Secondary wall NAC binding element (SNBE), a key cis-acting element required for target gene activation by secondary wall NAC master switches

Abstract

The biosynthesis of secondary walls in vascular plants requires the coordinated regulation of a suite of biosynthetic genes, and this coordination has recently been shown to be executed by the secondary wall NAC (SWN)-mediated transcriptional network. In Arabidopsis, five SWNs, including SND1, NST1/2 and VND6/7, function as master transcriptional switches to activate their common targets and consequently the secondary wall biosynthetic program. A recent report by Zhong et al.¹ revealed that SWNs bind to a common cis-acting element, namely secondary wall NAC binding element (SNBE), which is composed of an imperfect palindromic 19-bp consensus sequence, (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T). Genome-wide analysis of direct targets of SWNs showed that SWNs directly activate the expression of not only many transcription factors but also a battery of genes involved in secondary wall biosynthesis, cell wall modification and programmed cell death, the promoters of which all contain multiple SNBE sites. The functional significance of the SNBE sites is further substantiated by our current in planta expression study demonstrating that representative SNBE sequences from several SWN direct target promoters are sufficient to drive the expression of the GUS reporter gene in secondary wall-forming cells. The identification of the SWN DNA binding element (SNBE) and the SWN direct targets marks an important step forward toward the dissection of the transcriptional network regulating the biosynthesis of secondary walls, the most abundant biomass produced by land plants.

One of the pivotal innovations for vascular plants to conquer the terrestrial habitat was their ability to construct secondary cell walls, which allowed vascular plants to build reinforced xylem conduits for fluid conduction and mechanical support.² Secondary walls are mainly composed of cellulose, hemicelluloses and lignin, the biosynthesis of which requires the coordinated expression of a battery of biosynthetic genes and a concerted action of their encoded proteins. It has been shown that in Arabidopsis, the coordinated expression of secondary wall biosynthetic genes is regulated by a group of closely-related secondary wall NAC master switches (collectively called SWNs), including SND1, NST1/2 and VND6/7.^3,4 SND1 and NST1 function redundantly in the regulation of secondary wall thickening in fibers,^5–7 whereas VND6/7 are involved in secondary wall deposition in vessels.⁸ NST1 and NST2 play essential roles in the secondary wall biosynthesis during the formation of endothecium in anthers.⁹

Analysis of SND1-regulated downstream transcription factors revealed a complex transcriptional network composed of transcription factors positioned at several regulatory levels.⁴ In this network, several transcription factors, including MYB46, MYB83, MYB103, SND3 and KNAT7, are direct targets of SND1 and other SWNs, including NST1/2 and VND6/7.¹⁰ Of these SWN direct targets, MYB46 and MYB83 act redundantly as second-level master regulators of secondary wall biosynthesis.^11,12 Furthermore, several SWN-regulated downstream transcription factors, including MYB58, MYB63 and MYB85, are key regulators specific for lignin biosynthesis.^10,13,14 The SWN-mediated transcriptional network governing secondary wall biosynthesis is most likely conserved in vascular plants because homologs of SWNs and their downstream transcription factors are present in diverse vascular plants and some of them have been demonstrated to be functional orthologs of their Arabidopsis counterparts.^4,15–17 The finding that SWNs are master transcriptional switches controlling secondary wall biosynthesis offers an unprecedented opportunity to dissect the molecular mechanism underlying the transcriptional regulation of secondary wall formation. The recent report by Zhong et al.¹ marks an important advance toward this direction by successful mapping of the SWN binding elements and uncovering an array of additional SWN direct targets, including not only transcription factors but also many genes involved in secondary wall biosynthesis, cell wall modification and programmed cell death.

Based on the previous finding that SND1 binds to a 24-bp sequence in the promoter of its direct target, MYB46,¹¹ Zhong et al.¹ set out to map the precise SND1 binding site. Using mutational analysis coupled with the electrophoretic mobility shift assay (EMSA), Zhong et al.¹ revealed the consensus SND1 binding sequence, (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C) GN(A/C/T)(A/T) and designated this 19-bp imperfect palidromic sequence as secondary wall NAC binding element (SNBE) (Fig. 1A). It was found that SNBEs are present in the promoters of all direct target genes of SND1 and VND7. Subsequent EMSA and transactivation experiments have demonstrated that representative SNBE sequences from the promoters of these target genes are bound and activated by not only SND1 and VND7 but also other SWNs, including NST1/2 and VND6.¹ These findings indicate that SWNs activate the expression of their common direct target genes through binding to the SNBE sites in their promoters.

Figure 1.

