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. 2008 Aug;147(4):1710-22.
doi: 10.1104/pp.108.120238. Epub 2008 Jun 26.

An abscisic acid-induced protein, HVA22, inhibits gibberellin-mediated programmed cell death in cereal aleurone cells

Woei-Jiun Guo  1 , Tuan-Hua Ho
Affiliations

An abscisic acid-induced protein, HVA22, inhibits gibberellin-mediated programmed cell death in cereal aleurone cells

Woei-Jiun Guo et al. Plant Physiol. 2008 Aug.

Erratum in

  • Plant Physiol. 2008 Oct;148(2):1182. David Ho, Thun-Hua [corrected to Ho, Tuan-Hua]

Abstract

Plant HVA22 is a unique abscisic acid (ABA)/stress-induced protein first isolated from barley (Hordeum vulgare) aleurone cells. Its yeast homolog, Yop1p, functions in vesicular trafficking and in the endoplasmic reticulum (ER) network in vivo. To examine the roles of plant HVA22, barley HVA22 was ectopically expressed in barley aleurone cells. Overexpression of HVA22 proteins inhibited gibberellin (GA)-induced formation of large digestive vacuoles, which is an important aspect of GA-induced programmed cell death in aleurone cells. The effect of HVA22 was specific, because overexpression of green fluorescent protein or another ABA-induced protein, HVA1, did not lead to the same effect. HVA22 acts downstream of the transcription factor GAMyb, which activates programmed cell death and other GA-mediated processes. Moreover, expression of HVA22:green fluorescent protein fusion proteins showed network and punctate fluorescence patterns, which were colocalized with an ER marker, BiP:RFP, and a Golgi marker, ST:mRFP, respectively. In particular, the transmembrane domain 2 was critical for protein localization and stability. Ectopic expression of the most phylogenetically similar Arabidopsis (Arabidopsis thaliana) homolog, AtHVA22D, also resulted in the inhibition of vacuolation to a similar level as HVA22, indicating function conservation between barley HVA22 and some Arabidopsis homologs. Taken together, we show that HVA22 is an ER- and Golgi-localized protein capable of negatively regulating GA-mediated vacuolation/programmed cell death in barley aleurone cells. We propose that ABA induces the accumulation of HVA22 proteins to inhibit vesicular trafficking involved in nutrient mobilization to delay coalescence of protein storage vacuoles as part of its role in regulating seed germination and seedling growth.

