Dissertations / Theses on the topic 'Alfalfa plant'

Phosphoenolpyruvate carboxylase (PEPC) was examined during cold acclimation of seedlings of the freezing-tolerant cultivar (Medicago sativa ssp falcata cv Anik) and the relatively freezing-sensitive cultivar (Medicago sativa cv Trek) of alfalfa. With four days of cold acclimation, PEPC activity increased to 3.5-fold and 2-fold the control levels in Anik and Trek, respectively. This was associated with an increase in the level of a 110 kD PEPC protein and a decrease in the amount of a 120 kD PEPC polypeptide in both cultivars. The role of reversible phosphorylation in regulating PEPC activity was demonstrated by in vitro phosphorylation and dephosphorylation, which caused partial activation and deactivation of PEPC, respectively. In vivo phosphorylation experiments revealed that the 110 kD PEPC subunit is phosphorylated on serine residue(s) during cold acclimation in Anik but not in Trek. Increased PEPC activity could account for the 70% increase in the non-autotrophic or dark fixation of carbon observed in cold acclimated Anik seedlings. A possible role for dark carbon fixation in the cold-induced development of freezing tolerance is through the production of NADPH. Such a source of reducing power may be required for the repair of cold-induced damage and restoration of normal cellular functions.

Cold-acclimation-specific (CAS) gene expression was examined by screening a cDNA library prepared from poly(A)$ sp+$ RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three distinct CAS cDNA clones, pSM784, pSM2201, and pSM2358 were isolated. The genes corresponding to all three clones are coordinately induced by cold. Expression of these genes is not triggered by other stress treatments such as heat shock, water stress, wounding, or treatment with exogenous ABA. A positive correlation was observed between the level of expression of each gene and the degree of freezing tolerance of four alfalfa cultivars.<br>A full-length cDNA clone for the most abundantly-expressed gene, cas18 was isolated and sequenced. The deduced polypeptide, CAS18, is relatively small (167 amino acids), is highly hydrophillic, rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/Dehydrin gene family--proteins which accumulate in response to water stress or abscisic acid (ABA). The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4 and 1.0 kb in cold acclimated seedlings and cell cultures. The clone described here, Acs784, corresponds to the 1.0 kb transcript.<br>Expression of this gene is 30-fold greater in cold-acclimated cells than in nonacclimated cells after one week of low temperature treatment. Return to room temperature (deacclimation) results in the rapid disappearance of the three transcripts within just 5 hours. Studies of nuclear "run-on" transcription and transcript stability show that low temperature regulates the expression of cas18 at both the transcriptional and post-transcriptional levels.

Three plant growth regulators (two for growth enhancement, one for growth inhibition) were applied to several consecutive cuttings of alfalfa during the period of May-August in the Blythe, CA, area. All treatments resulted in reduced tonnage compared to the untreated check, although application of prohexadione calcium (active ingredient in the growth inhibition plant growth regulator) did result in increase in alfalfa quality when applied in May. Subsequent applications during the summer of this material did not result in a quality class increase for alfalfa hay production. Treatments of growth enhancement chemistries did result in less tonnage, however, data indicate that this reduction may be due to nutritional needs of alfalfa not being supplied during periods of increased growth. Supplemental nutrition along with the growth enhancement chemistries has not been tested to determine resulting alfalfa yields.

The performance of an alfalfa (Medicago sativa L.) population has been improved by recurrent selection for germination salt tolerance. However, recurrent selection may lead to increased inbreeding. Since alfalfa is subject to severe inbreeding depression, accumulation of inbreeding during the selection process may negatively affect performance. This experiment was designed to determine the effects of inbreeding on germination performance in alfalfa under saline and non-saline conditions. The germination performance of seed having three different levels of inbreeding as examined. No adverse effects of inbreeding were observed in non-saline conditions. Consistent (but nonsignificant) declining trends were observed in germination percentage in saline conditions as the level of inbreeding increased. Proportionately larger declines were observed between generations in germination speed and early seedling vigor. These results suggest heterozygosity in alfalfa may be maintained simultaneously while recurrent selection for germination salt tolerance is conducted. Moreover, reducing inbreeding during recurrent selection for germination salt tolerance may be more successful if germination speed index or early seeding vigor are used for the measurement.

Gene expression in two different genotypes of alfalfa, salt-tolerant and salt-sensitive, was examined by studying differences in protein products coded for by poly(A+) RNA isolated from shoot and root tissue. Plants were grown in hydroponics under unstressed or salt-stressed conditions. Two salinity levels (low salt: 30 mM NaCl and 6 mM CaCl2 and high salt: 133 mM NaCl and 27 mM CaCl2) and one unstressed control were applied. The salt-tolerant genotype showed higher biomass accumulation than the salt-sensitive genotype under both control and salt-stressed conditions. The difference in biomass accumulation between the two genotypes was greatest at the highest salt level. The effect of salt stress on gene expression was studied via in vitro translation of poly (A+) RNA with (35S) -methionine. The labeling pattern was similar in all treatments when analyzed by one dimensional SDS-PAGE. However, a two dimensional analysis (isoelectric focusing followed by SDS-PAGE) showed that salt-stress induced a number of new proteins and repressed several others.

Plant growth regulators were aerially applied on April 26, 1996, to two alfalfa fields, one (Cibola) in its first year of production and the other field (CUF 101) in its third year. The first year field was approximately 50% through the cutting cycle and the third year field was approximately 30% through the cutting cycle when applications were made. No statistical differences were noted in hay tonnage the first cutting after application. Yield increases due to treatments were noted in the second cutting and maximum increases ranged from 277 -461 lbs of hay per acre. No yield or quality differences were noted the third cutting after application. The two fields differed in their response. The lowest rates of plant growth regulators produced higher yields in the first year field, but these treatment rates had greatly lowered hay quality the previous cutting. These effects were not noticed in the third year stand field. Increased hay tonnage was noted in the third year field from the 16 oz/acre rates, but was not evident in the first year field. Hay quality was usually highest in the check the first two cuttings after treatment. It is unknown if the differences noted between the two fields are due to different age of plants, variety and/or stage of growth when treatments were applied.

