NAC Transcription Factor Family Research Articles

Expression analysis of CsNAC30 gene in cucumber under drought stress

This study performed a bioinformatics analysis of the CsNAC30 gene and examined its expression levels in cucumber leaves and roots under drought stress induced by PEG-6000. The bioinformatics analysis showed that the open reading frame (ORF) of CsNAC30 is 1254 bp, encoding a protein consisting of 417 amino acids. This protein is relatively unstable and hydrophilic, lacks transmembrane regions, and contains 49 phosphorylation sites. It also features a conserved NAM domain at the N-terminus. Homology and phylogenetic analyses indicate that CsNAC30 is a member of the NAC transcription factor family and is most closely related to the melon CMeNAC105 and the winter melon BhNAC96. Quantitative real-time PCR (qPCR) revealed that CsNAC30 expression is induced by drought stress, with relative levels increasing in both leaves and roots following PEG treatment. Expression peaked at 6 h in leaves and 9 h in roots before decreasing. These findings suggest that CsNAC30 plays a role in the cucumber’s response to drought stress.

Transcriptome, hormonal, and secondary metabolite changes in leaves of DEFENSE NO DEATH 1 (DND1) silenced potato plants

DEFENSE NO DEATH 1 (DND1) is a cyclic nucleotide-gated ion channel protein. Earlier, it was shown that the silencing of DND1 in the potato (Solanum tuberosum L.) leads to resistance to late blight, powdery mildew, and gray mold diseases. At the same time, however, it can reduce plant growth and cause leaf necrosis. To obtain knowledge of the molecular events behind the pleiotropic effect of DND1 downregulation in the potato, metabolite and transcriptome analyses were performed on three DND1 silenced lines of the cultivar ‘Désirée.’ A massive increase in the salicylic acid content of leaves was detected. Concentrations of jasmonic acid and chlorogenic acid and their derivatives were also elevated. Expression of 1866 genes was altered in the same way in all three DND1 silenced lines, including those related to the synthesis of secondary metabolites. The activation of several alleles of leaf rust, late blight, and other disease resistance genes, as well as the induction of pathogenesis-related genes, was detected. WRKY and NAC transcription factor families were upregulated, whereas bHLHs were downregulated, indicating their central role in transcriptome changes. These results suggest that the maintenance of the constitutive defense state leads to the reduced growth of DND1 silenced potato plants.

Mucilage secretion from the root cap requires the NAC family transcription factor BEARSKIN2.

The root cap secretes mucilage and sheds border cells (border-like cells, BLCs) in Arabidopsis (Arabidopsis thaliana). These mucilage and root cap-derived cells form a defensive barrier against soil pathogens. BEARSKIN1 (BRN1) and BRN2 are 2 homologous NAM, ATAF1/2, and CUC2 (NAC) family transcription factors of Arabidopsis, and mucilage secretion is inhibited in the brn1/2 double mutant. BRN1 and BRN2 are also involved in the expression of a pectin-digesting enzyme, POLYGALACTURONASE (RCPG), that facilitates BLC shedding. To further explore the connection between mucilage secretion and BLC shedding, we examined mucilage production in Arabidopsis lines displaying altered BLC detachment. Inactivation of BRN2 blocked mucilage synthesis and secretion, while inactivation of BRN1 and RCPG did not. Interestingly, RCPG sorted into mucilage-carrying vesicles budding from the Golgi and inhibited mucilage secretion in brn2-delayed BLC detachment. The root cap of a germinating seedling is initially covered with a cuticle, which is replaced by mucilage from BLCs as the seedling begins to shed these cells. Ectopic expression of RCPG in germinating seedlings caused early BLC formation and accelerated the cuticle-to-mucilage transition, indicating that RCPG expression and mucilage secretion are co-regulated. Furthermore, brn2 roots exhibited slower growth and increased cell death when subjected to salt or osmotic stress. Our research suggests that BRN2-mediated mucilage secretion contributes to BLC release to build an extracellular defense zone surrounding the root cap.

A novel AP2/ERF transcription factor, NtERF10, positively regulates plant height in tobacco

Ethylene response factors have been shown to be involved in the effects of plant developmental processes and to regulate stress tolerance. The aim of this study was to recognize the regulatory mechanisms of ethylene response factors on tobacco plant height. In this study, a gene-edited mutant (ERF10-KO) and wild type (WT) were utilized as experimental materials. Transcriptome and metabolome analyses were used to investigate the regulatory mechanism of NtERF10 gene editing on plant height in tobacco. Here, through the analysis of differentially expressed genes (DEGs), 2051 genes were upregulated and 1965 genes were downregulated. We characterized the different ERF10-KO and WT plant heights and identified key genes for photosynthesis, the plant hormone signal transduction pathway and the terpene biosynthesis pathway. NtERF10 was found to affect the growth and development of tobacco by regulating the expression levels of the PSAA, PSBA, GLY17 and GGP3 genes. Amino acid metabolism was analyzed by combining analyses of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). In addition, we found that members of the bHLH, NAC, MYB, and WRKY transcription factor families have vital roles in regulating plant height. This study not only provides important insights into the positive regulation of the ethylene response factor NtERF10 on plant height during plant growth and development but also provides new research ideas for tobacco molecular breeding.

Construction of Gene Expression Profiles of the NAC Transcription Factor Family in Sugar Beet Under Abiotic Stress

Construction of Gene Expression Profiles of the NAC Transcription Factor Family in Sugar Beet Under Abiotic Stress

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.

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 NAM, ATAF1/2, and CUC2 (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.