Starch Properties and Morphology of Eight Floury Endosperm Mutants in Rice.

Besides increasing grain yield, improving rice (Oryza sativa L.) quality has been paid more and more attention recently. Cooking and eating quality (CEQ) is an important indicator of rice quality. Since CEQs are quantitative traits and challenging for measurement, efforts have mainly focused on two major genes, Wx and SSIIa. Chalkiness and floury endosperm significantly affect the eating quality of rice, leading to noticeable changes in CEQ. Due to the easily observable phenotype of floury endosperm, cloning single gene mutations that cause floury endosperm and evaluating changes in CEQs indirectly facilitate the exploration of the minor genes controlling CEQ. In this study, eight mutants with different degrees of floury endosperm, generated through ethylmethane sulfonate (EMS) mutagenesis, were analyzed. These mutants exhibited wide variation in starch morphology and CEQs. Particularly, the z2 mutant showed spherical starch granules significantly increased rapid visco analyzer (RVA) indexes and urea swelling, while the z4 mutant displayed extremely sharp starch granules and significantly decreased RVA indexes and urea swelling compared to the wild type. Additionally, these mutants still maintained correlations with certain RVA profiles, suggesting that the genes PUL, which affect these indexes, may not undergo mutation. Cloning these mutated genes in the future, especially in z2 and z4, will enhance the genetic network of rice eating quality and hold significant importance for molecular marker-assisted breeding to improve rice quality.

Ethanol suppresses rice seed germination through inhibiting ROS signaling.

Ethanol is frequently used not only as priming but also as a solvent to dissolve hardly water-soluble phytohormones gibberellic acid (GA3) and abscisic acid (ABA) in seed germination. However, the molecular and physiological mechanisms of ethanol's impact on seed germination remain elusive. In this report, we investigated how ethanol affected reactive oxygen species (ROS) during rice seed germination. Ethanol at a concentration of 3.5% (v/v) inhibited 90% seed germination, which was almost reversed by H2O2. H2O2 contents in embryos were reduced by ethanol after 18 h imbibition. Antioxidant enzymes assays revealed that only superoxide dismutase (SOD) activities in seed embryos were lowered by ethanol, in line with the suppressed mRNA expression of SOD genes during imbibition. Additionally, compared to the mock condition, ethanol increased ABA contents but decreased GA (GA1 and GA3) in seed embryos, resulting in disharmonizing GA/ABA balance. Conceivably ethanol induced transcription of OsNCEDs, the key genes for ABA biosynthesis, and OsABA8ox3, a key gene for ABA catabolism. Furthermore, ethanol promoted ABA signaling by upregulating ABA receptor genes and ABA-responsive element (ABRE)-binding protein/ABRE-binding factors during imbibition. Overall, our results demonstrate that lowering of H2O2 levels due to suppressed SOD activities in rice germinating seed embryos is the decisive factor for ethanol-induced inhibition of seed germination, and GA/ABA balance and ABA signaling also play important roles in ethanol's inhibitory impact on seed germination.

The role of PPR proteins in posttranscriptional regulation of organelle components in plants.

Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants. They are sequence-specific RNA-binding proteins and play key roles in posttranscriptional processes within organelles. Their combined actions have profound effects on chloroplast photosynthetic electron transport chain and mitochondrial respiratory chain, affecting photosynthesis and respiration respectively, and ultimately on yield, fertility, and grain quality. Over the past decade, much has been learned about the molecular functions of these proteins on plant growth and development. However, due to the large size of this protein family, the functions of most members remain largely unknown. Here, we summarize the molecular mechanisms of PPR proteins functions on organelle genes, and effects on development of organelles and plants. Problems that need to be resolved are also identified. This article will provide a theoretical basis for understanding the functions of PPR protein family and genetic improvements of grain yield and quality.

Disruption of a Upf1-like helicase-encoding gene OsPLS2 triggers light-dependent premature leaf senescence in rice.

