Identification of a β-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.)

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing β-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an ～4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS–PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K_m and V_max values of TAG were 31.7 μM and 0.25 μkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific β-glucosidase hydrolyzing TAG, which releases the physiologically active TA.     

Expression of α-expansin genes in young seedlings of rice (Oryza sativa L.)

 Rice is the only cereal in which germination and coleoptile elongation occur in hypoxia or anoxia. Little is known of the molecular basis directly underlying coleoptile cell extension. In this paper, we describe the expression of α-expansin genes in embryos during seed development and young seedlings grown under various oxygen concentrations. The genes Os-EXP2 and Os-EXP1 were predominantly expressed in the developing seeds, mainly in newly developed leaves, coleoptiles, and seminal roots. These expansins expressed in the developing seeds may give cells the potential to expand after seed imbibition begins. In coleoptiles, Os-EXP4 and Os-EXP2 mRNAs were greatly induced by submergence, while they were weakly detected in aerobic or anoxic conditions. Under submerged soil conditions, the signals hybridized with probes Os-EXP4 and Os-EXP2 in coleoptiles were strongest when coleoptiles elongated in the water layer. These data show that expansin gene expression is highly correlated with coleoptile elongation in response to oxygen concentrations. The Os-EXP4 gene was also expressed in leaves, mesocotyls, and coleorhizas of young seedlings. The growth of these tissues was also correlated with the presence of expansins. Therefore, the evidence derived from this study clearly demonstrates that expansins are indispensable for the growing tissues of rice seedlings. Received: 23 December 1999 / Accepted: 24 February 2000     

Chromosomal localization and genomic organization of α-amylase genes in rice (Oryza sativa L.)

Summary Genes for -amylase, alcohol dehydrogenase, andEm, an ABA-regulated gene expressed late in embryogenesis, were localized on rice chromosomes by the analysis of primary trisomies. The validity of the mapping approach was confirmed usingAdh-1 as a control. TheAdh-1 gene has previously been assigned to chromosome 11 using conventional techniques. In this study we confirm this assignment and report an additional locus for alcohol dehydrogenase (Adh-2) on chromosome 9. The -amylase genes were located on chromosomes 1, 2, 6, 8, and 9 while theEm gene was mapped to chromosome 5. To facilitate trisomic analysis and correlation of cloned genes with bands observed on Southern blots, a nomenclature for the rice -amylase genes has been proposed. In addition to mapping nine cloned -amylase genes, we have identified two previously uncloned -amylase genes as part of this study. Polymorphism for -amylase genes belonging to each of the three subfamilies was observed between M202 and IR36. The maximum degree of polymorphism was found among genes belonging to the RAmy3 subfamily, which also has the most diverse group of genes.     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Involvement of glutathione in heat shock– and hydrogen peroxide–induced cadmium tolerance of rice (Oryza sativa L.) seedlings

The role of reduced glutathione (GSH) in heat shock (HS)- and H₂O₂-induced protection of rice (Oryza sativa L., cv. Taichung 1) seedlings from Cd stress was investigated. HS- and H₂O₂-pretreatment resulted in an increase in GSH content in leaves of rice seedlings. Addition of exogenous GSH under non-HS conditions, which resulted in an increase in GSH in leaves, enhanced subsequent Cd tolerance of rice seedlings. Pretreatment with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, which effectively inhibited GSH content induced by HS and H₂O₂, reduced subsequent Cd tolerance. Furthermore, the effect of BSO on HS- and H₂O₂-induced GSH accumulation and toxicity by subsequent Cd stress can be reversed by the addition of GSH. The time-course analyses of HS in rice seedlings demonstrated that the accumulation of H₂O₂ preceded the increase in GSH. Based on the data obtained in this study, it could be concluded that the early accumulation of H₂O₂ during HS signals the increase in GSH content, which in turn protects rice seedlings from oxidative damage caused by Cd.     

Extent of heterosis in rice (Oryza sativa L.) under cold stress conditions — yield and its components

Summary The nature and extent of heterosis for grain yield and its component characters was estimated in a seven-parent F₁-diallel cross in rice under the cold stress conditions of Palampur, representing sub-temperate climate of the mid hills of Himachal Pradesh. The highest heterotic effects for grain yield were observed in Himalaya 1/Phul Patas 72 and China 988/Himdhan hybrids, which gave 60.36 and 32.48% heterosis, respectively, over mid parent, 38.76 and 26.86% heterosis, respectively, over high parent and 32.30 and 26.86% heterosis, respectively, over the best variety, Himdhan. Heterosis for grain yield in these crosses was due to an increase in tiller number, panicle length, spikelets/panicle and 1,000-grain weight. Large number of crosses exhibited significant heterosis for high spikelet sterility and the majority of them had significant heterosis for late flowering, taking more than 120 days to flower, which resulted in lack of heterosis for grain yield in such crosses due to cold stress at the reproductive stage.Part of the thesis submitted by the senior author to Himachal Pradesh Agricultural University, Palampur (H.P.) India, in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Agriculture     

Impact of epistasis and QTL×environment interaction on the developmental behavior of plant height in rice (Oryza sativa L.)

QTLs with epistatic effects and environmental interaction effects for the developmental behavior of plant height in rice were studied by conventional and conditional methods for quantitative trait loci (QTLs) by mapping with a doubled-haploid population of 123 lines from IR64/Azucena in three environments. The results showed that epistatic effects were important and most epistasis could be detected only by conditional QTL mapping, while most non–epistatic QTLs could be detected by both conventional and conditional methods. Many modificative QTLs showed only epistatic effects without their own additive effects at some stages. QTL×environment (QE) interaction effects were detected more often than QTL main effects for plant-height behavior, which might indicate that gene expression could be greatly affected by the environment. No QTLs had effects during the whole of ontogeny. Conditional QTL mapping might be a valid way to reveal dynamic gene expression for the development of quantitative traits, especially for epistatic effects. Received: 19 May 2000 / Accepted: 27 October 2000     

The difference of potassium dynamics between yellowish red soil and yellow cinnamon soil under rapeseed (Brassica napus L.)–rice (Oryza sativa L.) rotation

To increase the use efficiency of potassium (K) fertilizer, special attention was paid to the dynamics of soil K in the root zone and non-root zone. Difference in K dynamics between yellowish red soil and yellow cinnamon soil under rapeseed (Brassica napus L.)rice (Oryza sativa L.) rotation was studied using a rhizobox system. Results showed that soil water soluble K (Sol-K) and exchangeable K (Ex-K) in the root zone of both soils were reduced in the early stage of rapeseed growth. Along with plant growth and K uptake, soil Sol-K in the inner (0–20 mm to root zone), middle (20–40 mm) and outer (40–60 mm) compartments of the non-root zone of yellowish red soil migrated towards the root zone. As a result, soil Ex-K was transformed into Sol-K. The changes in soil Sol-K and Ex-K in the non-root zone of yellow cinnamon soil were similar to yellowish red soil, and soil non-exchangeable K (Nonex-K) in the root zone also decreased significantly. In the early stage of rice growth, waterlogging promoted diffusion of soil Sol-K from non-root zone to root zone and transformation of Ex-K into Sol-K. Along with the growth of rice and K uptake, soil Ex-K in each compartment of yellowish red soil decreased significantly. Soil Sol-K and Ex-K in the yellow cinnamon soil declined to a certain extent, and then remained unchanged, while soil Nonex-K kept on decreasing. It revealed that the plants first absorbed K in the root zone, of which K reserve was replenished by a gradual diffusion of K from the non-root zone. The closer to the root zone, the greater the contribution to K uptake by plants. Within one rotation cycle, Ex-K and Sol-K in yellowish red soil were the main forms of K available to the plants, and little Nonex-K could be absorbed. However, in the yellow cinnamon soil, Nonex-K was the main form of K available to the plants, followed by Ex-K and Sol-K.     

Genetic basis of ovicidal response to whitebacked planthopper (Sogatella furcifera Horváth) in rice (Oryza sativa L.)

Rice (Oryza sativa L.) ovicidal response to the whitebacked planthopper (Sogatella furcifera Horváth) is characterized by formation of watery lesions and production of an ovicidal substance benzyl benzoate, which results in high egg mortality of whitebacked planthopper. A gene with ovicidal activity to whitebacked planthopper, designated Ovc, and four ovicidal quantitative trait loci (QTLs), qOVA-1-3, qOVA-4, qOVA-5-1 and qOVA-5-2 were identified using near isogenic lines with reciprocal genetic backgrounds of a non-ovicidal Indica variety IR24 and an ovicidal Japonica variety Asominori. Ovc and the four QTLs were mapped on chromosomes 6, 1, 4, 5 and 5, respectively. Ovc is the first gene identified that kills insect eggs in plants. The Asominori allele at Ovc was essential for increasing egg mortality and responsible for production of benzyl benzoate and formation of watery lesions. The Asominori alleles at qOVA-1-3, qOVA-5-1 and qOVA-5-2 increased egg mortality in the presence of Ovc. In contrast, the Asominori allele at qOVA-4 suppressed egg mortality, indicating that qOVA-4 caused transgressive segregation for egg mortality. It was concluded that Ovc and four ovicidal QTLs accounted for the majority of the phenotypic variance for the ovicidal response to whitebacked planthopper in Asominori.     

