Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules

Host plant glutamine synthetase (GS) has been purified 100-fold from N₂-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody-containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N₂-fixing alfalfa nodules.

Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts.

     

Development of Bacteroids in Alfalfa (Medicago sativa) Nodules

The morphology, acetylene reduction capability, and nucleic acid content of bacteroids in different regions of alfalfa (Medicago sativa var. Buffalo) nodules were studied by electron microscopy, gas chromatography, and laser flow microfluorometry, respectively. Bacteroids in the nodule tips were small (1 to 2.5 micrometers in length), had low nucleic acid content, and contained distinct central nucleoids. These bacteroids were comparatively inactive in acetylene reduction in situ. Bacteroids in the middle regions of alfalfa nodules were greatly enlarged (5 to 7 micrometers in length), had relatively high nucleic acid content, and did not possess central nucleoids. The bacteroids were very active in acetylene reduction. Bacteroids in the basal nodule region also were enlarged and without distinct nucleoid regions, but had relatively low nucleic acid content and low in situ acetylene-reducing activity.     

大孔吸附树脂对苜蓿皂苷的吸附和解吸附作用(英文)

采用5种不同极性的树脂(AB-8、S-8、NKA-9、D-101和X-5)来评价对苜蓿皂苷的吸附和解吸附作用,其中中等极性的AB-8树脂对苜蓿皂苷具有最大的吸附量,用55%~65%的乙醇溶液能有效地将吸附的皂苷洗脱下来。当苜蓿粗提物量和AB-8树脂量为1∶1时,树脂的吸附量达到饱和。采用AB-8树脂,用90%乙醇洗脱,苜蓿提取物的最大解吸附量为108.4 mg/g干重树脂。通过大孔树脂吸附和解吸附,将90%乙醇洗脱液浓缩,皂苷含量(53%)是苜蓿粗提物含量(5.68%)的9倍。结果表明,AB-8大孔吸附树脂可用于苜蓿皂苷的大规模制备。     

Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest

Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.     

Nitrogen Fixation in an Establishing Alfalfa (Medicago sativa L.) Ley in Sweden, Estimated by Three Different Methods

In an investigation of the ability of alfalfa to fix nitrogen under field conditions in Scandinavia, N₂ fixation during the establishment year ranged between 7.85 and 10.37 g of N m⁻², depending on the method used. The methods used were an in situ acetylene reduction method, a ¹⁵N isotope dilution method using two reference crops, and a total-N difference method. The dynamics of nitrogenase activity in relation to plant development was studied by using the acetylene reduction method. Also, the diurnal variation in N₂ fixation at the field site was studied with the acetylene reduction method; no diurnal change was detected, which is explained by the fact that the nodules within the soil were protected against short-term fluctuations in temperature. The significant amount of nitrogen fixed by alfalfa during its first year even at northern latitudes suggests that this crop offers an alternative to conventional field management of heavily fertilized nonlegume leys.     

根癌农杆菌介导的苜蓿体胚转化

以苜蓿体细胞胚胎作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究该转化体系的最佳实验参数。实验结果显示,负压处理10min和共培养5d时表达率最高(可达17．4％)。以这一转化方法分别对带有3种不同启动于的表达载体进行比较,发现由CMV35S启动于驱动的GUS基因的瞬时表达率可达82．7％,Ubil启动于驱动的可达57．8％,而Actl启动于驱动的则未见表达。     

Rhizobium meliloti Mutants Defective in Symbiotic Nitrogen Fixation Affect the Oxygen Gradient in Alfalfa (Medicago sativa) Root Nodules

Rhizobium meliloti bacteroids carrying mutations in either fdxNor fixX isolated from alfalfa root nodules were shown to containthe nitrogenase proteins NifH, NifD and NifK. In contrast toan in vitro system of N₂-fixation based on R. meliloti wild-typebacteroids, nitrogenase activity could not be restored in crudeextracts of these mutant bacteroids by the addition of an artificialelectron donor, indicating that the nitrogenase proteins werepresent but not functional. ESR-studies revealed that both mutantslacked the FeMo-cofactor of nitrogenase. To analyse the roleof free O₂ on the damage of the nitrogenase components and theFeMo-cofactor in these mutant bacteroids, microelectrode studiesof O₂ concentrations and gradients within alfalfa root noduleswere carried out. R. meliloti mutants defective in other genesnecessary for symbiotic N₂-fixation were also included in thisstudy. Four distinct types of O₂ gradients were defined by theapparent presence or absence of an O₂ diffusion barrier andby the minimum internal O₂ concentration. These data clearlydemonstrated the influence of the microsymbiont on the O₂ gradientswithin the nodules. Nodules induced by Rm0540, an R. melilotimutant with altered exopolysaccharide production, which is notable to infect plant cells, did not contain an O₂ diffusionbarrier. In contrast, nodules containing a mutant defectivein dicarboxylate transport (dctA-), produced an O₂ gradientsimilar to the wild-type. Microelectrode measurements revealedH₂ concentrations in alfalfa wild-type nodules comparable tosoyabean, whereas no hydrogen could be detected in nodules harbouringthe dctA mutant or any other mutant strain. Key words: Nitrogen fixation, Rhizobium meliloti bacteroids, ferredoxin-like proteins, microelectrode studies     

Isolation and Identification of Endogenous Cytokinins from Alfalfa (Medicago sativa L)

Endogenous cytokinins in alfalfa were isolated, and identified by mass spectrometry, trans- Ribosylzeatin (RZ) and ribosyldihydrozeatin (DHRZ) were identified from the root, and the combined content (benzyladenine equivalent) was estimated to be approximately 0.5/μg/100 g of fresh weight, eis- and trans-KL were identified from the stems and leaves. The relative content of the m-isomer was approximately five times greater than that of the trans-isomer.     

Detection of Norspermidine and Norspermine in Medicago sativa L. (Alfalfa)

Shoot meristem tissues of alfalfa, Medicago sativa L., were found by high performance liquid chromatography analyses to contain the uncommon polyamines, norspermidine and norspermine. The chemical structures of norspermidine and norspermine, purified from alfalfa, were confirmed by comparison of mass spectra with those from authentic standards. The discovery of norspermidine and norspermine in alfalfa implicates the presence of at least two biosynthetic enzymes, a polyamine oxidase and a previously uncharacterized aminopropyltransferase.     

CO(2) Fixation in Opuntia Roots

Nonautotrophic CO₂ metabolism in Opuntia echinocarpa roots was studied with techniques of manometry and radiometry. The roots were grown in a one-quarter strength nutrient solution for several days; the distal 2 cm was used for physiological studies. The roots assimilated significant quantities of ¹⁴CO₂ and appeared to show a crassulacean-type acid metabolism with respect to quality and quantity. Most of the ¹⁴C activity was associated with the distal portion of the elongating root indicating correlation with metabolic activity. The ¹⁴CO₂ assimilation was comparable to a crassulacean leaf succulent, but 3 times greater than that found for stem tissue of the same Opuntia species.

The rates of O₂ and CO₂ exchange and estimated CO₂ fixation were 180, 123, and 57 μl/g per hour. A respiratory quotient of 0.66 was found.

The products of ¹⁴CO₂ fixation were similar in most respects to reported experiments with leaf succulents. Equilibration of the predominant malic acid with isocitric, succinic, and fumaric acids was not evident. The latter observation was interpreted as metabolic isolation of the fixation products rather than poor citric acid cycle activity.

A rapid turnover of the fixed ¹⁴CO₂ was measured by following decarboxlyation kinetics and by product analysis after a postincubation period. The first order rate constant for the steady state release was 4.4 × 10⁻³ min⁻¹ with a half-time of 157.5 minutes. Amino acids decayed at a more rapid rate than organic acids.

     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析

FTL(F-box Triple LRR protein)是F-box蛋白家族的成员,具有F-box保守结构域,在植物抵御逆境胁迫过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsFTL基因,该基因的全长1422 bp,编码473个氨基酸。该蛋白含有1个F-box结构域及3个LRR重复。系统进化分析表明,MsFTL与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein亲缘关系最近。两者蛋白序列比对发现共有11个差异位点。在低温、盐、干旱以及外源ABA处理下,MsFTL基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农杆菌介导法转化烟草。对经过抗性筛选、PCR和Real-time PCR验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD活性,CAT活性高于野生型,MDA含量低于野生型。本研究表明,MsFTL基因在提高植物对低温胁迫的抗性方面具有重要的作用。     

Photoperiodic Effects on the Emanation of Volatiles from Alfalfa (Medicago sativa L.) Florets

Alfalfa (Medicago sativa L.) plants acclimated to photoperiods of 18 hours light, 6 hour dark in plant growth chambers exhibited a daily cyclic pattern of floret volatile emanation with a maximum emanation of about 6.5 nanograms of hydrocarbons/floret·30 minutes. This maximum was reached about 6 to 8 hours into the light period. After 8 hours of light, emanation of volatiles decreased rapidly to less than 0.1 ng/floret·30 min even though light and temperature remained constant. Under continuous illumination, only a small increase of volatile emanation occurred during the following 24 hours. It appeared that a dark period was necessary to promote floret volatile emanation. Floret volatile emanation was drastically affected for at least 7 days following a photoperiod change. A photoperiod change caused 6-fold concentration oscillations every 2 hours. The results are interpreted on the basis of a very active floral metabolism controlled by photoperiodically induced rhythms.     

Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in Alfalfa Seed Production Areas in the United States

The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.     

Alfalfa (Medicago sativa L.) seed yield in relation to phosphorus fertilization and honeybee pollination

This investigation was conducted at the Agricultural and Veterinary Training and Research Station, King Faisal University, Al-Ahsa, Saudi Arabia, during the alfalfa growing season in 2014. The study aimed to evaluate the impact of phosphorus fertilization and honeybee pollination on alfalfa seed production. The experiment was divided into 9 treatments of open pollination, honeybee pollination, and non-pollination with three different levels (0, 300 or 600 kg P₂O₅/ha/year) of triple super phosphate. All vegetative growth attributes of Hassawi alfalfa were significantly higher in the non-insect pollination plots, while the yield and yield component traits were significantly higher with either open pollination or honeybee pollination in parallel with the increasing level of phosphorus fertilizer up to 600 kg P₂O₅/ha/year in light salt-affected loamy sand soils. There was no seed yield in Hassawi alfalfa without insect pollination. Therefore, placing honeybee colonies near the fields of Hassawi alfalfa and adding 600 kg P₂O₅/ha/year can increase seed production.     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.