Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa)

Abstract

AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass.

Keywords: Apoplast; CYP19-4; Oryza sativa L.; cold stress tolerance; immunophilin; increased tillering.

Fig. 1.

Multiple sequence alignment and phylogenetic relationships among monocot CYP19-4 homologues. (A) Alignment of the mature regions (excluding the N-terminal targeting sequences) of the deduced amino acid sequences of O. sativa CYP19-4 and CYP19-4 homologues from other monocot species. The sequences were aligned using ClustalW2 and visualized with GeneDoc version 2.7. Amino acids necessary for CsA binding as determined for human CyPA are marked with asterisks. Secondary structural features (α-helix and β-sheets) are indicated. The backgrounds indicate amino acid similarity: black, 90%; dark grey, 80%; light grey, 40%. (B) Phylogenetic tree of CYP19-4 homologues from monocots. Phylogenetic analysis based on the alignment in (A) was carried out using MEGA5.2.

Fig. 2.

Expression patterns of OsCYP19-4 in various tissues, developmental stages, and abiotic stress conditions. (A) Expression of OsCYP19-4 in various tissues and developmental stages, as detected by RT-PCR and qRT-PCR. cDNA templates from endosperm (En), root (Ro), shoot (Sh), stem (St), and leaf (Le) were used for amplification. OsActin1 was used as a control. (B) qRT-PCR showing the expression of OsCYP19-4 in 10-day-old rice plants treated with 4 °C (cold), drought, 100mM H₂O₂, and 100 μM cadmium for 0, 1, 3, 6, 12, 24, and 48 hours. The relative expressions were normalized to that of OsActin1. Error bars denote the SE of three biological replicates. Asterisks indicate significant differences between no treatment (0) and each stress treatment time point (Studentʼs t test; **P<0.01).

Fig. 3.

Subcellular localization of OsCYP19-4. (A) Merged images of GFP fluorescence and chloroplast autofluorescence from epidermal cells and protoplasts of N. benthamiana infected with Agrobacterium GV3101 harbouring OsCYP19-4-GFP, OsCYP19-4ΔSP-GFP, and OsCYP19-4SP-GFP. Asterisks indicate several points of GFP fluorescence near the plasma membrane. (B) Merged confocal laser scanning images of GFP and FM4-64 fluorescence in N. benthamiana epidermal cells transiently expressing OsCYP19-4-GFP. The open white arrowheads in the insets indicate OsCYP19-4-GFP protein in the apoplast (green) imaged in the presence of 8.2 μM FM4-64 plasma membrane-staining dye (red). (C) The effect of BFA on the intracellular localization of OsCYP19-4. Transgenic Arabidopsis seedlings expressing OsCYP19-4-GFP were treated with DMSO (upper) or the same volume of 50 μM BFA (lower) for 3h and then co-treated with 5 μM FM4-64 for 15min. Arabidopsis transgenic seedlings with the empty pCAMBIA1302 vector were used as a control for the BFA compartment assay. Scale bars=20 μm.

Fig. 4.

PPIase activity assay using recombinant OsCYP19-4 mature protein in vitro. (A) Expression and purification of recombinant mature OsCYP19-4 (OsCYP19-4ΔSP) in Escherichia coli. Expression of OsCYP19-4ΔSP in E. coli was induced by treatment with IPTG for 4h and the resulting proteins were analysed by 12% SDS-PAGE. Un, un-induced; In, induced by 1mM IPTG for 4h; Pu, purified protein; His, anti-His immunoblot. (B) A protease-coupled assay was used to measure PPIase activity. The prolyl cis-trans isomerization of the tetrapeptide substrate (Suc-Ala-Phe-Pro-Phe-2,4-difluoroanilide) was reflected by an increase in absorbance at 390nm. The curves represent isomerization of the Suc-AFPF-pNA substrate at 10 °C over the course of 300 s in the absence of OsCYP19-4 (Blank) and in the presence of 50 or 100nM recombinant OsCYP19-4 protein. For the inhibition of OsCYP19-4 PPIase activity, 1 μM CsA was incubated with 100nM OsCYP19-4.

Fig. 5.

Enhanced tolerance to cold stress in OsCYP19-4-overexpressing rice plants. (A) Two-week-old wild-type and transgenic OsCYP19-4-overexpressing rice seedlings before exposure to 4 °C treatment under dark conditions for 4 d. (B) Wild-type and OsCYP19-4-overexpressing rice seedlings after 4 °C treatment in the dark for 4 d and recovery at 28 °C for 10 d. Scale bars=5cm. (C) Survival rate after cold treatment and recovery. Data are mean±SE of three biological replicates; 20 plants were counted for each of four independent lines. Asterisks indicate statistically significant differences (Student’s t test; *P<0.05) between wild-type and transgenic lines.

Fig. 6.

Relative EL between OsCYP19-4-overexpressing and wild-type plants under cold stress. Two-week-old rice seedlings were exposed to 4 °C for the indicated number of days and the relative EL of leaf cells was monitored. Data represent mean±SE from three different experiments. Asterisks indicate significant differences between wild-type and transgenic lines (Studentʼs t test; *P<0.05, **P<0.01).

Fig. 7.

Overexpression of OsCYP19-4 in transgenic rice plants confers increased tillering capacity and grain yield. (A) Thirteen-week-old wild-type and OsCYP19-4-overexpressing plants. Plants were grown in a growth chamber for 4 weeks at the seedling stage and then five plants were transferred to a pot in a greenhouse for 9 weeks (Scale bar=17cm). (B) Number of tillers in 13-week-old wild-type and OsCYP19-4-overexpressing plants. Tillers were counted in 20 plants of each line. Data are mean±SE of two replicates. (C) Number of spikes in 32-week-old wild-type and OsCYP19-4 transgenic plants. Data represent mean±SD (n=10). (D) Total weight of grains from harvested spikes in 32-week-old wild-type and OsCYP19-4-overexpressing transgenic plants. Data represent mean±SD (n=10). WT1–4, different wild-type plants; OE1–4, independent OsCYP19-4-overexpressing transgenic lines. Asterisks indicate significant differences between wild-type and transgenic lines (Student’s t test; *P<0.05, **P<0.01).