A novel rice PR10 protein, RSOsPR10, specifically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway

Plant roots have important roles not only in absorption of water and nutrients, but also in stress tolerance such as desiccation, salt, and low temperature. We have investigated stress-response proteins from rice roots using 2-dimensional polyacrylamide-gel electrophoresis and found a rice protein, RO-292, which was induced specifically in roots when 2-week-old rice seedlings were subjected to salt and drought stress. The full-length RO-292 cDNA was cloned, and was determined to encode a protein of 160 amino acid residues (16.9 kDa, pI 4.74). The deduced amino acid sequence showed high similarity to known rice PR10 proteins, OsPR10a/PBZ1 and OsPR10b. RO-292 mRNA accumulated rapidly upon drought, NaCl, jasmonic acid and probenazole, but not by exposure to low temperature or by abscisic acid and salicylic acid. The RO-292 gene was also up-regulated by infection with rice blast fungus. Interestingly, induction was observed almost exclusively in roots, thus we named the gene RSOsPR10 (root specific rice PR10). The present results indicate that RSOsPR10 is a novel rice PR10 protein, which is rapidly induced in roots by salt, drought stresses and blast fungus infection possibly through activation of the jasmonic acid signaling pathway, but not the abscisic acid and salicylic acid signaling pathway.     

Hydraulic conductivity of rice roots

A pressure chamber and a root pressure probe technique have been used to measure hydraulic conductivities of rice roots (root Lp(r) per m(2) of root surface area). Young plants of two rice (Oryza sativa L.) varieties (an upland variety, cv. Azucena and a lowland variety, cv. IR64) were grown for 31-40 d in 12 h days with 500 micromol m(-2) s(-1) PAR and day/night temperatures of 27 degrees C and 22 degrees C. Root Lp(r) was measured under conditions of steady-state and transient water flow. Different growth conditions (hydroponic and aeroponic culture) did not cause visible differences in root anatomy in either variety. Values of root Lp(r) obtained from hydraulic (hydrostatic) and osmotic water flow were of the order of 10(-8) m s(-1) MPa(-1) and were similar when using the different techniques. In comparison with other herbaceous species, rice roots tended to have a higher hydraulic resistance of the roots per unit root surface area. The data suggest that the low overall hydraulic conductivity of rice roots is caused by the existence of apoplastic barriers in the outer root parts (exodermis and sclerenchymatous (fibre) tissue) and by a strongly developed endodermis rather than by the existence of aerenchyma. According to the composite transport model of the root, the ability to adapt to higher transpirational demands from the shoot should be limited for rice because there were minimal changes in root Lp(r) depending on whether hydrostatic or osmotic forces were acting. It is concluded that this may be one of the reasons why rice suffers from water shortage in the shoot even in flooded fields.     

淹水和干旱条件下水稻根系形成的QTLs及候选基因分析

为了研究不同水分条件下组成型根系性状和适应性根系性状的遗传机制,利用由IR64／Azucena发展的双单倍体（DH）群体分析了淹水和干旱条件下水稻幼苗种子根长（SRL）、不定根数（ARN）、总根干重（RW）及其对应的相对参数（干旱和淹水条件下根系性状的比值）的QTLs。淹水与干旱条件下检测到一个共同的种子根长QTL和一个共同的总根干重QTL。同时对前人发表的遗传群体定位的根系性状QTLs进行比较分析,检测到几个共同的根系性状QTLs。对与细胞伸长、分裂相关的候选基因进行了定位,其中4个细胞壁相关的ESTs（OsEXP2,OsEXP4,EXT和Xet）被定位在与不同水分条件下检测出的根系性状QTLs的相同区间。     

Mapping QTLs and candidate genes for rice root traits under different water-supply conditions and comparative analysis across three populations

To investigate the genetic factors underlying constitutive and adaptive morphological traits of roots under different water-supply conditions, a recombinant inbred line (RIL) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 249 molecular markers, was used in cylindrical-pot experiments. Eighteen QTLs were detected for seminal root length (SRL), adventitious root number (ARN), and lateral root length (LRL) and lateral root number (LRN) on the seminal root at a soil depth of from 3 to 6 cm under flooding and upland conditions. One identical QTL was detected under both flooding and upland conditions. The relative parameters under the two water-supply conditions were also used for QTL analysis. Five QTLs for upland induced variations in the traits were detected with the positive alleles from Azucena. A comparative analysis was performed for the QTLs detected in this study and those reported from two other populations with Azucena as a parent. Several identical QTLs for root elongation were found across the three populations with positive alleles from Azucena. Candidate genes were screened from ESTs and cDNA-AFLP clones for comparative mapping with the detected QTLs. Two genes for cell expansion, OsEXP2 and endo-1,4--D-glucanase EGase, and four cDNA-AFLP clones from root tissues of Azucena, were mapped on the intervals carrying the QTLs for SRL and LRL under upland conditions, respectively.Communicated by H.C. Becker     

