Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin,Expansion and Divergence

Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes.     

Control of Cell Proliferation,Organ Growth,and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.     

Cell Cycle Control in Arabidopsis

Although the basic mechanism of cell cycle control is conservedamong eukaryotes, its regulation differs in each type of organism.Plants have unique developmental features that distinguish themfrom other eukaryotes. These include the absence of cell migration,the formation of organs throughout the entire life-span fromspecialized regions called meristems, and the potency of non-dividingcells to re-enter the cell cycle. The study of plant cell cyclecontrol genes is expected to contribute to the understandingof these unique developmental phenomena. The principal regulatorsof the eukaryotic cell cycle, the cyclin-dependent kinases (CDKs)and cyclins, are conserved in plants. This review focuses oncell cycle regulation in the plant Arabidopsis thaliana . Whileexpression of one Arabidopsis CDK gene, Cdc2aAt, was positivelycorrelated with the competence of cells to divide, expressionof a mitotic-like cyclin, cyc1At, was almost exclusively confinedto dividing cells. The expression of the Arabidopsis -type cyclinsappears to be an early stage in the response of plant cellsto external and internal stimuli. Arabidopsis thaliana (L.) Heynh.; cell cycle; CDK; cyclin; plant development; plant hormone     

Genome-wide analysis of gene expression during early Arabidopsis flower development

Detailed information about stage-specific changes in gene expression is crucial for the understanding of the gene regulatory networks underlying development. Here, we describe the global gene expression dynamics during early flower development, a key process in the life cycle of a plant, during which floral patterning and the specification of floral organs is established. We used a novel floral induction system in Arabidopsis, which allows the isolation of a large number of synchronized floral buds, in conjunction with whole-genome microarray analysis to identify genes with differential expression at distinct stages of flower development. We found that the onset of flower formation is characterized by a massive downregulation of genes in incipient floral primordia, which is followed by a predominance of gene activation during the differentiation of floral organs. Among the genes we identified as differentially expressed in the experiment, we detected a significant enrichment of closely related members of gene families. The expression profiles of these related genes were often highly correlated, indicating similar temporal expression patterns. Moreover, we found that the majority of these genes is specifically up-regulated during certain developmental stages. Because co-expressed members of gene families in Arabidopsis frequently act in a redundant manner, these results suggest a high degree of functional redundancy during early flower development, but also that its extent may vary in a stage-specific manner.     

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.     

Significance of Population Size on the Fixation of Nonsynonymous Mutations in Genes Under Varying Levels of Selection Pressure

Previous studies observed a higher ratio of divergences at nonsynonymous and synonymous sites (ω = d_N/d_S) in species with a small population size compared to that estimated for those with a large population size. Here we examined the theoretical relationship between ω, effective population size (N_e), and selection coefficient (s). Our analysis revealed that when purifying selection is high, ω of species with small N_e is much higher than that of species with large N_e. However the difference between the two ω reduces with the decline in selection pressure (s → 0). We examined this relationship using primate and rodent genes and found that the ω estimated for highly constrained genes of primates was up to 2.9 times higher than that obtained for their orthologous rodent genes. Conversely, for genes under weak purifying selection the ω of primates was only 17% higher than that of rodents. When tissue specificity was used as a proxy for selection pressure we found that the ω of broadly expressed genes of primates was up to 2.1-fold higher than that of their rodent counterparts and this difference was only 27% for tissue specific genes. Since most of the nonsynonymous mutations in constrained or broadly expressed genes are deleterious, fixation of these mutations is influenced by N_e. This results in a higher ω of these genes in primates compared to those from rodents. Conversely, the majority of nonsynonymous mutations in less-constrained or tissue-specific genes are neutral or nearly neutral and therefore fixation of them is largely independent of N_e, which leads to the similarity of ω in primates and rodents.     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.     

Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants

We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein.     

Slow Co-Evolution of the MAGO and Y14 Protein Families Is Required for the Maintenance of Their Obligate Heterodimerization Mode

The exon junction complex (EJC) plays important roles in RNA metabolisms and the development of eukaryotic organisms. MAGO (short form of MAGO NASHI) and Y14 (also Tsunagi or RBM8) are the EJC core components. Their biological roles have been well investigated in various species, but the evolutionary patterns of the two gene families and their protein-protein interactions are poorly known. Genome-wide survey suggested that the MAGO and Y14 two gene families originated in eukaryotic organisms with the maintenance of a low copy. We found that the two protein families evolved slowly; however, the MAGO family under stringent purifying selection evolved more slowly than the Y14 family that was under relative relaxed purifying selection. MAGO and Y14 were obliged to form heterodimer in a eukaryotic organism, and this obligate mode was plesiomorphic. Lack of binding of MAGO to Y14 as functional barrier was observed only among distantly species, suggesting that a slow co-evolution of the two protein families. Inter-protein co-evolutionary signal was further quantified in analyses of the Tol-MirroTree and co-evolution analysis using protein sequences. About 20% of the 41 significantly correlated mutation groups (involving 97 residues) predicted between the two families was clade-specific. Moreover, around half of the predicted co-evolved groups and nearly all clade-specific residues fell into the minimal interaction domains of the two protein families. The mutagenesis effects of the clade-specific residues strengthened that the co-evolution is required for obligate MAGO-Y14 heterodimerization mode. In turn, the obliged heterodimerization in an organism serves as a strong functional constraint for the co-evolution of the MAGO and Y14 families. Such a co-evolution allows maintaining the interaction between the proteins through large evolutionary time scales. Our work shed a light on functional evolution of the EJC genes in eukaryotes, and facilitates to understand the co-evolutionary processes among protein families.     

