Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.     

A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.     

Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members

Lignin is an important biopolymer that is deposited in secondary cell walls of plant cells (e.g., tracheary elements) and in response to stresses such as wounding. Biosynthesis of lignin monomers occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters, subsequently reduced to the corresponding monolignols (hydroxycinnamoyl alcohols). 4CL is encoded by a family of four genes in Arabidopsis thaliana (At4CL1-At4CL4), which are developmentally regulated and co-expressed with other phenylpropanoid genes. We investigated in detail the wound-induced expression of At4CL1-At4CL4, and found that At4CL1 and At4CL2 mRNA accumulation follows biphasic kinetics over a period of 72 h, while At4CL4 expression is rapidly activated for a period of at least 12 h before declining. In order to localize cis-regulatory elements involved in the developmental and wound-induced regulation of the At4CL gene family members, At4CL promoter-beta-glucuronidase (GUS) reporter gene fusions were constructed and transferred into Arabidopsis plants. Analysis of these plants revealed that the promoter fragments direct discrete and distinct patterns of expression, some of which did not recapitulate expected patterns of wound-induced expression. The locations of regulatory elements associated with the At4CL2 gene were investigated in detail using a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the gene fused to GUS. Positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene were located both in the promoter and transcribed regions of the At4CL2 gene.An erratum to this article can be found at     

A comprehensive expression analysis of the Arabidopsis proline-rich extensin-like receptor kinase gene family using bioinformatic and experimental approaches

The Arabidopsis proline-rich extensin-like receptor kinase (PERK) family consists of 15 predicted receptor kinases. A comprehensive expression analysis was undertaken to identify overlapping and unique expression patterns within this family relative to their phylogeny. Three different approaches were used to study AtPERK gene family expression, and included analyses of the EST, MPSS and NASCArrays databases as well as experimental RNA blot analyses. Some of the AtPERK members were identified as tissue-specific genes while others were more broadly expressed. While in some cases there was a good association between these different expression patterns and the position of the AtPERK members in the kinase phylogeny, in other cases divergence of expression patterns was seen. The PERK expression data identified by the bioinformatics and experimental approaches were found generally to show similar trends and supported the use of data from large-scale expression studies for obtaining preliminary expression data. Thus, the bioinformatics survey for ESTs and microarrays is a powerful comprehensive approach for obtaining a genome-wide view of genes in a multigene family.     

Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector.

TcP2beta ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2beta genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2beta is located within the SIRE element, while in the other loci it is positioned within the first 50bases upstream of the AUG with an AG acceptor site at position -12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2beta H1. 8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71-76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.     

Distinctive features of plant organs characterized by global analysis of gene expression in Arabidopsis.

The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).     

Identification and expression analysis of twelve members of the nucleobase-ascorbate transporter (NAT) gene family in Arabidopsis thaliana

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments. This classification nicely correlates with the patterns of organ- and tissue-specific expression during the whole life cycle of A. thaliana. Interestingly, expression of two members of clade III, AtNAT7 and AtNAT8, was found to be up-regulated in undifferentiated tissues such as callus or tumors produced by Agrobacterium tumefaciens. Clade V comprises AtNAT12 possessing a hydrophilic N-terminal extension. Transient expression of green fluorescent protein (GFP) fusions in different systems showed that AtNAT12 along with AtNAT7 and -8 are located in the plasma membrane. Mutations in any of the AtNAT genes do not induce phenotypic alterations. The absence of obvious mutant phenotypes in single but also in double and triple mutants suggests a high degree of functional redundancy between AtNAT genes, but might also point to redundant functions provided by genes or pathways unrelated to the AtNATs.     

