Sorghum stem aerenchyma formation is regulated by SbNAC_D during internode development

Sorghum bicolor is a drought-resilient C4 grass used for production of grain, forage, sugar, and biomass. Sorghum genotypes capable of accumulating high levels of stem sucrose have solid stems that contain low levels of aerenchyma. The D-locus on SBI06 modulates the extent of aerenchyma formation in sorghum stems and leaf midribs. A QTL aligned with this locus was identified and fine-mapped in populations derived from BTx623*IS320c, BTx623*R07007, and BTx623*Standard broomcorn. Analysis of coding polymorphisms in the fine-mapped D-locus showed that genotypes that accumulate low levels of aerenchyma encode a truncated NAC transcription factor (Sobic.006G147400, SbNAC_d1), whereas parental lines that accumulate higher levels of stem aerenchyma encode full-length NAC TFs (SbNAC-D). During vegetative stem development, aerenchyma levels are low in nonelongated stem internodes, internode growing zones, and nodes. Aerenchyma levels increase in recently elongated internodes starting at the top of the internode near the center of the stem. SbNAC_D was expressed at low levels in nonelongated internodes and internode growing zones and at higher levels in regions of stem internodes that form aerenchyma. SbXCP1, a gene encoding a cysteine protease involved in programmed cell death, was induced in SbNAC_D genotypes in parallel with aerenchyma formation in sorghum stems but not in SbNAC_d1 genotypes. Several sweet sorghum genotypes encode the recessive SbNAC_d1 allele and have low levels of stem aerenchyma. Based on these results, we propose that SbNAC_D is the D-gene identified by Hilton (1916) and that allelic variation in SbNAC_D modulates the extent of aerenchyma formation in sorghum stems.