específicas de cada especie aparecen mediante al menos dos mecanismos: el establecimiento de nuevas interacciones de la DELLA, y el acceso a nuevos promotores diana a través de interactores conservados. En resumen, proponemos que las DELLAs son proteínas intrínsecamente promiscuas, con propiedades de "hub" en virtualmente todas las plantas, y la conservación de sus dianas transcripcionales depende en gran medida de la evolución de sus interactores. La conservación de las propiedades de "hub" de las proteínas DELLA las convierte en dianas biotecnológicas ideales, ya que la mayoría del conocimiento generado en una especie podría ser fácilmente adaptado a otras especies relativamente lejanas.
[CA] Les proteïnes DELLA són elements centrals de la ruta de senyalització per gibberel·lines (GAs), on actuen com a repressors de les respostes a GAs. En angiospermes, s'ha observat que les DELLAs interaccionen amb centenars de factors de transcripció i altres reguladors transcripcionals, modulant d'aquesta manera l'expressió gènica. Per tant, la participació generalitzada de les GAs al llarg del cicle vital de les plantes és una conseqüència directa de la promiscuïtat de les proteïnes DELLA i del seu rol com a reguladors transcripcionals clau. Tot i que les DELLAs es troben en totes les plantes terrestres, només són regulades per GAs en traqueofites, en les quals s'han centrat la majoria dels estudis anteriors. El treball ací presentat pretén desxifrar en quin punt de l'evolució les DELLAs van adquirir les característiques moleculars que les converteixen en "hubs", i quins avantatges biològics podrien estar relacionats amb l'evolució de les DELLAs. En el primer capítol, descrivim anàlisis comparatius de xarxes de co-expressió gèniques associades a DELLA en espècies vasculars i no vasculars, i proposem que les DELLAs tenen un paper crític en la conformació de panorames transcripcionals. Des de la seua aparició en l'ancestre de les plantes terrestres, van connectar múltiples programes transcripcionals que serien independents sense elles, van millorar l'eficiència de la transmissió d'informació i augmentar el nivell de complexitat en la regulació transcripcional. També observem que aquest efecte es va incrementar després de la seua integració en la senyalització per GAs. En el segon capítol, proporcionem proves experimentals més sòlides que estenen aquesta conclusió. Usant una combinació de rastrejos de doble híbrid en rent dirigits, amb DELLAs de diferents posicions en el llinatge vegetal, i complementació heteròloga en plantes d'Arabidopsis i Marchantia, vam mostrar que la promiscuïtat és una característica conservada en totes les proteïnes DELLA examinades; la qual cosa suggereix que aquesta propietat pot haver estat codificada en la DELLA ancestral, i després es va mantenir al llarg de l'evolució, amb episodis de co-evolució entre les DELLAs i els seus interactors. Finalment, la comparació de dianes transcripcionals de les DELLAs en diferents espècies mostra la cridanera conservació d'un petit conjunt de funcions regulades per DELLAs en plantes vasculars i no vasculars -incloent la resposta a factors de estrès-, mentre que anàlisis comparatius de promotors indiquen que les dianes específiques de cada espècie apareixen mitjançant al menys dos mecanismes: l'establiment de noves interaccions de la DELLA, i l'accés a nous promotors diana a través d'interactors conservats. En resum, proposem que les DELLAs són proteïnes intrínsecament promíscues, amb propietats de "hub" en virtualment totes les plantes, i la conservació de les seues dianes transcripcionals depèn en gran mesura de l'evolució dels seus interactors. La conservació de les propietats de "hub" de les proteïnes DELLA les converteix en dianes biotecnològiques ideals, ja que la majoria del coneixement generat en una espècie podria ser fàcilment adaptat a altres espècies relativament llunyanes.
[EN] DELLA proteins are central elements of the gibberellin (GA) signaling pathway, where they act as repressors of GA responses. In angiosperms, DELLAs have been shown to interact with hundreds of transcription factors and other transcriptional regulators, thereby modulating gene expression. Hence, the widespread involvement of GAs along the plant life cycle is a direct consequence of the promiscuity of DELLA proteins and their role as key transcriptional regulators. Although DELLAs can be found in all land plants, they are only regulated by GAs in tracheophytes, where most of the previous studies have been focused. The work presented here aims to decipher at which point in evolution did DELLAs acquired the molecular features that render them as 'hubs', and what biological advantages could be related with DELLA evolution. In the first chapter, we describe comparative analyses of DELLA-associated gene co-expression networks in vascular and non-vascular species and propose that DELLAs have a critical role in the conformation of transcriptional landscapes. Upon their emergence in the ancestor of land plants, they connected multiple transcriptional programs that would be independent without them, improved the efficiency of information transmission and increased the level of complexity in transcriptional regulation. We also observed that this effect was enhanced after their integration in GA signaling. In the second chapter, we provide stronger experimental evidence that extends this conclusion. Using a combination of targeted yeast two-hybrid screenings with DELLAs from different positions in the plant lineage, and heterologous complementation in Arabidopsis and Marchantia plants, we show that promiscuity is a conserved feature in all the examined DELLA proteins, which suggests that this property might have been encoded in the ancestral DELLA, and then maintained along evolution, with episodes of co-evolution between DELLAs and their partners. Finally, comparison of DELLA transcriptional targets in different species shows a striking conservation of a small set of functions regulated by DELLAs in vascular and non-vascular plants -including the response to stress factors-, while comparative promoter analysis indicates that species-specific DELLA targets emerge through at least two mechanisms: establishment of novel DELLA interactions, and the access by conserved partners to new target promoters. In summary, we propose that DELLAs are intrinsically promiscuous proteins, with hub properties in virtually all land plants, and the conservation of their transcriptional targets largely depends on the evolution of their interactors. The conservation of the hub properties of DELLA proteins makes them ideal biotechnological targets, as most of the knowledge generated in one species could be readily adapted to other relatively distant species.
Esta tesis doctoral ha sido posible gracias a un contrato predoctoral FPU del Ministerio de Educación (FPU2014-01941).
Briones Moreno, A. (2020). Evolution of DELLA proteins as transcriptional hubs in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/159378
TESIS

