NAC Transcription Factor Family Research Articles

A Transcriptomic Analysis Sheds Light on the Molecular Regulation of Wood Formation in Populus trichocarpa during Drought Stress

Wood is an abundant and essential renewable resource whose production is threatened in some parts of the world by drought. A better understanding of the molecular mechanisms underlying wood formation during drought is critical to maintaining wood production under increasingly adverse environmental conditions. In this study, we investigated wood formation in black cottonwood (Populus trichocarpa) during drought stress. The morphological changes during drought stress in P. trichocarpa included the wilting and drooping of leaves, stem water loss, and a reduction in whole plant biomass. The water embolism rate indicated that the water transport in stems was blocked under drought conditions. An anatomical analysis of the xylem and cambium revealed that drought stress changed the structure of vessel cells, increased lignin accumulation, and decreased the cambium cell layers. We subsequently identified 12,438 and 9156 differentially expressed genes from stem xylem and cambium tissues under well-watered and drought conditions, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that these genes were mainly involved in hormone signal transduction and amino sugar and nucleotide sugar metabolism. To further explore the molecular mechanism of wood formation in response to drought, we analyzed the expression patterns of the genes involved in lignin, cellulose, and hemicellulose biosynthesis in xylem and the genes involved in cambial activity in the cambium. To better understand the regulatory networks governing xylem development and cambium activity in response to drought, we analyzed the MYB (138), AP2 (130), bHLH (89), and NAC (81) transcription factor families to shed light on the interactions between the TFs in these families and the genes they regulate. Identifying the key genes that regulate wood formation in P. trichocarpa during drought provides a genetic foundation for further research on the molecular regulatory networks and physiology underpinning wood formation during drought stress.

Pangenome characterization and analysis of the NAC gene family reveals genes for Sclerotinia sclerotiorum resistance in sunflower (Helianthus annuus).

Sunflower (Helianthus annuus) is one of the most important economic crops in oilseed production worldwide. The different cultivars exhibit variability in their resistance genes. The NAC transcription factor (TF) family plays diverse roles in plant development and stress responses. With the completion of the H. annuus genome sequence, the entire complement of genes coding for NACs has been identified. However, the reference genome of a single individual cannot cover all the genetic information of the species. Considering only a single reference genome to study gene families will miss many meaningful genes. Apangenome-wide surveyand characterization of the NAC genes in sunflower species were conducted. In total, 139 HaNAC genes are identified, of which 114 are core and 25 are variable. Phylogenetic analysis of sunflower NAC proteins categorizes these proteins into 16 subgroups. 138 HaNACs are randomly distributed on 17 chromosomes. SNP-based haplotype analysis shows haplotype diversity of the HaNAC genes in wild accessions is richer than in landraces and modern cultivars. Ten HaNAC genes in the basal stalk rot (BSR) resistance quantitative trait loci (QTL) are found. A total of 26 HaNAC genes are differentially expressed in response to Sclerotinia head rot (SHR). A total of 137 HaNAC genes are annotated in Gene Ontology (GO) and are classified into 24 functional groups. GO functional enrichment analysis reveals that HaNAC genes are involved in various functions of the biological process. We identified NAC genes in H. annuus (HaNAC) on a pangenome-wide scale and analyzed S. sclerotiorum resistance-related NACs. This study provided a theoretical basis for further genomic improvement targeting resistance-related NAC genes in sunflowers.

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis

Freezing stress can seriously affect plant growth and development, but the mechanisms of these effects and plant responses to freezing stress require further exploration. Here, we identified a NAC-family transcription factor (TF), NAC056, that can promote freezing tolerance in Arabidopsis. NAC056 mRNA levels are strongly induced by freezing stress in roots, and the nac056 mutant exhibits compromised freezing tolerance. NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor (CBF) pathway genes. Interestingly, we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants; therefore, NAC056–CBF1–NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress. In addition, 35S::NAC056 transgenic plants show enhanced freezing tolerance, which is partially reversed in the cbfs triple mutant. Thus, NAC056 confers freezing tolerance through the CBF pathway, mediating plant responses to balance growth and freezing stress tolerance.

