OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C₃₀–C₃₂ aldehydes and C₃₀ primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.     

OsLEA3-2, an Abiotic Stress Induced Gene of Rice Plays a Key Role in Salt and Drought Tolerance

Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses.     

Activation of the <Emphasis Type="Italic">WUS</Emphasis> gene induces ectopic initiation of floral meristems on mature stem surface in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.     

A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

Background

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.

Results

Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R ²/R ² _cv/R ² _ev and RPD values for a perfect prediction capacity.

Conclusions

Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

     

Edaphic factors and plant–insect interactions: direct and indirect effects of serpentine soil on florivores and pollinators

Edaphic factors can lead to differences in plant morphology and tissue chemistry. However, whether these differences result in altered plant–insect interactions for soil-generalist plants is less understood. We present evidence that soil chemistry can alter plant–insect interactions both directly, through chemical composition of plant tissue, and indirectly, through plant morphology, for serpentine-tolerant Mimulus guttatus (Phrymaceae). First, we scored floral display (corolla width, number of open flowers per inflorescence, and inflorescence height), flower chemistry, pollinator visitation and florivory of M. guttatus growing on natural serpentine and non-serpentine soil over 2 years. Second, we conducted a common garden reciprocal soil transplant experiment to isolate the effect of serpentine soil on floral display traits and flower chemistry. And last, we observed arrays of field-collected inflorescences and potted plants to determine the effect of soil environment in the field on pollinator visitation and florivore damage, respectively. For both natural and experimental plants, serpentine soil caused reductions in floral display and directly altered flower tissue chemistry. Plants in natural serpentine populations received fewer pollinator visits and less damage by florivores relative to non-serpentine plants. In experimental arrays, soil environment did not influence pollinator visitation (though larger flowers were visited more frequently), but did alter florivore damage, with serpentine-grown plants receiving less damage. Our results demonstrate that the soil environment can directly and indirectly affect plant–mutualist and plant–antagonist interactions of serpentine-tolerant plants by altering flower chemistry and floral display.     

Floral display size in comfrey, Symphytum officinale L. (Boraginaceae): relationships with visitation by three bumblebee species and subsequent seed set

The fecundity of insect-pollinated plants may not be linearly related to the number of flowers produced, since floral display will influence pollinator foraging patterns. We may expect more visits to plants with more flowers, but do these large plants receive more or fewer visits per flower than small plants? Do all pollinator species respond in the same way? We would also expect foragers to move less between plants when the number of flowers per plant are large, which may reduce cross-pollination compared to plants with few flowers. We examine the relationships between numbers of inflorescence per plant, bumblebee foraging behaviour and seed set in comfrey, Symphytum officinale, a self-incompatible perennial herb. Bumblebee species differed in their response to the size of floral display. More individuals of Bombus pratorum and the nectar-robbing B.?terrestris were attracted to plants with larger floral displays, but B. pascuorum exhibited no increase in recruitment according to display size. Once attracted, all bee species visited more inflorescences per plant on plants with more inflorescences. Overall the visitation rate per inflorescence and seed set per flower was independent of the number of inflorescences per plant. Variation in seed set was not explained by the numbers of bumblebees attracted or by the number of inflorescences they visited for any bee species. However, the mean seed set per flower (1.18) was far below the maximum possible (4 per flower). We suggest that in this system seed set is not limited by pollination but by other factors, possibly nutritional resources.     

Physiological role of rice germin-like protein 1 (OsGLP1) at early stages of growth and development in <Emphasis Type="Italic">indica</Emphasis> rice cultivar under salt stress condition

Since their discovery, germin and germin-like proteins (GLPs) were found to be associated with salt stress along with other physiological roles. Although a number of GLP family members showed spatio-temporal changes in expressional up-regulation or down-regulation upon exposure to salt stress across plant species, very little is known about any rice GLP member in relation to salt stress. Rice germin-like protein 1 (OsGLP1), belongs to “Cupin” superfamily, is a plant glycoprotein and is associated with the plant cell wall. Our previous studies on endogenous down-regulation of OsGLP1 in rice and heterologous expression in tobacco documented that the OsGLP1 possessing superoxide dismutase activity is involved in cell wall cross-linking and fungal disease resistance in plants. In the present study, the transgenic rice lines having reduced OsGLP1 expression were analyzed in advanced generation for deciphering the involvement of OsGLP1 under salt stress. OsGLP1 gene-silencing construct integated transgenic lines were confirmed by Southern hybridization and RNA-interfernce (RNAi) mediated gene-silencing of the transgenic rice lines was confirmed by northern blot analysis. The expression of endogenous OsGLP1 protein level was found to be reduced in salt sensitive indica rice cultivar Badshahbhog following salt stress. Additionally, the RNAi-mediated OsGLP1 gene-silencing in transgenic rice lines resulted improved salt tolerance as compared to the untransformed ones during seed germination, initial establishment, early seedling growth and callus proliferation. Salt tolerance nature of the OsGLP1 gene-silenced plants at early stages of growth and development depicted the negative correlation between the OsGLP1 expression and salt tolerance of rice.     

Cloning,transformation and expression of cell cycle-associated protein kinase OsWee1 in indica rice (Oryza sativa L.)

The development process of seed in plants is a cycle of cells which occur gradually and regularly. One of the genes involved in controling this stage is the Wee1 gene. Wee1 encode protein kinase which plays an important role in phosphorylation, inactivation of cyclin-dependent kinase 1 (CDK1)-cyclin (CYC) and inhibiting cell division at mitotic phase. The Overexpression of Wee1 leads to delaying entry into mitotic phase, resulting in enlargement of cell size due to suppression of cell division. Accordingly, the cloning and overexpressing of Wee1 in rice plant is important aim of this research in achieving better quantity and quality of future rice. The main objective of this present study is to cloning and generate transgenic rice plants overexpressing of Wee1 gene. Wee1 was isolated from cDNA of indica rice (Oryza sativa), called OsWee1. The full length of OsWee1 was 1239?bp in size and successfully inserted into plant expression vector pRI101ON. Seven-day-old rice seedlings were prepared for transformation of OsWee1 gene using Agrobacterium-mediated transformation method. Four positive transgenic lines were identified through the presence of kanamycin resistance gene (nptII) using genomic PCR analysis. Southern blot analysis result provides evidence that four independent rice transformants contained one to three rearranged transgene copies. Further screening in transgenic rice generation is needed in order to obtain stable expression of OsWee1.