KEY MESSAGE: The OsPLS2 locus was isolated and cloned by map-based cloning that encodes a Upf1-like helicase. Disruption of OsPLS2 accelerated light-dependent leaf senescence in the rice mutant of ospls2. Leaf senescence is a very complex physiological process controlled by both genetic and environmental factors, however its underlying molecular mechanisms remain elusive. In this study, we report a novel Oryza sativa premature leaf senescence mutant (ospls2). Through map-based cloning, a G-to-A substitution was determined at the 1st nucleotide of the 13th intron in the OsPLS2 gene that encodes a Upf1-like helicase. This mutation prompts aberrant splicing of OsPLS2 messenger and consequent disruption of its full-length protein translation, suggesting a negative role of OsPLS2 in regulating leaf senescence. Wild-type rice accordingly displayed a progressive drop of OsPSL2 protein levels with age-dependent leaf senescence. Shading and light filtration studies showed that the ospls2 phenotype, which was characteristic of photo-oxidative stress and reactive oxygen species (ROS) accumulation, was an effect of irritation by light. When continuously exposed to far-red light, exogenous H2O2 and/or abscisic acid (ABA), the ospls2 mutant sustained hypersensitive leaf senescence. In consistence, light and ROS signal pathways in ospls2 were activated by down-regulation of phytochrome genes, and up-regulation of PHYTOCHROME-INTERACTING FACTORS (PIFs) and WRKY genes, all promoting leaf senescence. Together, these data indicated that OsPLS2 played an essential role in leaf senescence and its disruption triggered light-dependent leaf senescence in rice.

A 22-bp deletion in OsPLS3 gene encoding a DUF266-containing protein is implicated in rice leaf senescence.

Key message The OsPLS3 locus was isolated by map-based cloning that encodes a DUF266-containing protein. OsPLS3 regulates the onset of leaf senescence in rice. Glycosyltransferases (GTs) are one of the most important enzyme groups required for the modification of plant secondary metabolites and play a crucial role in plant growth and development, however the biological functions of most GTs remain elusive. We reported here the identification and characterization of a novel Oryza sativa premature leaf senescence mutant (ospls3). Through map-based cloning strategy, we determined that 22-bp deletion in the OsPLS3 gene encoding a domain of unknown function 266 (DUF266)-containing protein, a member of GT14-like, underlies the premature leaf senescence phenotype in the ospls3 mutant. The OsPLS3 mRNA levels progressively declined with the age-dependent leaf senescence in wild-type rice, implying a negative role of OsPLS3 in regulating leaf senescence. Physiological analysis, and histochemical staining and transmission electron microscopy assays indicated that the ospls3 mutant accumulated higher levels of ethylene and reactive oxygen species than its wild type. Furthermore, the ospls3 mutant showed hypersensitivity to exogenous 1-aminocyclopropane-1-carboxylic acid, H2O2 and high level of cytokinins. Our results indicated that the DUF266-containing gene OsPLS3 plays an important role in the onset of leaf senescence, in part through cytokinin and ethylene signaling in rice.

Involvement of CAT in the detoxification of HT-induced ROS burst in rice anther and its relation to pollen fertility.

KEY MESSAGE: HT-induced ROS burst in developing anther is closely related to the lowered CAT activity as the result of the markedly suppressed OsCATB transcript, thereby causing severe fertility injury for rice plants exposed to HT at meiosis stage. The reproductive stage of rice plants is highly sensitive to heat stress. In this paper, different rice cultivars were used to investigate the relationship of HT-induced floret sterility with reactive oxygen species (ROS) detoxification in rice anthers under well-controlled climatic conditions. Results showed that high temperature (HT) exposure significantly enhanced the ROS level and malondialdehyde (MDA) content in developing anther, and the increase in ROS amount in rice anther under HT exposure was closely associated with HT-induced decline in the activities of several antioxidant enzymes. For various antioxidant enzymes, SOD and CAT were more susceptible to the ROS burst in rice anther induced by HT exposure than APX and POD, in which SOD and CAT activity in developing anther decreased significantly by HT exposure, whereas APX activity was relatively stable among different temperature regimes. HT-induced decrease in CAT activity was attributable to the suppressed transcript of OsCATB. This occurrence was strongly responsible for HT-induced increase in ROS level and oxidative-damage in rice anther, thereby it finally caused significant reduction in pollen viability and floret fertility for the rice plants exposed to HT during meiosis. Exogenous application of 1000 µM salicylic acid (SA) may alleviate HT-induced reduction in pollen viability and floret fertility, concomitantly with the increased CAT activity and reduced ROS level in rice anther.