Effect of foliar spray of zinc on chloroplast β-carbonic anhydrase expression and enzyme activity in rice (Oryza sativa L.) leaves

The β-carbonic anhydrase (β-CA) is regarded as a zinc-containing enzyme involved in photosynthesis. Here, the rice plants of cv. N22 were treated with foliar spray at a Zn²⁺ concentration range from 0–35.0 mM during the tillering stage. The β-CA expression in the treated leaves was quantitatively determined by RT-qPCR and gel-based immunoblotting techniques, and its enzyme activity and relative chlorophyll concentration were measured. Results indicated that exogenous zinc could benefit rice plants at the tillering stage, particularly chloroplast β-CA with a fourfold enhancement in gene expression and a 14.6 % increase in its activity by treating the rice leaves with the 7.0-mM Zn²⁺ concentration, thereby promoting photosynthesis by a 19.4 % increase in relative chlorophyll concentration per unit leaf area. Results also showed that the application of Zn²⁺ at a concentration exceeding 7.0 mM could result in leaf senescence, and in some cases leaf hurts with significant inactivation (decreasing by approximately 70 %) of β-CA enzyme. It could be concluded that the application of 7.0-mM Zn²⁺ benefits rice plants at the tillering stage. The β-CA activity was associated with the catalytic microenvironment, thus providing an indicator for physiological response to exogenous zinc in rice.     

Phytosiderophores revisited: 2′-deoxymugineic acid-mediated iron uptake triggers nitrogen assimilation in rice (Oryza sativa L.) seedlings

Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 μM of the chemically synthesized phytosiderophore 2′-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.     

Three genetic systems controlling growth, development and productivity of rice (Oryza sativa L.): a reevaluation of the ‘Green Revolution’

The Green Revolution (GR-I) included worldwide adoption of semi-dwarf rice cultivars (SRCs) with mutant alleles at GA20ox2 or SD1 encoding gibberellin 20-oxidase. Two series of experiments were conducted to characterize the pleiotropic effects of SD1 and its relationships with large numbers of QTLs affecting rice growth, development and productivity. The pleiotropic effects of SD1 in the IR64 genetic background for increased height, root length/mass and grain weight, and for reduced spikelet fertility and delayed heading were first demonstrated using large populations derived from near isogenic IR64 lines of SD1. In the second set of experiments, QTLs controlling nine growth and yield traits were characterized using a new molecular quantitative genetics model and the phenotypic data of the well-known IR64/Azucena DH population evaluated across 11 environments, which revealed three genetic systems: the SD1-mediated, SD1-repressed and SD1-independent pathways that control rice growth, development and productivity. The SD1-mediated system comprised 43 functional genetic units (FGUs) controlled by GA. The SD1-repressed system was the alternative one comprising 38 FGUs that were only expressed in the mutant sd1 backgrounds. The SD1-independent one comprised 64 FGUs that were independent of SD1. GR-I resulted from the overall differences between the former two systems in the three aspects: (1) trait/environment-specific contributions; (2) distribution of favorable alleles for increased productivity in the parents; and (3) different responses to (fertilizer) inputs. Our results suggest that at 71.4 % of the detected loci, a QTL resulted from the difference between a functional allele and a loss-of-function mutant, whereas at the remaining 28.6 % of loci, from two functional alleles with differentiated effects. Our results suggest two general strategies to achieve GR-II (1) by further exploiting the genetic potential of the SD1-repressed and SD1-independent pathways and (2) by restoring the SD1-mediated pathways, or ‘back to the nature’ to fully exploit the genetic diversity of those loci in the SD1-mediated pathways which are virtually inaccessible to most rice-breeding programs worldwide that are exclusively based on sd1.     

Identification of quantitative trait loci associated with small brown planthopper (Laodelphax striatellus Fallén) resistance in rice (Oryza sativa L.)

F(2) and BC(1) populations derived from the cross between 02428 / Rathu Heenati were used to investigate small brown planthopper (SBPH) resistance. Using the F(2) population, three QTLs for antixenosis against SBPH were located on chromosomes 2, 5 and 6, and accounted for 30.75% of the phenotypic variance; three QTLs for antibiosis against SBPH were detected on chromosomes 8, 9 and 12. qSBPH5-c explaining 7.21% of phenotypic variance for antibiosis was identified on chromosome 5 using the BC(1) population. A major QTL, qSBPH12-a1, explained about 40% of the phenotypic variance, and a minor QTL, qSBPH4-a, was detected by the SSST method in both the F(2) and BC(1) populations. The QTLs indentified in the present study will be useful for marker assisted selection of SBPH resistance in rice.     

