DROUGHT-INDUCED UNKNOWN PROTEIN 1 positively modulates drought tolerance in cultivated alfalfa(Medicago sativa L.)

Alfalfa is the most widely cultivated perennial legume forage crop worldwide.Drought is one of the major environmental factors influencing alfalfa productivity.However,the molecular mechanisms underlying alfalfa responses to drought stress are still largely unknown.This study identified a drought-inducible gene of unknown function,designated as Medicago sativa DROUGHT-INDUCED UNKNOWN PROTEIN 1(MsDIUP1).MsDIUP1 was localized to the nucleus,chloroplast,and plasma membranes.Overexpression of MsDIUP1 in Arabidopsis resulted in increased tolerance to drought,with higher seed germination,root length,fresh weight,and survival rate than in wild-type(WT)plants.Consistently,analysis of MsDIUP1 over-expression(OE)alfalfa plants revealed that MsDIUP1 also increased tolerance to drought stress,accompanied by physiological changes including reduced malondialdehyde(MDA)content and increased osmoprotectants accumulation(free proline and soluble sugar),relative to the WT.In contrast,disruption of MsDIUP1 expression by RNA interference(RNAi)in alfalfa resulted in a droughthypersensitive phenotype,with a lower chlorophyll content,higher MDA content,and less osmoprotectants accumulation than that of the WT.Transcript profiling of alfalfa WT,OE,and RNAi plants during drought stress showed differential responses for genes involved in stress signaling,antioxidant defense,and osmotic adjustment.Taken together,these results reveal a positive role for MsDIUP1 in regulating drought tolerance.

Photosynthesis and Transpiration Characters of Alfalfa (Medicago sativa) and Their Relationship with Relevant Factors during Branching Stage

[ Objective] The paper presents the diumal changes of photosynthesis and transpiration of different alfalfa varieties and their relationship with the associated physiological and ecological factors during branching stage, so as to provide a basis for the development, utilization, and breed- ing of alfalfa. [ Method] Under natural conditions, the diurnal changes of net photosynthetic rate （Pn）, transpiration rate （Tr）, the relevant physio- logical factors including leaf temperature （TI）, stomatal conductance （Gs） and intemal COn concentration （Ci）, as well as the relevant physiologi- cal factors including photosynthetic available radiation （PAR）, CO2 concentration in field （Ca） and air temperature （Ta） were measured in four al- falfa varieties （Algonguin, WL323 HQ, WL414, and Millionaire）. The water use efficiency （WUE） and light use efficiency （LUE） were calculated, and the correlation among them was also analyzed. [Result] The Pn, Tr, PAR and Ta of the four varieties appeared to vary in a single-peak curve; the sequence of WUE was WL323 HQ ~ Algonguin ~ WL414 ~ Millionaire; there was no significant difference in LUE of the four alfalfa varieties; coef- ficient analysis showed that Pn was mainly affected by PAR, Gs, and Ci, while Tr by PAR and Ta. [ Conclusion] WL323 HQ is the variety with high Pn, high WUE and low Tr, and it has strong adaptability to drought. In four alfalfa varieties, PAR, Ta, Gs, and TI are the primary determining fac- tors while Ca and Ci the limiting factors of Tr; Gs is the primary determining factor while Ci the limiting factor of Pn.

Degradation of TCP in Soil Planted with Alfalfa(Medicago sativa L.)

