Effect of nodules on dehydration response in alfalfa (Medicago sativa L.)

Drought is a key abiotic stress that negatively affects growth and development as well as symbiotic nitrogen fixation in alfalfa (Medicago sativa L.). To understand whether nodulation would affect drought stress response in alfalfa, we analyzed the lipid peroxidation, activities of antioxidant enzymes including superoxide dismutase (SOD), and catalase (CAT), contents of superoxide anion radical, non-enzymatic antioxidants including reduced glutathione (GSH) and proline, total protein, and soluble sugar in dehydration-stressed alfalfa. Three-month-old alfalfa plants without nodule, with active nodules, or with inactive nodules were dehydrated for 0, 1, 2, 4, 6, 8, and 10 h. We found that roots and leaves from plants with nodules, especially with active nodules, showed less lipid peroxidation which was associated with higher CAT activities and higher levels of GSH. Roots and leaves with active nodules also accumulated less free proline and soluble sugar compared to plants without nodules, suggesting that proline and soluble sugar may have a limited role in osmotic adjustment in these plants. The results suggested that active nodules may have a positive effect on drought stress tolerance in alfalfa.     

Quantitative ovule sterility in Medicago sativa

 Ovule sterility was found to be associated with callose deposition in B17, a plant with low fertility from the alfalfa cv Blazer XL. The site of callose deposition, which began during embryo-sac development and affected 81% of the ovules in mature florets, at random positions in the ovary, appeared to be the embryo-sac wall or the integumentary tapetum. The fertile ovules of B17 transmitted the ovule-sterility trait to the progenies, thereby demonstrating a sporophytic genetic control. B17 was crossed with P13, a Peruvian plant with 5% callosized ovules, to generate reciprocal F₁ populations, and an F₁ plant (91% callosized ovules) was used to obtain the backcross populations. B17 was also crossed to unrelated, highly fertile, plants. S₁ progenies from B17 and P13 were also studied. All the progeny populations displayed continuous variation for the percentage of sterile ovules, supporting a polygenic control. Narrow-sense heritability estimated by offspring-midparent regression was 0.85. Reduced transmission of the sterility trait through the pollen is hypothesized to explain the difference between reciprocal crosses. Six progeny plants showing 100% callosized ovules proved to be female-sterile. Ovule sterility could be an important component of the generally observed low realized seed potential in alfalfa. Received: 2 March 1998 / Accepted: 28 May 1998     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.

Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

Gibberellin and Protease Activity in Medicago sativa

During germination of the lucerne seed, proteolytic and gibberellinic activity increases. Exogenous GA added to the solution in which the seed was germinated caused an increment in proteolytic activity. CCC inhibited the biosynthesis of GA in the seed- ling, and had no effect on the proteases. This indicates that there are other factors Hum GA responsible for the increment in proteolytic activity during Medicago germination.     

Influence of parental genotype on plastid inheritance in Medicago sativa

Research using chlorophyll-deficient mutants has shown that plastids are inherited biparentally in Medicago sativa L. (alfalfa). Variation in plastid transmission behavior was observed among crosses in earlier studies, but it was not determined whether this variation was under genetic control. In my research, genetic analyses of the frequencies of normal (G), chlorophyll-deficient (CD), and sectored (G and CD) progenies produced from G x CD crosses demonstrated that plastid inheritance patterns in alfalfa are influenced by both maternal and paternal genotypes. A strong paternal bias in plastid transmission existed in the majority of crosses despite the potential developmental disadvantages associated with paternally contributed CD plastids. The high frequencies of uniparental progenies suggest that genetic control of plastid inheritance in alfalfa may be exerted through effects on the number and distribution of maternal and paternal plastids early in embryo development.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Effect of Light and Benzyladenine on Dark-Treated Growing Rice (Oryza sativa) Leaves II. Changes in Peroxidase Activity

Changes in peroxidase activity were studied in the attachedfirst leaf of dark-treated Oryza sativa L. cv. Bala seedlingsin response to benzyladenine and light treatments during laterperiods of leaf growth, prior to maturation. Darkness causeda mild decrease in peroxidase activity; but in illuminated leaves,the enzyme activity was stable at all times. There was a sharprise in peroxidase activity in dark-treated leaves upon lightor benzyladenine application, irrespective of the time of treatment.Benzyladenine treatment to illuminated leaves also caused arise in peroxidase activity. Exogenous hydrogen peroxide, glycolateand amizol resulted in a rise in peroxidase activity, whichwas further enhanced by benzyladenine treatment in both lightand dark incubated leaves. Proline maintained chlorophyll levels,whereas hydroxyproline caused chlorophyll degradation. Benzyladenineenhanced the proline effect and counteracted the hydroxyprolineeffect on chlorophyll. Both proline and hydroxyproline increasedperoxidase activity in the leaves of light and dark incubatedseedlings, and the enzyme activity further increased after benzyladeninetreatment. (Received December 7, 1984; Accepted May 8, 1985)     

Root system of Medicago sativa and Medicago truncatula: drought effects on carbon metabolism

Plant and Soil - Changes in snow cover can influence root growth and distribution of herbaceous species in water limiting desert ecosystems. However, how the growth of root systems of herbaceous...     