In mono- und eudikotylen Pflanzen kodiert eine Genfamilie (RpoT, RNA-Polymerase des T3/T7-Typs) mitochondriale und plastidäre RNA-Polymerasen (RNAP), die den ungeraden T-Phagen-Polymerasen ähneln. RpoT-Gene von Angiospermen sind gut charakterisiert, während aus tiefer abzweigenden Pflanzenspecies bisher lediglich die Gene aus dem Moos Physcomitrella beschrieben wurden. Um einen Beitrag zur Aufklärung der molekularen Evolution der RpoT-Polymerasen im Pflanzenreich zu liefern und um Erkenntnisse über die potentielle Bedeutung von multiplen Phagen-Typ (RNAP) in Pflanzen zu gewinnen, wurden die RpoT-Gene aus dem Lycophyten Selaginella moellendorffii und aus dem basalen Angiosperm Nuphar advena identifiziert und charakterisiert. Selaginella moellendorffii (Moosfarn)-Trace-Sequenzdaten mit hoher Ähnlichkeit zu RpoT-Sequenzen von Angiospermen wurden benutzt, um das full-length SmRpoT-Gen und die entsprechende cDNA zu isolieren. Die SmRpoT-mRNA ist 3542 nt lang und weist einen offenen Leserahmen von 3006 nt auf, der für ein putatives Protein aus 1002 Aminosäuren mit einer molekularen Masse von 113 kDa kodiert. Das SmRpoT-Gen besteht aus 19 Exons und 18 Introns, die in ihren Positionen mit denen aus den Angiosperm- und Physcomitrella-Genen konserviert sind. Mittels Southernblot-Analyse wurde nachgewiesen, dass S. moellendorffii ein single-copy RpoT-Gen kodiert. Für das N-terminale Transitpeptid von SmRpoT konnte gezeigt werden, dass es bei transienter Expression in Arabidopsis- und Selaginella-Protoplasten den Transport von GFP (green fluorescent protein) exclusiv in Mitochondrien vermittelt. In N. advena wurden mittels Screening einer BAC-Bibliothek drei RpoT-Gene identifiziert. Sowohl die genomischen als auch die cDNA-Sequenzen wurden aufgeklärt. Die NaRpoT-mRNAs kodieren putative Polypeptide von 996, 990 und 985 Aminosären. Alle drei Gene besitzen 19 Exons und 18 Introns, die in ihren Positionen mit denen der RpoT-Gene aus Selaginella und allen anderen Landpflanzen konserviert sind. Die kodierten Proteine weisen auf Aminosäureebene einen hohen Konservierungsgrad auf, einschließlich aller essentiellen Regionen und Aminosäurereste, die für die T7-RNAP bekannt sind. Die N-terminalen Transitpeptide zweier der kodierten RNAP, NaRpoTm1 und NaRpoTm2, vermittelten den Import von GFP exclusiv in Mitochondrien, während die dritte Polymerase, NaRpoTp, in Chloroplasten importiert wurde. Interessanterweise muß die Translation der NaRpoTp-mRNA an einem CUG-Codon initiiert werden, um ein funktionelles Protein mit plastidärem Transitpeptid zu erhalten. Die N. advena RpoTp-RNAP ist somit neben AGAMOUS aus Arabidopsis und der RpoTp-RNAP aus Nicotiana, ein weiteres Beispiel für jene selten vorkommenden pflanzlichen mRNAs, deren Translation exclusiv an nicht-AUG-Codons initiiert wird. Die Rekonstruktion von phylogenetischen Bäumen resultierte in unterschiedlichen Positionen für die Selaginella- und Nuphar-Polymerasen: Im Gegensatz zu der RpoT-Polymerase aus S. moellendorffii und denen aus Physcomitrella, die in den phylogenetischen Analysen Schwesterpositionen zu allen anderen Phagentyp-RNAP der Angiospermen einnehmen, clusterten die Nuphar-RpoTs zusammen mit den deutlich separierten mitochondrialen (NaRpoTm1 und NaRpoTm2) und plastidären (NaRpoTp) Polymerasen. Selaginella kodiert eine einzige mitochondriale RNAP, während Nuphar zwei mitochondriale und eine plastidäre RNAP besitzt. Die Identifizierung einer Plastiden-lokalisierten Phagentyp-RNAP in diesem basalen Eudikotylen, die ortholog zu allen anderen RpoT-Enzymen der Blütenpflanzen ist, läßt darauf schließen, daß die Acquisition einer nukleär kodierten plastidären RNAP, die noch in den Lycopoden fehlt, nach der Trennung der Leucopoden von allen anderen Tracheophyten erfolgte. Eine “dual-targeting” RNAP (mitochondrial und plastidär lokalisiert), wie sie in Eudikotylen, nicht jedoch in Monokotylen vorkommt, wurde weder in Selaginella noch in Nuphar nachgewiesen, vermutlich ist sie ein evolutionäres Novum von eudikotylen Pflanzen wie Arabidopsis.
In mono- and eudicot plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species until recently were limited to the moss Physcomitrella. To elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we identified and characterized RpoT genes in the lycophyte Selaginella moellendorffii and the basal eudicot Nuphar advena. Selaginella moellendorffii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3,006 nt, encoding a putative protein of 1,002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. Using Southern blot analysis, it was shown that S. moellendorffii encodes a single RpoT gene. The N-terminal transit peptide of SmRpoT was shown to confer targeting of green fluorescent protein (GFP) exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. In Nuphar advena three RpoT genes were identified by BAC library screening. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those from S. moellendorffii and the RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of GFP exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp polymerase, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. Reconstruction of phylogenetic trees revealed different positions of the RpoTs from the lycophyte Selaginella and the basal eudicot Nuphar. In contrast to the RpoTs of S. moellendorffii and those of the moss Physcomitrella, which are according to the phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. Selaginella encodes a single mitochondrial RNAP, whereas Nuphar harbors two mitochondrial and one plastid phage-type polymerases. Identification of a plastid localized phage-type RNAP in this basal eudicot, orthologous to all other RpoTp enzymes of flowering plants, suggests that the acquisition of a nuclear encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastid RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar or Selaginella suggesting that its occurrence is an evolutionary novelty of eudicotyledoneous plants like Arabidopsis.

The onset of flowering time in a plant is extremely significant when evaluating population success. Floral growth, seed production, and dispersal are all dependent upon flowering time. Flowering early (and hence longer) increases the prospect of pollination but typically reduces vegetative growth and yields fewer/smaller flowers. Flowering late (and hence shorter) guarantees more/bigger flowers but carries the risk of insufficient pollination. This fundamental trade-off between growth and flowering time suggests that there may be an optimal time to initiate flowering. In this thesis, we consider a deterministic hybrid integrodifferential model where we represent the growing season in continuous time and the time between seasons as a discrete map. We track the evolution of flowering time, as a phenotype, by explicitly considering it as a variable in our model. The model is analyzed from two different viewpoints: (1) by mutual invasion analysis in the sense of adaptive dynamics; and (2) by deriving equations for the mean trait value and total population density when flowering time is considered to be Gamma-distributed. In both cases evolution to an intermediary flowering time was observed.

Individuals of Eriogonum abertianum Torr. (Polygonaceae) flower in spring, or following onset of summer rains, or both. Within populations flowering time is mainly environmentally determined: there is little genetic variance for flowering time, and experimental moisture limitation significantly delays flowering. In the field a Sonoran Desert population experienced significantly more mortality during the foresummer droughts, and had a significantly greater proportion of spring-flowering plants, than a Chihuahuan Desert population. Greenhouse experiments suggest a genetic basis for differences in size and time of flowering between these populations. Fossil and biogeographic evidence support an adaptive interpretation of earlier flowering in the Sonoran Desert. A model of selection comparing spring-plus-summer flowering with spring-only flowering suggests that expected summer fecundity may not offset the risk of foresummer mortality in the Sonoran population. Rather than switching to a spring-only habit as predicted by the model, the species' range ends where summer rainfall declines abruptly. The invariance of the spring-plus-summer habit is not explained by the demographic, historical, or genetic data. Plants which live for more than a year in the wild have offspring which, in the greenhouse, live longer than the offspring of the general population. This suggests a genetic basis for the occasional observed perennation. Analysis of a quantitative genetic model suggests that when adult survivorship is low, selection will generally reduce perennation. The annual habit is thus likely to persist even in the presence of genetic variation for perennation. Optimal control models of plant carbon allocation are extended to include within-season mortality and allometric growth constraints. When parameters are varied in numerical experiments, resulting predictions for easily measurable characters (e.g., time to first flower) often vary only slightly; most differences are in fitness, suggesting that satisfactory empirical tests may be difficult to conduct. Arbitrary mortality functions can optimally lead to multiple flowering episodes, and this can depend sensitively on parameter values. Optimal trajectories with allometric constraints are divided into a period of vegetative growth and another period of mixed growth.

The ecology and evolution of pathogens are key factors in predicting the severity and spread of disease, as well as treatment outcomes. However, the effects of multiple trophic levels that include host, microbial competitors and viruses are typically overlooked. In this thesis I develop our understanding of bacteria-phage coevolution, microbial dispersal and the role of the microbiome in disease. The results of these experiments have direct implications for phage therapy: the use of bacteriophages to treat bacterial infections. Firstly, I explore the risks of phage application in the environment and draw parallels with the misuse of antibiotics in selecting for bacterial resistance. I then demonstrate that the evolution of resistance to phages in a plant pathogenic bacterium is context-dependent. Notably, I find a fitness cost in plant infections that is absent when the bacteria are cultured solely in the laboratory. I then characterize four novel phages and use a simple laboratory based assay to predict their potential as phage therapy agents in an agricultural context. Next I show that reservoir species of plant hosts can affect the evolution of virulence, when bacteria are passaged on both a focal and distant host, but find no evidence of local adaptation. I also show that the evolution of such traits can occur in a parallel manner at the genetic level. I then determine a compositional shift in the microbiota associated with the symptoms of bleeding canker disease in Horse Chestnut trees across the length of the UK. Finally, I find an age-elated decline in bacterial species richness and evidence for niche-assembly theories by investigating bacterial dispersal in UK Oak trees in a single woodland.

