NAC Genes Research Articles

Unveiling Key Genes and Unique Transcription Factors Involved in Secondary Cell Wall Formation in Pinus taeda.

Pinus taeda is a key timber species, and extensive research has been conducted on its wood formation. However, a comprehensive investigation into the biosynthetic pathways of lignin, cellulose, and hemicellulose in P. taeda is lacking, resulting in an incomplete understanding of secondary cell wall (SCW) formation in this species. In this study, we systematically analyzed transcriptomic data from previously published sources and constructed detailed pathways for lignin, cellulose, and hemicellulose biosynthesis. We identified 188 lignin-related genes and 78 genes associated with cellulose and hemicellulose biosynthesis. An RT-qPCR highlighted 15 key lignin biosynthesis genes and 13 crucial genes for cellulose and hemicellulose biosynthesis. A STEM analysis showed that most essential enzyme-coding genes clustered into Profile 14, suggesting their significant role in SCW formation. Additionally, we identified seven NAC and six MYB transcription factors (TFs) from atypical evolutionary clades, with distinct expression patterns from those of the previously characterized NAC and MYB genes, indicating potentially unique functions in SCW formation. This research provides the first comprehensive overview of lignin, cellulose, and hemicellulose biosynthetic genes in P. taeda and underscores the importance of non-canonical NAC and MYB TFs, laying a genetic foundation for future studies on SCW regulatory mechanisms.

EMBRYONIC FLOWER 1 regulates male reproduction by repressing the jasmonate pathway downstream transcription factor MYB26.

The evolutionarily conserved Polycomb repressive complexes (PRC) mediate genome-wide transcriptional silencing and regulate a plethora of development, as well as environmental responses in multicellular organisms. The PRC2-catalyzed trimethylation of lysine 27 on histone H3 (H3K27me3) is recognized by reader-effector modules of PRC1 to implement gene repression. Here, we report that the Arabidopsis (Arabidopsis thaliana) H3K27me3 effector EMBRYONIC FLOWER 1 (EMF1) interacts with and constrains the R2R3 DNA binding transcription factor MYB26 by a eudicot-conserved motif in the stamen. MYB26 activates the transcription of two NAC domain genes, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, whose encoded proteins mediate anther secondary cell thickening in jasmonate (JA)-regulated stamen maturation. In this process, the transcriptional activity of MYB26 is negatively modulated by the JAZ-PRC repressive complex to precisely regulate the expression of NST1 and NST2. Disruption of EMF1 repression stimulates MYB26, leading to the excessive transcription of the two NAC genes and male sterility. Our results reveal a novel mechanism in polycomb-mediated gene silencing and illustrate that the plant Polycomb complex regulates stamen development by preventing the hypersensitivity of JA responses in male reproduction.

Capsicum annuum NAC4 (CaNAC4) Is a Transcription Factor with Roles in Biotic and Abiotic Stresses.

Transcription factors (TFs) regulate gene expression by binding to DNA. The NAC gene family in plants consists of crucial TFs that influence plant development and stress responses. The whole genome of Capsicum annuum shows over 100 NAC genes (CaNAC). Functional characteristics of the most CaNAC TFs are unknown. In this study, we identified CaNAC4, a novel NAC TF in C. annuum. CaNAC4 expression increased after inoculation with the pathogens, Xanthomonas axonopodis pv. vesicatoria race 3 and X. axonopodis pv. glycines 8ra, and following treatment with the plant hormones, salicylic acid and abscisic acid. We investigated the functional characteristics of the CaNAC4 gene and its roles in salt tolerance and anti-pathogen defense in transgenic Nicotiana benthamiana. For salt stress analysis, the leaf discs of wild-type and CaNAC4-transgenic N. benthamiana plants were exposed to different concentrations of sodium chloride. Chlorophyll loss was more severe in salt stress-treated wild-type plants than in CaNAC4-transgenic plants. To analyze the role of CaNAC4 in anti-pathogen defense, a spore suspension of Botrytis cinerea was used to infect the leaves. The disease caused by B. cinerea gradually increased in severity, and the symptoms were clearer in the CaNAC4-transgenic lines. We also investigated hypersensitive response (HR) in CaNAC4-transgenic plants. The results showed a stronger HR in wild-type plants after infiltration with the apoptosis regulator, BAX. In conclusion, our results suggest that CaNAC4 may enhance salt tolerance and act as a negative regulator of biotic stress in plants.

