Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield.

Generating a new variety of plant with erect-leaf is a critical strategy to improve rice grain yield, as plants with this trait can be dense-planted. The erect-leaf is a significant morphological trait partially regulated by Brassinosteroids (BRs) in rice plants. So far, only a few genes can be used for molecular breeding in rice. Here, we identified OsBAK1 as a potential gene to alter rice architecture. Based on rice genome sequences, four closely related homologs of Arabidopsis BAK1 (AtBAK1) gene were amplified. Phylogenetic analysis and suppression of a weak Arabidopsis mutant bri1-5 indicated that OsBAK1 (Os08g0174700) is the closest relative of AtBAK1. Genetic, physiological, and biochemical analyses all suggest that the function of OsBAK1 is conserved with AtBAK1. Overexpression of a truncated intracellular domain of OsBAK1, but not the extracellular domain of OsBAK1, resulted in a dwarfed phenotype, similar to the rice BR-insensitive mutant plants. The expression of OsBAK1 changed important agricultural traits of rice such as plant height, leaf erectness, grain morphologic features, and disease resistance responses. Our results suggested that a new rice variety with erect-leaf and normal reproduction can be generated simply by suppressing the expression level of OsBAK1. Therefore, OsBAK1 is a potential molecular breeding tool for improving rice grain yield by modifying rice architecture.

Over-expression of OsUGE-1 altered raffinose level and tolerance to abiotic stress but not morphology in Arabidopsis.

OsUGE-1 is known to be induced by various abiotic stresses, but its exact function in plants is unclear. In the present study, OsUGE-1 was over-expressed in Arabidopsis, transgenic plants conferred tolerance to salt, drought and freezing stress without altering plant morphology. In addition, transgenic plants showed a higher level of the soluble sugar raffinose than did wild-type plants. Our results suggest that elevated level of raffinose with over-expressed OsUGE-1 resulted in enhanced tolerance to abiotic stress. Thus, the gene may be applied to improve tolerance to abiotic stress in crops.

Transgenic rice plants ectopically expressing AtBAK1 are semi-dwarfed and hypersensitive to 24-epibrassinolide.

Brassinosteroids (BRs) are endogenous plant hormones essential for plant growth and development. Brassinosteroid insensitive1 (BRI1)-assocaiated receptor kinase (BAK1) is one of the key components in the BR signal transduction pathway due to its direct association with the BR receptor, BRI1. Although BRI1 and its orthologs have been identified from both dicotyledonous and monocotyledonous plants, less is known about BAK1 and its orthologs in higher plants other than Arabidopsis. This article provides the first piece of evidence that AtBAK1 can greatly affect growth and development of rice plants when ectopically expressed, suggesting that rice may share similar BR perception mechanism via BRI1/BAK1 complex. Interestingly, transgenic rice plants displayed semi-dwarfism and shortened primary roots. Physiological analysis and cell morphology assay demonstrated that the observed phenotypes in transgenic plants were presumably caused by hypersensitivity to endogenous levels of BRs, different from BR insensitive and deficient rice mutants. Consistently, several known BR inducible genes were also upregulated in transgenic rice plants, further suggesting that BAK1 was able to affect BR signaling in rice. On the other hand, the transgenic plants generated by overproducing AtBAK1 may potentially have agricultural applications because the dwarfed phenotype is generally resistant to lodging, while the fertility remains unaffected.

Cloning and expression of a novel cDNA encoding a mannose-specific jacalin-related lectin from Oryza sativa.

Lectin plays an important role in defense signaling in plants. A few genes of this family have been cloned. Here we report on a mannose-specific jacalin-related lectin in rice. Using sequence information of wheat gene VER2, which we had previously cloned, we were able to amplify a cDNA of OsJAC1 from Oryza sativa by RT-PCR. The cDNA of OsJAC1 was 1172 bp and contained a 921-bp open reading frame (ORF) encoding dirigent (amino acids 26-139) and jacalin (amino acids 175-305) domains of 306 amino acids. Comparison of the OsJAC1 sequence with those of other lectins (jacalin) from rice, wheat and other species revealed that OsJAC1 had the 12 amino acid positions conserved in all mannose-binding lectins. Semi-quantitative RT-PCR revealed that OsJAC1 expression was present in stems, leaves and young spikes but not young roots; the expression was high in leaves and low in stems and spikes. And methyl jasmonate could induce the expression of OsJAC1. To test the activity of OsJAC1, the jacalin domain at the C-terminal was expressed in E. coli. BL21 (DE3). The purified recombinant protein could agglutinate red blood cells of rabbit, and the agglutination activity was strongly inhibited by mannose compared with other carbohydrates. These results indicate that lectin with dirigent and jacalin domains exists in rice as well as wheat. This is the first report of cDNA cloning of mannose-binding jacalin-related lectin with a dirigent domain in N-terminal region from O. sativa.