The SNBE sequences are capable of driving the GUS reporter gene expression in secondary wall-forming cells. (A) The top part shows the representative SNBE sequences from the promoters of several SWN direct targets, including transcription factors (MYB46, MYB83, SND3, MYB103, KNAT7) and hydrolases involved in programmed cell death [a ribonuclease (RNS3) and a xylem-specific cysteine protease (XCP1)]. The critical nucleotides in the SNBE sequences are shaded. The lower part shows the tracheary element responsive elements (TERE) from two xylem-specific cysteine proteases (XCP1 and XCP2) together with the consensus TERE sequence. (B) Diagram of the GUS reporter gene driven by three copies of SNBE sequence from various SWN direct target promoters as shown in (A). The GUS reporter constructs in a binary vector were introduced into Arabidopsis and at least 30 transgenic plants in the first generation were examined for GUS expression. CaMV, cauliflower mosaic virus; NosT, nopaline synthase terminator. (C and D) Cross sections of stems from transgenic plants expressing the GUS reporter gene driven by five copies of XCP1-TERE (C) and XCP2-TERE (D) showing the absence of GUS staining. (E to J) Cross sections of stems from transgenic plants expressing the GUS reporter gene driven by three copies of representative SNBE sequence from MYB46 (E and F), MYB83 (G), SND3 (H), MYB103 (I) and KNAT7 (J) showing the GUS staining in secondary wall-forming cells, including xylem and interfascicular fibers. (K and L) Cross sections of stems from transgenic plants expressing the GUS reporter gene driven by 3 copies of SNBE sequence from RNS3 (K) and XCP1 (L) showing the specific GUS staining in the xylem. if, interfascicular fiber; xy, xylem. Scale bar in (C) = 125 µm for (C–L).

To further substantiate the functional significance of the SNBE sites, we investigated the in planta expression patterns of the GUS reporter gene driven by three tandem repeats of various SNBEs (Fig. 1A and B). We tested several SNBEs from a number of representative SWN direct target genes, including transcription factors (MYB46, MYB83, SND3, MYB103 and KNAT7) and programmed cell death genes (RNS3 and XCP1). Since SWNs are specifically expressed in secondary wall-forming cells, it is expected that the SNBE-driven GUS reporter gene is also expressed in the same tissue types as SWNs. Examination of transgenic Arabidopsis plants expressing the GUS reporter gene driven by these SNBE sequences revealed that the SNBEs from MYB46, MYB83, SND3, MYB103 and KNAT7 all directed GUS expression in secondary wall-forming cells in the stems (Fig. 1E–J), including both xylem and interfascicular fibers, an expression pattern similar to that of SND1.¹ On the other hand, the SNBEs from RNS3 and XCP1 directed GUS expression only in xylem cells (Fig. 1K and L), a pattern identical to that of the xylemspecific SWNs, VND6 and VND7. This in planta functional study of the SNBE sites supports the conclusion that SWNs bind to the SNBE sequences in the promoters of their direct target genes and thereby activate their expression in the secondary wall-forming cells.

It was previously reported that the TERE sequences were responsible for the xylem-specific expression of a number of cell wall modification and programmed cell death genes.¹⁸ We therefore also examined the expression patterns of the GUS reporter gene driven by five tandem repeats of TEREs from the promoters of two xylem-specific cysteine proteases XCP1 and XCP2. It was found that the TEREs were unable to drive the GUS reporter gene expression in xylem cells in the Arabidopsis stems (Fig. 1C and D), suggesting that the xylem-specific expression of XCP1/2 is not mediated through TEREs.

The identification of the consensus SNBE sequence and the demonstration that they can direct gene expression in secondary wall forming cells mark a significant step forward toward the dissection of the SWN-mediated transcriptional network regulating secondary wall biosynthesis. It is noteworthy that although different SWNs are expressed in specific secondary wall forming cells, such as SND1 in fibers and VND6/7 in vessels, respectively,^5,8,10 they all bind to the same SNBE sequences albeit with different affinities toward various SNBE sequence variants.¹ It is apparent that plants evolved to have multiple SWNs, probably via duplication and diversification, to regulate secondary wall thickening in different cell types without drastic alterations in their DNA binding specificity. This conservation of the DNA binding specificity also imply that the same SNBE sites might have been evolved when vascular plants first acquired the ability to construct secondary walls and that these sites are evolutionarily conserved due to strong selection pressure. This possibility is consistent with the previous findings that SWN homologs from other species, such as poplar and Eucalyptus, are able to activate the secondary wall biosynthetic program when overexpressed in Arabidopsis,^4,15 most likely through binding to the SNBE sites. It will be interesting to further investigate whether SWN homologs from different lineages of vascular plants are functional orthologs of the Arabidopsis SWNs and whether their DNA binding specificities are all conserved.

Secondary walls are the major components of wood biomass, which has recently been considered to be an important source for biofuel production in addition to its traditional uses in a variety of applications, such as construction, pulping and paper-making, and direct burning for energy. Uncovering the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis will help us design novel strategies for improvement of wood quantity and quality, such as manipulating the biosynthetic pathways of individual secondary wall components by altering one or a few key transcriptional switches. Available evidence indicates that the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis are well conserved across plant species,⁴ and therefore it is expected that the knowledge on the transcriptional control of secondary wall biosynthesis learned from one species will be applicable to others, such as biofuel crops, for engineering biomass specifically tailored for biofuel production.