Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Proteomic analysis of salt stress-responsive proteins in rice root

Salt stress is one of the major abiotic stresses in agriculture worldwide. We report here a systematic proteomic approach to investigate the salt stress-responsive proteins in rice (Oryza sativa L. cv. Nipponbare). Three-week-old seedlings were treated with 150 mM NaCl for 24, 48 and 72 h. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis. More than 1100 protein spots were reproducibly detected, including 34 that were up-regulated and 20 down-regulated. Mass spectrometry analysis and database searching helped us to identify 12 spots representing 10 different proteins. Three spots were identified as the same protein, enolase. While four of them were previously confirmed as salt stress-responsive proteins, six are novel ones, i.e. UDP-glucose pyrophosphorylase, cytochrome c oxidase subunit 6b-1, glutamine synthetase root isozyme, putative nascent polypeptide associated complex alpha chain, putative splicing factor-like protein and putative actin-binding protein. These proteins are involved in regulation of carbohydrate, nitrogen and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. This study gives new insights into salt stress response in rice roots and demonstrates the power of the proteomic approach in plant biology studies.     

Differential regulation of aquaporins, small GTPases and V-ATPases proteins in rice leaves subjected to drought stress and recovery

Mechanisms of drought tolerance are complex, interacting, and polygenic. This paper describes patterns of gene expression at precise physiological stages of drought in 35-day-old seedlings of Oryza sativa cv. Nipponbare. Drought was imposed gradually for up to 15 days, causing abscisic acid levels to rise and growth to cease, and plants were then re-watered. Proteins were identified from leaf samples after moderate drought, extreme drought, and 3 and 6 days of re-watering. Label-free quantitative shotgun proteomics resulted in identification of 1548 non-redundant proteins. More proteins were down-regulated in early stages of drought but more were up-regulated as severe drought developed. After re-watering, there was notable down regulation, suggesting that stress-related proteins were being degraded. Proteins involved in signalling and transport became dominant as severe drought took hold but decreased again on re-watering. Most of the nine aquaporins identified were responsive to drought, with six decreasing rapidly in abundance as plants were re-watered. Nine G-proteins appeared in large amounts during severe drought and dramatically degraded once plants were re-watered. We speculate that water transport and drought signalling are critical elements of the overall response to drought in rice and might be the key to biotechnological approaches to drought tolerance.     

Physiological and genetic analyses of aluminium tolerance in rice, focusing on root growth during germination

Aluminium (Al) ion limits root growth of plants in acidic soils, and rice exhibits the highest level of Al-tolerance among graminous crops. To elucidate Al-tolerance mechanisms in rice, response to Al was compared between rice (Oryza sativa L., cv. Nipponbare) and wheat (Triticum aestivum L., cv. ET8), focusing on seminal root growth at seedling stage and germination stage. At seedling stage, rice and wheat were similarly sensitive to Al in both dose- and time-dependent manner during a 24-h Al exposure. On the contrary, at germination stage, rice was more tolerant to Al than wheat, and wheat roots displayed the loss of plasma membrane integrity more extensively than rice. A rice mutant exhibiting Al hypersensitivity at germination stage was obtained by screening 42,840 R2 progeny derived from the regenerated R0 plants of Nipponbare and thereafter confirmation of the mutant phenotype in R3 progeny. At germination stage, root growth of the mutant was strongly inhibited in the presence of Al but not in the absence of Al. However, at seedling stage, root growth of the mutant and wild type was similarly tolerant to Al. Taken together, we conclude that rice possesses Al-tolerant function that is under genetic control and specifically operates for root growth at germination stage, making rice more tolerant to Al than wheat.     

Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa

MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important regulatory roles in both animals and plants, miRNA genes have been intensively studied in animals, but not in plants. In this study, we adopted a homology search approach to identify homologs of previously validated plant miRNAs in Arabidopsis thaliana and Oryza sativa. We identified 20 potential miRNA genes in Arabidopsis and 40 in O. sativa, providing a relatively complete enumeration of family members for these miRNAs in plants. In addition, a greater number of Arabidopsis miRNAs (MIR168, MIR159 and MIR172) were found to be conserved in rice. With the novel homologs, most of the miRNAs have closely related fellow miRNAs and the number of paralogs varies in the different miRNA families. Moreover, a probable functional segment highly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family was identified. These results support a model of variegated miRNA regulation in plants, in which miRNAs with different functional elements on their pre-miRNA fold-backs can differ in their function or regulation, and closely related miRNAs can be diverse in their specificity or competence to downregulate target genes. It appears that the sophisticated regulation of miRNAs can achieve complex biological effects through qualitative and quantitative modulation of gene expression profiles in plants.     

