Duplication of centromeric histone H3 (HTR12) gene in Arabidopsis halleri and A. lyrata, plant species with multiple centromeric satellite sequences

Arabidopsis halleri and lyrata have three different major centromeric satellite sequences, a unique finding for a diploid Arabidopsis species. Since centromeric histones coevolve with centromeric satellites, these proteins would be predicted to show signs of selection when new centromere satellites have recently arisen. We isolated centromeric protein genes from A. halleri and lyrata and found that one of them, HTR12 (CENP-A), is duplicated, while CENP-C is not. Phylogenetic analysis indicates that the HTR12 duplication occurred after these species diverged from A. thaliana. Genetic mapping shows that HTR12 copy B has the same genomic location as the A. thaliana gene; the other copy (A, at the other end of the same chromosome) is probably the new copy. To test for selection since the duplication, we surveyed diversity at both HTR12 loci within A. lyrata. Overall, there is no strong evidence for an "evolutionary arms race" causing multiple replacement substitutions. The A. lyrata HTR12B sequences fall into three classes of haplotypes, apparently maintained for a long time, but they all encode the same amino acid sequence. In contrast, HTR12A has low diversity, but many variants are amino acid replacements, possibly due to independent selective sweeps within populations of the species.     

Selection on amino acid substitutions in Arabidopsis

Studies of nucleotide diversity have found an excess of low-frequency amino acid polymorphisms segregating in Arabidopsis thaliana, suggesting a predominance of weak purifying selection acting on amino acid polymorphism in this inbreeding species. Here, we investigate levels of diversity and divergence at synonymous and nonsynonymous sites in 6 circumpolar populations of the outbreeding Arabidopsis lyrata and compare these results with A. thaliana, to test for differences in mutation and selection parameters across genes, populations, and species. We find that A. lyrata shows an excess of low-frequency nonsynonymous polymorphisms both within populations and species wide, consistent with weak purifying selection similar to the patterns observed in A. thaliana. Furthermore, nonsynonymous polymorphisms tend to be more restricted in their population distribution in A. lyrata, consistent with purifying selection preventing their geographic spread. Highly expressed genes show a reduced ratio of amino acid to synonymous change for both polymorphism and fixed differences, suggesting a general pattern of stronger purifying selection on high-expression proteins.     

A role for F-actin in hexokinase-mediated glucose signaling

HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.     

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.Key words: Arabidopsis, F-actin, glucose signaling, hexokinase, hTalin, mitochondria, polysomes, protoplasts, transient expression assay, fluorescence microscopy     

Genome-wide comparison of nucleotide-binding site-leucine-rich repeat-encoding genes in Arabidopsis

Plants, like animals, use several lines of defense against pathogen attack. Prominent among genes that confer disease resistance are those encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) proteins. Likely due to selection pressures caused by pathogens, NB-LRR genes are the most variable gene family in plants, but there appear to be species-specific limits to the number of NB-LRR genes in a genome. Allelic diversity within an individual is also increased by obligatory outcrossing, which leads to genome-wide heterozygosity. In this study, we compared the NB-LRR gene complement of the selfer Arabidopsis thaliana and its outcrossing close relative Arabidopsis lyrata. We then complemented and contrasted the interspecific patterns with studies of NB-LRR diversity within A. thaliana. Three important insights are as follows: (1) that both species have similar numbers of NB-LRR genes; (2) that loci with single NB-LRR genes are less variable than tandem arrays; and (3) that presence-absence polymorphisms within A. thaliana are not strongly correlated with the presence or absence of orthologs in A. lyrata. Although A. thaliana individuals are mostly homozygous and thus potentially less likely to suffer from aberrant interaction of NB-LRR proteins with newly introduced alleles, the number of NB-LRR genes is similar to that in A. lyrata. In intraspecific and interspecific comparisons, NB-LRR genes are also more variable than receptor-like protein genes. Finally, in contrast to Drosophila, there is a clearly positive relationship between interspecific divergence and intraspecific polymorphisms.     

