Guohua Chai

Genome-Wide Identification, Evolutionary Expansion, and Expression Profile of Homeodomain-Leucine Zipper Gene Family in Poplar (Populus trichocarpa)

Background Homeodomain-leucine zipper (HD-ZIP) proteins are plant-specific transcriptional factors known to play crucial roles in plant development. Although sequence phylogeny analysis of Populus HD-ZIPs was carried out in a previous study, no systematic analysis incorporating genome organization, gene structure, and expression compendium has been conducted in model tree species Populus thus far. Principal Findings In this study, a comprehensive analysis of Populus HD-ZIP gene family was performed. Sixty-three full-length HD-ZIP genes were found in Populus genome. These Populus HD-ZIP genes were phylogenetically clustered into four distinct subfamilies (HD-ZIP I–IV) and predominately distributed across 17 linkage groups (LG). Fifty genes from 25 Populus paralogous pairs were located in the duplicated blocks of Populus genome and then preferentially retained during the sequential evolutionary courses. Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus HD-ZIP gene family. Microarray analysis has shown that 21 Populus paralogous pairs have been differentially expressed across different tissues and under various stresses, with five paralogous pairs showing nearly identical expression patterns, 13 paralogous pairs being partially redundant and three paralogous pairs diversifying significantly. Quantitative real-time RT-PCR (qRT-PCR) analysis performed on 16 selected Populus HD-ZIP genes in different tissues and under both drought and salinity stresses confirms their tissue-specific and stress-inducible expression patterns. Conclusions Genomic organizations indicated that segmental duplications contributed significantly to the expansion of Populus HD-ZIP gene family. Exon/intron organization and conserved motif composition of Populus HD-ZIPs are highly conservative in the same subfamily, suggesting the members in the same subfamilies may also have conservative functionalities. Microarray and qRT-PCR analyses showed that 89% (56 out of 63) of Populus HD-ZIPs were duplicate genes that might have been retained by substantial subfunctionalization. Taken together, these observations may lay the foundation for future functional analysis of Populus HD-ZIP genes to unravel their biological roles.

Comprehensive analysis of CCCH zinc finger family in poplar (Populus trichocarpa)

BackgroundCCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date.ResultsIn the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X11-C-X6-C-X3-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs (81 of 90, 90 %) are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue-specific expression and responses to drought stress treatment of 12 selected Populus CCCH genes.ConclusionsThis study provides the first systematic analysis of the Populus CCCH proteins. Comprehensive genomic analyses suggested that segmental duplications contribute significantly to the expansion of Populus CCCH gene family. Transcriptome profiling provides first insights into the functional divergences among members of Populus CCCH gene family. Particularly, some CCCH genes may be involved in wood development while others in drought tolerance regulation. Our results presented here may provide a starting point for the functional dissection of this family of potential RNA-binding proteins.

Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar (Populus trichocarpa)

Calcium-dependent protein kinases (CDPKs) are Ca2+-binding proteins known to play crucial roles in Ca2+ signal transduction pathways which have been identified throughout plant kingdom and in certain types of protists. Genome-wide analysis of CDPKs have been carried out in Arabidopsis, rice and wheat, and quite a few of CDPKs were proved to play crucial roles in plant stress responsive signature pathways. In this study, a comprehensive analysis of Populus CDPK and its closely related gene families was performed, including phylogeny, chromosome locations, gene structures, and expression profiles. Thirty Populus CDPK genes and twenty closely related kinase genes were identified, which were phylogenetically clustered into eight distinct subfamilies and predominately distributed across fifteen linkage groups (LG). Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus CDPK gene family. Furthermore, microarray analysis showed that a number of Populus CDPK and its closely related genes differentially expressed across disparate tissues and under various stresses. The expression profiles of paralogous pairs were also investigated to reveal their evolution fates. In addition, quantitative real-time RT-PCR was performed on nine selected CDPK genes to confirm their responses to drought stress treatment. These observations may lay the foundation for future functional analysis of Populus CDPK and its closely related gene families to unravel their biological roles.

CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase, Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed(1[C][W])

Disruption of a glucomannan synthase alters cellulose crystallinity and spatial distribution, yielding thinner adherent mucilage with increased density in seeds of Arabidopsis. Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.

R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time

In plants, the R2R3-MYB gene family contains many pairs of paralogous genes, which play the diverse roles in developmental processes and environmental responses. The paper reports the characterization of 81 pairs of Populus R2R3-MYB genes. Chromosome placement, phylogenetic, and motif structure analyses showed that these gene pairs resulted from multiple types of gene duplications and had five different gene fates. Tissue expression patterns revealed that most duplicated genes were specifically expressed in the tissues examined. qRT-PCR confirmed that nine pairs were highly expressed in xylem, of which three pairs (PdMYB10/128, PdMYB90/167, and PdMYB92/125) were further functionally characterized. The six PdMYBs were localized to the nucleus and had transcriptional activities in yeast. The heterologous expression of PdMYB10 and 128 in Arabidopsis increased stem fibre cell-wall thickness and delayed flowering. In contrast, overexpression of PdMYB90, 167, 92, and 125 in Arabidopsis decreased stem fibre and vessel cell-wall thickness and promoted flowering. Cellulose, xylose, and lignin contents were changed in overexpression plants. The expression levels of several genes involved in secondary wall formation and flowering were affected by the overexpression of the six PdMYBs in Arabidopsis. This study addresses the diversity of gene duplications in Populus R2R3-MYBs and the roles of these six genes in secondary wall formation and flowering control.

Poplar PdMYB221 is involved in the direct and indirect regulation of secondary wall biosynthesis during wood formation

Wood is formed by the successive addition of secondary xylem, which consists of cells with a conspicuously thickened secondary wall composed mainly of cellulose, xylan and lignin. Currently, few transcription factors involved in the direct regulation of secondary wall biosynthesis have been characterized in tree species. Here, we show that PdMYB221, a poplar ortholog of the Arabidopsis R2R3-MYB transcription factor AtMYB4, directly regulates secondary wall biosynthesis during wood formation. PdMYB221 is predominantly expressed in cells of developing wood, and the protein it encodes localizes to the nucleus and acts as a transcriptional repressor. Ectopic expression of PdMYB221 resulted in reduced cell wall thicknesses of fibers and vessels in Arabidopsis inflorescence stems. The amounts of cellulose, xylose, and lignin were decreased and the expression of key genes synthesizing the three components was suppressed in PdMYB221 overexpression plants. Transcriptional activation assays showed that PdMYB221 repressed the promoters of poplar PdCESA7/8, PdGT47C, PdCOMT2 and PdCCR1. Electrophoretic mobility shift assays revealed that PdMYB221 bound directly to the PdCESA8, PdGT47C, and PdCOMT2 promoters. Together, our results suggest that PdMYB221 may be involved in the negative regulation of secondary wall formation through the direct and indirect suppression of the gene expression of secondary wall biosynthesis.

Arabidopsis C3H14 and C3H15 have overlapping roles in the regulation of secondary wall thickening and anther development

Plant tandem CCCH zinc finger (TZF) proteins play diverse roles in developmental and adaptive processes. Arabidopsis C3H14 has been shown to act as a potential regulator of secondary wall biosynthesis. However, there is lack of direct evidence to support its functions in Arabidopsis. It is demonstrated here that C3H14 and its homologue C3H15 redundantly regulate secondary wall formation and that they additionally function in anther development. Plants with double, but not single, T-DNA mutants for C3H14 or C3H15 have few pollen grains and thinner stem secondary walls than the wild type. Plants homozygous for c3h14 and heterozygous for c3h15 [c3h14 c3h15(±)] have slightly thinner secondary walls than plants heterozygous for c3h14 and homozygous for c3h15 [c3h14(±) c3h15], and c3h14(±) c3h15 have lower fertility. Overexpression of C3H14 or C3H15 led to increased secondary wall thickness in stems and the ectopic deposition of secondary walls in various tissues, but did not affect anther morphology. Transcript profiles from the C3H14/15 overexpression and c3h14 c3h15 plants revealed marked changes in the expression of many genes associated with cell wall metabolism and pollen formation. Subcellular localization and biochemical analyses suggest that C3H14/15 might function at both the transcriptional and post-transcriptional levels.

Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar

Wood biomass is mainly made of secondary cell walls, whose formation is controlled by a multilevel network. The tandem CCCH zinc finger (TZF) proteins involved in plant secondary wall formation are poorly understood. Two TZF genes, PdC3H17 and PdC3H18, were isolated from Populus deltoides and functionally characterized in Escherichia coli, tobacco, Arabidopsis and poplar. PdC3H17 and PdC3H18 are predominantly expressed in cells of developing wood, and the proteins they encode are targeted to cytoplasmic foci. Transcriptional activation assays showed that PdMYB2/3/20/21 individually activated the PdC3H17 and PdC3H18 promoters, but PdMYB3/21 were most significant. Electrophoretic mobility shift assays revealed that PdMYB3/21 bound directly to the PdC3H17/18 promoters. Overexpression of PdC3H17/18 in poplar increased secondary xylem width and secondary wall thickening in stems, whereas dominant repressors of them had the opposite effects on these traits. Similar alteration in secondary wall thickening was observed in their transgenic Arabidopsis plants. qRT-PCR results showed that PdC3H17/18 regulated the expression of cellulose, xylan and lignin biosynthetic genes, and several wood-associated MYB genes. These results demonstrate that PdC3H17 and PdC3H18 are the targets of PdMYB3 and PdMYB21 and are an additional two components in the regulatory network of secondary xylem formation in poplar.

Cell wall polysaccharide distribution in Miscanthus lutarioriparius stem using immuno-detection

Key messageCell wall polysaccharides’ occurrences in two internodes of different development stages inM. lutarioripariusstem were analyzed and three major differences between them were identified by cell wall polysaccharide probes.AbstractDeposition and modification of cell wall polysaccharides during stem development affect biomass yield of the Miscanthus energy crop. The distribution patterns of cell wall polysaccharides in the 2nd and the 11th internodes of M. lutarioriparius stem were studied using in situ immunofluorescence assay. Crystalline cellulose and xylan were present in most of the stem tissues except phloem, where xyloglucan was the major composition of hemicellulose. The distribution of pectin polysaccharides varied in stem tissues, particularly in vascular bundle elements. Xylogalacturonan, feruloylated-1,4-β-d-galactan and (1,3)(1,4)-β-glucans, however, were insufficient for antibodies binding in both internodes. Furthermore, the distribution of cell wall polysaccharides was differentiated in the two internodes of M. lutarioriparius. The significant differences in the pattern of occurrence of long 1,5-α-l-arabinan chain, homogalacturonan and fucosylated xyloglucans epitope were detected between the two internodes. In addition, the relationships between probable functions of polysaccharides and their distribution patterns in M. lutarioriparius stem cell wall were discussed, which would be helpful to understand the growth characteristics of Miscanthus and identify potential targets for either modification or degradation.

Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3

Root hairs are tip-growing long tubular outgrowths of specialized epidermal cells, and are important for nutrient and water uptake and interaction with the soil microflora. Here we characterized two poplar cellulose synthase-like D (CSLD) genes, PdCSLD5 and PdCSLD6, the most probable orthologs to the Arabidopsis AtCSLD3/KOJAK gene. Both PdCSLD5 and PdCSLD6 are strongly expressed in roots, including in the root hairs. Subcellular localization experiments showed that these two proteins are located not only in the polarized plasma membrane of root hair tips, but also in Golgi apparatus of the root hair and non-hair-forming cells. Overexpression of these two poplar genes in the atcsld3 mutant was able to rescue most of the defects caused by disruption of AtCSLD3, including root hair morphological changes, altered cell wall monosaccharide composition, increased non-crystalline β-1,4-glucan and decreased crystalline cellulose contents. Taken together, our results provide evidence indicating that PdCSLD5 and PdCSLD6 are functionally conserved with AtCSLD3 and support a role for PdCSLD5 and PdCSL6 specifically in crystalline cellulose production in poplar root hair tips. The results presented here also suggest that at least part of the mechanism of root hair formation is conserved between herbaceous and woody plants.