Ethylene-induced stem growth of deepwater rice is correlated with expression of gibberellin- and abscisic acid-biosynthetic genes

Ethylene decreases the content of endogenous abscisic acid (ABA) and increases the level of bioactive gibberellin A₁ (GA₁) in the submerged internodes of deepwater rice. During partial submergence, internodes of deepwater rice undergo rapid elongation as a result of ethylene accumulation in the internodal lacunae. In anin vitro experiment using stem sections from deepwater rice, treatment with 5 μL L^-1 ethylene promoted stem growth by up to 3.2-foId times over air treatment. Expression patterns were analyzed for genes that encode GA- and ABA-biosynthesis enzymes to determine any possible molecular basis for the changes observed in GA₁ and ABA contents as a result of ethylene action. Expression of theOsGA20ox2 andOsGA20ox4 genes, which encode GA 20-oxidase, and of theOsGA3ox2 gene, which encodes the enzyme that converts GA₂₀ to CA₁, was up-regulated, whereas that of three ABA-biosynthetic genes —OsNCED1, OsNCED2, andOsNCEDS-was down-regulated in the presence of ethylene. These results indicate that GA and ABA contribute equally to the submergence-or ethylene-induced stem elongation of deepwater rice via the coordinated and opposite regulation of biosynthesis.     

Expression of expansin genes in strawberry varieties with contrasting fruit firmness.

Fruit softening is associated with cell wall disassembly mediated by the action of a complex set of enzymes and proteins. Expansins, a group of proteins with unknown enzymatic activity, are proposed to be involved in this process. In order to study the involvement of expansins in strawberry fruit softening we have analyzed the expression level of five expansin mRNAs (FaEXP1, FaEXP2, FaEXP4, FaEXP5 and FaEXP6) in the cultivars "Selva", "Camarosa" and "Toyonaka", which differ in fruit firmness during ripening. We have found a correlation between mRNA expression levels and fruit firmness for FaEXP1, FaEXP2 and FaEXP5. For these three mRNAs we have observed higher expression levels in the softest cultivar (Toyonaka) than in the other two firmer cultivars (Selva and Camarosa) at the beginning of ripening. This correlation was not found in the case of FaEXP4 and FaEXP6, although both genes displayed a different expression pattern in the three cultivars analyzed. Western-blot analysis revealed that the accumulation of expansin proteins begins earlier in the softest cultivar during ripening.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.     

Expansins in deepwater rice internodes.

Cell walls of deepwater rice (Oryza sativa L.) internodes undergo long-term extension (creep) when placed under tension in acidic buffers. This is indicative of the action of the cell wall-loosening protein expansin. Wall extension had a pH optimum of around 4.0 and was abolished by boiling. Acid-induced extension of boiled cell walls could be reconstituted by addition of salt-extracted rice or cucumber cell wall proteins. Cucumber expansin antibody recognized a single protein band of 24.5-kD apparent molecular mass on immunoblots of rice cell wall proteins. Expansins were partially purified by concanavalin A affinity chromatography and sulfopropyl (SP) cation-exchange chromatography. The latter yielded two peaks with extension activity (SP20 and SP29), and immunoblot analysis showed that both of these active fractions contained expansin of 24.5-kD molecular mass. The N-terminal amino acid sequence of SP20 expansin is identical to that deduced from the rice expansin cDNA Os-EXP1. The N-terminal amino acid sequence of SP29 expansin matches that deduced from the rice expansin cDNA Os-EXP2 in six of eight amino acids. Our results show that two expansins occur in the cell walls of rice internodes and that they may mediate acid-induced wall extension.     

Expression of an expansin is associated with endosperm weakening during tomato seed germination

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 microM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.     

Regulation of expansin gene expression affects growth and development in transgenic rice plants

To investigate the in vivo functions of expansins, we generated transgenic rice plants that express sense and antisense constructs of the expansin gene OsEXP4. In adult plants with constitutive OsEXP4 expression, 12% of overexpressors were taller and 88% were shorter than the average control plants, and most overexpressors developed at least two additional leaves. Antisense plants were shorter and flowered earlier than the average control plants. In transgenic plants with inducible OsEXP4 expression, we observed a close correlation between OsEXP4 protein levels and seedling growth. Coleoptile and mesocotyl length increased by up to 31 and 97%, respectively, in overexpressors, whereas in antisense seedlings, they decreased by up to 28 and 43%, respectively. The change in seedling growth resulted from corresponding changes in cell size, which in turn appeared to be a function of altered cell wall extensibility. Our results support the hypothesis that expansins are involved in enhancing growth by mediating cell wall loosening.     