Unveiling the Genomic Symphony: Identification Cultivar-Specific Genes and Enhanced Insights on Sweet Sorghum Genomes Through Comprehensive superTranscriptomic Analysis.

Sorghum (Sorghum bicolor (L.) Moench) is a multipurpose crop grown for food, fodder, and bioenergy production. Its cultivated varieties, along with their wild counterparts, contribute to the core genetic pool. Despite the availability of several re-sequenced sorghum genomes, a variable portion of sorghum genomes is not reported during reference genome assembly and annotation. The present analysis used 223 publicly available RNA-seq datasets from seven sweet sorghum cultivars to construct superTranscriptome. This approach yielded 45,864 Representative Transcript Assemblies (RTAs) that showcased intriguing Presence/Absence Variation (PAV) across 15 published sorghum genomes. We found 301 superTranscripts were exclusive to sweet sorghum, including 58 de novo genes encoded core and linker histones, zinc finger domains, glucosyl transferases, cellulose synthase, etc. The superTranscriptome added 2,802 new protein-coding genes to the Sweet Sorghum Reference Genome (SSRG), of which 559 code for different transcription factors (TFs). Our analysis revealed that MULE-like transposases were abundant in the sweet sorghum genome and could play a hidden role in the evolution of sweet sorghum. We observed large deletions in the D locus and terminal deletions in four other NAC encoding loci in the SSRG compared to its wild progenitor (353) suggesting non-functional NAC genes contributed to trait development in sweet sorghum. Moreover, superTranscript-based methods for Differential Exon Usage (DEU) and Differential Gene Expression (DGE) analyses were more accurate than those based on the SSRG. This study demonstrates that the superTranscriptome can enhance our understanding of fundamental sorghum mechanisms, improve genome annotations, and potentially even replace the reference genome.

ArNAC148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13

Acer rubrum, an ornamental tree known for its stunning autumn colors, has an elusive molecular mechanism that governs its leaf senescence. We performed the genome-wide analysis of NAC transcription factor genes and PYRABACTIN RESISTANCE1-LIKE (PYLs) and found that ArNAC148 and ArPYL13 were significantly upregulated in senescing leaves as compared to mature leaves. Subcellular localization studies confirmed the nuclear localization of ArNAC148 and the cytoplasmic localization of ArPYL13. Electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that ArNAC148 directly binds to the promoter of ArPYL13. Luciferase reporter assays further showed that ArNAC148 activates the transcription of ArPYL13. The transient expression of ArNAC148 and ArPYL13 in tobacco leaves promoted chlorophyll degradation, increased H2O2 level, MDA contents, and electrolyte leakage in response to abscisic acid (ABA). Moreover, the virus-induced gene silencing of ArNAC148 and ArPYL13 in A. rubrum produced results that were opposite to those observed in transient expression experiments. Our findings suggest that ArNAC148 induces leaf senescence by directly activating the transcription of ArPYL13, providing insights into the ABA-mediated regulatory mechanisms governing leaf senescence in A. rubrum. This study offers new perspectives for researchers to explore the roles of NAC and PYL genes in regulating leaf senescence in woody ornamental plants.

Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa.

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence.

A Combined Metabolome and Transcriptome Reveals the Lignin Metabolic Pathway during the Developmental Stages of Peel Coloration in the 'Xinyu' Pear.

