Stress-induced higher vein density in the C3-C4 intermediate Moricandia suffruticosa under drought and heat stress

The evolution of C4 photosynthesis involved multiple anatomical and physiological modifications, yet our knowledge of the genetic regulation involved remains elusive. In this study, systematic analyses were conducted comparing the C3-C4 intermediate Moricandia suffruticosa and its C3 relative Brassica napus (rapeseed). We found that the leaves of M. suffruticosa had significantly higher vein density than those of B. napus, and the vein density was further increased in M. suffruticosa under drought and heat stress. Moreover, the bundle sheath distance, as the mean distance from the outer wall of one bundle sheath to the outer wall of an adjacent one, decreased and the number of centripetal chloroplasts in bundle sheath cells was found to be altered in M. suffruticosa leaves under drought and heat treatments. These results suggest that abiotic stress can induce a change in an intermediate C3-C4 anatomy towards a C4-like anatomy in land plants. By integrating drought and heat factors, co-expression network and comparative transcriptome analyses between M. suffruticosa and B. napus revealed that inducible auxin signaling regulated vascular development, and autophagy-related vesicle trafficking processes were associated with this stress-induced anatomical change. Overexpressing three candidate genes, MsERF02, MsSCL01, and MsDOF01, increased leaf vein density and/or enhanced photosynthetic assimilation and drought adaptability in the transgenic lines. The findings of this study may improve our understanding of the genetic regulation and evolution of C4 anatomy.