This study sought to determine whether NaCl altered the plasma membrane proteins in alfalfa exhibiting differential NaCl concentrations, and whether caso4 modified the responses. Two alfalfa cultivars, Centurion and Condor, were grown in 0.5 strength Hoagland solution in a greenhouse. The cultivars were exposed to 0, 88, and 132 mM of NaCl alone and mixed with caso4 .H20 at 7 and 14 Mm caso4 for 3, 9, and 60 days. In experiment 1, roots were dried to determine their Na, Ca, K, and Mg concentration. The results were similar to previous reports in which CaS04 alleviated the salt stress by increasing K and Mg levels and reducing Na. In experiment 2, after proteins of the plasma membrane were isolated and their purity was determined by vanadate, ATPase activity showed a significant increase in the presence of calcium. In addition, total plasma membrane protein was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Salt treatments induced both quantitative and qualitative changes in proteins. These changes were affected by the length of exposure to treatment solution or the ability of the plants to adapt to the salt stress.

During the present studies we attempted to answer the following questions: (i) Does low temperature alter the phosphorylation level of proteins in isolated nuclei? (ii) Does the nuclear phosphoprotein population change during a prolonged exposure of seedlings to cold? (iii) Do heat-stable proteins accumulate in the nucleus during a prolonged exposure of seedlings to cold? (iv) Are the answers to the above three questions related to freezing tolerance? A possible relationship between the observed cold-induced changes in phosphoproteins and the level of freezing tolerance was explored by comparing the results of experiments conducted on two cultivars (Apica and Trek) of alfalfa (Medicago sativa L.) which markedly differ in their capacity for cold acclimation.<br>We show that the phosphorylation level of several nuclear proteins is subject to rapid and reversible enhancement by low temperature. Several phosphoproteins were found to be constitutively present in the nucleus of both cultivars. The cold-induced stimulation of the phosphorylation of many of these proteins was much greater in the relatively freezing tolerant cultivar Apica than in the relatively freezing sensitive cultivar Trek. Population of nuclear phosphoproteins was found to be considerably more complex in Apica than in Trek. During a prolonged exposure of the seedlings to 4$ sp circ$C, additional phosphoproteins were imported into the nucleus of Apica seedlings but not those Trek.<br>Some heat-stable proteins were constitutively present in the nucleus of both cultivars. However during the 4-day cold treatment, a large accumulation of several additional heat-stable proteins was observed in the tolerant, but not the sensitive, cultivar. (Abstract shortened by UMI.)

Backcross populations (BC) from Medicago sativa L. x M. dzhawakhetica Bordz. hybrids were analyzed for chromosome number, fertility and morphological characteristics. Previously obtained F1 hybrids were recovered when diploid (2n = 2x = 16) M. sativa was crossed with tetraploid (2n = 4x = 32) M. dzhawakhetica. Resulting F1 hybrids were triploid (2n = 3x = 24), completely male sterile and had low levels of female fertility. Subsequent populations were obtained by successive backcrossing to unrelated (4x) M. sativa clones. The BC1 plants were pentaploid (2n = 5x = 40) and both male and female fertile. BC2 populations had chromosome numbers ranging from 2n = 32 to 48, and most plants (94% were male and female fertile. BC3 populations were tetraploid (2n = 32) or near tetraploid (2n = 33) and were morphologically similar to M. sativa. Preliminary analysis of mitochondrial nucleic acids by agarose gel electrophoresis, indicated biparental inheritance of this organelle in the F1 hybrids; however, further analysis provided inconclusive results.

Three greenhouse experiments were conducted with three phenotypes of alfalfa (Medicago sativa L.) obtained from a potassium (K)-deficient field and with their diallel crosses grown on low K soil. The first experiment was conduc ted to study the partitioning and broad-sense heritability of K a nd sodium (Na) between leaves and stems of the three phenotypes which were deficient in K and exhibited normal (N), marginal chlorotic (M), and white spot chlorotic (W) leaflets. The second experiment was conducted to study the partitioning of K and Na in leaves, stems, and roots as influenced by 32 alfalfa crosses obtained from diallel crossing of the mother plants of the three phenotypes. The objectives of the third experiment were to study the effects and interactions of nine alfalfa crosses and three soil K and Na levels on transpiration, biomass, and elemental composition of alfalfa components. The three phenotypes showed no variations in their leaf and stem K concentrations but varied in their ability to partition Na between the leaves and stems. Phenotype M accumulated more Na compared to N and W phenotypes. The Na trait was highly heritable in the broad sense. The K and Na concentrations varied among the diallel crosses. Crosses with M as the maternal parent had high Na concentrations in leaves while stems and roots accumulated lesser amounts. In contrast, the remaining crosses had higher Na concentrations in roots and lower and least amounts in stems and leaves, respectively. Significant genetic variation among alfalfa crosses from a single cultivar was observed for transpiration, biomass production, plant water-use efficiency, elemental concentrations, and K utilization efficiency. Leaf and stem biomass and K concentrations in alfalfa components increased in response to increasing soil K levels. The Na concentrations in stems and roots fell in response to increasing soil K levels and increased in response to Na application. The K utilization efficiency of alfalfa increased with increase in soil Na levels, indicating partial Na substitution for K. The differences among alfalfa phenotypes and crosses from a single cultivar in their Na accumulation and translocation were thought to be governed by plant genetics rather than the direct effect of K availability.

