SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis

Abstract

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.

Figure 1.

Gene Expression and Phylogenetic Analyses of NAC Genes. (A) Preferential expression of a group of NAC genes in stems. The expression data are derived from the AtGenExpress project. Leaves and roots are from 7-day-old plants, and stems are from second internodes of 21-day-old plants. (B) Phylogenetic analysis of stem-associated NAC genes together with other characterized NAC genes. The branch lengths of the tree are proportional to divergence. The 0.1 scale represents 10% change. Bootstrap values are shown in percentages at nodes. (C) RT-PCR analysis showing the expression of SND1 in stems but not in other organs. The expression of the EF1α gene was used as an internal control. (D) Cross section of a stem showing the areas (circled) microdissected by laser for RT-PCR analysis. if, interfascicular fiber; pi, pith cell; xy, xylem bundle. (E) RT-PCR analysis of laser-microdissected cells showing the expression of SND1 in interfascicular fibers and xylem cells but not in pith cells.

Figure 2.

Developmental Gene Expression Pattern and Transcriptional Activation Analysis of SND1. The expression of the SND1 gene during different stages of stem development was studied using the GUS reporter gene. Transgenic Arabidopsis plants expressing SND1:GUS were examined for GUS activity. For subcellular localization, green fluorescent protein (GFP)–tagged SND1 was expressed in carrot protoplasts and its subcellular location was examined with a laser confocal microscope. For transcriptional activation analysis, full-length or partial sequence of SND1 fused with the GAL4 DNA binding domain was expressed in yeast and tested for the activation of expression of the His3 and LacZ reporter genes. if, interfascicular fiber; xy, xylem. Bars = 150 μm in (A) to (D) and 14 μm in (E) for (E) to (H). (A) Cross section of a rapidly elongating internode showing GUS staining in developing interfascicular fiber cells. (B) Cross section of an internode near the cessation of elongation showing GUS staining in both interfascicular fibers and metaxylem. (C) Cross section of a nonelongating internode showing intensive GUS staining in both interfascicular fibers and metaxylem, both undergoing massive secondary wall thickening. (D) Cross section of a mature internode showing high GUS staining in secondary xylem but weaker staining in interfascicular fibers. (E) High magnification of a stem section showing GUS staining in xylary fiber cells but absent in developing vessels (arrows) in metaxylem. (F) and (G) Differential interference contrast (DIC) image (F) and the corresponding fluorescent signals (G) of a carrot cell expressing GFP alone. (H) and (I) DIC image (H) and the corresponding fluorescent signals (I) of a carrot cell expressing SND1-GFP. Note that SND1-GFP is targeted to the nucleus. (J) Transactivation analysis of different regions of SND1 fused with the GAL4 DNA binding domain in yeast. Note that the full-length and C-terminal region of SND1 were able to activate the expression of His3 and LacZ reporter genes.

Figure 3.

Effects of Dominant Repression of SND1 on Secondary Wall Thickening in Fibers. The full-length SND1 cDNA was fused in-frame with the dominant EAR repression sequence and transformed into Arabidopsis plants. The phenotypes of the transgenic plants were examined. co, cortex; if, interfascicular fiber; ve, vessel; xf, xylary fiber. Bars = 75 μm in (C) and (D), 35 μm in (E), (F), (I), and (J), and 2.4 μm in (G) for (G), (H), (K), and (L). (A) RT-PCR analysis showing expression of the SND1 repressor (SND1-SRDX) in the stems of three representative transgenic Arabidopsis lines. The expression of the endogenous SND1 gene (SND1) is shown for comparison. (B) Wild type plant (left) and a transgenic Arabidopsis plant expressing the SND1 repressor (right). (C) and (D) Longitudinal sections of the interfascicular region of stems showing the similar length of fiber cells in the wild type (C) and the SND1 repressors (D). (E) and (F) Cross sections of the interfascicular region showing that the interfascicular fibers of SND1 repressors (F) had thin walls compared with those of the wild type (E). (G) and (H) Transmission electron micrographs of interfascicular fiber walls of the wild type (G) and the SND1 repressors (H). (I) and (J) Cross sections of the vascular bundle region of the wild type (I) and the SND1 repressors (J). (K) and (L) Transmission electron micrographs of xylem cells showing that although the wall thickness of vessels was not changed, that of the xylary fibers was reduced severely in the SND1 repressors (L) compared with the wild type (K).

Figure 4.

