A comparative molecular-physiological study of submergence response in lowland and deepwater rice

Survival of rice (Oryza sativa) upon an extreme rise of the water level depends on rapid stem elongation, which is mediated by ethylene. A genomic clone (OS-ACS5) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon short- and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety. Under hypoxic conditions, abscisic acid and gibberellin had a reciprocal opposite effect on the activity of OS-ACS5. Gibberellin up-regulated and abscisic acid down-regulated OS-ACS5 mRNA accumulation. Growth experiments indicated that lowland rice responded to submergence with a burst of growth early on, but lacked the ability to sustain elongation growth. Sustained growth, characteristic for deepwater rice, was correlated with a prolonged induction of OS-ACS5. In addition, a more pronounced capacity to convert ACC to ethylene, a limited ACC conjugation, and a high level of endogenous gibberellin(20) were characteristic for the deepwater variety. An elevated level of OS-ACS5 messenger was found in cv IR36 plants treated with exogenous ACC. This observation was concomitant with an increase in the capacity of converting ACC to ethylene and in elongation growth, and resulted in prolonged survival. In conclusion, OS-ACS5 is involved in the rapid elongation growth of deepwater rice by contributing to the initial and long-term increase in ethylene levels. Our data also suggest that ACC limits survival of submerged lowland rice seedlings.     

A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.     

Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 degrees C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.     

Flash flooding resistance of rice genotypes of Oryza sativa L., O. glaberrima Steud., and Interspecific hybridization progeny

In the costal area and inland valleys of Guinea, flash floods occur frequently following heavy rain during the rainy season. Young rice seedlings are particularly vulnerable to submergence stress due to insufficient height and carbohydrate reserves. In an attempt to characterize the physiological responses of young seedlings to flash flood, a wide genetic base including O. sativa, O. glaberrima, and interspecific hybridization progenies (IHP) was used.

Twelve day-old seedlings were submerged for 7 days, and plant height, dry matter accumulation (DMA), and lodging were measured. Upland rice (O. sativa) showed greater shoot elongation, larger reduction in DMA during submergence, and higher lodging, which lead to low flash flooding resistance (FFR). The physiological traits of most O. glaberrima and upland rice (O. sativa) to flash flood were opposite to those of submergence tolerant cultivars, as evidenced from the results of a principal component analysis. The physiological response of Saligbeli was different to other O. glaberrima genotypes in terms of FFR. Saligbeli exhibited enhanced shoot elongation with the increase in DMA during submergence. These features seemed to be a unique way to cope with submergence. Some IHP adapted to lowland cultivation showed traits similar to Saligbeli. The vigorous growth of Saligbeli and IHP underwater should be further investigated for improving FFR in rice.     

Proteomic analysis of rice leaf sheath during drought stress

Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice. To investigate the initial response of rice to drought stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to drought conditions from 2 to 6 days, and proteins were extracted from leaf sheaths, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 2 to 6 days, 10 proteins increased in abundance and the level of 2 proteins decreased. The functional categories of these proteins were identified as defense, energy, metabolism, cell structure, and signal transduction. In addition to drought stress, accumulations of protein were analyzed under several different stress conditions. The levels of an actin depolymerizing factor, a light harvesting complex chain II, a superoxidase dismutase and a salt-induced protein were changed by drought and osmotic stresses, but not cold or salt stresses, or abscisic acid treatment. The effect of drought stress on protein in the leaf sheaths of drought-tolerant rice cultivar was also analyzed. The light harvesting complex chain II and the actin depolymerizing factor were present at high levels in a drought-tolerant rice cultivar before stress application. With drought stress, actin depolymerizing factor was expressed in leaf blades, leaf sheaths, and roots. These results suggest that actin depolymerizing factor is one of the target proteins induced by drought stress.     

Anaerobiosis and plant growth hormones induce two genes encoding 1-aminocyclopropane-1-carboxylate synthase in rice (Oryza sativa L.).

