Large-scale genome duplications and paralog divergence in fish

Based on fish genomic studies, we review mechanisms of divergence in duplicated genes (paralogs), resulted in small (“subfunctionalization”) or large (“neofunctionalization”) changes in paralogs. Gene divergence occurs due to several processes, such as non-synonymous substitutions, exon-intron structure rearrangement, and alterations in regulatory regions, which cause differential temporal or spatial expression of paralogous gene copies during ontogenesis.     

Alternative splicing of flowering regulatory gene LFY in Arabidopsis thaliana

为研究拟南芥成花调控基因LFY,我们采用RT-PCR方法分离克隆了三种选择性剪接的片段,分别命名为LFY1239,LFY1263和LFY1275.序列分析表明LFY1263包含一个大小为1 263 bp的开放阅读框,与之前报道的LFY基因片段大小相同,而LFY1239在第一外显子的3'端缺失了36 bp,LFY1275在第一内含子的3'末端插入了12 bp.对几种片段表达部位的分析显示,LFY1239只能在营养生长期的莲座叶中表达,而LFY1263和LFY1275在营养生长期和花期的花器官和莲座叶中都可以检测到,并且,LFY1263呈现出主导地位,LFY1275与LFY1263表达的比例表现为花器官高于莲座叶,该比例的变化可能预示着与成花调控有关.     

Hydrogen peroxide-induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.     

Association of glutathione with flowering in Arabidopsis thaliana

In order to study the relationship between GSH and flowering, wild-type and late-flowering mutant, fca-1, of Arabidopsis thaliana were treated with L-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, under long-day conditions. BSO treatment of the fca-1 mutant starting at 17 d after imbibition promoted flowering. However, when the treatment was started at 12 d after imbibition, BSO treatment at 10(-4) M resulted in an inhibition of flowering. This inhibitory effect of BSO on flowering was abolished by GSH treatment at 10(-4) M, although GSH treatment at an increased concentration of 10(-3) M clearly delayed flowering. In contrast, BSO treatment of wild-type plants starting at 12 d after imbibition promoted flowering, whose effect was abolished by GSH application. In the fca-1 mutant, whose endogenous GSH levels were high, chilling treatment lowered the GSH levels and promoted flowering, as was the case in the BSO treatment. An A. thaliana mutant, cad2-1, which has a defect in GSH biosynthesis also exhibited late flowering. The late-flowering phenotype of this mutant tended to be strengthened by BSO and abolished by GSH treatment. These results suggest that flowering is associated with the rate of GSH biosynthesis and/or the levels of GSH in A. thaliana.     

Characterization of the genome of Arabidopsis thaliana

The small crucifer Arabidopsis thaliana has many useful features as an experimental organism for the study of plant molecular biology. It has a four-week life-cycle, only five chromosomes and a genome size less than half that of Drosophila. To characterize the DNA sequence organization of this plant, we have randomly selected 50 recombinant lambda clones containing inserts with an average length of 12,800 base-pairs and analyzed their content of repetitive and unique DNA by various genome blot, restriction digestion and RNA blot procedures. The following conclusions can be drawn. The DNA represented in this random sample is composed predominantly of single-copy sequences. This presumably reflects the organization of the Arabidopsis genome as a whole and supports prior conclusions reached on the basis of kinetics of DNA reassociation. The DNA that encodes the ribosomal RNAs constitutes the only major class of cloned nuclear repetitive DNA. It consists of approximately 570 tandem copies of a heterogeneous 9900-base-pair repeat unit. There is an average of approximately 660 copies of the chloroplast genome per cell. Therefore, the chloroplast genome constitutes the major component of the repetitive sequences found in A. thaliana DNA made from whole plants. The inner cytosine residue in the sequence C-C-G-G is methylated more often than the outer in the tandem ribosomal DNA units, whereas very few differences in the methylation state of these two cytosine residues are detected in unique sequences.     

Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T₁ lines were obtained, and a 3:1 segregation ratio was obtained in the T₂ generation of each line. In the T₃ generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.     

Transient gene expression and influence of promoters on foreign gene expression in Arabidopsis thaliana

Summary The influence of a variety of parameters was investigated on polyethylene glycol (PEG)-mediated transient nptII and gus gene expression in mesophyll protoplasts of Arabidopsis thaliana ecotype, Estland, in order to develop a suitable transient gene expression system. The investigation revealed that a combination of 20% PEG, incubation time of 15 min, 20–30 μg plasmid concentration per ml along with 50 μg carrier DNA m/l, and inclusion of calcium and magnesium ions during transfection followed by a culture period of 24 h registered maximum NPTII activity. Of the various promoters used for driving expression of the gus gene, the ubiquitin promoter from A. thaliana was the most efficient followed by 35S promoter of the CaMV and the actin promoter of rice. For comparison, similar studies in protoplasts of rice, wheat, and Brassica also revealed the differences in strength of these promoters. Arabidopsis ubiquitin promoter was the most effective in Brassica, and the rice actin1 promoter was the most effective in rice and wheat.     

