Catalase associated with antagonistic changes of abscisic acid and gibberellin response, biosynthesis and catabolism is involved in eugenol-inhibited seed germination in rice.

KEY MESSAGE: The inhibitory effect of eugenol on rice germination is mediated by a two-step modulatory process: Eugenol first regulates the antagonism of GA and ABA, followed by activation of catalase activity. The natural monoterpene eugenol has been reported to inhibit preharvest sprouting in rice. However, the inhibitory mechanism remains obscure. In this study, simultaneous monitoring of GA and ABA responses by the in vivo GA and ABA-responsive dual-luciferase reporter system showed that eugenol strongly inhibited the GA response after 6 h of imbibition, whereas eugenol significantly enhanced the ABA response after 12 h of imbibition. Gene expression analysis revealed that eugenol significantly induced the ABA biosynthetic genes OsNCED2, OsNCED3, and OsNCED5, but notably suppressed the ABA catabolic genes OsABA8ox1 and OsABA8ox2. Conversely, eugenol inhibited the GA biosynthetic genes OsGA3ox2 and OsGA20ox4 but significantly induced the GA catabolic genes OsGA2ox1 and OsGA2ox3 during imbibition. OsABI4, the key signaling regulator of ABA and GA antagonism, was notably induced before 12 h and suppressed after 24 h by eugenol. Moreover, eugenol markedly reduced the accumulation of H2O2 in seeds after 36 h of imbibition. Further analysis showed that eugenol strongly induced catalase activity, protein accumulation, and the expression of three catalase genes. Most importantly, mitigation of eugenol-inhibited seed germination was found in the catc mutant. These findings indicate that catalase associated with antagonistic changes of ABA and GA is involved in the sequential regulation of eugenol-inhibited seed germination in rice.

CFM6 is an Essential CRM Protein Required for the Splicing of nad5 Transcript in Arabidopsis Mitochondria.

Plant chloroplast RNA splicing and ribosome maturation (CRM)-domain-containing proteins are capable of binding RNA to facilitate the splicing of group I or II introns in chloroplasts, but their functions in mitochondria are less clear. In the present study, Arabidopsis thaliana CFM6, a protein with a single CRM domain, was expressed in most plant tissues, particularly in flower tissues, and restricted to mitochondria. Mutation of CFM6 causes severe growth defects, including stunted growth, curled leaves, delayed embryogenesis and pollen development. CFM6 functions specifically in the splicing of group II intron 4 of nad5, which encodes a subunit of mitochondrial complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in cfm6 also led to the loss of complex I activity and to its improper assembly. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.

The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Transcriptome analysis reveals potential roles of a barley ASR gene that confers stress tolerance in transgenic rice.

Control of gene expression and induction of cellular protection mechanisms are two important processes that plants employ to protect themselves against abiotic stresses. ABA-, stress, and ripening-induced (ASR) proteins have been identified to participate in such responses. Previous studies have proposed that these proteins can act as transcription factors and as molecular chaperones protecting transgenic plants against stresses; however a gene network regulated by ASRs has not been explored. To expand our knowledge on the function of these proteins in cereals, we present the functional characterization of a barley ASR gene. Expression of HvASR5 was almost ubiquitous in different organs and responded to ABA and to different stress treatments. When expressed ectopically, HvASR5 was able to confer drought and salt stress tolerance to Arabidopsis thaliana and to improve growth performance of rice plants under stress conditions. A transcriptomic analysis with two transgenic rice lines overexpressing HvASR5 helped to identify potential downstream targets and understand ASR-regulated cellular processes. HvASR5 up-regulated the expression of a distinct set of genes associated with stress responses, metabolic processes (particularly carbohydrate metabolism), as well as reproduction and development. These data, together with the confirmed nuclear and cytoplasmic localization of HvASR5, further support the hypothesis that HvASR5 can also carry out roles as molecular protector and transcriptional regulator.

The transcription factor OsbHLH035 mediates seed germination and enables seedling recovery from salt stress through ABA-dependent and ABA-independent pathways, respectively.

