Molecular Characterization of the Acyl-CoA-Binding Protein Genes Reveals Their Significant Roles in Oil Accumulation and Abiotic Stress Response in Cotton.

Members of the acyl-CoA-binding protein (ACBP) gene family play vital roles in diverse processes related to lipid metabolism, growth and development, and environmental response. Plant ACBP genes have been well-studied in a variety of species including Arabidopsis, soybean, rice and maize. However, the identification and functions of ACBP genes in cotton remain to be elucidated. In this study, a total of 11 GaACBP, 12 GrACBP, 20 GbACBP, and 19 GhACBP genes were identified in the genomes of Gossypium arboreum, Gossypium raimondii, Gossypium babardense, and Gossypium hirsutum, respectively, and grouped into four clades. Forty-nine duplicated gene pairs were identified in Gossypium ACBP genes, and almost all of which have undergone purifying selection during the long evolutionary process. In addition, expression analyses showed that most of the GhACBP genes were highly expressed in the developing embryos. Furthermore, GhACBP1 and GhACBP2 were induced by salt and drought stress based on a real-time quantitative PCR (RT-qPCR) assay, indicating that these genes may play an important role in salt- and drought-stress tolerance. This study will provide a basic resource for further functional analysis of the ACBP gene family in cotton.

Genome-wide characterization of the UDP-glycosyltransferase gene family reveals their potential roles in leaf senescence in cotton.

Uridine diphosphate (UDP) glycosyltransferases (UGTs) involved in many metabolic processes and are essential for plant growth and development. Although UGTs proteins have been studied in many plants, the biological functions of UGT genes in cotton leaf senescence are still unknown. In the present study, we performed a genome-wide survey and identified 157 GrUGT, 152 GaUGT and 261 GHUGT genes in Gossypium raimondii, G. arboreum, and G. hirsutum, respectively, that were classified into 15 groups. Analysis of protein motif and gene structure demonstrated that structural and functional conservation occurred within same groups but diverged among the different groups. Gene duplication analysis indicated the different duplication ways happened between tetraploid G. hirsutum and the two diploid species. Whole genome or segmental duplications played a main role in the expansion of the GHUGT family in cotton, and experienced purifying selection during the long evolutionary process in cotton. Cis-acting regulatory elements analysis indicated that they were associated with complex hormone regulatory networks and the stress response. Additionally, to identify GHUGT candidate genes responsive to leaf senescence, we analyzed the expression patterns of GHUGT genes using our transcriptome data from two cultivars of upland cotton with contrasting tolerance to leaf senescence. Subsequently, gene expression profiling based on real-time quantitative PCR showed that selected GHUGT candidate genes might be involved in ABA and JA regulation. Through further functional verification, silencing GHUGT116 gene via VIGS (Virus-induced gene silencing) delayed dark-induced leaf senescence. Overall, the results provide useful and valuable information for understanding the evolution of cotton UGTs genes and the function in leaf senescence.

Genome-Wide Identification and Expression Analyses of the Cotton AGO Genes and Their Potential Roles in Fiber Development and Stress Response.

Argonaute proteins (AGOs) are indispensable components of RNA silencing. However, systematic characterization of the AGO genes have not been completed in cotton until now. In this study, cotton AGO genes were identified and analyzed with respect to their evolution and expression profile during biotic and abiotic stresses. We identified 14 GaAGO, 14 GrAGO, and 28 GhAGO genes in the genomes of Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum. Cotton AGO proteins were classified into four subgroups. Structural and functional conservation were observed in the same subgroups based on the analysis of the gene structure and conserved domains. Twenty-four duplicated gene pairs were identified in GhAGO genes, and all of them exhibited strong purifying selection during evolution. Moreover, RNA-seq analysis showed that most of the GhAGO genes exhibit high expression levels in the fiber initiation and elongation processes. Furthermore, the expression profiles of GhAGO genes tested by quantitative real-time polymerase chain reaction (qPCR) demonstrated that they were sensitive to Verticillium wilt infection and salt and drought stresses. Overall, our results will pave the way for further functional investigation of the cotton AGO gene family, which may be involved in fiber development and stress response.

Transcriptional Differences in Peanut (Arachis hypogaea L.) Seeds at the Freshly Harvested, After-ripening and Newly Germinated Seed Stages: Insights into the Regulatory Networks of Seed Dormancy Release and Germination.

