CRISPR/Cas9-mediated mutation in auxin efflux carrier OsPIN9 confers chilling tolerance by modulating reactive oxygen species homeostasis in rice

Abstract

Phytohormone auxin plays a vital role in plant development and responses to environmental stresses. The spatial and temporal distribution of auxin mainly relies on the polar distribution of the PIN-FORMED (PIN) auxin efflux carriers. In this study, we dissected the functions of OsPIN9, a monocot-specific auxin efflux carrier gene, in modulating chilling tolerance in rice. The results showed that OsPIN9 expression was dramatically and rapidly suppressed by chilling stress (4°C) in rice seedlings. The homozygous ospin9 mutants were generated by CRISPR/Cas9 technology and employed for further research. ospin9 mutant roots and shoots were less sensitive to 1-naphthaleneacetic acid (NAA) and N-1-naphthylphthalamic acid (NPA), indicating the disturbance of auxin homeostasis in the ospin9 mutants. The chilling tolerance assay showed that ospin9 mutants were more tolerant to chilling stress than wild-type (WT) plants, as evidenced by increased survival rate, decreased membrane permeability, and reduced lipid peroxidation. However, the expression of well-known C-REPEAT BINDING FACTOR (CBF)/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1 (DREB)-dependent transcriptional regulatory pathway and Ca2+ signaling genes was significantly induced only under normal conditions, implying that defense responses in ospin9 mutants have probably been triggered in advance under normal conditions. Histochemical staining of reactive oxygen species (ROS) by 3′3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) showed that ospin9 mutants accumulated more ROS than WT at the early stage of chilling stress, while less ROS was observed at the later stage of chilling treatment in ospin9 mutants. Consistently, antioxidant enzyme activity, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), improved significantly during the early chilling treatments, while was kept similar to WT at the later stage of chilling treatment, implying that the enhanced chilling tolerance of ospin9 mutants is mainly attributed to the earlier induction of ROS and the improved ROS scavenging ability at the subsequent stages of chilling treatment. In summary, our results strongly suggest that the OsPIN9 gene regulates chilling tolerance by modulating ROS homeostasis in rice.

Overexpression of OsPIN9 Impairs Chilling Tolerance via Disturbing ROS Homeostasis in Rice

“…Recently, Manna et al (2022) [63] reported that salt and drought treatment significantly induced OsPIN9 expression, indicating the potential role of OsPIN9 in abiotic stress adaptation. In line with this, our previous study showed that mutation of OsPIN9 by CRISPR/Cas9 technology confers enhanced chilling tolerance in rice [64]. However, whether and how OsPIN9 overexpression regulates rice chilling tolerance is unknown.…”

Deficiency of Auxin Efflux Carrier OsPIN1b Impairs Chilling and Drought Tolerance in Rice

“…These results suggested that IAA and OsPIN genes could affect each other to regulate plant growth and development. PIN is associated with abiotic stress adaptation [33][34][35][36], and abiotic stress treatments influence OsPIN genes' expression. Cold stress inhibited the expression of OsPIN1c, OsPIN1d, OsPIN9, OsPIN10 and OsPIN10b in rice roots, and paralogous OsPIN genes displayed a similar expression profile under cold stress [34], implying that they might act synergistically to modulate rice cold tolerance.…”

“…PAT is probably implicated in plant cold adaptation [28]. Recently, we reported that OsPIN9, another auxin efflux carrier, is involved in regulating rice chilling tolerance by modulating ROS homeostasis [33,34]. However, whether OsPIN1b is involved in cold adaptation is still elusive.…”

“…Plant chilling tolerance is tightly associated with ROS homeostasis in plant cells [33,62]. ospin1b mutants are more sensitive to chilling stress; we wondered whether the chilling treatment influences ROS homeostasis in ospin1b mutants.…”

“…Further research showed that cold tolerance is positively associated with GNOM, a SEC7 containing ARF-GEF, which participates in modulating the endosomal trafficking of auxin efflux carriers [29]. Our previous reports showed that low temperature differentially regulates the expression of OsPIN genes in rice roots, among which OsPIN9 is gradually suppressed under chilling treatment; further experiments indicated that OsPIN9 negatively regulates rice chilling tolerance [32][33][34], which provides a potential target for breeding cold-resistant crops by CRISPR/Cas9 technology. PAT is also involved in regulating drought tolerance.…”