The B2 domain of VIVIPAROUS1 is bi-functional and regulates nuclear localization and transactivation

Abstract

The transcriptional regulator VIVIPA-ROUS1 (VP1) is composed of four functional domains that control different aspects of gene expression during seed development. The B2 domain is required for its role as a transcriptional activator, functioning at the site of transcription and/or for its transport into the nucleus. Previous work showed that the B2 domain was required for transactivation of the Em promoter. We demonstrate that VP1::GFP localizes to the nucleus of barley (Hordeum vulgare) aleurone cells, but when B2 is deleted, nuclear accumulation is lost. However, the B2 domain itself is not sufficient for nuclear localization of GFP::GUS. Using point mutagenesis on the putative NLS within B2, we show that the VP1::GFP still accumulates in the nucleus. Utilizing a comparative approach, through the alignment of B2 domains from various VP1/ABI3 proteins, oincluding the ABI3 orthologs from Physcomitrella patens, revealed the involvement of other conserved amino acids. Mutating VP1 at the conserved threonine on the N-terminal side of the putative NLS and at a conserved arginine-glutamine-arginine sequence on the C-terminal side prevented nuclear localization of VP1. A single amino acid change, from alanine to threonine, within this NLS found in the Arabidopsis abi3-7 mutant prevents transcription of AtEm1 and AtEm6 in vivo. We show that this same mutation in VP1 prevents transactivation of the Em-GUS reporter in barley aleurone but does not interfere with nuclear localization. Our data demonstrate that the B2 domain of VP1 is bifunctional in nature regulating both nuclear localization and transactivation.

Transcription factors regulating the progression of monocot and dicot seed development

“…In the monocots, maize and barley, ABA and certain seed factors control the auto-regulated TF genes VP1 and ABI5 [49]. VP1 also upregulates ABI5 [51]. Apart from ABA, other unknown factors also regulate ABI4 [56].…”

“…VP1/ABI3 is an activator of embryo maturation and a repressor of germination through independent domains. Its B2 domain trans-activates other seed genes, such as OPAQUE2 in maize [50,51]. The N-terminal domain is essential for the ABA-mediated response, while the B3 domain has been implicated in controlling the expression of other seedspecific genes by binding to the RY element of their promoters [52].…”

A Developmental Switch of Gene Expression in the Barley Seed Mediated by HvVP1 (Viviparous-1) and HvGAMYB Interactions

“…B, Distribution of the conserved motifs among the deduced protein sequences in the dendrogram (A), found by means of MEME analysis. A, B1, B2, and B3 correspond to conserved domains, as described by Nakamura and Toyama (2001) and Marella and Quatrano (2007). To investigate the activation capacity of HvVP1 in yeast (Saccharomyces cerevisiae), a series of constructs containing different regions of the HvVP1 ORF was prepared (Fig.…”

Regulation of the ABA-responsive Em promoter by ABI3 in the moss Physcomitrella patens

“…7 The B1 domain is involved in physical interaction with the bZIP transcription factor ABI5 that binds to the ACGT core-containing ABA-responsive element (ABRE). [8][9][10] The B2 domain contains a putative nuclear localization signal (NLS), 11 is responsible for ABA-dependent activation through ABRE, [12][13][14][15] and also facilitates interaction with ABI5. 12 In addition, ABI3/VP1 transactivates gene promoters through the B3 DNA binding domain that recognizes the RY (or Sph) elements 7,16 found in the 5' regions of MAT genes, such as C1 in maize or 2S albumin genes in Arabidopsis and Brassica napus.…”