Seed dormancy and germination are the two important traits related to plant survival, reproduction and crop yield. To understand the regulatory mechanisms of these traits, it is crucial to clarify which genes or pathways participate in the regulation of these processes. However, little information is available on seed dormancy and germination in peanut. In this study, seeds of the variety Luhua No.14, which undergoes nondeep dormancy, were selected, and their transcriptional changes at three different developmental stages, the freshly harvested seed (FS), the after-ripening seed (DS) and the newly germinated seed (GS) stages, were investigated by comparative transcriptomic analysis. The results showed that genes with increased transcription in the DS vs FS comparison were overrepresented for oxidative phosphorylation, the glycolysis pathway and the tricarboxylic acid (TCA) cycle, suggesting that after a period of dry storage, the intermediates stored in the dry seeds were rapidly mobilized by glycolysis, the TCA cycle, the glyoxylate cycle, etc.; the electron transport chain accompanied by respiration was reactivated to provide ATP for the mobilization of other reserves and for seed germination. In the GS vs DS pairwise comparison, dozens of the upregulated genes were related to plant hormone biosynthesis and signal transduction, including the majority of components involved in the auxin signal pathway, brassinosteroid biosynthesis and signal transduction as well as some GA and ABA signal transduction genes. During seed germination, the expression of some EXPANSIN and XYLOGLUCAN ENDOTRANSGLYCOSYLASE genes was also significantly enhanced. To investigate the effects of different hormones during seed germination, the contents and differential distribution of ABA, GAs, BRs and IAA in the cotyledons, hypocotyls and radicles, and plumules of three seed sections at different developmental stages were also investigated. Combined with previous data in other species, it was suggested that the coordination of multiple hormone signal transduction nets plays a key role in radicle protrusion and seed germination.

Systematic analysis of NAC transcription factors in Gossypium barbadense uncovers their roles in response to Verticillium wilt.

As one of the largest plant-specific gene families, the NAC transcription factor gene family plays important roles in various plant physiological processes that are related to plant development, hormone signaling, and biotic and abiotic stresses. However, systematic investigation of the NAC gene family in sea-island cotton (Gossypium babardense L.) has not been reported, to date. The recent release of the complete genome sequence of sea-island cotton allowed us to perform systematic analyses of G. babardense NAC GbNAC) genes. In this study, we performed a genome-wide survey and identified 270 GbNAC genes in the sea-island cotton genome. Genome mapping analysis showed that GbNAC genes were unevenly distributed on 26 chromosomes. Through phylogenetic analyses of GbNACs along with their Arabidopsis counterparts, these proteins were divided into 10 groups (I-X), and each contained a different number of GbNACs with a similar gene structure and conserved motifs. One hundred and fifty-four duplicated gene pairs were identified, and almost all of them exhibited strong purifying selection during evolution. In addition, various cis-acting regulatory elements in GbNAC genes were found to be related to major hormones, defense and stress responses. Notably, transcriptome data analyses unveiled the expression profiles of 62 GbNAC genes under Verticillium wilt (VW) stress. Furthermore, the expression profiles of 15 GbNAC genes tested by quantitative real-time PCR (qPCR) demonstrated that they were sensitive to methyl jasmonate (MeJA) and salicylic acid (SA) treatments and that they could be involved in pathogen-related hormone regulation. Taken together, the genome-wide identification and expression profiling pave new avenues for systematic functional analysis of GbNAC candidates, which may be useful for improving cotton defense against VW.

[Analysis of the peanut transgenic offspring with depressing AhFAD2 gene].

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

Molecular characterization of a stress-induced NAC gene, GhSNAC3, from Gossypium hirsutum.

NAC genes, specific to plants, play important roles in plant development as well as in response to biotic and abiotic stresses. Here, a novel gene encoding a NAC domain, named as GhSNAC3, was isolated from upland cotton (Gossypium hirsutum L.). Sequence analyses showed that GhSNAC3 encodes a protein of 346 amino acids with an estimated molecular mass of 38.4 kDa and pI of 8.87. Transient localization assays in onion epidermal cells confirmed GhSNAC3 is a nuclear protein. Transactivation studies using a yeast system revealed that GhSNAC3 functions as a transcription activator. Quantitative real-time polymerase chain reaction analysis indicated that GhSNAC3 was induced by high salinity, drought and abscisic acid treatments. We overexpressed GhSNAC3 in tobacco by using Agrobacterium-mediated transformation. Transgenic lines produced longer primary roots and more fresh weight under salt and drought stresses as compared to wild-type plants. Collectively, our results indicated that overexpression of GhSNAC3 in tobacco can enhance drought and salt tolerances.

Seed-Specific Expression of AtLEC1 Increased Oil Content and Altered Fatty Acid Composition in Seeds of Peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the major oil crops and is the fifth largest source of plant oils in the world. Numerous genes participate in regulating the biosynthesis and accumulation of the storage lipids in seeds or other reservoir organs, among which several transcription factors, such as LEAFY COTYLEDON1 (AtLEC1), LEC2, and WRINKLED1 (WRI1), involved in embryo development also control the lipid reservoir in seeds. In this study, the AtLEC1 gene was transferred into the peanut genome and expressed in a seed-specific manner driven by the NapinA full-length promoter or its truncated 230-bp promoter. Four homozygous transgenic lines, two lines with the longer promoter and the other two with the truncated one, were selected for further analysis. The AtLEC1 mRNA level and the corresponding protein accumulation in different transgenic overexpression lines were altered, and the transgenic plants grew and developed normally without any detrimental effects on major agronomic traits. In the developing seeds of transgenic peanuts, the mRNA levels of a series of genes were upregulated. These genes are associated with fatty acid (FA) biosynthesis and lipid accumulation. The former set of genes included the homomeric ACCase A (AhACC II), the BC subunit of heteromeric ACCase (AhBC4), ketoacyl-ACP synthetase (AhKAS II), and stearoyl-ACP desaturase (AhSAD), while the latter ones were the diacylglycerol acyltransferases and oleosins (AhDGAT1, AhDGAT2, AhOle1, AhOle2, and AhOle3). The oil content and seed weight increased by 4.42-15.89% and 11.1-22.2%, respectively, and the levels of major FA components including stearic acid, oleic acid, and linoleic acid changed significantly in all different lines.

Natural variations in stearoyl-acp desaturase genes affect the conversion of stearic to oleic acid in maize kernel.

KEY MESSAGE: We identified 11 SAD genes, and mined their natural variations associated with the conservation of stearic to oleic acid, especially ZmSAD1 supported by both the QTL and an expression QTL. Maize oil is generally regarded as a healthy vegetable oil owing to its low abundance of saturated fatty acids. Stearoyl-ACP desaturase (SAD) is a key rate-limiting enzyme for the conservation of stearic (C18:0) to oleic (C18:1) acid. Here, 11 maize SAD genes were identified to have more divergent functions than Arabidopsis SAD genes. The genomic regional associations in a maize panel including 508 inbred lines identified 6 SAD genes significantly associated (P < 0.01) with the C18:0/C18:1 ratio or the level of C18:0 or C18:1, one gene of which co-localized with a quantitative trait locus (QTL) and 5 of which co-localized with an expression QTL. ZmSAD1, supported by both the QTL and an expression QTL, had the largest effect on C18:0/C18:1. One nonsynonymous single-nucleotide polymorphism in exon 3 and one 5-bp insertion/deletion in the 3' untranslated region were further shown to contribute to the natural variation in C18:0/C18:1 according to ZmSAD1-based association mapping. Finally, selection tests of ZmSAD1 in teosinte, regular maize, and high-oil maize indicated that ZmSAD1 was not a selection target during the process of maize domestication and high-oil maize development. These results will guide the manipulation of the ratio between saturated and unsaturated fatty acids in maize.

Cloning and Characterization of 5' Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5' flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5' terminal and/or 3' terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65 bp proximal promoter region and the 52 bp 5' UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

Identification of expressed resistance gene analogs from peanut (Arachis hypogaea L.) expressed sequence tags.

Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two polymerase chain reaction-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.

Identification of expressed resistance gene-like sequences by data mining in 454-derived transcriptomic sequences of common bean (Phaseolus vulgaris L.).

BACKGROUND: Common bean (Phaseolus vulgaris L.) is one of the most important legumes in the world. Several diseases severely reduce bean production and quality; therefore, it is very important to better understand disease resistance in common bean in order to prevent these losses. More than 70 resistance (R) genes which confer resistance against various pathogens have been cloned from diverse plant species. Most R genes share highly conserved domains which facilitates the identification of new candidate R genes from the same species or other species. The goals of this study were to isolate expressed R gene-like sequences (RGLs) from 454-derived transcriptomic sequences and expressed sequence tags (ESTs) of common bean, and to develop RGL-tagged molecular markers. RESULTS: A data-mining approach was used to identify tentative P. vulgaris R gene-like sequences from approximately 1.69 million 454-derived sequences and 116,716 ESTs deposited in GenBank. A total of 365 non-redundant sequences were identified and named as common bean (P. vulgaris = Pv) resistance gene-like sequences (PvRGLs). Among the identified PvRGLs, about 60% (218 PvRGLs) were from 454-derived sequences. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that PvRGLs were actually expressed in the leaves of common bean. Upon comparison to P. vulgaris genomic sequences, 105 (28.77%) of the 365 tentative PvRGLs could be integrated into the existing common bean physical map. Based on the syntenic blocks between common bean and soybean, 237 (64.93%) PvRGLs were anchored on the P. vulgaris genetic map and will need to be mapped to determine order. In addition, 11 sequence-tagged-site (STS) and 19 cleaved amplified polymorphic sequence (CAPS) molecular markers were developed for 25 unique PvRGLs. CONCLUSIONS: In total, 365 PvRGLs were successfully identified from 454-derived transcriptomic sequences and ESTs available in GenBank and about 65% of PvRGLs were integrated into the common bean genetic map. A total of 30 RGL-tagged markers were developed for 25 unique PvRGLs, including 11 STS and 19 CAPS markers. The expressed PvRGLs identified in this study provide a large sequence resource for development of RGL-tagged markers that could be used further for genetic mapping of disease resistant candidate genes and quantitative trait locus/loci (QTLs). This work also represents an additional method for identifying expressed RGLs from next generation sequencing data.