Photoperiodism refers to the organism’s ability to detect and respond to seasonal changes in the daily duration of light and dark and thus constitutes one of the most significant and complex examples of the interaction between the organism and its environment. This thesis attempts to describe the prevalence of variation in a photoperiodic response, its adaptive value, and its putative genetic basis in a common cruciferous weed, Capsella bursa-pastoris (Brassicaceae). Furthermore, the thesis presents a first comprehensive comparative overview of the circadian clock mechanism in an early land plant, Physcomitrella patens (Bryophyta), thus providing insights into the evolution of the plant circadian system. In an introductory survey of global gene expression changes among early- and late flowering accessions of C. bursa-pastoris we found an enrichment of genes involved in photoperiodic response and regulation of the circadian clock. Secondly, by phenotyping circadian rhythm variation in a worldwide sample of accessions with known flowering time, we detected robust latitudinal clines in flowering time and circadian period length, which constitute strong indications of local adaptation to photoperiod in the shaping of flowering time variation in this species. In an attempt to elucidate putative genetic causes for the correlated variation between circadian rhythm and flowering time, we found that sequence variation and diverged expression in components regulating light input to the clock, PHYTOCHROME B (PHYB) and DE-ETIOLATED 1 (DET1) make them strong candidate genes. Finally, we present a comparative study of circadian network topology in the moss P. patens. Phylogenetic analyses and time series expression studies of putative clock homologues indicated that several core clock genes present in vascular plants appeared to be lacking in the moss. Consequently, while the clock mechanism in higher plants constitutes at least a three-loop system of interacting components, the moss clock appears to comprise only a single loop. We conclude that C. bursa-pastoris is a highly suitable model system for the further elucidation of the molecular variation that influences adaptive change in natural plant populations. Furthermore, we believe that the continuing study of the seemingly less complex circadian network of P. patens not only can provide insights into the evolution of the plant circadian system, but also may help to clarify some of the remaining issues of the circadian clock mechanism in higher plants.

L’évolution convergente des formes de croissance est un phénomène fondamental reliant l’écologie et l’évolution des plantes. Remarquablement illustré dans plusieurs systèmes insulaires, ce phénomène n’a jamais été identifié en Nouvelle-Calédonie, pourtant connue pour la richesse et l’originalité de sa flore. Par une approche combinant architecture des plantes, traits fonctionnels, taxonomie, phylogénie et données environnementales, cette thèse analyse l’histoire évolutive de la monocaulie, une forme de croissance mal connue, en Nouvelle-Calédonie. Les monocaules sont des plantes autoportantes ligneuses dont les fonctions majeures sont assurées par une seule tige apparente. En Nouvelle-Calédonie, elles sont représentées par 182 espèces dicotylédones appartenant à 41 genres et 30 familles et sont gravement menacées d’extinction. L’évolution répétée de la monocaulie en Nouvelle-Calédonie, issue d’au moins 31 événements d’apparition, est l’un des cas les plus remarquables de convergence en milieu insulaire. Dans le genre Atractocarpus, la monocaulie est apparue récemment deux à trois fois via diverses réductions des branches en inflorescences, montrant l’importance des processus hétérochroniques dans l’évolution des formes de croissance. La monocaulie est fortement corrélée à plusieurs traits démontrant des contraintes majeures dans la coordination fonctionnelle. L’évolution de la monocaulie est fortement associée aux forêts denses humides et au substrat ultramafique, et semble avoir contribué à la diversification des lignées par des phénomènes de partitionnement de niche. La remarquable convergence de la monocaulie en Nouvelle-Calédonie peut s’expliquer par quatre hypothèses majeures liées (i) à la structure particulière des forêts denses humides (en lien avec les cyclones) favorisant l’exploration unidirectionnelle de l’espace, (ii) aux contraintes édaphiques liées aux substrats ultramafiques favorisant la paupérisation architecturale, (iii) à l’absence historique de grands brouteurs, auxquels les monocaules sont particulièrement sensibles, et (iv) à la persistance des forêts denses humides lors des épisodes glaciaires (servant de refuges pour ces espèces sensibles) et leur expansion post-glaciaire (fournissant de nombreuses opportunités écologiques)
The convergent evolution in growth habit is a fundamental phenomenon linking plant ecology and evolution. Remarkably illustrated in island biotas, this phenomenon has never been identified in the original and megadiverse New Caledonian biodiversity hotspot. Through an approach combining plant architecture, functional traits, taxonomy, phylogeny and environmental data, this thesis analyses the evolutionary history of the scarcely known monocaulous growth habit in New Caledonia. Monocauls are self-supporting woody plants whose cardinal functions rely on a single visible stem. In New Caledonia, they are represented by 182 dicotyledonous species belonging to 41 genera and 30 families and are critically endangered. The repeated evolution of the monocaulie in New Caledonia, resulting from at least 31 independent events, is one of the most remarkable cases of convergence in insular environments. In the genus Atractocarpus (Rubiaceae), monocauly evolved recently two to three times through branch reductions into inflorescences, emphasizing the importance of heterochronic processes in the evolution of growth habit. Monocauly is strongly correlated with several traits illustrating major constraints in functional coordination. The evolution of monocauly is strongly associated with rainforests and ultramafic substrate, and seems to have contributed to the diversification of lineages by niche partitioning. The remarkable convergence toward monocauly in New Caledonia can be explained by four major hypotheses: (i) the structural features of rainforests (related to cyclone frequency and intensity) favoring unidirectional exploration of space, (ii) the edaphic constraints associated with ultramafic substrate favoring architectural pauperization, (iii) the historical absence of large native browsers to which monocauls are particularly sensitive, and (iv) the persistence of rainforest during – and spread-out after – glacial episodes that served as refugia and further provided ecological opportunities

Methyl halides (CH3Cl, CH3Br and CH3I) are a group of volatile organic compounds which contribute to natural ozone degradation in the atmosphere. Plants are important emitters of these compounds due to the activity of halide/thiol methyltransferases (HTMTs), however, the function of HTMTs or methyl halide production is not known. In Arabidopsis thaliana, one HTMT is primarily responsible for the production of methyl halides and encoded by the HARMLESS TO OZONE LAYER (HOL) gene. In this study, an A. thaliana hol mutant and 35S::HOL lines were used to investigate the function of HOL. No support was found for the hypothesis that HOL contributes to salt stress tolerance via the disposal of excessive amounts of halides. On the contrary, increased HOL activity made 35S::HOL plants more susceptible towards salt stress. Despite the toxicity of methyl halides, differences in HOL activity in hol mutant and 35S::HOL plants did not affect the performance of insect herbivores, nor did it alter the microbial diversity of the rhizosphere in these lines. Microarray analysis of WT and hol mutant plants pointed to a function of HOL in starch/carbon metabolism and stress response pathways in A. thaliana. Brassica crops are significant emitters of methyl halides. A HOL-homologous gene (BraA.HOL.a) was identified in Brassica rapa. It was confirmed that this gene contributes to methyl halide production in B. rapa since braA.hol.a mutants had significantly reduced emission levels compared to WT. HOL-homologous genes were also found in various plant species throughout the plant kingdom including the moss Physcomitrella patens, which was shown to produce CH3Br. These data show that methyl halide production is an ancient mechanism in land plants. Moreover, phylogenetic analysis revealed a clear separation between HTMTs from glucosinolate (GL)-containing plants and HTMTs from eudicots without GLs supporting the hypothesis of a novel function of HTMTs in the order Brassicales.