BACKGROUND: Many transcription factors (TFs), such as those in the basic helix-loop-helix (bHLH) family, are important for regulating plant growth and plant responses to abiotic stress. The expression of OsbHLH035 is induced by drought and salinity. However, its functional role in rice growth, development, and the salt response is still unknown. RESULTS: The bHLH TF OsbHLH035 is a salt-induced gene that is primarily expressed in germinating seeds and seedlings. Stable expression of GFP-fused OsbHLH035 in rice transgenic plants revealed that this protein is predominantly localized to the nucleus. Osbhlh035 mutants show delayed seed germination, particularly under salt-stress conditions. In parallel, abscisic acid (ABA) contents are over-accumulated, and the expression of the ABA biosynthetic genes OsABA2 and OsAAO3 is upregulated; furthermore, compared with that in wild-type (WT) seedlings, the salt-induced expression of OsABA8ox1, an ABA catabolic gene, in germinating Osbhlh035 mutant seeds is downregulated. Moreover, Osbhlh035 mutant seedlings are unable to recover from salt-stress treatment. Consistently, sodium is over-accumulated in aerial tissues but slightly reduced in terrestrial tissues from Osbhlh035 seedlings after salt treatment. Additionally, the expression of the sodium transporters OsHKT1;3 and 1;5 is reduced in Osbhlh035 aerial and terrestrial tissues, respectively. Furthermore, genetic complementation can restore both the delayed seed germination and the impaired recovery of salt-treated Osbhlh035 seedlings to normal growth. CONCLUSION: OsbHLH035 mediates seed germination and seedling recovery after salt stress relief through the ABA-dependent and ABA-independent activation of OsHKT pathways, respectively.

Multiple Patterns of Regulation and Overexpression of a Ribonuclease-Like Pathogenesis-Related Protein Gene, OsPR10a, Conferring Disease Resistance in Rice and Arabidopsis.

An abundant 17 kDa RNase, encoded by OsPR10a (also known as PBZ1), was purified from Pi-starved rice suspension-cultured cells. Biochemical analysis showed that the range of optimal temperature for its RNase activity was 40-70°C and the optimum pH was 5.0. Disulfide bond formation and divalent metal ion Mg2+ were required for the RNase activity. The expression of OsPR10a::GUS in transgenic rice was induced upon phosphate (Pi) starvation, wounding, infection by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), leaf senescence, anther, style, the style-ovary junction, germinating embryo and shoot. We also provide first evidence in whole-plant system, demonstrated that OsPR10a-overexpressing in rice and Arabidopsis conferred significant level of enhanced resistance to infection by the pathogen Xoo and Xanthomona campestris pv. campestris (Xcc), respectively. Transgenic rice and Arabidopsis overexpressing OsPR10a significantly increased the length of primary root under phosphate deficiency (-Pi) condition. These results showed that OsPR10a might play multiple roles in phosphate recycling in phosphate-starved cells and senescing leaves, and could improve resistance to pathogen infection and/or against chewing insect pests. It is possible that Pi acquisition or homeostasis is associated with plant disease resistance. Our findings suggest that gene regulation of OsPR10a could act as a good model system to unravel the mechanisms behind the correlation between Pi starvation and plant-pathogen interactions, and also provides a potential application in crops disease resistance.

Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice.