[Progress in research on plant stem cell organizer gene WUSCHEL].

The WUS (WUSCHEL) gene encodes a transcription factor that specifies the adjacent cells to be stem cell. The WUS dependent signal systems have been found in different tissues recent years. The feedback loop between the CLV and WUS genes maintains the stem cell self-renewal and apical dominance in shoot apical meristem. In the embryonic meristem, the expression of CLV3 depends on WUS only. During post-embryo development, both WUS and STM are needed for CLV3 expression and the triggering of organogenesis. The expression of AG in floral meristem is activated by co-existence of WUS and LFY, AG acts as a negative regulator of WUS expression to downregulate the WUS level. The signal system established by WUS is also involved in ovule development. The somatic embryogenesis can be promoted efficiently by WUS, especially in the presence of auxin. The results of prevoius works indicated that cell competence to WUS activity is related to microenvironment and the combination of WUS signal with different environmental factors could activate different downstream genes.

[Functional analysis of vernalization-related gene VER17 in flower development using antisense RNA strategy in winter wheat].

To understand the function of vernalization-related gene VER17 in winter wheat (Triticum aestivum L. cv. Jingdong No.1), an antisense RNA strategy was used. The antisense VER17 with a vector pBI121 was constructed and transformed into winter wheat by using the pollen-tube-pathway method. Fourteen independent transgenic plants transformed with antisense VER17 and five control transformants transformed with pBI121 blank vector were obtained and confirmed by GUS histochemical assay and PCR-Southern blot analysis. Phenotypes of T(0) and T(1) transgenic plants showed that the plants of the antisense VER17 transgenic lines degenerated top or basal spikelets and had delayed flowering time, which suggested that the VER17 gene functions in accelerating flowering and the development of the top or basal spikelets and the stamen which were impacted by vernalization treatment.

Cloning and characteristics of an allene oxide synthase gene (TaAOS) of winter wheat.

Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase pathway which leads to the formation of jasmonic acid (JA). A full length cDNA of TaAOS was cloned in winter wheat (Triticum aestivum L. cv. Jinghua No.3) seedlings. The open reading frame encompassed 1410 bp encoding a polypeptide of 470 amino acids with calculated molecular mass of 51.9 kD. Southern blot analysis suggested there are three copies of the gene in wheat genome. The TaAOS mRNA could be strongly induced by exogenous JA, and the highest level JA was observed after a 10 h induction. In situ RNA hybridization of seedling indicated preferential gene expression in young leaves, especially in the parenchyma cells around the vascular bundles, and the hybridization also showed that exogenous La(3+) could not suppress the expression of TaAOS induced by JA.

Basic helix-loop-helix transcription factor from wild rice (OrbHLH2) improves tolerance to salt- and osmotic stress in Arabidopsis.

Salt stress adversely affects plant growth and development. Some plants reduce the damage of high-salt stress by expressing a series of salt-responsive genes. Studies of the molecular mechanism of the salt-stress response have focused on the characterization of components involved in signal perception and transduction. In the present work, we cloned and characterized a basic helix-loop-helix (bHLH) encoding gene, OrbHLH2, from wild rice (Oryza rufipogon), which encodes a homologue protein of ICE1 in Arabidopsis. OrbHLH2 protein localized in the nucleus. Overexpression of OrbHLH2 in Arabidopsis conferred increased tolerance to salt and osmotic stress, and the stress-responsive genes DREB1A/CBF3, RD29A, COR15A and KIN1 were upregulated in transgenic plants. Abscisic acid (ABA) treatment showed a similar effect on the seed germination or transcriptional expression of stress-responsive genes in both wild type and OrbHLH2-overexpressed plants, which implies that OrbHLH2 does not depend on ABA in responding to salt stress. OrbHLH2 may function as a transcription factor and positively regulate salt-stress signals independent of ABA in Arabidopsis, which provides some useful data for improving salt tolerance in crops.

