Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and Arabidopsis plants

Tao Li,Yumin Zhang,Ying Liu,Xudong Li,Guanglong Hao,Qinghui Han,Lynnette M.A Dirk,A Bruce Downie,Yong-Ling Ruan,Jianmin Wang,Guoying Wang,Tianyong Zhao

doi:10.1074/jbc.ra120.013948

Abstract

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of RAFS from maize (or corn, Zea mays) (ZmRAFS) is induced by drought, heat, cold, and salinity stresses. We found that zmrafs mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants. This indicated that ZmRAFS and its product raffinose contribute to plant drought tolerance. ZmRAFS overexpression in Arabidopsis enhanced drought stress tolerance by increasing myo-inositol levels via ZmRAFS-mediated galactinol hydrolysis in the leaves due to sucrose insufficiency in leaf cells and also enhanced raffinose synthesis in the seeds. Supplementation of sucrose to detached leaves converted ZmRAFS from hydrolyzing galactinol to synthesizing raffinose. Taken together, we demonstrate that ZmRAFS enhances plant drought tolerance through either raffinose synthesis or galactinol hydrolysis, depending on sucrose availability in plant cells. These results provide new avenues to improve plant drought stress tolerance through manipulation of the raffinose anabolic pathway.

Highlights

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress
We demonstrated that maize zmrafs mutants lacking raffinose and hyperaccumulating galactinol but with wild type (WT) amounts of sucrose and myo-inositol, were more sensitive to drought stress than null segregant lines in two maize inbred lines
When galactinol was saturating (15.9 mM), regardless of the sucrose concentration, the amount of myo-inositol that was generated was always greater than that of raffinose produced; the ratio of myoinositol to raffinose gradually decreased as the sucrose concentration was increased (Fig. 5B). These results show that the relative amount of the two substrates, sucrose and galactinol determines whether ZmRAFS predominantly hydrolyzed galactinol unproductively or predominantly completed the synthesis of raffinose, suggesting that greater raffinose production in Arabidopsis leaves expressing ZmRAFS may be possible by boosting substrate amounts

Summary

RESULTS

ZmRAFS transcription was rapidly and dramatically up-regulated in leaves when the V3 stage (three leaves stage) seedlings were treated with heat shock, dehydration or subjected to increased salt within 2-3 h, but returned to background levels with prolonged treatment (Fig. 1A). The raffinose content in the ZmGOLS2/ZmRAFS-1 and -2 plants was significantly greater than that, of the control plants, and that of ZmGOLS2 plants when sucrose treated (Fig. 6G) despite the seemingly different ratios of protein of the two enzymes accumulated in those plants (Fig. S4) These results are consistent with the greater accumulation of raffinose in the seeds of the ZmRAFS plants (19), these data suggested that infiltration of sucrose to the detached leaves of ZmGOLS2/ZmRAFS-expressing Arabidopsis plants provided sufficient sucrose substrate for ZmRAFS to complete the transferase reaction after hydrolyzing galactinol and subsequently, to accumulate raffinose. By characterization of the maize knock out mutant (zmrafs) and ZmRAFS-expressing Arabidopsis plants, we found that raffinose, but not galactinol, directly and positively regulates plant drought stress tolerance

DISCUSSION

EXPERIMENTAL PROCEDURES

CONFLICT OF INTEREST