Fractal analysis on root systems of rice plants in response to drought stress

A fractal analytical method was used to examine the developmental responses of root systems in upland rice genotype CT9993-5-10-1-M (japonica) and lowland genotype IR62266-42-6-2 (indica) (abbreviated as CT9993 and IR62266, respectively) to soil water stress. The root systems were grown for one month in root boxes with 25 cm in length, 2 cm in width and 40 cm in depth, which were filled with soil. The root systems were sampled by following the needle-pinboard method, and then spread on the transparent plastic films with nets after carefully washing out the soils. The two-dimensional images of root systems were digitized by using a scanner. The digitized images were used for analysis based on fractal geometry with the box-counting method. The reductions in shoot dry weight, photosynthesis rate and transpiration rate of IR62266 by soil drought were greater than those of CT9993. The change of fractal parameters in response to soil moisture conditions differed between the two rice genotypes. The values of fractal abundance (FA) and fractal dimension (FD) in well-watered IR62266 plants were larger than in CT9993. The value of FA of IR62266 was decreased more by drought stress than that of CT9993, indicating that the volume of soils explored by the whole root systems of CT9993 was maintained or less decreased under drought stress in comparison to IR62266. Moreover, the values of FD tended to increase in CT9993 while it tended to decrease in IR62266 in response to drought. These root responses detected by the fractal analysis in CT9993 may be advantageous for its extracting more water from drying soils, which explains its better growth under drought-stressed condition.     

Candidate genes for drought tolerance and improved productivity in rice (Oryza sativa L.)

Candidate genes are sequenced genes of known biological action involved in the development or physiology of a trait. Twenty-one putative candidate genes were designed after an exhaustive search in the public databases along with an elaborate literature survey for candidate gene products and/or regulatory sequences associated with enhanced drought resistance. The downloaded sequences were then used to design primers considering the flanking sequences as well. Polymerase chain reaction (PCR) performed on 10 diverse cultivars that involvedJaponica, Indica and local accessions, revealed 12 polymorphic candidate genes. Seven polymorphic candidate genes were then utilized to genotype 148 individuals of CT9993 × IR62266 doubled haploid (DH) mapping population. The segregation data were tested for deviation from the expected Mendelian ratio (1:1) using a Chi-square test (<1%). Based on this, four candidate genes were assessed to be significant and the remaining three, as non-significant. All the significant candidate genes were biased towards CT9993, the female parent in the DH mapping population. Single-marker analysis strongly associated (<1%) them to different traits under both well-watered and low-moisture stress conditions. Two candidate genes,EXP15 andEXP13, were found to be associated with root number and silicon content in the stem respectively, under both well-watered and low-moisture stress conditions     

Proteome response of Elymus elongatum to severe water stress and recovery

Tall wheatgrass (Elymus elongatum Host) is a drought-tolerant, cool-season forage grass native to Iran. A proteomic approach has been applied to identify mechanisms of drought responsiveness and tolerance in plants undergoing vegetative stage drought stress and then recovery after rewatering. Uniformed clones were reproduced from a parent plant collected from Brojen (central region of Iran). Clones were grown in pots and drought was initiated by withholding water for 16 d. The leaf samples were taken in triplicate from both stressed/rewatered plants and continuously watered controls at five times: (i) 75% FC, (ii) 50% FC, (iii) 25% FC, (iv) 3 d after rewatering, and (v) 14 d after rewatering. Changes in the proteome pattern of shoots were studied using two-dimensional gel electrophoresis. Following the 16 d water stress, both shoot dry weight and leaf width decreased up to 67% compared with the well-watered plants, whereas proline content increased up to 20-fold. Leaf relative water contents (RWC) also declined from 85% to 24%. Out of about 600 protein spots detected on any given two-dimensional gel, 58 protein spots were reproducibly and significantly changed during drought stress and recovery. Only one protein (abscisic acid- and stress-inducible protein) showed significant changes in expression and position in response to severe drought. The fifty-eight responsive proteins were categorized in six clusters including two groups of proteins specifically up- and down-regulated in response to severe drought stress. Eighteen proteins belonging to these two groups were analysed by liquid chromatography tandem mass spectrometry leading to the identification of 11 of them, including the oxygen-evolving enhancer protein 2, abscisic acid- and stress-inducible protein, several oxidative stress tolerance enzymes, two small heat shock proteins, and Rubisco breakdown. The results suggest that E. elongatum may tolerate severe drought stress by accumulating proline and several proteins related to drought-stress tolerance. Recovery after rewatering might be another mechanism by which plants tolerate erratic rainfall in semi-arid regions.     

