Arabidopsis Thaliana NAC Research Articles

Genome-wide identification and characterization of NAC genes in Brassica juncea var. tumida.

BackgroundNAC (NAM, ATAF1/2, and CUC2) transcription factors play an important role in plant growth and development. However, in tumorous stem mustard (Brassica juncea var. tumida), one of the economically important crops cultivated in southwest China and some southeast Asian countries, reports on the identification of NAC family genes are lacking. In this study, we conducted a genome-wide investigation of the NAC family genes in B. juncea var. tumida, based on its recently published genome sequence data.MethodsThe NAC genes were identified in B. juncea var. tumida using the bioinformatics approach on the whole genome level. Additionally, the expression of BjuNAC genes was analyzed under high- and low-temperature stresses by quantitative real-time PCR (qRT-PCR).ResultsA total of 300 BjuNAC genes were identified, of which 278 were mapped to specific chromosomes. Phylogenetic analysis of B. juncea var. tumida, Brassica rapa, Brassica nigra, rice and Arabidopsis thaliana NAC proteins revealed that all NAC genes were divided into 18 subgroups. Furthermore, gene structure analysis showed that most of the NAC genes contained two or three exons. Conserved motif analysis revealed that BjuNAC genes contain a conserved NAM domain. Additionally, qRT-PCR data indicated that thirteen BjuNAC genes with a varying degree of up-regulation during high-temperature stress. Conversely, four BjuNAC genes (BjuNAC006, BjuNAC083, BjuNAC170 and BjuNAC223) were up-regulated and two BjuNAC genes (BjuNAC074 and BjuNAC295) down-regulated under low temperature, respectively. Together, the results of this study provide a strong foundation for future investigation of the biological function of NAC genes in B. juncea var. tumida.

Arabidopsis NAC Transcription Factor JUNGBRUNNEN1 Exerts Conserved Control Over Gibberellin and Brassinosteroid Metabolism and Signaling Genes in Tomato.

The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripening-related genes, and leads to an increase in the levels of various amino acids (mostly proline, β-alanine, and phenylalanine), γ-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.

Subgroup-specific intrinsic disorder profiles of arabidopsis NAC transcription factors: Identification of functional hotspots

Protein intrinsic disorder (ID), referring to the lack of a fixed tertiary structure, is significant in signaling and transcription. We recently characterized ID in 6 phylogenetically representative Arabidopsis thaliana NAC transcription factors. Their transcription regulatory domains are mostly disordered but contain short, functionally important regions with structure propensities known as molecular recognition features. Here, we analyze for NAC subgroup-specific ID patterns. Some subgroups, such as the VND subgroup implicated in secondary cell wall biosynthesis, and the NAP/SHYG subgroup have highly conserved ID profiles. For the stress-associated ATAF1 subgroup and the CUC/ORE1 subgroup involved in development, only sub clades have similar ID patterns. For similar ID profiles, conserved molecular recognition features and sequence motifs represent likely functional determinants of e.g. transcriptional activation and interactions. Based on our analysis, we suggest that ID profiling of regulatory proteins in general can be used to guide identification of interaction partners of network proteins.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences. Co-expression analysis across publicly available microarray experiments identified 25 genes co-expressed with ATAF1. The promoter regions of ATAF1 co-expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis.

Characterising the role of the Eucalyptus grandis SND2promoter in secondary cell wall biosynthesis

