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.

Background: Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.

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-b-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.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008;Wang et al., 2012;Pauly et al., 2013). Each of these polysaccharides is composed of a b-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of a-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986;Iiyama et al., 1994;Obel et al., 2007;Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the b-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Some...

BackgroundHomeodomain-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 FindingsIn 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.ConclusionsGenomic 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.

Calcium-dependent protein kinases (CDPKs) are Ca(2+)-binding proteins known to play crucial roles in Ca(2+) 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.

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor CRY2 regulates photoperiodic flowering in the experimental model plant Arabidopsis thaliana. However, it is unclear whether genetic variations affecting cryptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two cryptochromes in soybean, GmCRY1a and GmCRY2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) exhibit a strong latitudinal cline in photoperiodic flowering. Similar to their Arabidopsis counterparts, both GmCRY1a and GmCRY2a affected blue light inhibition of cell elongation, but only GmCRY2a underwent blue light-and 26S proteasome-dependent degradation. However, in contrast to Arabidopsis cryptochromes, soybean GmCRY1a, but not GmCRY2a, exhibited a strong activity promoting floral initiation, and the level of protein expression of GmCRY1a, but not GmCRY2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmCRY1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmCRY1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmCRY1a protein play an important role in determining latitudinal distribution of soybeans.blue light ͉ cryptochrome ͉ photoperiodism ͉ photoreceptor

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.

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys(2)/His(2)-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis.