Arabidopsis ERF4 and MYB52 Transcription Factors Play Antagonistic Roles in Regulating Homogalacturonan De-methylesterification in Seed Coat Mucilage

Abstract

One-sentence summary: Arabidopsis ERF4 and MYB52 transcription factors interact 18 and play antagonistic roles in regulating homogalacturonan de-methylesterification 19 related genes in the seed coat mucilage.ABSTRACT 24 The Arabidopsis (Arabidopsis thaliana) seed coat mucilage is a specialized cell wall with 25 pectin as its major component. Pectin is synthesized in the Golgi apparatus with 26 homogalacturonan fully methylesterified, but it must undergo de-methylesterification by 27 pectin methylesterase (PME) after being secreted into the cell wall. This reaction is 28 critical for pectin maturation, but the mechanisms of its transcriptional regulation remain 29 largely unknown. Here, we show that the Arabidopsis ERF4 transcription factor 30 positively regulates pectin de-methylesterification during seed development and directly 31 suppresses the expression of PME INHIBITOR13 (PMEI13), 14, 15 and 32 SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). The erf4 mutant seeds showed 33 repartitioning of mucilage between soluble and adherent layers as a result of decreased 34 PME activity and increased degree of pectin methylesterification. ERF4 physically 35 associates with and antagonizes MYB52 in activating PMEI6, 14 and SBT1.7 and 36 MYB52 also antagonizes ERF4 activity in the regulation of downstream targets. Gene 37 expression studies revealed that ERF4 and MYB52 have opposite effects on pectin 38 de-methylesterification. Genetic analysis indicated that the erf4-2 myb52 double mutant 39 seeds show mucilage phenotype similar to wild-type. Taken together, this study 40 demonstrates that ERF4 and MYB52 antagonize each other's activity to maintain the 41 appropriate degree of pectin methylesterification, expanding our understanding of how 42 pectin de-methylesterification is fine-tuned by the ERF4-MYB52 transcriptional complex 43 in the seed mucilage. 44 130 6 stress (Liu et al., 2017). ERF4 is expressed ubiquitously in Arabidopsis (Winter et al., 131 2007), suggesting that it has other functions in addition to those mentioned above. For 132 example, its regulatory role in cell wall biology has not been reported yet. 133Here, we provide evidence that ERF4 positively regulates pectin de-methylesterification 134 in the seed mucilage. We find a repartitioning of seed mucilage polysaccharides in the 135 erf4 seeds under vigorous shaking conditions which can be attributed to an increase in 136 DM. ERF4 binds to the cis-regulatory elements and directly suppresses the expression of 137 PMEI13, 14, 15 and SBT1.7. Moreover, ERF4 and MYB52 were found to interact, and 138 play completely opposite roles in pectin de-methylesterification by antagonizing each 139 other's transcriptional activity, which is confirmed by gene expression and genetic 140 evidence. Overall, these findings provide a fine-tuned mechanism where ERF4 and 141 MYB52 antagonistically control mucilage modification related genes. 142 RESULTS 143Expression pattern of ERF4 is correlated with seed mucilage deposition 144 Previous studies revea...