Functional characterization of evolutionarily divergent 4-coumarate:coenzyme a ligases in rice

Abstract

4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis. 4CL has been much studied in dicotyledons, but its function is not completely understood in monocotyledons, which display a different monolignol composition and phenylpropanoid profile. In this study, five members of the 4CL gene family in the rice (Oryza sativa) genome were cloned and analyzed. Biochemical characterization of the 4CL recombinant proteins revealed that the rice 4CL isoforms displayed different substrate specificities and catalytic turnover rates. Among them, Os4CL3 exhibited the highest turnover rate. No apparent tissue-specific expression of the five 4CLs was observed, but significant differences in their expression levels were detected. The rank in order of transcript abundance was Os4CL3 > Os4CL5 > Os4CL1 > Os4CL4 > Os4CL2. Suppression of Os4CL3 expression resulted in significant lignin reduction, shorter plant growth, and other morphological changes. The 4CL-suppressed transgenics also displayed decreased panicle fertility, which may be attributed to abnormal anther development as a result of disrupted lignin synthesis. This study demonstrates that the rice 4CLs exhibit different in vitro catalytic properties from those in dicots and that 4CL-mediated metabolism in vivo may play important roles in regulating a broad range of biological events over the course of rice growth and development.

Figure 1.

Gene structure and phylogenetic analysis of the rice 4CL family. A, Schematic structure of rice 4CL genes on chromosomes. Black bars represent the chromosomes. Exons are represented as dark gray arrows, and introns are indicated by light gray lines between exons. The lengths of exons and introns are indicated by the scale bar. The five 4CL gene loci are as follows: Os4CL1, Os08g14760; Os4CL2, Os02g46970; Os4CL3, Os02g08100; Os4CL4, Os06g44620; Os4CL5, Os08g34790. B, Unrooted phylogenetic tree of 4CL isoforms from four representative species was constructed with bootstrap values after 1,000 trials. The scale bar corresponds to 0.05 amino acid substitutions per position in the sequence. 4CLs from dicot species were clustered in two clades, classified as type I and type II 4CLs. Four 4CLs from rice and the moss P. patens are grouped in separate clusters. Plant species are as follows: Os, Oryza sativa; At, Arabidopsis thaliana; Ptr, Populus trichocarpa; Pp, Physcomitrella patens.

Figure 2.

Expression profiles of rice 4CLs over the course of growth. Total RNAs were isolated from roots, culms, leaf blades, leaf sheaths, and panicles of rice plants at 3 weeks, 5 weeks, and 10 weeks old. Transcripts of rice 4CLs were determined using quantitative reverse transcription-PCR analysis. The rice ACTIN1 gene was used as a reference for normalization. A, Os4CL1. B, Os4CL2. C, Os4CL3. D, Os4CL4. E, Os4CL5. The results are means ± se of independent triplicate assays.

Figure 3.

Activity determination of the Os4CL3 promoter and in situ localization of Os4CL3 expression. Localization of Os4CL3 expression was investigated through promoter-GUS assays and in situ hybridization. A, GUS staining of the promoter-GUS activity in a seedling. The inset shows a cross section of the root area indicated in the box. B, GUS staining of a cross section of stem. C, GUS staining of a leaf blade. D, GUS staining of a flower. The insets show pistils and anthers. E and F, In situ hybridization of stem cross sections (6-week-old plants) with an Os4CL3-specifc antisense probe (E) and with an Os4CL3-specific sense probe (F). An, Anther; CF, cortical fiber; E, epidermis; Ex, exodermis; Ov, ovary; Sti, stigma; Sty, style; VB, vascular bundle. Bars = 500 μm.

Figure 4.

Phenotype of the Os4CL3-suppressed transgenic rice. Wild-type (WT) and Os4CL3-suppressed transgenic (Os4CL3AS) rice were grown in a phytotron. Morphology was systematically recorded from seedling to mature stages. A, Ten-day-old plants. B, Seventy-day-old plants. C, Mature rice grain. D, Measurement of the tensile strength of a rice stem. N represents the mechanical force unit in Newton. Error bars represent se of triple sample measurements (P < 0.01, by t test). Bars = 50 mm in A, 10 cm in B, and 7 mm in C.