Background NAC and MYB transcription factors (TFs) have been shown to play prominent roles in the regulation of plant developmental processes. Two Arabidopsis thaliana NAC domain TFs (AtSND2, AtSND3) and one MYB domain TF (AtMYB103) were shown to be downstream targets of two master regulators of xylem fibre cell development, NST1 and SND1 [1,2]. These TFs were able to induce the expression of a GUS reporter gene under the control of the AtCesA8 promoter [3], indicating that they may be involved in the regulation of cellulose biosynthesis in the secondary cell walls of A. thaliana xylem fibres. It is hypothesized that putative orthologs of these TFs will also play important roles in regulating fibre secondary cell wall biosynthesis in woody plants such as Eucalyptus grandis. The transcriptional network regulating wood fibre development is uncharacterized in E. grandis, therefore it would be beneficial to identify upstream components of this transcriptional network in E. grandis. In this ongoing study we aim to identify TFs which bind to the promoter of the putative ortholog of AtSND2 in E. grandis (EgSND2) and possibly also to the promoters of the orthologs of AtSND3 and AtMYB103. In parallel, we are characterizing the detailed heterologous expression patterns of the EgSND2, EgSND3 and MYB103 promoter regions in A. thaliana plants. This work forms part of an effort to elucidate the transcriptional network regulating wood fibre development in E. grandis. Methods A reverse BLAST approach was used to identify candidate orthologs of AtSND2, AtSND3 and AtMYB103 in Eucalyptus, Populus and Vitis. In silico cis-element analysis was performed on the promoter regions of the putative orthologs to identify previously characterised and novel cis-elements. The 1.5 kb regions upstream of the translational start site (TSS) of EgSND2, EgSND3 and EgMYB103 were isolated from E. grandis genomic DNA. The amplified fragments were cloned into pMDC162, a GUS reporter vector, and introduced into A. thaliana Col-O plants for heterologous GUS expression analysis. The same reporter constructs were also transformed directly into the vascular cambium of potted E. grandis plants to determine endogenous promoter activity by Induced Somatic Sector Analysis (ISSA, [4]). Qualitative GUS reporter analyses were performed on the EgSND2promoter::GUS, EgSND3promoter::GUS and EgMYB103promoter::GUS constructs in Arabidopsis plants at 1, 3 and 6 weeks. A 500 bp truncation upstream of the TSS of EgSND2 was generated and cloned into the pHIS2.1 vector along with the 1.5 kb EgSND2 promoter sequence for use in yeast onehybrid (Y1-H) screening. Candidate proteins which bind to the cloned promoter sequences will be identified using Y1-H analysis and further characterised.

VASCULAR‐RELATED NAC‐DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation

The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), acts as a key regulator of xylem vessel differentiation. In order to identify direct target genes of VND7, we performed global transcriptome analysis using Arabidopsis transgenic lines in which VND7 activity could be induced post-translationally. This analysis identified 63 putative direct target genes of VND7, which encode a broad range of proteins, such as transcription factors, IRREGULAR XYLEM proteins and proteolytic enzymes, known to be closely associated with xylem vessel formation. Recombinant VND7 protein binds to several promoter sequences present in candidate direct target genes: specifically, in the promoter of XYLEM CYSTEINE PEPTIDASE1, two distinct regions were demonstrated to be responsible for VND7 binding. We also found that expression of VND7 restores secondary cell wall formation in the fiber cells of inflorescence stems of nst1 nst3 double mutants, as well as expression of NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3, however, the vessel-type secondary wall deposition was observed only as a result of VND7 expression. These findings indicated that VND7 upregulates, directly and/or indirectly, many genes involved in a wide range of processes in xylem vessel differentiation, and that its target genes are partially different from those of NSTs.

Global Analysis of Direct Targets of Secondary Wall NAC Master Switches in Arabidopsis

We report the genome-wide analysis of direct target genes of SND1 and VND7, two Arabidopsis thaliana NAC domain transcription factors that are master regulators of secondary wall biosynthesis in fibers and vessels, respectively. Systematic mapping of the SND1 binding sequence using electrophoretic mobility shift assay and transactivation analysis demonstrated that SND1 together with other secondary wall NACs (SWNs), including VND6, VND7, NST1, and NST2, bind to an imperfect palindromic 19-bp consensus sequence designated as secondary wall NAC binding element (SNBE), (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T), in the promoters of their direct targets. Genome-wide analysis of direct targets of SND1 and VND7 revealed that they directly activate the expression of not only downstream transcription factors, but also a number of non-transcription factor genes involved in secondary wall biosynthesis, cell wall modification, and programmed cell death, the promoters of which all contain multiple SNBE sites. SND1 and VND7 directly regulate the expression of a set of common targets but each of them also preferentially induces a distinct set of direct targets, which is likely attributed to their differential activation strength toward SNBE sites. Complementation study showed that the SWNs were able to rescue the secondary wall defect in the snd1 nst1 mutant, indicating that they are functionally interchangeable. Together, our results provide important insight into the complex transcriptional program and the evolutionary mechanism underlying secondary wall biosynthesis, cell wall modification, and programmed cell death in secondary wall-containing cell types.

VND-INTERACTING2, a NAC Domain Transcription Factor, Negatively Regulates Xylem Vessel Formation in Arabidopsis

The Arabidopsis thaliana NAC domain transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) acts as a master regulator of xylem vessel differentiation. To understand the mechanism by which VND7 regulates xylem vessel differentiation, we used a yeast two-hybrid system to screen for proteins that interact with VND7 and identified cDNAs encoding two NAC domain proteins, VND-INTERACTING1 (VNI1) and VNI2. Binding assays demonstrated that VNI2 effectively interacts with VND7 and the VND family proteins, VND1-5, as well as with other NAC domain proteins at lower affinity. VNI2 is expressed in both xylem and phloem cells in roots and inflorescence stems. The expression of VNI2 overlaps with that of VND7 in elongating vessel precursors in roots. VNI2 contains a predicted PEST motif and a C-terminally truncated VNI2 protein, which lacks part of the PEST motif, is more stable than full-length VNI2. Transient reporter assays showed that VNI2 is a transcriptional repressor and can repress the expression of vessel-specific genes regulated by VND7. Expression of C-terminally truncated VNI2 under the control of the VND7 promoter inhibited the normal development of xylem vessels in roots and aerial organs. These data suggest that VNI2 regulates xylem cell specification as a transcriptional repressor that interacts with VND proteins and possibly also with other NAC domain proteins.

The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling

TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.

A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence

The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.

VASCULAR‐RELATED NAC‐DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots

The Arabidopsis thaliana NAC domain transcription factor, vascular-related NAC-DOMAIN7 (VND7), plays a pivotal role in regulating the differentiation of root protoxylem vessels. In order to understand the mechanisms underscoring the function of VND7 in vessel differentiation in more detail, we conducted extensive molecular analyses in yeast (Saccharomyces cerevisiae), Arabidopsis, and Nicotiana tabacum L. cv. Bright Yellow 2 (tobacco BY-2) cells. The transcriptional activation activity of VND7 was confirmed in yeast and Arabidopsis, and the C-terminal region was shown to be required for VND7 transcriptional activation. Expression of the C-terminus-truncated VND7 protein under the control of the native VND7 promoter resulted in inhibition of the normal development of metaxylem vessels in roots and vessels in aerial organs, as well as protoxylem vessels in roots. The expression pattern of VND7 overlapped that of VND2 to VND5 in most of the differentiating vessels. Furthermore, a yeast two-hybrid assay revealed the ability of VND7 to form homodimers and heterodimers with other VND proteins via their N-termini, which include the NAC domain. The heterologous expression of VND7 in tobacco BY-2 cells demonstrated that the stability of VND7 could be regulated by proteasome-mediated degradation. Together these data suggest that VND7 regulates the differentiation of all types of vessels in roots and shoots, possibly in cooperation with VND2 to VND5 and other regulatory proteins.

Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses

Jasmonic acid (JA) is an important phytohormone that regulates plant defense responses against herbivore attack, pathogen infection and mechanical wounding. In this report, we provided biochemical and genetic evidence to show that the Arabidopsis thaliana NAC family proteins ANAC019 and ANAC055 might function as transcription activators to regulate JA-induced expression of defense genes. The role of the two NAC genes in JA signaling was examined with the anac019 anac055 double mutant and with transgenic plants overexpressing ANAC019 or ANAC055. The anac019 anac055 double mutant plants showed attenuated JA-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) and LIPOXYGENASE2 (LOX2) expression, whereas transgenic plants overexpressing the two NAC genes showed enhanced JA-induced VSP1 and LOX2 expression. That the JA-induced expression of the two NAC genes depends on the function of COI1 and AtMYC2, together with the finding that overexpression of ANAC019 partially rescued the JA-related phenotype of the atmyc2-2 mutant, has led us to a hypothesis that the two NAC proteins act downstream of AtMYC2 to regulate JA-signaled defense responses. Further evidence to substantiate this idea comes from the observation that the response of the anac019 anac055 double mutant to a necrotrophic fungus showed high similarity to that of the atmyc2-2 mutant.

SND1, a NAC Domain Transcription Factor, Is a Key Regulator of Secondary Wall Synthesis in Fibers ofArabidopsis

Secondary walls in fibers and tracheary elements constitute the most abundant biomass produced by plants. Although a number of genes involved in the biosynthesis of secondary wall components have been characterized, little is known about the molecular mechanisms underlying the coordinated expression of these genes. Here, we demonstrate that the Arabidopsis thaliana NAC (for NAM, ATAF1/2, and CUC2) domain transcription factor, SND1 (for secondary wall-associated NAC domain protein), is a key transcriptional switch regulating secondary wall synthesis in fibers. We show that SND1 is expressed specifically in interfascicular fibers and xylary fibers in stems and that dominant repression of SND1 causes a drastic reduction in the secondary wall thickening of fibers. Ectopic overexpression of SND1 results in activation of the expression of secondary wall biosynthetic genes, leading to massive deposition of secondary walls in cells that are normally nonsclerenchymatous. In addition, we have found that SND1 upregulates the expression of several transcription factors that are highly expressed in fibers during secondary wall synthesis. Together, our results reveal that SND1 is a key transcriptional activator involved in secondary wall biosynthesis in fibers.