A rice YABBY gene, OsYABBY4, preferentially expresses in developing vascular tissue.

Developmental gene families have diversified during land plant evolution. The primary role of YABBY gene family is promoting abaxial fate in model eudicot, Arabidopsis thaliana. However recent results suggest that roles of YABBY genes are not conserved in the angiosperms. In this paper, a rice YABBY gene was isolated, and its expression patterns were analyzed in detail. Sequence characterization and phylogenetic analyses showed the gene is OsYABBY4, which is group-classified into FIL/YAB3 subfamily. Beta-glucuronidase reporter assay and in situ analysis consistently revealed that OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, predominantly in the phloem tissue, suggesting that the function of the rice gene is different from those of its counterparts in eudicots. OsYABBY4 may have been recruited to regulate the development of vasculature in rice. However, transgenic Arabidopsis plants ectopically expressing OsYABBY4 behaved very like those over-expressing FIL or YAB3 with abaxialized lateral organs, suggesting the OsYABBY4 protein domain is conserved with its Arabidopsis counterparts in sequences. Our results also indicate that the functional diversification of OsYABBY4 may be associated with the divergent spatial-temporal expression patterns, and YABBY family members may have preserved different expression regulatory systems and functions during the evolution of different kinds of species.

Heterotrimeric G protein alpha subunit is involved in rice brassinosteroid response.

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways. The null mutation of RGA (rice heterotrimeric G protein alpha subunit), which encodes the alpha subunit of heterotrimeric G protein in rice, causes severe dwarfism and reduced responsiveness to gibberellic acid in rice. However, less is known about heterotrimeric G protein in brassinosteroid (BR) signaling, one of the well-understood phytohormone pathways. In the present study, we used root elongation inhibition assay, lamina inclination assay and coleoptile elongation analysis to demonstrated reduced sensitivity of d1 mutant plants (caused by the null mutation of RGA) to 24-epibrassinolide (24-epiBL), which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development. Moreover, RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner. Our results show that RGA is involved in rice brassinosteroid response, which may be beneficial to elucidate the molecular mechanisms of G protein signaling and provide a novel perspective to understand BR signaling in higher plants.

Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana.

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.

FPF1 transgene leads to altered flowering time and root development in rice.

AtFPF1 (FLOWERING PROMOTING FACTOR 1) is a gene that promotes flowering in Arabidopsis. An expression vector containing AtFPF1 driven by a Ubi-1 promoter was constructed. The gene was introduced into rice callus by Agrobacterium-mediated transformation and fertile plants were obtained. The presence of AtFPF1 in rice plants was confirmed by PCR, Southern and Northern blot analyses, as well as by beta-glucuronidase assay. The results showed that, as in Arabidopsis, AtFPF1 reduced flowering time in rice. Furthermore, introduction of AtFPF1 enhanced adventitious root formation but inhibited root growth in rice during the seedling stage. The results suggest that AtFPF1 promotes flowering time in both dicots and monocots, and plays a role in the initiation of adventitious roots in rice.

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity.

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.

Vernalization-induced flowering in wheat is mediated by a lectin-like gene VER2.

A vernalization-related gene VER2 was isolated from winter wheat ( Triticum aestivum L.) using a differential screening approach. The deduced VER2 is a lectin-like protein of 300 amino acids, which contains the presence of a jacalin-like GWG domain. RNA in situ hybridization results demonstrated that VER2 gene expression is restricted to the marginal meristems of immature leaves in vernalized wheat seedlings. No hybridization signal was detected in the epidermal tissue and vascular bundles. However, "devernalization" resulted in the silencing of VER2 gene activity. The gene expression pattern of VER2 induced by jasmonate was similar to that induced by vernalization. Antisense inhibition of VER2 in transgenic wheat showed that heading and maturation time were delayed up to 6 weeks compared with non-transformed wheat and the pBI121empty-vector-transformed wheat. Tissue degeneration at the top of the spike was also noticed in the antisense inhibited transgenic wheat. These results suggest that VER2 plays an important role in vernalization signaling and spike development in winter wheat.