Constitutive Expression of miR408 Improves Biomass and Seed Yield in Arabidopsis

Abstract

miR408 is highly conserved among different plant species and targets transcripts encoding copper-binding proteins. The function of miR408 in reproductive development remains largely unclear despite it being known to play important roles during vegetative development in Arabidopsis. Here, we show that transgenic Arabidopsis plants overexpressing MIR408 have altered morphology including significantly increased leaf area, petiole length, plant height, flower size, and silique length, resulting in enhanced biomass and seed yield. The increase in plant size was primarily due to cell expansion rather than cell proliferation, and was consistent with higher levels of myosin gene expression and gibberellic acid (GA) measured in transgenic plants. In addition, photosynthetic rate was significantly increased in the MIR408-overexpressing plants, as manifested by higher levels of chloroplastic copper content and plastocyanin (PC) expression. In contrast, overexpression of miR408-regulated targets, Plantacyanin and Laccase 13, resulted in reduced biomass production and seed yield. RNA-sequencing revealed that genes involved in primary metabolism and stress response were preferentially enriched in the genes upregulated in MIR408-overexpressing plants. These results indicate that miR408 plays an important role in regulating biomass and seed yield and that MIR408 may be a potential candidate gene involved in the domestication of agricultural crops.

Keywords: biomass; copper; miR408; photosynthesis; seed yield.

FIGURE 1

Effects of miR408 overexpression on Arabidopsis development. (A) Wild type (left) and miR408-overexpressing plants (MIR408-OX; right) grown in soil for 2 weeks. (B,C) Leaf area (B) petiole length (C) of individual leaves of the wild type and MIR408-OX plants grown in soil for 2 weeks (n = 15). (D) Dry weight was determined for 2-week-old plants grown in soil (n = 20). (E) Comparison of flowers (upper, scale bar = 2 mm) and silique (lower, scale bar = 1 cm) between the wild type and MIR408-OX plants. (F) Seed morphology of the wild type and MIR408-OX plants, scale bar = 500 μm. (G) Seeds from more than 15 plants were collected and quantified. Data are means ± SD from n biological repeats. ^∗p < 0.01, ^∗∗p < 0.05 vs. wild type, by two-tailed Student’s t-test.

FIGURE 2

Cell size and number. (A) Epidermal cells of the first rosette leaf from the wild type (upper) and MIR408-OX plants (lower). The scale bars represent 50 μm. (B) Epidermal cell area of the first rosette leaf (n = 50). (C) The number of cells per leaf (n = 10). (D) Epidermal cells of the first leaf petiole from the wild type (upper) and MIR408-OX (lower) plants. The scale bars represent 50 μm. (E) Cell length of the first leaf petiole epidermal cell (n = 50). (F) The number of cell from the first leaf petiole (n = 10). Data are means ± SD from n biological repeats. ^∗p < 0.01 vs. the wild type, by two-tailed Student’s t-test.

FIGURE 3

Expression levels of myosin and GA biosynthetic genes in the wild type and MIR408-OX transgenic plants. (A,B) Quantitative RT-PCR analysis of mRNA levels for myosin genes (A) and GA biosynthetic genes (B) using the rosette leaves from the plants grown in soil for 2 weeks. Actin2 was used as an internal control. Data are means ± SD of three biological experiments. (C) Levels of endogenous GAs in wild type and MIR408-OX plants. Data are means ± SD of three technical repeats. ^∗p < 0.01, ^∗∗p < 0.05 vs. the wild type, by two-tailed Student’s t-test.

FIGURE 4

Enhanced photosynthesis by overexpressing MIR408. (A) Comparison of net photosynthetic rate between the wild type and MIR408-OX transgenic plants grown in soil for 2 weeks (n = 5). (B) The net photosynthetic rate is positively correlated with stomatal conductance (G_s) and negatively correlated with internal CO₂ concentration (C_i) (n = 5). (C) Glucose content in the shoots of plants grown in soil for 2 weeks (n = 3). (D) Immunoblot analysis of PETE (PC) protein levels in the wild type and MIR408-OX transgenic plants. Values represent PETE2 levels normalized against the loading control RPT5 using Image J software and set to one for wild type. Data are means ± SD from n biological repeats. ^∗p < 0.01, ^∗∗p < 0.05 vs. the wild type, by two-tailed Student’s t-test.

FIGURE 5

Characterization of genes with altered levels of expression in the MIR408-OX transgenic plants. (A) Differentially expressed genes between the wild type and MIR408-OX plants. (B) Correlation between RNA-sequencing and qRT-PCR data on the selected genes. Pearson correlation was calculated using data points representing Log₂ transformed transcript level ratios of wild type and MIR408-OX plants. (C) Enriched representative GO terms (p < 0.001) in the biological category that associated with induced (left) and repressed (right) genes in the MIR408-OX transgenic plants. (D) Enriched pathways associated with the differentially expressed genes by KEGG analysis.

FIGURE 6

Biomass and seed yield traits of Plantacyanin (ARPN) and LAC13 overexpressors. (A) Confirmation of miR408 targeting on ARPN and LAC13 by 5′-RACE. The complementary mRNA and miRNA sequences are shown with shaded boxes. Vertical arrows mark the sequenced cleavage sites with the frequency of clones shown. (B) RNA gel blot analysis for wild type and ARPN-OX or LAC13-OX transgenic lines. Actin2 served as loading control. (C) Stereomicroscopy images of siliques obtained from self-pollinated wild type, ARPN-OX, and ARPN-OX/LAC13-OX (D-OX) parental plants. Red arrowheads indicate abnormal ovules. (D) Comparison of silique development on 6-week-old plants of the wild type, MIR408-OX, ARPN-OX, LAC13-OX, and D-OX genotypes. (E) Seeds from different genotypes were collected and quantified (n = 15). Data are means ± SD. Genotypes labeled with the same letters have no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).