Orientadores: Michel Georges Albert Vincentz, Renato Vicentini dos Santos
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O sequenciamento de um número crescente de genomas completos tem transformado a biologia. Mais especificamente, no campo da biologia evolutiva, tem se tornado possível endereçar perguntas centrais sobre o funcionamento ultimato dos mecanismos de transformação genética, com potencial impacto em todos os campos da biologia, assim como na filosofia. Esta tese está dividida em dois aspectos importantes da evolução de genomas: o processo de duplicação e fixação de genes duplicados, que é a base do surgimento de famílias multigênicas, e a evolução de redes de regulação, que determinam as relações de causalidade nos processos celulares. Os dois aspectos se relacionam à evolução da complexidade, tanto no que tange o conteúdo gênico dos seres vivos quanto nas interações mecanistica entre os genes via seus produtos (RNAs e proteínas basicamente). No primeiro aspecto abordamos a evolução de dois mecanismos biológicos que depende da ação integrada entre proteínas de famílias distintas: o mecanismo de síntese e degradação do polissacarídeo de parede celular xiloglucano, e o ciclo das chaperonas calreticulina/calnexina envolvidas no controle de qualidade de proteínas sintetizadas no retículo endoplasmático. Nossos trabalhos mostraram que uma forma primordial de xiloglucano, mais simples, surgiu antes da conquista do meio terrestre pela linhagem das plantas, ao contrário do que se imaginava, e que o ciclo calreticulina/calnexina é produto da subfuncionalização em eucariotos basais de uma chaperona ancestral, além do surgimento de funções específicas na família da calreticulina em plantas terrestres. O interesse em evolução de famílias multigênicas nos levou a desenvolver um método (Phylexpress) para análise de ortologia em larga escala, bem como permitir a integração de dados de expressão na tentativa de entender a dinâmica evolutiva da expressão gênica em famílias multigênicas. Utilizamos nosso método para revisitar o conteúdo gênico dos ESTs públicos de cana-de-açúcar, como prova de conceito, numa análise comparativa com o proteoma predito de sorgo. Nossos resultados mostram uma cobertura em termos de ortólogos para apenas ~58% do proteoma predito de sorgo em contrates com estimativas anteriores, com métodos mais simples, que chegaram a 90% do proteoma hipotético de cana. Para abordar a dinâmica evolutiva de redes de regulação, realizamos medições, em escala genômica, das alterações nos níveis de mRNAs de plântulas de sorgo e arroz em resposta a tratamentos de curta duração (2hrs) com sinais exógenos de ABA (hormônio vegetal) e dos açúcares glicose e sacarose. Utilizamos dados públicos e experimentalmente comparáveis de Arabidopsis thaliana em resposta aos mesmos sinais para realizar comparações que revelassem respostas conservadas ou divergentes entre ortólogos. Além disso, buscamos entender a dinâmica evolutiva das respostas transcricionais num contexto de duplicação gênica em famílias multigênicas, onde há diversos genes potencialmente redundantes do ponto de vista bioquímico/estrutural. Nossa abordagem sugere que redes de regulação gênica em eucariotos complexos evoluem majoritariamente de forma neutra, pois parecem apresentar uma taxa de divergência constante, que independe da rede (disparada por cada um dos diferentes sinais) e das espécies envolvidas. Nossos dados são complementares e potencialmente confirmadores de modelos recentes de evolução não-adaptativa em redes de regulação gênica. Concluímos que a evolução da complexidade em sistemas biológicos está parcialmente ligada à diminuição da eficiência da seleção, causada majoritariamente por números populacionais efetivos restritivos presentes nas linhagens de eucariotos complexos (vertebrados e plantas terrestres)
Abstract: The availability of complete sequences of a growing number of genomes is transforming biology. More specifically, in the field of evolutionary biology, it became possible to address central questions on the ultimate mechanisms underlying genetic changes. It has a broad impact on biology and philosophy as well. This thesis deals with two important aspects of genome evolution: the process of gene duplication and fixation of duplicated genes, which is the basis of the origins of multigenic families, and the evolution of genetic regulatory networks that determines the causality of the cellular processes. Both aspects are related to the evolution of complexity regarding the gene content of living forms and the mechanistic interaction between the gene products (mainly RNAs and proteins). In the first aspect we studied the evolution of two biological mechanisms depending on the integrated function of proteins from distinct families. The mechanism of synthesis and remobilization of xyloglucan, a plant cell wall polysaccharide, and the calreticulin/calnexin cycle of protein folding that takes place in the endoplasmic reticulum. Our work showed that a primordial form of xyloglucan already existed before the land conquest by plants. We propose that the calreticulin/calnexin cycle is the product of subfuncionalization of an ancestral eukaryotic chaperone, and plants evolved specific calreticulin functions due to gene duplication. Our interest in the evolution of multigenic families impelled the development of Phylexpress, a method dedicated to large-scale orthology analyses. It can integrate expression data in the context of multigenic families with the goal of understand the evolutionary dynamics of gene expression. We used Phylexpress to revisit the gene content of the publicly available sugarcane ESTs as a proof of concept. Our results showed that the ESTs sampled orthologs for just ~58% of the predict sorghum proteome, in contrast with previous estimations acconting for 90% of the hypotethical sugarcane proteome. In order to approach the evolutionary dynamics of regulatory networks, we measured global changes in gene expression of sorghum and rice plantlets in response to short-term treatments (2hrs) with exogenous ABA (plant hormone) and the sugars glucose and sucrose. We took public data from comparable experiments using Arabidopsis thaliana in order to unravel conserved and divergent responses across orthologs. Furthermore, we analyzed the evolutionary change in transcriptional responses in a context of gene duplications in multigenic families, leading to a set of potentially redundant genes in terms of biochemical/structural properties. Our approach suggests that gene regulatory networks in complex eukaryotes evolve mainly neutrally, in a constant rate that is independent of the analyzed network (triggered by each one of the signals) and the species. Our data is complementary and potentially confirmatory of recent models of nonadaptive evolution in regulatory networks. We concluded that the evolution of the complexity in biological systems is partially connected to the attenuation of the efficiency, mainly due to low effective population sizes present in the lineages that gave rise to complex eukaryotes (vertebrates and land plants)
Doutorado
Genetica Vegetal e Melhoramento
Doutor em Genetica e Biologia Molecular

In my thesis I aim to improve our phylogenetic and evolutionary knowledge of two ancient and distantly related groups of aquatic flowering plants, Hydatellaceae and Alismatales. While the phylogeny of monocots has received fairly intense scrutiny for two decades, some parts of its diversification have been less frequently investigated. One such lineage is the order Alismatales, which defines one of the deepest splits in monocot evolution. Many families of Alismatales are aquatic or semi-aquatic, and they have been implicated in historical discussions of monocot origins. I evaluate inter-familial relationships in the order, considering a suite of 17 plastid genes for 31 Alismatales taxa for all 13 recognized families. This study improves on our understanding of, and confidence in, higher-order Alismatales relationships. I also uncovered convergent gene loss of plastid-encoded subunits for the NADH dehydrogenase complex. I then expand monocot coverage outside Alismatales by including unpublished and newly sequenced data for other orders. This large-scale sample facilitated a re-evaluation of monocot phylogeny and molecular dating, the latter using 25 fossil constraints. Previously included in the monocot order Poales, Hydatellaceae are a small family of ephemeral aquatics relatively recently found to be the sister group of water lilies (Cabombaceae and Nymphaeaceae). I present the first molecular phylogeny of the family and evaluate aspects of the family's morphological evolution. I show how sexual system shifts are associated with shifts in other reproductive traits. I also infer a temporal scale for Hydatellaceae diversification using a two-step Bayesian approach. I use the resulting dated tree to address biogeographic patterns and aspects of niche evolution. I show that its "Gondwanan" distribution is the result of long-distance dispersal and not continental rifting, and demonstrate strong phylogenetic niche conservatism in the family. These studies expand our understanding of evolution in Hydatellaceae, and provide a substantial update to our understanding of Alismatales (and more generally monocot) phylogeny and divergence times.

Nepenthes pitchers are highly specialised insect traps. Adaptations for trapping include a viscoelastic fluid (uncommon), slippery wax crystals (common) and downward-pointing cells on the inner pitcher wall (ubiquitous), as well as the pitcher rim (peristome, present in almost all species) which is only slippery when wet. ‘Knock-out’ manipulations of individual trap components showed that in the elongate form of N. rafflesiana, the waxy inner pitcher wall was most important whereas the (wax-free) typical form relied mainly on the peristome. Thus they pursued different trapping strategies. In both varieties, the pitcher fluid was highly efficient at retaining prey, regardless of viscoelasticity. The ‘peristome aquaplaning’ mechanism implies that trapping success depends on environmental factors. Combined measurements of capture efficiency, meteorological data and peristome surface wetness showed that capture efficiency varied dramatically and was determined by rain, condensation, and peristome nectary secretion. Prey capture, trapping efficiency, nectar secretion and odour of N. rafflesiana (typical form) also increased significantly over the first two weeks after pitcher opening, while fluid pH and viscoelasticity decreased. The data showed that trapping is a dynamic, age-dependent process and support the importance of ‘peristome aquaplanning’ but not of the fluid’s viscoelasticity for prey capture. Pitcher characteristics vary considerably between Nepenthes species. I recorded morphological parameters from 58 species and mapped them onto published phylogenetic trees. Interspecific variation of trap characteristics indicates the presence of distinct trapping strategies that might have evolved in response to different ecological and environmental factors.

