Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.     

Comprehensive sequence and expression profile analysis of PEX11 gene family in rice

PEX11 gene family has been shown to be involved in peroxisome biogenesis but very little is known about this gene family in rice. Here we show that five putative PEX11 genes (OsPEX11-1-5) present in rice genome and each contain three conserved motifs. The PEX11 sequences from rice and other species can be classified into three major groups. Among the five rice PEX11 genes, OsPEX11-2 and -3 are most likely duplicated. Expression profile and RT-PCR analysis suggested that the members of PEX11 family in rice had differential expression patterns: OsPEX11-1 and OsPEX11-4 had higher expression levels in leaf tissues than in the other tissues, OsPEX11-2 was detected only in germinated seeds, OsPEX11-3 was expressed predominantly in endosperm and germinated seeds, and OsPEX11-5 was expressed in all the tissues investigated. We also observed that the rice PEX11 genes had differential expression patterns under different abiotic stresses. OsPEX11-1 and OsPEX11-4 were induced by abscisic acid (ABA), hydrogen peroxide (H2O2), salt and low nitrogen stress conditions. OsPEX11-3 was responsive to ABA and H2O2 treatments, and OsPEX11-5 was responsive to ABA, H2O2, and salt treatments. However, OsPEX11-2 had no response to any of the stresses. Our results suggest that the rice PEX11 genes have diversification not only in sequences but also in expression patterns under normal and various stress conditions.     

Mouse keratin 19: complete amino acid sequence and gene expression during development

The complete amino acid sequence of the mouse keratin 19 (K19) was determined from a partial sequence of cDNA isolated from a mouse (day 10.5) embryo library and an amplified genomic fragment. Analysis of the sequence reveals strong evolutionary conservation with other K19s. Examination of the expression of the gene encoding K19 (K19) during development using an RNase protection assay reveals it is expressed in extra-embryonic tissues by day 8.5 and in the embryo proper by at least day 9.5. Furthermore, the K19 gene is induced in differentiating F9 embryonal carcinoma cells. These results indicate that K19 is another keratin, in addition to the K8-K18 pair, which is synthesized early during mouse development. Finally, Southern analysis of the K19 gene reveals that it is found as a unique copy in the mouse genome, in contrast to what is found in humans, which have at least one processed pseudogene.     

Sequence and expression analysis of the thioredoxin protein gene family in rice

Thioredoxin (Trx) proteins play important biological functions in cells by changing redox via thioldisulfied exchange. This system is especially widespread in plants. Through database search, we identified 30 potential Trx protein-encoding genes (OsTrx) in rice (Oryza sativa L.). An analysis of the complete set of OsTrx proteins is presented here, including chromosomal location, conserved motifs, domain duplication, and phylogenetic relationships. Our findings suggest that the expansion of the Trx gene family in rice, in large part, occurred due to gene duplication. A comprehensive expression profile of Trx genes family was investigated by analyzing the signal data of this family extracted from the whole genome microarray analysis of Minghui 63 and Zhenshan 97, two indica parents, and their hybrid Shanyou 63, using 27 different tissues representing the entire life cycle of rice. Results revealed specific expression of some members at germination transition as well as the 3-leaf stage during the vegetative growth phase of rice. OsTrx genes were also found to be differentially up- or down-regulated in rice seedlings subjected to treatments of phytohormones and light/dark conditions. The expression levels of the OsTrx genes in the different tissues and under different treatments were also checked by RT-PCR analysis. The identification of OsTrx genes showing differential expression in specific tissues among different genotypes or in response to different environmental cues could provide a new avenue for functional analyses in rice.     

Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags

Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.     

Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development

A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined. The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb. Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons. There are eight introns in both the human and the hamster des genes. Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved. A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns. These sequences all belong to the human AluI family. When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al. [Cell 43 (1985) 327-338] for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes [Minty and Kedes, Mol. Cell. Biol. 6 (1986) 2125-2136] in the 5'-flanking region. The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene. Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.     

Phosphoenolpyruvate carboxykinase in Arabidopsis: changes in gene expression, protein and activity during vegetative and reproductive development

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in different tissues of Arabidopsis thaliana throughout its vegetative and reproductive growth. The A. thaliana genome contains two PEPCK genes (PCK1 and PCK2), and these are predicted to generate 73,404 and 72,891 Da protein products, respectively. Both genes were transcribed in a range of tissues; however, PCK1 mRNA appeared to be more abundant and was present in a wider range of tissues. PEPCK protein was present in flowers, fruit, developing seed, germinating seed, leaves, stems and roots. Two PEPCK polypeptides, of approximately 74 and approximately 73 kDa were detected by immunoblotting, and these may arise from PCK1 and PCK2, respectively. PEPCK was abundant in cotyledons during post-germinative growth, and this is consistent with its well established role in gluconeogenesis. PEPCK was also abundant in sink tissues, such as young leaves, in developing flowers, fruit and seed. Immunohistochemistry and in situ hybridization showed that PEPCK was present in the nectaries, stigma, endocarp of the fruit wall and in tissues involved in the transfer of assimilates to the developing ovules and seeds, such as the vasculature and seed coat. The potential functions of PEPCK in A. thaliana are discussed.     