BACKGROUND: Anthocyanin accumulates in many plant tissues or organs, in rice for example leading to red, purple red and purple phenotypes for protection from damage by biotic and abiotic stresses and for reproduction. Purple leaf, leaf sheath, stigma, pericarp, and apiculus are common in wild rice and landraces and occasionally found in modern cultivars. No gene directly conferring anthocyanin deposited in a purple leaf sheath has yet been isolated by using natural variants. An F2 population derived from ssp. japonica cv. Tainung 72 (TNG72) with purple leaf sheath (PSH) crossed with ssp. indica cv. Taichung Sen 17 (TCS17) with green leaf sheath (GSH) was utilized to isolate a gene conferring leaf sheath color. RESULTS: By positional cloning, 10-and 3-bp deletions in the R2R3 Myb domain of OsC1 were uncovered in GSH varieties TCS17 and Nipponbare, respectively. Allelic diversity, rather than gene expression levels of OsC1, might be responsible for anthocyanin accumulation. Parsimony-based analysis of genetic diversity in 50 accessions, including cultivars, landraces, and A-genome wild rice, suggests that independent mutation occurred in Asian, African, South American, and Australian species, while O. meridionalis had a divergent sequence. OsC1 was thought of as a domestication related gene, with up to 90 % reduction of genetic diversity in GSH; however, no values from three tests showed significant differences from neutral expectations, implying that OsC1 had not been subjected to recent selection. Haplotype network analysis revealed that species from different continents formed unique haplotypes with no gene flow. Two major groups of haplotypes corresponding to 10-bp deletion and other sequences were formed in Asian rice, including O. rufipogon, O. nivara and O. sativa. Introgressions of OsC1 between subspecies through natural and artificial hybridization were not rare. Because artificial and natural selection imposed admixture on rice germplasm in Taiwan, the genealogy of OsC1 might not be congruent with the current distribution of alleles through lineage diversification. CONCLUSION: OsC1 is responsible for purple leaf sheath, and much new information about OsC1 is provided e.g., new alleles, non-domestication syndrome, and incongruence of genealogy with geographic distribution.

A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

Early life exposure to a rodent carcinogen propiconazole fungicide induces oxidative stress and hepatocarcinogenesis in medaka fish.

Conazole pollution is an emerging concern to human health and environmental safety because of the broad use of conazole fungicides in agriculture and medicine and their frequent occurrence in aquifers. The agricultural pesticide propiconazole has received much regulatory interest because it is a known rodent carcinogen with evidence of multiple adverse effects in mammals and non-targeted organisms. However, the carcinogenic effect and associated mechanism of propiconazole in fish under microgram-per-liter levels of environmental-relevant exposure remains unclear. To explore whether early life of propiconzaole exposure would induce oxidative stress and latent carcinogenic effects in fish, we continuously exposed larvae of wild type or p53(-/-) mutant of medaka fish (Oryzias latipes) to propiconazole (2.5-250µg/L) for 3, 7, 14 or 28 days and assessed liver histopathology and/or the oxidative stress response and gene expression during exposure and throughout adulthood. Propiconazole dose-dependently induced reactive oxygen species (ROS) level, altered homeostasis of antioxidant superoxide dismutase, catalase and glutathione S-transferase and caused lipid and protein peroxidation during early life exposure in wild type medaka. Such exposure also significantly upregulated gene expression of the cytochrome P450 CYP1A, but marginally suppressed that of tumor suppressor p53 in adults. Furthermore, histopathology revealed that p53(-/-) mutant medaka with early life exposure to propiconazole showed increased incidence of hepatocarcionogensis, as compared to the p53(-/-) control group and wild type strain. We demonstrated that propiconazole can initiate ROS-mediated oxidative stress and induce hepatic tumorigenesis associated with CYP1A- and/or p53 -mediated pathways with the use of wild type and p53(-/-) mutant of medaka fish. The toxic response of medaka to propiconazole is compatible with that observed in rodents.

Hygromycin B-induced cell death is partly mediated by reactive oxygen species in rice (Oryza sativa L.).

The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O2(·-), H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.

Gene knockout of glutathione reductase 3 results in increased sensitivity to salt stress in rice.