Isolation, Characterization, and Expression Analysis of NAC Transcription Factor from Andrographis paniculata (Burm. f.) Nees and Their Role in Andrographolide Production.

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant's leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.

Comprehensive in silico characterization of NAC transcription factor family of Pinellia ternata and functional analysis of PtNAC66 under high-temperature tolerance in transgenic Arabidopsis thaliana

Pinellia ternata, a valuable Chinese herb, suffers yield reduction due to “sprout tumble” under high temperatures. However, the mechanisms underlying its high-temperature stress remain poorly understood. NAM, ATAF1/2, and CUC2 (NAC) transcription factors regulate plant tissue growth and abiotic stress. Hence, there has been no comprehensive research conducted on NAC transcription factors in P. ternata. We identified 98 PtNAC genes unevenly distributed across 13 chromosomes, grouped into 15 families via phylogenetic analysis. Gene expression analysis revealed diverse expression patterns of PtNAC genes in different tissue types. Further studies revealed that PtNAC5/7/17/35/43/47/57/66/86 genes were highly expressed in various tissues of P. ternata and induced by heat stress, among which PtNAC66 was up-regulated at the highest folds induced by heat temperature. PtNAC66 is a nuclear protein that can selectively bind to the cis-responsive region NACRS but lacks the ability to activate transcription in yeast. For further research, PtNAC66 was cloned and transgenic Arabidopsis was obtained. PtNAC66 overexpression increased high-temperature tolerance compared to wild-type plants. Transcriptome profiling demonstrated that overexpression of PtNAC66 led to significant modification of genes responsible for regulating binding, catalytic activity, transcription regulator activity and transporter activity response genes. Additionally, PtNAC66 was found to bind the promoters of CYP707A3, MYB102 and NAC055, respectively, and inhibited their expression, affecting the high-temperature stress response in Arabidopsis. Our research established the foundation for functional studies of PtNAC genes in response to high-temperature forcing by characterizing the P. ternata NAC gene family and examining the biological role of PtNAC66 in plant high-temperature tolerance.

Genome-wide identification and characterization of NAC transcription factor family members in Trifolium pratense and expression analysis under lead stress

BackgroundThe NAC TF family is widely involved in plant responses to various types of stress. Red clover (Trifolium pratense) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response.ResultsIn this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula. Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean (Glycine max). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover.ConclusionThese results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.

VqNAC44 enhances stilbene synthesis and disease resistance in Chinese wild grape by interacting with VqMYB15

As significant phytoalexins, stilbene compounds can improve the stress resistance of grapes under biotic and abiotic stress conditions and have biological effects such as antitumour, antioxidant, immune regulation and cardiovascular protection activities in humans. RESVERATROL SYNTHASE (RS), also known as STILBENE SYNTHASE (STS), is the critical enzyme regulating stilbene synthesis and has been identified in a few plant species. However, the regulatory mechanisms of stilbene synthesis are uncertain. In this study, an NAC family transcription factor from Vitis quinquangularis, named VqNAC44, was characterized as an indirect regulator of stilbene synthesis. It is worth noting that VqNAC44 did not bind to the STS promoter nor did it interact with the STS protein but interacted with the MYB transcription factor VqMYB15. This interaction between VqMYB15 and VqNAC44 was validated by a yeast two-hybrid assay and bimolecular fluorescence complementation. Overexpressing VqNAC44 in Arabidopsis thaliana significantly increased its tolerance to biotic and abiotic stresses. Transient overexpression of VqNAC44 and VqMYB15 in grape leaves resulted in increased expression of the STS gene and increased production of stilbene compounds. The experimental results confirmed that VqNAC44 regulated stilbene synthesis by interacting with VqMYB15, thereby enhancing the plant stress resistance.

Whole-Transcriptome Sequencing Reveals the Global Molecular Responses and NAC Transcription Factors Involved in Drought Stress in Dendrobium catenatum.