Physiological, genetic, and molecular characterization of a high-Cd-accumulating rice cultivar, Jarjan

Cadmium (Cd) in rice is a major source of Cd intake for people on a staple rice diet. The mechanisms underlying Cd accumulation in rice plant are still poorly understood. Here, we characterized the physiology and genetics of Cd transport in a high-Cd-accumulating cultivar (Jarjan) of rice (Oryza sativa). Jarjan showed 5- to 34-fold higher Cd accumulation in the shoots and grains than the cultivar Nipponbare, when it was grown in either a non-Cd-contaminated or a Cd-contaminated soil. A short-term uptake experiment showed no significant difference in Cd uptake by the roots between the two cultivars. However, Jarjan translocated 49% of the total Cd taken up to the shoots, whereas Nipponbare retained most of the Cd in the roots. In both concentration- and time-dependent experiments, Jarjan showed a superior capacity for root-to-shoot translocation of Cd. These results indicate that the high-Cd-accumulation phenotype in Jarjan results from efficient translocation of Cd from roots to shoots. Genetic analysis using an F(2) population derived from Jarjan and Nipponbare revealed that plants showing high- and low-Cd-accumulation phenotypes segregated in a 1:3 ratio, indicating that high accumulation in Jarjan is controlled by a single recessive gene. Furthermore, we isolated OsHMA3, a gene encoding a tonoplast-localized Cd transporter from Jarjan. The OsHMA3 protein was localized in all roots cells, but the sequence has a mutation leading to loss of function. Therefore, failure to sequester Cd into the root vacuoles by OsHMA3 is probably responsible for high Cd accumulation in Jarjan.     

Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings

Changes in abscisic acid (ABA) contents in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl2, the ABA content rapidly increased in the leaves and roots of Cd-tolerant cultivar (cv. Tainung 67, TNG67) but not in the Cd-sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 caused by Cd was less than that of TNG67. Exogenous application of ABA reduced transpiration rate, decreased Cd content, and enhanced Cd tolerance of TN1 seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone, reduced ABA accumulation, increased transpiration rate and Cd content, and decreased Cd tolerance of TNG67 seedlings. Fluridone effect on Cd toxicity of TNG67 seedlings was reversed by the application of ABA. The roles of endogenous ABA in Cd tolerance of rice seedlings are discussed and suggested.     

Inheritance of Oryza sativa endornavirus in F1 and F2 hybrids between japonica and indica rice

We have found a 14 kbp double-stranded RNA (dsRNA) in many cultivars of japonica rice (Oryza sativa L.) but not in any cultivars of indica rice. This dsRNA is an RNA replicon with plasmid-like properties and is proposed to be a novel dsRNA virus, Oryza sativa endornavirus (OSV). Reciprocal crosses between the OSV-carrier japonica variety (Nipponbare) and the OSV-free indica variety (IR 26 or Kasalath) were performed to investigate whether OSV can be transmitted to F1 hybrids. When IR 26 and Nipponbare were used, efficient transmission of OSV from ova (93%) and pollen (89%) was observed. When Kasalath and Nipponbare were used, the OSV transmission efficiency to F1 progeny was 68% from ova and 20% from pollen. The transmission of OSV to F2 progeny plants was also complicated, showing non-Mendelian inheritance. These results suggest that the dsRNA replicon (OSV) is unstable in indica rice plants.     

Analysis of collinear regions of Oryza AA and CC genomes

Comparative analyses of genome structure and sequence of closely related species have yielded insights into the evolution and function of plant genomes. A total of 103,844 BAC end sequences delegated -73.8 Mb of O. officinalis that belongs to the CC genome type of the rice genus Oryza were obtained and compared with the genome sequences office cultivar, O. sativa ssp.japonica cv. Nipponbare. We found that more than 45% of O. officinalis genome consists of repeat sequences, which is higher than that of Nipponbare cultivar. To further investigate the evolutionary divergence of AA and CC genomes, two BAC-contigs of O. officinalis were compared with the collinear genomic regions of Nipponbare. Of 57 genes predicted in the AA genome orthologous regions, 39 had orthologs in the regions of the CC genome. Alignment of the orthologous regions indicated that the CC genome has undergone expansion in both genic and intergenic regions through primarily retroelement insertion. Particularly, the density of RNA transposable elements was 17.95% and 1.78% in O. officinalis and O. sativa, respectively. This explains why the orthologous region is about 100 kb longer in the CC genome in comparison to the AA genome.     

Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.