Genome wide gene expression in artificially synthesized amphidiploids of Arabidopsis

The merging of two different genomes occurs during the formation of amphidiploids, and the merged regulatory networks have the potential to generate a new gene expression pattern. We examined the genome-wide gene expression of two newly synthesized amphidiploids between Arabidopsis thaliana and the related species Arabidopsis lyrata subsp. lyrata and Arabidopsis halleri subsp. gemmifera. 1,137 (4.7%) and 1,316 (5.4%) of probesets showed differential gene expression in A. thaliana-A. halleri and A. thaliana-A. lyrata hybrids respectively, compared to the mid parent value and of these, 489 were in common. Genes that differed in expression between the parental lines tended to have an expression level in both hybrids differing from the mid parent value. In contrast to protein coding genes, there is little differential expression of transposons. Genes in the categories of chloroplast-targeted and response to stress were overrepresented in the non-additively expressed genes in both amphidiploids. As these genes have the potential to contribute directly to the plant phenotype, we suggest that rapid changes of gene expression in amphidiploids might be important for producing greater biomass.     

Kinetic and proteomic analyses of <Emphasis Type="Italic">S</Emphasis>-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Summary. Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.     

Expression analysis of Arabidopsis thaliana small secreted protein genes

Small proteins secreted to the extracellular matrix in plants regulate many physiological activities, including pathogen response, material transport, and morphogenesis, but the functions of most small secreted proteins have not been elucidated except for some well-known small secreted proteins. To predict the functions and physiological roles of unidentified small secreted proteins, information on their expression patterns is valuable. Here, we report expression analysis of Arabidopsis thaliana small secreted protein (ATSP) genes that encode proteins possessing a signal peptide at N-terminal, and protein sizes were less than 100 amino acid residues. By promoter:reporter experiments, we examined the expression of 122 ATSPs, including 47 unannotated ATSPs that do not have any discernable motifs, in tissues and at the cellular level in Arabidopsis seedlings, and floral organs. As a result, 79 ATSP genes were expressed in various regions of the seedlings, and 37 ATSP genes were specifically expressed.     

高等植物己糖激酶基因研究进展

己糖激酶(HXK)具有催化己糖磷酸化的作用,是植物体呼吸代谢过程中的关键酶之一。近十几年的研究发现,HXK在植物的糖感知和糖信号转导过程中扮演重要的角色。目前GenBank已登录28种高等植物的HXK同源基因,其在不同物种中均以多基因家族形式存在。HXK基因家族多数成员包括9个外显子,编码492-522个氨基酸。HXK亚细胞定位研究发现,植物HXK家族成员主要分布于线粒体,少数成员存在于细胞质、叶绿体和质体基质中。植物HXK基因家族大部分成员在不同器官或组织中均有表达,但是拟南芥(Arabidopsis thaliana)AtHKL3和水稻(Oryza sativa)OsHXK10仅在花中表达。高等植物部分HXK不仅影响植物生长发育,还调控植物激素信号转导以及调节植物花青素合成途径中相关基因表达。应用MEGA 4.0软件对18个物种HXK基因氨基酸序列构建系统进化树,HXK基因序列聚为7小支,聚类关系能反映不同基因结构和功能的差异。     

High diversity due to balancing selection in the promoter region of the Medea gene in Arabidopsis lyrata

Molecular imprinting is the differential expression and/or silencing of alleles according to their parent of origin [1, 2]. Conflicts between parents, or parents and offspring, should cause "arms races," with accelerated evolution of the genes involved in imprinting. This should be detectable in the evolution of imprinting genes' protein sequences and in the promoter regions of imprinted genes. Previous studies, however, found no evidence of more amino acid substitutions in imprinting genes [1, 3]. We have analyzed sequence diversity of the Arabidopsis lyrata Medea (MEA) gene and divergence from the A. thaliana sequence, including the first study of the promoter region. In A. thaliana, MEA is imprinted, with paternal alleles silenced in endosperm cells [4, 5], and also functions in the imprinting machinery [4, 6]; MEA protein binding at the MEA promoter region indicates self-regulated imprinting [7-9]. We find the same paternal MEA allele silencing in A. lyrata endosperm but no evidence for adaptive evolution in the coding region, whereas the 5' flanking region displays high diversity, with distinct haplotypes, suggesting balancing selection in the promoter region.     

