多年生黑麦草的逆境生理研究进展

在研究和借鉴国内外最新研究成果的基础上,系统论述和报道了多年生黑麦草在水分、温度以及重金属等逆境胁迫下的生理变化,展望了多年生黑麦草在逆境生理方面的研究趋势,指出多年生黑麦草的抗性机理以及相关分子生物学方面的研究将成为未来研究的重点和热点,为今后深入开展多年生黑麦草的研究和利用提供科学依据。     

Development of a genomic microsatellite library in perennial ryegrass (Lolium perenne) and its use in trait mapping

Background and Aims: Perennial ryegrass (Lolium perenne) is one of the key forageand amenity grasses throughout the world. In the UK it accountsfor 70 % of all agricultural land use with an estimated farmgate value of £6 billion per annum. However, in termsof the genetic resources available, L. perenne has lagged behindother major crops in Poaceae. The aim of this project was thereforethe construction of a microsatellite-enriched genomic libraryfor L. perenne to increase the number of genetic markers availablefor both marker-assisted selection in breeding programmes andgene isolation. Methods: Primers for 229 non-redundant microsatellite markers were designedand used to screen two L. perenne genotypes, one amenity andone forage. Of the 229 microsatellites, 95 were found to showpolymorphism between amenity and forage genotypes. A selectionof microsatellite primers was selected from these 95 and usedto screen two mapping populations derived from intercrossingand backcrossing the two forage and amenity grass genotypes. Key Results and Conclusions: The utility of the resulting genetic maps for analysis of thegenetic control of target traits was demonstrated by the mappingof genes associated with heading date to linkage groups 4 and7.     

Effects of sheath tube length on leaf development in perennial ryegrass (Lolium perenne L.)

Position in and contribution of leaf laminae to the canopy of forage grasses are important both in determining herbage growth rates and intake rate by grazing animals. These canopy characteristics are controlled by the way dry matter is apportioned between sheath and lamina in growing leaves. The objective of this work was to determine how the development of individual leaves is affected by altering the effective length of the psuedostem tube, on the assumption that the light environment within the tube varied. The development of a leaf from initiation at the apex to maturity was followed by successive destructive dissections of tillers. Vertical incisions were made in the pseudostem of each tiller to three different depths. The three treatments imposed were — no incision (control), moderate and severe incision of the sheath length. Destructive harvests of tillers followed 3, 6, 12 and 24 days after imposition of treatments. Incision resulted in the length of the monitored leaf being reduced significantly at all harvests, and differentiation of the sheath beginning earlier. The length reduction reflected a reduction in both cell size and cell number and the effects were evident at the earliest harvest. The data support the theory that leaf size and timing of onset of sheath development are influenced by the environment of the developing leaf. The present results indicate that sheath tube length affects leaf development and suggests that the effects are substantially explained by a direct light effect on the location and depth of the elongation zone.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Kinetics of nitrate and ammonium absorption and accompanying H+ fluxes in roots of Lolium perenne L. and N2-fixing Trifolium repens L.