Glutathione reductase (GR) is one of important antioxidant enzymes in plants. This enzyme catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) with the accompanying oxidation of NADPH. Previously, we showed that salt-stress-responsive GR3 is a functional protein localized in chloroplasts and mitochondria in rice. To learn more about the role of GR3 in salt-stress tolerance, we investigated the response to 100 mM NaCl treatment in wild-type rice (WT); GR3 knockout mutant of rice (gr3); and the functional gr3-complementation line (C1). Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitously expressed in all tissues except the sheath at heading stage. GR3 promoter-GUS was expressed in the vascular cylinder and cortex of root tissues in rice seedlings, vascular tissue of nodes, embryo and aleurone layer of seeds, and young flowers. Under both normal and salt-stress conditions, total GR activity was decreased by 20 % in gr3. Oxidative stress, indicated by malondialdehyde content, was greater in gr3 than the WT under salt stress. As compared with the WT, gr3 was sensitive to salt and methyl viologen; it showed inhibited growth, decreased maximal efficiency of photosystem II, decreased GSH and GSSG contents, and the ratio of GSH to GSSG. Conversely, the gr3-complementation line C1 rescued the tolerance to methyl viologen and salinity and recovered the growth and physiological damage caused by salinity. These results reveal that GR3 plays an important role in salt stress tolerance by regulating the GSH redox state in rice.

Three novel alleles of FLOURY ENDOSPERM2 (FLO2) confer dull grains with low amylose content in rice.

Rice is a major food source for much of the world, and expanding our knowledge of genes conferring specific rice grain attributes will benefit both farmer and consumer. Here we present novel dull grain mutants with a low amylose content (AC) derived from mutagenesis of Oryza sativa, ssp. japonica cv. Taikeng 8 (TK8). Positional cloning of the gene conferring the dull grain phenotype revealed a point mutation located at the acceptor splice site of intron 11 of FLOURY ENDOSPERM2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein. Three novel flo2 alleles were identified herein, which surprisingly conferred dull rather than floury grains. The allelic diversity of flo2 perturbed the expression of starch synthesis-related genes including OsAGPL2, OsAGPS2b, OsGBSSI, OsBEI, OsBEIIb, OsISA1, and OsPUL. The effect of the flo2 mutations on the physicochemical properties of the grain included a low breakdown, setback, and consistency of rice, indicating a good elasticity and soft texture of cooked rice grains. The effects of FLO2, combined with the genetic background of the germplasm and environmental effects, resulted in a variety of different amylose content levels, grain appearance, and physicochemical properties of rice, providing a host of useful information to future grain-quality research and breeding.

A novel soybean (Glycine max) gene encoding a family 3 ß-glucosidase has high isoflavone 7-O-glucoside-hydrolyzing activity in transgenic rice.

A previous study demonstrated that purified Glycine max ß-glucosidase (GmBGL) could hydrolyze glucosyl isoflavone to the aglyconic form. This study reports the cloning and functional characterization of a soybean cDNA encoding the ß-glucosidase. GmBGL was isolated by use of a purified soybean N-terminal amino acid sequence and conserved sequences of ß-glucosidase genes from other plants. Sequence analysis of GmBGL revealed an open reading frame of 1884 bp encoding a polypeptide of 627 amino acids with a calculated molecular mass of 69 kDa. Phylogenetic analysis classified the GmBGL into the glycosyl hydrolase 3 family. In soybean, the GmBGL transcript was predominantly accumulated in roots and leaves. To examine the enzymatic activity and substrate specificity, GmBGL was ectopically expressed in transgenic rice. Purified GmBGL protein from transgenic rice could catalyze the hydrolysis of genistin and daidzin to produce genistein and daidzein, respectively, which confirmed GmBGL as a functional ß-glucosidase with isoflavone glucoside-hydrolyzing activity. This paper reveals that GmBGL is a key enzyme in transforming glucosyl isoflavones to aglycones in soybean, which may help in genetic manipulation of aglycone-rich soybean seeds.

Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening.

By oligo microarray expression profiling, we identified a rice RING zinc-finger protein (RZFP), OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. As compared with the wild type, rice and Arabidopsis with OsRZFP34 overexpression showed increased relative stomata opening even with ABA treatment. Furthermore, loss-of-function mutation of OsRZFP34 and AtRZFP34 (At5g22920), an OsRZFP34 homolog in Arabidopsis, decreased relative stomata aperture under nonstress control conditions. Expressing OsRZFP34 in atrzfp34 reverted the mutant phenotype to normal, which indicates a conserved molecular function between OsRZFP34 and AtRZFP34. Analysis of water loss and leaf temperature under stress conditions revealed a higher evaporation rate and cooling effect in OsRZFP34-overexpressing Arabidopsis and rice than the wild type, atrzfp34 and osrzfp34. Thus, stomata opening, enhanced leaf cooling, and ABA insensitivity was conserved with OsRZFP34 expression. Transcription profiling of transgenic rice overexpressing OsRZFP34 revealed many genes involved in OsRZFP34-mediated stomatal movement. Several genes upregulated or downregulated in OsRZFP34-overexpressing plants were previously implicated in Ca(2+) sensing, K(+) regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.

