NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity

Three distinct pattern elements of the silique are thought to contribute to its dehiscence: a separation layer, cells with a secondary wall adjacent to the separation layer, and a valve endocarp layer with secondary wall. However, the role of the secondary wall has not been proven, and the factors that regulate its formation in siliques remain to be characterized. We show here that secondary wall formation in siliques is necessary for dehiscence, and that two plant-specific transcription factors, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1 and NST3), regulate its formation in siliques of Arabidopsis. The promoters of the NST1 and NST3 genes were active in the valve endocarp layer and in cells surrounding vascular vessels in the replum, and NST1 promoter activity only was faintly detectable at valve margins. In nst1 mutants, specific loss of secondary walls was evident at valve margins, while nst1 nst3 double mutants lacked secondary walls in all parts of the siliques, with the exception of vascular vessels. These siliques were similarly indehiscent. The promoters of two tissue-identity genes, INDEHISCENT (IND) and SHATTERPROOF2 (SHP2), were as active in the nst1 nst3 mutant as in the wild-type. Moreover, the ectopic secondary wall formation that occurs in the fruitfull (ful) mutant was absent in the ful nst1 double mutant. We propose that secondary walls in valve margins are required for dehiscence, and that NST1 and NST3 regulate their formation in siliques in a partially redundant manner after the establishment of tissue identity.