Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

紫花苜蓿(Medicago sativa)对干旱胁迫的光合生理响应

紫花苜蓿是重要的豆科牧草,具有较强的抗旱性,然而干旱仍是制约紫花苜蓿生产的主要逆境因子。通过盆栽试验,以抗旱性强弱不同的两种紫花苜蓿为试验材料,对干旱胁迫下紫花苜蓿的光合生理进行较为系统的研究,结果表明：（1）干旱胁迫下两种紫花苜蓿叶片净光合速率（Pn）、蒸腾速率（E）、气孔导度（Gs）、叶绿素含量（Chl）都有不同幅度的下降;叶绿体超微结构遭到破坏。相对于抗旱性弱的苜蓿,抗旱性强的苜蓿随干旱胁迫程度的加深,净光合速率下降较慢,叶绿体的外形及基粒结构受到的影响较小。（2）轻度干旱胁迫下气孔限制是两种紫花苜蓿P。降低的主要因素,中度和重度干旱胁迫下非气孔限制是Pn降低的主要因素。（3）对叶绿素荧光参数的研究表明：干旱胁迫下两种紫花苜蓿PSⅡ反应中心光化学效率（F／F=）、PSⅡ潜在活性（Fv／Fo）降低。总体上抗旱性强的紫花苜蓿Fv／Fm和Fv／Fo下降幅度小,PSⅡ利用光能的能力及PSⅡ的潜在活性均较强。PsⅡ光化学淬灭系数（qP）、非光化学淬灭系数（qN）的变化表现为干旱胁迫下两种紫花苜蓿qP值降低、qN值升高,总体上抗旱性强的紫花苜蓿qP降低的幅度低且qN升高幅度大,表明抗旱性强的紫花苜蓿PSⅡ反应中心电子传递活性受到的影响小,光合机构的损伤程度低。     

水分对苜蓿叶片光合特性的影响

采用田间试验, 对每茬灌水3次(W₃)、2次(W₂)、1次(W₁)和不灌水(W₀)四种条件下的土壤水分, 苜蓿(Medicago sativa)叶片的叶绿素荧光参数、气孔导度(G_s)、净光合速率(P_n)和蒸腾速率(T_r)进行测定。结果表明, 灌水提高了苜蓿叶片的原初光能转换效率(F_v/F_m)、P_n和T_r, 并随着灌水量的增加而增加。苜蓿叶片的F_v/F_m、P_n和T_r的日均值与土壤含水量均呈极显著正相关关系。苜蓿叶片的P_n与F_v/F_m和光合有效辐射(PAR)的乘积呈正相关关系。灌水还改变了苜蓿叶片P_n的日变化格局。灌水较多的处理(W₃和W₂), 苜蓿叶片没有出现光合“午休”现象,P_n的日变化趋势呈现“单峰”型。而灌水较少和不灌水的处理(W₁和W₀), 苜蓿叶片出现了明显的光合“午休”现象, 其P_n的日变化进程呈现“双峰”型。在相同的水分条件下, 初花期苜蓿叶片的P_n高于再生期的, T_r则相反。     

Symbiosis between the root-nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa root-nodule bacteria (Sinorhizobium meliloti) were obtained from nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (T isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant root-nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and T) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25–68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the symbiotic efficiency of the isolates under standard conditions but did not correlate with the source of root-nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that the strains of root-nodule bacteria forming efficient symbioses under salinization conditions can be found.     

干旱胁迫下紫花苜蓿根系形态变化及与水分利用的关系

采用盆栽实验方法研究了紫花苜蓿(品种:陇东和阿尔冈金)根系形态、生物量、蒸腾耗水量等对持续干旱的反应及与水分利用效率(WUE)间的关系,以期揭示紫花苜蓿对干旱胁迫的适应机制。结果表明:干旱胁迫使得紫花苜蓿根系形态特征在年季间、茬次间和品种间发生了显著变化,主要表现为主根伸长生长受到抑制、主根直径变细、侧根和根系总长度伸长生长则被促进、根系表面积和直径≥1mm的侧根数目显著增加、根系生物量下降,这是紫花苜蓿对干旱逆境的适应策略,但这种适应性存在限度。另一方面,干旱胁迫条件下紫花苜蓿草产量和蒸腾耗水量也因生长年限、茬次和品种的不同而呈现不同程度的降低。紫花苜蓿根系形态性状(总根长、根系生物量与根冠比)与植株水分利用效率间具有显著的相关性,其中根重对水分效率的影响是第一位的。WUE在根系形态与冠层水分消耗的协同变化下得到有限提高。对干旱的耐性最终表现为第2年第1年、第1茬和第2茬第3茬、陇东阿尔冈金。     

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.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

Nitrogen fixation, stomatal response and transpiration in Medicago sativa, Trifolium repens and T. subterraneum under water stress and recovery

Stomatal behaviour, transpiration and nitrogen fixation were investigated in Medicago sativa L. (cvs. Tierra de Campos and Aragon, Hidalgo-Maynar 1966), Trifolium repens L. (cv. Aberystwyth S-184) and Trifolium subterraneum L. (cv. Clare) subjected to drought by withholding water and then to three days’ recovery after rewatering. Dawn leaf water potential was measured with pressure chamber, stomatal response with a diffusion porometer and nitrogen fixation by using acetylene reduction technique. At low water potentials, the leaf resistance was higher in Medicago than in Trifolium. As water stress developed all species decreased their transpiration, T. subterraneum being the one most affected by moderate deficits. During water stress ‘Tierra de Campos’ always maintained higher acetylene reduction levels than ‘Aragon’ and the Trifolium species, except for the lowest water potentials. During recovery from water stress only ‘Tierra de Campos’ reached predeficit transpiration rates. In ‘Tierra de Campos’ acetylene reduction recovery after rewatering was more rapid and intense than in ‘Aragon’. It is concluded that, of the plants investigated, ‘Tierra de Campos’ was best adapted to water deficits.     

水分胁迫下紫花苜蓿和高粱种子萌发特性及幼苗耐旱性

利用PEG溶液(水势梯度:-0.1～-0.5MPa)模拟水分胁迫,研究了紫花苜蓿(品种:阿尔冈金和陇东)和高粱(品种:抗四)种子的萌发能力的变化及对萌发环境的最低水分需求,并进行种间差异比较.结果显示:PEG水分胁迫通过限制种子有效水分的吸收而抑制了其萌发,且随着胁迫强度的增加,萌发能力减弱,主要表现在:萌发率、吸水速率、萌发活力、萌发胁迫指数等随胁迫强度的增加而下降,根芽比则随之增加.另一方面,种子群体萌动、萌发和出苗达50%概率时间随胁迫强度的增加而越发延迟,且各阶段对环境临界水势的需求不同,出苗阶段最为严格,说明种子出苗过程对环境水分胁迫最为敏感,耐旱能力最弱.相比之下,在同等胁迫条件下,高粱种子的萌发能力较苜蓿种子受到影响较小,各个阶段对环境水势的需求也相对较为宽松.因此,苜蓿苗期对干旱胁迫的忍耐能力不及高粱,且出苗过程中对环境水分条件的需求存在品种间差异.     

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.