Observation of the development of leaf vein and stomata and identification candidate transcription factors related to vein/stoma development in grapevine leaf (Vitis vinifera L.)

The leaf functions through photosynthesis and determines the yield and quality of the grape (Vitis vinifera L.). Understanding the morphology, anatomy, and molecular regulation of leaf development can provide insight into the function of the leaf. In the present study, the microstructure of the vein and stomata was observed using an improved tissue clearing method, and differently expressed transcription factors (TFs) underlying leaf development were determined by transcriptome analysis in the ‘Pinot Noir’ grape. The process of stomata development from a single epidermal cell to a mature stomata cell was observed clearly under light microscopy by improving the method of tissue clearing. The onset of stomata formation in the leaf tip occurred prior to that in the central leaf, and developing stomata were observed in the center of the leaves at different development stages except for L5, when only mature stomata were present. The major vein was initiated before leaf unfolding (L1) and numbers of vessels increased underlying leaf growth. The shape of the vessel presented tapering. Freely ending veins were not observed until stages L4 and L5, and the vessels were different from other order veins in shape and ways of connection. Development-dependent differences between stomata and vein suggested differences in function during leaf development. A total of 445 TFs involved in leaf morphology and anatomy were identified. The members of the bHLH family (SCRM) and MYB family (MYB88) can contribute to stomata patterning during leaf development. Members of the TF families of ARF (ARF5), Dof (TMO6 and HCA2), HD-Zip III (HB14), and GRAS (SCR, SCL14, and SCL28) were potential regulators involved in vein development. The grape orthologs of WOX9, TCP4, and GRF1-9 can participate in leaf outgrowth and expansion. These results will be helpful for our further study of the molecular regulation of leaf development and the relation between leaf structure and function, which can be applied to improve grape production.