Dendrobium catenatum is a highly drought-tolerant herb, which usually grows on cliffs or in the branches of trees, yet the underlying molecular mechanisms for its tolerance remain poorly understood. We conducted a comprehensive study utilizing whole-transcriptome sequencing approaches to investigate the molecular response to extreme drought stress in D. catenatum. A large number of differentially expressed mRNAs, lncRNAs, and circRNAs have been identified, and the NAC transcription factor family was highly enriched. Meanwhile, 46 genes were significantly up-regulated in the ABA-activated signaling pathway. In addition to the 89 NAC family members accurately identified in this study, 32 members were found to have different expressions between the CK and extreme drought treatment. They may regulate drought stress through both ABA-dependent and ABA-independent pathways. Moreover, the 32 analyzed differentially expressed DcNACs were found to be predominantly expressed in the floral organs and roots. The ceRNA regulatory network showed that DcNAC87 is at the core of the ceRNA network and is regulated by miR169, miR393, and four lncRNAs. These investigations provided valuable information on the role of NAC transcription factors in D. catenatum's response to drought stress.

A telomere-to-telomere reference genome of ficus (Ficus hispida) provides new insights into sex determination.

A high-quality reference genome is indispensable for resolving biologically essential traits. Ficus hispida is a dioecious plant. A complete Ficus reference genome will be crucial for understanding their sex evolution and important biological characteristics, such as aerial roots, mutualistic symbiosis with ficus-wasps, and fruiting from old stems. Here, we generated a telomere-to-telomere (T2T) genome for F. hispida using PacBio HiFi and Oxford Nanopore Ultra-long sequencing technologies. The genome contiguity and completeness has shown improvement compared with the previously released genome, with the annotation of six centromeres and 28 telomeres. We have refined our previously reported 2-Mb male-specific region into a 7.2-Mb genomic region containing 51 newly predicted genes and candidate sex-determination genes AG2 and AG3. Many of these genes showed extremely low expression, likely attributed to hypermethylation in the gene body and promoter regions. Gene regulatory networks (GRNs) revealed that AG2 and AG3 are related to the regulation of stamen development in male flowers, while the AG1 gene is responsible for regulating female flowers' defense responses and secondary metabolite processes. Comparative analysis of GRNs showed that the NAC, WRKY, and MYB transcription factor families dominate the female GRN, whereas the MADS and MYB transcription factor families are prevalent in the male GRN.

Comprehensive genomic characterisation of the NAC transcription factor family and its response to drought stress in Eucommia ulmoides.

The NAC transcription factor family enhances plant adaptation to environmental challenges by participating in signalling pathways triggered by abiotic stressors and hormonal cues. We identified 69 NAC genes in the Eucommia ulmoides genome and renamed them according to their chromosomal distribution. These EuNAC proteins were clustered into 13 sub-families and distributed on 16 chromosomes and 2 scaffolds. The gene structures suggested that the number of exons varied from two to eight among these EuNACs, with a multitude of them containing three exons. Duplicated events resulted in a large gene family; 12 and four pairs of EuNACs were the result of segmental and tandem duplicates, respectively. The drought-stress response pattern of 12 putative EuNACs was observed under drought treatment, revealing that these EuNACs could play crucial roles in mitigating the effects of drought stress responses and serve as promising candidate genes for genetic engineering aimed at enhancing the drought stress tolerance of E. ulmoides. This study provides insight into the evolution, diversity, and characterisation of NAC genes in E.ulmoides and will be helpful for future characterisation of putative EuNACs associated with water deficit.

An insertion of transposon in DcNAP inverted its function in the ethylene pathway to delay petal senescence in carnation (Dianthus caryophyllus L.).

