Transcription factor OsNF-YB9 regulates reproductive growth and development in rice.

MAIN CONCLUSION: OsNF-YB9 controls heading by affecting expression of regulators of flowering. It affects the development of the reproductive meristem by interacting with MADS1 and controlling expression of hormone-related genes. Nuclear Factor-Y (NF-Y) family of transcription factors takes part in many aspects of growth and development in eukaryotes. They have been classified into three subunit classes, namely, NF-YA, NF-YB and NF-YC. In plants, this transcription factor family is much diverged and takes part in several developmental processes and stress. We investigated NF-Y subunit genes of rice (Oryza sativa) and found OsNF-YB9 as the closest homologue of LEAFY COTYLEDON1. OsNF-YB9 delayed the heading date when ectopically expressed in rice. Expression of several heading date regulating genes such as Hd1, Ehd1, Hd3a and RFT1 were altered. OsNF-YB9 overexpression also resulted in morphological defects in the reproductive organs and led to pseudovivipary. OsNF-YB9 interacted with MADS1, a key regulator of floral development. This NF-Y subunit acted upstream to several transcription factors as well as signalling proteins involved in brassinosteroid and gibberellic acid metabolism and cell cycle. OsNF-YB9 and OsNF-YC12 interacted in planta and the latter also delayed heading in rice upon overexpression suggesting its involvement in a similar pathway. Our data provide new insights into the rice heading date pathway integrating these OsNF-Y subunit members to the network. These features can be exploited to improve vegetative growth and yield of rice plants in future.

Stage-specific reprogramming of gene expression characterizes Lr48-mediated adult plant leaf rust resistance in wheat.

Wheat genotype CSP44 carrying a recessive gene Lr48 exhibits adult plant resistance (APR; incompatible reaction) but gives a compatible reaction (susceptibility) at the seedling stage against leaf rust. A comparative gene expression analysis involving cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative PCR (qPCR) was carried out for incompatible and compatible reactions in the genotype CSP44. cDNA-AFLP analysis was conducted using RNA samples that were isolated from flag leaves following inoculation with leaf rust race 77-5 (the most virulent race) and also after mock inoculation. As many as 298 of a total of 493 expressed transcript-derived fragments (TDFs) exhibited differential expression (262 upregulated and 36 downregulated). Of these 298 TDFs, 48 TDFs were eluted from gels, re-amplified, cloned, and sequenced. Forty two of these 48 TDFs had homology with known genes involved in the following biological processes: energy production, metabolism, transport, signaling, defense response, plant-pathogen interaction, transcriptional regulation, translation, and proteolysis. The functions of the remaining six TDFs could not be determined; apparently, these represented some novel genes. The qPCR analysis for 18 TDFs (with known and unknown functions, but showing major differences in expression) was conducted using RNA isolated from the seedlings as well as from the adult plants. The expression of at least 11 TDFs was induced and that of 4 other TDFs attenuated or remained near normal in adult plants following leaf rust inoculations. The remaining three TDFs had non-specific/developmental stage-specific expression. Functional annotation of TDFs that were upregulated suggest that the APR was supported by transient recruitment and reprogramming of processes like perception and recognition of pathogen effector by receptors, followed by CDPK and MAPK signaling, transport, metabolism, and energy release.

OsMED14_2, a tail module subunit of Mediator complex, controls rice development and involves jasmonic acid.

The Mediator complex is essential for eukaryotic transcription, yet its role and the function of its individual subunits in plants, especially in rice, remain poorly understood. Here, we investigate the function of OsMED14_2, a subunit of the Mediator tail module, in rice development. Overexpression and knockout of OsMED14_2 resulted in notable changes in panicle morphology and grain size. Microscopic analysis revealed impact of overexpression on pollen maturation, reflected by reduced viability, irregular shapes, and aberrant intine development. OsMED14_2 was found to interact with proteins involved in pollen development, namely, OsMADS62, OsMADS63 and OsMADS68, and its overexpression negatively affected the expression of OsMADS68 and the expression of other genes involved in intine development, including OsCAP1, OsGCD1, OsRIP1, and OsCPK29. Additionally, we found that OsMED14_2 overexpression influences jasmonic acid (JA) homeostasis, affecting bioactive JA levels, and expression of OsJAZ genes. Our data suggest OsMED14_2 may act as a regulator of JA-responsive genes through its interactions with OsHDAC6 and OsJAZ repressors. These findings contribute to better understanding of the Mediator complex's role in plant traits regulation.

Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis.

Rice MADS29 has recently been reported to cause programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development. However, analyses involving OsMADS29 protein expression domains and characterization of OsMADS29 gain-of-function and knockdown phenotypes revealed novel aspects of its function in maintaining hormone homeostasis, which may have a role in the development of embryo and plastid differentiation and starch filling in endosperm cells. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signalling and biosynthesis.

