The sucrose synthase gene family in Populus: structure, expression, and evolution

Sucrose synthase is a key enzyme in sucrose metabolism in plant cells, and it is involved in the synthesis of cell wall cellulose. Although the sucrose synthase gene (SUS) family in the model plants Arabidopsis thaliana has been characterized, little is known about this gene family in trees. This study reports the identification of two novel SUS genes in the economically important poplar tree. These genes were expressed predominantly in mature xylem. Using molecular cloning and bioinformatics analysis of the Populus genome, we demonstrated that SUS is a multigene family with seven members that each exhibit distinct but partially overlapping expression patterns. Of particular interest, three SUS genes were preferentially expressed in the stem xylem, suggesting that poplar SUSs are involved in the formation of the secondary cell wall. Gene structural and phylogenetic analyses revealed that the Populus SUS family is composed of four main subgroups that arose before the separation of monocots and dicots. Phylogenetic analyses associated with the tissue- and organ-specific expression patterns. The high intraspecific nucleotide diversity of two SUS genes was detected in the natural population, and the π _nonsyn/π _syn ratio was significantly less than 1; therefore, SUS genes appear to be evolving in Populus, primarily under purifying selection. This is the first comprehensive study of the SUS gene family in woody plants; the analysis includes genome organization, gene structure, and phylogeny across land plant lineages, as well as expression profiling in Populus.     

Expression and nucleotide diversity of the poplar <Emphasis Type="Italic">COBL</Emphasis> gene

The COBRA gene encodes a putative glycosyl-phosphatidylinositol-anchored protein that regulates cellulose deposition and oriented cell expansion in the plant cell wall. This study reports the identification of PtCOBL4, a first COBRA gene from the tree Populus tomentosa. The full-length cDNA of PtCOBL4 was isolated from a xylem cDNA library. The deduced protein sequence shares 72.7% identity with Arabidopsis AtCOBL4 protein involved in secondary cell wall deposition. Analysis of differential tissue expression by real-time polymerase chain reaction (PCR) indicated that PtCOBL4 is expressed predominantly in the mature xylem zone. By using the sequenced whole genome and DNA microarray data of Populus, we demonstrated that COBRA is a multigene family of 11 members, each of which exhibit different tissue-specific expression patterns. To evaluate the functional consequences of nucleotide polymorphisms in the PtCOBL4 locus, the patterns of variation in a 2,002-bp region of the gene were surveyed in 40 unrelated individuals representative of almost the entire natural range of P. tomentosa. Sixty-one single-nucleotide polymorphisms (SNPs) were identified at a frequency of one SNP per 32.8 bp of sequence, giving an estimated nucleotide diversity of π _T = 0.00800 and θ _w = 0.00716. Within coding regions, nonsynonymous diversity (π _nonsyn = 0.00285) was markedly lower than synonymous diversity (π _syn = 0.02128); the π _nonsyn/π _syn ratio was 0.13, significantly less than 1, indicating that the synonymous sites were subject to strong purifying selection. These results provide the necessary foundation for improving the quantity and quality of cellulose via genetic engineering or by candidate-gene-based association genetics in P. tomentosa.     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

An extensive analysis of R2R3-MYB regulatory genes from Fagus crenata

Using pairs of degenerate primers, we conducted a polymerase chain reaction to amplify the partial R2R3 domains of a majority of the R2R3-MYB family genes from Fagus crenata and identified a total of 85 independent gene fragments. By phylogenetic analysis of the deduced amino acid sequences, we found that many of the beech genes clustered with members from Arabidopsis, suggesting that these members represent beech orthologs of Arabidopsis. Some of the orthologous relationships became more evident when the complete gene structures were compared. Further, a large number of genes formed an additional and expanding cluster, independent from the other subgroups. These members were further compared with the Populus and Vitis family genes. In the epidermal cell fate clade, expansion of the beech family genes was comparable with those of the Populus and Vitis families, but the number of genes present in every subclade fluctuated extensively. Beech genes were abundant in the general flavonoid pathway regulation and TT2-related subclades; no beech gene was included in the anthocyanin-related subclade. Further analysis of the newly amplified regulatory genes to elucidate their functions may clarify the role of these genes in the evolution of plant species.     

Unbiased estimation of symmetrical directional mutation pressure from protein-coding DNA

The most generally applicable procedure for obtaining estimates of the symmetrical, or strand-nonspecific, directional mutation pressure (μ_D) on protein-coding DNA sequences is to determine the G+C content at synonymous codon sites (P _syn), and to divide P _syn by twice the arithmetic mean of the G+C content at synonymous codon sites of a large number of randomly generated, synonymously coding DNA sequences (P _syn). Unfortunately, the original procedure yields biased estimates of P _syn and μ_D and is computationally expensive. We here present a fast procedure for estimating unbiased μ_D values. The procedure employs direct calculation of P _syn (≈P _syn) and two normalization procedures, one for P _syn≤P _syn and another for P _syn≥P _syn. The normalization removes a bias sometimes caused by codons specifying arginine, asparagine, isoleucine, and leucine. Consequently, comparison of protein-coding genes that are translated using different genetic codes is facilitated. Received: 5 May 1995 / Accepted: 30 November 1995     

The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family,defined by the adjacent,region-specific homeotic gene spalt

 We report the full coding sequence of a new Drosophila gene, spalt-related, which is homologous and adjacent to the region-specific homeotic gene, spalt. Both genes have three widely spaced sets of C₂H₂ zinc finger motifs, but spalt-related encodes a fourth pair of C-terminal fingers resembling the Xenopus homologue, Xsal-1. The degrees of sequence divergence among all three members of this family are comparable, suggesting that the Drosophila genes originated from an ancient gene duplication. The spalt-related gene is expressed with quantitative variations from mid-embryogenesis (8–12 h) to the adult stage, but not in ovaries or early embryos. Expression is localized to limited parts of the body, including specific cell populations in the nervous system. In the wing disc, spalt and spalt-related are expressed in indistinguishable domains; in the nervous system and some other organs the expression patterns extensively overlap but are not identical, indicating that the genes have partially diverged in terms of developmental regulation. A characteristic central set of zinc fingers specifically binds to an A/T-rich consensus sequence, defining some DNA binding properties of this ancient family of nuclear factors. Received: 31 July 1996 / Accepted: 4 September 1996     

BAHD superfamily of acyl-CoA dependent acyltransferases in <Emphasis Type="Italic">Populus</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by “in silico” northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members’ evolutionary relationships and gene functions implicated in plant growth, development and metabolism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene

‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male parental line, ‘Krymka’, with an ACC oxidase (CMACO-1) gene in antisense orientation is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation analysis in the T₁ generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon male parental lines have potential for use in the production of ‘Galia’ F₁ hybrids with improved shelf life.     

The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression

Background  

Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus.     

Search for nucleotide diversity patterns of local adaptation in dehydrins and other cold-related candidate genes in Scots pine (Pinus sylvestris L.)

Nucleotide variation at several cold candidate genes including seven members of the dehydrin gene family was surveyed in haplotypes of Scots pine (Pinus sylvestris) sampled in populations showing divergence for cold tolerance in Europe. Patterns of nucleotide diversity, linkage disequilibrium, and frequency spectrum of alleles were compared between north and south populations to search for signs of directional selection potentially underlying adaptation to cold. Significant differentiation between populations in allelic frequency or haplotype structure was detected at dhn1, dhn3, and abaH loci. Allelic dimorphism with no evidence of haplotype clustering by geographical distribution was found at dhn9. An excess of fixed non-synonymous mutations as compared to the outgroup P. pinaster pine species was found at dhn1. Differences in nucleotide polymorphisms were found between the members of the Kn class of dehydrin upregulated during cold acclimation (average π_sil = 0.004) as compared to the SKn class (average π_sil = 0.024). The multilocus nucleotide diversity at silent sites (θ _W = 0.009) was moderate compared to other conifer species, but higher than previous estimates for Scots pine. There was an excess of rare and high frequency derived variants as revealed by significantly negative multilocus value of Tajima’s D (D = −0.72, P < 0.01) and negative mean value of Fay and Wu H statistics (H = −0.50). The level of linkage disequilibrium decayed rapidly with an average expected r ² of 0.2 at about 200 bp. Overall, there was a positive correlation between polymorphism and divergence at ten loci when outgroup sequence was available. The discovered polymorphism will be used for further evaluation of the adaptive role of genes through association mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression analyses of AtMRP4 , a novel MRP -like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated. Received: 20 February 1998 / Accepted: 9 March 1998     

Footprints of intragenic recombination at HLA loci

 To evaluate the effect of balancing selection and intragenic recombination (or gene conversion) at six individual HLA loci, synonymous nucleotide diversity in different exon groups is examined within (π_w) and between (π_b) allelic lineages that may be defined by either serological or DNA sequence differences. Both π values are high in exons which encode for the peptide binding region (PBR) and tend to decrease in other exons. The value of π_w is significantly smaller than that of π_b in any exon of any locus. However, even π_w is much greater than nucleotide diversity at non-HLA loci. These observations provide additional strong evidence for the operation of balancing selection in PBR-encoding exons and its indirect effects on polymorphism at linked neighboring regions. It appears that allelic lineages have generally evolved in isolation but the linkage relationships within and between exons are incomplete throughout the long evolutionary history. To quantify intragenic recombination and account for the large discrepancy between the HLA and non-HLA diversity, a population genetics model is analyzed with special reference to the evolution of modern humans. The analysis suggests that the recombination rate between two sites 1000 base pairs apart is about 10^–5 per generation and that the effective size of human populations (equivalent roughly to the number of breeding individuals in a randomly mating population) has dropped from 10⁵ to 10⁴ in most of the Quaternary. One possibility for this reduction is discussed. Received: 11 August 1997 / Revised: 8 October 1997