Gene family evolution in green plants with emphasis on the origination and evolution of Arabidopsis thaliana genes

Gene family size variation is an important mechanism that shapes the natural variation for adaptation in various species. Despite its importance, the pattern of gene family size variation in green plants is still not well understood. In particular, the evolutionary pattern of genes and gene families remains unknown in the model plant Arabidopsis thaliana in the context of green plants. In this study, eight representative genomes of green plants are sampled to study gene family evolution and characterize the origination of A. thaliana genes, respectively. Four important insights gained are that: (i) the rate of gene gains and losses is about 0.001359 per gene every million years, similar to the rate in yeast, Drosophila, and mammals; (ii) some gene families evolved rapidly with extreme expansions or contractions, and 2745 gene families present in all the eight species represent the ‘core’ proteome of green plants; (iii) 70% of A. thaliana genes could be traced back to 450 million years ago; and (iv) intriguingly, A. thaliana genes with early origination are under stronger purifying selection and more conserved. In summary, the present study provides genome‐wide insights into evolutionary history and mechanisms of genes and gene families in green plants and especially in A. thaliana.     

Comprehensive Expression Analysis of Rice Armadillo Gene Family During Abiotic Stress and Development

Genes in the Armadillo (ARM)-repeat superfamily encode proteins with a range of developmental and physiological processes in unicellular and multicellular eukaryotes. These 42 amino acid, long tandem repeat-containing proteins have been abundantly recognized in many plant species. Previous studies have confirmed that Armadillo proteins constitute a multigene family in Arabidopsis. In this study, we performed a computational analysis in the rice genome (Oryza sativa L. subsp. japonica), and identified 158 genes of Armadillo superfamily. Phylogenetic study classified them into several arbitrary groups based on a varying number of non-conserved ARM repeats and accessory domain(s) associated with them. An in-depth analysis of gene expression through microarray and Q-PCR revealed a number of ARM proteins expressing differentially in abiotic stresses and developmental conditions, suggesting a potential roles of this superfamily in development and stress signalling. Comparative phylogenetic analysis between Arabidopsis and rice Armadillo genes revealed a high degree of evolutionary conservation between the orthologues in two plant species. The non-synonymous and synonymous substitutions per site ratios (Ka/Ks) of duplicated gene pairs indicate a purifying selection. This genome-wide identification and expression analysis provides a basis for further functional analysis of Armadillo genes under abiotic stress and reproductive developmental condition in the plant lineage.     

Plant CDK inhibitors: studies of interactions with cell cycle regulators in the yeast two-hybrid system and functional comparisons in transgenic Arabidopsis plants

. The cyclin-dependent kinase (CDK) inhibitors ICK1 and ICK2 have been shown to inhibit plant CDK activity in vitro, and the expression of ICK1 was able to inhibit cell division in the plant and modify plant growth and morphology. In order to characterize other ICK1-related inhibitor genes and understand possible differences among plant CDK inhibitors, the interactions of plant CDK inhibitors with cell cycle regulators were analysed in the yeast two-hybrid system and their functions were compared in transgenic Arabidopsis plants. Yeast two-hybrid results indicate that there are likely two groups of plant CDK inhibitors. The A-group inhibitors ICK1, ICK2, ICK6 and ICK7 interact with Cdc2a and three D-type cyclins (D1, D2 and D3), while the B-group inhibitors ICK4, ICK5 and ICKCr interact with D-type cyclins but not with Arabidopsis Cdc2a. ICK1 (A-group), and ICK4 and ICKCr (B-group) were expressed separately in transgenic Arabidopsis plants. Overexpression of the three inhibitor genes resulted in plants of a smaller size with serrated leaves and modified flowers. These plants also had reduced nuclear DNA content (polyploidy), suggesting that expression of these inhibitors affected endoreduplication. Further, there were apparent differences in the strength of effect among the inhibitors. These results provide the first evidence on the CDK inhibitory function for ICK4 and ICKCr. They also suggest that these CDK inhibitors play important roles in cell division and plant growth.