Expression dynamics of metabolic and regulatory components across stages of panicle and seed development in indica rice.

Carefully analyzed expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the spatial and temporal expression profiles of rice genes in 19 stages of vegetative and reproductive development. We could verify expression of 22,980 genes in at least one of the tissues. Differential expression analysis with respect to five vegetative tissues and preceding stages of development revealed reproductive stage-preferential/-specific genes. By using subtractive logic, we identified 354 and 456 genes expressing specifically during panicle and seed development, respectively. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1) and male meiosis (P3), the genes involved in jasmonic acid and phenylpropanoid biosynthesis were significantly upregulated. P6 stage of panicle, containing mature gametophytes, exhibited enrichment of transcripts involved in homogalacturonon degradation. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of three panicle-specific genes, OsAGO3, OsSub42, and RTS, and an early seed-specific gene, XYH, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development.

The Mediator complex in plants: structure, phylogeny, and expression profiling of representative genes in a dicot (Arabidopsis) and a monocot (rice) during reproduction and abiotic stress.

The Mediator (Med) complex relays regulatory information from DNA-bound transcription factors to the RNA polymerase II in eukaryotes. This macromolecular unit is composed of three core subcomplexes in addition to a separable kinase module. In this study, conservation of Meds has been investigated in 16 plant species representing seven diverse groups across the plant kingdom. Using Hidden Markov Model-based conserved motif searches, we have identified all the known yeast/metazoan Med components in one or more plant groups, including the Med26 subunits, which have not been reported so far for any plant species. We also detected orthologs for the Arabidopsis (Arabidopsis thaliana) Med32, -33, -34, -35, -36, and -37 in all the plant groups, and in silico analysis identified the Med32 and Med33 subunits as apparent orthologs of yeast/metazoan Med2/29 and Med5/24, respectively. Consequently, the plant Med complex appears to be composed of one or more members of 34 subunits, as opposed to 25 and 30 members in yeast and metazoans, respectively. Despite low similarity in primary Med sequences between the plants and their fungal/metazoan partners, secondary structure modeling of these proteins revealed a remarkable similarity between them, supporting the conservation of Med organization across kingdoms. Phylogenetic analysis between plant, human, and yeast revealed single clade relatedness for 29 Med genes families in plants, plant Meds being closer to human than to yeast counterparts. Expression profiling of rice (Oryza sativa) and Arabidopsis Med genes reveals that Meds not only act as a basal regulator of gene expression but may also have specific roles in plant development and under abiotic stress conditions.

Analysis of rice nuclear-localized seed-expressed proteins and their database (RSNP-DB).

Nuclear proteins are primarily regulatory factors governing gene expression. Multiple factors determine the localization of a protein in the nucleus. An upright identification of nuclear proteins is way far from accuracy. We have attempted to combine information from subcellular prediction tools, experimental evidence, and nuclear proteome data to identify a reliable list of seed-expressed nuclear proteins in rice. Depending upon the number of prediction tools calling a protein nuclear, we could sort 19,441 seed expressed proteins into five categories. Of which, half of the seed-expressed proteins were called nuclear by at least one out of four prediction tools. Further, gene ontology (GO) enrichment and transcription factor composition analysis showed that 6116 seed-expressed proteins could be called nuclear with a greater assertion. Localization evidence from experimental data was available for 1360 proteins. Their analysis showed that a 92.04% accuracy of a nuclear call is valid for proteins predicted nuclear by at least three tools. Distribution of nuclear localization signals and nuclear export signals showed that the majority of category four members were nuclear resident proteins, whereas other categories have a low fraction of nuclear resident proteins and significantly higher constitution of shuttling proteins. We compiled all the above information for the seed-expressed genes in the form of a searchable database named Rice Seed Nuclear Protein DataBase (RSNP-DB) https://pmb.du.ac.in/rsnpdb . This information will be useful for comprehending the role of seed nuclear proteome in rice.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Development of an inducible male-sterility system in rice through pollen-specific expression of l-ornithinase (argE) gene of E. coli.

In the present investigation, an inducible male-sterility system has been developed in the rice. In order to introduce inducible male-sterility, the coding region of l-ornithinase (argE) gene of E. coli was fused to the Oryza sativa indica pollen allergen (OSIPA) promoter sequence which is known to function specifically in the pollen grains. Transgenic plants were obtained with argE gene and the transgenic status of plants was confirmed by PCR and Southern blot analyses. RT-PCR analysis confirmed the tissue-specific expression of argE in the anthers of transgenic rice plants. Transgenic rice plants expressing argE, after application of N-acetyl-phosphinothricin (N-ac-PPT), became completely male-sterile owing to the pollen-specific expression of argE. However, argE-transgenic plants were found to be self fertile when N-ac-PPT was not applied. Normal fertile seeds were obtained from the cross pollination between male-sterile argE transgenics and untransformed control plants, indicating that the female fertility is not affected by the N-ac-PPT treatment. These results clearly suggest that the expression of argE gene affects only the male gametophyte but not the gynoecium development. Induction of complete male-sterility in the rice is a first of its kind, and moreover this male- sterility system does not require the deployment of any specific restorer line.