Relationship of ROS accumulation and superoxide dismutase isozymes in developing anther with floret fertility of rice under heat stress.

High temperature (HT) at meiosis stage is one of most important environment constraint affecting spikelet fertility and rice yield. In this paper, the effects of HT exposure at meiosis stage on the ROS (reactive oxygen species) accumulation, various superoxide dismutase (SOD, EC1.15.1.11) isozymes in developing anther, and its relationship with HT-induced decline in pollen viability and floret fertility were investigated by using four rice cultivars differing in heat tolerance under well-controlled climatic condition. Results showed that HT exposure significantly increased ROS level and malondialdehyde (MDA) content in rice anther, and this occurrence was strongly responsible for the HT-induced decline in pollen viability and harmful effect of HT adversity on floret fertility. However, the increased extent of ROS concentration in rice anther under HT exposure was greatly variable, depending on both the intensity and duration of HT exposure and different rice cultivars used. The SOD and CAT activities of HT-sensitive cultivars decreased more profoundly than those of HT-tolerant cultivars under the same HT regimes. Among various types of SOD enzymes, Cu/Zn-SODa expressed highly in rice anther and responded sensitively to HT exposure, while Cu/Zn-SODb expressed weakly in rice anther and preferentially in rice leaves. HT exposure suppressed the expression of Cu/Zn-SODa in developing anther, which was closely associated with the down-regulated transcripts of cCu/Zn-SOD1 gene. Hence, Cu/Zn-SODa may play a central role in the regulation of total SOD activity and ROS detoxification in rice anther as affected by HT exposure at meiosis stage.

A single cytosine deletion in the OsPLS1 gene encoding vacuolar-type H+-ATPase subunit A1 leads to premature leaf senescence and seed dormancy in rice.

Leaf senescence is a programmed developmental process orchestrated by many factors, but its molecular regulation is not yet fully understood. In this study, a novel Oryza sativa premature leaf senescence mutant (ospls1) was examined. Despite normal development in early seedlings, the ospls1 mutant leaves displayed lesion-mimics and early senescence, and a high transpiration rate after tillering. The mutant also showed seed dormancy attributable to physical (defect of micropyle structure) and physiological (abscisic acid sensitivity) factors. Using a map-based cloning approach, we determined that a cytosine deletion in the OsPLS1 gene encoding vacuolar H(+)-ATPase subunit A1 (VHA-A1) underlies the phenotypic abnormalities in the ospls1 mutant. The OsPSL1/VHA-A1 transcript levels progressively declined with the age-dependent leaf senescence in both the ospls1 mutant and its wild type. The significant decrease in both OsPSL1/VHA-A1 gene expression and VHA enzyme activity in the ospls1 mutant strongly suggests a negative regulatory role for the normal OsPLS1/VHA-A1 gene in the onset of rice leaf senescence. The ospls1 mutant featured higher salicylic acid (SA) levels and reactive oxygen species (ROS) accumulation, and activation of signal transduction by up-regulation of WRKY genes in leaves. Consistent with this, the ospls1 mutant exhibited hypersensitivity to exogenous SA and/or H2O2 Collectively, these results indicated that the OsPSL1/VAH-A1 mutation played a causal role in premature leaf senescence through a combination of ROS and SA signals. To conclude, OsPLS1 is implicated in leaf senescence and seed dormancy in rice.