A Bayesian model for gene family evolution.

BACKGROUND: A birth and death process is frequently used for modeling the size of a gene family that may vary along the branches of a phylogenetic tree. Under the birth and death model, maximum likelihood methods have been developed to estimate the birth and death rate and the sizes of ancient gene families (numbers of gene copies at the internodes of the phylogenetic tree). This paper aims to provide a Bayesian approach for estimating parameters in the birth and death model. RESULTS: We develop a Bayesian approach for estimating the birth and death rate and other parameters in the birth and death model. In addition, a Bayesian hypothesis test is developed to identify the gene families that are unlikely under the birth and death process. Simulation results suggest that the Bayesian estimate is more accurate than the maximum likelihood estimate of the birth and death rate. The Bayesian approach was applied to a real dataset of 3517 gene families across genomes of five yeast species. The results indicate that the Bayesian model assuming a constant birth and death rate among branches of the phylogenetic tree cannot adequately explain the observed pattern of the sizes of gene families across species. The yeast dataset was thus analyzed with a Bayesian heterogeneous rate model that allows the birth and death rate to vary among the branches of the tree. The unlikely gene families identified by the Bayesian heterogeneous rate model are different from those given by the maximum likelihood method. CONCLUSIONS: Compared to the maximum likelihood method, the Bayesian approach can produce more accurate estimates of the parameters in the birth and death model. In addition, the Bayesian hypothesis test is able to identify unlikely gene families based on Bayesian posterior p-values. As a powerful statistical technique, the Bayesian approach can effectively extract information from gene family data and thereby provide useful information regarding the evolutionary process of gene families across genomes.

Identification and analysis of common bean (Phaseolus vulgaris L.) transcriptomes by massively parallel pyrosequencing.

BACKGROUND: Common bean (Phaseolus vulgaris) is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. RESULTS: We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt). These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC) end sequences, and a total of 21% of the unigenes (12,724) including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs) and transcription factors were also identified in this study. CONCLUSIONS: This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and will aid in the development of molecular markers that can be used for tagging genes of interest. Additionally, these sequences will provide a means for better annotation of the on-going common bean whole genome sequencing.

[Cloning and characterization of a transcription factor ZmNAC1 in maize (Zea mays)].

NAC transcription factors are a family of functionally diverse proteins. They are unique to plants and play an important role in regulation of plant growth and development, hormone regulation and responses to various stresses. A cDNA encoding the NAC-like gene homologue was isolated from maize (Zea mays L.) by RT-PCR and designated ZmNAC1 (GenBank Accession No. EU224278). Sequence analysis showed that cDNA of ZmNAC1 was 1,029 bp long and contained a single open reading frame (ORF, 26 to approximately 907 bp). The predicted ZmNAC1 protein has 293 amino acids with an estimated molecular mass of 32.3 kDa and an isoelectric point of 8.65. RT-PCR analysis showed that the expression of ZmNAC1 was induced by low temperature, PEG, salt, and ABA, respectively. These results suggest that ZmNAC1 may play important roles in biotic and abiotic resistance pathways. This is the first NAC-like gene reported in maize.

Proteomic analysis of early germs with high-oil and normal inbred lines in maize.

High-oil maize as a product of long-term selection provides a unique resource for functional genomics. In this study, the abundant soluble proteins of early developing germs from high-oil and normal lines of maize were compared using two-dimensional gel electrophoresis (2-DGE) in combination with mass spectrometry (MS). More than 1100 protein spots were detected on electrophoresis maps of both high-oil and normal lines by using silver staining method. A total of 83 protein spots showed significant differential expression (>two-fold change; t-test: P < 0.05) between high-oil and normal inbred lines. Twenty-seven protein spots including 25 non-redundant proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Functional categorization of these proteins was carbohydrate metabolism, cytoskeleton, protein metabolism, stress response, and lipid metabolism. Three such proteins involved in lipid metabolism, namely putative enoyl-ACP reductase (ENR), putative stearoyl-ACP desaturase (SAD) and putative acetyl-CoA C-acyltransferase (ACA), had more abundant expressions in high-oil lines than in normal. At the mRNA expression level, SAD, ENR and ACA were expressed at significantly higher levels in high-oil lines than in normal. The results demonstrated that high expressions of SAD, ENR and ACA might be associated to increasing oil concentration in high-oil maize. This study represents the first proteomic analysis of high-oil maize and contributes to a better understanding of the molecular basis of oil accumulation in high-oil maize.