OsTCP19 influences developmental and abiotic stress signaling by modulating ABI4-mediated pathways.

Class-I TCP transcription factors are plant-specific developmental regulators. In this study, the role of one such rice gene, OsTCP19, in water-deficit and salt stress response was explored. Besides a general upregulation by abiotic stresses, this transcript was more abundant in tolerant than sensitive rice genotypes during early hours of stress. Stress, tissue and genotype-dependent retention of a small in-frame intron in this transcript was also observed. Overexpression of OsTCP19 in Arabidopsis caused upregulation of IAA3, ABI3 and ABI4 and downregulation of LOX2, and led to developmental abnormalities like fewer lateral root formation. Moreover, decrease in water loss and reactive oxygen species, and hyperaccumulation of lipid droplets in the transgenics contributed to better stress tolerance both during seedling establishment and in mature plants. OsTCP19 was also shown to directly regulate a rice triacylglycerol biosynthesis gene in transient assays. Genes similar to those up- or downregulated in the transgenics were accordingly found to coexpress positively and negatively with OsTCP19 in Rice Oligonucleotide Array Database. Interactions of OsTCP19 with OsABI4 and OsULT1 further suggest its function in modulation of abscisic acid pathways and chromatin structure. Thus, OsTCP19 appears to be an important node in cell signaling which crosslinks stress and developmental pathways.

Identification of Novel SNP in Promoter Sequence of TaGW2-6A Associated with Grain Weight and Other Agronomic Traits in Wheat (Triticum aestivum L.).

TaGW2 is an orthologue of rice gene OsGW2, which encodes E3 RING ubiquitin ligase and controls the grain size in rice. In wheat, three copies of TaGW2 have been identified and mapped on wheat homoeologous group 6 viz. TaGW2-6A, TaGW2-6B and TaGW2-6D. In the present study, using as many as 207 Indian wheat genotypes, we identified four SNPs including two novel SNPs (SNP-988 and SNP-494) in the promoter sequence of TaGW2-6A. All the four SNPs were G/A or A/G substitutions (transitions). Out of the four SNPs, SNP-494 was causal, since it was found associated with grain weight. The mean TGW (41.1 g) of genotypes with the allele SNP-494_A was significantly higher than mean TGW (38.6 g) of genotypes with the allele SNP-494_G. SNP-494 also regulates the expression of TaGW2-6A so that the wheat genotypes with SNP-494_G have higher expression and lower TGW and the genotypes with SNP-494_A have lower expression but higher TGW. Besides, SNP-494 was also found associated with grain length-width ratio, awn length, spike length, grain protein content, peduncle length and plant height. This suggested that gene TaGW2-6A not only controls grain size, but also controls other agronomic traits. In the promoter region, SNP-494 was present in 'CGCG' motif that plays an important role in Ca2+/calmodulin mediated regulation of genes. A user-friendly CAPS marker was also developed to identify the desirable allele of causal SNP (SNP-494) for use in marker-assisted selection for improvement of grain weight in wheat. Using four SNPs, five haplotypes were identified; of these, Hap_5 (G_A_G_A) was found to be a desirable haplotype having significantly higher grain weight (41.13g) relative to other four haplotypes (36.33-39.16 g).

Use of methylation filtration and C(0)t fractionation for analysis of genome composition and comparative genomics in bread wheat.

We investigated the compositional and structural differences in sequences derived from different fractions of wheat genomic DNA obtained using methylation filtration and C(0)t fractionation. Comparative analysis of these sequences revealed large compositional and structural variations in terms of GC content, different structural elements including repeat sequences (e.g., transposable elements and simple sequence repeats), protein coding genes, and non-coding RNA genes. A correlation between methylation status [determined on the basis of selective inclusion/exclusion in methylation-filtered (MF) library] of different repeat elements and expression level was observed. The expression levels were determined by comparing MF sequences with expressed sequence tags (ESTs) available in the public domain. Only a limited overlap among MF, high C(0)t (HC), and ESTs was observed, suggesting that these sequences may largely either represent the low-copy non-transcribed sequences or include genes with low expression levels. Thus, these results indicated a need to study MF and HC sequences along with ESTs to fully appreciate complexity of wheat gene space.

Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.