Detection of Quantitative Trait Loci for Yield and Drought Tolerance Traits in Soybean Using a Recombinant Inbred Line Population

To investigate the genetic basis of drought tolerance in soybean ( Glycine max L. Merr.) a recombinant inbred population with 184 F_2:7:11 lines developed from a cross between Kefeng1 (drought tolerant) and Nannong1138-2 (drought sensitive) were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci (QTLs) were identified: 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and D1b, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.     

Changes in chloroplast ultrastructure in leaves of drought-stressed maize inbred lines

Chloroplasts are commonly the site of the earliest abiotic injury visible in plant ultrastructure. In this study, six inbred lines of maize (Zea mays L.) were used to analyze changes in the ultrastructure of chloroplasts and related physiological parameters under conditions of drought stress simulated by 20% polyethylene glycol 6000 (?0.6 MPa) for two days. Chloroplasts of three maize lines proved to be more sensitive. They showed changes in the ultrastructure in response to drought, including damage of thylakoid membranes, an increase in the number and size of plastoglobuli, swelling of thylakoid membranes both stromal and granal, disorganization of the thylakoid membrane system, an obvious increase in the intrathylakoid space, and a decrease in the length-to-width ratio and area of chloroplasts. In addition, the contents of malondialdehyde increased markedly in the sensitive lines. Contrary to the sensitive lines, stable structures and shapes of chloroplasts were observed in the drought-resistant lines; it could be considered as an advantage contributing to drought tolerance in the plants. In addition, the drought index of leaf fresh mass (LMDI) in the drought-sensitive lines was ≤ 0.5, which was also associated with a lower content of leaf chlorophyll. In contrast, drought tolerance coincided with lesser growth reduction, and higher LMDI and leaf chlorophyll content.     

Hydraulic regulation strategies for whole-plant water balance of two maize inbred lines differing in drought resistance under short-term osmotic stress

The conservation of water in agriculture requires an understanding of the mechanisms of plant–water relations. This study aimed to reveal hydraulic regulation strategies of maize (Zea mays L.) for maintaining the plant water balance during drought. The water relations of two maize inbred lines (Tian4 and 478) that differ in their resistance to drought in the field were investigated under well-watered conditions and osmotic stress induced with 10 % PEG 6000. The leaf transpiration rate and leaf water potential of 478 varied diurnally, but remained constant in Tian4, which is more drought resistant. Tian4 plants showed morphological, anatomical and physiological advantages that protected them from foliar water loss. The strategies of leaf hydraulics to regulate leaf water balance during the day and during short-term osmotic stress also differed between Tian4 and 478. The leaf hydraulic conductivity of Tian4 and 478 increased temporarily, but their root hydraulic conductivities were reduced under osmotic stress. However, the root hydraulic conductivity of Tian4 subsequently recovered. Lower and rapidly reduced leaf transpiration and the ability of root hydraulics to recover from short-term osmotic stress can help explain the strategies for plant water balance of drought-tolerant maize.     

Genetic analysis of drought tolerance in maize by molecular markers. II. Plant height and flowering

Drought is a serious agronomic problem, and one of the most important factors contributing to crop yield loss. In maize grown in temperate areas, drought stress occurs just before and during the flowering period; consequently, tolerance to water stress in this species is largely determined by events that occur at or shortly after flowering. The purposes of our investigation were: (1)?to identify the chromosomal regions where factors conferring drought tolerance for traits related to plant development and flowering are located and (2)?to compare these regions with those carrying QTLs controlling these traits, in order to get indirect information on the genetic and physiological basis of maize response to water stress. To this aim, we performed a linkage analysis between the expression of male and female flowering time, anthesis-silking interval (ASI), plant height and molecular markers. The experiment was carried out under two environmental conditions, well-watered and water-stressed, on a maize population of 142 recombinant inbred lines obtained by selfing the F₁ between lines B73 and H99 and genotyped by RFLP, microsatellites (SSR) and AFLP markers, for a total of 153 loci. Linkage analysis revealed that, for male flowering time and plant height, most of the QTLs detected were the same under control and stress conditions. In contrast, with respect to female flowering time and ASI diverse QTLs appeared to be expressed either under control conditions or under stress. All of the QTLs conferring tolerance to drought were located in a different chromosome position as compared to the map position of the factors controlling the trait per se. This suggests that plant tolerance, in its different components, is not attributable to the presence of favourable allelic combinations controlling the trait but is based on physiological characteristics not directly associated with the control of the character.     

