OsCPK29 interacts with MADS68 to regulate pollen development in rice.

Pollen development and its germination are obligatory for the reproductive success of flowering plants. Calcium-dependent protein kinases (CPKs, also known as CDPKs) regulate diverse signaling pathways controlling plant growth and development. Here, we report the functional characterization of a novel OsCPK29 from rice, which is mainly expressed during pollen maturation stages of the anther. OsCPK29 exclusively localizes in the nucleus, and its N-terminal variable domain is responsible for retaining it in the nucleus. OsCPK29 knockdown rice plants exhibit reduced fertility, set fewer seeds, and produce collapsed non-viable pollen grains that do not germinate. Cytological analysis of anther semi-thin sections during different developmental stages suggested that pollen abnormalities appear after the vacuolated pollen stage. Detailed microscopic study of pollen grains further revealed that they were lacking the functional intine layer although exine layer was present. Consistent with that, downregulation of known intine development-related rice genes was also observed in OsCPK29 silenced anthers. Furthermore, it has been demonstrated that OsCPK29 interacts in vitro as well as in vivo with the MADS68 transcription factor which is a known regulator of pollen development. Therefore, phenotypic observations and molecular studies suggest that OsCPK29 is an important regulator of pollen development in rice.

bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice.

Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142OEplants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes.

Rice OsiSAP7 negatively regulates ABA stress signalling and imparts sensitivity to water-deficit stress in Arabidopsis.

Stress associated protein (SAP) genes in plants regulate abiotic stress responses. SAP gene family consists of 18 members in rice. Although their abiotic stress responsiveness is well established, the mechanism of their action is poorly understood. OsiSAP7 was chosen to investigate the mechanism of its action based on the dual nature of its sub-cellular localization preferentially in the nucleus or sub-nuclear speckles upon transient expression in onion epidermal cells. Its expression was down-regulated in rice seedlings under abiotic stresses. OsiSAP7 was localized evenly in the nucleus under unstressed conditions and in sub-nuclear speckles on MG132 treatment. OsiSAP7 exhibits E3 ubiquitin ligase activity in vitro. Abiotic stress responses of OsiSAP7 were assessed by its overexpression in Arabidopsis under the control of a stress inducible promoter rd29A. Stress response assessment was done at seed germination and advanced stages of development. Transgenics were ABA insensitive at seed germination stage and sensitive to water-deficit stress at advanced stage as compared to wild type (WT). They were also impaired in ABA and stress-responsive gene expression. Our study suggests that OsiSAP7 acts as a negative regulator of ABA and water-deficit stress signalling by acting as an E3 ubiquitin ligase.

Spatial and temporal activity of upstream regulatory regions of rice anther-specific genes in transgenic rice and Arabidopsis.

Upstream regulatory regions (URRs) of rice anther-specific genes, namely OSbHLH (coding for basic helix-loop-helix-containing protein) and OSFbox (F-box protein encoding gene), selected from the microarray data have been cloned to control expression of GUS and GFP reporter genes in stably transformed rice. Quantitative real time PCR analysis shows maximum transcript accumulation of these two genes in the meiotic anthers. Analysis of OSbHLH and OSFbox URRs by PLACE database reveal the presence of known pollen-specific cis elements. The URRs of both OSbHLH and OSFbox genes have maximum activity in the meiotic anther stage in rice, but confer constitutive expression in the heterologous dicot system, Arabidopsis, indicative of monocot specificity. Another rice gene (OSIPK; with homology to genes encoding calcium-dependent protein kinases) URR already reported to have anther-specific activity in Arabidopsis and tobacco also confers anther-specific expression in rice and is active in the pollen tubes, suggesting it belongs to the category of late expressed genes. The spatial activity of three URRs has also been analysed by histochemical evaluation of GUS activity in different anther cells/tissues. The activity of OSIPK URR in rice is strongest among the three URRs.

A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis.

OSIPP3 gene (coding for pectin methylesterase inhibitor protein) was isolated from a pre-pollinated inflorescence-specific cDNA library by differential screening of stage-specific libraries from Oryza sativa. OSIPP3 is present in the genome of rice as a single copy gene. OSIPP3 gene was expressed exclusively in the pre-pollinated spikelets of rice. Upstream regulatory region (URR) of OSIPP3 was isolated and a series of 5'-deletions were cloned upstream of GUS reporter gene and were used to transform Arabidopsis. OSIPP3_del1 and del2 transgenic plants showed GUS expression in root, anther and silique, while OSIPP3_del3 showed GUS activity only in anthers and siliques. Pollen-specific expression was observed in case of plants harboring OSIPP3_del4 construct. It can, therefore, be concluded that the OSIPP3 URR between -178 and +108 bp is necessary for conferring pollen-specific expression in Arabidopsis.

Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants.

• The inbuilt mechanisms of plant survival have been exploited for improving tolerance to abiotic stresses. Stress-associated proteins (SAPs), containing A20/AN1 zinc-finger domains, confer abiotic stress tolerance in different plants, however, their interacting partners and downstream targets remain to be identified. • In this study, we have investigated the subcellular interactions of rice SAPs and their interacting partner using yeast two-hybrid and fluorescence resonance energy transfer (FRET) approaches. Their efficacy in improving abiotic stress tolerance was analysed in transgenic Arabidopsis plants. Regulation of gene expression by genome-wide microarray in transgenics was used to identify downstream targets. • It was found that the A20 domain mediates the interaction of OsSAP1 with self, its close homolog OsSAP11 and a rice receptor-like cytoplasmic kinase, OsRLCK253. Such interactions between OsSAP1/11 and with OsRLCK253 occur at nuclear membrane, plasma membrane and in nucleus. Functionally, both OsSAP11 and OsRLCK253 could improve the water-deficit and salt stress tolerance in transgenic Arabidopsis plants via a signaling pathway affecting the expression of several common endogenous genes. • Components of a novel stress-responsive pathway have been identified. Their stress-inducible expression provided the protection against yield loss in transgenic plants, indicating the agronomic relevance of OsSAP11 and OsRLCK253 in conferring abiotic stress tolerance.

Characterization of a pollen-preferential gene OSIAGP from rice (Oryza sativa L. subspecies indica) coding for an arabinogalactan protein homologue, and analysis of its promoter activity during pollen development and pollen tube growth.

During differential screening of inflorescence-specific cDNA libraries from Oryza sativa indica, an arabinogalactan protein (OSIAGP) cDNA (586 bp) expressing preferentially in the inflorescence has been isolated. It encodes an arabinogalactan protein of 59 amino acids (6.4 kDa) with a transmembrane domain and a secretory domain at the N terminus. The protein shows homology with AGP23 from Arabidopsis, and its homologue in japonica rice is located on chromosome 6. OSIAGP transcripts also accumulate in shoots and roots of rice seedling grown in the dark, but light represses expression of the gene. Analysis of a genomic clone of OSIAGP revealed that its promoter contains several pollen-specificity and light-regulatory elements. The promoter confers pollen-preferential activity on gus, starting from the release of microspores to anther dehiscence in transgenic tobacco, and is also active during pollen tube growth. Analysis of pollen preferential activity of the promoter in the transgenic rice system revealed that even the approximately 300 bp fragment has activity in pollen and the anther wall and further deletion down to approximately 100 bp completely abolishes this activity, which is consistent with in-silico analysis of the promoter. Arabinogalactan proteins have been shown to be involved in the cell elongation process. The homology of OSIAGP with AGP23 and the fact that seedling growth in the dark and pollen tube growth are events based on cell elongation strengthen the possibility of OSIAGP performing a similar function.

Genome analysis and genetic enhancement of tomato.

The Solanaceae is an important family of vegetable crops, ornamentals and medicinal plants. Tomato has served as a model member of this family largely because of its enriched cytogenetic, genetic, as well as physical, maps. Mapping has helped in cloning several genes of importance such as Pto, responsible for resistance against bacterial speck disease, Mi-1.2 for resistance against nematodes, and fw2.2 QTL for fruit weight. A high-throughput genome-sequencing program has been initiated by an international consortium of 10 countries. Since heterochromatin has been found to be concentrated near centromeres, the consortium is focusing on sequencing only the gene-rich euchromatic region. Genomes of the members of Solanaceae show a significant degree of synteny, suggesting that the tomato genome sequence would help in the cloning of genes for important traits from other Solanaceae members as well. ESTs from a large number of cDNA libraries have been sequenced, and microarray chips, in conjunction with wide array of ripening mutants, have contributed immensely to the understanding of the fruit-ripening phenomenon. Work on the analysis of the tomato proteome has also been initiated. Transgenic tomato plants with improved abiotic stress tolerance, disease resistance and insect resistance, have been developed. Attempts have also been made to develop tomato as a bioreactor for various pharmaceutical proteins. However, control of fruit quality and ripening remains an active and challenging area of research. Such efforts should pave the way to improve not only tomato, but also other solanaceous crops.

Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems.

Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants.

Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco.

Stress perception and signal transduction leading to tolerance involve a complex interplay of different gene products. We describe here the isolation and characterization of an intronless gene (OSISAP1) from rice encoding a zinc-finger protein that is induced after different types of stresses, namely cold, desiccation, salt, submergence, and heavy metals as well as injury. The gene is also induced by stress hormone abscisic acid. Overexpression of the gene in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed-germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings, and their developmental pattern. Thus, OSISAP1 seems to be an important determinant of stress response in plants.