A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

<Emphasis Type="Italic">ATHB23</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> class I homeodomain-leucine zipper gene,is expressed in the adaxial region of young leaves

Homeobox genes are essential regulators of plant development. ATHB23, a class I homeodomain leucine zipper gene of Arabidopsis, was found to be induced by treatment with the phytohormone gibberellin (GA). In order to clarify its role in development, we performed a histochemical analysis of transgenic plants containing a construct with a GUS::GFP reporter under the control of the 1.5 kb upstream region of ATHB23. The construct was mainly expressed in young leaves and the styles of flowers but not in mature leaves. Microscopic examination of young leaves revealed that it was expressed in the adaxial domain of leaf primordia and the rib meristem. Expression of ATHB23, like that of GA5 encoding GA 20-oxidase, was reduced in mutants related to adaxial-abaxial leaf polarity (phb-1d, se-2, and kan1 kan2). Reduced expression of the GUS::GFP reporter gene was also observed in an se-2 background. These results indicate that ATHB23 is under the control of GA and other activators such as PHB, and is involved in establishing polarity during leaf development.     

Identification and functional analysis of light-responsive unique genes and gene family members in rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa

MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important regulatory roles in both animals and plants, miRNA genes have been intensively studied in animals, but not in plants. In this study, we adopted a homology search approach to identify homologs of previously validated plant miRNAs in Arabidopsis thaliana and Oryza sativa. We identified 20 potential miRNA genes in Arabidopsis and 40 in O. sativa, providing a relatively complete enumeration of family members for these miRNAs in plants. In addition, a greater number of Arabidopsis miRNAs (MIR168, MIR159 and MIR172) were found to be conserved in rice. With the novel homologs, most of the miRNAs have closely related fellow miRNAs and the number of paralogs varies in the different miRNA families. Moreover, a probable functional segment highly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family was identified. These results support a model of variegated miRNA regulation in plants, in which miRNAs with different functional elements on their pre-miRNA fold-backs can differ in their function or regulation, and closely related miRNAs can be diverse in their specificity or competence to downregulate target genes. It appears that the sophisticated regulation of miRNAs can achieve complex biological effects through qualitative and quantitative modulation of gene expression profiles in plants.     

A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.     

The ankyrin repeat gene family in rice: genome-wide identification,classification and expression profiling

Ankyrin repeat (ANK) containing proteins comprise a large protein family. Although many members of this family have been implicated in plant growth, development and signal transduction, only a few ANK genes have been reported in rice. In this study, we analyzed the structures, phylogenetic relationship, genome localizations and expression profiles of 175 ankyrin repeat genes identified in rice (OsANK). Domain composition analysis suggested OsANK proteins can be classified into ten subfamilies. Chromosomal localizations of OsANK genes indicated nine segmental duplication events involving 17 genes and 65 OsANK genes were involved in tandem duplications. The expression profiles of 158 OsANK genes were analyzed in 24 tissues covering the whole life cycle of two rice genotypes, Minghui 63 and Zhenshan 97. Sixteen genes showed preferential expression in given tissues compared to all the other tissues in Minghui 63 and Zhenshan 97. Nine genes were preferentially expressed in stamen of 1 day before flowering, suggesting that these genes may play important roles in pollination and fertilization. Expression data of OsANK genes were also obtained with tissues of seedlings subjected to three phytohormone (NAA, GA3 and KT) and light/dark treatments. Eighteen genes showed differential expression with at least one phytohormone treatment while under light/dark treatments, 13 OsANK genes showed differential expression. Our data provided a very useful reference for cloning and functional analysis of members of this gene family in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

Comparative phylogenetic analysis of cystatin gene families from arabidopsis,rice and barley

The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.Electronic Supplementary Material Supplementary material is available for this article at     

Cytological analysis and genetic control of rice anther development

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte and haploid gametophyte generations.Rice (Oryza sativa) is the world's major staple food,and manipulation of pollen fertility is particularly important for the demands to increase rice grain yield.Towards a better understanding of the mechanisms controlling rice male reproductive development,we describe here the cytological changes of anther development through 14 stages,including cell division,differentiation and degeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form mature pollen grains through meiosis and mitosis.Furthermore,we compare the morphological difference of anthers and pollen grains in both monocot rice and eudicot Arabidopsis thaliana.Additionally,we describe the key genes identified to date critical for rice anther development and pollen formation.     