Protoplasts were isolated from mesophyll tissue, callus and seedling cotyledons of Medicago sativa L. cv. Regen S and the halophyte M. marina L. Cotyledon protoplasts of Regen S were cultured in protoplast and cell culture media used previously for alfalfa protoplast culture and in media that had been simplified. There were no differences in the plating efficiencies of protoplasts cultured in the simple and complex media, but cells produced in the latter were greener and they colonized sooner. Protoplasts of M. marina grew at one-half the rate of Regen S protoplasts. Etiolated cotyledon protoplasts of Regen S were fused at 31°C using a solution containing PEG, DMSO and calcium at high pH. The frequency of fusion was 16% of the surviving protoplasts. These methods for protoplast isolation, culture and fusion should be useful in the somatic hybridization of alfalfa and M. marina.

Glyphosate (N-(phosphonomethyl)glycine)-containing herbicides are a common and highly effective method to terminate alfalfa (Medicago sativa) stands. With the development and use of glyphosate-resistant (Roundup Ready®) alfalfa, this tool is no longer an option. The purpose of this research was to determine the optimal strategy to rotate from glyphosate-resistant alfalfa into silage corn (Zea mays). Studies were conducted in 2012 and 2013 at sites near Cache Junction and Cornish, Utah to determine the effect of tillage type and timing [fall conventional till (FCT), spring conventional till (SCT), fall strip-till (FST), spring strip-till (SST), and no-till (NT)], 2,4-D plus dicamba herbicide timing (fall, spring, in-crop, and a control), and N rate (0, 56, 112, and 224 kg N ha-1) on soil penetration resistance (PR), alfalfa re-growth, and corn emergence rate index (ERI), silage yield, quality, and economic return. The fall, spring, and in-crop herbicide timings across all tillage treatments reduced alfalfa stem count and biomass by at least 95% and 98%, respectively. Tillage reduced PR compared to NT to or near the depth of tillage. The ERI was significantly higher under FCT, SCT, and SST and when herbicides were applied in fall or spring. Silage yield, quality, and economic return were the highest when spring herbicide timing was used with all tillage types and timings and the fall herbicide timing under conventional tillage. Increasing N rates increased crude protein, milk ha-1, and dry matter yield. However, optimal yield and quality can be obtained with no additional N fertilizer. First-year silage corn yield, quality, and economic return can be optimized under fall or spring conventional till, strip-till, and no-till at the spring herbicide timing along with the fall herbicide timing for conventional tillage with no additional N fertilizer.

Alfalfa (Medicago sativa L.) is the most important forage crop in the United States and consistently produces high yields and quality, but harvest frequency is the most significant factor for maximizing forage yield and quality. The objective of this research was to determine forage yield, quality, and regrowth rate among new alfalfa cultivars under four different harvest frequencies. Some of these cultivars have been marketed as having rapid rates of regrowth after cutting to maximize the number of harvests per year. Five cultivars were placed under four harvest frequencies of 25, 30, 35, and 40 days in a split-plot design. There was a significant yield and regrowth rate effect across cultivars and harvest frequencies, but little forage quality effect during the two years of this research. These results confirm previous findings that a 35-d harvest frequency is optimal for forage yield, quality, and stand persistence.

To study the effect of NaCl stress on gene expression in alfalfa (Medicago sativa L.), greenhouse and laboratory experiments were conducted with 22 cultivars during germination and post-germination growth. The ability of alfalfa cultivars to germinate at the different NaCl concentrations was not related to their postgermination performance (salt tolerance) under those conditions. Genetic effects were evident for Na and Cl ion uptake and accumulation in alfalfa shoots and roots. The strategies of alfalfa cultivars to cope with NaCl stress includes exclusion of Na from shoots and Cl from roots or from the whole plant depending on cultivar and NaCl level. The reduction in shoot dry weight was not related to water stress or reduction in Ca, Mg, or chlorophyll concentrations. Results of this study indicated that the reduction in K ion uptake and more directly the toxic effects of high Na and Cl in plant tissues contributed to the reduction in shoot dry weight . The possible subpartitioning of Na and Cl ions between different tissues, cell types, cell components, and/or the different changes in protein structure and enzyme activity at the high Na and Cl concentrations, independent of total concentration of ions, might also contribute to differences in salt tolerance (shoot dry weight) among cultivars. The factors determining shoot dry weight in alfalfa grown under the different NaCl treatments of this study were not the same for all cultivars. Sodium chloride stress induced qualitative and quantitative changes in shoot and root proteins of alfalfa. These changes were dependent on cultivar and salt concentration, as well as length of exposure to salt stress.