Molecular markers linked to genes underlying seedling tolerance for ferrous iron toxicity

A double haploid (DH) population consisting of 123 lines derived from a japonica variety, Azucena, and an indica variety, IR64, and 100 BC1F1 (Azucena) lines were cultivated hydroponically using two treatments: one with excess Fe2+ at the concentration of 250 mg L-1 and a control with standard nutrient solution. Genotypic tolerance was evaluated using an index scale based on degree of leaf bronzing and relative decease in shoot dry weight (RDSDW) Toxic symptoms were not observed for Azucena and BClFl plants. In contrast, index values for the DH population indicated segregation for tolerance, and IR64 was moderately sensitive. Molecular marker loci associated with variations in index values and in RDSDW, and gene loci for tolerance were detected using 175 Markers mapped on all 12 chromosomes by single marker loci and interval mapping. Two gene loci were identified to be flanked by RG345 and RG381, and linked to RG810, respectively, on chromosome 1 for both index values and RDSDW. They explained 32% and 13% of the total variation in the index values, and 15% and 21 % in the RDSDW in the population, respectively. The variation in RDSDW was also explained by a locus linked to RG978 on chromosome 8 by about 10%. Comparison of the two marker genotypic class means indicated that the tolerant alleles were from Azucena at the first locus on chromosome 1 and the locus on chromosome 8, and that at the second locus on chromosome 1 from IR64.     

Effect of soil mechanical impedance on root growth of two rice varieties under field drought stress

Two upland rice varieties, Azucena and Bala, were screened for root growth under droughted and irrigated treatments in two field sites at the West Africa Rice Development Association (WARDA) experimental farm, Côte d’Ivoire, during the dry season of 1999/2000. The sites were chosen to represent contrasting soil profile penetration resistance (PR) characteristics on upland sites, although both were relatively impeding. The number of nodal root axes per unit area passing through horizontal transects (root density) was counted at 35, 56, 77 and 98 days after sowing (DAS) at 10 cm depth intervals. Azucena consistently maintained a greater root density than Bala and a greater proportion of Azucena roots grew to 30 cm depth (22.7% vs. 8.4% at 77 DAS). There was little detectable effect of water regime on root distribution but evidence of lower root numbers at depths below 20 cm in the higher PR site was revealed. A site by variety by soil depth interaction suggests that Azucena roots are more strongly affected by very high PR than those of Bala. PR between 0–30 cm depth increased greatly with decreasing soil water content during the drought as the soil dried. This increase is likely to have prevented or greatly impaired further nodal root growth within this layer. At 40 cm depth, PR was high (3–4 MPa) but did not increase during the drought. At this depth root growth rate was likely to be greatly reduced despite the availability of water. These results demonstrate that varietal differences in root morphology characterised in the laboratory can be also detected in impeding field soils as differences in the density of roots at depth. Relatively poor root growth in these fields in the absence of drought was probably due to the high mechanical impedance and/or the physiological stress of the plants in the dry season. Our results indicate that high mechanical impedance was a more fundamental constraint on root growth than soil water availability during the drought. Thus, varietal differences in root penetration ability might be very important for drought avoidance in soils of this type.     

High temperature stress and spikelet fertility in rice (Oryza sativa L.)