Differences in fennel seed responses to drought stress at the seed formation stage in sensitive and tolerant genotypes

To understand the effects of drought on fennel seed production and determine the underlying molecular processes, various fennel genotypes were exposed to drought stress. The yield and quality, including aromatic oil content, of fennel seeds were reduced by drought during seed development. To explore drought-induced biological processes in fennel, a label-free/gel-free proteomic analysis was performed. In Gaziantep and Tatmaj cultivars, which are sensitive and tolerant fennel genotypes, respectively, 106 and 92 drought-responsive proteins were identified. Comparison of protein-functional profiles indicated that proteins classified in stress, cell, and protein synthesis/degradation categories consisted important responsive mechanisms against drought stress. Pathway analysis visualized that the tricarboxylic acid cycle is important for both cultivars. In Tatmaj, moderate activation of proteins related to oxidative pentose phosphate pathway was detected along with an increase in photosynthesis-related proteins. Furthermore, cluster analysis of drought-responsive proteins using protein abundance at milky, dough, and mature stages identified protein homeostasis as a mechanism of drought tolerance in fennel. These results suggest that coordinated energy consumption and supply might be a drought-tolerance mechanism in fennel plants.

     

Proteomic analysis of rice leaf sheath during drought stress

Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice. To investigate the initial response of rice to drought stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to drought conditions from 2 to 6 days, and proteins were extracted from leaf sheaths, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 2 to 6 days, 10 proteins increased in abundance and the level of 2 proteins decreased. The functional categories of these proteins were identified as defense, energy, metabolism, cell structure, and signal transduction. In addition to drought stress, accumulations of protein were analyzed under several different stress conditions. The levels of an actin depolymerizing factor, a light harvesting complex chain II, a superoxidase dismutase and a salt-induced protein were changed by drought and osmotic stresses, but not cold or salt stresses, or abscisic acid treatment. The effect of drought stress on protein in the leaf sheaths of drought-tolerant rice cultivar was also analyzed. The light harvesting complex chain II and the actin depolymerizing factor were present at high levels in a drought-tolerant rice cultivar before stress application. With drought stress, actin depolymerizing factor was expressed in leaf blades, leaf sheaths, and roots. These results suggest that actin depolymerizing factor is one of the target proteins induced by drought stress.     

A single segment substitution line population for identifying traits relevant to drought tolerance and avoidance

A population of chromosome segment substitution lines was developed using KDML105 as the recurrent parent and one of DH212 (IR68586-F2-CA-143) or DH103 (IR68586-F2-CA-31) as the donor parent. The donor parents are part of a doubled haploid population from a cross between CT9993, an upland japonica accession, and IR62266, a lowland indica accession. Multiple QTL that are relevant to drought avoidance, drought tolerance and yield traits under drought stress were mapped in this doubled haploid population and the segments selected for the chromosome segment substitution lines were chosen to capture these QTL. The chromosome segment substitution line population was phenotyped under irrigated and mild drought stress conditions, which identified that many yield traits under drought stress had been introduced into the chromosome segment substitution lines.     

Identification of proteins associated with water-deficit tolerance in C4 perennial grass species, Cynodon dactylon×Cynodon transvaalensis and Cynodon dactylon

The study was conducted to examine differential proteomic responses to water-deficit stress in hybrid bermudagrass [Cynodon dactylon (L.) Pers. ×Cynodon transvaalensis Burtt Davy, cv. Tifway] and common bermudagrass (C. dactylon, cv. C299). Plants were exposed to water-deficit stress for 15 days by withholding irrigation in a growth chamber. Leaf electrolyte leakage increased and photochemical efficiency and relative water content declined under water-deficit stress, but the extent of changes in each of the physiological parameters for 'Tifway' was less pronounced than those for 'C299'. Total proteins of leaves were extracted from well-watered and water-deficit plants and separated by two-dimensional gel electrophoresis. Of the 750 protein spots reproducibly detected, 32 proteins had increases in the abundance and 22 proteins exhibited decreases in the abundance in at least one genotype under water-deficit stress. A significantly higher number of proteins were found to accumulate in 'Tifway' than in 'C299' and 16 proteins with increasing abundance were detected only in 'Tifway' under water-deficit stress. All stress-responsive proteins were subjected to mass spectrometry analysis, which were mainly involved in metabolism, energy, cell growth/division, protein synthesis and stress defense. Functional analysis of differential drought-responsive proteins between the two genotypes suggests that the superior water-deficit tolerance in 'Tifway' bermudagrass could be mainly associated with less severe decline in the abundance level of proteins involved in photosynthesis (chlorophyll a-b, ATP synthase subunit alpha, phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase/oxygenase) and greater increase in the abundance level of antioxidant defense proteins (superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase and peroxiredoxin), demonstrating that maintaining photosynthesis and active antioxidant defense mechanisms may play a critical role in C(4) grass adaptation to water-deficit stress.     

