Guang Qi

Comprehensive Analysis of NAC Domain Transcription Factor Gene Family in Populus trichocarpa

BackgroundNAC (NAM, ATAF1/2 and CUC2) domain proteins are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. NAC transcription factors comprise of a large gene family represented by more than 100 members in Arabidopsis, rice and soybean etc. Recently, a preliminary phylogenetic analysis was reported for NAC gene family from 11 plant species. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, conserved motifs, and expression profiling analysis has been presented thus far for the model tree species Populus.ResultsIn the present study, a comprehensive analysis of NAC gene family in Populus was performed. A total of 163 full-length NAC genes were identified in Populus, and they were phylogeneticly clustered into 18 distinct subfamilies. The gene structure and motif compositions were considerably conserved among the subfamilies. The distributions of 120 Populus NAC genes were non-random across the 19 linkage groups (LGs), and 87 genes (73%) were preferentially retained duplicates that located in both duplicated regions. The majority of NACs showed specific temporal and spatial expression patterns based on EST frequency and microarray data analyses. However, the expression patterns of a majority of duplicate genes were partially redundant, suggesting the occurrence of subfunctionalization during subsequent evolutionary process. Furthermore, quantitative real-time RT-PCR (RT-qPCR) was performed to confirm the tissue-specific expression patterns of 25 NAC genes.ConclusionBased on the genomic organizations, we can conclude that segmental duplications contribute significantly to the expansion of Populus NAC gene family. The comprehensive expression profiles analysis provides first insights into the functional divergence among members in NAC gene family. In addition, the high divergence rate of expression patterns after segmental duplications indicates that NAC genes in Populus are likewise to have been retained by substantial subfunctionalization. Taken together, our results presented here would be helpful in laying the foundation for functional characterization of NAC gene family and further gaining an understanding of the structure-function relationship between these family members.

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.

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.

Molecular cloning and expression analysis of 13 NAC transcription factors in Miscanthus lutarioriparius

Key messageThe 13MlNACgenes could respond to various abiotic stresses, suggesting their crucial roles in stress response. Overexpression ofMlNAC2inArabidopsisled to improved drought tolerance.AbstractNAC (NAM, ATAF1/2 and CUC2) proteins are plant-specific transcription factors that play crucial roles in plant development, growth and stress responses. In this study, 13 stress-responsive NAC genes were identified from Miscanthus lutarioriparius. Full-length cDNA sequences were obtained for 11 MlNAC genes, which were phylogenetically classified into six subfamilies. Sequence alignment revealed the highly conserved NAC domain in the N-terminus of these MlNACs, while the C-terminus was highly divergent. We performed quantitative real-time RT-PCR to examine the expression profiles of MlNAC genes in different tissues including root, rhizome, mature stem, young stem, leaf and sheath. The 13 MlNAC genes displayed distinct tissue-specific patterns in six tissues examined. To gain further insight into their roles in response to abiotic stresses, expressions of MlNAC genes were analyzed under different stresses and hormone treatments including salt, drought, cold, wounding, abscisic acid, Methyl jasmonate and salicylic acid. The 13 MlNAC genes could respond to at least five stress treatments, and over 100-fold variations in transcript levels of MlNAC1, MlNAC2, MlNAC4, and MlNAC12 were observed in salt, drought and MeJA treatments, which indicated that MlNACs play crucial roles in stress response. Crosstalk among various abiotic stress and hormone responses was also discussed based on the expression of MlNAC genes. Overexpression of MlNAC2 in Arabidopsis (Col-0) led to improved drought tolerance. The water loss rate was significantly lower, and the recovery rate after a 60-min dehydration stress treatment was significantly higher in the MlNAC2 overexpression lines than the control.

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.