Diversity of flowering responses in wild Arabidopsis thaliana strains

Although multiple environmental cues regulate the transition to flowering in Arabidopsis thaliana, previous studies have suggested that wild A. thaliana accessions fall primarily into two classes, distinguished by their requirement for vernalization (extended winter-like temperatures), which enables rapid flowering under long days. Much of the difference in vernalization response is apparently due to variation at two epistatically acting loci, FRI and FLC. We present the response of over 150 wild accessions to three different environmental variables. In long days, FLC is among those genes whose expression is most highly correlated with flowering. In short days, FRI and FLC are less important, although their contribution is still significant. In addition, there is considerable variation not only in vernalization response, but also in the response to differences in day length or ambient growth temperature. The identification of accessions that flower relatively early or late in specific environments suggests that many of the flowering-time pathways identified by mutagenesis, such as those that respond to day length, contribute to flowering-time variation in the wild. In contrast to differences in vernalization requirement, which are mainly mediated by FRI and FLC, it seems that variation in these other pathways is due to allelic effects at several different loci.     

Effects of gene expression on molecular evolution in Arabidopsis thaliana and Arabidopsis lyrata

We analyzed the complete genome sequence of Arabidopsis thaliana and sequence data from 83 genes in the outcrossing A. lyrata, to better understand the role of gene expression on the strength of natural selection on synonymous and replacement sites in Arabidopsis. From data on tRNA gene abundance, we find a good concordance between codon preferences and the relative abundance of isoaccepting tRNAs in the complete A. thaliana genome, consistent with models of translational selection. Both EST-based and new quantitative measures of gene expression (MPSS) suggest that codon preferences derived from information on tRNA abundance are more strongly associated with gene expression than those obtained from multivariate analysis, which provides further support for the hypothesis that codon bias in Arabidopsis is under selection mediated by tRNA abundance. Consistent with previous results, analysis of protein evolution reveals a significant correlation between gene expression level and amino acid substitution rate. Analysis by MPSS estimates of gene expression suggests that this effect is primarily the result of a correlation between the number of tissues in which a gene is expressed and the rate of amino acid substitution, which indicates that the degree of tissue specialization may be an important determinant of the rate of protein evolution in Arabidopsis.     

The shrunken genome of Arabidopsis thaliana

This paper examines macro and micro-level patterns of genome size evolution in the Brassicaceae. A phylogeny of 25 relatives of Arabidopsis thaliana was reconstructed using four molecular markers under both parsimony and Bayesian methods. Reconstruction of genome size (C value) evolution as a discrete character and as a continuous character was also performed. In addition, size dynamics in small chromosomal regions were assessed by comparing genomic clones generated for Arabidopsis lyrata and for Boechera stricta to the fully sequenced genome of A. thaliana. The results reveal a sevenfold variation in genome size among the taxa investigated and that the small genome size of A. thaliana is derived. Our results also indicate that the genome is free to increase or decrease in size across these evolutionary lineages without a directional bias. These changes are accomplished by insertions and deletions at both large and small-scales occurring mostly in intergenic regions, with repetitive sequences and transposable elements implicated in genome size increases. The focus upon taxa relatively closely related to the model organism A. thaliana, and the combination of complementary approaches, allows for unique insights into the processes driving genome size changes.     

Mutations in AP22.65 accelerate flowering in Arabidopsis thaliana

Identification of the gene(s) responsible for flowering time in Arabidopsis has significant implications. We used the T-DNA insertion library of Arabidopsis thaliana to screen an early-flowering mutant that exhibits accelerated flowering under short-day conditions. AP22.65, a novel flowering-time gene in that species, was isolated and identified via genome-walking and bioinformatics analysis. The flowering time of AP22.65-complementing plants was similar to that of the Col-0 wild type (WT). Conversely, its overexpression delayed flowering. Consistent with this phenotype, expression of AP22.65 was decreased in the ap22.65-1 mutant, recovered in AP22.65-complementing plants, and increased in AP22.65-overexpressing plants. Compared with the WT, expression levels of critical genes in different flowering pathways, i.e., SPY, FLC, GI, CO, FT, and LFY, were down-regulated in loss-of-function mutants. Expression of AP22.65 was distributed in flowers, siliques, rosette leaves, and whole seedlings. Therefore, this gene may be a negative regulator of Arabidopsis flowering.