Sand pear is the main cultivated pear species in China, and brown peel is a unique feature of sand pear. The formation of brown peel is related to the activity of the cork layer, of which lignin is an important component. The formation of brown peel is intimately associated with the biosynthesis and accumulation of lignin; however, the regulatory mechanism of lignin biosynthesis in pear peel remains unclear. In this study, we used a newly bred sand pear cultivar 'Xinyu' as the material to investigate the biosynthesis and accumulation of lignin at nine developmental stages using metabolomic and transcriptomic methods. Our results showed that the 30 days after flowering (DAF) to 50DAF were the key periods of lignin accumulation according to data analysis from the assays of lignin measurement, scanning electron microscope (SEM) observation, metabolomics, and transcriptomics. Through weighted gene co-expression network analysis (WGCNA), positively correlated modules with lignin were identified. A total of nine difference lignin components were identified and 148 differentially expressed genes (DEGs), including 10 structural genes (PAL1, C4H, two 4CL genes, HCT, CSE, two COMT genes, and two CCR genes) and MYB, NAC, ERF, and TCP transcription factor genes were involved in lignin metabolism. An analysis of RT-qPCR confirmed that these DEGs were involved in the biosynthesis and regulation of lignin. These findings further help us understand the mechanisms of lignin biosynthesis and provide a theoretical basis for peel color control and quality improvement in pear breeding and cultivation.

Genome-wide investigation and analysis of NAC transcription factor family in Populus tomentosa and expression analysis under salt stress.

The NAC transcription factor family is one of the largest families of TFs in plants, and members of NAC gene family play important roles in plant growth and stress response. Recent release of the haplotype-resolved genome assembly of P. tomentosa provide a platform for NAC protein genome-wide analysis. A total of 270 NAC genes were identified and a comprehensive overview of the PtoNAC gene family is presented, including gene promoter, structure and conserved motif analyses, chromosome localization and collinearity analysis, protein phylogeny, expression pattern, and interaction analysis. The results indicate that protein length, molecular weight, and theoretical isoelectric points of the NAC TF family vary, while gene structure and motif are relatively conserved. Chromosome mapping analysis showed that the P. tomentosa NAC genes are unevenly distributed on 19 chromosomes. The interchromosomal evolutionary results indicate 12 pairs of tandem and 280 segmental duplications. Segmental duplication is possibly related to amplification of P. tomentosa NAC gene family. Expression patterns of 35 PtoNAC genes from P. tomentosa subgroup were analysed under high salinity, and seven NAC genes were induced by this treatment. Promoter and protein interaction network analyses showed that PtoNAC genes are closely associated with growth, development, and abiotic and biotic stress, especially salt stress. These results provide a meaningful reference for follow-up studies of the functional characteristics of NAC genes in the mechanism of stress response and their potential roles in development of P. tomentosa.

Genome-wide analysis of NAC transcription factors and exploration of candidate genes regulating selenium metabolism in Broussonetia papyrifera.

Genome-wide identification revealed 79 BpNAC genes belonging to 16 subfamilies, and their gene structures and evolutionary relationships were characterized. Expression analysis highlighted their importance in plant selenium stress responses. Paper mulberry (Broussonetia papyrifera), a deciduous arboreal plant of the Moraceae family, is distinguished by its leaves, which are abundant in proteins, polysaccharides, and flavonoids, positioning it as a novel feedstock. NAC transcription factors, exclusive to plant species, are crucial in regulating growth, development, and response to biotic and abiotic stress. However, extensive characterization of the NAC family within paper mulberry is lacking. In this study, 79 BpNAC genes were identified from the paper mulberry genome, with an uneven distribution across 13 chromosomes. A comprehensive, genome-wide analysis of BpNACs was performed, including investigating gene structures, promoter regions, and chromosomal locations. Phylogenetic tree analysis, alongside comparisons with Arabidopsis thaliana NACs, allowed for categorizing these genes into 16 subfamilies in alignment with gene structure and motif conservation. Collinearity analysis suggested a significant homologous relationship between the NAC genes of paper mulberry and those in Morus notabilis, Ficus hispida, Antiaris toxicaria, and Cannabis sativa. Integrating transcriptome data and Se content revealed that 12 BpNAC genes were associated with selenium biosynthesis. Subsequent RT-qPCR analysis corroborated the correlation between BpNAC59, BpNAC62 with sodium selenate, and BpNAC55 with sodium selenite. Subcellular localization experiments revealed the nuclear functions of BpNAC59 and BpNAC62. This study highlights the potential BpNAC transcription factors involved in selenium metabolism, providing a foundation for strategically breeding selenium-fortified paper mulberry.