Evaluation of oxidative stress tolerance in maize (Zea mays L.) seedlings in response to drought

Seedlings of selected six genotypes of maize (Zea mays L.) differing in their drought sensitivity (LM5 and Parkash drought-tolerant and PMH2, JH3459, Paras and LM14 as drought-sensitive) were exposed to 72 h drought stress at two leaf stage. Alterations in their antioxidant pools combined with activities of enzymes involved in defense against oxidative stress were investigated in leaves. Activities of some reactive oxygen species (ROS)-scavenging enzymes, catalase (CAT) and ascorbate peroxidase (APX) were enhanced in tolerant genotypes in response to drought stress. Superoxide dismutase (SOD) activity was significantly decreased in sensitive genotypes, but remained unchanged in tolerant genotypes under stress. Peroxidase (POX) activity was significantly induced in tolerant, as well as sensitive genotypes. Imposition of stress led to increase in H2O2 and malondialdehyde (MDA, a marker for lipid peroxidation) content in sensitive genotypes, while in tolerant genotypes no change was observed. Significant increase in glutathione content was observed in sensitive genotypes. Ascorbic acid pool was induced in both tolerant and sensitive genotypes, but induction was more pronounced in tolerant genotypes. Significant activation of antioxidative defence mechanisms correlated with drought-induced oxidative stress tolerance was the characteristic of the drought tolerant genotypes. These studies provide a mechanism for drought tolerance in maize seedlings.     

Identification of functional genetic variations underlying drought tolerance in maize using SNP markers

Single nucleotide polymorphism (SNP) is a common form of genetic variation and popularly exists in maize genome. An Illumina GoldenGate assay with 1 536 SNP markers was used to genotype maize inbred lines and identified the functional genetic variations underlying drought tolerance by association analysis. Across 80 lines, 1 006 polymorphic SNPs (65.5% of the total) in the assay with good call quality were used to estimate the pattern of genetic diversity, population structure, and familial relatedness. The analysis showed the best number of fixed subgroups was six, which was consistent with their original sources and results using only simple sequence repeat markers. Pairwise linkage disequilibrium (LD) and association mapping with phenotypic traits investigated under water-stressed and well-watered regimes showed rapid LD decline within 100-500 kb along the physical distance of each chromosome, and that 29 SNPs were associated with at least two phenotypic traits in one or more environments, which were related to drought-tolerant or drought-responsive genes. These drought-tolerant SNPs could be converted into functional markers and then used for maize improvement by marker-assisted selection.     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Variety-specific response of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) leaf mitochondria to drought stress

The main objective of the present work was to examine leaf respiratory responses to dehydration and subsequent recovery in three varieties of winter wheat (Triticum aestivum L.) known to differ in their level of drought tolerance. Under dehydration, both total respiration and salicylhydroxamic acid (SHAM)-resistant cytochrome (Cyt) pathway respiration by leaf segments decreased significantly compared with well-watered plants. This decrease was more pronounced in the drought-sensitive Sadovo and Prelom genotypes. In contrast, the KCN-resistant SHAM-sensitive alternative (Alt) pathway became increasingly engaged, and accounted for about 80% of the total respiration. In the drought-tolerant Katya variety, increased contribution of the Alt pathway was accompanied by a slight decrease in Cyt pathway activity. Respiration of isolated leaf mitochondria also showed a variety-specific drought response. Mitochondria from drought-sensitive genotypes had low oxidative phosphorylation efficiency after dehydration and rewatering, whereas the drought-tolerant Katya mitochondria showed higher phosphorylation rates. Morphometric analysis of leaf ultrastructure revealed that mitochondria occupied approximately 7% of the cell area in control plants. Under dehydration, in the drought-sensitive varieties this area was reduced to about 2.0%, whereas in Katya it was around 6.0%. The results are discussed in terms of possible mechanisms underlying variety-specific mitochondrial responses to dehydration.     