Overexpression of SND1 Affects Overall Plant Development. The full-length SND1 cDNA driven by the CaMV 35S promoter was expressed in transgenic Arabidopsis plants. (A) RT-PCR analysis showing SND1 overexpression in the seedlings (top panel) and stems (bottom panel) of several representative lines of transgenic plants. (B) Seedlings of the wild type (left) and an SND1 overexpressor (right). (C) Leaves of the wild type (left) and SND1 overexpressors (middle and right). (D) Adult plants of the wild type (left) and an SND1 overexpressor (right). (E) Flowers of the wild type (left) and an SND1 overexpressor (right).

Figure 5.

Overexpression of SND1 Induces Ectopic Deposition of Lignified Secondary Walls in Epidermal and Mesophyll Cells of Leaves. Leaves of 3-week-old transgenic plants were observed with a confocal microscope for secondary walls and lignin autofluorescence, and differential interference contrast (DIC) and confocal images were collected. Bars = 42 μm in (A) to (F) and 21 μm in (G) to (J). (A) and (B) DIC image (A) and lignin autofluorescent signals (B) of a wild-type leaf showing the helical secondary wall thickening in veins. (C) and (D) DIC image (C) and lignin autofluorescent signals (D) of leaf epidermis of SND1 overexpressors showing the massive ectopic secondary wall thickening. (E) and (F) DIC image (E) and lignin autofluorescent signals (F) of leaf mesophyll cells of SND1 overexpressors showing the ectopic deposition of secondary walls. (G) and (H) High magnification of (C) and (D) showing the band-like secondary wall thickening in epidermis. (I) and (J) High magnification of (E) and (F) showing the reticulated secondary wall thickening in mesophyll cells.

Figure 6.

SND1 Overexpression Causes Ectopic Deposition of Lignified Secondary Walls in Normally Nonsclerenchymatous Cells of Flowers and Stems. For visualization of lignified secondary walls, tissues were stained with phloroglucinol-HCl before observation with a microscope. co, cortex; ep, epidermis; if, interfascicular fiber; ve, vessel; vn, vascular strand; xf, xylary fiber; xy, xylem. Bars = 92 μm in (A) and (B), 32 μm in (C), 160 μm in (D) and (E), 53 μm in (F) and (G), and 4.4 μm in (H) to (K). (A) Wild-type carpel showing lignin staining in vascular strands. (B) Carpel of SND1 overexpressors showing ectopic deposition of lignified secondary walls in parenchyma cells. (C) High magnification of a carpel of SND1 overexpressors showing the helical secondary wall thickening. (D) and (E) Cross sections of stems showing ectopic lignin staining in the epidermis of SND1 overexpressors (E) compared with the wild type (D). (F) and (G) Lignin staining of the epidermal peel of stems showing ectopic deposition of thick secondary walls (inset) in SND1 overexpressors (G) compared with the wild type (F). (H) and (I) Transmission electron micrographs of interfascicular fiber walls of the wild type (H) and SND1 overexpressors (I). (J) and (K) Transmission electron micrographs of xylem cells of the wild type (J) and SND1 overexpressors (K).

Figure 7.

Real-Time Quantitative PCR Analysis of Genes Induced by SND1 Overexpression. (A) Relative expression level of genes involved in the biosynthesis of cellulose (CesA7 and CesA8), xylan (FRA8), and lignin (CCoAOMT and 4CL1) and genes involved in programmed cell death (XCP1, XCP2, and BFN1) in the seedlings of SND1 overexpressors compared with the wild type (control). The expression level of each gene in the wild type is set to 1. Error bars represent se of three replicates. (B) Relative expression levels of fiber-associated transcription factors in the seedlings of SND1 overexpressors compared with the wild type (control).

Figure 8.

Detection of Cellulose and Xylan in Ectopically Deposited Secondary Walls in Leaves and Stems of SND1 Overexpressors. Thin sections were stained with Calcoflour White for the detection of cellulose or probed with the LM10 xylan monoclonal antibody for the detection of xylan. co, cortex; ep, epidermis; if, interfascicular fiber; xy, xylem. Bars = 186 μm in (A) to (F) and 102 μm in (G) to (L). (A) and (B) Toluidine blue staining of leaf sections showing thick walls of epidermal cells in SND1 overexpressors (B) compared with the wild type (A). (C) and (D) Calcoflour White staining of cellulose in leaf sections of the wild type (C) and SND1 overexpressors (D). (E) and (F) Detection of xylan in leaf sections of the wild type (E) and SND1 overexpressors (F). (G) and (H) Toluidine blue staining of stem sections of the wild type (G) and SND1 overexpressors (H). (I) and (J) Calcoflour White staining of cellulose in stem sections of the wild type (I) and SND1 overexpressors (J). The inset shows a high magnification of epidermis with intensive cellulose staining in walls. (K) and (L) Detection of xylan in stem sections of the wild type (K) and SND1 overexpressors (L). The inset shows a high magnification of epidermis with intensive xylan staining in walls.