The plant hormone ethylene is believed to be responsible for the ability of rice to grow in the deepwater regions of Southeast Asia. Ethylene production is induced by hypoxia, which is caused by flooding, because of enhanced activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in the ethylene biosynthetic pathway. We have cloned three divergent members, (OS-ACS1, OS-ACS2, and OS-ACS3), of a multigene family encoding ACC synthase in rice. OS-ACS1 resides on chromosome 3 and OS-ACS3 on chromosome 5 in the rice genome. The OS-ACS1 and OS-ACS3 genes are induced by anaerobiosis and indoleacetic acid (IAA) + benzyladenine (BA) + LiCl treatment. The anaerobic induction is differential and tissue specific; OS-ACS1 is induced in the shoots, whereas OS-ACS3 is induced in the roots. These inductions are insensitive to protein synthesis inhibitors, suggesting that they are primary responses to the inducers. All three genes are actually induced when protein synthesis is inhibited, indicating that they may be under negative control or that their mRNAs are unstable. The OS-ACS1 gene was structurally characterized, and the function of its encoded protein (M(r) = 53 112 Da, pI 8.2) was confirmed by expression experiments in Escherichia coli. The protein contains all eleven invariant amino acid residues that are conserved between aminotransferases and ACC synthases cloned from various dicotyledonous plants. The amino acid sequence shares significant identity to other ACC synthases (69-34%) and is more similar to sequences in other plant species (69% with the tomato LE-ACS3) than to other rice ACC synthases (50-44%). The data suggest that the extraordinary degree of divergence among ACC synthase isoenzymes within each species arose early in plant evolution and before the divergence of monocotyledonous and dicotyledonous plants.     

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.     

The Expression Pattern of the Tonoplast Intrinsic Protein gamma-TIP in Arabidopsis thaliana Is Correlated with Cell Enlargement

The vacuolar membrane (tonoplast) contains an abundant intrinsic protein with six membrane-spanning domains that is encoded by a small gene family. Different isoforms of tonoplast intrinsic protein (TIP) are expressed in different tissues or as a result of specific signals. Using promoter-β-glucuronidase (GUS) fusions and in situ hybridization, we have examined the expression of γ-TIP in Arabidopsis thaliana. GUS staining of plants transformed with promoter-GUS fusions showed that γ-TIP gene expression is high in recently formed tissues of young roots. In the shoot, γ-TIP gene expression was highest in the vascular bundles of stems and petioles, as well as in the stipules and in the receptacle of the flower. No GUS activity was detected in root or shoot meristems or in older tissues, suggesting temporal control of γ-TIP gene expression associated with cell elongation and/or differentiation. In situ hybridization carried out with whole seedlings confirmed that in root tips, γ-TIP mRNA was present only in the zone of cell elongation just behind the apical meristem. In seedling shoots, mRNA abundance was also found to be correlated with cell expansion. These results indicate that γ-TIP may be expressed primarily at the time when the large central vacuoles are being formed during cell enlargement.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Analysis of rice Act1 5' region activity in transgenic rice plants.

The 5' region of the rice actin 1 gene (Act1) has been developed as an efficient regulator of foreign gene expression in transgenic rice plants. To determine the pattern and level of rice Act1 5' region activity, transgenic rice plants containing the Act1 5' region fused to a bacterial beta-glucuronidase (Gus) coding sequence were generated. Two independent clonal lines of transgenic rice plants were analyzed in detail. Quantitative analysis showed that tissue from these transgenic rice plants have a level of GUS protein that represents as much as 3% of total soluble protein. We were able to demonstrate that Act1-Gus gene expression is constitutive throughout the sporophytic and gametophytic tissues of these transgenic rice plants. Plants from one transgenic line were analyzed for the segregation of GUS activity in pollen by in situ histochemical staining, and the inheritance and stability of Act1-Gus expression were assayed in subsequently derived progeny plants.     

Short-Term Complete Submergence of Rice at the Tillering Stage Increases Yield

Flooding is a major threat to agricultural production. Most studies have focused on the lower water storage limit in rice fields, whereas few studies have examined the upper water storage limit. This study aimed to explore the effect of waterlogging at the rice tillering stage on rice growth and yield. The early-ripening late japonica variety Yangjing 4227 was selected for this study. The treatments included different submergence depths (submergence depth/plant height: 1/2 (waist submergence), 2/3 (neck submergence), and 1/1 (complete submergence)) and durations (1, 3, and 5 d). The control group was treated with the conventional alternation of drying and wetting. The effects of waterlogging at the tillering stage on root characteristics, dry matter production, nitrogen and phosphorus accumulation, yield, yield components, and 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene expression were explored. Compared with the control group, the 1/1 group showed significant increases in yield, seed-setting rate, photosynthetically efficient leaf area, and OS-ACS3 gene expression after 1 d of submergence. The grain number per panicle, dry weight of the aboveground and belowground parts, and number of adventitious roots also increased. Correlation analysis revealed a significant positive correlation between the panicle number and nitrogen content; however, no significant correlation was found for phosphorus content. If a decrease in rice yield of less than 10% is acceptable, half, 2/3, and complete submergence of the plants can be performed at the tillering stage for 1-3 d; this treatment will increase the space available for rice field water management/control and will improve rainfall resource utilization.