Novel resources to investigate leaf plasmodesmata formation in C₃ and C₄ monocots

Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. More productive and photosynthetically efficient C₄ plants form more PD at the mesophyll (M)-bundle sheath (BS) interface in their leaves than their less efficient C₃ relatives. In C₄ leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO₂ concentrating mechanism, which increases the CO₂ partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C₃ and C₄ leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C₃) and Setaria viridis (setaria, C₄) with fluorescent protein-tagged PD to build the first spatiotemporal atlas of leaf pit field (cluster of PD) density in monocots without the need for electron microscopy. Across leaf development, setaria had consistently more PD connections at the M-BS wall interface than rice while the difference in M-M pit field density varied. While light was a critical trigger of PD formation, cell type and function determined leaf pit field density. Complementary temporal mRNA sequencing and gene co-expression network analysis revealed that the pattern of pit field density correlated with differentially expressed PD-associated genes and photosynthesis-related genes. PD-associated genes identified from our co-expression network analysis are related to cell wall expansion, translation and chloroplast signalling.