[Objective] The research aimed to study the degradation of 2,4,6-trichlorophenol （TCP） in soil planted with alfalfa （Medicago sativa L.）,as well as to provide references for the Chlorophenols phytoremediation technology in the practical application.[Method] By the use of pot culture experiment in greenhouse,the phytoremediation effect of alfalfa on TCP-contaminated soil,the growth conditions of alfalfa,as well as the effect of TCP on the activity of soil polyphenol oxidase,dehydrogenase and catalase were studied.[Result] After the alfalfa was grown for 75 d,the TCP content in soil of three different concentrations treatments （low,middle and high） decreased dramatically within 15 d,and then the decreasing rate was gradually slow; on the 30^th d of cultivation,the fresh weight of treated alfalfa showed no significant difference with the control （P〈0.05）,indicating that TCP in soil had inhibition effect on the growth of alfalfa; alfalfa could significantly enhance the activities of polyphenol oxidase,dehydrogenase and catalase,thus raising the degradation capability of soil plants and microorganisms on pollutants in soil.[Conclusion] There results indicated that alfalfa could enhance the degradation rate of organics in the contaminated soil and enhance soil enzyme activity,so the alfalfa could be used for the bioremediation of TCP contaminated soil.

Genetic Diversity of Tunisian and Chinese Alfalfa (Medicago sativa L.) Revealed by RAPD and ISSR Markers

Medicago sativa L.) is one of the most important forage crops in the world. The genetic variability analysis of 19 alfalfa populations collected from three sites in South Tunisia (Gabes, Kebili, Tozeur) and 1 from North West China were carried out using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Five RAPD primers amplified 44 bands of which 22 were polymorphic;and five ISSR primers amplified 51 bands of which 33 were polymorphic. The percentage of polymorphic bands detected by RAPD and ISSR was 50% and 64.7%, respectively. The resolving power (Rp) varied between 0.6 and 4.1 with an average of 2.02 for RAPD marker and between 0.7 and 6.5 with an average of 2.28 for ISSR marker. However the Average Informativeness band (AvIb) was ranged from 0.2 to 0.9 with an average of 0.5 in RAPD marker and from 0.29 to 0.7 with an average of 0.624 in ISSR marker. The RAPD marker revealed less within population genetic diversity than ISSR marker. Although Cluster (UPGMA) and Correspondence Factorial Analyses (CFA) indicate that populations’ clustering made independently both from the geographical origin.

Comparative transcriptome analyses reveal that the MsNST1 gene affects lignin synthesis in alfalfa(Medicago sativa L.)

As the second most abundant natural polymer,accounting for approximately 30%of the organic carbon in the biosphere,lignin plays an essential role in plant development.However,a high lignin content affects the nutritional quality of alfalfa(Medicago sativa L.),the most widely cultivated perennial legume forage crop.Histological analysis indicated that G-lignin and S-lignin were present in the stem,leaf,and petiole of alfalfa,and the deposition of lignin increased gradually in descending internodes.Neutral detergent fiber(NDF),acid detergent fiber(ADF),and acid detergent lignin(ADL)contents continually increased from the top to the bottom of the stem,and ADL content showed a similar trend in leaves.Alfalfa leaves and stems from five different nodes(1,2,4,6,and 8)were used as materials to investigate molecular regulatory mechanisms in lignin synthesis by RNA sequencing.Respectively 8074 and 7752 differentially expressed genes(DEGs)were identified in leaves and stems,and 1694 DEGs were common to the two tissues.‘‘Phenylpropanoid biosynthesis”was the most enriched pathway in both leaves and stems,and 134 key regulatory genes in lignin synthesis were identified by a weighted gene co-expression network analysis.The NAC family transcription factor MsNST1 gene was highly expressed in old leaf and stem tissues.The deposition pattern of G-and S-lignin differed among M.truncatula wild-type,nst1 mutants,and overexpression lines,and the transcription levels of lignin synthesis genes such as HCT,F5H,and COMT in these three materials also differed.These results suggest that MsNST1 affects lignin synthesis in alfalfa.These findings provide a genetic basis and abundant gene resources for further study of the molecular mechanisms of lignin synthesis,laying a foundation for low-lignin alfalfa breeding research.