In future climates, greater heat tolerance at anthesis will be required in rice. The effect of high temperature at anthesis on spikelet fertility was studied on IR64 (lowland indica) and Azucena (upland japonica) at 29.6 degrees C (control), 33.7 degrees C, and 36.2 degrees C tissue temperatures. The objectives of the study were to: (i) determine the effect of temperature on flowering pattern; (ii) examine the effect of time of day of spikelet anthesis relative to a high temperature episode on spikelet fertility; and (iii) study the interactions between duration of exposure and temperature on spikelet fertility. Plants were grown at 30/24 degrees C day/night temperature in a greenhouse and transferred to growth cabinets for the temperature treatments. Individual spikelets were marked with paint to relate fertility to the time of exposure to different temperatures and durations. In both genotypes the pattern of flowering was similar, and peak anthesis occurred between 10.30 h and 11.30 h at 29.2 degrees C, and about 45 min earlier at 36.2 degrees C. In IR64, high temperature increased the number of spikelets reaching anthesis, whereas in Azucena numbers were reduced. In both genotypesor=33.7 degrees C at anthesis caused sterility. In IR64, there was no interaction between temperature and duration of exposure, and spikelet fertility was reduced by about 7% per degrees C>29.6 degrees C. In Azucena there was a significant interaction and spikelet fertility was reduced by 2.4% degrees Cd-1 above a threshold of 33 degrees C. Marking individual spikelets is an effective method to phenotype genotypes and lines for heat tolerance that removes any apparent tolerance due to temporal escape.     

QTLs and epistasis for aluminum tolerance in rice (Oryza sativa L.) at different seedling stages

To investigate the genetic background for aluminum (Al) tolerance in rice, a recombinant inbred (RI) population, derived from a cross between an Al-sensitive lowland indica rice variety IR1552 and an Al-tolerant upland japonica rice variety Azucena, was used in culture solution. A molecular linkage map, together with 104 amplified fragment length polymorphism (AFLP) markers and 103 restriction fragment length polymorphism (RFLP) markers, was constructed to map quantitative trait loci (QTLs) and epistatic loci for Al tolerance based on the segregation for relative root length (RRL) in the population. RRL was measured after stress for 2 and 4 weeks at a concentration of 1mM of Al³⁺ and a control with a pH 4.0, respectively. Two QTLs were detected at both the 2nd and the 4th weeks on chromosomes 1 and 12 from unconditional mapping, while the QTL on chromosome 1 was only detected at the 2nd stress week from conditional mapping. The effect of the QTL on chromosome 12 was increased with an increase of the stress period from 2 to 4 weeks. The QTL on chromosome 1 was expressed only at the earlier stress, but its contribution to tolerance was prolonged during growth. At least one different QTL was detected at the different stress periods. Mean comparisons between marker genotypic classes indicated that the positive alleles at the QTLs were from the Al-tolerant upland rice Azucena. An important heterozygous non-allelic interaction on Al tolerance was found. The results indicated that tolerance in the younger seedlings was predominantly controlled by an additive effect, while an epistatic effect was more important to the tolerance in older seedlings; additionally the detected QTLs may be multiple allelic loci for Al tolerance and phosphorus-uptake efficiency, or for Al and Fe²⁺ tolerance. Received: 29 July 1999 / Accepted: 13 October 1999     

Evaluation of near-isogenic lines of rice introgressed with QTLs for root depth through marker-aided selection

Drought is one of the main abiotic constraints in rice. A deep root system contributes efficiently to maintaining the water status of the crop through a stress period. After identifying QTLs affecting root parameters in a doubled-haploid (DH) population of rice derived from the cross IR64/Azucena, we started a marker-assisted backcross program to transfer the Azucena allele at four QTLs for deeper roots (on chromosomes 1, 2, 7 and 9) from selected DH lines into IR64. We selected the backcross progenies strictly on the basis of their genotypes at the marker loci in the target regions up to the BC₃F₂. We assessed the proportion of alleles remaining from Azucena in the non-target areas of the BC₃F₂ plants, which was in the range expected for the backcross stage reached. Twenty nine selected BC₃F₃ near-isogenic lines (NILs) were developed and compared to IR64 for the target root traits and three non-target traits in replicated experiments. Of the three tested NILs carrying target 1, one had significantly improved root traits over IR64. Three of the seven NILs carrying target 7 alone, as well as three of the eigth NILs carrying both targets 1 and 7, showed significantly improved root mass at depth. Four of the six NILs carrying target 9 had significantly improved maximum root length. Five NILs carrying target 2 were phenotyped, but none had a root phenotype significantly different from that of IR64. A re-analysis of the initial data with the composite interval mapping technique revealed two linked QTLs with opposite effects in this area. Some NILs were taller than IR64 and all had a decreased tiller number because of a likely co-introgression of linked QTLs. The usefulness of NILs, the efficiency of marker-aided selection for QTLs and the relationship between root traits are discussed. The NILs with an improved root system will permit testing the importance of root depth for water-limited environments. Received: 17 July 2000 / Accepted: 20 October 2000     

