Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.)

Background  

Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Optimization of isolation and storage of sperm cells from pollen of perennial ryegrass (Lolium perenne L.)

Summary A procedure for isolating sperm cells of perennial ryegrass (Lolium perenne L.) was developed. The sperm cells were released from the pollen grains by osmotic shock, with the right combination of pH and osmolality being important for optimal release. Various combinations of vitamins E, C and fetal calf serum were tested with the aim of improving yield and long-term viability, and their possible mode of action as important components for improvement of these two parameters is discussed. Under optimized conditions, a yield of 12% was established, and the storage time after which 50% of the sperm cells were still viable was improved to 60 h. Cytological observations demonstrated that sperm cells of perennial ryegrass are true protoplasts, which may allow future fusion experiments to be carried out.     

Effects of the fungal endophyte, Neotyphodium lolii, on net photosynthesis and growth rates of perennial ryegrass (Lolium perenne) are independent of In Planta endophyte concentration

• Background and Aims Neotyphodium lolii is a fungal endophyteof perennial ryegrass (Lolium perenne), improving grass fitnessthrough production of bioactive alkaloids. Neotyphodium speciescan also affect growth and physiology of their host grasses(family Poaceae, sub-family Pooideae), but little is known aboutthe mechanisms. This study examined the effect of N. lolii onnet photosynthesis (P_n) and growth rates in ryegrass genotypesdiffering in endophyte concentration in all leaf tissues. • Methods Plants from two ryegrass genotypes, Nui D andNui UIV, infected with N. lolii (E+) differing approx. 2-foldin endophyte concentration or uninfected clones thereof (E–)were grown in a controlled environment. For each genotype xendophyte treatment, plant growth rates were assessed as tilleringand leaf extension rates, and the light response of P_n, darkrespiration and transpiration measured in leaves of young (30–45d old) and old (>90 d old) plants with a single-chamber openinfrared gas-exchange system. • Key Results Neotyphodium lolii affected CO₂-limited ratesof P_n, which were approx. 17 % lower in E+ than E– plants(P < 0·05) in the young plants. Apparent photon yieldand dark respiration were unaffected by the endophyte (P >0·05). Neotyphodium lolii also decreased transpiration(P < 0·05), but only in complete darkness. There wereno endophyte effects on P_n in the old plants (P > 0·05).E+ plants grew faster immediately after replanting (P < 0·05),but had approx. 10 % lower growth rates during mid-log growth(P < 0·05) than E– plants, but there was noeffect on final plant biomass (P > 0·05). The endophyteeffects on P_n and growth tended to be more pronounced in NuiUIV, despite having a lower endophyte concentration than NuiD. • Conclusions Neotyphodium lolii affects CO₂ fixation,but not light interception and photochemistry of P_n. The impactof N. lolii on plant growth and photosynthesis is independentof endophyte concentration in the plant, suggesting that theendophyte mycelium is not simply an energy drain to the plant.However, the endophyte effects on P_n and plant growth are stronglydependent on the plant growth phase.     

模拟喀斯特不同土壤生境下黑麦草对水分胁迫的生长和光合生理响应

以多年生草本黑麦草为对象,模拟喀斯特生境土壤特征设置浅而宽(Shallow and wide, SW:30×30×5 cm³)和深而窄(Deep and narrow, DN:10×10×45 cm³)两种土壤容器,以正常降水量为对照供水(W100%),设减水50%(W50%)和减水70%(W30%)共3种水分处理进行盆栽实验,探究了两种不同土壤生境中土壤水分变化对黑麦草生长及光合生理的影响,以进一步理解喀斯特地区植物的适应对策。结果显示:(1)SW生境对水分变化敏感,随供水减少土壤含水量显著下降。轻度减水下植物叶面积增大,光合速率提高,地上部分生长得到促进,但水分严重减少对其生长和光合生理有抑制作用,但地上质量分数和水分利用效率却显著升高;(2)DN生境保水能力较好,随供水减少土壤水分含量下降较为平缓。叶片相对含水量、气孔导度和比叶面积在各水分处理之间差异不显著,但严重减水条件下总生物量、地上质量分数和水分利用效率均有回升。研究表明:浅而宽生境中植物倾向于通过提高地上部分的生长,保持较高的光合速率,并向地上部分分配较多生物量来应对水分胁迫;...     