Mapping of genes controlling aluminum tolerance in rice: comparison of different genetic backgrounds

Aluminum toxicity is the main factor limiting the productivity of crop plants in acid soils, particularly in the tropics and subtropics. In this study, a doubled-haploid population derived from the rice ( Oryza sativa L.) breeding lines CT9993 and IR62266 was used to map genes controlling Al tolerance. A genetic linkage map consisting of 280 DNA markers (RFLP, AFLP and SSR) was constructed to determine the position and nature of quantitative trait loci (QTLs) affecting Al tolerance. Three characters - control root length (CRL), Al-stressed root length (SRL) and root length ratio (RR) - were evaluated for the DH lines and the parents at the seedling stage in nutrient solution. A total of 20 QTLs controlling root growth under Al stress and control conditions were detected and distributed over 10 of the 12 rice chromosomes, reflecting multigenic control of these traits. The two QTLs of largest effect, qALRR-1-1 and qALRR-8 for root length ratio (a measurement of Al tolerance) were localized on chromosomes 1 and 8, respectively. Three other QTLs in addition to qALRR-8 were apparently unique in the CT9993 x IR62266 mapping population, which may explain the high level of Al tolerance in CT9993. Comparative mapping identified a conserved genomic region on chromosome 1 associated with Al tolerance across three rice genetic backgrounds. This region provides an important starting point for isolating genes responsible for different mechanisms of aluminum tolerance and understanding the genetic nature of this trait in rice and other cereals.     

Response of proteome and morphological structure to short‐term drought and subsequent recovery in Cucumis sativus leaves

Drought is the primary limitation to plant growth and yield in agricultural systems. Cucumber (Cucumis sativus) is one of the most important vegetables worldwide and has little tolerance for water deficit. To understand the drought stress response strategy of this plant, the responses of cucumber to short‐term drought and rewatering were determined in this study by morphological structure and proteomic analyses. The leaf relative water content was significantly decreased under drought, and the cell structure was altered, while rewatering obviously alleviated the symptoms of water shortage and cell damage. A total of 320 and 246 proteins exhibiting significant abundance changes in response to drought and recovery, respectively, were identified. Our proteome analysis showed that 63 co‐regulated proteins were shared between drought and rewatering, whereas most of the responsive proteins were unique. The proteome is adjusted through a sequence of regulatory processes including the biosynthesis of secondary metabolites and the glutathione metabolism pathway, which showed a high correlation between protein abundance profile and corresponding enzyme activity. Drought and recovery regulated different types of proteins, allowing plants to adapt to environmental stress or restore growth, respectively, which suggests that short‐term drought and recovery are almost fully uncoupled processes. As an important component of the antioxidant system in plants, glutathione metabolism may be one of the main strategies for regulating antioxidant capacity during drought recovery. Our results provide useful information for further analyses of drought adaptability in cucumber plants.     

Photosynthetic responses and acclimation of two castor bean cultivars to repeated drying–wetting cycles

To investigate the responses of castor bean to repeated drying–wetting cycles (RDWC), morpho-physiological parameters of two cultivars (Jiaxiang 2 and Hangbi 8) were determined by a pot experiment under well-watered control and RDWC. RDWC inhibited plant growth and leaf development, decreased water loss rate (WLR), and enhanced leaf mass per area (LMA) and chlorophyll content as indicated by spectral reflectance indices for both cultivars. Photosynthesis was inhibited by progressive drought stress but quickly recovered after rewatering for each cycle. Both cultivars exhibit a similar pattern of acclimation to RDWC: (1) higher LMA and lower WLR, (2) increased photosynthetic capacity under drought stress with increasing cycle numbers, (3) quick recovery and over-compensation for photosynthesis after rewatering, and (4) increased chlorophyll content. Jiaxiang 2 shows a high capacity for water preservation under drought stress and an over-compensation for photosynthesis after rewatering compared with Hangbi 8.     

