Heat stress screening of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) seedlings for acquired thermotolerance

Heat can be one of the major abiotic stresses that adversely affect crop production worldwide at different stages of development. As field screening for heat tolerance can be inconsistent and seasonally-limited, it is important to develop a reliable protocol under controlled conditions that allows simultaneous screening of multiple genotypes. The objective of this research was to develop a straightforward laboratory protocol using acquired thermotolerance (ATT) in peanut seedlings as a measure of one mechanism of heat stress tolerance. Sixteen genotypes, including selected accessions of the US peanut minicore collection along with standard checks, were evaluated for acquired themotolerance in two independent experiments. A change in the temperature sensitivity of chlorophyll accumulation was used as an indicator of acquired thermotolerance. Pre-incubation at 38°C for 4 h before the 30-min 50°C challenge triggered the acquired thermotolerance system of the leaf disks, resulting in chlorophyll accumulation upon exposure to light. There was considerable variation among genotypes for ATT in both experiments. Genotypic ranking for mean ATT values were highly correlated (0.949) in both experiments. The effect of seed weight on ATT was not significant. This method is relatively simple and inexpensive and can be used to screen a large number of genotypes.     

Chlorophyll fluorescence emission as a reporter on cold tolerance in Arabidopsis thaliana accessions

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around −15°C. This, together with the method''s easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.Key words: chlorophyll fluorescence, cold acclimation, electrolyte leakage, high-throughput screening, natural accessions     

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.     

Heat susceptibility of grain filling in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) linked with rapid chlorophyll loss during a 3-day heat treatment

Brief heat events (1–3 days, >30 °C) commonly reduce wheat (Triticum aestivum L.) grain size and consequently yield. To identify mechanisms of tolerance to such short heat events, 36 wheat genotypes were treated under day/night temperatures of 37 °C/27 °C for 3-days in a growth chamber, at 10 days after anthesis, and a range of developmental, chlorophyll and yield-related traits monitored. The degree of flag leaf chlorophyll loss during the treatment was the variable that showed the highest correlation to grain weight loss (r = 0.63; p < 0.001), identifying chlorophyll stability during this brief period as a potential determinant or indicator of grain weight stability under heat. Variables summarizing the combined during- and post-heat chlorophyll losses showed similar or lower correlations with heat tolerance of grain filling, despite the fact that genotypes varied in their ability to resume normal chlorophyll loss rates after the heat treatment. Additionally, heat tolerance of grain size showed no correlation with grain filling duration or traits relating to utilization of stem carbon reserves under heat stress. Measurement of chlorophyll loss over a forecasted heat wave was thereby identified as a potential basis for developing tools to help breeders select heat tolerant genotypes.     

Physiological and morphological characteristics of chickpea accessions under low temperature stress

Economically important crop chickpea (Cicer arietinum L.) is sensitive to chilling stress, and breeding for chilling tolerance is the economic option even in countries with a high risk for drought and heat stresses. In this study, we have analyzed chilling-induced responses of ten chickpea accessions under field and growth-chamber conditions in order to screen, using phenotypic and physiological methods, for chilling tolerance. The field data analysis revealed that there were significant differences between accessions in their cold tolerance. The percent survival and cold-tolerance scores were the most important indices describing genotype tolerance to low temperature under field conditions; they can be used to assess chickpea cold tolerance. During environmentally controlled testing, the effects of low temperature regimes (−10°C for 15 and 30 min) were studied and cold tolerance was measured by electrolyte leakage from damaged leaves. The analysis of field data and cold treatments showed that two accessions, Sel 95Th1716 and Sel 96Th11439, grouped in one cluster, are good cold-tolerant genotypes (showing low scores for cold tolerance and electrolyte leakage). In comparison with ILC 8262, released as cold-tolerant accession, these genotypes showed more tolerance. Flip 00-6C, ILC 533, and Jam were less tolerant to cold stress. Thus, we have shown that as well as field studies, short-term cold treatment and electrolyte leakage assay can be used to evaluate low temperature tolerance of chickpea profitably in a short time.     

