Molecular and physiological characterization of AIP1, encoding the acetolactate synthase regulatory subunit in rice.

Flooding deprives plants of oxygen and thereby causes severe stress by interfering with energy production, leading to growth retardation. Enzymes and metabolites may help protect plants from waterlogging and hypoxic environmental conditions. Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids (BCAAs), providing the building blocks for proteins and various secondary metabolites. Additionally, under energy-poor conditions, free BCAAs can be used as an alternative energy source by mitochondria through a catabolic enzyme chain reaction. In this study, we characterized ALS-INTERACTING PROTEIN 1 (OsAIP1), which encodes the regulatory subunit of ALS in rice (Oryza sativa). This gene was expressed in all parts of the rice plant, and its expression level was significantly higher in submerged and low-oxygen environments. Rice transformants overexpressing OsAIP1 showed a higher survival rate under hypoxic stress than did non-transgenic control plants under the same conditions. The OsAIP1-overexpressing plants accumulated increased levels of BCAAs, demonstrating that OsAIP1 is an important factor in the hypoxia resistance mechanism. These results suggest that ALS proteins are part of a defense mechanism that improves the tolerance of plants to low-oxygen environments.

AIP1, Encoding the Small Subunit of Acetolactate Synthase, Is Partially Responsible for Resistance to Hypoxic Stress in Arabidopsis thaliana.

Flooding is a significant stress to land plants, depriving them of essential oxygen. Plants have evolved diverse strategies with variable success to survive flooding. Similar strategies have been described in organisms from other kingdoms. Several fungal species can successfully survive a low-oxygen environment by increasing their branched-chain amino acid (BCAA) contents. BCAAs may act as alternative electron acceptors in the respiratory chain under an oxygen-limited environment. The key and first enzyme for BCAA biosynthesis is acetolactate synthase (ALS). We identified two homologous genes encoding the small subunit of ALS in Arabidopsis (Arabidopsis thaliana). We determined that ALS INTERACTING PROTEIN1 (AIP1), which encodes the small subunit of ALS, is strongly expressed in all organs and highly expressed under submergence and low-oxygen stresses. We also showed that the overexpression of AIP1 confers tolerance to low-oxygen stress. These results indicate that ALS may play an essential role under prolonged flooding or oxygen deficiency in Arabidopsis.

Molecular and physiological characterization of AtHIGD1 in Arabidopsis.

Flooding is a principal stress that limits plant productivity. The sensing of low oxygen levels (hypoxia) plays a critical role in the signaling pathway that functions in plants in flooded environments. In this study, to investigate hypoxia response mechanisms in Arabidopsis, we identified three hypoxia-related genes and subjected one of these genes, Arabidopsis thaliana HYPOXIA-INDUCED GENE DOMAIN 1 (AtHIGD1), to molecular characterization including gene expression analysis and intracellular localization of the encoded protein. AtHIGD1 was expressed in various organs but was preferentially expressed in developing siliques. Confocal microscopy of transgenic plants harboring eGFP-tagged AtHIGD1 indicated that AtHIGD1 is localized to mitochondria. Importantly, plants overexpressing AtHIGD1 exhibited increased resistance to hypoxia compared to wild type. Our results represent the first report of a biological function for an HIGD protein in plants and indicate that AtHIGD1 is a mitochondrial protein that plays an active role in mitigating the effects of hypoxia on plants.

Cuticle ultrastructure, cuticular lipid composition, and gene expression in hypoxia-stressed Arabidopsis stems and leaves.

KEY MESSAGE: An increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis and may allow plants to cope with oxygen deficiency. The hydrophobic cuticle layer consisting of cutin polyester and cuticular wax is the first barrier to protect the aerial parts of land plants from environmental stresses. In the present study, we investigated the role of cuticle membrane in Arabidopsis responses to oxygen deficiency. TEM analysis showed that the epidermal cells of hypoxia-treated Arabidopsis stems and leaves possessed a thinner electron-translucent cuticle proper and a more electron-dense cuticular layer. A reduction in epicuticular wax crystal deposition was observed in SEM images of hypoxia-treated Arabidopsis stem compared with normoxic control. Cuticular transpiration was more rapid in hypoxia-stressed leaves than in normoxic control. Total wax and cutin loads decreased by approximately 6-12 and 12-22%, respectively, and the levels of C29 alkanes, secondary alcohols, and ketones, C16:0 ω-hydroxy fatty acids, and C18:2 dicarboxylic acids were also prominently reduced in hypoxia-stressed Arabidopsis leaves and/or stems relative to normoxic control. Genome-wide transcriptome and quantitative RT-PCR analyses revealed that the expression of several genes involved in the biosynthesis and transport of cuticular waxes and cutin monomers were downregulated more than fourfold, but no significant alterations were detected in the transcript levels of fatty acid biosynthetic genes, BCCP2, PDH-E1α, and ENR1 in hypoxia-treated Arabidopsis stems and leaves compared with normoxic control. Taken together, an increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis. The present study elucidates one of the cuticle-related adaptive responses that may allow plants to cope with low oxygen levels.