Kinetic parameters for NH₄⁺ and NO₃^? uptake were measured in intact roots of Lolium perenne and actively N₂-fixing Trifolium repens. Simultaneously, net H⁺ fluxes between the roots and the root medium were recorded, as were the net photosynthetic rate and transpiration of the leaves. A Michaelis–Menten-type high-affinity system operated in the concentration range up to about 500 mmol m^?3 NO₃^? or NH₄⁺. In L. perenne, the V_max of this system was 9–11 and 13–14 μmol g^?1 root FW h^?1 for NO₃^? and NH₄⁺, respectively. The corresponding values in T. repens were 5–7 and 2 μmol g^?1 root FW h^?1. The K_m for NH₄⁺ uptake was much lower in L. perenne than in T. repens (c. 40 compared with 170 mmol m^?3), while K_m values for NO₃^? absorption were roughly similar (around 130 mmol m^?3) in the two species. There were no indications of a significant efflux component in the net uptake of the two ions. The translocation rate to the shoots of nitrogen derived from absorbed NO₃^?-N was higher in T. repens than in L. perenne, while the opposite was the case for nitrogen absorbed as NH₄⁺. Trifolium repens had higher rates of transpiration and net photosynthesis than L. perenne. Measurements of net H⁺ fluxes between roots and nutrient solution showed that L. perenne absorbing NO₃^? had a net uptake of H⁺, while L. perenne with access to NH₄⁺ and T. repens, with access to NO₃^? or NH₄⁺, in all cases acidified the nutrient solution. Within the individual combinations of plant species and inorganic N form, the net H⁺ fluxes varied only a little with external N concentration and, hence, with the absorption rate of inorganic N. Based on assessment of the net H⁺ fluxes in T. repens, nitrogen absorption rate via N₂ fixation was similar to that of inorganic N and was not down-regulated by exposure to inorganic N for 2 h. It is concluded that L. perenne will have a competitive advantage over T. repens with respect to inorganic N acquisition.     

Nitrogen deficiency increases the residence time of respiratory carbon in the respiratory substrate supply system of perennial ryegrass

Plant respiration draws on substrate pools of different functional/biochemical identity. Little is known about the effect of nitrogen deficiency on those pools' sizes, half-lives and relative contribution to respiration, and consequently, of carbon residence time in respiratory metabolism. Here we studied how nitrogen fertilization affects the respiratory carbon supply system of shoots and roots of Lolium perenne , a perennial grass. Plants grown at two nitrogen supply levels in continuous light were labelled with ¹³CO₂/¹²CO₂ for intervals ranging from 1 h to 1 month. The rate and isotopic composition of shoot, root and plant respiration were measured, and the time-courses of tracer incorporation into respired CO₂ were analysed by compartmental modelling. Nitrogen deficiency reduced specific respiration rate by 30%, but increased the size of the respiratory supply system by 30%. In consequence, mean residence time of respiratory carbon increased with nitrogen deficiency (4.6 d at high nitrogen and 9.2 d at low nitrogen supply). To a large extent, this was due to a greater involvement of stores with a long half-life in respiratory carbon metabolism of nitrogen-deficient plants. At both nitrogen supply levels, stores supplying root respiration were primarily located in the shoot, probably in the form of fructans.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.     

Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering

Simulated swards of perennial ryegrass ( Lolium perenne ) growing in 1-m³ soil blocks in the glasshouse were either well watered or deprived of water for 57 d and then rewatered. The first aim was to measure effects of drought on sugar (water-soluble carbohydrate) composition of laminae and sheaths of mature laminae, and bases and laminae of young (growing) leaves. The second aim was to use pulse labelling with ¹⁴CO₂ to follow the partitioning of recently-fixed assimilates, and the assembly and consumption of reserve sugars (fructans). Over the last 7 d of drought growth almost stopped, old leaves died faster than they were replaced, and total sugar (which had doubled in concentration during drought) was rapidly consumed. Leaf laminae had lower content of total sugars and of large fructan (DP>5) than did growing bases and sheaths. Drought greatly reduced the rate at which sugar was exported from the laminae to the sheaths and growing leaf bases, and the rate at which it was converted to fructan. Nevertheless, fructan accumulated over the first 50 d of drought. Rewatering did not result in depolymerization and remobilization of sugars that had been formed during the last 7 d of drought, but stimulated their further assembly into high-DP fructans. Our hypothesis, that accumulation of neo-kestose (a DP-3 fructan) in droughted laminae was a symptom of sugar remobilization just before death, was disproved. It is concluded that sugar reserves contribute to drought resistance only under extreme conditions. The specific role of fructan in dry environments might be to improve regrowth when drought is relieved, rather than to enhance growth during drought.     

