The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression

Summary The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from –982 to –634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 F1 gene and of several highly expressed plant genes.     

Chloroplast ribonucleoprotein-like proteins of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis> are not involved in RNA stability and RNA editing

Many RNA recognition motif (RRM)-containing proteins are known to exist in chloroplasts. Major members of the RRM protein family, which are chloroplast ribonucleoproteins (cpRNPs), have been investigated in seed plants, including tobacco and Arabidopsis thaliana, but never in early land plants, such as bryophytes. In this study, we surveyed RRM proteins encoded in the moss Physcomitrella patens genome and predicted 25 putative chloroplast RRM proteins. Among them, two RRM-containing proteins, PpRBP2a and PpRBP2b, resembled cpRNPs and were thus referred to as cpRNP-like proteins. However, knockout mutants of either one or two PpRBP2 genes exhibited a wild type-like phenotype. Unlike Arabidopsis cpRNPs, the levels of mRNA accumulation in chloroplasts were not affected in the PpRBP2 knockout mutants. In addition, the efficiency of RNA editing was also not altered in the mutants. This suggests that PpRBP2a and 2b may be functionally distinct from Arabidopsis cpRNPs.     

Genome-Wide Identification,Biochemical Characterization,and Expression Analyses of the YTH Domain-Containing RNA-Binding Protein Family in Arabidopsis and Rice

RNA-binding proteins are critical to RNA metabolism in cells and, thus, play important roles in diverse biological processes. In the present study, we identified the YTH domain-containing RNA-binding protein (RBP) family in Arabidopsis thaliana and rice at the molecular and biochemical levels. A total of 13 and 12 genes were found to encode YTH domain-containing RBPs in Arabidopsis and rice and named as AtYTH01–13 and OsYTH01–12, respectively. The phylogeny, chromosomal location, and structures of genes and proteins were analyzed. Electrophoretic mobility shift assays demonstrated that recombinant AtYTH05 protein could bind to single-stranded RNA in vitro, demonstrating that the YTH proteins have RNA-binding activity. Analyses of publicly available microarray data, gene expression by qRT-PCR, and AtYTH05 promoter activity indicate that the Arabidopsis AtYTHs and rice OsYTHs genes have distinct and diverse expression patterns in different tissues and developmental stages, showing tissue- and developmental-specific expression patterns. Furthermore, analyses of publicly available microarray data also indicate that many of the Arabidopsis AtYTHs and rice OsYTHs genes might be involved in responses to various abiotic and biotic stresses as well as in response to hormones. Our data demonstrate that the YTH family proteins are a novel group of RBPs and provide useful clues to define their biological functions of this RBP family in plants.     

A betaine aldehyde dehydrogenase gene from <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis> enhances tolerance of <Emphasis Type="Italic">Arabidopsis</Emphasis> to high salt and drought stresses

YUCCA is an important enzyme which catalyzes a key rate-limiting step in the tryptophan-dependent pathway for auxin biosynthesis and implicated in several processes during plant growth and development. Genome wide analyses of YUCCA genes have been performed in Arabidopsis, rice, tomato, and Populus, but have never been characterized in soybean, one of the most important oil crops in the world. In this study, 22 GmYUCCA genes (GmYUCCA1-22) were identified and named based on soybean whole-genome sequence. Phylogenetic analysis of YUCCA proteins from Glycine max, Arabidopsis, Oryza sativa, tomato, and Populus euphratica revealed that GmYUCCA proteins could be divided into four subfamilies. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that GmYUCCA genes have diverse expression patterns in different tissues and under various stress treatments. Compared to the wild type (WT), the transgenic GmYUCCA5 Arabidopsis plants displayed downward curling of the leaf blade margin, evident apical dominance, higher plant height, and shorter length of siliques. Our results provide a comprehensive analysis of the soybean YUCCA gene family and lay a solid foundation for further experiments in order to functionally characterize these gene members during soybean growth and development.     

Comparative genomics and reverse genetics analysis reveal indispensable functions of the serine acetyltransferase gene family in Arabidopsis

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis

Summary The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 181 and 182 desaturation, respectively;fab1, which is elevated in 160 and decreased in 181; andfab2, which is elevated in 180 and decreased in 181. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.     

Genomic localization of AtRE1 and AtRE2, copia-type retrotransposons,in natural variants of Arabidopsis thaliana

Retrotransposons are ubiquitous components of plant genomes. They affect genome organization, and can also affect the expression patterns of neighboring genes. Retrotransposons are therefore important elements for changing genomic information. To understand the evolution of the Arabidopsis genome, we examined the distribution of certain retrotransposons, AtRE1s and AtRE2s, in the genomes of 12 natural variants (accessions) of Arabidopsis thaliana. AtRE1 and AtRE2 are copia-type retrotransposons that are potentially active. Their copy numbers are low, and they are absent from the genomes of some accessions. We detected four loci with AtRE1s inserted in six accessions, and one locus with an insertion of a solo-LTR-like sequence derived from AtRE1 in two accessions. Seven loci with AtRE2s inserted were detected on eight accessions. These loci were distributed in euchromatic regions of chromosomes 1, 2, 3, and 4. The AtRE1 and AtRE2 sequences at some loci identified in this study have not been recorded in the database of the 1001 Genome project. The sequences of AtRE1s and those of AtRE2s in different accessions and at different loci were highly conserved. There was a complete or almost complete conservation of sequences of both long terminal repeats in each AtRE1 and in each AtRE2. These results suggest that AtRE1 and AtRE2 appeared quite recently in the Arabidopsis genome. Furthermore, sequence comparisons of AtRE1 and AtRE2 loci among accessions revealed the possibility that large deletions containing entire sequences of AtRE1 and AtRE2 have occurred in some accessions.     

Comprehensive analysis of CCCH-type zinc finger gene family in citrus (Clementine mandarin) by genome-wide characterization

The CCCH-type zinc finger proteins comprise a large gene family of regulatory proteins and are widely distributed in eukaryotic organisms. The CCCH proteins have been implicated in multiple biological processes and environmental responses in plants. Little information is available, however, about CCCH genes in plants, especially in woody plants such as citrus. The release of the whole-genome sequence of citrus allowed us to perform a genome-wide analysis of CCCH genes and to compare the identified proteins with their orthologs in model plants. In this study, 62 CCCH genes and a total of 132 CCCH motifs were identified, and a comprehensive analysis including the chromosomal locations, phylogenetic relationships, functional annotations, gene structures and conserved motifs was performed. Distribution mapping revealed that 54 of the 62 CCCH genes are unevenly dispersed on the nine citrus chromosomes. Based on phylogenetic analysis and gene structural features, we constructed 5 subfamilies of 62 CCCH members and integrative subfamilies from citrus, Arabidopsis, and rice, respectively. Importantly, large numbers of SNPs and InDels in 26 CCCH genes were identified from Poncirus trifoliata and Fortunella japonica using whole-genome deep re-sequencing. Furthermore, citrus CCCH genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various stress conditions. Our comprehensive analysis of CleC3Hs is a valuable resource that further elucidates the roles of CCCH family members in plant growth and development. In addition, variants and comparative genomics analyses deepen our understanding of the evolution of the CCCH gene family and will contribute to further genetics and genomics studies of citrus and other plant species.