Characterization of an Arabidopsis thaliana Homologue of the Nuclear Export Receptor CAS by its Interaction with Importinα

Abstract: We have previously described four genes encoding different Importin α-like proteins from Arabidopsis thaliana . Here we describe the putative nuclear export receptor for Importin α. Using protein interaction assays in the yeast two-hybrid system, we characterized an Arabidopsis protein showing high similarity to human CAS, the nuclear export receptor for Importin α. Arabidopsis CAS specifically bound to four different plant Importin α proteins but not to proteins containing leucine-rich nuclear export signals (NESs) that are recognized by Exportin 1 (XPO1/CRM1). Like all members of the Importin β family, Arabidopsis CAS also interacted with the regulatory GTPase Ran. Deletion of 15 amino acid residues from the amino terminus of CAS abolished binding of Importin α, but did not influence the interaction with the GTPase Ran. We found two regions of Importin α1 that profoundly influence the binding to CAS: the amino terminal Importin beta-binding (IBB) domain and the carboxy terminus. Whereas the IBB domain did not directly bind to CAS, but might rather affect the interaction through conformational changes within the Importin α protein, the carboxy terminal domain strongly interacted with CAS.     

Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.     

Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.     

Comparative gene mapping in Arabidopsis lyrata chromosomes 1 and 2 and the corresponding A. thaliana chromosome 1: recombination rates, rearrangements and centromere location

To add detail to the genetic map of Arabidopsis lyrata, and compare it with that of A. thaliana, we have developed many additional markers in the A. lyrata linkage groups, LG1 and LG2, corresponding to A. thaliana chromosome 1. We used a newly developed method for marker development for single nucleotide polymorphisms present in gene sequences, plus length differences, to map genes in an A. lyrata family, including variants in several genes close to the A. thaliana centromere 1, providing the first data on the location of an A. lyrata centromere; we discuss the implications for the evolution of chromosome 1 of A. thaliana. With our larger marker density, large rearrangements between the two Arabidopsis species are excluded, except for a large inversion on LG2. This was previously known in Capsella; its presence in A. lyrata suggests that, like most other rearrangements, it probably arose in the A. thaliana lineage. Knowing that marker orders are similar, we can now compare homologous, non-rearranged map distances to test the prediction of more frequent crossing-over in the more inbreeding species. Our results support the previous conclusion of similar distances in the two species for A. lyrata LG1 markers. For LG2 markers, the distances were consistently, but non-significantly, larger in A. lyrata. Given the two species' large DNA content difference, the similarity of map lengths, particularly for LG1, suggests that crossing-over is more frequent across comparable physical distances in the inbreeder, A. thaliana, as predicted.     

拟南芥和琴叶拟南芥中MADS-box基因的比较进化分析

MADS-box基因编码一类转录因子。在被子植物中,MADS-box基因对于营养生长和生殖发育都有重要的调控作用,是植物体(特别是花序、花和果实)的正常发育所不可或缺的。为了理解近缘物种在遗传基础上的异同,我们对拟南芥(Arabidopsis thaliana)和琴叶拟南芥(A.lyrata)基因组中MADS-box基因的拷贝数目和进化式样进行了比较分析。通过搜索公共数据库,我们在拟南芥和琴叶拟南芥中分别鉴定出了106和115个基因。系统发育分析的结果表明,这些基因属于I型和II型MADS-box基因。在两个物种分化之后,II型基因的拷贝数目变化不大,I型基因则经历了多次独立的基因丢失和获得事件。通过比较这些基因在染色体上的排列,我们不但鉴定出了存在微共线性的基因组区段,而且发现新基因产生的主要机制是串联重复和散在重复。分子进化的研究进一步表明,I型和II型基因在进化式样上存在着显著差异:II型基因在进化中一般都受到了较强的选择压力,而I型基因大多受到的选择压力较弱。本研究将为深入理解近缘物种在基因和基因组层面上的异同、探讨物种分化和生物多样性形成的机制等问题提供新思路。     

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.