Analysis of Genetic Relationship among Medicago sativa,Medicago falcate and Trigonella foenum-graecum Using ISSR

[Objective] This study aimed to analyze the genetic relationships among Medicago sativa,Medicago falcata and Trigonella foenum-graecum.[Method] ISSR technique was adopted to determine their genetic relationships.[Result] M.sativa,M.falcate and T.foenum-graecum had a broad genetic base.T.foenum-graecum shared closer relationship with M.falcata rather than M.sativa.The study on relationship between M.sativa and T.foenum-graecum was advantageous to identify disputable transition types.But a boundary should be found to identify species to be M.sativa or T.foenum-graecum.[Conclusion] This study will provide reference for identifying some disputable transition types.

Study on Physiological Activity of the Resistance of Medicago sativa to Thrips

[Objective] The experiment explored of resistance of different Medicago sativa through synthetic variations of all physiological indexes and the correlation analysis between physiological activity and damage degree after thrips damage. [Method] According to studying physiological activity (POD activity, SOD activity, PPO activity, PAL activity, MDA content and free proline content) of alfalfa in alfalfa resistance to different number of thrips, the correlation between physiological activity and resistance was investigated. [Result] It was shown that there were negative correlations among POD, SOD, PPO, PAL, MDA, free praline and the number of thrips, and there were negative correlations among POD, SOD, PPO, PAL, MDA, free praline and CAT. The content changes of POD,SOD,PPO,CAT,MDA and free proline in high resistant alfalfa were slow and PAL was quick and the activities of PPO,PAL,CAT were high, and the activities of POD、SOD were low. [Conclusion] The research provided theoretical basis and materials for identifying resistance varieties of alfalfa to thrip.

Bioinformatics Analysis and Homology Modeling Study of Protein Disulfide Isomerase(mPDI) from Medicago sativa L.

pdi gene from Medicago sativa L. ,encoding Protein Disulfide Isomerase（ mPDI ）, has been cloned and sequenced. According to the mRNA and amino acid sequence, the character of mPDI such as the physical and chemical properties, hydrophilicity/hydrophobicity, signal peptide, secondary structure, coiled coil, transmembrane domains, O-glycogylation site, active site, subcellular localization, functional structural domains and three-dimensional structure were analyzed by a series of bioinformatics software. The results showed that mPDI was a hydrophobic and stable protein with 3 coiled coils, 30-glycogylation sites, 2 structural domains of thioredoxin, 2 active sites of thioredoxin, and located in rough endoplasmic reticulum. It has 512 amino acids, the theoretical pl is 4.98, and signal peptide located in 1-24AA. In the secondary structure, a-helix, random coil, extended chain is 26.37%, 53.32%, 20.31% respectively. The validation of modeling accords with the stereochemistry.

Research on Phytoremediation of Heavy Metal Pollution in River Sediment by Medicago sativa L.

[Objective] The aim was to study the phytoremediation of heavy metal pollution in river sediment by Medicago sativa L.,so as to provide reliable references for the phytoremediation of heavy metal pollution in river sediment.[Method] The air-dried,screened and mixed sediment was put in rectangular PVC box（0.6 m×0.5 m×0.4 m） with seepage vent at the bottom,and the water holding capacity（WHC） of sediment was kept at 30%-60% by deionized water.The seeds of Medicago sativa L.were sown in April 2010,and seedlings were thinned after 7 d.Samples were collected from rhizosphere soil every 30 d,and were used to determine the content of heavy metals,bacteria quantity and enzyme activity in sediment.In addition,the accumulation of heavy metals in the roots,stems and leaves of plant was measured after harvest in October.[Result] Different parts of Medicago sativa L.varied in accumulation capacity to different heavy metals.The accumulation amount of Zn in Medicago sativa L.was the highest,especially in roots.Meanwhile,the accumulation amount of heavy metals like Ni,Cr,Cu and Pb in roots was higher than that of stems and leaves.In contrast,Mn was mainly accumulated in leaves and its amount accounted for 42.47% of the total amount in plant.Besides,the accumulation amount of all heavy metals was the lowest in stems.Ni,Cr,Cu and Pb could be degraded more effectively than Mn,and increasing the planting time and sowing times of crop was beneficial to the degradation of heavy metals.After planted Medicago sativa L.,the quantity of microorganisms in sediment went up obviously,and dehydrogenase activity also showed an increaseing trend.[Conclusion] Medicago sativa L.has certain restoring effect on Zn,Ni,Cr,Cu and Pb,and could be used to restore heavy metal pollution in river sediment.