Knowledge of relationships between nutrient levels, yield, maturity, and environmental influences on alfalfa (Medicago sativa L.) are necessary to estimate when to harvest alfalfa to maximize quality and yield. Objectives were to document the change in nutrient content, yield, and growth stage of alfalfa grown in Utah as it matures and to develop a simple model to predict optimal harvest date. The study involved three locations in major alfalfa producing regions in Utah. Samples were collected from three commonly grown alfalfa varieties between 26 April and 26 September in 1987. Maximum and minimum levels of crude protein (CP) observed were 32.8% to 16.2%, acid detergent fiber (ADF) 39.4% to 14.0%, and dry matter (OM) 31.7% to 14.3%. Maximum yield of 7.0 Mg ha-1 for a single harvest was observed. The growth stage and average yield in Mg ha-1 for all varieties and harvests collected were: prebud 3.6; midbud 4.2; and late bud to early bloom 4.9. As alfalfa matured CP% declined, ADF% increased, and DM% increased. Criteria used to estimate optimal harvest date was achieving not less than 20.0% CP, at least 29.0% ADF, but not more than 31.0% ADF. The estimated optimal harvest date was determined 63.3% of the time by not exceeding 31.0% ADF. Early to midbud were characteristic growth stages of the estimated optimal harvest date occurring 34.4% and 41.0% respectively. Midbud stage was characterized by elongation of the peduncle at second and third axillary bud positions. Accumulated growing degree hours (AGDH) were calculated using the ASYMCUR modeling concept. Height models were developed by averaging AGDH at 5 cm increments of shoot height. Models developed from the Nephi site were used to predict data from other sites. These models made about 37% acceptable predictions ranging from 0-100%. The general model made 23% acceptable predictions, variety models 47%, harvest models 43%, and 36% from specific data models. The general model, with a coefficient of variation (CV) of 14.6%, made fewer acceptable predictions than specific models having CV of 11.1% and 6.0%. Height models generated using the ASYMCUR concept were inefficient in predicting growth of alfalfa. This may be due to inaccurate estimates of when regrowth began, inaccurate weather data, and a variety of temperature related stresses which reduces the growth rate of alfalfa per unit of growing degree hours. Development of stress factor in cosine equations, improved data collection, and additional model generation and testing, could reduce variability and increasing percentage of acceptable predictions.

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.

This research was conducted in order to provide information about the effect of sound waves in the growth settings of alfalfa (also known as lucerne) and lentils. Humans have researched about the effect of sound on humans and other animals since the beginning of our time. One of humanity’s oldest research on effects of sound is human communication. In order to expand the research of the effect of sound, this thesis expands to the area of plants. The method used consisted of playing Korobeiniki – also known as the theme song from Tetris – recorded in two versions. One version was played in piano and the second version was played in sine waves, to compare the two different frequency spectrums’ effect on plant growth. Test groups were based on the version that was played; the first group was exposed to piano while the second one was exposed to sine waves. One control group without music was used as well. Among two test groups and one control group, alfalfa and lentils grew in three batches in each group. With each batch having a unique result, the research resulted in a non-generalisable fashion, which suggests further research is vital in order to provide a conclusion which is based on more of a quantitative outcome.

To identify a model legume experimental system for studying low temperature signaling and cold acclimation, cold-induced expression and regulation of homologues of alfalfa (Medicago sativa) cold acclimation-specific genes cas15 and cas30 were examined in M. arborea (relatively frost tolerant) and M. truncatula (relatively frost sensitive). Both cas15 and cas30 genes are present in the genomes of both species but whereas both genes are cold-induced in M. arborea, only cas15 is induced in M. truncatula. Cold-induced expression of these genes is inhibited by calcium chelators and channel blockers and by the membrane fluidizer benzyl alcohol. Treatment of leaves with dimethylsulfoxide, a membrane rigidifier, induced both genes at 25°C. A cold-activated MAP kinase activity was expressed in both species. These results suggest that M. truncatula, an annual, self-pollinated species may be successfully used as model experimental systems in studies of cold signaling and role of cas genes in cold acclimation in legumes.

