The actin multigene family in Populus: organization, expression and phylogenetic analysis

Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of π_syn = 0.01205 was detected, and the π_non-syn/π_syn ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.     

Genome-Wide Identification of the Invertase Gene Family in Populus

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.     

The syringaldazine-oxidizing peroxidase PXP 3-4 from poplar xylem: cDNA isolation,characterization and expression

The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases, we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar to those of the corresponding protein from poplar xylem (enzymatic properties, isoelectric point, and migration in a native gel). PXP 3-4 was expressed in the stem and in the root xylem. The data demonstrate that PXP 3-4 (and/or PXP 5) are present in differentiating xylem, supporting a function in secondary cell wall formation.     

Characterization of genes with tissue-specific differential expression patterns in Populus

Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

Variation in gene transcription and protein for key enzymes of carbohydrate metabolism in poplar xylem with respect to growth phase

We compared gene expression levels for enzymes of carbohydrate metabolism in the twig xylem of two Populus species with the seasonal levels of starch and soluble sugars (sucrose, glucose, and fructose) and relative levels of the enzymes. Plants of Populus deltoides Bartr. ex Marsh and P. balsamifera L., 3–4 years old, were grown outside in Lubbock, TX, USA in 43 L pots. The xylem in the middle portion of the twigs was sampled during the dormant period (November–February), at bud break (for P. balsamifera), and during the growth flush (April–July). The gene expression for ADP-glucose pyrophosphorylase (AGPase), sucrose synthase (SuSy), and sucrose-phosphate synthase (SPS) generally coincided with the levels of the carbohydrates in whose metabolism these enzymes are involved. Gene expression for AGPase and its protein levels were high when the xylem starch content was high (growing period). However, P. balsamifera maintained high AGPase levels in dormant and growing twigs, unlike P. deltoides whose dormant twigs had low AGPase and low gene expression. Compared to growing twigs, gene expression for SuSy and SPS and their protein levels were higher in dormant twigs when soluble sugar content was higher. No down-regulation of these genes appears to occur when pools of the associated carbohydrates are high. Contrary to our expectation, the gene expression for β-amylase was highest in growing twigs when starch content was high. High β-amylase gene expression in growing twigs may be involved in maintaining a sufficient level of soluble sugars for growth through possibly controlling the extent of starch accumulation.     

Gene expression in tension wood and bast fibres

Tension wood is produced in the xylem of some angiosperm trees, such as poplar (Populus spp.), whereas bast fibers are phloem-derived cells best known from annual crops, such as flax (Linum usitatissimum L.). Despite their different origins, secondary walls of both tension wood and bast fibers share distinctive properties, including an abundance of axially oriented, crystalline cellulose produced in a distinctive gelatinous-type layer. Because of these unique properties, tension wood and phloem fibers have separately been the subject of at least nine previously published gene or protein profiling studies. Here we review these experiments with a focus on those genes, whose expression distinguishes both tension wood and bast fibers from the more predominant types of xylem found elsewhere in the stem. Notable among these is an evolutionarily distinctive group of fasciclin-like arabinogalactan proteins (FLA) and a putative rhamnogalacturonan lyase.     

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.     

Identification of candidate genes in Arabidopsis and Populus cell wall biosynthesis using text-mining, co-expression network analysis and comparative genomics

Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of biofuels from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidence supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database, and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional characterization in relation to cell wall biosynthesis.