Primary Study on ISSR Molecular Makers of Resistant Gene against Sclerotinia trifoliorum in Medicago sativa L.

[ Objective] The paper was to study ISSR molecular makers of resistant gene against Sclerotinia trifoliorum in Medicago sativa L. [ Method] Using mi- crosatellite markers （ISSR） molecular maker technology, combined with bulked-segregant analysis （BSA） method, the molecular makers for gene linkage with re- sistance against S. trifoliorum were screened from five resistant plants and seven susceptible plants. Leaf in vitro inoculation method was used to carry out resistant verification on 94 hybrid plants in Ft generation of high resistant No. 83 ~ high susceptible No. 4. [ Result] Among 93 ISSR primers, 35 primers could produce clear and stable amplification bands, and six of them could produce 9 specific bands between resistant and susceptible DNA pools. Resistance verification result showed that 825 - 1400, 831 - 1480, 850 - 1800, 858 - 1600, 866 - 1900, 888 - 1400 could be used as ISSR molecular makers of the resistant gene against S. trifoliorum in M. sativa. [Conclusion] The results provided basis for the further research on mapping,, cloning and genetically modified of resistant gene against S. trifoliorum in M. sativa.

Expression of Cold Resistant Gene CAS19 of Gongnong No.2 Medicago sativa L. in Tobacco

[Objective] The aim was to study the expression of cold resistant gene CAS19 of Gongnong No.2 Medicago sativa L. in tobacco. [Method] A pair of primers was designed according to nucleotide sequences of cold resistant gene CAS19 of M. sativa,and then RT-PCR was used to amplify the protein gene of CAS19,which was then cloned into pMD18-T vector and subcloned into expression vector PBI121. The recombination expression plasmid PBCAS was constructed. And then it was transferred into tobacco genome via Agrobacterium,and Southern-blotting analysis was used for detecting transgenic plants. [Result] CAS19 gene was integrated into the tobacco genome and highly expressed. [Conclusion] This study had provided theoretical basis for further exploring the expression mechanism of cold resistant gene CAS19 in tobacco.

In vitro Evaluation of Seed Germination in Twelve Alfalfa Cultivars under Salt Stress

Alfalfa(Medicago sativa L.),when exposed to abiotic stress such as salinity,suffers significant losses in yield and productivity.The present study evaluated the salinity tolerance of 12 alfalfa cultivars in vitro using five concentrations of sodium chloride(NaCl),ranging from 0 to 250 mmol L^(−1).The results obtained in the current study revealed that the Saudi cultivars,Kasimi and Hassawi,and the German cultivar(Berlin)had the highest salinity tolerance in terms of germination percentage(GP),corrected germination rate index(CGRI),days to reach 50%germination(GT_(50)),and ability to form cotyledonary and true leaves.Under mmol L^(−1) NaCl,the Saudi cultivar Kasimi cultivar showed GP,CGRI,and GT_(50) of 55.20%,123.15,and 3.77 days,respectively.Similarly,the German cultivar(Berlin)showed GP,CGRI,and GT_(50) of 50.06%,86.61,and 5.17 days,respectively.These findings might reveal a pivotal aspect in salt tolerance in alfalfa.Our results will help to select salt-tolerant alfalfa cultivars that could thrive in arid and semi-arid areas with salinity problems.

Effects of EDTA and Zn Stress on Physiological Characteristics of Medicago sativa L.