Petal senescence is the final stage of flower development. Transcriptional regulation plays key roles in this process. However, whether and how post-transcriptional regulation involved is still largely unknown. Here, we identified an ethylene-induced NAC family transcription factor DcNAP in carnation (Dianthus caryophyllus L.). One allele, DcNAP-dTdic1, has an insertion of a dTdic1 transposon in its second exon. The dTdic1 transposon disrupts the structure of DcNAP and causes alternative splicing, which transcribes multiple domain-deleted variants (DcNAP2 and others). Conversely, the wild type allele DcNAP transcribes DcNAP1 encoding an intact NAC domain. Silencing DcNAP1 delays and overexpressing DcNAP1 accelerates petal senescence in carnation, while silencing and overexpressing DcNAP2 have the opposite effects, respectively. Further, DcNAP2 could interact with DcNAP1 and interfere the binding and activation activity of DcNAP1 to the promoters of its downstream target ethylene biosynthesis genes DcACS1 and DcACO1. Lastly, ethylene signalling core transcriptional factor DcEIL3-1 can activate the expression of DcNAP1 and DcNAP2 in the same way by binding their promoters. In summary, we discovered a novel mechanism by which DcNAP regulates carnation petal senescence at the post-transcriptional level. It may also provide a useful strategy to manipulate the NAC domains of NAC transcription factors for crop genetic improvement.

NACs, generalist in plant life.

Plant-specific NAC proteins constitute a major transcription factor family that is well-known for its roles in plant growth, development, and responses to abiotic and biotic stresses. In recent years, there has been significant progress in understanding the functions of NAC proteins. NAC proteins have a highly conserved DNA-binding domain; however, their functions are diverse. Previous understanding of the structure of NAC transcription factors can be used as the basis for their functional diversity. NAC transcription factors consist of a target-binding domain at the N-terminus and a highly versatile C-terminal domain that interacts with other proteins. A growing body of research on NAC transcription factors helps us comprehend the intricate signalling network and transcriptional reprogramming facilitated by NAC-mediated complexes. However, most studies of NAC proteins have been limited to a single function. Here, we discuss the upstream regulators, regulatory components and targets of NAC in the context of their prospective roles in plant improvement strategies via biotechnology intervention, highlighting the importance of the NAC transcription factor family in plants and the need for further research.

Integrated metabolic profiling and transcriptome analysis of Lonicera japonica flowers for chlorogenic acid, luteolin and endogenous hormone syntheses

The active ingredients of many medicinal plants are the secondary metabolites associated with the growth period. Lonicera japonica Thunb. is an important traditional Chinese medicine, and the flower development stage is an important factor that influences the quality of medicinal ingredients. In this study, transcriptomics and metabolomics were performed to reveal the regulatory mechanism of secondary metabolites during flowering of L. japonica. The results showed that the content of chlorogenic acid (CGA) and luteolin gradually decreased from green bud stage (Sa) to white flower stage (Sc), especially from white flower bud stage (Sb) to Sc. Most of the genes encoding the crucial rate-limiting enzymes, including PAL, C4H, HCT, C3′H, F3′H and FNSII, were down-regulated in three comparisons. Correlation analysis identified some members of the MYB, AP2/ERF, bHLH and NAC transcription factor families that are closely related to CGA and luteolin biosynthesis. Furthermore, differentially expressed genes (DEGs) involved in hormone biosynthesis, signalling pathways and flowering process were analysed in three flower developmental stage.

Transcriptome features of stone cell development in weevil-resistant and susceptible Sitka spruce.

Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.

Ectopic Expression of PsnNAC090 Enhances Salt and Osmotic Tolerance in Transgenic Tobacco.