[ES] Las modificaciones químicas post-transcripcionales implican un nuevo nivel de modulación de la expresión génica. Al comienzo de esta Tesis, algunos componentes del complejo de metilación del nitrógeno en posición 6 de la adenosina (m6A) habían sido caracterizados en plantas. Sin embargo, a diferencia de mamíferos y levadura, ninguno de los 13 homólogos de AlkB (atALKBH1-10B) - potenciales desmetilasas (o erasers) - y las 13 proteínas de la familia YTH (ECT1- 11, AT4G11970 y CPSF30) - potenciales proteínas de reconocimiento de m6A (o readers) - identificadas en el genoma de Arabidopsis se habían caracterizado funcionalmente. Además, varios estudios describen la presencia de m6A en RNAs de virus de mamíferos y las diferentes funciones que desempeña esta modificación en la regulación de esas infecciones. No obstante, no se ha estudiado la posible implicación de este mecanismo molecular en las infecciones virales de plantas. El descubrimiento de la interacción entre la CP del virus del mosaico de la alfalfa (AMV) y una proteína de Arabidopsis (atALKBH9B) con homología a una eraser humana fue el punto de partida de esta Tesis. En este trabajo se confirma esta interacción, y se demuestra que atALKBH9B también puede reconocer los RNAs virales. Los resultados revelan que atALKBH9B tiene la capacidad de desmetilar m6A a partir de moléculas de RNA monocatenario in vitro. Esta proteína se acumula en gránulos citoplasmáticos que se colocalizan con siRNA bodies y se asocian a P-bodies, lo que sugiere que su actividad podría estar relacionada con el silenciamiento y/o degradación de mRNA. Por otro lado, ensayos preliminares muestran que los RNAs del AMV, el virus del mosaico del pepino (CMV), el virus de la arruga del nabo (TCV) y el virus del mosaico de la coliflor (CaMV) se metilan durante la infección en Arabidopsis. Además, para AMV y CMV, los resultados fueron corroborados por UPLC-PDA-Tof-MS y los sitios m6A a lo largo de los RNAs del AMV fueron identificados mediante MeRIP-seq. Los resultados presentados confirman que la relación m6A/A a lo largo de los RNAs virales aumenta en plantas atalkbh9b en comparación con las silvestres, mientras que la traducción y/o replicación se ven afectadas y el movimiento sistémico a los tallos florales está prácticamente bloqueado. A diferencia de la CP de AMV, la de CMV no interacciona con atALKBH9B por Y2H y, como ocurre con el resto de virus analizados (CMV, TCV y CaMV), su ciclo de infección no se ve afectado en plantas atalkbh9b. Además, la secuenciación de mRNA realizada en este trabajo revela que la infección por AMV induce algunos genes de Arabidopsis pertenecientes a la maquinaria m6A, MTA, MTB, VIR y ECT5. De acuerdo con el efecto antiviral dependiente de m6A para el AMV y teniendo en cuenta que ECT2, ECT3 y ECT4 fueron recientemente caracterizadas como readers citoplasmáticas, la supresión del módulo ECT2/ECT3/ECT5 aumenta significativamente los títulos sistémicos de AMV y CMV. El efecto antiviral de ECT2 sobre AMV parece estar modulado por su unión directa a los residuos de m6A presentes en los RNAs virales, ya que un mutante de ECT2 defectuoso en el reconocimiento de m6A pierde la actividad antiviral que sí presenta la proteína original y no es capaz de arrastrar RNAs virales in vivo. Por otro lado, acorde a la localización previamente descrita para ECT2 y ECT4 y la capacidad de ECT2 para experimentar una fase similar al gel in vitro, la expresión transitoria de ECT5 muestra un patrón citoplasmático con formación de agregados. Se propone que, como se ha descrito para las proteínas YTH de mamíferos, la interacción entre las ECTs y el RNA polimetilado (en este caso, RNA viral) promovería la formación de gránulos de estrés y, en consecuencia, reduciría las tasas de traducción y replicación viral. En resumen, en este trabajo se caracteriza la primera m6A eraser de plantas, atALKBH9B, y, por primera vez, se describe la influencia del mecanismo de metilación m6A en las infecciones virales de plantas.<br>[EN] Post-transcriptional chemical modifications entail a new level of gene expression modulation. At the beginning of this Thesis, some components of the N6-adenosine methylation (m6A) complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) - putative demethylases (or erasers) - and 13 proteins of the YTH family (ECT1-11, AT4G11970 and CPSF30) had been identified in the Arabidopsis genome. However, unlike mammals and yeast, no functional roles had been described for any of these proteins. Besides, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses and the critical roles that this modification plays regulating viral infections. However, the potential relevance of this molecular mechanism on plant viral infections remained fully unexplored. The discovery of the interaction between the AMV CP and an Arabidopsis protein (atALKBH9B) with similarity to a human eraser was the starting point of this Thesis. Here, this interaction is confirmed and it is demonstrated that atALKBH9B can also recognize the viral RNAs. Furthermore, the obtained results prove that atALKBH9B has the capability of demethylating m6A from single-stranded RNA molecules in vitro. This protein was observed to accumulate in cytoplasmic granules that colocalize with siRNA-bodies and associate to P-bodies, suggesting that atALKBH9B activity could be related to mRNA silencing and/or decay processes. On the other hand, preliminary assays show that viral RNAs of AMV, Cucumber mosaic virus (CMV), Turnip crinkle virus (TCV) and Cauliflower mosaic virus (CaMV) become methylated during infection in Arabidopsis. Besides, for AMV and CMV, the results were corroborated by UPLC-PDA-Tof-MS and m6A sites along the RNAs of AMV were identified through MeRIP-seq approach. The results presented here confirm that m6A/A ratio along viral RNAs is increased in atalkbh9b plants compared to wild type, whereas translation and/or replication are impaired and systemic movement to the floral stems is practically blocked. In contrast to AMV, CMV CP does not interact with atALKBH9B by Y2H and, as it occurs with the rest of the assayed viruses (CMV, TCV and CaMV), its infection cycle is not affected in atalkbh9b plants. Furthermore, the mRNA-seq analysis performed in this Thesis reveals that some Arabidopsis factors belonging to the m6A machinery, MTA, MTB, VIR and ECT5 genes, are upregulated upon AMV infection. Consistent with the m6A-dependent antiviral effect for AMV and considering that ECT2, ECT3 and ECT4 were recently characterized as cytoplasmic m6A readers, mutations of ECT2/ECT3/ECT5 Arabidopsis module significantly increase AMV and CMV systemic titers. The antiviral effect of ECT2 on AMV seems to be modulated via its direct binding to the m6A residues presented in the viral RNAs, since an ECT2 mutant defective in m6A recognition loses wild type antiviral activity and is not able to pull down viral RNAs in vivo. On the other hand, according to the previous subcellular localization described for ECT2 and ECT4 and the ability of ECT2 to undergo gel-like phase in vitro, the transitory expression of ECT5 displays a cytoplasmic pattern with the formation of some aggregates. As found for mammal YTH proteins, the interaction between ECTs and poly-methylated RNA (in this case viral RNA) is proposed to promote the formation of stress granules and, consequently, reduce viral translation and replication rates. In summary, in this work, atALKBH9B is reported as the first m6A eraser identified in plants and, for the first time, it is described the influence of m6A methylation mechanism in plant viral infections.<br>[CA] Les modificacions químiques post-transcripcionals impliquen un nou nivell de modulació de l'expressió gènica. Al començament d'esta Tesi, s'havien caracteritzat alguns components del complex de metilació del nitrogen en posició 6 de la adenosina (m6A) en plantes. No obstant això, a diferència de mamífers i llevat, cap dels 13 homòlegs d'AlkB (atALKBH1-10B) - potencials desmetilases (o erasers) - i les 13 proteïnes de la família YTH (ECT1- 11, AT4G11970 i CPSF30) - potencials proteïnes de reconeixement de m6A (o readers) - identificades en el genoma d'Arabidopsis s'havien caracteritzat funcionalment. A més, diversos estudis han descrit la presència de residus m6A en RNAs de virus de mamífers i les diferents funcions que exercix esta modificació en la regulació de les infeccions virals. No obstant això, no s'ha estudiat la possible implicació d'este mecanisme molecular en les infeccions virals de plantes. El descobriment de la interacció entre la CP del virus del mosaic de l'alfals (AMV) i una proteïna d'Arabidopsis (atALKBH9B) amb homologia a una eraser humana va ser el punt de partida d'esta Tesi. En este treball es confirma esta interacció, i es demostra que atALKBH9B també pot reconéixer els RNAs virals. Els resultats revelen que atALKBH9B té la capacitat de desmetilar m6A a partir de molècules de RNA monocatenari in vitro. Esta proteïna s'acumula en grànuls citoplasmàtics que es colocalitzen amb siRNA bodies i s'associen a P-bodies, la qual cosa suggerix que l'activitat atALKBH9B podria estar relacionada amb els processos de silenciament i/o degradació de mRNA. D'altra banda, assajos preliminars mostren que els RNAs virals de l'AMV, el virus del mosaic del cogombre (CMV), el virus de l'arruga del nap (TCV) i el virus del mosaic de la coliflor (CaMV) es metilen durant la infecció en Arabidopsis. A més, per AMV i CMV els resultats van ser confirmats per UPLC-PDA-Tof-MS i els llocs m6A al llarg dels RNAs d'AMV s'identificaren mitjançant MeRIP-seq. Els resultats presentats confirmen que la relació m6A/A al llarg dels RNAs virals augmenta en les plantes atalkbh9b en comparació amb les silvestres, mentre que la traducció i/o replicació es veuen afectades i el moviment sistèmic a les tiges florals està pràcticament bloquejat. A diferència de la CP d'AMV, la de CMV no interacciona amb atALKBH9B per Y2H i, com ocorre amb la resta dels virus analitzats (CMV, TCV i CaMV), el seu cicle d'infecció no es veu afectat en plantes atalkbh9b. A més, la seqüenciació de mRNA realitzada en este treball revela que la infecció per AMV indueix alguns gens d'Arabidopsis pertanyents a la maquinària m6A, MTA, MTB, VIR i ECT5. D'acord amb l'efecte antiviral dependent de m6A per a l'AMV i tenint en compte que ECT2, ECT3 i ECT4 van ser recentment caracteritzades com readers citoplasmàtiques, la supressió del mòdul ECT2/ECT3/ECT5 augmenta significativament els títols sistèmics d'AMV i CMV. L'efecte antiviral d'ECT2 sobre AMV sembla estar modulat a través de la seua unió directa als nucleòtids m6A presents en els RNAs virals, ja que un mutant de la proteïna ECT2 defectuós en el reconeixement de m6A perd l'activitat antiviral que sí que presenta la proteïna original i no és capaç d'arrossegar RNAs virals in vivo. D'altra banda, d'acord amb la localització subcel·lular descrita prèviament per a ECT2 i ECT4 i la capacitat d'ECT2 per a experimentar una fase similar al gel in vitro, l'expressió transitòria d'ECT5 mostra un patró citoplasmàtic amb la formació d'agregats. Es proposa que, com s'ha descrit per a les proteïnes YTH de mamífers, la interacció entre les ECTs i el RNA polimetilat (en aquest cas, RNA viral) promouria la formació de grànuls d'estrès i, en conseqüència, reduiria les taxes de traducció i replicació viral. En resum, en este treball es caracteritza la primera m6A eraser de plantes, atALKBH9B, i, per primera vegada, es descriu la influència de l'mecanisme de metilació M6A en les infeccions virals de plantes.<br>Martínez Pérez, M. (2020). Involvement of the host RNA N6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155976<br>TESIS