Genomic analysis and expression investigation of caleosin gene family in Arabidopsis

Caleosin is a common lipid-droplet surface protein, which has the ability to bind calcium. Arabidopsis (Arabidopsis thaliana) is considered a model organism in plant researches. Although there are growing researches about caleosin in the past few years, a systemic analysis of caleosins in Arabidopsis is still scarce. In this study, a comprehensive investigation of caleosins in Arabidopsis was performed by bioinformatics methods. Firstly, eight caleosins in Arabidopsis are divided into two types, L-caleosin and H-caleosin, according to their molecular weights, and these two types of caleosin have many differences in characteristics. Secondly, phylogenetic tree result indicates that L-caleosin may evolve from H-caleosin. Thirdly, duplication pattern analysis shows that segmental and tandem duplication are main reasons for Arabidopsis caleosin expansion with the equal part. Fourthly, the expression profiles of caleosins are also investigated in silico in different organs and under various stresses and hormones. In addition, based on promoter analysis, caleosin may be involved in calcium signal transduction and lipid accumulation. Thus, the classification and expression analysis of caleosin genes in Arabidopsis provide facilities to the research of phylogeny and functions in this gene family.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

Phylogenetic and expression analysis of the glutamate-receptor-like gene family in Arabidopsis thaliana

The ionotropic glutamate receptor (iGluR) gene family has been widely studied in animals and is determined to be important in excitatory neurotransmission and other neuronal processes. We have previously identified ionotropic glutamate receptor-like genes (GLRs) in Arabidopsis thaliana, an organism that lacks a nervous system. Upon the completion of the Arabidopsis genome sequencing project, a large family of GLR genes has been uncovered. A preliminary phylogenetic analysis divides the AtGLR gene family into three clades and is used as the basis for the recently established nomenclature for the AtGLR gene family. We performed a phylogenetic analysis with extensive annotations of the iGluR gene family, which includes all 20 Arabidopsis GLR genes, the entire iGluR family from rat (except NR3), and two prokaryotic iGluRs, Synechocystis GluR0 and Anabaena GluR. Our analysis supports the division of the AtGLR gene family into three clades and identifies potential functionally important amino acid residues that are conserved in both prokaryotic and eukaryotic iGluRs as well as those that are only conserved in AtGLRs. To begin to investigate whether the three AtGLR clades represent different functional classes, we performed the first comprehensive mRNA expression analysis of the entire AtGLR gene family. On the basis of RT-PCR, all AtGLRs are expressed genes. The three AtGLR clades do not show distinct clade-specific organ expression patterns. All 20 AtGLR genes are expressed in the root. Among them, five of the nine clade-II genes are root-specific in 8-week-old Arabidopsis plants.     

苹果柠檬酸合酶3基因家族成员鉴定及表达分析

柠檬酸合酶(citrate synthase 3, CS3)是细胞代谢途径中的关键酶之一,其活性调节着生物体的物质和能量代谢过程。本研究旨在从苹果全基因组中鉴定CS3基因家族成员,并进行生物信息学和表达模式分析,为研究苹果CS3基因的潜在功能提供理论基础。利用BLASTp基于GDR数据库鉴定苹果CS3家族成员,通过Pfam、SMART、MEGA5.0、clustalx.exe、ExPASy Proteomics Server、MEGAX、SOPMA、MEME和WoLF PSORT等软件分析CS3蛋白序列基本信息、亚细胞定位情况、结构域组成、系统进化关系以及染色体定位情况。利用酸含量的测定和实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术检测苹果6个CS3的组织表达和诱导表达特性。苹果CS3基因家族包含6个成员,这些CS3蛋白包括473−608个不等的氨基酸残基,等电点分布在7.21−8.82。亚细胞定位结果显示CS3蛋白分别定位在线粒体和叶绿体。系统进化分析可将其分为3类,各亚家族基因数量分别为2个。染色体定位结果显示,CS3基因分布在苹果不同的染色体上。蛋白二级结构以a-螺旋为主,其次是无规则卷曲,b-转角所占比例最小。筛选的6个家族成员在不同苹果组织中均有表达,整体表达趋势从高到低依次为MdCS3.4相对表达含量最高,MdCS3.6次之,其他家族成员相对表达量依次为MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5。qRT-PCR结果显示,MdCS3.1和MdCS3.3基因在酸含量较低的‘成纪1号’果肉中相对表达量最高,酸含量较高的‘艾斯达’果肉中MdCS3.2和MdCS3.3基因相对表达量最高。因此,本研究对不同苹果品种中CS3基因相对表达量进行了检测,并分析了其在苹果果实酸合成过程中的作用。结果表明,CS3基因在不同苹果品种中的相对表达量存在差异,为后续研究苹果品质形成机制提供了参考。     

