Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW during leaf patterning in C4 grasses

Author summaryThe correct patterning of cell-types within organs is required to ensure appropriate morphology and physiology in any organism. In grasses, two distinct cellular patterns develop in internal leaf tissues depending on the type of photosynthesis that is carried out. Previous work suggested that the transcription factor SCARECROW (SCR) has different patterning roles in leaves of rice and maize which carry out 'C-3' versus 'C-4' photosynthesis, respectively. SCR patterns epidermal cell-types in rice leaves and internal leaf tissues in maize. Here we generate scr mutations in another C-4 plant, Setaria viridis, and reveal patterning defects in both the epidermis and inner leaf tissues. This observation provides information about the evolutionary trajectory of the SCR patterning pathway in grasses but also raises the question of how the inner leaf patterning component is distinguished from the epidermal role. Through the identification of genetic interactions between SCR and a second transcription factor NAKED-ENDOSPERM (NKD) we demonstrate that combined SCR/NKD function patterns inner leaf tissues in the C-4 grasses maize and S. viridis but not in the C-3 grass rice. We propose that patterning of epidermal cell-types represents the ancestral role for SCR in grass leaves and that as C-4 grasses evolved, the gene was recruited along with NKD to pattern inner leaf cell types. Some C-4 species have retained the ancestral function whereas others such as maize have not. Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C-3 or C-4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C-3 species veins are separated by a greater number of intervening mesophyll cells than in derived C-4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C-4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C-3 species rice. Here we show that SCR is required for inner leaf patterning in the C-4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C-4 but not C-3 grasses. Together our results provide insight into the evolution of cell patterning in grass leaves and demonstrate a novel patterning role for IDD genes in C-4 leaves.

Automated summary

This study reveals the role of the SCARECROW gene in C-4 plants, showing that SCR and NAKED-ENDOSPERM together regulate the differentiation of internal leaf tissues. The authors found that SCR is involved in the patterning of internal leaf tissues in C-4 plants, with NKD enhancing its function. This study sheds light on the evolutionary process of cell patterning in grass leaves.