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

Abstract

The accumulation of storage compounds in the starchy endosperm of developing cereal seeds is highly regulated at the transcriptional level. These compounds, mainly starch and proteins, are hydrolyzed upon germination to allow seedling growth. The transcription factor HvGAMYB is a master activator both in the maturation phase of seed development and upon germination, acting in combination with other transcription factors. However, the precise mechanism controlling the switch from maturation to germination programs remains unclear. We report here the identification and molecular characterization of Hordeum vulgare VIVIPAROUS1 (HvVP1), orthologous to ABA-INSENSITIVE3 from Arabidopsis thaliana HvVP1 transcripts accumulate in the endosperm and the embryo of developing seeds at early stages and in the embryo and aleurone of germinating seeds up to 24 h of imbibition. In transient expression assays, HvVP1 controls the activation of Hor2 and Amy6.4 promoters exerted by HvGAMYB. HvVP1 interacts with HvGAMYB in Saccharomyces cerevisiae and in the plant nuclei, hindering its interaction with other transcription factors involved in seed gene expression programs, like BPBF. Similarly, this interaction leads to a decrease in the DNA binding of HvGAMYB and the Barley Prolamine-Box binding Factor (BPBF) to their target sequences. Our results indicate that the HvVP1 expression pattern controls the full Hor2 expression activated by GAMYB and BPBF in the developing endosperm and the Amy6.4 activation in postgerminative reserve mobilization mediated by GAMYB. All these data demonstrate the participation of HvVP1 in antagonistic gene expression programs and support its central role as a gene expression switch during seed maturation and germination.

Figure 1.

A, Phylogenetic tree with the deduced amino acid sequences of the VP1 genes from distinct barley cultivars (cv Igri, cv Morex, and cv Haruna Nijo), with wheat (TaVP1), T. turgidum (TtVP1), T. monococum (TmVP1), B. distachyon (BdVP1), and rice (OsVP1), and with AtABI3 from Arabidopsis. Bootstrapping values are specified at the branches. 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).

Figure 2.

A, HvVP1 transcript splicing model. Exons I to VI are indicated as black bars. Motifs A, B1, B2, and B3 are as in Figure 1. B, Functional assay in yeast for the activation capacity of the HvVP1 protein. Different constructs (1–5) were generated as fusions to the GAL4 DNA-binding domain (black box at the N terminus), and the activation capacity was assayed in two alternative reporter systems: β-Galactosidase (β-Gal) activity (LacZ reporter gene; Miller’s units [M.U.] in liquid medium) and HIS3 (growth capacity in His-depleted [His⁻] medium).

Figure 3.

HvVP1 expression analyses by northern blot and mRNA in situ hybridization analyses in barley developing (A) and germinating (B) seeds. A.1, Northern-blot analysis. Eight micrograms of total RNA from developing endosperms (from 5, 10, 15, and 20 dap) and immature (I; 25 dap) and mature (M) embryos was loaded in each lane and hybridized to an HvVP1 gene-specific probe. A.2, HvVP1 transcript localization in longitudinal sections of developing (25 dap) seeds by mRNA in situ hybridization B.1, Northern-blot analysis in germinating aleurone (16, 24, 48, and 72 hoi) and germinating embryo (8, 16, and 24 hoi). B.2, HvVP1 transcript localization in germinating barley seeds (36 hoi) by mRNA in situ hybridization assay. Left and right images correspond to hybridizations with HvVP1 antisense and sense probes, respectively. The ethidium bromide-stained ribosomal RNA images are included as loading controls. e, Endosperm; em, embryo; p, pericarp; s, scutellum.

Figure 4.

Transactivation assays using as effectors the TFs HvVP1 and HvGAMYB. The Hor2 and Amy6.4 gene promoters driving the expression of the uidA gene (GUS activity) were used as reporters. A, Schematic representation of the effector and reporter constructs used in the analyses. B, Cobombardment in barley developing endosperms of the effector and reporter combinations indicated. C, The effector and reporter constructs designated were cobombarded in germinating aleurones from barley kernels. The relative amounts of reporter and effector plasmids used in these assays correspond to a 1:1 ratio. Values are means ± se of three independent replicates.

Figure 5.

Protein-protein interaction by Y2H assays. A, The structures of HvGAMYB and HvVP1 are depicted, showing the locations of functionally relevant domains within the proteins. B, HvGAMYB (complete ORF) or HvVP1 N⁴⁶⁴ (the 464 N-terminal amino acid residues) expressed as translational fusions to the GAL4 DNA-binding domain (BD) and activation domain (AD), respectively. C and D, Interaction capacity assayed by measuring the β-Gal activity (LacZ reporter gene; M.U. in liquid medium; C) and by evaluating the yeast growth capacity in a His-depleted medium (reporter gene HIS3) with increasing concentrations of 3-aminotriazole (3-AT; D).

Figure 6.

In planta interaction of HvVP1 and HvGAMYB. A, Structures of gene constructs used in bimolecular fluorescence complementation assays using particle gun transformation of onion epidermal cells. B, GFP reconstruction in the nuclei of cells transformed with a combination of the constructs described above (b). Bright-field images (a) and 4′,6-diamidino-2-phenylindole staining (c) was used to reveal nuclei within the cells.

Figure 7.

Protein-protein interactions of HvVP1, GAMYB, and BPBF in Y3H experiments and the effect of HvVP1 on GAMYB and BPBF DNA-binding capacities. A, Schematic representation of the constructs used in the Y3H assays, where the BPBF ORF was translationally fused to the GAL4 DNA-binding domain (BD) in the pBridge plasmid harboring HvVP1 under the control of a Met-repressible promoter. The GAMYB ORF was translationally fused to the GAL4 activation domain (AD) in pGAD424. B, Schematic representation of the constructs used for the Y3H assays and quantification of β-Gal activity (expressed as M.U.) in liquid assays using the constructs represented above. The proteins expressed in Met-depleted medium are indicated at right. C, EMSAs of HvGAMYB and BPBF proteins in the absence (−) or presence (+) of the HvVP1 protein. Incubations were done with a specific ³²P-labeled probe containing a target site for the corresponding TFs: GLM (G-box-like motif), BLZ2; PB, BPBF; MBS (MYB-binding site), GAMYB.

Figure 8.

Transactivation assays using, as effectors, the TFs HvVP1 and BPBF and, as reporter, the Hor2 gene promoter driving the expression of the uidA gene (GUS activity). A, Schematic representation of the effector and reporter constructs used in the analyses. B, Cobombardment experiments on barley developing endosperms using the indicated combinations of effector and reporter constructs. The relative amount of reporter-to-effector plasmids used in these assays corresponds to a 1:1 ratio. Values are means ± se of three independent replicates.

Figure 9.

Proposed model of the transcriptional regulation of SSP (Hor2) and hydrolase (Amy6.4) genes in barley seeds mediated by HvVP1 interacting with GAMYB, DOF (BPBF and SAD), BLZ2, and MR1 (MYBR1) TFs during the maturation and postgermination phases. The starchy endosperm and the embryo-plus-aleurone layer are considered separately. Relative gene expression levels are indicated on the y axis. The dotted vertical lines indicate the time of root emergence.