Дисертації з теми "Plant biochemical genetics"

A number of major crop species, such as allohexaploid wheat and autotetraploid potato are polyploid. Potato is the fourth most important crop in terms of production and has become an important food source in many countries. Therefore, the molecular analysis was directed towards investigating ways to develop markers to assist the potato breeding process; for example breeding for powdery scab disease resistance, and tolerance to cold induced sweetening. Polyploids have more possible genotypes per population, allele dosage effects and increased marker complexity compared to diploids. Potato is also outcrossing and therefore highly heterozygous. Various methods for detecting marker-trait associations including, linkage, quantitative trait locus (QTL) and association mapping were studied and protocols developed. A mapping population was produced and a number of traits were measured including powdery scab resistance. Powdery scab disease assays were carried out over six seasons and markers associated with disease resistance were identified. Markers associated with resistance to powdery scab were identified on chromosomes I, IV, V, VI, VIII and IX using analysis of variance (ANOVA). Linkage maps were produced for each parent of the population and QTL associated with resistance and susceptibility to disease were identified using interval mapping, which revealed QTL on chromosomes II, V, VII , VIII, IX and an unanchored linkage group. QTL were detected across years on regions of chromosomes VIII and IX. These QTL results had some overlap with the marker-trait associations that were identified using ANOVA analysis. Another marker identification technique was tested, known as association or linkage disequilibrium mapping. Alleles of candidate genes were tested for association with cold-induced sweetening using a germplasm collection. The alleles identified as important were of the apoplastic invertase and UGPase genes and a unique interaction between alleles of the apoplastic invertase and apoplastic invertase inhibitor was also detected. This thesis describes the first study into the genetics of powdery scab resistance and the markers identified as associated with resistance will be validated for use in a marker-assisted selection (MAS) programme. The tools and resources developed as part of this thesis are vital to the potato breeding programme that requires the identification of associated molecular markers.

Three isoenzyme systems (amylase, esterase and glutamate oxalo acetate transaminase) were examined in seeds of pea ( Pisum sativum L.) and showed clear variations in their band patterns on gel electro phoresis between different lines. The inheritance of these isoenzyme systems, and the location of their structural genes on the pea genome were investigated. Reciprocal crosses were made between lines, F2 seeds were analysed for segregation in the band patterns of the isoenzymes, and F2 plants were investigated to find linkage between the genes for these isoenzymes and genes for selected morphological markers. The results obtained showed that each of the investigated isoenzyme systems is genetically controlled by co-dominant alleles at a single locus. The gene for amylase ( Amy ) was found to be on chromosome 2, linked to the loci k and wb ( wb.. .9-k. . .25.. .Amy ). The gene for esterase ( Est ) was found to be linked to the gene Br (chromosome 4) but the exact location is uncertain because of a lack of morphological markers. The gene for glutamate oxaloacetate transaminase ( Got ) was found to be on chromosome 1 linked to the loci a and d ( a...24...Got...41.. d). Gel electrophoresis techniques have also been used to investigate genetically controlled variation in the major subunits (50,000 Mr) of vicilin, a storage protein of Pisum sativum L. The Fl protein band patterns were shown to be additive with respect to those of the parental lines and to be identical in reciprocal crosses. Genetic analysis of the F2 plants indicated that the 50,000 Mr vicilin subunits band pattern is controlled by a pair of co-dominant genes at a single locus. The F2 data were used to locate this major vicilin gene locus ( Vc-1 ) to chromoscane 7, closely linked to the r locus (for round and wrinkled seed surface). A third member of pea legumin gene family, denoted legB, has been sequenced using the "dideoxy chain termination" method with the M1 3 sequencing system. The complete nucleotide sequence showed that this gene has a general form typical of an eukaryotic gene. The homolgies between this gene and the previously published gene "legA" 'were estimated and showed strong homology between the two genes with eight amino acid substitutions and deletion of 14 bp in the third intron (IVS-3).The inheritance of ribosomal RNA (rRNA) genes in ( Pisum sativum L.) was investigated in a cross between two different lines, where length variation in rDNA fragments of Eco RI digests was observed. The results obtained showed that the rRNA genes are controlled by simple Mendelian system with "co-dominance" between alleles. In order to locate the rRNA gene sites to positions on the chromosomes, the segregation of ECO. RI restriction fragments of rDNA from F2 plants with respect to genes for selected morphological markers on chromosomes 4 and 7 (the chromosomes known to have nucleolus organizer regions) were tested. The F2 data showed no linkages between the selected markers and rRNA genes, therefore, in situ hybridization using rDNA radioactive probe ((^3)H- labelled rDNA clone, pHAI) and physical mapping procedures were used. The results obtained have located the rRNA gene sites to nucleolus organizer regions (satellite constrictions) at 138 and 60 map units from the centromeres of chromosomes 4 and 7, respectively.