The NAC transcription factor family is well known to play vital roles in plant development and stress responses. For this research, a salt-inducible NAC gene, PsnNAC090 (Po-tri.016G076100.1), was successfully isolated from Populus simonii × Populus nigra. PsnNAC090 contains the same motifs at the N-terminal end of the highly conserved NAM structural domain. The promoter region of this gene is rich in phytohormone-related and stress response elements. Transient transformation of the gene in the epidermal cells of both tobacco and onion showed that the protein was targeted to the whole cell including the cell membrane, cytoplasm and nucleus. A yeast two-hybrid assay demonstrated that PsnNAC090 has transcriptional activation activity with the activation structural domain located at 167-256aa. A yeast one-hybrid experiment showed that PsnNAC090 protein can bind to ABA-responsive elements (ABREs). The spatial and temporal expression patterns of PsnNAC090 under salt and osmotic stresses indicated that the gene was tissue-specific, with the highest expression level in the roots of Populus simonii × Populus nigra. We successfully obtained a total of six transgenic tobacco lines overexpressing PsnNAC090. The physiological indicators including peroxidase (POD) activity, superoxide dismutase (SOD) activity, chlorophyll content, proline content, malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were measured in three transgenic tobacco lines under NaCl and polyethylene glycol (PEG) 6000 stresses. The findings reveal that PsnNAC090 improves salt and osmotic tolerance by enhancing reactive oxygen species (ROS) scavenging and reducing membrane lipid peroxide content in transgenic tobacco. All the results suggest that the PsnNAC090 gene is a potential candidate gene playing an important role in stress response.

Genome-wide analyses of the NAC transcription factor family to reveal the potential candidate genes responding to powdery mildew in balsam pear

Genome-wide analyses of the NAC transcription factor family to reveal the potential candidate genes responding to powdery mildew in balsam pear

Comparative functional analysis of PdeNAC2 and AtVND6 in the tracheary element formation.

Tracheary elements (i.e. vessel elements and tracheids) are highly specialized, non-living cells present in the water-conducting xylem tissue. In angiosperms, proteins in the VASCULAR-RELATED NAC-DOMAIN (VND) subgroup of the NAC (NAM, ATAF1,2, and CUC2) transcription factor family (e.g. AtVND6) are required for the differentiation of vessel elements through transcriptional regulation of genes responsible for secondary cell wall formation and programmed cell death. Gymnosperms, however, produce only tracheids, the mechanism of which remains elusive. Here, we report functional characteristics of PdeNAC2, a VND homolog in Pinus densiflora, as a key regulator of tracheid formation. Interestingly, our molecular genetic analyses show that PdeNAC2 can induce the formation of vessel element-like cells in angiosperm plants, demonstrated by transgenic overexpression of either native or NAC domain-swapped synthetic genes of PdeNAC2 and AtVND6 in both Arabidopsis and hybrid poplar. Subsequently, genome-wide identification of direct target (DT) genes of PdeNAC2 and AtVND6 revealed 138 and 174 genes as putative DTs, respectively, but only 17 genes were identified as common DTs. Further analyses have found that PdeNAC2 does not control some AtVND6-dependent vessel differentiation genes in angiosperm plants, such as AtVRLK1, LBD15/30 and pit-forming Rho-like GTPases from plant (ROP) signaling genes. Collectively, our results suggest that different target gene repertoires of PdeNAC2 and AtVND6 may contribute to the evolution of tracheary elements.

The NAC transcription factor family in Eucommia ulmoides: Genome-wide identification, characterization, and network analysis in relation to the rubber biosynthetic genes.

The NAC transcription factor family is a large plant gene family, participating in plant growth and development, secondary metabolite synthesis, biotic and abiotic stresses responses, and hormone signaling. Eucommia ulmoides is a widely planted economic tree species in China that can produce trans-polyisoprene: Eucommia rubber (Eu-rubber). However, genome-wide identification of the NAC gene family has not been reported in E. ulmoides. In this study, 71 NAC proteins were identified based on genomic database of E. ulmoides. Phylogenetic analysis showed that the EuNAC proteins were distributed in 17 subgroups based on homology with NAC proteins in Arabidopsis, including the E. ulmoides-specific subgroup Eu_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 7, and multitudinous EuNAC genes contained two or three exons. Chromosomal location analysis revealed that the EuNAC genes were unevenly distributed on 16 chromosomes. Three pairs of genes of tandem duplicates genes and 12 segmental duplications were detected, which indicated that segmental duplications may provide the primary driving force of expansion of EuNAC. Prediction of cis-regulatory elements indicated that the EuNAC genes were involved in development, light response, stress response and hormone response. For the gene expression analysis, the expression levels of EuNAC genes in various tissues were quite different. To explore the effect of EuNAC genes on Eu-rubber biosynthesis, a co-expression regulatory network between Eu-rubber biosynthesis genes and EuNAC genes was constructed, which indicated that six EuNAC genes may play an important role in the regulation of Eu-rubber biosynthesis. In addition, this six EuNAC genes expression profiles in E. ulmoides different tissues were consistent with the trend in Eu-rubber content. Quantitative real-time PCR analysis showed that EuNAC genes were responsive to different hormone treatment. These results will provide a useful reference for further studies addressing the functional characteristics of the NAC genes and its potential role in Eu-rubber biosynthesis.

