an abscisic acid-induced protein, hva22, inhibits ......1991; xu et al., 1996; sivamani et al.,...

An Abscisic Acid-Induced Protein, HVA22, InhibitsGibberellin-Mediated Programmed Cell Death in CerealAleurone Cells1[W][OA]

Woei-Jiun Guo and Tuan-Hua David Ho2*

Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan

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 examinethe roles of plant HVA22, barley HVA22 was ectopically expressed in barley aleurone cells. Overexpression of HVA22 proteinsinhibited gibberellin (GA)-induced formation of large digestive vacuoles, which is an important aspect of GA-inducedprogrammed cell death in aleurone cells. The effect of HVA22 was specific, because overexpression of green fluorescent proteinor another ABA-induced protein, HVA1, did not lead to the same effect. HVA22 acts downstream of the transcription factorGAMyb, which activates programmed cell death and other GA-mediated processes. Moreover, expression of HVA22:greenfluorescent protein fusion proteins showed network and punctate fluorescence patterns, which were colocalized with an ERmarker, BiP:RFP, and a Golgi marker, ST:mRFP, respectively. In particular, the transmembrane domain 2 was critical for proteinlocalization 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 betweenbarley HVA22 and some Arabidopsis homologs. Taken together, we show that HVA22 is an ER- and Golgi-localized proteincapable of negatively regulating GA-mediated vacuolation/programmed cell death in barley aleurone cells. We propose thatABA induces the accumulation of HVA22 proteins to inhibit vesicular trafficking involved in nutrient mobilization to delaycoalescence of protein storage vacuoles as part of its role in regulating seed germination and seedling growth.

Abscisic acid (ABA) mediates various importantplant developmental and physiological processes andalso plant responses to stress conditions (Zeevaart andCreelman, 1988; Leung and Giraudat, 1998). In partic-ular, ABA is required for seed maturation programsand the maintenance of seed dormancy (Nambara andMarion-Poll, 2003; Gubler et al., 2005). The uniquefeature of de novo synthesis of hydrolytic enzymes inresponse to gibberellin (GA) and ABA has made thecereal aleurone cells an idea system for studying hor-mone signaling and action during seed germinationand seedling growth. In barley (Hordeum vulgare), morethan a dozen ABA-induced proteins have been isolatedfrom aleurone layers as part of the effort to study ABAaction in seed physiology (Shen et al., 1993). Amongthose, LEA (for late embryogenesis abundant) proteinshave been proposed to provide cell tolerance for seed

desiccation and environmental stresses (Bartels et al.,1991; Xu et al., 1996; Sivamani et al., 2000). However,little is known about how these proteins functionduring seed development and germination.

The barley HVA22 gene encodes one of these ABA-induced LEA proteins isolated from the aleuronetissue (Shen et al., 1993). Bioinformatic analysis hasrevealed that 354 HVA22 homologs are present indiverse eukaryotic organisms, including plants, mosses,yeast, and mammals (Supplemental Fig. S1). Thesehomologs share high amino acid sequence similarityin a conserved TB2/DP1 domain. Interestingly, nohomologs were found in prokaryotes to date (Supple-mental Fig. S1), suggesting that HVA22 is likely in-volved in eukaryote-specific functions.

The regulation of HVA22 expression during plantdevelopment is well characterized. In both barleyand Arabidopsis (Arabidopsis thaliana), transcripts ofHVA22 homologs in leaves are highly induced byABA, drought, cold, and salt stresses (Shen et al., 1993,2001; Chen et al., 2002). The ABA response complexlocated in the barley HVA22 gene promoter is neces-sary and sufficient in mediating the induction of thisgene by ABA (Shen and Ho, 1995). Moreover, expres-sion of the barley HVA22 gene is correlated with seeddormancy status (Shen et al., 2001). HVA22 mRNAgradually accumulates in the aleurone layer duringthe late stage of seed maturation (Shen et al., 2001),when high levels of endogenous ABA biosynthesisoccur to maintain seed dormancy (Karssen et al., 1983).In nondormant seeds, transcripts of barley HVA22 in

1 This work was supported by grants from Academia Sinica andthe National Science Council of Taiwan to T.-H.D.H. and by anAcademia Sinica postdoctoral fellowship to W.-J.G.

2 Present address: Department of Biology, Washington University,St. Louis, MO 63017.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Tuan-Hua David Ho ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.120238

1710 Plant Physiology, August 2008, Vol. 147, pp. 1710–1722, www.plantphysiol.org � 2008 American Society of Plant Biologists

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the embryo are degraded within 12 h after imbibition.In contrast, the HVA22 transcript level remains high indormant grains, in which GA treatment is able to breakdormancy with a concomitant decline of HVA22 tran-scripts. These results suggest that turnover of HVA22gene products in aleurone layers controls seed dor-mancy and germination (Shen et al., 1993). However,how plant HVA22 proteins function at the molecularand cellular levels in regulating seed germination isunknown.