Fine mapping of brown planthopper (Nilaparvata lugens Stål) resistance gene Bph28(t) in rice (Oryza sativa L.)

The brown planthopper (BPH; Nilaparvata lugens Stål) is one of the most destructive insect pests of rice (Oryza sativa L.) throughout the Asian rice-growing countries. DV85 is a BPH-resistant indica variety. A single dominance gene conferring resistance in DV85 was previously mapped on the long arm of chromosome 11. The objectives of this study were to investigate feeding behaviors of BPH on DV85 plants and fine-map the BPH resistance gene, here designated Bph28(t). A seedling bulk test was conducted to identify resistant plant reactionsvg to BPH feeding. The results showed that the resistance of DV85 functions by means of tolerance during BPH attack, rather than non-preference and antibiosis. For fine mapping, two F2 populations were developed by crossing DV85 with the susceptible japonica variety Kinmaze and indica 9311. A high-resolution genetic map harboring Bph28(t) was constructed and Bph28(t) was finally physically defined to an interval of 64.8 kb between markers Indel55 and Indel66. The fine-mapped Bph28(t) gene will facilitate marker-assisted gene pyramiding for BPH resistance.     

Salinity–sodicity induced changes in reproductive physiology of rice (Oryza sativa) under dense soil conditions

Seed set in rice under salinity stress is a major problem caused by a number of factors. The most important factor which is not explored yet in rice under field conditions is stigma receptivity and grain filling against salinity–sodicity stress. We conducted experiments to study the effect of salinity–sodicity on stigma receptivity and grain filling of rice (of different tolerance limits against salinity alone) under field conditions along with other parameters at reproductive phase. Our results showed that viability of pollens was reduced in all the cultivars under salinity–sodicity stress showing significant effect in the salt sensitive Basmati cultivars. The starch synthase activity (∝1-4 glucan-glucosyle transferases, EC 2.4.1.21) was inhibited more significantly in sensitive than in tolerant cultivars. In tolerant rice cultivars the degree of inhibition on all the parameters studied was less, thus showing better grain yield compared to sensitive cultivars. Results regarding stigma receptivity revealed that salinity–sodicity reduced F1 seed set when pollinator plants were grown under dense saline-sodic soil and pistillate plants under control soil. More deleterious effects were observed when both the pollinator and pistillate plants were grown under dense saline-sodic soil conditions. This reduction was highly significant in fine Basmati cultivars due to significant effect on stigma receptivity and starch synthase activity. The data suggest that stigma receptivity is more detrimental for seed set compared to pollen viability and other physiological parameters under dense saline-sodic conditions. In this study KS-282 proved to be the best cultivar for dense saline-sodic soil showing non-significant effect of salinity–sodicity induced changes in all physiological and biochemical functions, pollen viability, stigma receptivity coupled with higher activity of starch synthase towards grain filling and seed set, resulting in higher grain yield compared to all the test cultivars.     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Combined gene family characterization and RNA-Seq to study the response of β-ketoacyl-CoA synthase to abiotic stress in rice (Oryza sativa L.)

Plant Growth Regulation - β-ketoacyl-CoA synthase is a key enzyme in the biosynthesis of over-long-chain fattty acids; thus, it plays a crucial role in plant resistance to stress. Herein, 33...     

Do lignification and silicification of the cell wall precede silicon deposition in the silica cell of the rice (Oryza sativa L.) leaf epidermis?

Aims

Rice is a well-known silica-accumulating plant. The dumbbell-shaped silica bodies in the silica cells in rice leaf epidermis are formed via biosilicification, but the underlying mechanisms are largely unknown.

Methods

Leaves at different developmental stages were collected to investigate silica cell differentiation by analyzing structures and silicon localization in the silica cells.

Results

Exogenous silicon application increased both shoot and root biomass. When silicon was supplied, silica cells in the leaf epidermis developed gradually into a dumbbell-shape and became increasingly silicified as leaves aged. Silicon deposition in the silica cells was not completed until the leaf was fully expanded. Multiple lines of evidence suggest that lignification of silica cell walls precedes silicon deposition in the lumen of silica cells. The organized needle-like silica microstructures were formed by moulding the inner cell walls and filling up the lumen of the silica cell following leaf maturation.

Conclusions

Two processes were involved in silicon deposition: (1) the silica cell wall was lignified and silicified, and then (2) the silicon was deposited gradually in silica cells as leaves aged. Silica body formation was not completed until the leaf was fully mature.