QTLs and Candidate Genes for Rice Root Growth Under Flooding and Upland Conditions

To investigate the genetic factors underlying constitutive and adaptive root growth under different water-supply conditions, a double haploid (DH) population, derived from a cross between lowland rice variety IR64 and upland rice variety Azucena, with 284 molecular markers was used in cylindrical pot experiments. Several QTLs for seminal root length (SRL), adventitious root number (ARN) and total root dry weight (RW) respectively, under both flooding and upland conditions were detected. Two identical QTLs for SRL and RW were found under flooding and upland conditions. The relative parameters defined as the ratio of parameters under the two water-supply conditions were also used for QTL analysis. A comparative analysis among different genetic populations was performed for the QTLs for root traits and several consistent QTLs for root traits across genetic backgrounds were detected. Candidate genes for cell expansion and elongation were used for comparative mapping with the detected QTLs. Four cell wall-related expressed sequence tags (ESTs) for OsEXP2, OsEXP4, EXT and Xet were mapped on the intervals carrying the QTLs for root traits.     

Whole plant responses, key processes, and adaptation to drought stress: the case of rice

Most high-yielding rice cultivars developed for irrigated conditions, including the widely grown lowland variety IR64, are highly susceptible to drought stress. This limits their adoption in rainfed rice environments where there is a risk of water shortage during the growing season. Mapping studies using lowland-by-upland rice populations have provided limited information about the genetic basis of variation in yield under drought. One approach to simultaneously improve and understand rice drought tolerance is to generate backcross populations, select superior lines in managed stress environments, and then evaluate which features of the selected lines differ from the recurrent parent. This approach was been taken with IR64, using a range of tolerant and susceptible cultivars as donor parents. Yields of the selected lines measured across 13 widely contracting water environments were generally greater than IR64, but genotype-by-environment effects were large. Traits expected to vary between IR64 and selected lines are plant height, because many donors were not semi-dwarf types, and maturity, because selection in a terminal stress environment is expected to favour earliness. In these experiments it was found that some lines that performed better under upland drought were indeed taller than IR64, but that shorter lines with good yield under drought could also be identified. In trials where drought stress developed in previously flooded (lowland) fields, height was not associated with performance. There was little change in maturity with selection. Other notable differences between IR64 and the selected backcross lines were in their responses to applied ABA and ethylene in greenhouse experiments at the vegetative stage and in leaf rolling observed under chronic upland stress in the field. These observations are consistent with the hypothesis that adaptive responses to drought can effectively allow for improved performance across a broad range of water environments. The results indicate that the yield of IR64 under drought can be significantly improved by backcrossing with selection under stress. In target environments where drought is infrequent but significant in certain years, improved IR64 with greater drought tolerance would be a valuable option for farmers.     

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.     

Four QTL in Rice Associated with Broad Spectrum Resistance to Blast Isolates from Rice and Barley

Pyricularia grisea is the most destructive and cosmopolitan fungal pathogen of rice and it can also cause disease on other agriculturally important cereals. We determined the number, location and interaction of quantitative trait loci (QTL) associated with resistance to P. grisea isolates obtained from rice (THL142 and THL222) and barley (TH16 and THL80) grown in Thailand. The isolates showed a spectrum of virulence when used to inoculate a series of differentials. We used a reference blast resistance mapping population of rice (IR64 × Azucena). IR64 was highly resistant, and Azucena was highly susceptible, to all four isolates. The numbers of resistant vs. susceptible progeny suggest that the resistance of IR64 is determined by two or three genes with additive effects. The correlation coefficients for all pairwise comparisons of disease severity were high and highest between barley isolates and between rice isolates. Four QTL were detected, one on each of the following chromosomes 2, 8, 9 and 10. IR64 contributed resistance alleles at three of the QTL (chromosomes 2, 8 and 9). Azucena contributed the resistance allele at the QTL on chromosome 10 in response to inoculation with isolate THL142. The results of the QTL analysis support interpretation of the phenotypic frequency distributions regarding the number of genes determining resistance to the four isolates in this population. Our results are novel in adding blast isolates from barley to the catalogue of pathogen specificities to which a gene, or genes, from IR64 confer resistance.     