Cell Membrane Stability and Leaf Surface Wax Content as Affected by Increasing Water Deficits in Maize

A field experiment was conducted with a water-stressed treatmentand well-watered control using eight maize (Zea mays L.) cultivars.Effects of water deficits on cell membrane stability (CMS) measuredby the polyethylene glycol (PEG) test, leaf surface wax content,and relative growth rate were investigated. Cytoplasmic lipidcontent was also analysed. Cell membrane stability and leaf surface wax content increasedwith the degrees of stress. Tolerance to drought evaluated asincrease in CMS under water deficit conditions was well differentiatedbetween cultivars and was well correlated with a reduction inrelative growth rate under stress. A negative correlation wasfound between percentage injury in the PEG test and leaf surfacewax content. High phospholipid contents were observed in tissuesof drought tolerant cultivars under water deficit conditions. Key words: Cell membrane stability, cytoplasmic lipid, drought tolerance, leaf surface wax, relative growth rate     

Maize leaf temperature responses to drought: Thermal imaging and quantitative trait loci (QTL) mapping

Leaf temperature has been shown to vary when plants are subjected to water stress conditions. Recent advances in infrared thermography have increased the probability of recording drought tolerant responses more accurately. The aims of this study were to identify the effects of drought on leaf temperature using infrared thermography. Furthermore, the genomic regions responsible for the expression of leaf temperature variation in maize seedlings (Zea mays L.) were explored. The maize inbred lines Zong3 and 87-1 were evaluated using infrared thermography and exhibited notable differences in leaf temperature response to water stress. Correlation analysis indicated that leaf temperature response to water stress played an integral role in maize biomass accumulation. Additionally, a mapping population of 187 recombinant inbred lines (RILs) derived from a cross between Zong3 and 87-1 was constructed to identify quantitative trait loci (QTL) responsible for physiological traits associated with seedling water stress. Leaf temperature differences (LTD) and the drought tolerance index (DTI) of shoot fresh weight (SFW) and shoot dry weight (SDW) were the traits evaluated for QTL analysis in maize seedlings. A total of nine QTL were detected by composite interval mapping (CIM) for the three traits (LTD, RSFW and RSDW). Two co-locations responsible for both RSFW and RSDW were detected on chromosomes 1 and 2, respectively, which showed common signs with their trait correlations. Another co-location was detected on chromosome 9 between LTD and shoot biomass, which provided genetic evidence that leaf temperature affects biomass accumulation. Additionally, the utility of a thermography system for drought tolerance breeding in maize was discussed.     

应用实时荧光定量PCR技术分析玉米水分胁迫诱导基因的表达模式

应用实时荧光定量PCR(real-time quantitative PCR)技术,研究了玉米中10个水分胁迫诱导基因的相对表达量及表达模式。结果表明,在花丝中,除基因Mads和Grp外,其他8个基因均随干旱胁迫程度加重,相对表达强度增加;在幼穗中,除基因Mads外,其他9个基因均随干旱胁迫程度加重,相对表达强度增加。应用实时荧光定量PCR和宏阵列技术的研究表明,在水分胁迫条件下,基因Arf3、Mads、Trx和F15相对表达量较高,可能在玉米应答水分胁迫过程起更重要的作用。     

Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea)

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance. The nucleotide sequence, reported in this paper, have been submitted to GenBank under accession number AY274834.     

Genotypic variation in drought stress response and subsequent recovery of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Three wheat (Triticum aestivum L.) genotypes, Sadovo, Katya and Prelom, with different tolerance to drought were comparatively evaluated in terms of leaf respiratory responses to progressing dehydration and consecutive rewatering. Under drought stress, the respiration of all varieties gradually decreased, as the drought-tolerant Katya showed the most pronounced decline at earlier stages of dehydration. When water stress intensified, this genotype gave relatively stable respiration rates compared with the drought-sensitive varieties. Additionally, dehydrated Katya leaves displayed lower stomatal conductance and higher photosynthesis values, which resulted in greater water use efficiency during the dehydration period. Combination of drought stress and short-term changes in leaf temperature also induced genotype-specific response that differed from the response to drought only. Over the whole temperature range, the leaves of Katya exposed to dehydration for 14 days, showed higher respiration rates compared to the drought-sensitive varieties. The sensitive varieties maintained higher respiration rates under control conditions and mild dehydration, and very low rates under severe drought. In Katya, respiration and photosynthesis were fully restored from the stress within the first day of rewatering. The drought-sensitive genotypes displayed a considerably slower recovering capacity. The results are discussed in terms of possible physiological mechanisms underlying plant tolerance to drought.     

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.