Functional characterization of the Arabidopsis ubiquitin-specific protease gene family reveals specific role and redundancy of individual members in development

Ubiquitin-specific proteases (UBPs) are a highly conserved family of proteins in eukaryotes, and play critical roles in protein de-ubiquitination. Here we report a systematic genetic and expression profiling analysis of the UBP gene family in the Arabidopsis thaliana genome. Mutation analysis of 25 of the 27 member genes representing 13 of the 14 sub-families of the UBP gene family revealed that single-gene mutants of three genes in two sub-families exhibit visible phenotypes. Two of these three genes belonging to the UBP15 sub-family were selected for further characterization. The ubp15 mutants display narrower, serrated and flat rosette leaves, partially due to a defect in cell proliferation, as well as other phenotypes such as early flowering, weak apical dominance and reduced fertility, while the line over-expressing UBP15 shows opposite phenotypes. We demonstrated that UPB15 has UBP activity in vitro , and that this biochemical activity is essential for its in vivo function. A genetic interaction analysis among members of this sub-family revealed that UBP15 and UBP16, but not UBP17, have functional redundancy. Our data thus suggest that distinct UBPs, even within a closely related sub-family, can function in different developmental pathways. Although there are clearly functional redundancies among related sub-family members, those redundancies cannot be inferred simply based on the amino acid identity of the family members.     

Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis

Monocotyledons and dicotyledons are distinct, not only in their body plans and developmental patterns, but also in the structural features of their cell walls. The recent completion of the rice (Oryza sativa) genomic sequence and publication of the sequence data, together with the completed database of the Arabidopsis thaliana genome, provide the first opportunity to compare the full complement of cell-wall-related genes from the two distinct classes of flowering plants. We made this comparison by exploiting the fact that Arabidopsis and rice have type I and type II walls, respectively, and therefore represent the two extremes in terms of the structural features of plant cell walls. In this review article, we classify all cell-wall-related genes into 32 gene families, and generate their phylogenetic trees. Using these data, we can phylogenetically compare individual genes of particular interest between Arabidopsis and rice. This comparative genome approach shows that the differences in wall architecture in the two plant groups actually mirror the diversity of the individual gene families involved in the cell-wall dynamics of the respective plant species. This study also identifies putative rice orthologs of genes with well-defined functions in Arabidopsis and other plant species.     

Characterization and expression analysis of a serine acetyltransferase gene family involved in a key step of the sulfur assimilation pathway in Arabidopsis

Ser acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O-acetyl-Ser from L-Ser and acetyl-CoA, leading to synthesis of Cys. According to its position at the decisive junction of the pathways of sulfur assimilation and amino acid metabolism, SATases are subject to regulatory mechanisms to control the flux of Cys synthesis. In Arabidopsis (Arabidopsis thaliana) there are five genes encoding SATase-like proteins. Two isoforms, Serat3;1 and Serat3;2, were characterized with respect to their enzymatic properties, feedback inhibition by L-Cys, and subcellular localization. Functional identity of Serat3;1 and Serat3;2 was established by complementation of a SATase-deficient mutant of Escherichia coli. Cytosolic localization of Serat3;1 and Serat3;2 was confirmed by using fusion construct with the green fluorescent protein. Recombinant Serat3;1 was not inhibited by L-Cys, while Serat3;2 was a strongly feedback-inhibited isoform. Quantification of expression patterns indicated that Serat2;1 is the dominant form expressed in most tissues examined, followed by Serat1;1 and Serat2;2. Although Serat3;1 and Serat3;2 were expressed weakly in most tissues, Serat3;2 expression was significantly induced under sulfur deficiency and cadmium stress as well as during generative developmental stages, implying that Serat3;1 and Serat3;2 have specific roles when plants are subjected to distinct conditions. Transgenic Arabidopsis plants expressing the green fluorescent protein under the control of the five promoters indicated that, in all Serat genes, the expression was predominantly localized in the vascular system, notably in the phloem. These results demonstrate that Arabidopsis employs a complex array of compartment-specific SATase isoforms with distinct enzymatic properties and expression patterns to ensure the provision of Cys in response to developmental and environmental changes.