Alfalfa, Medicago sativa L., cultivars, breeding lines and germplasm releases (populations) and Rhizobium meliloti L. strains that exhibit nitrogen fixation efficiency and tolerance to salinity and drought stresses should enhance seedling establishment, increase yields, reduce nitrogen dependency on petroleum-based nitrogen fertilizers, and allow wider use of irrigated lands in semiarid and a rid regions. In vitro experiments were conducted to determine relative salt (sodium chloride -- NaCl) tolerance: l) during germination and early seedling growth of 229 alfalfa populations from North Africa, the Middle East and the United States, 2) survival and growth of 92 rhizobial strains obtained from different laboratories in the United States or isolated from host plants growing in saline and arid regions of the Intermountain west, 3) compatibility, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strains, and 4) survival, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strain tested under drought conditions. Results from screening of alfalfa at salinity levels of 0 to 3.2 S m-1 (0.1 S m-1 = 640 mg L-1 = 10 mM NaCl at 25° C) indicated 29 populations exhibited salt tolerance at 2.8 S m-1. At salinity levels of 0 to 7.2 S m-1, 14 rhizobial strains exhibited salt tolerance at 6.4 s m-1. Compatibility, nodulation and nitrogen fixation efficiency in the 29 alfalfa populations and the 14 rhizobial strains under controlled conditions showed that four of the rhizobial strains were highly compatible with all 29 populations. These 29 populations and four strains were then exposed to 0 to 1.6 S m-1 NaCl, with the result that nitrogen was fixed with highly compatible symbionts at 1.6 S m-1 NaCl. From these results, the six most highly compatible alfalfa populations and the best overall rhizobial strain were combined and tested under simulated drought stress. These populations were able to fix more nitrogen under drought stress (-0.76 MPa) (0.088 nmol seedling-1 s-1) than they did under salt stress (1.6 S m-1) (0.066 nmol seedling-1 s-1). The in vitro screening technique for acetylene reduction appears to be a useful tool for detecting physiological changes due to salinity and water stresses and for measuring seedling nitrogen fixation efficiency. Breeding for drought and salinity tolerance in conjunction with high nitrogen fix ing potential may be more realistic than breeding strictly for nitrogen fixation without regard for environmental adaptation.