Apparent absence of viruses in most symptomless field-grown sweet potato in Uganda

Heat tolerance of groundnut (Arachis hypogaea L.) genotypes was evaluated by solute leakage and chlorophyll fluorescence techniques in heat-hardened and non-hardened plants. To determine the appropriate hardening treatment, 1-month-old plants of two groundnut genotypes, ICGV 86707 and Chico were conditioned at five combinations of hardening (37°C) and non-hardening (30°C) air temperatures over a 5-day period. Heat injury, was assessed through measurements of electrolyte leakage after stressing leaf discs to 55°C for 15 min. The relative injury was significantly influenced by the conditioning temperatures and by the temperature during 24 h prior to measurement if those involved non-hardening conditions. Relative injury and chlorophyll fluorescence were measured after stressing leaves of six genotypes at a range of temperatures between 49°C and 55°C. Significant genotype × hardening treatment interactions were observed in relative injury and chlorophyll fluorescence. Chico was susceptible to heat stress, the relative injury test identified ICGV 86707 as tolerant, and the chlorophyll fluorescence test identified ICGV 86707 as tolerant under hardened conditions and ICGV 87358 as tolerant when non-hardened. When expressed as percentage of control values, the relative injury and chlorophyll fluorescence measurements over the 49–53°C stress temperature range were strongly correlated. Chlorophyll concentrations were increased by hardening in all genotypes except Chico. In Chico, chl_b concentration was decreased and the chl_a/b ratio increased by hardening, and chlorophyll concentrations were correlated with chlorophyll fluorescence parameters. Chlorophyll concentration may therefore provide an alternative means of screening for heat tolerance.     

NaCl induced morpho-biochemical and anatomical changes in mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.)

Mulberry is an economically important tree, used for feeding the silkworm Bombyx mori L. Effect of different levels of NaCl on growth and development of mulberry has been studied using five mulberry genotypes selected on the basis of their performance under in vitro salinity. The study while endorsing the efficacy of in vitro screening of axillary buds of mulberry for salt tolerance, showed genotypic variability in its response to salinity. Salinity reduced growth and development of all genotypes. However, the putative tolerant genotypes showed better performance than the putative susceptible genotypes. Under low salinity (<0.5% NaCl) salt tolerant genotypes showed an increase in chlorophyll and protein concentrations, while in susceptible genotypes both were reduced by 3–58% at 0.5% NaCl and 50–64% at 1.00% NaCl. Leaf thickness increased by 16% at 1.00% NaCl in C776 and reduced by 1.0% in Mandalaya. The increase in chlorophyll concentration and leaf thickness under high salinity can be considered as preliminary selection parameters for salt tolerance in mulberry. The study confirmed the efficacy of in vitro method for screening of large number of genotypes for salt tolerance in mulberry.     

棉花抗枯、黄萎病品种苗期耐低钾种质筛选研究

本研究采用蛭石栽培和营养液浇灌的方法,确立棉花品种苗期耐低钾筛选指标,利用这些指标,评价我国88个抗枯、黄萎病棉花品种资源苗期耐低钾能力,并筛选钾素利用率高的优异种质。结果表明,株高、地上部干物重、地下部干物重、总干物重、钾利用率、叶绿素含量在不同钾浓度处理和不同品种之间均存在显著差异,可以作为棉花苗期耐低钾能力的评价指标,而叶面积在不同品种和不同钾浓度处理之间不存在差异。利用筛选出的指标对88个品种进行耐低钾评价,结果供试品种主要被分为耐低钾基因型、耐低钾中间类型和非耐低钾基因型3类,分别包括21个、58个和6个品种。     

棉花抗枯、黄萎病品种苗期耐低钾种质筛选研究

本研究采用蛭石栽培和营养液浇灌的方法,确立棉花品种苗期耐低钾筛选指标,利用这些指标,评价我国88个抗枯、黄萎病棉花品种资源苗期耐低钾能力,并筛选钾素利用率高的优异种质。结果表明,株高、地上部干物重、地下部干物重、总干物重、钾利用率、叶绿素含量在不同钾浓度处理和不同品种之间均存在显著差异,可以作为棉花苗期耐低钾能力的评价指标,而叶面积在不同品种和不同钾浓度处理之间不存在差异。利用筛选出的指标对88个品种进行耐低钾评价,结果供试品种主要被分为耐低钾基因型、耐低钾中间类型和非耐低钾基因型三类,分别包括21个、58个和6个品种。     

Water-Deficit Tolerance and Field Performance of Transgenic Alfalfa Overexpressing Superoxide Dismutase

Transgenic alfalfa (Medicago sativa) expressing Mn-superoxide dismutase cDNA tended to have reduced injury from water-deficit stress as determined by chlorophyll fluorescence, electrolyte leakage, and regrowth from crowns. A 3-year field trial indicated that yield and survival of transgenic plants were significantly improved, supporting the hypothesis that tolerance of oxidative stress is important in adaptation to field environments.     