Comprehensive Transcriptome Analysis of Responses during Cold Stress in Wheat (Triticum aestivum L.).

Wheat production is often impacted by pre-winter freezing damage and cold spells in later spring. To study the influences of cold stress on wheat seedlings, unstressed Jing 841 was sampled once at the seedling stage, followed by 4 °C stress treatment for 30 days and once every 10 days. A total of 12,926 differentially expressed genes (DEGs) were identified from the transcriptome. K-means cluster analysis found a group of genes related to the glutamate metabolism pathway, and many genes belonging to the bHLH, MYB, NAC, WRKY, and ERF transcription factor families were highly expressed. Starch and sucrose metabolism, glutathione metabolism, and plant hormone signal transduction pathways were found. Weighted Gene Co-Expression Network Analysis (WGCNA) identified several key genes involved in the development of seedlings under cold stress. The cluster tree diagram showed seven different modules marked with different colors. The blue module had the highest correlation coefficient for the samples treated with cold stress for 30 days, and most genes in this module were rich in glutathione metabolism (ko00480). A total of eight DEGs were validated using quantitative real-time PCR. Overall, this study provides new insights into the physiological metabolic pathways and gene changes in a cold stress transcriptome, and it has a potential significance for improving freezing tolerance in wheat.

Identification of the NAC Transcription Factor Family during Early Seed Development in Akebia trifoliata (Thunb.) Koidz

This study aimed to gain an understanding of the possible function of NACs by examining their physicochemical properties, structure, chromosomal location, and expression. Being a family of plant-specific transcription factors, NAC (petunia no apical meristem and Arabidopsis thaliana ATAF1, ATAF2, and CUC2) is involved in plant growth and development. None of the NAC genes has been reported in Akebia trifoliata (Thunb.) Koidz (A. trifoliata). In this study, we identified 101 NAC proteins (AktNACs) in the A. trifoliata genome by bioinformatic analysis. One hundred one AktNACs were classified into the following twelve categories based on the phylogenetic analysis of NAC protein: NAC-a, NAC-b, NAC-c, NAC-d, NAC-e, NAC-f, NAC-g, NAC-h, NAC-i, NAC-j, NAC-k, and NAC-l. The accuracy of the clustering results was demonstrated based on the gene structure and conserved motif analysis of AktNACs. In addition, we identified 44 pairs of duplication genes, confirming the importance of purifying selection in the evolution of AktNACs. The morphology and microstructure of early A. trifoliata seed development showed that it mainly underwent rapid cell division, seed enlargement, embryo formation and endosperm development. We constructed AktNACs co-expression network and metabolite correlation network based on transcriptomic and metabolomic data of A. trifoliata seeds. The results of the co-expression network showed that 25 AtNAC genes were co-expressed with 233 transcription factors. Metabolite correlation analysis showed that 23 AktNACs were highly correlated with 28 upregulated metabolites. Additionally, 25 AktNACs and 235 transcription factors formed co-expression networks with 141 metabolites, based on correlation analysis involving AktNACs, transcription factors, and metabolites. Notably, AktNAC095 participates in the synthesis of 35 distinct metabolites. Eight of these metabolites, strongly correlated with AktNAC095, were upregulated during early seed development. These studies may provide insight into the evolution, possible function, and expression of AktNACs genes.