[Objectives]The paper was better understand the mechanisms of Zn plant uptake in the presence of EDTA and to evaluate the contributions of Zn-EDTA complexes to Zn uptake.[Methods]Three alfalfa cultivars were cultivated for 60 d before exposure to 0,250μg Zn and 250μg Zn+10μg EDTA per kg soil for 50 d.Zn concentrations in tissues were analyzed by flame atomic absorbance spectrometry.Subsequently,Zn amount per plant,translocation factor(TF)and bio-concentration factor(BCF)were calculated.Nonenzymatic compounds in tissues were analyzed spectrophotometrically.[Results]Application of Zn+EDTA expressively increased biomass of different tissues of three alfalfa cultivars.Among the three alfalfa cultivars,Medicago sativa ssp.displayed the highest Zn concentration in tissues,the largest Zn amount in aerial parts,and the highest BCF in aerial parts under Zn+EDTA exposure.Under Zn+EDTA stress,increases in free proline in roots,stem,and leaves of M.sativa ssp.were found.Inhibited O_(2)^(·-)production in stem and leaves,increases in soluble sugar,but decreases in soluble protein were observed in M.sativa ssp.[Conclusions]M.sativa ssp.is superior to other two cultivars for Zn phyto-remediation,and its well-coordinated physiological changes under Zn+EDTA exposure confer the great Zn tolerance of this cultivar.

Research Advances of Root Rot in Medicago sativa L.

As the most famous perennial leguminous forage in the world, Medicago sativa L. ranks the first in both yield and nutritional value, and can be used for many years once planted in field production. However, root rot has become a very important reason for yield decrease and plant de-cline of alfalfa due to long service life. According to the research progress at home and abroad, the main pathogen distribution, harms and control methods of root rot in M. sativa at home and abroad were systematically expounded, in order to provide a theoretical basis for further research of the disease.

Preliminary Study on the Mutagenic Effects of Space Flight on Alfalfa Seeds

[Objective] The study aimed to understand the mutagenic effects of space flight on alfalfa seeds.[Method] Seeds of three lines of alfalfa were carried into orbit by the satellite 'Shijian-8' for space fight,the indices including seed germination rate,plant height and growth rate,were measured after the seeds been retrieved.[Result] Remarkable mutagenic effects occurred on the flight seeds,which were mainly presented by abnormal cotyledon,extended variation range of plant height and growth rate.Nineteen plants with increased plant height were preliminarily screened from the T0 progenies,but whether the mutated trait could inherit should be confirmed in further study.Three flight lines of alfalfa are different in the percentages of abnormal cotyledon(10%-18%),variation range of plant height(increased 30%-150% compared to control),selected plant number with increased plant height(5-7 plants),suggesting that differences exits between the three lines in mutation efficiency.[Conclusion] Valuable mutated materials could be obtained by space flight and applied in modern agriculture.

宁南山区紫花苜蓿(Medicago sativa)土壤干层水分动态及草粮轮作恢复效应

以各类作物农田水分为对照，连续两年对宁南山区不同生长年限苜蓿深层土壤水分以及10年生苜蓿地耕翻后轮作不同年份作物农田的水分进行了测定。结果表明，随着苜蓿生长年限的增加，干层深度与厚度先增加后减小。3年生苜蓿干层深度为720cm，6年生干层最深可达1000cm以下，10年生干层深度为920cm，3—12年生苜蓿地0—700cm土层基本上均属于土壤干层范围。苜蓿地0—800cm土壤湿度随生长年限增加而降低，2004年测定的4、7年生和12年生苜蓿地0—700cm土层平均含水率分别为5．30％、5．22％和5．01％；2005年测定的3、6年生和10年生苜蓿地0—800cm土层湿度分别为6．26％、5．60％和5．27％；而800～1000cm土层湿度在一定年限后有恢复趋势。300cm为苜蓿地降水下渗的最大临界深度，300cm以下土壤干层一旦形成，将长期存在，7—12年生苜蓿300～700cm土层湿度仅维持在4．0％左右。苜蓿地和农田的土壤干层厚度与湿度有较大差异，草粮轮作可使苜蓿土壤干层水分基本恢复到农田湿度，而且轮作年份越长，土壤各层次水分恢复效果越好，10年生苜蓿轮作18年后土壤水分基本恢复到农田状态。