Evolution has a major influence on the plant cell wall and variation in primary cell wall (PCW) composition is known to exist between different angiosperm taxa. The PCWs of lower land plants have not been well studied. It is of interest to see what changes have taken place in PCW composition during plant evolution. One of the main qualitative variables within angiosperms is the presence of mixed-linkage b-(1(r)3),(1(r)4)-glucan (MLG) in gramineous monocots and apparent absence from non-gramineous monocots and dicots. Anomolously, the presence of MLG had been reported in the dicot Phaseolus aureus. In the present work, MLG was not detected (< 0.02% w/w of the cell wall) in P. aureus hypocotyls or in PCWs of all non-gramineous land plants tested (dicots, monocots, pteridophytes and bryophytes). One exception was Flagellaria guineensis, a close relative of the gramineous monocots, which on licheninase digestion produced the characteristic tri- and tetrasaccharide of MLG. MLG is therefore restricted to the Poaceae and some closely related members of the Poales. Xyloglucan was found in all land plants tested including bryophytes. Methylation analysis had indicated some of the glycosyl linkages typically found in xyloglucan are present in a cell wall polymer from the charophyte Nitella. However, Driselase-digestible xyloglucan was not detected (< 0.01% w/w of the cell wall) in Chara, a charophyte, thought to be closely related to land plants. In addition, PCWs of Chara, two other charophytes and Ulva lactuca were not digested to xyloglucan-derived oligosaccharides by cellulase or xyloglucan-specific endoglucanase. Land plants are thought to have organised from a single species of charophyte: it is therefore likely the putative ancestor, unlike other charophytes, had xyloglucan in its cell walls.

Plants produce a rich variety of natural products to face environmental constraints. Enzymes of the cytochrome P450 CYP98 family are key actors in the production of phenolic bioactive compounds. They hydroxylate phenolic esters for lignin biosynthesis in angiosperms, but also produce various other bioactive phenolics. We characterized CYP98s from a moss, a lycopod, a fern, a conifer, a basal angiosperm, a monocot and from two eudicots. We found that substrate preference of the enzymes has changed during evolution of land plants with typical lignin-related activities only appearing in angiosperms, suggesting that ferns, similar to lycopods, produce lignin through an alternative route. A moss CYP98 knock-out mutant revealed coumaroyl-threonate as CYP98 substrate in vivo and showed a severe phenotype. Multiple CYP98s per species exist only in the angiosperms, where we generally found one isoform presumably involved in the biosynthesis of monolignols, and additional isoforms, resulting from independent duplications, with a broad range of functions in vitro
Graduate
2017-08-31

Land plants are characterized by the alternation of two generations: the haploid gametophyte and the diploid sporophyte. As land plants evolved from bryophytes to vascular plants, the sporophyte became the dominant generation in the life cycle. The sporophytes of bryophytes are developmentally simple structures characterized by determinate growth. In contrast, the sporophytes of vascular plants diverged to become highly variable and often complex structures with indeterminate growth. KNOTTED1-LIKE HOMEOBOX (KNOX) genes encode homeodomain containing transcription factors that are key regulators of sporophyte development. KNOX genes are divided into two subclasses, class 1 and class 2. The critical role of class 1 KNOX genes in the apical growth of all extant land plants studied to date, suggests that modifications to class 1 KNOX gene function may have played an important role in sporophyte evolution. However, the nature of any such modifications is largely unknown. In this study, a number of cross-species complementation experiments were carried out to determine the extent to which class 1 KNOX gene function is conserved between different land plant groups. The role of KNOX genes in hornworts was also investigated because hornworts are believed to be the sister group to vascular plants. The work presented in this thesis demonstrates that the function of class 1 KNOX genes is conserved between lycophytes and angiosperms. In contrast, the function of class 1 KNOX genes appears to have diversified between bryophytes and vascular plants. Collectively, these results indicate increased complexity of class 1 KNOX gene function during the evolution of land plants.

Transcriptomic analyses from across eukaryotes indicate that most of the genome is transcribed at some point in the developmental trajectory of an organism. One class of these transcripts is termed long intergenic noncoding RNAs (lincRNAs). Recently, attention has focused on understanding the evolutionary dynamics of lincRNAs, particularly their conservation within genomes. Here, we take a comparative genomic and phylogenetic approach to uncover factors influencing lincRNA emergence and persistence in the plant family Brassicaceae, to which Arabidopsis thaliana belongs. We searched 10 genomes across the family for evidence of >5000 lincRNA loci from A. thaliana. From loci conserved in the genomes of multiple species, we built alignments and inferred phylogeny. We then used gene tree/species tree reconciliation to examine the duplication history and timing of emergence of these loci. Emergence of lincRNA loci appears to be linked to local duplication events, but, surprisingly, not whole genome duplication events (WGD), or transposable elements. Interestingly, WGD events are associated with the loss of loci for species having undergone relatively recent polyploidy. Lastly, we identify 1180 loci of the 6480 previously annotated A. thaliana lincRNAs (18%) with elevated levels of conservation. These conserved lincRNAs show higher expression, and are enriched for stress-responsiveness and cis-regulatory motifs known as conserved noncoding sequences (CNSs). These data highlight potential functional pathways and suggest that CNSs may regulate neighboring genes at both the genomic and transcriptomic level. In sum, we provide insight into processes that may influence lincRNA diversification by providing an evolutionary context for previously annotated lincRNAs.

Pollinator sharing in mixed species communities is expected to significantly contribute to mating patterns in contemporary populations but may also affect the evolutionary trajectory of traits associated with plant mating. In this thesis, I considered how the spring environment and pollinator sharing may contribute to the widespread convergence in traits among spring flowering species using comparative biology. The proposed correlation between a spring flowering phenology and white or light floral colour, fleshy fruits, woody growth forms and understory occupation is confirmed. In addition, I examined the effects of pollinator responses to community and population traits to determine the relative importance of inter- and intraspecific interactions in pollinator mediated reproductive success of a spring flowering species, Trillium grandiflorum. In this study, the reproductive success of T. grandiflorum was pollen limited. However, the magnitude of pollen limitation was influenced only by intraspecific density and varied independently of community diversity. The results of this thesis contribute significantly to our understanding of pollinator-mediated interactions in spring flowering communities but also highlight future avenues of investigation.

Plants produce a variety of small molecules, including those essential for survival in all conditions (primary metabolites) or for more ecologically specific conditions (secondary metabolites). While primary metabolic pathways are broadly shared among plants, secondary metabolism is under constant selective pressure towards chemical innovation, given the continual fluctuation of the environment. Thus, plant secondary metabolism - whose constituents number in the hundreds of thousands - is lineage-specific, highly structurally diverse, and ultimately of high value to medicine, agriculture, and industry. Efforts to optimize the production of specific metabolites or to discover new compounds remain difficult primarily due to inadequate understandings of the metabolic genes involved and how these genes are regulated. This work first examines co-regulation, a major form of organization by which plant secondary metabolic genes are organized. In response to the bacterial crop pathogen Pseudomonas syringae, Arabidopsis thaliana and its relatives in the mustard family produce numerous secondary metabolites from the amino acid tryptophan, including the antimicrobial compound camalexin. However, hundreds of biosynthetic genes of unknown function are also simultaneously upregulated. Using metabolic profiling and co-expression analysis, I helped to identify the complete biosynthetic pathway to the indole-3-carbonylnitriles (ICNs), a previously unknown class of compounds. When the cytochrome P450 gene CYP82C2 is mutated, biosynthesis of the compound 4-hydroxy-ICN (4OH-ICN) is abolished, and plant defense against P. syringae is impaired. Conversely, addition of 4OH-ICN to plants is sufficient to suppress bacterial growth. Next, this work examines the evolution of camalexin and 4OH-ICN metabolism. Cytochrome P450-directed secondary metabolism has been shown almost without exception to be evolutionarily derived from changes to enzymes with broad substrate specificity. By contrast, I observe through genetics, enzyme phylogenetic analysis, and transient expression assays that the ICN and camalexin biosynthetic pathways evolved from a common chemical substrate. In particular, changes to camalexin catalysis by the newly duplicated gene CYP71A12 led to the formation of ICN metabolism in several mustard species, although both compounds are directly derived from indole cyanohydrin. Furthermore, 40H-ICN is an extremely recently evolved metabolite, derived from a flurry of genic, epigenetic and transposon-mediated rearrangements of a yet-more recent gene duplicate (CYP82C2). These regulatory changes to CYP82C2 lead to its pathogen-inducibility solely in the species A. thaliana. I additionally identify WRKY33 and MYB51 as two sets of defense regulators that carefully fine-tune 40H-ICN metabolism by direct biosynthetic gene regulation. WRKY33 transcription factor, which is involved in the species-specific regulation of CYP82C2, is conserved throughout flowering plants, indicating that transcriptional recruitment is an important feature in the expansion of secondary metabolism. Finally, this work probes possible molecular functions of 40H-ICN and camalexin by exploring the molecular mechanisms underlying their secretion from roots and regulation of cell death processes. This study ultimately reveals that the proliferation of diverse chemical arsenals in plants is greatly aided by the regulatory capture of new and rapidly evolving genes by evolutionarily more stable transcription factors. Future emphases on transcriptional regulators of secondary metabolism may thus aid in the discovery of new secondary metabolic pathways on a more rapid scale.

The so-called creep strength enhanced ferritic (CSEF) 9-12% Cr steels have been identified as the most promising class of materials for some of the key components in ultra-supercritical fossil-fired power plants, including the main steam pipes, headers and superheater tubings. These steels are less costly, and they have a lower coefficient of thermal expansion and a higher thermal conductivity when compared with austenitic stainless steels, making them less susceptible to degradation through thermal fatigue. However, experience has shown that the weldments in these steels are particularly prone to premature creep failure, due to a localised form of cracking in the heat-affected zone {HAZl, which is referred to as Type IV cracking. The work presented in this thesis is concerned with the effects of residual stresses and constraint on Type IV cracking. In the first part of this work, the residual stresses in a 25.4 mm thick, 324 mm diameter pipe girth weld, made in a P91 steel pipe, have been measured in both the as-welded and post weld heat treated (PWHT) conditions using neutron diffraction, and compared with the corresponding metallurgical zones across each weld. It was found that the highest as-welded tensile stresses resided near the outer boundary of the HAZ, and towards t he weld root region and these were not fully relieved by the applied PWHT. In both conditions substantial tensile direct and hydrostatic stresses existed across the HAZ, including the fine-grained and intercritically-annealed regions, where premature Type IV creep failures manifest in 9-12% Cr steel welds. Compressive stresses were found in the weld metal coinciding with the last weld bead to be deposited. In the second part of the work, creep tests were conducted at 625°C on cross-weld and simulated fine-grained HAZ specimens. The contributions of specific influences on creep performance (such as residual stress, constraint and creep damage associated with relaxation of residual stresses during PWHT) were then systematically examined. It was found that the geometric constraint (introducing a triaxial stress state) was beneficial in improving creep rupture life and that residual stresses (of the order of 50 MPa) showed a. clear reduction in life. Moreover there was some evidence that residual stress relaxation associated with PWHT may introduce some creep damage. The digital image correlation (DIC) technique was applied to resolve tensile and time dependent creep deformation properties along the length of P91 cross-weld samples. The results demonstrated the capability of the Die technique for full field measurement of strain during both room-temperature-tensile and high temperature creep tests in the vicinity of welded joints, where the gradients in microstructure and mechanical properties can be steep.

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Receptores cinases (RLKs) compõem uma grande famíla de proteínas transmembrânicas que possuem funções importantes na propagação e percepção de sinais celulares nas plantas. Em Arabidopsis thaliana, a superfamília de RLK é composta de mais de 600 membros e vários destes, principalmente aqueles que possuem repetições ricas em leucina (LRR), são considerados excelentes alvos para manipulação molecular em cultivares superiores no intuito de aumentar a produtividade e a resistência contra estresses bióticos e abióticos. A subfamília LRRII é particularmente relevante neste aspecto uma vez que seus membros apresentam funções duplas tanto no desenvolvimento quanto na resposta de defesa da planta. Apesar da relevância desta superfamília e da recente finalização do sequenciamento do genoma de tomateiro, a superfamília de RLK de tomate ainda não se encontra caracterizada e são poucos os trabalhos que analisaram a função biológica de seus membros. Neste trabalho, foi construído um inventário completo dos membros da superfamília de RLK de tomate. Para identificar os membros da superfamília RLK em tomate, foi realizado uma análise filogenética utilizando a superfamília de RLK de Arabidopsis como modelo. Um total de 647 RLKs foram recuperados do genoma de tomate e estes encontravam- se organizados no mesmo clado das subfamílias de RLKs de Arabidopsis. Apenas oito das 58 subfamílias exibiram expansão/redução específica no número de menbros comparado com Arabidopsis e apenas seis RLKs foram específicos em tomate, indicando que os RLKs de tomate compartilham aspectos funcionais e estruturais com os RLKs de Arabidopsis. Também foi caracterizado a subfamília LRRII através de análises filogenéticos, genômico, expressão gênica e interação com o fator de virulência de begomovírus, o nuclear shuttle protein (NSP). Os membros da subfamília LRRII de tomate e Arabidopsis demonstraram-se altamente conservados tanto em sequência quanto em estrutura. No entanto, a maioria dos pares ortólogos não mostraram conservados em relação à expressão gênica, indicando que estes ortólogos tenham se divergido na função após a especiação do ancestral comum entre o tomate e Arabidopsis. Baseado no fato de que membros de RLKs de Arabidopsis (NIK1, NIK2, NIK3 e NsAK) interagem com o NSP de begomovirus, foi verificado se ortólogos de NIKs, BAK1 e NsAK interagem com o NSP de Tomato Yellow Spot Virus (ToYSV). Os ortólogos dos genes que interagem com o NSP em tomate, SlNIKs e SlNsAK, interagiram especificamente com NSP na levedura e demonstraram um padrão de expressão consistente com o padrão de infecção de geminivírus. Além de sugerir uma analogia funcional entre estes ortólogos, estes resultados confirmam a observação anterior de que as interações NSP-NIK não são específicos para um vírus ou para um hospedeiro. Portanto, a sinalização antiviral mediado por NIK provavelmente ocorre em tomate, sugerindo que NIKs de tomate sejam alvos potenciais para manipular a resistência contra begomovírus que infectam esta planta.
Receptor-like kinases (RLKs) represent a large family of transmembrane proteins that play important roles in cellular signaling perception and propagation in plants. In Arabidopsis thaliana, the RLK superfamily is made-up of over 600 proteins and many of these RLKs, mainly those bearing leucine-rich repeats (LRR), have been considered as excellent targets for engineering superior crops with enhancement of yield and resistance to biotic and abiotic stresses. The LRRII-RLK subfamily is particularly relevant due to the dual function of its members in both development and defense. In spite of the relevance of the RLK family and the completion of the tomato genome sequencing, the tomato RLK family has not been characterized and a framework for functional predictions of the members of the family is lacking. In this investigation we disclosed a complete inventory of the members of the tomato RLK family. To generate a complete list of all members of the tomato RLK superfamily, we performed a phylogenetic analysis using the Arabidopsis RLKs as a template. A total of 647 RLKs were identified in the tomato genome, which were organized into the same RLK subfamily clades as Arabidopsis. Only eight of 58 RLK subfamilies exhibited specific expansion/reduction compared to their Arabidopsis counterparts and only six proteins were lineage-specific in tomato, indicating that the tomato RLKs share functional and structural conservation with Arabidopsis. We also characterized the LRRII-RLK family by phylogeny, genomic analysis, expression profile and interaction with the virulence factor from begomoviruses, the nuclear shuttle protein (NSP). The LRRII subfamily members from tomato and Arabidopsis were highly conserved in both sequence and structure. Nevertheless, the majority of the orthologous pairs did not display similar conservation in the gene expression profile, indicating that these orthologs may have diverged in function after speciation of tomato and Arabidopsis common ancestor. Based on the fact that members of the Arabidopsis RLK superfamily (NIK1, NIK2, NIK3 and NsAK) interact with the begomovirus nuclear shuttle protein (NSP), we examined whether the tomato orthologs of NIK, BAK1 and NsAK genes interacted with NSP of Tomato Yellow Spot Virus (ToYSV). The tomato orthologs of NSP interactors, SlNIKs and SlNsAK, interacted specifically with NSP in yeast and displayed an expression pattern consistent with the pattern of geminivirus infection. In addition to suggesting a functional analogy between these phylogenetically classified orthologs, these results expand our previous observation that NSP-NIK interactions are neither virus-specific nor host-specific. Therefore, NIK-mediated antiviral signalling is also likely to operate in tomato, suggesting that tomato NIKs may be good targets for engineering resistance against tomato-infecting begomoviruses.

