Zheng-Hua Ye

Mutation of a Chitinase-Like Gene Causes Ectopic Deposition of Lignin, Aberrant Cell Shapes, and Overproduction of Ethylene

Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis

SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening. Of these, dominant repression of SND2, SND3, MYB103, MYB85, MYB52, MYB54, and KNAT7 significantly reduced secondary wall thickening in fiber cells. Overexpression of SND2, SND3, and MYB103 increased secondary wall thickening in fibers, and overexpression of MYB85 led to ectopic deposition of lignin in epidermal and cortical cells in stems. Furthermore, SND2, SND3, MYB103, MYB85, MYB52, and MYB54 were able to induce secondary wall biosynthetic genes. Direct target analysis using the estrogen-inducible system revealed that MYB46, SND3, MYB103, and KNAT7 were direct targets of SND1 and also of its close homologs, NST1, NST2, and vessel-specific VND6 and VND7. Together, these results demonstrate that a transcriptional network consisting of SND1 and its downstream targets is involved in regulating secondary wall biosynthesis in fibers and that NST1, NST2, VND6, and VND7 are functional homologs of SND1 that regulate the same downstream targets in different cell types.

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

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.

MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in Arabidopsis

It has previously been shown that SECONDARY WALL–ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a key transcription factor regulating secondary cell wall formation, including the biosynthesis of cellulose, xylan, and lignin. In this study, we show that two closely related SND1-regulated MYB transcription factors, MYB58 and MYB63, are transcriptional regulators specifically activating lignin biosynthetic genes during secondary wall formation in Arabidopsis thaliana. MYB58 and MYB63 are phylogenetically distinct from previously characterized MYBs shown to be associated with secondary wall formation or phenylpropanoid metabolism. Expression studies showed that MYB58 and MYB63 are specifically expressed in fibers and vessels undergoing secondary wall thickening. Dominant repression of their functions led to a reduction in secondary wall thickening and lignin content. Overexpression of MYB58 and MYB63 resulted in specific activation of lignin biosynthetic genes and concomitant ectopic deposition of lignin in cells that are normally unlignified. MYB58 was able to activate directly the expression of lignin biosynthetic genes and a secondary wall–associated laccase (LAC4) gene. Furthermore, the expression of MYB58 and MYB63 was shown to be regulated by the SND1 close homologs NST1, NST2, VND6, and VND7 and their downstream target MYB46. Together, our results indicate that MYB58 and MYB63 are specific transcriptional activators of lignin biosynthesis in the SND1-mediated transcriptional network regulating secondary wall formation.

The MYB46 Transcription Factor Is a Direct Target of SND1 and Regulates Secondary Wall Biosynthesis in Arabidopsis

We demonstrate that the Arabidopsis thaliana MYB46 transcription factor is a direct target of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), which is a key transcriptional activator regulating the developmental program of secondary wall biosynthesis. The MYB46 gene is expressed predominantly in fibers and vessels in stems, and its encoded protein is targeted to the nucleus and can activate transcription. MYB46 gene expression was shown to be regulated by SND1, and transactivation analysis demonstrated that SND1 as well as its close homologs were able to activate the MYB46 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments revealed that SND1 binds to the MYB46 promoter. Dominant repression of MYB46 caused a drastic reduction in the secondary wall thickening of fibers and vessels. Overexpression of MYB46 resulted in an activation of the biosynthetic pathways of cellulose, xylan, and lignin and concomitantly led to ectopic deposition of secondary walls in cells that are normally nonsclerenchymatous. In addition, the expression of two secondary wall–associated transcription factors, MYB85 and KNAT7, was highly upregulated by MYB46 overexpression. These results demonstrate that MYB46 is a direct target of SND1 and is another key player in the transcriptional network involved in the regulation of secondary wall biosynthesis in Arabidopsis.

Arabidopsis irregular xylem8 and irregular xylem9: Implications for the Complexity of Glucuronoxylan Biosynthesis

Mutations of Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9 were previously shown to cause a collapsed xylem phenotype and decreases in xylose and cellulose in cell walls. In this study, we characterized IRX8 and IRX9 and performed chemical and structural analyses of glucuronoxylan (GX) from irx8 and irx9 plants. IRX8 and IRX9 are expressed specifically in cells undergoing secondary wall thickening, and their encoded proteins are targeted to the Golgi, where GX is synthesized. 1H-NMR spectroscopy showed that the reducing end of Arabidopsis GX contains the glycosyl sequence 4-β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp, which was previously identified in birch (Betula verrucosa) and spruce (Picea abies) GX. This indicates that the reducing end structure of GXs is evolutionarily conserved in woody and herbaceous plants. This sequence is more abundant in irx9 GX than in the wild type, whereas irx8 and fragile fiber8 (fra8) plants are nearly devoid of it. The number of GX chains increased and the GX chain length decreased in irx9 plants. Conversely, the number of GX chains decreased and the chain length heterodispersity increased in irx8 and fra8 plants. Our results suggest that IRX9 is required for normal GX elongation and indicate roles for IRX8 and FRA8 in the synthesis of the glycosyl sequence at the GX reducing end.

A Katanin-like Protein Regulates Normal Cell Wall Biosynthesis and Cell Elongation

Fibers are one of the mechanical tissues that provide structural support to the plant body. To understand how the normal mechanical strength of fibers is regulated, we isolated an Arabidopsis fragile fiber (fra2) mutant defective in the mechanical strength of interfascicular fibers in the inflorescence stems. Anatomical and chemical analyses showed that the fra2 mutation caused a reduction in fiber cell length and wall thickness, a decrease in cellulose and hemicellulose contents, and an increase in lignin condensation, indicating that the fragile fiber phenotype of fra2 is a result of alterations in fiber cell elongation and cell wall biosynthesis. In addition to the effects on fibers, the fra2 mutation resulted in a remarkable reduction in cell length and an increase in cell width in all organs, which led to a global alteration in plant morphology. The FRA2 gene was shown to encode a protein with high similarity to katanin (hence FRA2 was renamed AtKTN1), a protein shown to be involved in regulating microtubule disassembly by severing microtubules. Consistent with the putative function of AtKTN1 as a microtubule-severing protein, immunolocalization demonstrated that the fra2 mutation caused delays in the disappearance of perinuclear microtubule array and in the establishment of transverse cortical microtubule array in interphase and elongating cells. Together, these results suggest that AtKTN1, a katanin-like protein, is essential not only for normal cell wall biosynthesis and cell elongation in fiber cells but also for cell expansion in all organs.

IFL1, a Gene Regulating Interfascicular Fiber Differentiation in Arabidopsis, Encodes a Homeodomain-Leucine Zipper Protein

Arabidopsis inflorescence stems develop extraxylary fibers at specific sites in interfascicular regions. The spatial specification of interfascicular fiber differentiation is regulated by the INTERFASCICULAR FIBERLESS1 (IFL1) gene because mutation of that gene abolishes the formation of normal interfascicular fibers in Arabidopsis stems. To understand further the role of IFL1 in the specification of fiber differentiation, we cloned the IFL1 gene by using a positional cloning strategy. Sequence analysis showed that the IFL1 gene encodes a transcription factor that has the same features as a family of homeodomain-leucine zipper (HD-ZIP) proteins found only in plants. The predicted IFL1 protein is composed of three distinct domains, including a 60-amino acid HD at the N terminus followed by a 28-amino acid ZIP motif and a 724-amino acid C-terminal region. A nuclear targeting assay showed that IFL1 is able to direct a β-glucuronidase fusion protein into the nucleus, which is consistent with IFL1s presumed function as a transcription factor. Gene expression analysis demonstrated that the IFL1 gene is expressed in the interfascicular regions in which fibers differentiate, which is consistent with its role in the control of interfascicular fiber differentiation. Furthermore, the IFL1 gene was shown to be expressed in the vascular regions, indicating its possible role in the regulation of vascular tissue formation. This possibility is supported by the observation that differentiation of both xylary fibers and vessel elements is altered in the vascular bundles of ifl1 mutants. Our results provide direct evidence that an HD-ZIP protein plays a role in the spatial control of fiber differentiation.

Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis.

Secondary walls are the major component of wood, and studies of the mechanisms regulating secondary wall synthesis is important for understanding the process of wood formation. We have previously shown that the NAC domain transcription factor SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a key regulator of secondary wall synthesis in fibers of Arabidopsis thaliana stems and dominant repression of SND1 leads to a reduction in secondary wall thickening in fibers. However, T-DNA knockout of the SND1 gene did not cause an alteration in secondary wall thickness, suggesting that other SND1 homologs may compensate for the loss of SND1 expression. Here, we studied the effects of simultaneous inhibition of SND1 and its homolog, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), on secondary wall synthesis in fibers. We show that simultaneous RNA interference (RNAi) inhibition of the expression of both SND1 and NST1 genes results in loss of secondary wall formation in fibers of stems. The fiber cells in the stems of SND1/NST1–RNAi plants lack all three major secondary wall components, including cellulose, xylan, and lignin, which is accompanied by a severe reduction in the expression of genes involved in their biosynthesis. In addition, inhibition of SND1 and NST1 leads to down-regulation of several fiber-associated transcription factor genes. Double T-DNA knockout mutations of SND1 and NST1 genes cause the same effects, as does simultaneous RNAi inhibition of SND1 and NST1. Our results provide first line evidence demonstrating that SND1 and NST1 function redundantly in the regulation of secondary wall synthesis in fibers.

Arabidopsis Fragile Fiber8, Which Encodes a Putative Glucuronyltransferase, Is Essential for Normal Secondary Wall Synthesis

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.