QTLs linked to leaf epicuticular wax,physio-morphological and plant production traits under drought stress in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Drought stress is the major constraint to rice (Oryza sativa L.) production and yield stability in rainfed ecosystems. Identifying genomic regions contributing to drought resistance will help to develop rice cultivars suitable for rainfed regions through marker-assisted breeding. Quantitative trait loci (QTLs) linked to leaf epicuticular wax, physio-morphological and plant production traits under water stress and irrigated conditions were mapped in a doubled haploid (DH) line population from the cross CT9993-5-10-1-M/IR62266-42-6-2. The DH lines were subjected to water stress during anthesis. The DH lines showed significant variation for epicuticular wax (EW), physio-morphological and plant production traits under stress and irrigated conditions. A total of 19 QTLs were identified for the various traits under drought stress and irrigated conditions in the field, which individually explained 9.6%–65.6% of the phenotypic variation. A region EM15_10-ME8_4-R1394A-G2132 on chromosome 8 was identified for leaf EW and rate of water loss i.e., time taken to reach 70% RWC from excised leaves in rice lines subjected to drought stress. A large effect QTL (65.6%) was detected on chromosome 2 for harvest index under stress. QTLs identified for EW, rate of water loss from excised leaves and harvest index under stress in this study co-located with QTLs linked to shoot and root-related drought resistance traits in these rice lines and might be useful for rainfed rice improvement.     

Rice height QTLs in KDML105 chromosome segment substitution lines

Rice is an important crop that is consumed by approximately half of the world's population on a regular basis. Plant height is an important characteristic with shorter rice often having higher lodging resistance and better soil nutrient utilization allowing for lower fertilizer use. We used a Chromosome Segment Substitution Line (CSSL) population generated by introgressing segments of CT9993 and IR62266 into KDML 105. We identified height QTLs on chromosomes 1 and 4. We performed whole genome sequencing of the parental lines and found that IR62266 has the deletion in Gibberellin 20-oxidase 2 corresponding to the semi-dwarf 1 locus. However, short height on chromosome 1 came from CT9993 with no mutation in Gibberellin 20-oxidase 2, or any known height genes. The height QTL on chromosome 4 contains mutations in Peroxisome biogenesis protein 6, which has been linked to a reduced growth phenotype in A. thaliana, making this a good candidate height gene.     

Saturation mapping of QTL regions and identification of putative candidate genes for drought tolerance in rice

We have developed 85 new markers (50 RFLPs, 5 SSRs, 12 DD cDNAs, 9 ESTs, 8 HSP-encoding cDNAs and one BSA-derived AFLP marker) for saturation mapping of QTL regions for drought tolerance in rice, in our efforts to identify putative candidate genes. Thirteen of the markers were localized in the close vicinity of the targeted QTL regions. Fifteen of the additional markers mapped, respectively, inside one QTL region controlling osmotic adjustment on chromosome 3 ( oa3.1) and 14 regions that affect root traits on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 10 and 12. Differential display was used to identify more putative candidate genes and to saturate the QTL regions of the genetic map. Eleven of the isolated cDNA clones were found to be derived from drought-inducible genes. Two of them were unique and did not match any genes in the GenBank, while nine were highly similar to cDNAs encoding known proteins, including a DnaJ-related protein, a zinc-finger protein, a protease inhibitor, a glutathione-S-transferase, a DNA recombinase, and a protease. Twelve new cDNA fragments were mapped onto the genetic linkage map; seven of these mapped inside, or in close proximity to, the targeted QTL regions determining root thickness and osmotic adjustment capacity. The gene I12A1, which codes for a UDP-glucose 4-epimerase homolog, was identified as a putative target gene within the prt7.1/brt7.1 QTL region, as it is involved in the cell wall biogenesis pathway and hence may be implicated in modulating the ability of rice roots to penetrate further into the substratum when exposed to drought conditions. RNAs encoding elongation factor 1, a DnaJ-related protein, and a homolog of wheat zinc-finger protein were more prominently induced in the leaves of IR62266 (the lowland rice parent of the mapping materials used) than in those of CT9993 (the upland rice parent) under drought conditions. Homologs of 18S ribosomal RNA, and mRNAs for a multiple-stress induced zinc-finger protein, a protease inhibitor, and a glutathione-S-transferase were expressed at significantly higher levels in CT9993 than in IR62266. Thus several genes involved in the regulation of DNA structure and mRNA translation were found to be drought-regulated, and may be implicated in drought resistance.Communicated by R. Hagemann     

QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress

Cell-membrane stability (CMS) is considered to be one of the major selection indices of drought tolerance in cereals. In order to determine which genomic region is responsible for CMS, 104 rice (Oryza sativa L.) doubled haploid (DH) lines derived from a cross between CT9993–5-10–1-M and IR62266-42–6-2 were studied in the greenhouse in a slowly developed drought stress environment. Drought stress was induced on 50-day-old plants by withholding water. The intensity of stress was assessed daily by visual scoring of leaf wilting and by measuring leaf relative water content (RWC). The leaf samples were collected from both control (well-watered) and stressed plants (at 60–65% of RWC), and the standard test for CMS was carried out in the laboratory. There was no significant difference (P>0.05) in RWC between the two parental lines as well as among the 104 lines, indicating that all the plants were sampled at a uniform stress level. However, a significant difference (P<0.05) in CMS was observed between the two parental lines and among the population. No significant correlation was found between CMS and RWC, indicating that the variation in CMS was genotypic in nature. The continuous distribution of CMS and its broad-sense heritability (34%) indicates that CMS should be polygenic in nature. A linkage map of this population comprising of 145 RFLPs, 153 AFLPs and 17 microsatellite markers was used for QTL analysis. Composite interval mapping identified nine putative QTLs for CMS located on chromosomes 1, 3, 7, 8, 9, 11 and 12. The amount of phenotypic variation that was explained by individual QTLs ranged from 13.4% to 42.1%. Four significant (P<0.05) pairs of digenic interactions between the detected QTLs for CMS were observed. The identification of QTLs for this important trait will be useful in breeding for the improvement of drought tolerance in rice. This is the first report of mapping QTLs associated with CMS under a natural water stress condition in any crop plants. Received: 8 September 1999 / Accepted: 13 October 1999     

水分胁迫及复水过程中小麦抗氧化酶的变化

对两个抗旱性不同的小麦品种进行水分胁迫和复水处理,研究其抗氧化酶活性的响应。在水分胁迫下,陇春-20的相对含水量高于优鉴-24,复水24h后,优鉴-24的相对含水量恢复较快且高于陇春-20。水分胁迫下,优鉴-24中H2O2含量增加迅速,而且各阶段含量均高于陇春-20,复水后两个品种的H2O2含量都下降,这表明优鉴-24在水分胁迫时受到更严重的氧化胁迫。采用温和胶电泳结合抑制剂实验发现小麦有3条Mn—SOD,一条Fe—SOD和Cu／Zn-SOD同工酶带,CAT同工酶有3条谱带。在水分胁迫和复水期间,优鉴-24的SOD和CAT活性高于陇春-20,随着水分胁迫程度的增加,两个品种的SOD和CAT活性都增强,复水后,优鉴-24的SOD活性继续增强,而陇春-20的Mn—SOD—3活性略微降低,Fe—SOD和Cu／Zn—SOD活性略微升高,陇春-20的CAT活性降低。水分胁迫诱导了Mn—SOD—1在优鉴-24及Mn—SOD-2和Fe—SOD在陇春-20中的表达。     

Selenium improves recovery of wheat seedlings at rewatering after drought stress

Rewatering after drought is beneficial to plants subjected to moderate drought stress, and selenium (Se) could increase the tolerance of plants to stressful environment. The role of Se in rewatering of drought-treated wheat seedlings (Triticum aestivum L., cv Hengmai5229) was studied. The objective was to elucidate whether Se could improve recovery of wheat seedlings at rewatering after drought stress. Drought stress induced a significant reduction in growth parameters, total chlorophyll and soluble protein contents, and increased the rate of superoxide radical (O ₂ ^·? ) production, MDA content, and the activities of peroxidase, catalase (CAT), and superoxide dismutase in wheat seedlings. Rewatering after drought did not significantly affect biomass accumulation of seedlings over drought treatment, although it decreased the rate of O ₂ ^·? production and MDA content. However, the combined treatment of rewatering and Se evidently promoted biomass accumulation of seedlings over drought treatment and rewatering alone; and the rate of O ₂ ^·? production, MDA content, soluble protein content and CAT activity were recovered to the control values. This indicates that Se improved recovery of wheat seedlings at rewatering after drought stress.