Isolation of Arabidopsis mutants lacking components of acquired thermotolerance

Acquired thermotolerance is a complex physiological phenomenon that enables plants to survive normally lethal temperatures. This study characterizes the temperature sensitivity of Arabidopsis using a chlorophyll accumulation bioassay, describes a procedure for selection of acquired thermotolerance mutants, and provides the physiological characterization of one mutant (AtTS02) isolated by this procedure. Exposure of etiolated Arabidopsis seedlings to 48 degrees C or 50 degrees C for 30 min blocks subsequent chlorophyll accumulation and is eventually lethal. Arabidopsis seedlings can be protected against the effects of a 50 degrees C, 30-min challenge by a 4-h pre-incubation at 38 degrees C. By the use of the milder challenge, 44 degrees C for 30 min, and protective pretreatment, mutants lacking components of the acquired thermotolerance system were isolated. Putative mutants isolated by this procedure exhibited chlorophyll accumulation levels (our measure of acquired thermotolerance) ranging from 10% to 98% of control seedling levels following pre-incubation at 38 degrees C and challenge at 50 degrees C. The induction temperatures for maximum acquired thermotolerance prior to a high temperature challenge were the same in AtTS02 and RLD seedlings, although the absolute level of chlorophyll accumulation was reduced in the mutant. Genetic analysis showed that the loss of acquired thermotolerance in AtTS02 was a recessive trait. The pattern of proteins synthesized at 25 degrees C and 38 degrees C in the RLD and AtTS02 revealed the reduction in the level of a 27-kD heat shock protein in AtTS02. Genetic analysis showed that the reduction of this protein level was correlated with the acquired thermotolerance phenotype.     

Overexpression of ethylene response factor <Emphasis Type="Italic">TERF2</Emphasis> confers cold tolerance in rice seedlings

Rice (Oryza sativa L.) is a warm-season plant exposed to various stresses. Low temperature is an important factor limiting extension of rice cultivation areas and productivity. Previously, we have demonstrated that tomato ERF protein TERF2 enhances freezing tolerance of transgenic tobacco and tomato plants. Herein, we report that overexpression of TERF2 enhances transgenic rice tolerance to cold without affecting growth or agronomic traits. Physiological assays revealed that TERF2 could not only increase accumulation of osmotic substances and chlorophyll, but also reduce reactive oxygen species (ROS) and malondialdehyde (MDA) content and decrease electrolyte leakage in rice under cold stress. Further analysis of gene expression showed that TERF2 could activate expression of cold-related genes, including OsMyb, OsICE1, OsCDPK7, OsSODB, OsFer1, OsTrx23, and OsLti6, in transgenic rice plants under natural condition or cold stress. Thus, our findings demonstrated that TERF2 modulated expression of stress-related genes and a series of physiological adjustments under cold stress, indicating that TERF2 might have important regulatory roles in response to abiotic stress in rice and possess potential utility in improving crop cold tolerance.     

Chlorophyll Fluorescence as a Possible Tool for Salinity Tolerance Screening in Barley (Hordeum vulgare L.)

The application of chlorophyll fluorescence measurements to screening barley (Hordeum vulgare L.) genotypes for salinity tolerance has been investigated. Excised barley leaves were cut under water and incubated with the cut end immersed in water or in a 100-mM NaCl solution, either in the dark or in high light. Changes in rapid fluorescence kinetics occurred in excised barley leaves exposed to the saline solution only when the incubation was carried out in the presence of high light. Fluorescence changes consisted of decreases in the variable to maximum fluorescence ratio and in increases in the relative proportion of variable fluorescence leading to point I in the Kautsky fluorescence induction curve. These relative increases in fluorescence at point I appeared to arise from a delayed plastoquinone reoxidation in the dark, since they disappeared after short, far-red illumination, which is known to excite photosystem I preferentially. We show that a significant correlation existed between some fluorescence parameters, measured after a combined salt and high-light treatment, and other independent measurements of salinity tolerance. These results suggest that chlorophyll fluorescence, and especially the relative fluorescence at point I in the Kautsky fluorescence induction curve, could be used for the screening of barley genotypes for salinity tolerance.     