Plant oils are used in food, fuel, and feedstocks for many consumer products, and so understanding the process by which they are made and modified will help us to make plant oils more healthy, useful, and sustainable. While some of the genes encoding the ER-localized enzymatic steps to triacylglycerol (TAG) have been well understood and documented, several are still in need of study. The glycerol-3-phosphate acyl transferase (GPAT) enzymatic activity is the first step in the pathway to TAG, and it acylates glycerol 3-phosphate to produce lysophosphatidic acid. GPAT9 (AT5G60620) is conserved across land plants and is homozygous lethal, indicating an essential function. Transcript level in knockdown mutants correlates with GPAT activity and with oil levels, and the protein interacts with other enzymes in the TAG biosynthesis pathway. These data suggest that GPAT9 encodes the main GPAT involved in membrane lipid and TAG synthesis. The phosphatidic acid phosphatase (PAP) step in TAG synthesis is responsible for the hydrolysis of inorganic phosphate from phosphatidic acid and creation of diacylglycerol (DAG). There are 13 putative PAPs in Arabidopsis which are homologous to known PAPs. Most of these are involved in other processes, including the plastidial lipid synthesis pathway and signaling pathways. The Arabidopsis gene LPPβ (At4g22550) is expressed in seed tissue, its protein product is localized to the ER, and it encodes PAP activity, indicating that it is a likely candidate for the PAP involved in oil synthesis. At the conclusion of this work, questions remain about the role of LPPβ in oil synthesis and which genes encode the major enzymes involved in the steps generating phosphatidylcholine and converting it back to DAG; but the main Kennedy Pathway enzymes generating TAG have been identified and characterized.

Thesis (MSc (Genetics. Plant Biotechnology)--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Glycogen was isolated from E. coli and analysed for the amount of phosphate present within it. It was confirmed that a significant proportion of the glucose residues were phosphorylated at the C6 position. This glycogen phosphate was found also in both glgb- (glycogen branching enzyme) and glgp- (glycogen phosphorylase enzyme) mutants, demonstrating that a mechanism for phosphate incorporation that does not involve GlgP alone, and which is capable of incorporating phosphate into linear glucans could exist. The degree of phosphorylation depended on the amount of phosphate present in the media, which less being incorporated in media where phosphate was reduced. Screening for glycogen phosphorylating genes using a E. coli genomic library in a functional expression system identified the malP gene as a possible candidate for incorporation of the phosphate at the C6 position. There was no difference, however, between the glycogen phosphate content of the mutant and wild type. Efforts were made to construct a malp-/glgp- double mutant, but these were unsuccessful. In addition the influence of plants and human proteins on yeast glycogen metabolism was also investigated. These proteins have been demonstrated to have an effect on starch or glycogen in humans, plant and E. coli, but the data from this study indicated that this was not the case in yeast.
AFRIKAANSE OPSOMMING: Glikogeen, wat geisoleer was uit E.coli was geanaliseer vir fosfaat inhoud daarin. Daar was gevind dat `n beduidende proporsie van die glukose residue gefosforileerd was op die C6 posisie. Hierdie gefosforileerde glikogeen was ook gevind in glg- (glikogeen vertakkingsensieme) en glgp- (glikogeen fosforileringsensieme) mutante wat daarop dui dat `n meganisme vir fosforilering bestaan was nie slegs aangewese is op die aktiwiteit van GlgP nie, en om fosfaat te inkorporeer in linêre glukane. Die graad van fosforilering was ook afhanklik van die hoeveelheid fosfaat teenwoordig in die medium, met gevolglik minder wat geinkorporeer kan word in medium waar fosfaat verminderd was. Seleksie-gebaseerde ondersoeking vir fosforileringsensieme van glikogeen deur gebruik te maak van E. coli genomiese biblioteke in `n funksionele uitdrukkingssisteem het die malP geen geidentifiseer as een van die moontlike kandidate wat verantwoordelik kan wees vir inkorporering van fosfaat in the C6 posisie. Daar was egter geen verskil in die fosfaat inhoud van glikogeen tussen die wilde tipe en die mutante. Pogings wat aangewend is om `n malp-/glgpdubbel mutant te konstrueer was onsuksesvol. Verder is die invloed van plant en mens proteine op gis glikogeen ook bestudeer. Vroeër is aangetoon dat hierdie proteine `n invloed op stysel en glikogeen het in mense, plante en E. coli, maar data van hierdie studie toon aan dat dit nie die geval in gis is nie.

