Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development

Abstract

Transcriptional regulation is one of the most important mechanisms controlling development and cellular functions in plants and animals. The Arabidopsis thaliana bHLH transcription factor (TF) DYSFUNCTIONL TAPETUM1 (DYT1) is required for normal male fertility and anther development and activates the expression of the bHLH010/bHLH089/bHLH091 genes. Here, we showed that DYT1 is localized to both the cytoplasm and nucleus at anther stage 5 but specifically to the nucleus at anther stage 6 and onward. The bHLH010/bHLH089/bHLH091 proteins have strong nuclear localization signals, interact with DYT1, and facilitate the nuclear localization of DYT1. We further found that the conserved C-terminal BIF domain of DYT1 is required for its dimerization, nuclear localization, transcriptional activation activity, and function in anther development. Interestingly, when the BIF domain of DYT1 was replaced with that of bHLH010, the DYT1 N -bHLH010 BIF chimeric protein shows nuclearpreferential localization at anther stage 5 but could not fully rescue the dyt1-3 phenotype, suggesting that the normal spatiotemporal subcellular localization of DYT1 is important for DYT1 function and/or that the BIF domains from different bHLH members might be functionally distinct. Our results support an important positive feedback regulatory mechanism whereby downstream TFs increase the function of an upstream TF by enhancing its nucleus localization through the BIF domain.

Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation

“…The basic helix-loop-helix (bHLH) transcriptional factor DYSFUNCTIONAL TAPETUM1 (DYT1) is among the earliest acting male-specific regulators and is required for the normal development of tapetum by regulating the normal expression of more than 1,000 anther genes, and dyt1 mutants form abnormal prematurely vacuolated tapetal cells and lack mature pollen (Zhang et al, 2006;Feng et al, 2012;Gu et al, 2014;Zhu et al, 2015). In addition, bHLH010, bHLH089, and bHLH091 together are required for normal tapetum development and transcriptome and pollen fertility, forming both feed-forward and feedback loops with DYT1 (Zhu et al, 2015;Cui et al, 2016). Another bHLH transcription factor, ABORTED MICROSPORES (AMS), is a key regulator of anther transcriptome, sporopollenin biosynthesis and secretion, and pollen wall formation (Sorensen et al, 2003;Xu et al, 2010;Feng et al, 2012;Ma et al, 2012;Xu et al, 2014).…”

Reply: The BIF Domain Is Structurally and Functionally Distinct from Other Types of ACT-Like Domains

“…However, there are substantial functional and structural differences (see below) between the C-terminal BIF/ACT-like domain of plant bHLH proteins (Feller et al, 2006;Kong et al, 2012;Cui et al, 2016) and the original ACT domain and previously reported ACT-like domain in other proteins (Chipman and Shaanan, 2001;Siltberg-Liberles and Martinez, 2009;Lang et al, 2014).…”

“…DYT1 expression and function are precisely regulated by a set of interactions: On one hand, protein products of DYT1 downstream genes bHLH010/089/091 interact and form a feedforward loop with DYT1 to control the proper expression of shared downstream anther genes (Zhu et al, 2015); on the other hand, bHLH010/bHLH089/bHLH091 facilitates the stage-dependent nuclear-specific localization of DYT1 in a positive feedback regulatory loop enhancing DYT1 function (Cui et al, 2016).…”

“…This C-terminal domain of DYT1 is required for the interaction with and the nuclear localization by bHLH010/ bHLH089/bHLH091, and transcriptional activation activity and in vivo function of DYT1 (Cui et al, 2016); this domain in DYT1 and homologs was named the BIF domain for bHLH protein interaction and function and also found in over one-third of Arabidopsis bHLH proteins (Cui et al, 2016).…”

“…By contrast, the BIF/ACT-like domains in plant bHLH proteins are predicted to share a bbabba structure, which differs from the babbab structure in the position of four out of six motifs (Felleret al, 2006;Kong et al, 2012;Cui et al, 2016). At the sequence level, there are very low levels of amino acid sequence identities, with only 4.6% between the ACT domain of 3-phosphoglyceratedehydrogenaseand the ACT-like domain of threonine deaminase, and only 6.6% between the ACT domain and the ACT-like RAM domain; these percentages are not statistically different from that between two random sequences.…”