Microarray and proteomic analysis of brassinosteroid- and gibberellin-regulated gene and protein expression in rice

Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze expression changes of genes and proteins at genome scale. In this review, we summarize rice functional genomic research by using microarray and proteomic approaches and our recent research results focusing on the comparison of cDNA microarray and proteomic analyses of BR- and GA-regulated gene and protein expression in rice. We believe our findings have important implications for understanding the mechanism by which BR and GA regulate the growth and development of rice.     

An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution.

The embryology of amphioxus has much in common with vertebrate embryology, reflecting a close phylogenetic relationship between the two groups. Amphioxus embryology is simpler in several key respects, however, including a lack of pronounced craniofacial morphogenesis. To gain an insight into the molecular changes that accompanied the evolution of vertebrate embryology, and into the relationship between the amphioxus and vertebrate body plans, we have undertaken the first molecular level investigation of amphioxus embryonic development. We report the cloning, complete DNA sequence determination, sequence analysis and expression analysis of an amphioxus homeobox gene, AmphiHox3, evolutionarily homologous to the third-most 3' paralogous group of mammalian Hox genes. Sequence comparison to a mammalian homologue, mouse Hox-2.7 (HoxB3), reveals several stretches of amino acid conservation within the deduced protein sequences. Whole mount in situ hybridization reveals localized expression of AmphiHox3 in the posterior mesoderm (but not in the somites), and region-specific expression in the dorsal nerve cord, of amphioxus neurulae, later embryos and larvae. The anterior limit to expression in the nerve cord is at the level of the four/five somite boundary at the neurula stage, and stabilises to just anterior to the first nerve cord pigment spot to form. Comparison to the anterior expression boundary of mouse Hox-2.7 (HoxB3) and related genes suggests that the vertebrate brain is homologous to an extensive region of the amphioxus nerve cord that contains the cerebral vesicle (a region at the extreme rostral tip) and extends posterior to somite four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genome-wide identification, phylogeny and expression analysis of the lipoxygenase gene family in cucumber

Plant lipoxygenase (LOX) is involved in growth and developmental control processes, through the biosynthesis of regulatory molecules and defense responses to pathogens, wounding and stress. To date, few LOX proteins and little tissue expression profiling have been reported in detail for cucumber (Cucumis sativus L.). Recent completion of the cucumber genome sequence now permits genome-wide analysis of the LOX gene family in cucumber as well as comparison with LOX in Arabidopsis and rice. We identified 23 candidate LOX genes in the cucumber genome; phylogenetic analysis indicated that these LOX members cluster into two groups, designated types 1 and 2, as expected from previous studies. Sequence analysis showed that five binding sites of iron, including two consensus histidines in the LOX domain, are highly conserved in the cucumber LOX proteins. Analysis of chromosomal localization and genome distribution suggested that tandem duplication and/or polyploidal duplication contributed to the expansion of the cucumber LOX gene family. Based on intron/exon structure analysis, only a few of the extant intron patterns existed in the ancestor of monocots and eudicots. Expression data showed widespread distribution of the cucumber LOX gene family within plant tissues, suggesting that they perform different functions in different tissues.     

Molecular analysis of early rice stamen development using organ-specific gene expression profiling

Elucidating the regulatory mechanisms of plant organ formation is an important component of plant developmental biology and will be useful for crop improvement applications. Plant organ formation, or organogenesis, occurs when a group of primordial cells differentiates into an organ, through a well-orchestrated series of events, with a given shape, structure and function. Research over the past two decades has elucidated the molecular mechanisms of organ identity and dorsalventral axis determinations. However, little is known about the molecular mechanisms underlying the successive processes. To develop an effective approach for studying organ formation at the molecular level, we generated organ-specific gene expression profiles (GEPs) reflecting early development in rice stamen. In this study, we demonstrated that the GEPs are highly correlated with early stamen development, suggesting that this analysis is useful for dissecting stamen development regulation. Based on the molecular and morphological correlation, we found that over 26 genes, that were preferentially up-regulated during early stamen development, may participate in stamen development regulation. In addition, we found that differentially expressed genes during early stamen development are clustered into two clades, suggesting that stamen development may comprise of two distinct phases of pattern formation and cellular differentiation. Moreover, the organ-specific quantitative changes in gene expression levels may play a critical role for regulating plant organ formation. Electronic Supplementary Material Supplementary material is available for this article at Xiao-Chun Lu, Hua-Qin Gong contributed equally to this work.