Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression

Abstract

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1(-). In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1(-) exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors' knowledge, this is the first functional characterization of CCR1 in a grass species.

Fig. 1.

ZmCCR1 gene structure and impact of the Mu mutation on CCR expression. (A) Exon and intron organization of the ZmCCR1 gene. Black boxes indicate exons and lines between boxes indicate introns. Insertion of the Mu element is indicated by an open arrowhead. The references for ZmCCR1 are as follows: gene ID 542463 in NCBI, 199139 in Maize GDB, and GRMZM2G131205 in maizesequence.org. (B) RT-PCR of CCR1 expression in piled-up internodes of 20-d-old wild-type and Zmccr1^– plants.

Fig. 2.

Histochemical staining of lignin in wild type (A, C, E) and Zmccr1^– (B, D, F). Light micrographs of transverse sections stained with phloroglucinol from the top (A, B) and bottom (C, D) parts of the ear-bearing internode. (E and F) Enlargement of sclerenchyma located around vascular bundles. Black arrow in (D) indicates xylem vessels stained red with phloroglucinol. Bars: 500 μm (A–D), 50 μm (E, F). P, parenchyma; S, sclerenchyma; E, epidermis; X, xylem vessels.

Fig. 3.

Immunolocalization of H unit lignins in wild type (A, C) and Zmccr1^– (B, D). Indirect immunofluorescence micrographs of resin-embedded ear-bearing internode sections. Sections were performed in the top (A, B) and bottom (C, D) portions of internodes and labelled with anti-H antibodies. White boxes in A, C, D: enlargement of sclerenchyma cells. Anti-H label is indicated in red. P, parenchyma; S, sclerenchyma; X, xylem vessels. Bar: 50 μm (A-D).

Fig. 4.

Distribution of vascular bundle surface area in internodes of wild type and Zmccr1^–. Vascular bundle size was measured from 100 μm sections from the basal portion of the ear-bearing internode. Sections were stained with phloroglucinol reagent and scanned. Using Image Pro software each vascular bundle located in the external zone of the internode was numerized for counting. A total of 122 and 110 vascular bundles for wild type and Zmccr1^– mutant respectively were counted. Black and grey bars on the histogram represent, respectively, wild-type and Zmccr1^– vascular bundle sizes.