MultiSyn: A Webtool for Multiple Synteny Detection and Visualization of User's Sequence of Interest Compared to Public Plant Species.

Information on multiple synteny between plants and/or within a plant is key information to understand genome evolution. In addition, visualization of multiple synteny is helpful in interpreting evolution. So far, some web applications have been developed to determine and visualize multiple homology regions at once. However, the applications are not fully convenient for biologists because some of them do not include the function of synteny determination but visualize the multiple synteny plots by allowing users to upload their synteny data by determining the synteny based only on BLAST similarity information, with some algorithms not designed for synteny determination. Here, we introduce a web application that determines and visualizes multiple synteny from two types of files, simplified browser extensible data and protein sequence file by MCScanX algorithm, which have been used in many synteny studies.

A novel rice protein family of OsHIGDs may be involved in early signalling of hypoxia-promoted stem growth in deepwater rice.

KEY MESSAGE: OsHIGDs was identified as a novel hypoxia-responsive protein family. Among them, OsHIGD2 is characterized as a mitochondrial protein and is related to hypoxia signalling through interacting with mitochondrial proteins of critical functions in reducing cell damages caused by hypoxia. Recent evidence supports ethylene as a key factor in modulating plant responses to submergence stress. Meanwhile, there has been general consent that ethylene is not the only signal for the submergence-induced stem growth. In this study, we confirmed that hypoxia also promotes stem elongation in deepwater rice even in the absence of ethylene. As components of ethylene-independent hypoxia signalling, five HIGD (hypoxia-induced gene domain) protein genes were identified. Among the genes, OsHIGD2 showed the fastest and strongest induction by hypoxia as well as submergence. Co-expression analysis indicated that OsHIGD2 had a simultaneous expression pattern with fermentation-related genes, such as ADH1 (alcohol dehydrogenase 1) and PDC2 (pyruvate decarboxylase 2). Transient expression of OsHIGD2 in leaf epidermal cells of Nicotiana benthamiana provided evidence that the protein is localized to mitochondria. We further identified OsHIGD2-interacting proteins through the yeast two-hybrid assay using OsHIGD2 as bait. As a result, three mitochondrial proteins were discovered that function in the regulation of redox potential or reduction of protein damages caused by reactive oxygen species. In this report, we propose that OsHIGD2 is a mitochondrial protein which takes part in the early stage of hypoxia signalling by interacting with proteins that are related to oxygen utilization.

Needle longevity, photosynthetic rate and nitrogen concentration of eight spruce taxa planted in northern Japan.

Growth characteristics of Picea glehnii Masters, P. jezoensis (Sieb. et Zucc) Carr., P. jezoensis var. hondoensis (Mayr) Rehder and P. shirasawae Hayashi from Japan, P. abies (L.) Karst. from Europe and P. glauca Voss, P. mariana Britt., Sterns and Pogg. and P. rubens Sarg. from North America were compared. The trees were grown in similar conditions at the Tomakomai Experimental Forest of Hokkaido University in northern Japan. Tree growth, needle biomass, longevity, photosynthetic rate, nitrogen concentration and specific leaf area (SLA) were measured, and photosynthetic nitrogen-use efficiency was calculated. Picea jezoensis, P. jezoensis var. hondoensis, P. abies and P. glauca had high growth rates, high photosynthetic rates in young needles, high needle nitrogen concentrations and short needle life spans. In contrast, P. glehnii, P. shirasawae, P. mariana and P. rubens had low growth and photosynthetic rates, low needle nitrogen concentrations, long needle life spans and maintained a high photosynthetic nitrogen-use efficiency in older needles. Examination of relationships between several growth parameters of the eight taxa revealed positive correlations between SLA and mass-based photosynthetic rate and between SLA and mass-based nitrogen concentration, whereas mass-based photosynthetic rate and mass-based nitrogen concentration were negatively correlated with needle longevity. The species differed greatly in growth characteristics despite being grown in similar conditions.

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.

Biochemical properties and localization of the beta-expansin OsEXPB3 in rice (Oryza sativa L.).

Alpha-expansins are bound to the cell wall of plants and can be solubilized with an extraction buffer containing 1 M NaCl. Localization of alpha-expansins in the cell wall was confirmed by immunogold labeling and electron microscopy. The subcellular localization of vegetative beta-expansins has not yet been studied. Using antibodies specific for OsEXPB3, a vegetative beta-expansin of rice (Oryza sativa L.), we found that OsEXPB3 is tightly bound to the cell wall and, unlike alpha-expansins, cannot be solubilized with extraction buffer containing 1 M NaCl. OsEXPB3 protein could only be extracted with buffer containing SDS. The subcellular localization of the OsEXPB3 protein was confirmed by immunogold labeling and electron microscopy. Gold particles were mainly distributed over the primary cell walls. Immunohistochemistry showed that OsEXPB3 is present in all regions of the coleoptile and root tissues tested.

Effect of soil acidification on the growth of Korean pine (Pinus koraiensis) seedlings in a granite-derived forest soil.

The growth of pine trees has diminished in recent years in industrial areas of Korea. Soil acidification is believed to be responsible. To study its effects, we grew seedlings of three-year-old Korean pine in brown forest soil derived from granite, which had been treated with an acid solution, for 182 days. The anion mol ratio in the solution was SO4(2-):NO3-:Cl-=5:3:2, which is the average in the total precipitation in Korea; six H+ ion concentrations in the soil were studied (0 (control), 10, 30, 60 and 90 mmol H+.kg-1). With increasing amounts of H+ added to the soil, the concentrations of Ca, Mg, K, Al and Mn increased, especially below a soil pH of 3.8. The concentrations of Ca, Mg and K in pine needles and stems increased with increasing H+ added to the soil, whereas their concentrations in the root decreased. Conversely, the concentration of N and P in each organ of the pine plant was higher in all treatments than in controls. Also, the concentrations of Al and Mn increased significantly in all organs of the plant with increasing H+. We also estimated the effect of deliberate soil acidification on tree growth, using the molar ratio (Ca+Mg+K)/Al as an indicator of soil acidification. A strong positive correlation was found between the total dry mass (TDM) of seedlings and the (Ca+Mg+K)/Al molar ratio calculated from the concentrations of water-soluble elements in soil (r=0.99, p<0.001). When the (Ca+Mg+K)/Al molar ratio reached 1.0, the relative TDM had fallen to 40%. These results show that deliberate soil acidification reduces the growth of the Korean pine less than it does the Red pine, which has been the dominant species in Korea.

Whole genome analysis of the OsGRF gene family encoding plant-specific putative transcription activators in rice (Oryza sativa L.).

OsGRF1 (Oryza sativa GROWTH-REGULATING FACTOR1) is a rice gene encoding a putative novel transcriptional regulator. We identified and characterized eleven homologs of OsGRF1 in the rice genome. All twelve OsGRF proteins have two highly conserved regions, the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains, and sequences reminiscent of transcription factors. OsGRF genes were preferentially expressed in young and growing tissues, and applied gibberellic acid (GA3) enhanced the expression of seven OsGRF genes. In situ hybridization showed high levels of OsGRF1 transcripts in the shoot apical meristem and in cells surrounding the vasculature of the intercalary meristem. In a GAL4-based yeast assay, the C-terminal region of OsGRF1 was found to have transactivation activity. These results indicate that OsGRF1 acts as a transcriptional activator. Based on the in situ expression pattern of OsGRF1, we postulate that it may be involved in regulating vegetative growth in rice.

The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis.

Previously, we identified a novel rice gene, GROWTH-REGULATING FACTOR1 (OsGRF1), which encodes a putative transcription factor that appears to play a regulatory role in stem elongation. We now describe the GRF gene family of Arabidopsis thaliana (AtGRF), which comprises nine members. The deduced AtGRF proteins contain the same characteristic regions--the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains--as do OsGRF1 and related proteins in rice, as well as features indicating a function in transcriptional regulation. Most of the AtGRF genes are strongly expressed in actively growing and developing tissues, such as shoot tips, flower buds, and roots, but weakly in mature stem and leaf tissues. Overexpression of AtGRF1 and AtGRF2 resulted in larger leaves and cotyledons, as well as in delayed bolting of the inflorescence stem when compared to wild-type plants. In contrast, triple insertional null mutants of AtGRF1-AtGRF3 had smaller leaves and cotyledons, whereas single mutants displayed no changes in phenotype and double mutants displayed only minor ones. The alteration of leaf growth in overexpressors and triple mutants was based on an increase or decrease in cell size, respectively. These results indicate that AtGRF proteins play a role in the regulation of cell expansion in leaf and cotyledon tissues.

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.