Rising fertilizer prices have led a return to the use of grass-legume mixtures to reduce N costs and improve pasture productivity. The objective of this study was to determine optimal species combinations of binary grass-legume mixtures to improve forage production and pasture nutritive value in irrigated pastures of the Intermountain West. The study was conducted at the Utah State University Intermountain Pature Research Facility near Lewiston, UT. Tall Fescue (TF), Orchardgrass (OG), and meadow brome (MB) were grown with alfalfa (ALF), birdsfoot trefoil (BFTF), and cicer milkvetch (CMV) in legume-grass mixes and monocultures at planting ratios of 25:75, 50:50, 75:25. Grass monocultures were fertilized with 0 (0 N), 67 (67 N), or 134 kg N ha-1 (134 N). Forage was harvested four times each season during 2011-2012. Forage of the mixtures and monocultures from the first and third harvests was analyzed for crude protein (CP) and neutral-detergent fiber (NDF). Average forage production of the unfertilized TF, MB, and OG monocultures was 11.03, 9.76, and 8.10 Mg ha-1, respectively. TF-ALF, OG-ALF, and MB-ALF grass-legume mixes averaged 24.0, 35.0, and 41.0% higher forage production than their respective unfertilized grass monocultures. The grass-legume mixtures with the highest CP were MB-ALF 159, TF-ALF 159, and TF-OG-159 g kg-1 and average 59, 43 and 51% higher than their respective unfertilized grass monocultures. Likewise, the mixtures with the lowest NDF were OG-ALF 453 g kg-1, OG-BFTF 469 g kg-1, and MB-ALF 480 g kg-1. These mixtures had 10, 7, and 18% lower NDF than their respective unfertilized grass monocultures. Individual harvests had similarly higher yields and CP, with lower NDF for the mixtures than the unfertilized grass monocultures. The grass-legume mixture with the 50:50 planting ratio were most productive and had high forage quality. The grass-legume mixtures had similar forage production as the grass monocultures at 134 kg N ha-1. The grass-legume mixtures also had higher CP and lower NDF than the grass monocultures. Cicer milkvetch did not perform well in irrigated pastures. Grass-legume mixtures with ALF and BFTF can be used to replace commercial N while increasing forage nutritive value.

A major problem in irrigated agriculture in the Western U.S. is the gradual accumulation of salinity in the plant root zone. These nonuniformly saline soils contain increasing amounts of salinity with depth, and salt accumulation is accelerated in situations where leaching is minimized. Root growth and thus plant yield is limited in these soils due to decreased water uptake. We studied the root growth of two subpopulations of alfalfa differing in their ability to produce fibrous roots to determine if altering root morphology would increase plant yield and water extraction, in an irrigated saline soil. Soil profiles for a control and three treatments with increasing salinity were packed in to PVC cylinders fitted with a flat window down one side for root measurements. A single alfalfa plant was grown from seed in each cylinder, and irrigated with water enriched primarily in sulfate salts. Alfalfa plants were grown for five successive harvests in a greenhouse, and water extraction was measured in the control and high Salinity treatment by time-domain reflectometry. Final electrical conductivities of the soil ranged from 3.0 to 23 dS m-1. The yield of the high fibrous root subpopulation was not reduced by the soil salinity by the fifth harvest, while that of the low fibrous subpopulation was reduced 22%. Root growth of the high fibrous subpopulation was significantly increased by as much as 54% in the upper 30 cm of the root zone, compared to that of the low fibrous subpopulation. Water extraction was higher in the upper, least saline portion of the root zone for the high fibrous root subpopulation. The results of this study support the use of alfalfa with increased fibrous root production under saline irrigation with minimal leaching.

[Truncated abstract] In southern Western Australia the replacement of deep-rooted native vegetation with annual species has resulted in rising water tables and increased salinity due to insufficient water use. The area has a Mediterranean-type climate where rainfall during summer is generally low but variable resulting in limited plant growth. However, if rainfall does occur it potentially can contribute to to the increased water excess or drainage by increasing the soil water content before the main drainage period in winter. The first study investigated factors controlling soil water content changes during the fallow (December to May) in annual farming systems. This was achieved by examining variation in available soil water storage to a depth of 1.0-1.5 m at three sites within 13 seasons. Reasons for the variation were examined using the Agricultural Production Systems Simulator (APSIM). This study also investigated the relationship between soil water content at the end of the fallow period (1 May) and the amount of drainage below 2.5 m by using APSIM coupled to historical weather records at three locations. At the end of the fallow a mean of 24 mm (or 25%) of rainfall during the fallow was retained in the soil. Losses of soil water during the fallow were due to evaporation (mean of 60 mm), transpiration from plant cover (mean of 12 mm) and drainage below the root zone and run off (combined mean of 13 mm). Soil water accumulation during the fallow period had a significant impact on simulated drainage under wheat in the following growing season. Every 1 mm increase in soil wetness at the end of the fallow resulted in a 0.7-1 mm increase in simulated drainage during the growing season. ... Variation in the water excess due to variation in rainfall was greater than the reduction in water excess due to lucerne. This makes the decisions about when to grow lucerne to reduce water excess difficult if livestock enterprises are less profitable than cropping enterprises. The findings of this PhD indicate that lucerne does have a place in Mediterranean-type environments because of its greater water use than current farming practices. However, its use needs to be strategic and the strategy will vary from region to region. For example, in the low rainfall region lucerne sowings need to be matched with high soil water contents and phase length will generally be short (2-3 years). In comparison at high rainfall regions lucerne will need to be grown for longer or combined with other strategies to increase water use.

