The transcription factors HvABI5 and HvVP1 are required for the abscisic acid induction of gene expression in barley aleurone cells

Abstract

The abscisic acid (ABA) response promoter complexes (ABRCs) of the HVA1 and HVA22 genes have been shown to confer ABA-induced gene expression in cereals. A barley basic domain/Leu zipper (bZIP) transcription factor, HvABI5, is able to recognize ABRCs in vitro in a sequence-specific manner and to transactivate ABRC-beta-glucuronidase reporter genes when introduced to barley aleurone cells via particle bombardment. This transactivation is dependent on the presence of another transcription factor, HvVP1, and cannot be blocked by the negative regulator abi1-1. Using the double-stranded RNA interference technique, we show that HvABI5 and HvVP1 are necessary for the ABA induction of gene expression but have no effect on another hormone-regulated process, the gibberellin-induced and ABA-suppressed expression of alpha-amylase. Our work indicates that although other typical plant bZIP transcription factors may bind ABRCs in vitro, HvABI5 is related to a subfamily of bZIPs responsible for the ABA induction of gene expression. Furthermore, HvABI5 and HvVP1 are not involved in the ABA suppression of gene expression.

Figure 1.

Comparison of Two New Barley bZIPs, HvABI5 and HvZIP1. (A) Schemes of the HvABI5 and HvZIP1 proteins. Boxes represent the positions of conserved regions among ABI5-like bZIPs (C1 to C4), the Pro-rich region, the basic DNA binding domain (BR), and the Leu zipper dimerization domain. (B) Comparison of the HvABI5 amino acid sequence with the sequences of other plant bZIPs. The amino acid sequence was deduced from the cDNA nucleotide sequence and was aligned with similar proteins found in the database: the rice TRAB1 protein and the Arabidopsis bZIPs AREB2 and ABI5. Identical residues are shaded. Conserved regions are underlined, and the basic region and the Leu zipper are double underlined. Asterisks denote potential phosphorylation sites.

Figure 2.

HvABI5 Binds ABRCs in Vitro in a Sequence-Specific Manner. (A) and (C) Partial nucleotide sequences of ABRC3 (A) and ABRC1 (C) mutant and synthetic versions used as competitors in electrophoretic mobility shift assays. (B) and (D) Electrophoretic mobility shift assays with recombinant HvABI5. A 124-bp fragment probe containing ABRC3 (B) or a 90-bp fragment probe containing ABRC1 (D) and 2 μg of HvABI5 protein were used in each assay. bact indicates the control binding reaction with bacterial protein from Escherichia coli that carried an empty expression vector. The molar excess of each competitor used (30- and 300-fold) is indicated at the top of each lane. Arrows indicate the DNA-protein complexes.

Figure 3.

HvABI5 and VP1 Are Sufficient to Transactivate ABRC-GUS. (A) Schemes of the reporter and effector constructs used in the transient expression assays. (B) The reporter construct ABRC3-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) the effector constructs Ubi-HvABI5, Ubi-HvZIP1, and 35S-VP1 using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (closed bars) or without (open bars) 20 μM ABA. (C) The reporter construct Amy32-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) the effector constructs Ubi-HvABI5 and 35S-VP1 using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (hatched bars) or without (open bars) 1 μM GA₃.

Figure 4.

A Dominant-Negative Form of HvABI5 Represses the ABA Induction of ABRC-GUS. (A) Schemes of the reporter and effector constructs used in the transient expression assays. (B) One microgram of the reporter construct ABRC3-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) increasing amounts of the effector construct Ubi-HvABI5-ΔN as indicated in the reporter:effector ratios. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (closed bars) or without (open bars) 20 μM ABA.

Figure 5.

HvABI5-RNAi Specifically Inhibits the ABA Induction of ABRC-GUS but Has No Effect on the ABA Suppression of α-Amylase Expression. (A) Schemes of the reporter and effector constructs used in the transient expression assays. (B) The reporter constructs ABRC3-GUS and ABRC1-GUS were cobombarded into barley embryoless half-seeds with (+) or without (−) the effector constructs Ubi-HvABI5-RNAi or Ubi-HvZIP1-RNAi using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (closed or checked bars) or without (open bars) 20 μM ABA. Incubation with the hormone was initiated 6 h after bombardment. (C) The reporter construct Amy32-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) the effector construct Ubi-HvABI5-RNAi using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (hatched bars) or without (open bars) 20 μM ABA or 1 μM GA₃. Incubation with the hormone was initiated 6 h after bombardment.

Figure 6.

ABA Induction of ABRC-GUS also Is Inhibited by HvVP1-RNAi. (A) Schemes of the reporter and effector constructs used in the transient expression assays. (B) The reporter constructs ABRC3-GUS and ABRC1-GUS were cobombarded into barley embryoless half-seeds with (+) or without (−) the effector construct Ubi-HvVP1-RNAi using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (closed or checked bars) or without (open bars) 20 μM ABA. Incubation with the hormone was initiated 8 h after bombardment. (C) The reporter construct Amy32-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) the effector construct Ubi-HvVP1-RNAi using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (hatched bars) or without (open bars) 20 μM ABA or 1 μM GA₃. Incubation with the hormone was initiated 8 h after bombardment.

Figure 7.

Expression of HvABI5 but Not HvVP1 Is Upregulated by ABA. (A) Reverse transcriptase–mediated PCR analysis of HvABI5 and HvVP1 expression in barley aleurones. Reactions with (+) or without (−) reverse transcriptase (RT) and with total RNA isolated from aleurone layers incubated with (ABA) or without (Control) 100 μM ABA for 12 h are shown. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Numbers at right represent fold of induction by ABA ± se (n = 7). (B) Abundance of HvABI5 (squares) and HvVP1 (triangles) from aleurones incubated with (closed symbols) or without (open symbols) ABA was examined within a linear range of 10-fold dilution of the starting template and normalized with respect to the actin products. (C) Scheme of a transcriptional GUS fusion with the promoter and first intron (thin line) of HvABI5 (HvABI5-GUS) used in transient expression assays. (D) The reporter construct HvABI5-GUS (2 μg) was cobombarded into barley embryoless half-seeds with 1 μg of the Ubi-LUC internal control construct. Bars indicate GUS activities ± se after 12 h of incubation of the bombarded seeds with (+) or without (−) 20 μM ABA.

Figure 8.

The HvABI5/VP1 Transactivation of ABRC-GUS Is Not Inhibited by abi1-1. (A) Schemes of the reporter and effector constructs used in the transient expression assays. (B) The reporter construct ABRC3-GUS was cobombarded into barley embryoless half-seeds with (+) or without (−) the effector constructs 35S-abi1-1, Ubi-HvABI5, and 35S-VP1 using 1 μg of each construct. Bars indicate GUS activities ± se after 24 h of incubation of the bombarded seeds with (closed bars) or without (open bars) 20 μM ABA.

Figure 9.

Model for the ABA Regulation of HVA1 and HVA22. ABA could regulate the expression of the stress-induced genes HVA1 and HVA22 by activating a protein complex composed of HvABI5 and HvVP1 gathered at the ABRCs. These two transcription factors are involved specifically in the ABA upregulatory pathway and act downstream of abi1.