多年生黑麦草对干旱胁迫下喀斯特异质生境的生长响应策略

在喀斯特地区异质性生境中,土壤水分含量、植物生存空间和养分状况等的综合作用共同影响植物的生理功能和生长发育。以多年生黑麦草(Lolium perenne L.)为研究材料,通过模拟同质(S-S:浅土-浅土)和异质(S-D:浅土-深土)两种小生境,探究3种施水量(对照:W₀、中度干旱:W₁、重度干旱:W₂)条件下植物对异质生境的适应,主要解决以下问题:异质生境下植物是否会主动寻求高水分、厚土壤、或者更宽阔的生长空间来提高其对异质性生境的适应?降雨量的改变是否会导致植物的适应策略发生改变?主要结果如下:(1)只有在重度干旱(W₂)条件下,异质性生境中黑麦草的株高、分蘖数以及地上、地下生物量和地上、根质量分数显著高于同质生境,而在对照(W₀)和中度干旱(W₁)条件下两种生境均无明显差异。(2)在对照降水(W₀)条件下,异质生境中黑麦草浅土区根系生物量、根组织密度较深土区少,比根长、比根面积较深土区大;随着施水量的减少,浅土区根系生物量占整个...     

芘对黑麦草根系几种低分子量有机分泌物的影响

植物根系释放分泌物与有机污染物的植物修复机制密切相关,研究具有有机污染物修复潜力植物在污染胁迫条件下的根系分泌物特征有助于揭示其修复机制.以多环芳烃修复研究中常用的黑麦草为材料(Lolium perenneL.)为材料,在营养液栽培方式下研究了在芘胁迫处理下,黑麦草根系几种低分子量有机物分泌特征.结果表明,黑麦草对芘具有较强的耐受能力,芘胁迫处理下,生物量无显著变化.黑麦草根系分泌的低分子量有机酸主要为草酸.3、6 mg/L和9 mg/L芘胁迫处理下,低分子量有机酸的组成无明显变化,但含量随芘胁迫处理浓度上升而显著增加(P＜0.05);总糖分泌量随着芘胁迫处理浓度升高而呈现先略微上升后下降的趋势,相对高峰值出现在芘胁迫处理浓度3 mg/L,但差异不显著;氨基酸分泌总量随着芘胁迫处理浓度的增加而显著增多,芘胁迫浓度在3、6 mg/L和9 mg/L时,根系氨基酸的分泌总量分别是空白的1.37、2.02倍和2.65倍,但根系分泌的氨基酸组成无明显变化,19种常见氨基酸分泌的数量变化情况却不相同,分泌量总体均随着芘胁迫处理浓度的提高而增加,其中苏氨酸、丝氨酸、脯氨酸、甘氨酸、丙氨酸、亮氨酸、组氨酸和鸟氨酸的分泌量显著增多,差异显著(P＜0.05).     

刈割后黑麦草生理保护作用对其补偿性生长的影响

通过对黑麦草(Lolium perenne L)在轻度、中度、重度、全割刈割处理6 d和12 d后,残留叶片和叶片再生部分生长速率,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活力,丙二醛、可溶性糖、脯氨酸含量的分析以揭示在刈割胁迫后叶片抗氧护酶活力和渗透调节物含量与其补偿性生长的关系,以及牧草耐刈性的生理调控机理。结果表明,轻度和中度及全割后叶片生长速率均高于对照,重度刈割低于对照。全割后叶片补偿性生长最明显、轻度和中度次之,重度刈割无补偿性生长。对照黑麦草叶片各部位抗氧化酶和渗透调节物含量不同,叶片顶部MDA含量较高,伴随着较高的SOD、CAT活力和较高的脯氨酸含量;叶片基部MDA含量最低,SOD、CAT活力及脯氨酸含量也较低。与对照相比,不同强度刈割6 d黑麦草再生叶和叶片平均MDA含量、SOD和CAT活力、可溶性糖和脯氨酸含量均较低。而不同强度刈割12 d,黑麦草再生叶和叶片平均MDA含量仍较低,但SOD和CAT活力增高,脯氨酸含量增加,POD活力和可溶性糖含量低于对照。这表明刈割在减少了叶面积,降低光合能力的同时,刈割伤害胁迫启动了牧草补偿性生长使残留叶片快速生长,而且残留叶片面积与其补偿生长速率成正相关。另外,虽然不同强度刈割下叶片补偿性生长速率不同,但不同强度刈割(12 d)均激活残留叶片抗氧化保护酶系统和促进脯氨酸积累。在补偿生长过程中,CAT和SOD能及时清除残留叶片中积累的氧自由基,维持较低的膜脂过氧化和细胞膜完整性,积累的脯氨酸能维护细胞水分平衡。因此,抗氧化酶(SOD和CAT)和渗透调节物(脯氨酸)在黑麦草刈割后受伤部位快速自愈及残留叶片快速补偿生长中起重要生理保护作用。     

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP^® (AFLP^® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.     