Divergence of the expression and subcellular localization of CCR4-associated factor 1 (CAF1) deadenylase proteins in Oryza sativa.

Deadenylation, also called poly(A) tail shortening, is the first, rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. The CCR4-NOT complex, containing the two key components carbon catabolite repressor 4 (CCR4) and CCR4-associated factor 1 (CAF1), is a major player in deadenylation. CAF1 belongs to the RNase D group in the DEDD superfamily, and is a protein conserved through evolution from yeast to humans and plants. Every higher plant, including Arabidopsis and rice, contains a CAF1 multigene family. In this study, we identified and cloned four OsCAF1 genes (OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, rOsCAF1G, and rOsCAF1H, all exhibited 3'-5' exonuclease activity in vitro. Point mutations in the catalytic residues of each analyzed recombinant OsCAF1 proteins were shown to disrupt deadenylase activity. OsCAF1A and OsCAF1G mRNA were found to be abundant in the leaves of mature plants. Two types of OsCAF1B mRNA transcript were detected in an inverse expression pattern in various tissues. OsCAF1B was transient, induced by drought, cold, abscisic acid, and wounding treatments. OsCAF1H mRNA was not detected either under normal conditions or during most stress treatments, but only accumulated during heat stress. Four OsCAF1-reporter fusion proteins were localized in both the cytoplasm and nucleus. In addition, when green fluorescent protein fused with OsCAF1B, OsCAF1G, and OsCAF1H, respectively, fluorescent spots were observed in the nucleolus. OsCAF1B fluorescent fusion proteins were located in discrete cytoplasmic foci and fibers. We present evidences that OsCAF1B colocalizes with AtXRN4, a processing body marker, and AtKSS12, a microtubules maker, indicating that OsCAF1B is a component of the plant P-body and associate with microtubules. Our findings provide biochemical evidence that OsCAF1 proteins may be involved in the deadenylation in rice. The unique expression patterns of each OsCAF1 were observed in various tissues when undergoing abiotic stress treatments, implying that each CAF1 gene in rice plays a specific role in the development and stress response of a plant.

Organ- and stress-specific expression of the ASR genes in rice.

KEY MESSAGE: Rice ASR genes respond distinctly to abscisic acid, dehydration and cold stress. Their tissue-specific expression provides new hints about their possible roles in plant responses to stress. Plant ASR proteins have emerged as an interesting distinct group of proteins with apparent roles in protecting cellular structures as well as putative regulators of gene expression, both important responses of plants to environmental stresses. Regardless of the possible functions proposed by different studies, little is known about their role in cereals. To further understand the function of these proteins in the Gramineae, we investigated the expression pattern of the six ASR genes present in the rice genome in response to ABA, stress conditions and in different organs. Although transcription of most OsASRs is transiently enhanced by ABA treatment, the genes present a differential response under cold and drought stress as well as specific expression in certain tissues and organs. Analysis of their promoters reveals regulatory cis-elements associated to hormonal, sugar and stress responses. The promoters of two genes, OsASR1 and OsASR5, direct the expression of the GUS reporter gene especially to leaf vascular tissue in response to dehydration and low temperature. In control conditions, a GUS reporter assay also indicates specific expression of these two genes in roots, anthers and seed scutellar tissues. These results provide new clues about the possible role of ASRs in plant stress responses and development.

Abscisic acid- and stress-induced highly proline-rich glycoproteins regulate root growth in rice.

In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX1PX2 motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane.

Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice.

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53 % identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shock stress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance.