MYB75 functions in regulation of secondary cell wall formation in the Arabidopsis inflorescence stem

Abstract

Deposition of lignified secondary cell walls in plants involves a major commitment of carbon skeletons in both the form of polysaccharides and phenylpropanoid constituents. This process is spatially and temporally regulated by transcription factors, including a number of MYB family transcription factors. MYB75, also called PRODUCTION OF ANTHOCYANIN PIGMENT1, is a known regulator of the anthocyanin branch of the phenylpropanoid pathway in Arabidopsis (Arabidopsis thaliana), but how this regulation might impact other aspects of carbon metabolism is unclear. We established that a loss-of-function mutation in MYB75 (myb75-1) results in increased cell wall thickness in xylary and interfascicular fibers within the inflorescence stem. The total lignin content and S/G ratio of the lignin monomers were also affected. Transcript profiles from the myb75-1 inflorescence stem revealed marked up-regulation in the expression of a suite of genes associated with lignin biosynthesis and cellulose deposition, as well as cell wall modifying proteins and genes involved in photosynthesis and carbon assimilation. These patterns suggest that MYB75 acts as a repressor of the lignin branch of the phenylpropanoid pathway. Since MYB75 physically interacts with another secondary cell wall regulator, the KNOX transcription factor KNAT7, these regulatory proteins may form functional complexes that contribute to the regulation of secondary cell wall deposition in the Arabidopsis inflorescence stem and that integrate the metabolic flux through the lignin, flavonoid, and polysaccharide pathways.

Figure 1.

Expression pattern of the MYB75 in mature Arabidopsis plant and stems. A, qPCR analysis showing the predominant expression of MYB75 in stems. The expression level of MYB75 is relative to actin and is expressed in arbitrary units. Error bars represent se of three biological replicates. B, GUS expression pattern in different organs of mature MYB75::GUS plants: in leaf midrib (a), in the epidermal cells of the flower (b), in the epidermis of the silique (c), and in cortex and in vascular bundles in the cross section of lower inflorescence stem (d).

Figure 2.

Nuclear localization and transcriptional activity of MYB75. A, Protoplast transfected with 35S:MYB75-YFP. Left, DIC image; middle, YFP channel; right, merged images. B, Effector and reporter constructs used in the transfection assays. C, Transcriptional activity of MYB75. Effector genes were made by fusing GD in-frame with MYB75. Effector gene plasmid DNA was cotransfected with reporter gene Gal4:GUS plasmid DNA into Arabidopsis leaf mesophyll protoplasts. GUS activity was assayed after the transfected protoplasts were incubated in darkness for 20 to 22 h. Shown are means ± se of three replicates. MYB75 is a weak transcriptional activator.

Figure 3.

Secondary wall thickening in fibers and vessels in myb75-1 plants. The bottom inflorescence stem of 8-week-old plants were used for examination of secondary walls in fibers and vessels. A, Toluidine Blue staining of the cross sections in mutants and the wild type (WT; top; as indicated). Higher magnification of the Toluidine Blue-stained sections (bottom; as indicated). myb75-1 show thickened cell wall compared to the wild type. B, TEM of the wild type, myb75-1, and MYB75(o/x). myb75-1 show thickened interfascicular fibers. C, Measurements of secondary cell wall thickness in the wild type and mutants (as indicated; μm). The wall thickness was measured from transmission electron micrographs of fibers and vessels. Data are means (μm) ± se from 75 cells. if, Interfascicular fiber; ve, vessel; xf, xylary fiber. Bars = 50 and 100 μm (as indicated) in A and 5 μm in B.

Figure 4.

Secondary cell wall-associated gene expression in lower stems of myb75-1 plants. A, qRT-PCR analysis of the expression of lignin biosynthetic genes in lower inflorescence stems of MYB75 overexpresser [MYB75(o/x)] and loss-of-function mutants (myb75-1) compared with the wild type (WT). The expression levels of most of the genes in the lignin pathway were examined. 4CL1, 4-Coumarate-CoA ligase 1; HCT, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase; C3H1, p-coumarate 3-hydroxylase 1. B, qRT-PCR analysis of the expression of secondary wall biosynthetic genes in lower inflorescence stems of MYB75 overexpresser [MYB75(o/x)] and loss-of-function mutants (myb75-1) compared with the wild type. The expression levels of genes involved in the biosynthesis of cellulose in primary cell wall (CesA1, CesA3, and CesA6), cellulose in secondary cell wall (CesA4, CesA7, and CesA8), and xylan (FRA8, IRX8, and IRX9) were examined. Error bars represent se of three replicates.

Figure 5.

In vitro protein-protein interactions among potential secondary cell wall-associated transcription factors and MYB75 as determined by yeast two-hybrid assay. Known interactions (shown in dark-gray box; Zimmermann et al., 2004) were used as a positive control, and empty vectors were cotransformed to be used as a negative control.