Disentangling CO2 fluxes: direct measurements of mesocosm‐scale natural abundance 13CO2/12CO2 gas exchange, 13C discrimination,and labelling of CO2 exchange flux components in controlled environments

A ¹³C/¹²C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO₂ exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO₂ with known δ¹³C (δ_CΟ2?2.6 or ?46.8‰). The system did not fractionate C isotopes and no extraneous CO₂ leaked into chambers. The on‐line ¹³C discrimination (Δ) of ryegrass stands in light was independent of δ_CΟ2 when δ_CΟ2 was constant. The δ of CO₂ exchanged by the stands in light (δ_Nd) and darkness (δ_Rn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand‐level. However, Δ decreased by ～ 10‰ when δ_CΟ2 was switched from ?46.8 to ?2.5‰, and increased by ～ 10‰ following a shift from ?2.6 to ?46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non‐photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO₂ indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ～ 60% of total respiration and having a mean residence time of 3.6 d.     

Morphological compatibility of white clover and perennial ryegrass cultivars grown under two nitrate levels in flowing solution culture

The effects of nitrate (NO3-) supply on shoot morphology, vertical distribution of shoot and root biomass and total nitrogen (N) acquisition by two perennial ryegrass (Lolium perenne L.) cultivars (AberElan and Preference) and two white clover (Trifolium repens L.) cultivars (Grasslands Huia and AberHerald) were studied in flowing nutrient culture. Cultivars were grown from seed as monocultures and the clovers inoculated with Rhizobium. The 6-week measurement period began on day 34 (grasses) and day 56 (clovers) when the NO3- supply was adjusted to either 2 mmol m-3 (low nitrogen, LN) or 50 mmol m-3 (high nitrogen, HN). These treatments were subsequently maintained automatically. Plants were harvested at intervals to measure their morphology and N content. Cultivars of both species differed significantly in several aspects of their response to NO3- supply. In the grasses, the LN treatment increased the root : shoot ratio of AberElan but did not affect the distribution of root length in the root profile. In contrast, this treatment changed the root distribution of Preference compared with HN, resulting in a larger proportion of root length being distributed further down the root profile. The morphology of white clover Grasslands Huia was for the most part unaffected by the level of NO3- supply. In contrast, AberHerald exhibited different growth strategies, with LN plants increasing their stolon weight per unit length at the expense of leaf production, leaf area and stolon length, whereas HN plants showed reduced stolon thickness, greater leaf area production and stolon length per plant. Cultivars with different morphological/physiological strategies in response to NO3- supply may be of value in the construction of 'compatible mixtures' aimed at reducing oscillations in sward clover content by extending the range of conditions that allow balanced coexistence of species to occur.     

The effect of handling on the yield of two populations of Lolium perenne selected for differences in mature leaf respiration rate

Two populations of Lolium perenne L. S23 (perennial ryegrass), selected for differences in mature leaf dark respiration, were used in a non-destructive indexing system for individual plants, to determine growth parameters. Population GL66, selected for high respiratory rates and low yield, responded strongly to the indexing treatment, when grown at low plant density. Dry weights of all plant parts decreased strongly, as did dry matter percentages of the leaf blades. At high density this population demonstrated the same trend, but additionally allocation to the shoot increased. In contrast, GL72, selected for low respiratory rates and a high yield, responded only at a high plant density. It is argued that there might be a relation between the dissimilar response of the two populations to mechanical influences and the presence of the genotypes of the low-yielding population in the parent variety. The results also emphasize that non-destructive growth analyses can only be used when their effects on the plants are known.     

