Chwan-Yang Hong

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible α-amylase gene (αAmy8) promoter, and extracellular phytase secretion directed by an αAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46–60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0–5.5 and 2.0–6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.

Effect of magnesium deficiency on antioxidant status and cadmium toxicity in rice seedlings

Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Magnesium (Mg) is known as one of the essential nutrients for plants. Mg deficiency in plants affects metabolic processes. Plants grown in the field may encounter several abiotic stresses, rather than a single stress. Thus, the relationship between Mg nutrition and Cd toxicity is of ecological importance. In this study, effects of Mg deficiency on antioxidant systems and Cd toxicity in rice seedlings were investigated. Mg deficiency significantly decreased Mg concentrations in shoot and roots of rice seedlings. However, fresh weight and dry weight of rice seedlings were not affected by Mg deficiency. The contents of ascorbate and glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in Mg-deficient leaves were higher than respective control leaves. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll, and induction of oxidative stress. Based on these criteria, we demonstrated that Mg deficiency protected rice seedlings from Cd stress. Moreover, chlorophyll destruction by paraquat was higher in detached leaves from Mg-sufficient than Mg-deficient seedlings. Cd concentration was higher in Mg-deficient shoot and roots than their respective control shoot and roots, suggesting that the protective effect of Mg deficiency against Cd toxicity is not due to reduction of Cd uptake. Moreover, we observed that Cd-decreased Fe and Zn contents in Mg-deficient seedlings were more pronounced than that in Mg-sufficient seedlings. Of particular interest is the finding that the increase in OsIRT1, OsZIP1, and OsZIP3 transcripts caused by Cd in Mg-deficient roots was greater than that in control roots.

The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings.

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl2 (5 microM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl2 treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or L-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice.

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.

Na+ but not Cl− or osmotic stress is involved in NaCl-induced expression of Glutathione reductase in roots of rice seedlings

Glutathione reductase (GR, EC 1,6.4.2) is an important reactive oxygen species-scavenging enzyme. The present study examined the relative importance of Na(+), Cl(-), and the osmotic component in NaCl-induced expression of Oryza sativa glutathione reductase (OsGR) genes in rice roots. Semi-quantitative RT-PCR was used to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of GR identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with NaCl. Treatment with 150 mM NaCl and 150 mM NaNO(3) affected OsGR2 and OsGR3 induction similarly, which suggests that Na(+) but not Cl(-) is required for the NaCl-induced expression of OsGR2 and OsGR3. We also show that Na(+) but not Cl(-) is required for NaCl-enhanced GR activity and hydrogen peroxide (H(2)O(2)) production in rice roots. In addition to its component of ion toxicity, salt concentration in soil results in an osmotic effect. Here, we show that OsGR2 and OsGR3 expression, GR activity, and H(2)O(2) content were not affected at a concentration of mannitol iso-osmotic with 150 mM NaCl. NaCl-induced OsGR2 and OsGR3 in rice roots could be associated with Na(+) but not an osmotic component.

Abscisic Acid- and Stress-Induced Highly Proline-Rich Glycoproteins Regulate Root Growth in Rice

A class of plasma membrane-localized and highly Pro-rich glycoproteins are essential for abscisic acid- and stress-mediated repression of rice root elongation. 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.

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 Ca2+ sensing, K+ regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.

Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of OsADF3 confers Arabidopsis Thaliana drought tolerance

BackgroundActin depolymerizing factors (ADFs) are small actin-binding proteins. Many higher-plant ADFs has been known to involve in plant growth, development and pathogen defense. However, in rice the temporal and spatial expression of OsADF gene family and their relationship with abiotic stresses tolerance is still unknown.ResultsHere we reported the first comprehensive gene expression profile analysis of OsADF gene family. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We also demonstrated that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3 were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was enhanced in lateral roots and root tips. Ectopically overexpressed OsADF3 conferred the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, primary root length and survival. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were upregulated in transgenic Arabidopsis under drought stress.ConclusionsThese results suggested that OsADF gene family may participate in plant abiotic stresses response or tolerance and would facilitate functional validation of other OsADF genes.

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 messageRiceASRgenes 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.AbstractPlant 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.