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.

Genome-Wide Identification of NAC Family Genes in Oat and Functional Characterization of AsNAC109 in Abiotic Stress Tolerance.

The plant-specific NAC gene family is one of the largest transcription factor families, participating in plant growth regulation and stress response. Despite extensive characterization in various plants, our knowledge of the NAC family in oat is lacking. Herein, we identified 333 NAC genes from the latest release of the common oat genome. We provide a comprehensive overview of the oat NAC gene family, covering gene structure, chromosomal localization, phylogenetic characteristics, conserved motif compositions, and gene duplications. AsNAC gene expression in different tissues and the response to various abiotic stresses were characterized using RT-qPCR. The main driver of oat NAC gene family expansion was identified as segmental duplication using collinearity analysis. In addition, the functions of AsNAC109 in regulating abiotic stress tolerance in Arabidopsis were clarified. This is the first genome-wide investigation of the NAC gene family in cultivated oat, which provided a unique resource for subsequent research to elucidate the mechanisms responsible for oat stress tolerance and provides valuable clues for the improvement of stress resistance in cultivated oat.

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.

An integrative analysis of Vitis vinifera L. NAC genes in response to Botrytis cinerea

Botrytis cinerea, a pathogenic necrotizing fungus, induces diseases in grapevines such as grey mold, pre- and post-harvest decay, and a reduction in fruit quality. Plant biologists focus on Vitis vinifera L. NAC genes (VvNAC) due to their crucial role in the plant defense system against diverse stresses. The objective of this study was to conduct an integrative analysis of VvNAC genes, investigating their responses to B. cinerea through an in silico method followed by an in vitro approach. The goal was to establish a connection between the outcomes of both approaches, potentially offering complementary insights. The in silico approach involved the characterization of 74 VvNAC genes within the grapevine's genome. This encompassed the identification of subcellular localization, transmembrane helices, evolutionary rates, promoter analysis, protein-protein interactions, and expression patterns in response to B. cinerea infection followed by a quantitative real-time PCR (qRT-PCR). A noteworthy discovery from our analysis revealed that 10 different VvNAC genes have been linked to B. cinerea tolerance. This finding has the potential to serve as a model for future breeding programs.

Epigenomic mechanism regulating the quality and ripeness of apple fruit with differing harvest maturity.

Harvest maturity significantly affects the quality of apple fruit in post-harvest storage process. Although the regulatory mechanisms underlying fruit ripening have been studied, the associated epigenetic modifications remain unclear. Thus, we compared the DNA methylation changes and the transcriptional responses of mature fruit (MF) and immature fruit (NF). There were significant correlations between DNA methylation and gene expression. Moreover, the sugar contents (sucrose, glucose, and fructose) were higher in MF than in NF, whereas the opposite pattern was detected for the starch content. The expression-level differences were due to DNA methylations and ultimately resulted in diverse fruit textures and ripeness. Furthermore, the higher ethylene, auxin, and abscisic acid levels in MF than in NF, which influenced the fruit texture and ripening, were associated with multiple differentially expressed genes in hormone synthesis, signaling, and response pathways (ACS, ACO, ZEP, NCED, and ABA2) that were regulated by DNA methylations. Multiple transcription factor genes involved in regulating fruit ripening and quality via changes in DNA methylation were identified, including MIKCC-type MADS-box genes and fruit ripening-related genes (NAP, SPL, WRKY, and NAC genes). These findings reflect the diversity in the epigenetic regulation of gene expression and may be relevant for elucidating the epigenetic regulatory mechanism underlying the ripening and quality of apple fruit with differing harvest maturity.

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.

Phosphorylation of birch BpNAC90 improves the activation of gene expression to confer drought tolerance.