The biophysical basis of elongation growth in internodes of deepwater rice

Partial submergence induces rapid internodal elongation in deepwater rice (Oryza sativa L., cv Habiganj Aman II). We measured in vivo extensibility, tissue tension, hydraulic conductance and osmotic potential in the region of cell elongation in the uppermost internode. The in vivo extensibility of the internode, measured by stretching of living tissue with a custom-made constant stress extensiometer, rose rapidly following submergence of the plant. Both the elastic (E_el) and plastic (E_pl) extensibility increased when growth of the internode was induced. The submerged internode displayed tissue tension (elastic outward bending of longitudinally split internode sections); in air-grown control internodes, no such bending occurred. The hydraulic conductance, estimated from the kinetics of tissue shrinkage in 0.5 molar mannitol and subsequent swelling in distilled water, was not changed by submergence. The osmotic potential, measured with a dew-point hygrometer using frozen-thawed tissue, was only 18% less negative in the submerged internode than in the air-grown control. This indicates that osmoregulation takes place in rapidly elongating rice internodes. We suggest that the rapid expansion of the newly formed internodal cells of submerged plants is controlled by the yielding properties (E_pl) of the cell walls. Experiments with excised stem sections indicate that gibberellin is involved in increasing the E_pl of the elongating cell walls.     

Expression of OsBiP4 and OsBiP5 is highly correlated with the endoplasmic reticulum stress response in rice

Binding protein (BiP) is a chaperone protein involved in the folding of secretory proteins in the ER lumen. OsBiP1 is constitutively expressed in various tissues, whereas the expression of OsBiP4 and OsBiP5 (OsBiP4&5) is not detected in any tissue under normal conditions. However, expression of OsBiP4&5 was highly and specifically activated under ER stress conditions induced by DTT treatment, OsBiP1 knockdown, OsBiP1 overexpression, OsIRE1 overexpression, or various exogenous recombinant proteins in transgenic rice. In contrast, OsBiP4&5 did not accumulate in OsIRE1 knockdown transgenic rice even after DTT treatment. When the subcellular localization of OsBiP4&5 was investigated in seed endosperm cells under the ER stress condition, OsBiP4&5 were localized to the ER, but did not participate in ER-derived protein body (PB-I) formation in a different manner to OsBiP1. These results indicate that OsBiP4&5 levels were positively correlated with stress levels in the ER. Taken together, these results suggest that OsBiP4&5 are ER stress-related BiP proteins that are regulated by OsIRE1/OsbZIP50 pathway and that they may have a distinct function from that of OsBiP1 in rice.     

Characterization of genes encoding ABA 8'-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.)

Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.     

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo.

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

水稻扩展蛋白家族的生物信息学分析

扩展蛋白是植物细胞壁的重要组成部分,具有松驰细胞壁和增加细胞壁柔韧性的作用,在植物的生长发育及抗性等方面起到重要的作用。水稻的全基因组序列统计分析显示,水稻扩展蛋白基因家族包含58个成员,分属于A(34)、B(19)、LA(4)和LB(1)4个亚家族,分布在水稻10条染色体上的58个位点,且同一亚家族成员有成簇存在的现象。扩展蛋白基因长度范围为687~1128 bp,编码蛋白质具有保守的结构域,以及保守的半胱氨酸和色氨酸残基。多数情况下,亚家族成员之间的氨基酸一致率小于35%,而同一亚家族成员之间的氨基酸一致率大于35%。在内含子、外显子组成模式上,水稻扩展蛋白呈现明显的亚家族特异性,除个别基因以外,A类基因含有1或2个内含子,B类含有3个内含子,LA和LB类含有4个内含子。密码子使用统计显示,与其他物种相比,水稻中的扩展蛋白具有更多的密码子使用偏好性,有26个高频密码子存在。研究结果展示了水稻扩展蛋白基因家族的基本信息,为深入研究扩展蛋白基因的功能、探讨物种间的进化关系奠定基础。     

Anatomical analysis of growth and developmental patterns in the internode of deepwater rice

Submergence of the stem induces rapid internodal elongation in deepwater rice (Oryza sativa L. cv. Habiganj Aman II). A comparative anatomical study of internodes isolated from airgrown and partially submerged rice plants was undertaken to localize and characterize regions of growth and differentiation in rice stems. Longitudinal sections were examined by light and scanning-electron microscopy. Based on cell-size analysis, three zones of internodal development were recognized: a zone of cell division and elongation at the base of the internode, designated the intercalary meristem (IM); a zone of cell elongation without concomitant cell division; and a zone of cell differentiation where neither cell division nor elongation occur. The primary effects of submergence on internodal development were a threefold increase in the number of cells per cell file resulting from a decrease in the cell-cycle time from 24 to 7 h within the IM; an expansion of the cell-elongation zone from 5 to 15 mm leading to a threefold greater final cell length; and a suppression of tissue differentiation as indicated by reduced chlorophyll content and a lack of secondary wall formation in xylem and cortical sclerenchyma. These data indicate that growth of deepwater-rice internoes involves a balance between elongation and differentiation of the stem. Submergence shifts this balance in favor of growth.Abbreviations GA gibberellin - IM intercalary meristem