A test was conducted to evaluate the effect that renovating a weak alfalfa stand had upon yields in one field in the Yuma Valley. Results indicated that yields were not significantly different in the renovated vs. the not renovated plots. Under the conditions present in this test, there appeared to be no yield advantage to renovation during the first year.

Furadan 4F was applied to alfalfa regrowth stubble following the first harvest of 1998 to examine treatment effects of alfalfa weevils, aphids, resultant plant growth, and threecornered alfalfa hoppers. Furadan 4F reduced larval numbers throughout the period that larvae were present (25 days after treatment). Little effect was noted for adult weevil numbers in field. Aphid numbers were also reduced by the treatment, and alfalfa stems in Furadan 4F plots were significantly taller at 21 days post treatment. Little, if any, effect from the Furadan 4F treatment was noted for adult threecornered alfalfa hopper populations during the five months following application.

EPTC IO percent granules, Trifluralin 10 percent granules and Poast were evaluated for southern sandbur (cenchrus echinatus) control. Trifluralin resulted in variable control ranging from 90 percent down to 55 percent. EPTC produced good but sometimes varied control when applied in February, prior to the germination of the weeds, and repeated in April, June and July. Poast was effective postemergence only when applied to small seedlings. It was ineffective when applied to established sandbur.

Alfalfa irrigation is often withheld as a water conservation measure in Arizona. The objective of this research was to test the hypothesis that alfalfa yield and stand can be permanently damaged by withholding irrigation water. Irrigation was terminated during the summer (July through October) or winter (November through February) on a Superstition sand in Yuma. Summer irrigation termination reduced plant density from 4 to 1.5 plants per square foot and reduced hay yield from 0.76 to 0.27 tons per acre per cutting after irrigation was resumed. Winter irrigation termination had no residual effect on yield or plant density. Termination of alfalfa irrigation can permanently damage stands and reduce yield on sandy soil.

Alfalfa irrigation is often withheld as a water conservation measure in Arizona. The objective of this research was to test the hypothesis that alfalfa can be permanently damaged by withholding irrigation water. Irrigation was terminated during the summer (July or August through September) or summer through winter (July or August through mid -March) on a Casa Grande sandy loam at Maricopa. Irrigation termination at Maricopa was not detrimental to stand although hay yields were depressed from 1.47 to 1.28 tons per acre following the second cycle of summer irrigation termination and from 1.57 to 1.23 tons per acre per cutting following summer through winter irrigation termination. Alfalfa water use was reduced by irrigation termination even after irrigation was resumed. Total nonstructural carbohydrates in the roots were never less than the control during irrigation termination but decreased relative to normally irrigated alfalfa when irrigation resumed.

Membrane proteins specific to cold acclimation were studied in alfalfa (Medicago falcata L. cv Anik) seedlings and cell cultures. They were identified following separation on polyacrylamide gels and localized as far as possible to specific membranes by fractionation on continuous sucrose gradient and analysis of marker enzyme assays.<br>With cold acclimation, certain membrane proteins from seedlings showed subtle changes (mainly increases) in their steady-state amount and in their net synthesis rate. Most of these changes were in proteins with molecular weights below 100 kDa and were associated with light fractions of the sucrose gradient, favoring a Golgi, endoplasmic reticulum or tonoplast location for the proteins. Preliminary work done on membrane proteins from cell cultures showed more pronounced changes with cold acclimation. Most of the changes were in proteins with molecular weights below 100 kDa and were associated with heavy fractions of the sucrose gradient. Since they are easier to harvest and to manipulate, cell cultures appear to be the better system to use in future studies.

There has been limited research on the rooting patterns of alfalfa. This information would be important in developing new germplasm sources that are more water efficient, salt tolerant and drought tolerant. Ten alfalfa varieties were evaluated for possible genetic control of rooting traits. Seedlings were grown hydroponically in a growth chamber. The cultivars evaluated showed a significant statistical difference for width of lateral root system and number of lateral roots. The variation in seedling rooting patterns is sufficient to warrant a selection program for improved lateral rooting patterns.

A test was conducted to evaluate the effect of two seed treatments on seven varieties of alfalfa. The treatments included Rhizocote, Rhizocote plus Apron plus Rovral and untreated seed. Significantly fewer seedlings emerged for the coated than the uncoated seed when planted on a pound for pound basis. First cutting alfalfa yields were neither increased nor decreased as a result of the seed treatments.

Twentynine insecticide treatments were applied on Feb. 7, 2002, to evaluate the effects of chemistry and rates on cowpea aphids and associated parasitic wasps. Data were also able to be collected on blue alfalfa aphids and alfalfa weevil larvae. At seven days post treatment fewest cowpea aphids were noted in plots treated with Furadan, the 3.84 oz./acre rate of WarriorT, and several of the higher rates of Lorsban 4E. Organophosphate insecticides had fewer cowpea aphids on the whole than did pyrethroid insecticides. All insecticide treatments had significantly fewer parasitic wasps than the untreated check, although these populations may correspond to aphid populations. All insecticide treatments had fewer blue alfalfa aphids early in the study, with very good control noted from the 3.84 oz./acre rate of WarriorT and the combination of 8 oz. of Lorsban 4E + 2 oz./acre of Baythroid 2. Treatments containing Baythroid 2 had the fewest blue alfalfa aphids at 44 days post treatment. Treatments which had at least 70% fewer weevils than the untreated check at 12 days post treatment included Furadan, all treatments that included any formulation of Baythroid, and the high rates of WarriorT and Pounce 3.2 EC, and the 0.088 rate of F1785 50DF.