Gene family structure,expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees

Background  

Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups.     

Carotenoid biosynthesis genes in rice: structural analysis, genome-wide expression profiling and phylogenetic analysis

Carotenoids, important lipid-soluble antioxidants in photosynthetic tissues, are known to be completely absent in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthesis genes, have been performed to get carotenoid-enriched rice grain. Study of genes involved in their biosynthesis can provide further information regarding the abundance/absence of carotenoids in different tissues. We have identified 16 and 34 carotenoid biosynthesis genes in rice and Populus genomes, respectively. A detailed analysis of the domain structure of carotenoid biosynthesis enzymes in rice, Populus and Arabidopsis has shown that highly conserved catalytic domains, along with other domains, are present in these proteins. Phylogenetic analysis of rice genes with Arabidopsis and other characterized carotenoid biosynthesis genes has revealed that homologous genes exist in these plants, and the duplicated gene copies probably adopt new functions. Expression of rice and Populus genes has been analyzed by full-length cDNA- and EST-based expression profiling. In rice, this analysis was complemented by real-time PCR, microarray and signature-based expression profiling, which reveal that carotenoid biosynthesis genes are highly expressed in light-grown tissues, have differential expression pattern during vegetative/reproductive development and are responsive to stress.     

Calcineurin B-like proteins in rice can bind with calcium ion and associate with the Arabidopsis CIPK family members

Arabidopsis calcineurine B-like proteins (AtCBLs) are known to sense Ca²⁺ signals induced by a variety of extracellular stimuli such as cold, drought, low K⁺ concentration, and high salinity. The Ca²⁺-bound AtCBLs then associate with and activate a group of serine/threonine protein kinases (CIPKs) to transmit the signals further downstream. In this study, we isolated three rice cDNA clones (OsCBL1, 3, and 5), which respectively encode a polypeptide very similar to the AtCBL family. Deduced amino acid sequence analysis revealed that each OsCBL contains canonical EF-hand Ca²⁺-binding motifs. In fact, our electrophoresis mobility shift assays showed that both OsCBL1 and OsCBL3 proteins purified from Escherichia coli via the GST-fusion expression system can bind with Ca²⁺ in vitro. Using the yeast two-hybrid system and pull-down assays, we also demonstrated that OsCBLs, just like AtCBLs, can interact with the Arabidopsis CIPKs. In addition, deletion analyses indicated that the C-terminal nonkinase domain of the CIPK family is responsible for the interaction with OsCBLs. Taken together, these findings strongly suggest that OsCBLs are very likely to be the rice orthologs of AtCBLs, which may be involved in the various stress responses.     

The Arabidopsis Athb-8, -9 and genes are members of a small gene family coding for highly related HD-ZIP proteins

We report the isolation and characterization of two Arabidopsis homeobox genes highly related to the Athb-8 gene. The full-length cDNAs encode proteins of 841 and 852 amino acids which we have designated Athb-9 and -14, respectively. Athb-8, -9 and -14 are members of a small family of HD-Zip proteins (HD-ZIP III) characterized by a HD-Zip motif confined to the N-terminus of the polypeptide. The spatial organization of the HD-Zip domain of Athb-8, -9 and -14 is different from that of the Athb-1 (a member of the HD-ZIP I family) and Athb-2 (a member of the HD-ZIP II family) HD-Zip domains. DNA binding analysis performed with random-sequence DNA templates showed that the Athb-9 HD-Zip (HD-Zip-9) domain, but not the Athb-9 HD alone, binds to DNA. The HD-Zip-9 domain recognizes a 11 bp pseudopalindromic sequence (GTAAT(G/C)ATTAC), as determined by selecting high-affinity binding sites from random-sequence DNA. Moreover, gel retardation assays demonstrated that the HD-Zip-9 domain binds to DNA as a dimer. These data support the notion that the HD-ZIP III domain interacts with DNA recognition elements in a fashion similar to the HD-ZIP I and II domains.