The NAC transcription factors (TFs) play important roles in mediating abiotic stress tolerance; however, the mechanism is still not fully known. Here, an NAC gene (BpNAC90) from a gene regulatory network of Betula platyphylla (birch) that responded to drought was characterized. Overexpression and knockout of BpNAC90 displayed increased and reduced drought tolerance, respectively, relative to wild-type (WT) birch. BpNAC90 binds to different DNA motifs to regulate target genes in conferring drought tolerance, such as Eomes2, ABRE and Tgif2. BpNAC90 is phosphorylated by drought stress at Ser 205 by birch SNF1-related protein kinase 2 (BpSRK2A). Mutated BpNAC90 (termed S205A) with abolished phosphorylation, was transformed into birch for overexpression. The transgenic S205A plants displayed significantly reduced drought tolerance compared with plants overexpressing BpNAC90, but still showed increased drought tolerance relative to WT birch. At the same time, S205A showed a decreased capability to bind to motifs and reduced activation of target gene expression, which contributed to the reduced drought tolerance. Additionally, BpSRK2A and BpNAC90 can be induced by drought stress and form a complex to phosphorylate BpNAC90. The results together indicated that phosphorylation of BpNAC90 is necessary in conferring drought tolerance in birch.

The NAC gene family in the halophyte Limonium bicolor: Identification, expression analysis, and regulation of abiotic stress tolerance

NAC transcription factors regulate plant growth, development, and stress responses. However, the number, types, and biological functions of Limonium bicolor LbNAC genes have remained elusive. L. bicolor secretes excessive salt ions through salt glands on its stems and leaves to reduce salt-induced damage. Here, we identified 63 NAC members (LbNAC1–63) in L. bicolor, which were unevenly distributed across eight chromosomes. Cis-elements in the LbNAC promoters were related to growth and development, stress responses, and phytohormone responses. We observed strong colinearity between LbNACs and GmNACs from soybean (Glycine max). Thus, LbNAC genes may share similar functions with GmNAC genes. Expression analysis indicated that 16 LbNAC genes are highly expressed in roots, stems, leaves, and flowers, whereas 17 LbNAC genes were highly expressed throughout salt gland development, suggesting that they may regulate this developmental stage. Silencing LbNAC54 in L. bicolor decreased salt gland density, salt secretion from leaves, and overall salt tolerance. In agreement, genes related to salt gland development were significantly downregulated in LbNAC54-silenced lines. Our findings shed light on LbNAC genes and help elucidate salt gland development and salt secretion in L. bicolor. Our data also provide insight into NAC functions in halophytes.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

Characterization of NAC Gene Family in Ammopiptanthus mongolicus and Functional Analysis of AmNAC24, an Osmotic and Cold-Stress-Induced NAC Gene.

The NAC family of transcription factors (TFs) is recognized as a significant group within the plant kingdom, contributing crucially to managing growth and development processes in plants, as well as to their response and adaptation to various environmental stressors. Ammopiptanthus mongolicus, a temperate evergreen shrub renowned for its remarkable resilience to low temperatures and drought stress, presents an ideal subject for investigating the potential involvement of NAC TFs in stress response mechanisms. Here, the structure, evolution, and expression profiles of NAC family TFs were analyzed systematically, and a cold and osmotic stress-induced member, AmNAC24, was selected and functionally characterized. A total of 86 NAC genes were identified in A. mongolicus, and these were divided into 15 groups. Up to 48 and 8 NAC genes were generated by segmental duplication and tandem duplication, respectively, indicating that segmental duplication is a predominant mechanism in the expansion of the NAC gene family in A. mongolicus. A considerable amount of NAC genes, including AmNAC24, exhibited upregulation in response to cold and osmotic stress. This observation is in line with the detection of numerous cis-acting elements linked to abiotic stress response in the promoters of A. mongolicus NAC genes. Subcellular localization revealed the nuclear residence of the AmNAC24 protein, coupled with demonstrable transcriptional activation activity. AmNAC24 overexpression enhanced the tolerance of cold and osmotic stresses in Arabidopsis thaliana, possibly by maintaining ROS homeostasis. The present study provided essential data for understanding the biological functions of NAC TFs in plants.

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.