Changes in water status and osmolyte contents in leaves and roots of olive plants (Olea europaea L.) subjected to water deficit

Two-year-old olive trees (Olea europaea L., cv. Coratina) were subjected to a 15-day period of water deficit, followed by 12 days of rewatering. Water deficit caused decreases in predawn leaf water potential (Ψ_w), relative water content and osmotic potential at full turgor (Ψ _π100) of leaves and roots, which were normally restored upon the subsequent rewatering. Extracts of leaves and roots of well-watered olive plants revealed that the most predominant sugars are mannitol and glucose, which account for more than 80% of non-structural carbohydrates and polyols. A marked increase in mannitol content occurred in tissues of water-stressed plants. During water deficit, the levels of glucose, sucrose and stachyose decreased in thin roots (with a diameter <1 mm), whereas medium roots (diameter of 1–5 mm) exhibited no differences. Inorganic cations largely contribute to Ψ _π100 and remained stable during the period of water deficit, except for the level of Ca²⁺, which increased of 25% in water-stressed plants. The amount of malate increased in both leaves and roots during the dry period, whereas citrate and oxalate decreased. Thin roots seem to be more sensitive to water deficit and its consequent effects, while medium roots present more reactivity and a higher osmotic adjustment. The results support the hypothesis that the observed decreases in Ψ_w and active osmotic adjustment in leaves and roots of water-stressed olive plants may be physiological responses to tolerate water deficit.     

干旱胁迫下内生真菌感染对黑麦草叶内几种同工酶的影响

以内生真菌感染(endophyte-infected,EI)与不感染(endophyte-free,EF)的黑麦草(Lolium perenne L．)种子建立实验种群,分别对其施加长时间不同强度的干旱胁迫,通过比较黑麦草体内过氧化物酶(POD)、超氧化物歧化酶(SOD)、多酚氧化酶(PPO)活性及其同工酶谱的变化以探讨保护酶系统在内生真菌——植物共生体的抗旱性方面所作的贡献。研究结果表明,水分胁迫和内生真菌对黑麦草3种酶的影响不仅表现在总量上而且表现在同工酶的酶谱及各区带的酶活力上。就总酶活力而言,EI和EF植株中POD、SOD和PPO的活性均随着干旱胁迫强度的增加而增加,进一步将EI和EF植株的酶活力进行比较,发现与EF植株相比,EI植株中POD和PPO的活性相对较低,而SOD的活性相对较高。从同工酶的谱带数量和强弱来看,POD同工酶各区带活力均随干旱胁迫强度的增加而增加,EI植株叶片增加的幅度高于EF叶片,而且EI叶片在重度胁迫下出现了1条新带SOD同工酶各区带活力在EI叶片中有随干旱胁迫增加而增加的趋势,而在EF叶片中有些区带酶活力增强,有些区带酶活力减弱,且EI叶片在中度胁迫下出现了1条新带;PPO同工酶随干旱胁迫的增强,EI和EF叶片均表现为有些区带酶活力增强,有些区带酶活力减弱。总之,内生真菌的感染虽然没有显著提高宿主植物黑麦草POD、SOD和PPO的活性,但使宿主黑麦草对干旱胁迫的反应更为迅速,其中既包括POD、SOD等酶活力的迅速升高,也包括新酶带的产生。     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Physiological responses of the legume model Medicago truncatula cv. Jemalong to water deficit

In Medicago truncatula Gaertn. cv. Jemalong plants some mechanisms involved in drought resistance were analysed in response to a progressive water deficit imposed by suppression of soil irrigation. Withholding water supply until the soil had reached one-half of its maximum water content had no significant effect on leaf RWC, gas exchanges or chlorophyll fluorescence parameters. Under severe drought conditions, the plants resistance to water shortage involved mainly drought avoidance mechanisms through a decrease in stomatal conductance. The consequent decrease in the internal CO₂ concentration (C_i) should have limited the net CO₂ fixation (A). Since A decreased slightly more than C_i under severe water deficit, non-stomatal limitations of photosynthesis may have also occurred. Analysis of A/C_i curves showed reduced carboxylation efficiency due to limitations in RuBP regeneration and Rubisco activity, confirming the presence of non-stomatal limitations of photosynthesis. Drought tolerance mechanisms involving osmotic adjustment and an increase in cell membrane integrity were also present. Altogether, these mechanisms allowed M. truncatula cv. Jemalong plants to still maintain a quite elevated level of net CO₂ fixation rate under severe water deficit conditions. These results may contribute to identify useful physiological traits for breeding programs concerning drought adaptation in legumes.