Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.     

Spatial and temporal regulation of the forisome gene <Emphasis Type="Italic">for1</Emphasis> in the phloem during plant development

Forisomes are protein aggregates found uniquely in the sieve elements of Fabaceaen plants. Upon wounding they undergo a reversible, calcium-dependent conformational switch which enables them to act as cellular stopcocks. Forisomes begin to form in young sieve elements at an early stage of metaphloem differentiation. Genes encoding forisome components could therefore be useful as markers of early sieve element development. Here we present a comprehensive analysis of the developmental expression profile of for1, which encodes such a forisome component. The for1 gene is highly conserved among Fabaceaen species and appears to be unique to this phylogenetic lineage since no orthologous genes have been found in other plants, including Arabidopsis and rice. Even so, transgenic tobacco plants expressing reporter genes under the control of the for1 promoter display reporter activity exclusively in immature sieve elements. This suggests that the regulation of sieve element development is highly conserved even in plants where mature forisomes have not been detected. The promoter system could therefore provide a powerful tool for the detailed analysis of differentiation in metaphloem sieve elements in an unexpectedly broad range of plant species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

BAHD superfamily of acyl-CoA dependent acyltransferases in <Emphasis Type="Italic">Populus</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by “in silico” northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members’ evolutionary relationships and gene functions implicated in plant growth, development and metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens

Mitogen-activated protein kinase (MAPK) cascades play important roles in diverse developmental and physiological processes of plants, including pathogen-induced defense responses. Although at least 17 rice MAPKs have been identified and more than half of these MAPK genes have been shown to be pathogen or elicitor responsive, the exact role of most of the MAPKs in host-pathogen interaction is unknown. Here we report that OsMPK6 is an important regulator in rice disease resistance. Suppressing OsMPK6 or knocking out of OsMPK6 enhanced rice resistance to different races of Xanthomonas oryzae pv. oryzae, causing bacterial blight, one of the most devastating diseases of rice worldwide. The resistant plants showed increased expression of a subset of defense-responsive genes functioning in the NH1 (an Arabidopsis NPR1 orthologue)-involved defense signal transduction pathway. These results suggest that OsMPK6 functions as a repressor to regulate rice defense responses upon bacterial invasion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A transcriptome‐wide study on the microRNA‐ and the Argonaute 1‐enriched small RNA‐mediated regulatory networks involved in plant leaf senescence

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence‐associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)‐enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1‐enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1‐enriched sRNAs and the miRBase‐registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1‐enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above‐identified target genes at protein level were validated as regulated by 17 AGO1‐enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1‐enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1‐enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1‐enriched sRNAs in rice, indicating species‐specific functions of these sRNA–target pairs. These results could advance our understanding of the sRNA‐involved molecular processes modulating leaf senescence.     

Conservation of dual-targeted proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis> and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. V. Morgante and R. A. O. Rodrigues contributed equally to this work.     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Constitutive expression of two apple (Malus x domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis

Fruit trees, such as apple (Malus × domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level. A genome-wide search revealed significant conservation among autophagy genes in yeast and Arabidopsis. Up till now, however, there is no report about rice autophagy associated genes. Here we cloned OsAtg8 and OsAtg4 from Oryza sativa and detected their expression patterns in various tissues. Immunoblotting analysis showed that carboxyl terminus of OsAtg8 can be cleaved in yeast cell. Mutation analysis revealed that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacted with OsAtg4, and this interaction was not affected by the conserved Gly117 mutation. Our results demonstrate that Atg8 conjugation pathway is conserved in rice and may play important roles in rice autophagy.     

Overexpression of the rFCA RNA Recognition Motif Affects Morphologies Modifications in Rice (Oryza sativa L.)

RNA recognition motifs as important regulators of gene expression are highly conserved in animals and plants. The FCA floral promotion gene in Arabidopsis encodes a protein, containing two RNA recognition motifs (RRM) and a WW protein interaction domain. Here we isolated FCA cDNA from rice. FCA in rice (rFCA) was homologous to FCA-gamma of Arabidopsis and contained conserved domains. To investigate the function of RRM domain, fragment RRM1 and RRM2 of rFCA were introduced into rice subspecies Oryza sativa L. subsp. Indica var. 9311 and another rice subspecies Oryza sativa L. subsp. Japonica var. zhonghua11 transformation. Two transgenic lines exhibited similar phenotypes, flowering time delay, seed size and cell volume of transgenic plants was increased. These results showed that constitutive expression of RRMs could regulate cellular size. The patterns of overexpression of two RRM domains and their similar morphologies indicate they may play a same role.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

The floral volatile,methyl benzoate,from snapdragon (<Emphasis Type="Italic">Antirrhinum majus</Emphasis>) triggers phytotoxic effects in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Previously it has been shown that the floral scent of snapdragon flowers consists of a relatively simple mixture of volatile organic compounds (VOCs). These compounds are thought to be involved in the attraction of pollinators; however, little is known about their effect on other organisms, such as neighboring plants. Here, we report that VOCs from snapdragon flowers inhibit Arabidopsis root growth. Out of the three major snapdragon floral volatiles, myrcene, (E)-β-ocimene, and methyl benzoate (MB), MB was found to be primarily responsible for the inhibition of root growth. Ten micromoles MB reduced root length by 72.6%. We employed a microarray approach to identify the MB target genes in Arabidopsis that were responsible for the root growth inhibition phenotype in response to MB. These analyses showed that MB treatment affected 1.33% of global gene expression, including cytokinin, auxin and other plant-hormone-related genes, and genes related to seed germination processes in Arabidopsis. Accordingly, the root growth of cytokinin (cre1) and auxin (axr1) response mutants was less affected than that of the wild type by the volatile compound: roots of the treated mutants were reduced by 45.1 and 56.2%, respectively, relative to untreated control mutants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.