Small brown planthopper resistance loci in wild rice (Oryza officinalis).

Host-plant resistance is the most practical and economical approach to control the rice planthoppers. However, up to date, few rice germplasm accessions that are resistant to the all three kinds of planthoppers (1) brown planthopper (BPH; Nilaparvata lugens Stål), (2) the small brown planthopper (SBPH; Laodelphax striatellus Fallen), and (3) the whitebacked planthopper (WBPH, Sogatella furcifera Horvath) have been identified; consequently, the genetic basis for host-plant broad spectrum resistance to rice planthoppers in a single variety has been seldom studied. Here, one wild species, Oryza officinalis (Acc. HY018, 2n = 24, CC), was detected showing resistance to the all three kinds of planthoppers. Because resistance to WBPH and BPH in O. officinalis has previously been reported, the study mainly focused on its SBPH resistance. The SBPH resistance gene(s) was (were) introduced into cultivated rice via asymmetric somatic hybridization. Three QTLs for SBPH resistance detected by the SSST method were mapped and confirmed on chromosomes 3, 7, and 12, respectively. The allelic/non-allelic relationship and relative map positions of the three kinds of planthopper resistance genes in O. officinalis show that the SBPH, WBPH, and BPH resistance genes in O. officinalis were governed by multiple genes, but not by any major gene. The data on the genetics of host-plant broad spectrum resistance to planthoppers in a single accession suggested that the most ideally practical and economical approach for rice breeders is to screen the sources of broad spectrum resistance to planthoppers, but not to employ broad spectrum resistance gene for the management of planthoppers. Pyramiding these genes in a variety can be an effective way for the management of planthoppers.

Expression patterns of defense genes in resistance of the panicles exserted from the caulis and from the tillers to neck blast in rice.

The rice variety Xiushui227 is resistant to neck blast in the panicles exserted from the caulis but susceptible in the panicles from the tillers, however, the other variety Xiushui09 is susceptible to neck blast in the panicles from the caulis but resistant in the panicles from the tillers. These two varieties were used to analyze the expression patterns of defense genes in the panicles from the caulis and the first first-class tiller at the preliminary heading stage, after inoculating the necks in vitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-ß-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1 and PR5 could participate in defending Xiushui227 against neck blast in the panicles from the caulis. All defense genes used in this study except Cht-1, PR10a, and PPO may play roles in defending Xiushui09 against neck blast in the panicles from the tillers.

Expression of defense genes and antioxidant defense responses in rice resistance to neck blast at the preliminary heading stage and full heading stage.

The rice variety Xiushui227 is resistant to neck blast at three crucial panicle stages (the booting stage, the preliminary heading stage (PHS), and the full heading stage (FHS)) that controlling neck blast. The other rice variety Liangyou6326 is susceptible to neck blast at these three panicle stages. These two varieties were used to analyze the expression patterns of defense genes and antioxidant defense responses at the PHS and FHS, after inoculating the necks in vitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-ß-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1, CHS and PPO could mainly play important roles in the resistance to neck blast at the PHS in Xiushui227, and CHS and PPO may primarily participate in fighting back against to neck blast at the FHS. Conversely, PR1a, Cht-1, PR4, PR10a, CHS, LOX-RLL, and PPO could chiefly play participate in defending Liangyou6326 against neck blast at the FHS, and PR5, PAL, and POX22.3 may be primarily involved in fighting back against to neck blast at the PHS. Furthermore, the antioxidant enzymes superoxide dismutase, peroxidase, and catalase may mainly participate in defending Xiushui227 against neck blast at the PHS and defending Liangyou6326 against neck blast at the FHS, respectively. Therefore oxidative damage is less at the PHS in Xiushui227 and at the FHS in Liangyou6326, respectively.