This dissertation describes the characterization of a novel transposable element isolated from the plant pathogenic fungus Magnaporthe grisea. The sequence of MGR583, a previously reported repeated DNA fragment, was completed and shown to have features characteristic of non-LTR retroelements (LINEs). These include an element length of 5.9 kb, the lack of flanking long terminal repeats, the presence of short (6-13 bp) direct repeats flanking many element copies, and two principal open reading frames (ORFs). The first ORF is 570 amino acids in length and contains homology to the gag ORFs found in many retroelements. The second ORF is 1,295 amino acids in length and has strong homology to reverse transcriptases (RT) ORFs found in non-LTR retroelements (LINEs). In accordance with these results, the name of the repeat was changed to MGL for Magnaporthe grisea LINE. Analysis of the 3' terminus of MGL showed 90% homology to the 3' terminus of Mg-SINE, suggesting an evolutionary relationship between these two elements. A survey of the distribution of MGL in populations of M. grisea showed the element to be present in all isolates tested. Copy number was not uniform between isolates, with approximately fifty copies present in rice isolates and between less than 10 and up to 50 copies in the 17 non-rice isolates tested. A PCR-based assay was designed and used to screen M. grisea isolates for polymorphic MGL insertion loci. Thirteen polymorphic MGL insertions were scored and used to construct a phylogenetic tree that included 11 non-rice isolates and 20 rice isolates. The results strongly suggested that development of virulence on rice was a single event correlated with the acquisition of virulence on several other grass species. In addition, the observation that rearrangements occurred at one of the insertion loci in some rice isolate strains support the proposal that there is considerable plasticity in the genomes of these isolates. Finally, a yeast transposon ( Tyl) system was used to express and test the second ORF for RT activity. No activity was detected for any of the MGL RT constructs tested.

Protein-bound and free lysine contributed to the total lysine content of maize endosperm, and both of these can be significantly increased by the opaque-2 (o2) mutation. Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly correlated with the protein-bound lysine content of the endosperm. However, the biological basis of this correlation is unknown. The mechanism by which the free amino acid content, including free lysine, is increased by the o2 mutation is also poorly understood. Understanding the biological basis of these traits could provide new insights for improving maize nutritional quality. A maize genetic linkage map consisting of 83 DNA simple sequence repeat markers was created for two inbred lines (Oh51Ao2 and Oh545o2) that differ in elongation factor 1A and free amino acid content. Quantitative trait locus analysis was performed and identified two significant loci that accounted for 25% of the variance for elongation factor 1A content. One of them was linked with a cluster of 22-kD α-zein genes on the short arm of chromosome 4; the other locus was on the long arm of chromosome 7 and may be linked to the 27-kD γ-zein genes. Quantification of protein and mRNA levels of the major storage proteins suggested that a higher level of α-zein gene expression co-segregates with higher elongation factor 1A content. Furthermore, measurement of protein body size and density predicted a greater protein body surface area (80% higher) in Oh51Ao2 than Oh545o2, and this may partially explain the higher level of elongation factor 1A in Oh51 Ao2 by creating a more extensive cytoskeletal network. Quantitative trait locus analysis of free amino acid content identified four loci accounting for about 46% of the variation for this trait. One locus on the long arm of chromosome 2 is tightly linked to monofunctional aspartate kinase and a bifunctional aspartate kinase-homoserine dehydrogenase gene. Biochemical characterization of these enzymes indicated the aspartate kinase in Oh545o2 is less sensitive to lysine than that in Oh51 Ao2. Consequently, aspartate kinase 2 is the more promising gene involved in this quantitative locus.

Flavonoids are plant secondary metabolites well known for many key roles in the life cycle of plants. They also can affect human health. Citrus paradisi is known to produce several glucosylated flavonoids and these compounds are glucosylated by enzymes known as glucosyltransferases (GTs). The focus of this research was to optimize the heterologous expression, enrichment, and biochemical characterization of grapefruit putative GT protein, PGT2, and to test the hypothesis that PGT2 is a flavonoid GT. Results showed detectable amounts of activity with quercetin, a flavonol; however, activity was lower than what would be expected if this enzyme were a flavonol-specific GT. In an additional aspect of this study, bioinformatics were used to test the hypothesis that additional putative GT clones could be identified using the harvEST database.

Bibliography: leaves 207-220. Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.

Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xvii, 163 p.; also includes graphics (some col.). Includes bibliographical references (p. 148-163).

Successful reproduction in plants is a highly-regulated process reliant on the integration of both endogenous and external cues. Different accessions of the model plant Arabidopsis thaliana have been collected, including those with a winter annual or rapid-cycling flowering habit. Natural variation and mutant screens have enabled many flowering time genes to be identified. A key regulator of flowering is FLOWERING LOCUS C (FLC). FLC is a repressor of flowering and is regulated by a number of genes, including those in the autonomous and FRIGIDA-mediated pathways. Of particular interest are FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) and the autonomous pathway members, FCA and FY. FRI and FRL 1 promote FLC expression making them dominant repressors of flowering. FRI is proposed to initiate chromatin remodelling at the FLC locus leading to increased FLC expression. Once elevated, FLC levels are maintained until plants undergo an extended period of cold, therefore flowering occurs in spring. In contrast, FCA and FY promote flowering by repressing FLC expression. FCA has also been identified as a receptor of the plant hormone abscisic acid (ABA). Upon binding to FCA, ABA is proposed to disrupt/inhibit the FCA:FY interaction which results in delayed flowering. To characterise the FCA:ABA interaction and identify the ABA binding site, a number of truncated FCA proteins were utilised. Initially a FCA:FY GST-pulldown was used to identify the ABA binding site. However, when ABA failed to inhibit the FCA:FY interaction a direct binding assay using [�H]-ABA was employed. Another Arabidopsis ABA receptor, G-protein coupled receptor 2 (GCR2), was used as a positive control in these binding assays. Both FCA and GCR2 failed to bind [�H]-ABA suggesting a broader issue with the binding assay. The identification of FCA and GCR2 as ABA receptors can be attributed to the quality of the protein assayed, the sensitivity of the binding assay and the subsequent data analysis. This study resulted in the retraction of the original paper (Razem et at, 2006) reporting FCA as an ABA receptor. To investigate the molecular mechanism by which FRI and FRL1 act as positive regulators of FLC expresion, a biochemical approach was taken. FRI and FRL1 have no known homology to any other protein or domain and the only method for assessing protein function is through plant complementation experiments. In the absence of sequence homology, or a timely functional assay, a classical approach was taken to produce soluble protein for analysis. Truncation of predicted regions of disorder and expression, solubility and stability screens produced soluble protein of reasonable purity. This allowed characterisation of the biochemical properties of FRI and FRL1. Interaction studies between FRI and FRL1, and the zinc finger protein SUPRESSOR OF FRIGIDA 4 (SUF4), were also carried out. Polyclonal antibodies against FRI and FRL1, made during this study, were useful for protein detection in these experiments. The interaction studies, together with plant complementation experiments, suggest that the C-terminus of FRI is essential for protein function, while the N-terminus improves FRI activity. These findings provide a better understanding of how the components of the proposed "FRI-complex" may interact to promote FLC expression.

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

Il mais rappresenta una delle colture più diffuse nel Nord Italia. Negli ultimi anni si è assistito ad un notevole incremento della superficie a mais legato soprattutto all'uso dell'insilato di mais come substrato per la produzione di biogas. Per questo motivo è necessario avere il maggior numero di informazioni possibili per migliorare le performance della coltura. In letteratura ci sono molti studi che valutano l’impatto dell’ambiente e del management sulle caratteristiche chimico nutrizionali e sul potenziale metanigeno del foraggio di mais ma non ci sono lavori che considerano entrambi i parametri. L’obiettivo della tesi è stato quello di investigare come il danno da grandine , la genetica e il momento di raccolta possano influenzare la composizione della pianta intera tradotta come potenziale metanigeno e valore nutrizionale del foraggio.
In the Po Valley the maize crop represent one of the most cultivated plant used for cattle feeding but in the last 10 years it is also used as biogas substrate. Considering the importance of this cultivation, there is the continuous need to obtain information about this plant with the aim to improve the crop performance. There are numerous studies investigating the impact of environmental aspects and management practices on chemical and nutritional composition, and methane production in the literature but very few that evaluate those parameters together. The general objective of this thesis is to investigate how hail damage, type of hybrid and the harvesting date affect the whole plant composition. To accomplish this, two specific objectives are posed: i) verify the effects of hail damage levels on yield, chemical and nutritional feature as well as on BMP of maize grown in the Po Valley; and ii) to evaluate the value of different hybrids for animal nutrition and methane production in anaerobic fermenters and as delaying harvesting after the usual stage of maturity affects these features. It was also aimed to verify if chemical composition and in vitro digestibility tests could allow to estimate methane yield potential in maize whole plant.

by Yuan Dingyang.
"September 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 206-232).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

Much is known about how organs acquire their identity, yet we are only beginning to learn how their shape is regulated. Recent work has elucidated the role of coordinated cell division & expansion in determining plant organ shape. For instance, in Antirrhinum, leaf shape is affected in the cincinnata (cin) mutant because of an alteration in the cell division pattern. CIN codes for a TCP transcription factor and controls cell proliferation. It is unclear how exactly CIN-like genes regulate leaf morphogenesis. We have taken biochemical and genetic approach to understand the TCP function in general and the role of CIN-like genes in leaf morphogenesis in Antirrhinum and Arabidopsis. Targets of CINCINNATA To understand how CIN controls Antirrhinum leaf shape, we first determined the consensus target site of CIN as GTGGTCCC by carrying out RBSS assay. Mutating each of this target sequence, we determined the core binding sequence as TGGNCC. Hence, all potential direct targets of CIN are expected to contain a TGGNCC sequence. Earlier studies suggested that CIN activates certain target genes that in turn repress cell proliferation. To identify these targets, we compared global transcripts of WT and cin leaves by differential display PCR and have identified 18 unique, differentially expressed transcripts. To screen the entire repertoire of differentially expressed transcripts, we have carried out extensive micro-array analysis using 44K Arabidopsis chips as well as 13K custom-made Antirrhinum chips. Combining the RBSS data with the results obtained from the micro-array experiments, we identified several targets of CIN. In short, CIN controls expression of the differentiation-specific genes from tip to base in a gradient manner. In cin, such gradient is delayed, thereby delaying differentiation. We also find that gibberellic acid, cytokinin and auxin play important role in controlling leaf growth. Genetic characterization of CIN-homologues in Arabidopsis Arabidopsis has 24 TCP genes. Our work and reports from other groups have shown that TCP2, 4 and 10 are likely to be involved in leaf morphogenesis. These genes are controlled by a micro RNA miR319. To study the role of TCP4, the likely orthologue of CIN, we generated both stable and inducible RNAi lines. Down-regulation of TCP4 transcript resulted in crinkly leaves, establishing the role of TCP4 in leaf shape. To study the function of TCP2, 4 & 10 in more detail, we isolated insertion mutants in these loci. The strongest allele of TCP4 showed embryonic lethal phenotype, indicating a role for TCP4 in embryo growth. All other mutants showed mild effect on leaf shape, suggesting their redundant role. Therefore, we generated and studied various combinations of double and triple mutants to learn the concerted role of these genes on leaf morphogenesis. To further study the role of TCP4 in leaf development, we generated inducible RNAi and miRNA-resistant TCP4 transgenic lines and carried out studies with transient down-regulation and up-regulation of TCP4 function. Upon induction, leaf size increased in RNAi transgenic plants whereas reduced drastically in miR319 resistant lines, suggesting that both temporal & spatial regulation of TCP4 is required for leaf development. Biochemical characterization of TCP domain To study the DNA-binding properties of TCP4, random binding site selection assay (RBSS) was carried out and it was found that TCP4 binds to a consensus sequence of GTGGTCCC. By patmatch search and RT-PCR analysis, we have shown that one among 74 putative targets, EEL (a gene involved in embryo development), was down regulated in the RNAi lines of TCP4. This suggests that EEL could be the direct target of TCP4. We have tested this possibility in planta by generating transgenic lines in which GUS reporter gene is driven by EEL upstream region with either wild type or mutated TCP4 binding site. GUS analysis of embryos shows that transgenic with mutated upstream region had significantly reduced reporter activity in comparison to wild type, suggesting that EEL is a direct target of TCP4. We have further shown that TCP4 also binds to the upstream region of LOX2, a gene involved in Jasmonic acid (JA) biosynthesis (in collaboration with D. Weigel, MPI, Tubingen, Germany). TCP domain has a stretch of basic residues followed by a predicted helix-loop-helix region (bHLH), although it has little sequence homology with canonical bHLH proteins. This suggests that TCP is a novel and uncharacterized bHLH domain. We have characterized DNA-binding specificities of TCP4 domain. We show that TCP domain binds to the major groove of DNA with binding specificity comparable to that of bHLH proteins. We also show that helical structure is induced in the basic region upon DNA binding. To determine the amino acid residues important for DNA binding, we have generated point mutants of TCP domain that bind to the DNA with varied strength. Our analysis shows that the basic region is important for DNA binding whereas the helix-loop-helix region is involved in dimerization. Based on these results, we have generated a molecular model for TCP domain bound to DNA (in Collaboration with Prof. N. Srinivasan, IISc, Bangalore). This model was validated by further site-directed mutagenesis of key residues and in vitro assay. Functional analysis of TCP4 in budding yeast To assess TCP4 function in regulation of eukaryotic cell division, we have introduced TCP4 in S. cerevisiae under the GAL inducible promoter. TCP4 induction in yeast cells always slowed down its growth, indicative of its detrimental effect on yeast cell division. Flow cytometry analysis of synchronized cells revealed that TCP4 arrests yeast cell division specifically at G1→S boundary. Moreover, induced cells showed distorted cell morphology resembling shmoo phenotype. Shmooing is a developmental process which usually happened when the haploid cells get exposed to the cells of opposite mating type and get arrested at late G1 phase due to the inhibition of cdc28-cln2 complex. This suggested that TCP4-induced yeast cells are arrested at late G1 phase probably by the inhibition of cdc28-cln2 complex. To further investigate how TCP4 induce G1→S arrest, we carried out microarray analysis and found expression of several cell cycle markers significantly altered in TCP4-induced yeast cells. Studies on crinkly1, a novel leaf mutant in Arabidopsis To identify new genes involved in leaf morphogenesis, we have identified crinkly1 (crk1), a mutant where leaf shape and size are altered. We observed that crk1 also makes more number of leaves compared to wild type. Phenotypic analysis showed that crk1 leaf size is ~5 times smaller than that of wild type. Scanning electron microscopy (SEM) showed that both cell size and number are reduced in the mutant leaf, which explains its smaller size. We have mapped CRK1 within 3 cM on IV chromosome.

Bibliography: leaves 207-220.
220 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1996

Bibliography: leaves 207-220.
220 leaves : ill. ; 30 cm.
Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1996

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.

Plant biomass is the major source of available carbohydrates in Nature. The walls of plant cells are mainly composed by homo- and heteropolysaccharides, usually found intertwined with each other and making a highly complex and heterogeneous structure, which displays a strong resistance to biological degradation. Still, many microorganisms are able to breakdown this complex structure and use the available sugars as carbon and energy source. Several saprophytic bacteria, including Bacillus subtilis, release a high number of saccharolytic enzymes capable of cleaving most polysaccharides in plant cell walls, and are then able to transport and metabolize the resulting mono- and oligosaccharides. Thus, transport is a key step in sugar utilization. Here, we characterize four ABC-type transporters involved in the uptake of arabino- and galactooligosaccharides, and galacturonic acid oligomers and/or rhamnose-galacturonic acid disaccharides. We show that MsmX is the sole ATPase responsible for energizing these four ABC importers, thus playing a key role in pectin mobilization by B. subtilis. AraN, the solute-binding protein (SBP) from the ABC importer, AraNPQ, is shown by in vivo and in vitro analyses to bind arabinooligosaccharides. However, AraN displays a significantly lower affinity than that previously reported for other SBPs. In addition, AraN also binds cello-, xylo- and maltooligosaccharides with similar affinity but is shown to be non-essential for growth in the presence of these oligosaccharides. We speculate that binding of these sugars may prevent arabinooligosaccharides uptake when more favorable carbon sources, such as glucose-rich cello- and maltodextrins, are available. The mechanisms of expression of abn2, a gene encoding an endo-arabinanase, were investigated and we provide evidence that this AraR-independent gene is temporally regulated. The transition state regulator AbrB and the master sporulation regulator Spo0A are implicated in the regulation of abn2. Finally, we demonstrate that a metallic cluster close to the catalytic site of Abn2 is essential for its arabinanase activity.