缺磷对黑麦草-内生真菌共生体生长和根中酚含量的影响

培养液中缺磷或内生真菌感染对黑麦草地上部生长的影响不显著,但二者的交互作用却有着显著影响。随着磷浓度的下降,感染内生真菌的黑麦草显示出较强的生长优势,其根长和根中酚类物质的含量显著增加。     

Effect of phosphate rock,coal combustion by-product,lime, and cellulose on ryegrass in an acidic soil

Remediation of soil acidity is crucial for increasing crop production and improving environmental quality of acid infertile soils. Soil incubation and greenhouse pot experiments were carried out to examine the interactions between phosphate rock (PR), coal combustion by-product (BP), dolomitic lime (L), and cellulose (C) in an acidic soil and their effects on ryegrass (Lolium perenne L. cv Linn) growth. BP and PR application increased plant P content and dry matter yield (DMY) of shoots and roots by improving soil Ca availability and reducing Al toxicity. Application of BP at low rates (5 to 10 g BP kg^-1) with PR appeared to decrease both plant P content and DMY compared to PR application alone. The reduced DMY is due to an increased Al concentration in soil solution as a result of displacement of sorbed Al by Ca of BP. Increases in DMY were obtained by addition of lime along with PR and BP at low rates or by increasing BP application rates above 15 g kg^-1. This improved plant response was likely related to alleviation of Al toxicity by CaCO₃ contained in the BP. In addition to raising the pH to an acceptable level for plant growth, the dolomitic lime supplied needed Mg for plants, thereby maintaining a good balance between available Ca and Mg for plants in the BP- and PR-amended soils. The addition of cellulose to the BP- and PR-amended soils reduced water-soluble Al and increased DMY. Plant growth increased PR dissolution by 2.4 to 243% in a soil with low available P. Use of BP at moderate rates with PR and dolomitic lime appears to be the best combination in increasing crop yields on infertile acidic soils.     

Effect of leaf age and nitrogen fertilisation on sporulation of crown rust (Puccinia coronata var. lolii) on perennial ryegrass (Lolium perenne L.)

Individual leaves of perennial ryegrass cv. Aberystwyth S23 of two leaf ages and at two levels of nitrogen fertilisation were point inoculated with Puccinia coronata f.sp. coronata in a growth chamber. In general, there was no significant difference in the lifespan of inoculated versus control leaves. However, the higher rate of nitrogen extended leaf lifespan more markedly in rusted than in control leaves. Uredospore production varied according to leaf age: colonies on juvenile leaves produced three times as many spores as those on mature leaves.     

Peptide conjugates of benzene and toluene metabolites in English ryegrass

Biotransformation of [1-6-¹⁴C]benzene and [1-¹⁴C]toluene in English ryegrass (Lolium perenne L.) seedlings was investigated. Vapors of these compounds were absorbed by the leaves of this plant. Benzene and toluene were oxidized, forming phenol and benzoic acid, respectively. A portion of phenol and benzoic acid was bound by low-molecular-weight peptides forming conjugates. A qualitative amino acid composition of the peptides involved in the conjugation was determined. After removing plants from the atmosphere containing [1-6-¹⁴C]benzene and [1-¹⁴C]toluene, the radioactivity of the conjugates gradually decreased. This process was accompanied by the evolution of ¹⁴CO₂, indicating the breakdown of these conjugates. Radioactive compounds thus formed were oxidized, yielding carbon dioxide. A portion of phenol and benzoic acid, along with peptide conjugation, was subjected to further oxidative transformations up to disruption of the aromatic ring. By this pathway, nonvolatile carboxylic acids, such as muconic, fumaric, succinic, malic, malonic, glycolic, and glyoxylic, were formed. Using electron microscopy, a damaging effect of benzene on the cell ultrastructure of English ryegrass leaves was shown, and this toxic effect depended on the benzene concentration.     

Genotyping by RAD sequencing enables mapping of fatty acid composition traits in perennial ryegrass (Lolium perenne (L.))

Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially α‐linolenic acid, an omega‐3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high‐sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction‐associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n‐6) on LGs 2 and 5; and α‐linolenic acids (C18:3n‐3) on LG 1 were identified. Two candidate genes (a lipase and a beta‐ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n‐6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.