Sensitivity to Abscisic Acid and Osmoticum Changes During Embryogenesis of Alfalfa (Medicago sativa)

Developing embryos of alfalfa can be placed into nine stagesof development according to their morphological characteristics.The intact seeds do not germinate when removed from the podand placed on water until at least stage VI, and complete germinabilityis not achieved until the seeds are at the stage when they startto undergo maturation desiccation. Isolated embryos are germinableas early as stage III, and will germinate within 24 h on Murashigeand Skoog medium containing 3% sucrose. Osmoticum can inhibitembryo germination, but only proper osmotic conditions can maintaindevelopment in vitro; development does not occur at germination-inhibitingconcentrations of abscisic acid. The sensitivity to abscisicacid and osmoticum changes with stage of development. Early-stageembryos have the highest abscisic acid sensitivity and thisdeclines to the extent that mature dry embryos require a highconcentration (1?0 mol m^–3) to prevent their germination.Sensitivity of the embryos to osmoticum is maximum at stageVII of development. The combined inhibitory effect of abscisicacid and osmoticum on germination during development is greaterthan their individual effects. This combined effect is stage-dependent.Thus studies on the effects of abscisic acid and osmoticum onembryogenesis and associated synthetic events should be expectedto vary according to the sensitivity to these agents at differentstages of development. Key words: Medicago sativa, embryogenesis, abscisic acid, osmoticum, seed development     

陇东野生紫花苜蓿的核型分析

采用0.15%秋水仙素和0.002mol/L 8-羟基喹啉的混合液对陇东野生紫花苜蓿萌发种子的根尖预处理3h,室温条件下用1%果胶酶和1%纤维素酶酶解1.5h,得到清晰染色体制片.核型分析结果显示,陇东野生紫花苜蓿染色体数目为32条,具有1对随体,为2B核型,染色体核型组成公式为2n=32=30m(2SAT)+2sm,染色体长度组成公式为2n=2L+18M2+8M1+4S.     

Organspezifische multiple Formen der Glutamatdehydrogenase in Medicago sativa

Summary The alteration of the multiple forms of NAD-dependent glutamic dehydrogenase (GDH) during the development of Medicago sativa is investigated by means of polyacrylamide electrophoresis. Seed germination is accompanied by a characteristic change of the GDH-isoenzyme pattern. Seeds contain seven isoenzymes, which gradually decrease in number during germination. At the same time a pattern of new isoenzymes becomes visible. The seed pattern is called GDH-I and the later appearing pattern GDH-II. GDH-I is characteristic for the cotyledons, whereas GDH-II is the typical pattern of the root system. Shoots produce a mixed pattern composed of the GDH-II isoenzymes as well as some GDH-I isoenzymes.These isoenzyme patterns are organ specific. No qualitative change occurs during further development of the plants and during growth in the presence of different inorganic and organic N-sources in the culture medium.All the individual isoenzymes are found predominantly in the particulate fraction. They represent stable forms which are not altered by variation of the conditions of enzyme extraction or during enzyme purification. Re-electrophoresis of the individual isoenzymes following elution from the polyacrylamide gels reveals only one specific band. The molecular weights of all the distinctive isoenzymes are identical.There is some evidence that the different isoenzymes represent conformational forms of one enzyme, and it is postulated that the GDH-I isoenzymes are correlated to a normal metabolic (or catabolic) function of the enzyme, whereas the GDH-II isoenzymes are responsible for a primarily anabolic function of glutamic dehydrogenase.     

Non random chloroplast DNA hypervariability in Medicago sativa

Two hypervariable regions of the alfalfa (Medicago sativa L) chloroplast genome were used to describe levels of genetic relatedness among populations. PCR primers were developed to amplify the hypervariable regions. The frequency of occurrence of fragments of like size between populations was used to develop a measure of genetic relatedness. Relationships among 135 alfalfa accessions were investigated with principal component and cluster analyses, based on the genetic distance measures. Distinct clusters were taken as an indication of genetically distinct lineages. The populations investigated represented collections from world regions defined as the centers of origin of specific alfalfa germplasm sources, or else represented collections of introduced, and naturally adapted, accessions from agriculturally advanced regions. In general, this analysis indicated that the accessions from regions of origin of germplasm sources were largely homogeneous, while accessions from areas of introduction were much more diverse. In some cases, the accessions from a region of origin formed distinct clusters, suggesting that divergence has resulted in genetically distinct lines persisting in the original region of origin. Investigation of the stability of the marker fragments through vegetatively, and sexually, propagated plants indicated stable transmission through the sexual phase. However, one of the two regions underwent a deletion of 145 bp of one copy of a tandemly repeated 146 bp region in the equivalent of 80 years of vegetative growth. Received: 18 November 1999 / Accepted: 31 March 2000     

Cytomixis in pollen mother cells of Medicago sativa L

Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many plant species, but not in Medicago sativa spp. In the present study we report the identification of a few cytomictic alfalfa plants. Those plants, the "mother plants," were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S(1) and F(1) plants, and on controls. The S(1) and F(1) plants, like the mother plants, were found to be cytomictic. Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F(1) progenies. Variability of cytomixis in the F(1) plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability.     

Effect of sulphur fertilization on the nitrogen-sulphur relationships in alfalfa (Medicago sativa L. Pers.)

Summary Distribution of different nitrogen and sulphur fractions and N:S ratios in alfalfa (Medicago sativa L. Pers.) without and with S fertilization has been studied with a green house experiment. With increasing rates of applied S, the concentrations of total N, protein N, total S, protein S and total soluble S in the plant increased but those of non-protein N fractions (total soluble N, amino acid N, amide N, nitrate N) decreased. Thus the adequate supply of S in alfalfa increases the protein synthesis by accelerating protein metabolism. Sulphur application narrowed total N:total S ratios and widened protein N:protein S ratios. The data indicate that one part sulphur was required for every 11 to 12 parts of nitrogen to insure maximum production of protein. re]19750305     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.