不同品种紫花苜蓿(Medicago sativa)的化感效应研究

用砂培法研究紫花苜蓿(Medicago sativa)的六个品种苗期地上部分水浸提液对鸭茅(Dactylis glomerataL.)的化感效应。结果表明,紫花苜蓿六个品种茎叶水浸提液对鸭茅的化感效应在供试品种间差异显著。游客、苜蓿对鸭茅的种子发芽及幼苗生长有抑制作用,但游客却对鸭茅的苗干重有促进作用;三得利和赛迪对鸭茅的种子发芽、根长及根干重有抑制作用,却对苗生长的影响无显著差异。此外,供试紫花苜蓿浸提液对鸭茅的化感作用,随着浓度的升高,抑制作用随之增强。

Callus Induction of Alfalfa and Evaluation of Callus Tolerance to Acid and Aluminum

Medicago is an important grass in the legume family,which is suitable to be grown in neutral or alkalescent soils,and hence,can be widely spread in south of China with low pH value.It is one of major objectives to cultivate acid-aluminum tolerant species.In the research,6 high-quality species with high-yielding potential were taken as materials to make evaluation on acid-aluminum tolerance by cell-culture technique,and the results showed that tolerance capacity from high to low was GT13R＞Muge 701=Muge 702＞Acrora＞AC-3＞Sheshou No.2=Medicago sativa.

紫花苜蓿(Medicago sativa L.)Tify基因家族的鉴定、生物信息学及表达模式分析

Tify基因家族是植物特有的基因家族,在植物生长发育、响应逆境胁迫和信号转导等方面发挥着重要作用。为了研究紫花苜蓿中Tify基因家族及其在非生物胁迫下的响应,本研究在紫花苜蓿中鉴定出84个Tify基因并对其进行染色体定位、基因结构分析等方面的生物信息学分析,并随机挑选Ms Tify6、Ms Tify32、MsTify46、MsTify48、Ms Tify65五个基因进行盐、碱、干旱胁迫下的RT-qPCR分析。理化性质及结构分析表明:除MsTify1、Ms Tify2、MsTify3外的其余81个基因在除7号染色体外的7条染色体上呈不均匀分布。RT-qPCR结果显示了MsTify基因对盐、碱、干旱等非生物胁迫有明显的响应,在调控逆境胁迫中发挥着关键性作用。为后续研究Tify基因提供了新的理论基础。

Changes of Proline Content,Activity,and Active Isoforms of Antioxidative Enzymes in Two Alfalfa Cultivars Under Salt Stress

The plants of two elfalfa （Medicago sativa L.） cultivars differing in salt tolerance were subjected to three salt treatments, 70, 140, and 210 mM NaCl for 7 days. Root, shoot, and leaf growths were inhibited by increased salt treatments in both cultivars, and at 140 and 210 mM salt treatments, Zhongmu 1 had significantly higher root, shoot, and leaf dry weights per plant than Deft. The malondialdehyde （MDA） accumulation in Deft was considerably greater than in Zhongmu 1, indicating a higher degree of lipid peroxidation at 140 and 210 mM salt treatments. The changes in the activity and active isoforms of antioxidant enzymes such as superoxide dismutase （SOD, EC 1.15.1.1）, catalase （CAT, EC 1.11.1.6）, peroxidase （POD, EC 1.11.1.7）, and ascorbate peroxidase （APOX, EC 1.11,1.11）, accumulation of free proline, and rate of lipid peroxidation in leaves of two alfalfa cultivars were also investigated. After stress, the activity and active isoforms of antioxidative enzymes were altered and the extent of alteration varied between the cultivar Deft and Zhongmu 1. The proline accumulation in Deft was considerably greater than in Zhongmu 1 at 210 mM salt treatment. This indicated that proline accumulation may be the result, instead of the cause, of salt tolerance.