The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity

We previously reported that rice blast fungus or jasmonic acid induced the expression of rice pathogenesis-related class 10 (JIOsPR10) proteins (Kim et al. 2003, 2004). However, no further studies have been carried out to examine the expression, localization, and enzymatic activity of this protein in either developmental tissues or in tissues under abiotic stress conditions. In this study, rice JIOsPR10 was examined by Western blot analysis, immunolocalization, and biochemical assays. Western blots revealed that the JIOsPR10 protein was expressed in developmental tissues, including in flower and root. The protein was also expressed under abiotic stresses, such as occurs during senescence and wounding. Using immunohistochemical techniques, we determined that expression of JIOsPR10 was localized to the palea of flower, in the exodermis, and inner part of the endodermis of the root. In senescencing tissues of leaf and coleoptiles, its expression was localized in vascular bundles. The RNase activity using JIOsPR10 recombinant protein was determined and abolished after treatment with DTT in a native in-gel assay. To test this, we created JIOsPR10 mutant proteins containing serine substitutions of amino acids C81S, C83S, or both and examined their RNase activities. The activity of the C83S mutant was decreased in the agarose gel assay compared to the wild type. Taken together, we hypothesize that the JIOsPR10 protein possesses RNase activity that is sensitive to DTT, suggesting the importance of the disulfide bonding between cysteine residues and that it might play a role in constitutive self-defense mechanisms in plants against biotic and abiotic stresses.     

Silencing of PR-10-like proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis> results in an antagonistic induction of other PR proteins and in an increased tolerance upon infection with the oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

Recent studies on the root proteome of Medicago truncatula (Gaertn.) showed an induction of pathogenesis-related (PR) proteins of the class 10 after infection with the oomycete pathogen Aphanomyces euteiches (Drechs.). To get insights into the function of these proteins during the parasitic root-microbe association, a gene knockdown approach using RNAi was carried out. Agrobacterium rhizogenes-mediated transformation of M. truncatula roots led to a knockdown of the Medicago PR10-1 gene in transgenic in vitro root cultures. Proteomic analyses of the MtPr10-1i root cultures showed that MtPr10-1 was efficiently knocked down in two MtPr10-1i lines. Moreover, five additional PR-10-type proteins annotated as abscisic acid responsive proteins (ABR17s) revealed also an almost complete silencing in these two lines. Inoculation of the root cultures with the oomycete root pathogen A. euteiches resulted in a clearly reduced colonization and thus in a suppressed infection development in MtPr10-1i roots as compared to that in roots of the transformation controls. In addition, MtPr10-1 silencing led to the induction of a new set of PR proteins after infection with A. euteiches including the de novo induction of two isoforms of thaumatin-like proteins (PR-5b), which were not detectable in A. euteiches-infected control roots. Thus, antagonistic induction of other PR proteins, which are normally repressed due to PR-10 expression, is supposed to cause an increased resistance of M. truncatula upon an A. euteiches in vitro infection. The results were also further confirmed by detecting increased PR-5b induction levels in 2-D gels of a previously analyzed M. truncatula line (F83.005-9) exhibiting increased A. euteiches tolerance associated with reduced PR-10 induction levels.     

Comparison of QTLs for rice seedling morphology under different water supply conditions

The variation of seedling characteristics under different water supply conditions is strongly associated with drought resistance in rice (Oryza sativa L.) and a better elucidation of its genetics is helpful for improving rice drought resistance. Ninety-six doubled-haploid (DH)rice lines of an indica and japonica cross were grown in both flooding and upland conditions and QTLs for morphological traits at seedling stage were examined using 208 restriction fragment length polymorphism (RFLP) and 76 microsatellite (SSR) markers. A total of 32 putative QTLs were associated with the four seedling traits: average of three adventitious root lengths (ARL), shoot height (SH), shoot biomass (SW), and root to shoot dry weight ratio (RSR). Five QTLs detected were the same under control and upland conditions. The ratio between the mean value of the seedling trait under upland and flooding conditions was used for assessing drought tolerance. A total of six QTLs for drought tolerance were detected. Comparative analysis was performed for the QTLs detected in this case and those reported from two other populations with the same upland rice variety Azucena as parent. Several identical QTLs for seedling elongation across the three populations with the positive alleles from the upland rice Azucena were detected, which suggests that the alleles of Azucena might be involved in water stress-accelerated elongation of rice under different genetic backgrounds. Five cell wall-related candidate genes for OsEXP1, OsEXP2, OsEXP4, EXT, and EGase were mapped on the intervals carrying the QTLs for seedling traits.