Figure 5.

Lignin analysis and 4CL expression in transgenic rice. Rice culms at 3 months of age were sectioned and stained for lignin with phloroglucinol-HCl and for cellulose with calcofluor. A, Lignin staining in wild-type rice. B, Cellulose staining in wild-type rice. C, Lignin staining in Os4CL3AS transgenics. D, Cellulose staining in Os4CL3AS transgenics. Insets show closeup images of the vascular cells indicated in each box. Bars = 100 μm, and bars in insets = 50 μm. E, Klason lignin content in the course of growth from 10 d to 3.5 months. F, Transcripts from five 4CLs were measured in wild-type and Os4CL3AS transgenic plants at 2 weeks of age. After normalization using ACTIN1 as a reference, relative transcript abundance in the Os4CL3AS transgenics was calculated as a percentage of that in the wild type. Error bars represent se of three independent replicates. E, Epidermis; Pa, parenchyma cells; Ph, phloem; Sc, sclerenchyma cells; WT, wild type; Xy, xylem.

Figure 6.

Microscopic analysis of rice flower morphology at the heading stage. Morphology of flowers from the wild type and Os4CL3AS transgenics was characterized. A, Spikelet of the wild type. Bar = 2 mm. B, Anther of the wild type. Bar = 500 μm. C, Spikelet of 4CL3AS. Bar = 2 mm. D, Anther of 4CL3AS. Bar = 500 μm. E and G, Scanning electron microscopy (SEM) images of the anther surface of the wild type. Bars = 50 μm in E and 10 μm in G. F and H, SEM images of the anther surface of 4CL3AS. Bars = 50 μm in F and 10 μm in H. I and J, SEM images of pistils of the wild type (I) and 4CL3AS (J). Bars = 500 μm. K and L, 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining of pollen grains from the wild type (K) and 4CL3AS (L). Bars = 50 μm. M and N, DAPI staining of pollen grains from the wild type (M) and 4CL3AS (N). Bars = 50 μm. An, Anther; Lo, locule; MP, mature pollen; Ov, ovary; Pa, palea; Pi, pistil; St, stamen; Sti, stigma; Sty, style.

Figure 7.

Anther development in rice from stage 10 to 13. Rice anthers were transverse sectioned. The sections were observed with toluidine blue staining and under UV illumination. Four stages of anther development from the wild-type and Os4CL3AS transgenics were compared. In A to H, images were photographed with toluidine blue staining; in I to P, images were photographed under UV illumination. A to D and I to L, Anther development in the wild type from stage 10 to 13 with toluidine blue staining (A–D) and under UV illumination (I–L). E to H and M to P, Anther development in Os4CL3AS transgenics from stage 10 to 13 with toluidine blue staining (E–H) and under UV illumination (M–P). At stage 10, both the wild type (A and I) and Os4CL3AS (E and M) did not show obvious morphological differences in anther structures. At stage 11, the anther wall middle layer and endothecium degenerated, and typical falcate pollen grains were formed (B and J) and distorted locules in Os4CL3AS transgenic plants were observed (E and N). At stage 12, the endothecium underwent drastic secondary thickening and lignification, as indicated in striated U-shaped bands (C) and strong lignin UV illumination (K) in the wild-type anthers, whereas the endothecium secondary thickening and lignification were affected in Os4CL3AS transgenics (G and O). At stage 13, the anther dehiscence occurred in the wild type (D and L) but not in the abnormal anthers of Os4CL3AS transgenics (H and P). C, Connective tissue; E, epidermis; En, endothecium; MP, mature pollen; Msp, microspore; St, stomium; T, tapetum; V, vascular bundle. Bars = 100 μm.