Los terpenos constituyen el mayor grupo de metabolitos secundarios, siendo componentes de las glándulas de aceites esenciales, de las flores y de las resinas defensivas de plantas aromáticas, a los que proporcionan sus aromas y sabores característicos. Los terpenos volátiles se asocian a la defensa de muchas especies de plantas, animales y microorganismos contra depredadores, patógenos y competidores. Por otra parte, estos compuestos parecen servir como señales para atraer a los polinizadores y agentes dispersores de semillas, así como a depredadores de plagas. El estudio de compuestos orgánicos volátiles emitidos durante el desarrollo del fruto y después del desafío con diferentes agentes bióticos puede ayudar a conocer las interacciones de los frutos carnosos no sólo con vertebrados dispersores y depredadores, sino también con insectos y microorganismos. Los frutos carnosos son particularmente ricos en volátiles. En los frutos cítricos, los monoterpenos son los principales componentes de las glándulas del aceite esencial de la cáscara (flavedo), siendo el D-limoneno el más abundante (hasta 95% en la naranja). Esta característica hace que los cítricos sean un buen sistema modelo para el estudio de la función de los terpenos en los frutos. La biología molecular moderna permite la realización de experimentos para comprobar la función de terpenos por medio del uso de organismos transformados genéticamente en los que se han manipulado los niveles de acumulación de dichos compuestos. En este trabajo, se ha utilizado un plásmido que alberga el cDNA completo del gen de una limoneno sintasa de cítricos (CiTMTSE1) en orientación antisentido (AS) o sentido (S) para modificar la expresión y la acumulación de D-limoneno en plantas de naranjo dulce (Citrus sinensis L. Osb.). La acumulación de D-limoneno en las frutas AS se redujo drásticamente pero la acumulación de otros terpenos también se modificó, afectando a compuestos tales como alcoholes monoterpenos, cuya concentración se incrementó en la cáscara de las frutas. Las plantas transformadas fueron morfológicamente indistinguibles de las plantas control (WT) y de las plantas transformadas con el vector vacío (EV). Los frutos transgénicos fueron desafiados con un insecto plaga y con diferentes patógenos para probar si la alteración de los niveles de acumulación de estos volátiles daba como resultado una mejora en la respuesta del flavedo frente a plagas y patógenos. Los machos de la mosca mediterránea de la fruta (Ceratitis capitata) expuestos a las frutas AS y EV en ensayos en túnel de viento fueron significativamente más atraídos por el aroma de los frutos control EV. En otros experimentos de desafío con el hongo de la podredumbre verde Penicillium digitatum y la bacteria causante de la cancrosis de los cítricos Xanthomonas axonopodis subsp. citri, las frutas transgénicas con un contenido reducido de D-limoneno mostraron elevada resistencia a estos patógenos. El alto contenido en D-limoneno en la cáscara de naranjas maduras puede ser una señal para la atracción de plagas y microorganismos que podrían estar involucrados en la facilitación del acceso a la pulpa de los frugívoros dispersores de semillas. El análisis de la expresión génica global en el flavedo de las frutas transgénicas vinculó la disminución de D-limoneno y la reducción de la expresión de genes del metabolismo de monoterpenos con la activación de la expresión de genes implicados en inmunidad innata, incluyendo factores de transcripción, genes de quinasas implicadas en la entrada de Ca2+ en la célula y genes implicados en la activación de las cascadas de MAPKs, con la consiguiente activación de la ruta de señalización de ácido jasmónico (JA), lo que provocó la activación del metabolismo de JA y un aumentó drástico de la acumulación de JA en la cáscara de la naranja tras el desafío con P. digitatum, lo que explicaría la resistencia al menos a hongos necrotrofos observada en las frutas. Estos resultados indican que la acumulación de D-limoneno en la cáscara de la naranja estaría implicada en la interacción trófica entre las frutas, insectos y microorganismos, lo cual proporciona una visión mucho más amplia de las funciones de los terpenos en la naturaleza. También representa una alternativa muy prometedora para incrementar la resistencia o tolerancia de las plantas frente a patógenos y plagas.
Terpenes, the largest group of secondary metabolites, are well known as constituents of essential oils, floral scents and defensive resins of aromatic plants, to which they impart their characteristic aromas and flavors. Terpene volatiles defend many species of plants, animals and microorganisms against predators, pathogens and competitors. Moreover, those compounds seem to serve as advertisements to attract pollinators and seed-dispersal agents as well as pest predators. The study of VOCs emitted during fruit development and after challenge with different biotic agents may help to determine the interactions of fleshy fruits not only with legitimate vertebrate dispersers and predators, but also with insects and microorganisms. Fleshy fruits are particularly rich in volatiles. In citrus fruits, monoterpenes are the main components of the essential oil glands of the peel, being D-limonene the most abundant one (up to 95% in orange fruits). This characteristic makes citrus a good model system for studying the function of terpenes in plants. Modern molecular biology now enable experiments to test terpenoid function by the use of genetically transformed organisms in which terpene levels have been manipulated. In this work, a plasmid harboring the complete cDNA of a citrus limonene synthase gene (CiTMTSE1) in antisense (AS) or sense (S) orientation was used to modify the expression and accumulation of D-limonene of sweet orange (Citrus sinensis L. Osb) plants. D-limonene accumulation in AS fruits was dramatically reduced but the accumulation of other terpenoids was also modified, such as monoterpene alcohols, whose concentration increased in the peel of fruits. Genetically transformed plants were morphologically indistinguishable from wild-type (WT) and empty vector (EV) control plants. Transgenic fruits were challenged against a pest and different pathogens to test whether volatile profile alteration results in an improvement in the response of the fruit flavedo against them. Males of the Mediterranean fruit fly (Ceratitis capitata) exposed to AS fruits versus EV in wind tunnel assays were significantly more attracted to the odor of EV control fruits. In separate experiments with the green mould rot of citrus fruits and citrus canker caused by Penicillium digitatum and Xanthomonas axonopodis subsp. citri, respectively, transgenic fruits with a reduced content in D-limonene showed resistance to both pathogens. High D-limonene content in mature orange peels may be a signal for attractiveness of pests and microorganisms which might be likely involved in facilitating the access to the pulp of seed dispersal frugivores. A global gene expression analysis of the flavedo of AS transgenic fruits linked the decrease of D-limonene and monoterpene metabolism to the up-regulation of genes involved in the innate immunity response, including transcription factors together with Ca2+ entry into the cell and activation of MAPK cascades, contributing to activation of jasmonic acid (JA) signaling, which triggered the up-regulation of JA metabolism and drastically increased the accumulation of JA in orange peels upon fungal challenge, explaining the resistance to necrotrophic fungi observed in AS fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms and provide a much more comprehensive view of roles of terpenes in nature. It also represents a very promising alternative for increasing resistance or tolerance of plants to pathogens.
Rodríguez Baixauli, AM. (2013). Genetic engineering of plant volatiles in fleshy fruits: pest repellency and disease resistance through D-limonene downregulation in transgenic orange plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31655
TESIS