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Figures

Figure 1.
Figure 1.
Vacuolation of barley aleurone cells is regulated by GA and ABA treatment. A, Scheme of the gene construct. The gray arrow represents the ORF of DesRed (not drawn to scale), the expression of which is driven by the maize ubiquitin promoter, Ubi1. B, Confocal micrographs of transformed aleurone cells. Embryoless half-seeds were transformed with DesRed to observe the morphology of PSVs. Cells were examined 48 h after incubation in buffers containing no hormone (Control), 1 μm GA (GA), or 1 μm GA plus 20 μm ABA (GA+ABA). C, Percentage of vacuolated cells in transformed aleurone tissues. Aleurone tissues were bombarded as described for B. Cells vacuolated were scored, and the percentage of vacuolated cells was calculated relative to total cells observed (see “Materials and Methods”). The results represent averages ± se (n = 6).
Figure 2.
Figure 2.
Barley HVA22 inhibits PCD/vacuolation in aleurone cells in the presence of GA. A, Schemes of gene constructs. Gray arrows represent the ORF (not drawn to scale), the expression of which is driven by the maize ubiquitin promoter, Ubi1. B, Confocal micrographs of aleurone cells cobombarded with DesRed and a construct expressing HVA22 or vector. Samples were preincubated in control buffer for 24 h, then transferred to buffer containing no hormone (Control) or 1 μm GA (GA). After another 48 h, aleurone cells were observed with a confocal microscope. C, Magnified images of representative cells from B. D, Percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6).
Figure 3.
Figure 3.
Barley HVA22 inhibits PCD/vacuolation downstream of GAMyb in aleurone cells. A, Schemes of gene constructs. Gray arrows represent the orientation of genes or gene fragments (not drawn to scale). The expression of these constructs is driven by the maize ubiquitin promoter. B, Percentage of vacuolated cells in transformed aleurone tissues under hormone-free conditions. The reporter DesRed was cobombarded with empty vector, or GAMyb, or GAMyb and GAMybRNAi, or GAMyb and HVA22 as indicated. After 48 h of incubation in control buffer, percentage of vacuolated cells was calculated as described for Figure 1C. C, Percentage of vacuolated cells in transformed aleurone tissues with GA treatment. The reporter DesRed was cobombarded with constructs expressing HVA22 or GAMybRNAi, vector only, or various combinations as indicated. The aleurone tissues were preincubated in the control buffer for 48 h, then transferred to buffer containing no hormone or 1 μm GA. After another 48 h of incubation, percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6).
Figure 4.
Figure 4.
GAMybRNAi is effective at blocking GA-induced α-amylase expression in aleurone cells. A, Schemes of gene constructs. Gray arrows represent the orientation of genes or gene fragments (not drawn to scale). Except that GUS expression is driven by the α-amylase32 promoter, expression of other genes is under the control of the maize ubiquitin promoter, Ubi1. B and C, GUS activity produced by transformed aleurone tissues under control conditions or GA treatment. The reporter constructs, Amy∷GUS and Ubi∷LUC, were cobombarded with empty vector, constructs expressing GAMyb or GAMybRNAi, or various combinations as indicated. Bars indicate relative GUS activity ± se (see “Materials and Methods”) after 24 h of incubation in buffer containing no hormone or 1 μm GA.
Figure 5.
Figure 5.
HVA22RNAi does not block ABA-inhibited PCD/vacuolation in aleurone cells. A, Schemes of gene constructs. Gray arrows represent the orientation of genes or gene fragments (not drawn to scale). B and C, Percentage of vacuolated cells in transformed aleurone tissues. The reporter DesRed was cobombarded with empty vector, constructs expressing HVA22 or HVA22RNAi, or various combinations as indicated. Samples were preincubated in control buffer for 48 h, then transferred to buffer containing no hormone (Control), 1 μm GA, or 1 μm GA and 20 μm ABA. After another 48 h, aleurone cells were observed by confocal microscopy. Percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6).
Figure 6.
Figure 6.
Localization of HVA22:GFP in barley aleurone cells. A, Schemes of gene constructs. Gray arrows indicate ORF of HVA22 (not drawn to scale), fused in frame with GFP protein at the N terminus. B, Percentage of vacuolated cells in transformed aleurone cells. The reporter DesRed was cobombarded with a construct expressing HVA22:GFP or GFP alone. Samples were preincubated in control buffer for 24 h, then transferred to buffer containing no hormone (Control) or 1 μm GA. After another 48 h, aleurone cells were observed by confocal microscopy. Percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6). C, Confocal micrographs of aleurone cells observed in B. Representative cells cobombarded DesRed with a construct expressing HVA22:GFP (a, b, c, g, h, i) or GFP (d, e, f, j, k, l) were demonstrated. Fluorescence from green (a, d, g, j), red (b, e, h, k), and both (c, f, i, l) channels was shown.
Figure 7.
Figure 7.
Colocalization of HVA22:GFP with Golgi and ER markers in barley aleurone cells. Confocal micrographs of aleurone cells cobombarded with HVA22:GFP and BiP:RFP (A–D) or ST-mRFP (E–H) are shown. Fluorescence from green (A and E), red (B and F), and both (C and G) channels was observed after 48 h of transformation under control conditions. D and H show pixel intensities of green (green trace) and red (red trace) fluorescence along the transects in the left panels. The blue arrowheads indicate the coalignment of intensity peaks. The green stars and orange circles represent the start and end positions of the transacts.
Figure 8.
Figure 8.
Transmembrane domains are necessary for HVA22 function. A, Schemes of gene constructs. White boxes indicate predicted transmembrane domains of HVA22 protein. Black boxes and arrows indicate the same N-terminal and C-terminal regions used in these constructs. B, Percentage of vacuolated cells in transformed aleurone tissues. The reporter DesRed was cobombarded with empty vector, a construct expressing HVA22, or constructs expressing various HVA22 truncated forms (ΔTM1, ΔTM2, and ΔTM3) into aleurone cells. Samples were preincubated in control buffer for 24 h, then transferred to buffer containing no hormone (Control) or 1 μm GA. After another 48 h, aleurone cells were observed by confocal microscopy. Percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6). C, Confocal micrographs of transformed cells expressing HVA22:GFP (a–c), ΔTM1:GFP (e–g), ΔTM2:GFP (i–k), and ΔTM3:GFP (m–o). The intensity profiles of green and red fluorescence from representative cells expressing HVA22:GFP (d), ΔTM1:GFP (h), ΔTM2:GFP (l), and ΔTM3:GFP (p) are also shown.
Figure 9.
Figure 9.
Effects of overexpressing Arabidopsis AtHVA22 homologs on GA-induced PCD/vacuolation in barley aleurone cells. A, Schemes of gene constructs. Gray arrows represent the ORF (not drawn to scale), whose expression is driven by the maize ubiquitin promoter, Ubi1. B, Percentage of vacuolated cells in transformed aleurone tissues. The reporter DesRed was cobombarded with empty vector or constructs expressing Arabidopsis AtHVA11A, AtHVA11B, AtHVA11C, AtHVA11D, or AtHVA11E. Samples were preincubated in control buffer for 24 h, then transferred to buffer containing no hormone (Control) or 1 μm GA. After another 48 h, aleurone cells were observed with a confocal microscope. Percentage of vacuolated cells was calculated as described for Figure 1C. The results represent averages ± se (n = 6).
Figure 10.
Figure 10.
Scheme of factors and pathways involved in GA/ABA-regulated PCD/vacuolation in aleurone cells. A repressor, SLN1, and an activator, GAMyb, are important regulators in the GA-mediated PCD and the associated PSV vacuolation. ABA negatively regulates PCD both upstream and downstream of GAMyb. The ABA-induced HVA22 is involved in the inhibition downstream of GAMyb.
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References

    1. Audhya A, Desai A, Oegema K (2007) A role for Rab5 in structuring the endoplasmic reticulum. J Cell Biol 178 43–56 - PMC - PubMed
    1. Bartels D, Engelhardt K, Roncarati R, Schneider K, Rotter M, Salamini F (1991) An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein. EMBO J 10 1037–1043 - PMC - PubMed
    1. Bethke PC, Libourel IG, Aoyama N, Chung YY, Still DW, Jones RL (2007) The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy. Plant Physiol 143 1173–1188 - PMC - PubMed
    1. Bethke PC, Lonsdale JE, Fath A, Jones RL (1999) Hormonally regulated programmed cell death in barley aleurone cells. Plant Cell 11 1033–1046 - PMC - PubMed
    1. Brands A, Ho TH (2002) Function of a plant stress-induced gene, HVA22: synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic. Plant Physiol 130 1121–1131 - PMC - PubMed

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