bHLH010/089 Transcription Factors Control Pollen Wall Development via Specific Transcriptional and Metabolic Networks in Arabidopsis thaliana

Abstract

The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089–like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Environmental regulation of male fertility is mediated through Arabidopsis transcription factors bHLH89, 91, and 10

“…These bhlh89,91 pollen abnormalities under low light are also reminiscent of the abcg9,abcg31 mutant in which pollen grains shrivel and clump together upon exposure to air ( Choi et al , 2014 ). These two ATP binding cassette (ABC) transporters, involved in the deposition of steryl glycosides on the pollen coat, are down-regulated in the bhlh010 bhlh089 mutant ( Lai et al , 2022 ) and, according to our own data, appear to be regulated by bHLH89/91 in response to light ( Supplementary Table S3 ). Lai et al demonstrated that ABCG9 is directly activated by bHLH89.…”

“…Recently Lai et al (2022) demonstrated that bHLH89 and bHLH10 are required for normal formation of the pollen exine and intine, through positive regulation of the phospholipid and flavonol metabolic pathways. The data we present herein suggest that bHLH91, in addition to bHLH89 and bHLH10, might also play an important role in regulating pollen wall development, specifically in response to altered environmental conditions, with both bhlh89,10 and bhlh89,91 double mutants exhibiting defective pollen wall patterning under low light conditions ( Fig.…”

“…These tapetum defects are similar to those observed in the male-sterile dyt1 and ams mutants, which have large, vacuolated tapetum layers ( Sorensen et al , 2003 ; Zhang et al , 2006 ; Xu et al , 2010 ); dyt1 mutant meiocytes have thinner callose walls as in the bhlh089 bhlh010 mutant ( Zhang et al , 2006 ), and the ams mutant has irregular sized tapetum cells which exhibit delayed degradation ( Xu et al , 2010 ). Beyond tapetal defects, the bhlh089 bhlh010 double mutant pollen has thinner and more uneven exine and intine layers ( Lai et al , 2022 ).…”

“…Despite the high sequence similarity and functional redundancy indicated by the bhlh89 , 91 , and 10 mutant phenotypes, these bHLHs have been shown to play distinct roles in activating downstream transcription ( Cui et al , 2016 ; Lai et al , 2022 ). Different combinations of bHLH89/91/10 with DYT1 showed differential transcriptional activation; the DYT1–bHLH89 heterodimer, for example, bound to the MYB35 promoter to strongly activate expression, whereas DYT1–bHLH10 and DYT1–bHLH91 heterodimers showed lower activation capabilities ( Cui et al , 2016 ).…”

Full‐length EFOP3 and EFOP4 proteins are essential for pollen intine development in Arabidopsis thaliana

“…CELLULOSE SYNTHASE‐LIKE B3 ( CSLB03 ) gene encodes a cellulose synthase‐like protein with glycosyltransferase activity, with a protein structure that is highly similar to that of CESA. The intine of cslb03 mutants was incomplete or even absent, and the germination rate of cslb03 pollen was severely reduced relative to its wild‐type (Bernal et al., 2008; Lai et al., 2022; Richmond & Somerville, 2000). UDP‐SUGAR PYROPHOSPHORYLASE (AtUSP) was shown to synthesize UDP‐glucuronic acid (UDP‐GlcA), a precursor of cell wall components such as pectin and hemicellulose.…”