This work is a union of molecular biology and evolutionary biology in addressing questions regarding the evolution of organisms and their genes. This project focuses on major groups of seed plants (angiosperms, and four gymnosperms groups: conifers, cycads, Ginkgo and Gnetales). These organisms diverged roughly three hundred million years ago and evolved independently since then. Therefore, morphological characters have limited power in resolving their phylogeny. With the advancement of molecular methods, sequences of genes have been widely used in addressing evolutionary relationships. Plant molecular phylogenies have been traditionally done using chloroplast gene rbcL and nuclear rRNA genes. While these molecular markers revolutionized plant systematics, they do not contain enough phylogenetic information to resolve the phylogeny of seed plants. I used normalized splits, as a measure from spectral analysis, in examining data sets and identifying genes and sites within genes that are more reliable for building phylogenies and combining data sets. I used the genes encoding the largest subunit of RNA polymerase I, II and III (rpa1, rpb1 and rpc1, respectively) as a new line of evidence from the nuclear genome to address seed plant phylogeny. These protein-coding genes refute the Anthophyte hypothesis (which relates Gnetales and angiosperms) and support the Gnepines hypothesis (which places Gnetales within conifers as a sister group to Pinaceae). I also examined other available data sets from chloroplast, mitochondrial and 18S rRNA genes. A combined data set of RNA polymerases and seven other genes strongly support the Gnepines hypothesis. Other branches of seed plant phylogeny are also well supported using this data set. Angiosperms and gymnosperms form monophyletic groups, cycads are at the base of gymnosperms, followed by Ginkgo and a branch leading to conifers and Gnetales. I also studied the rate and pattern of molecular evolution of rpa1, rpb1 and rpc1 genes. A gene phylogeny of these genes strongly support that they share a common ancestor and that rpb1 and rpc1 are closer to one another than either are to rpa1. The pattern of functional constraint in these genes clearly follows known structural properties of the RNA polymerases. Evolutionary rate analyses show that rpa1 evolves more rapidly at non-synonymous sites and at the amino acid level, followed by rpc1 and rpb1. Interestingly, the evolutionary rate of these genes (and the proteins they encode) is positively related to the amount of concerted evolution in the genes transcribed by each RNA polymerase. This suggests a possible impact of concerted evolution (generated by recombination) on the molecular evolution of the largest subunit of RNA polymerases.

Includes abstract.
Includes bibliographical references.
Comparative biologists have refined the synthesis of molecularly dated phylogenies and ecological data into an important tool to reconstruct the evolution of species and biomes, and to unravel the history and role of abiotic determinants of diversity patterns (fire, climate, tectonism). This has been extended into the cross-disciplinary, geobiological approach of 'geoecodynamics' has exploits the spatial fidelity of locally restricted organisms to unravel the temporal and spatial evolution of landforms. This research approach is adopted here across 11 plant clades representing six prominent plant families of the Cape flora (Asteraceae, Orchidaceae, Restionaceae, Cyperaceae, Poaceae and Proteaceae) to infer (i) the relative roles of climatic changes and neotectonic uplift in shaping the CFR since the Early Miocene, and to detemine (ii) whether contrasting evolutionary processes (adaptive versus non-adaptive) exhibit spatial structuring within the flora, given the complex topography of the region.

The genome of living organisms is constantly subjected to the environmental influences that result in different negative, negligible or positive impacts. The ability to maintain the genome integrity and simultaneously provide its flexibility is the main determinant for the evolutionary success of any species. One of the important aspects of genome maintenance is the precise regulation of the DNA repair machinery. Results reported here indicate the existence of a tight, age-dependent regulation of homologous recombination, one of the two main DNA double-strand break repair pathways. We show that recombination is influenced by conditions such as the change of temperature (cold or warm), day length, water availability (drought or overwatering stress) and salinity. These stresses not only influence the genome stability of stress-subjected generations but also change the recombination in subsequent generations. This indicates the possible involvement of homologous recombination in plant evolution and development of plant stress tolerance.
xiv, 121 leaves ; 29 cm.

Angiosperms are one of the most dominant groups on Earth, and have fundamentally changed global ecosystem patterns and function. Therefore, unravelling their evolutionary history is key to understanding how the world around us was formed, and how it might change in the future. In this thesis, I use genome-scale data to investigate the evolutionary patterns and timescale of angiosperms at multiple taxonomic levels, ranging from angiosperm-wide to genus-level data sets. I begin by using the largest combination of taxon and gene sampling thus far to provide a novel estimate for the timing of angiosperm origin in the Triassic period. Through a range of sensitivity analyses, I demonstrate that this estimate is robust to many important components of Bayesian molecular dating. I then explore tactics for phylogenomic dating using multiple molecular clocks. I evaluate methods for estimating the number and assignment of molecular clock models, and strategies for partitioning molecular clock models in analyses of multigene data sets. I also demonstrate the importance of critically evaluating the precision in age estimates from molecular dating analyses. Finally, I assess the utility of plastid data sets for resolving challenging phylogenetic relationships, focusing on Pimelea Banks & Sol. ex Gaertn. Through analysis of a multigene data set, sampled from many taxa, I provide an improved phylogeny for Pimelea and its close relatives. I then generate a plastome-scale data set for a representative sample of species to further refine the Pimelea phylogeny, and characterise discordant phylogenetic signals within their chloroplast genomes. The work in this thesis demonstrates the power of genome- scale data to address challenging phylogenetic questions, and the importance of critical evaluation of both methods and results. Future progress in our understanding of angiosperm evolution will depend on broader and denser taxon sampling, and the development of improved phylogenetic methods.

Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第15486号
人博第516号
新制||人||125(附属図書館)
21||人博||516(吉田南総合図書館)
27964
京都大学大学院人間・環境学研究科相関環境学専攻
(主査)准教授 瀬戸口 浩彰, 教授 松井 正文, 教授 加藤 眞, 准教授 市岡 孝朗
学位規則第4条第1項該当

Phytophthora infestans, the oomycete pathogen responsible for late blight of potato and tomato, is regarded as the biggest threat to global potato production and is thought to cost the industry around £6 billion annually. Traditionally, fungicides have been used to control the disease, but this is both economically and environmentally costly, as multiple chemical applications may be required during a single growing season. P. infestans has rapidly overcome genetic resistances introduced into cultivated potato from wild species. This provides the rationale for developing artificial resistance genes to create durable resistance to late blight disease.Phytophthora species secrete essential effectors into plant cells that target critical host cellular mechanisms to promote disease. One such P. infestans effector is AVR3a^KI which is recognised by the potato R3a protein, a member of the CC-NB-LRR type resistance gene family. However, the closely related virulent form, AVR3a^EM, which is homozygous in more than 70% of wild P. infestans isolates, evades this recognition. Domain swapping experiments have revealed that the LRR domain of R3a is involved in recognition of AVR3a^KI, as the CC-NB domain of an R3a-paralog which does not mediate recognition of AVR3a^KI, is able to induce a HR when combined with the LRR of wild-type R3a. However, a chimeric protein consisting of the CC-NB domain of a more distantly-related homolog of R3a and the LRR of domain of R3a, is unable to recognise AVR3a^KI, suggesting that function is achieved only when the different domains of an R protein are attuned to recognition and signalling. Gain-of-function variants of R3a (R3a*), engineered by an iterative process of error-prone PCR, DNA fragmentation, re-assembly of the leucine rich repeat (LRR)-encoding region of R3a, are able to recognise both forms of AVR3a. This gain-of-recognition is accompanied by a gain-of-mechanism, as shown by a cellular re-localisation from the cytoplasm to prevacuolar compartments upon perception of recognised effector forms. However, R3a* variants do not confer resistance to AVR3a^EM-carrying isolates of P. infestans.Future efforts will target the NB-ARC domain of R3a, in a bid to fine-tune the intra-cellular signalling of gain-of-recognition R3a* variants. It is hoped that a shuffled R3a* gene, capable of conferring resistance to P. infestans isolates harbouring AVR3a^EM, will provide durable late blight resistance when deployed in the field in combination with other mechanistically different R proteins.