Reduced Expression of PRX2/ ATPRX1, PRX8, PRX35, and PRX73 Affects Cell Elongation, Vegetative Growth, and Vasculature Structures in Arabidopsis thaliana

Abstract

Class III peroxidases (PRXs) are involved in a broad spectrum of physiological and developmental processes throughout the life cycle of plants. However, the specific function of each PRX member in the family remains largely unknown. In this study, we selected four class III peroxidase genes (PRX2/ATPRX1, PRX8, PRX35, and PRX73) from a previous genome-wide transcriptome analysis, and performed phenotypic and morphological analyses, including histochemical staining, in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi plants. The reduced mRNA levels of corresponding PRX genes in PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi seedlings resulted in elongated hypocotyls and roots, and slightly faster vegetative growth. To investigate internal structural changes in the vasculature, we performed histochemical staining, which revealed alterations in cell wall structures in the main vasculature of hypocotyls, stems, and roots of each PRXRNAi plant compared to wild-type (Col-0) plants. Furthermore, we found that PRX35RNAi plants displayed the decrease in the cell wall in vascular regions, which are involved in downregulation of lignin biosynthesis and biosynthesis-regulated genes' expression. Taken together, these results indicated that the reduced expression levels of PRX2/ATPRX1, PRX8, PRX35, and PRX73 affected hypocotyl and root elongation, vegetative growth, and the vasculature structures in hypocotyl, stem, and root tissues, suggesting that the four class III PRX genes play roles in plant developmental processes.

Keywords: PRX2/ATPRX1; PRX35; PRX73; PRX8; cell elongation; class III peroxidase; vasculature structure; vegetative growth.

Figure 1

The expression of each PRX gene in the four PRXRNAi plants. (a) Schematic diagram of PRX2RNAi, PRX8RNAi, PRX35RNAi, and PRX73RNAi constructs. LB, RB, 35S, PRX, and 35Ster indicate the T-DNA left border, T-DNA right border, cauliflower mosaic virus (CaMV) 35S promoter, each peroxidase ORF, and the CaMV 35S terminator, respectively. (b,c) Relative expression levels of target or other PRX genes in wild-type (Col-0) and respective PRXRNAi plants grown for 6 days. Relative expression levels measured by RT–qPCR in wild-type (Col-0) plants were defined as 1.0. Error bars indicate the standard error of the mean. The UBQ10 gene was used as an internal control. Asterisks indicate significant differences (p < 0.005, Student’s t-test). Note: ns–not significant.

Figure 2

Morphological changes in the four PRXRNAi plants at early developmental stages. (a) Phenotypes of 6-day-old wild-type (Col-0) and respective PRXRNAi seedlings grown in half-strength MS medium. White and orange triangles indicate hypocotyl and root ends, respectively. Scale bar = 1 cm. (b) Relative lengths of hypocotyls and roots in 6-day-old grown wild-type (Col-0) and respective PRXRNAi seedlings. Error bars indicate standard error of the mean (n = 20). Significant differences (p < 0.001, Student’s t-test) are indicated by asterisks. (c) Phenotypes of 2-week-old soil-grown wild-type (Col-0) and respective PRXRNAi plants before bolting. Scale bar = 1 cm.

Figure 3

Morphological changes in four PRXRNAi plants at late developmental stages. (a) Phenotypes of 30-day-old soil-grown wild-type (Col-0) and respective PRXRNAi plants. Scale bar = 2 cm. (b) Phenotypes of third to sixth rosette leaves of wild-type (Col-0) and respective PRXRNAi plants grown in soil for 30 days. Scale bar = 1 cm. (c) Lengths of the leaf petiole, leaf blade, and leaf width in the fifth and sixth rosette leaves of 30-day-old soil-grown plants. Error bars indicate standard error of the mean (n ≥ 53). Asterisks indicate significant differences (p < 0.001, Student’s t-test). (d) Bolting days of wild-type (Col-0) and respective PRXRNAi plants (n = 17). Asterisks indicate significant differences (p < 0.001, Student’s t-test). (e,f) Total chlorophyll and (e) anthocyanin (f) contents in rosette leaves of 45-day-old soil-grown wild-type (Col-0) and respective PRXRNAi plants (n = 10). Red squares shown in (e) indicate the chlorophyll a/b contents. Asterisks indicate significant differences (p < 0.005, Student’s t-test).

Figure 4

Transverse sections of basal main stems of wild-type (Col-0) and PRXRNAi plants. (a,b) Enlarged photos of the xylem (a) and interfascicular fiber (b) parts (upper right corner) in toluidine blue-stained sections of resin-embedded stems of soil-grown 6-week-old wild-type (Col-0) and respective PRXRNAi plants. White and yellow arrows indicate the xylem and interfascicular fibers, respectively. The phloem (Ph), xylem (Xy), pith (Pi), and interfascicular fiber (IF) parts are indicated. Scale bars = 50 μm. (c) Average total numbers of xylem cells and interfascicular fibers (left) along the vascular ring, and vascular bundles (right) in stems of soil-grown 6-week-old wild-type (Col-0) and respective PRXRNAi plants. Error bars indicate the standard error of the mean (n ≥ 10). Asterisks indicate significant differences (p < 0.005, Student’s t-test).

Figure 5

Transverse sections of hypocotyl and root regions in wild-type (Col-0) and PRX35RNAi seedlings. (a) Enlarged photos of vascular bundles (upper left corner) in toluidine blue-stained sections of resin-embedded upper hypocotyls in 6-day-old wild-type (Col-0) and PRX35RNAi seedlings. Scale bars are presented in photos. (b) Enlarged photos of vascular bundles (upper left corner) in toluidine blue-stained sections of resin-embedded upper hypocotyls in elongation zones of roots of whole 6-day-old seedlings. The red and blue boxes are magnified in (c,d). Scale bars are presented in photos. (c–e) Transmission electron microscopy (TEM) images of resin-embedded vascular bundles in the elongation zone of roots of the wild-type (Col-0) and PRX35RNAi seedlings shown in (b). Enlarged pictures of the red and blue boxes are shown in (c) and (d), respectively. White arrows indicate the abnormal part of the PRX35RNAi line. A comparison of the cell wall thicknesses of the cortexes between the wild-type (Col-0) and PRX35RNAi lines are represented in (e). The cortex (Co), endodermis (En), epidermis (Ep), metaphloem sieve element (MSe), metaxylem (Mx), procambium (Pc), pericycle (Pe), phloem (Ph), phloem companion cell (CC), protophloem pole pericycle cells (PPP), protophloem sieve element (PSe), protoxylem (Px), and xylem (Xy) cells are indicated. Scale bars are presented in photos.

Figure 6

Stained cross-sections, cell numbers, and expression levels of lignin biosynthesis and biosynthesis-regulated genes in the main stems between wild-type (Col-0) and PRX35RNAi plants. (a) Toluidine blue stained cross-sections of paraffin-embedded basal stem portions in 6-week-old wild-type (Col-0) and PRX35RNAi plants. White boxes shown in upper left corner in each photo are magnified. Scale bars are presented in photos. (b) Total cell numbers of xylem cells and interfascicular fibers along the vascular ring, and the numbers of epidermal cells and vascular bundles (VBs). (c,d) Transcript levels of lignin synthesis (c) and lignin synthesis-regulated genes (d) in different regions of stems of wild-type (Col-0) and PRX35RNAi plants, measured by RT–qPCR. Expression levels in wild-type (Col-0) plants were defined as 1.0. Error bars indicate the standard error of the mean. The UBQ10 gene was used as an internal control. Asterisks indicate significant differences (p < 0.005, Student’s t-test).