A comparison of hydroponic and soil-based screening methods to identify salt tolerance in the field in barley

Success in breeding crops for yield and other quantitative traits depends on the use of methods to evaluate genotypes accurately under field conditions. Although many screening criteria have been suggested to distinguish between genotypes for their salt tolerance under controlled environmental conditions, there is a need to test these criteria in the field. In this study, the salt tolerance, ion concentrations, and accumulation of compatible solutes of genotypes of barley with a range of putative salt tolerance were investigated using three growing conditions (hydroponics, soil in pots, and natural saline field). Initially, 60 genotypes of barley were screened for their salt tolerance and uptake of Na(+), Cl(-), and K(+) at 150 mM NaCl and, based on this, a subset of 15 genotypes was selected for testing in pots and in the field. Expression of salt tolerance in saline solution culture was not a reliable indicator of the differences in salt tolerance between barley plants that were evident in saline soil-based comparisons. Significant correlations were observed in the rankings of genotypes on the basis of their grain yield production at a moderately saline field site and their relative shoot growth in pots at EC(e) 7.2 [Spearman's rank correlation (rs)=0.79] and EC(e) 15.3 (rs=0.82) and the crucial parameter of leaf Na(+) (rs=0.72) and Cl(-) (rs=0.82) concentrations at EC(e) 7.2 dS m(-1). This work has established screening procedures that correlated well with grain yield at sites with moderate levels of soil salinity. This study also showed that both salt exclusion and osmotic tolerance are involved in salt tolerance and that the relative importance of these traits may differ with the severity of the salt stress. In soil, ion exclusion tended to be more important at low to moderate levels of stress but osmotic stress became more important at higher stress levels. Salt exclusion coupled with a synthesis of organic solutes were shown to be important components of salt tolerance in the tolerant genotypes and further field tests of these plants under stress conditions will help to verify their potential utility in crop-improvement programmes.     

An improvement of the diffusion model for assessment of drought stress in plant tissues

The article discusses an improvement of a previously developed method for assessment of ion leakage from plant tissues as a gauge of membrane and cell wall performance under stressful environment. It employs conductometric measurements of the ion efflux from leaves and their quantitative interpretation by a theoretical model based on the laws of diffusion. Experimental data are readily fit with the model and results are in accordance with relative water content of dehydrated barley (Hordeum vulgare) seedlings of two distinct cultivars. Some new parameters obtained from fitting are proposed as reliable indicators of the leaf status. They appear to be helpful in further distinguishing the behavior of two separate cellular structures with respect to their electrolyte permeability. It is concluded that the established method based on the kinetics of ion leakage is adequate for evaluation of contrasting genotypes under normal and stress conditions. Furthermore, it could be used as a simple and powerful tool for routine analysis and screening for drought tolerance in crops.     

Characteristics of chlorophyll fluorescence and antioxidative system in super-hybrid rice and its parental cultivars under chilling stress

Characteristics of chlorophyll fluorescence and antioxidative system were investigated in rice (Oryza sativa L.) super-hybrid Liangyoupeijiu (LYPJ), maternal cultivar Peiai64s, and paternal cultivar indica rice 9311 under chilling stress. During 6-d chilling treatment, chlorophyll content of all three genotypes was gradually declined. However, the decrease in photosystem 2 (PS 2) maximum photochemical efficiency (F_v/F_m) and quantum yield of PS 2 (Φ_PS2) was less expressive in LYPJ than in parental cultivars The electrolyte leakage and malondialdehyde content in all cultivars increased after chilling treatment, but LYPJ exhibited the least increasing tendency. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) were higher in LYPJ than in parental cultivars. The results demonstrated that tolerance to chilling stress in LYPJ might be adopted mostly from its maternal cultivar.