Studies with Saccharomyces cerevisiae (Yop1p) andXenopus (DP1) homologs of HVA22 reveal their poten-tial roles in vesicular trafficking. Yeast Yop1p is able tophysically interact with several Rab GTPases (Ypt1,Ypt6, Ypt7, and YIF1) and YIP1, which are involved inendoplasmic reticulum (ER)-to-Golgi transport inyeast (Yang et al., 1998; Calero et al., 2001; De Antoniet al., 2002). Deletion or overexpression of Yop1presulted in abnormal vesicle accumulation in yeastcells (Calero et al., 2001; Brands and Ho, 2002). Fur-thermore, Yop1p/DP1 proteins also interact with ERreticulon proteins (Rtn4/NogoA) to shape the ERnetwork in vivo (Voeltz et al., 2006; Hu et al., 2008).The simultaneous absence of Rtn4a and Yop1p resultedin disrupted tubular peripheral ER. Amino acid se-quence alignment between Yop1p and HVA22 homo-logs from barley and Arabidopsis indicated that plantHVA22 proteins contain structures similar to yeastYop1p, a short hydrophilic loop flanked by two hy-drophobic stretches (Brands and Ho, 2002). It is plau-sible that plant HVA22 proteins also play a role invesicle trafficking processes in plant cells.

How is vesicle trafficking related to seed germina-tion? Since HVA22 was first cloned from barley aleu-rone cells, we focused our initial studies on the role ofHVA22 in this particular tissue. The cereal aleuronelayer is a metabolically active tissue surrounding thenutrient-rich starchy endosperm. Upon germination,GA produced by the growing embryo induces theproduction and secretion of hydrolytic enzymes fromthe aleurone to the starchy endosperm (Fincher, 1989).These enzymes liberate sugars and amino acidsneeded for the growing embryo and seedlings (Jonesand Jacobsen, 1991). ABA blocks the production ofthese enzymes primarily at the transcriptional level(Lovegrove and Hooley, 2000) and inhibits germina-tion by limiting the availability of energy and nutrients(Garciarrubio et al., 1997; Graham, 2008). Living aleu-rone cells become dispensable after hydrolase secre-tion and die a few days after germination. Theprogrammed cell death (PCD) of cereal aleurone tissueis preceded by extensive vacuolation of protein stor-age vacuoles (PSVs). At the time of death, a singlelarge vacuole occupies virtually all of the cytoplasm(Bethke et al., 1999). Disassembly of PSVs is associatedwith extensive vesicle trafficking and fusion (Jonesand Price, 1970; Vitale and Hinz, 2005).

Based on the strong ABA induction of HVA22 inaleurone layers and its potential involvement in ves-icle transport, we reasoned that barley HVA22 may be

involved in GA/ABA-regulated aleurone cell PCDand the associated vacuolation of PSVs. In this study,fluorescent DesRed protein was transiently expressedin the aleurone cells to allow the visualization ofPSV morphologic changes. Our results indicate thatbarley HVA22 is an important downstream regulatoryprotein of ABA action, which is involved in the inhi-bition of GA-mediated PCD. The localization ofHVA22-GFP fusion protein in ER and the Golgi appa-ratus suggests that this protein functions in vesiculartrafficking.

RESULTS

GA and ABA Regulate PCD/Vacuolation in BarleyAleurone Cells

Early studies showed that GA treatment inducesPCD of barley aleurone protoplasts, which is coordi-nated with distinct vacuolation of PSVs, but ABAblocks this process. Therefore, the numbers and sizesof PSVs in the aleurone cells have been usedas semiquantitative markers of PCD (Bethke et al.,1999, 2007). To conveniently observe changes of PSVsin living cells, a red fluorescent protein, DesRed, wastransiently expressed in barley aleurone cells drivenby the constitutive maize (Zea mays) ubiquitin pro-moter (Fig. 1A). To eliminate the interference fromendogenous GA or ABA synthesized by embryo,barley half-seeds without embryos were used in thisstudy. Transformed aleurone tissues were incubated inbuffer containing no hormone or 1 mM GA for 48 h,then the transformed aleurone cells were observedwith a confocal microscope. The DesRed fluorescencedisappeared in the PSVs due to pH and proteolyticactivity (Zentella et al., 2002; Fluckiger et al., 2003; Fig.1B). Thus, PSVs appeared as dark intracellular bodieswhose sizes and numbers could be easily examined.

Many small PSVs were observed in the aleuronecells when incubated in the hormone-free controlbuffer (Fig. 1B, control). However, these vacuolescoalesced and became one large vacuole after 48 h oftreatment with 1 mM GA (Fig. 1B, GA). The enlarge-ment of storage vacuoles was significantly inhibitedwhen 20 mM ABA was added with GA at the same time(Fig. 1B, GA1ABA). To provide a quantitative analy-sis, cells containing only one to three large vacuoles (asshown in Fig. 1B, GA) were classified as vacuolatedcells. The percentage of vacuolated cells was calcu-lated relative to total observed transformed cells. Morethan 80% of observed cells became distinctly vacuo-lated after 48 h of GA treatment, and ABA significantlyinhibited the vacuolation to the control level (i.e. 20%–30%; Fig. 1C). These results demonstrate that transientexpression of a fluorescent marker, DesRed, provides asimple but efficient method to monitor the extent ofPCD-associated vacuolation of PSVs in barley aleu-rone cells, and the antagonistic effect of GA and ABAon this process can be quantified.

HVA22 Inhibits Vacuolation and Programmed Cell Death

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Overexpression of HVA22 Inhibits GA-Induced PCD

To examine if plant HVA22 affects GA-mediatedPCD, an effector construct to overexpress barleyHVA22 in aleurone cells was cobombarded with thereporter construct for DesRed expression (Fig. 2A).The expression of HVA22 open reading frame (ORF) isdriven by the maize ubiquitin promoter. Transformedbarley half-seeds were first incubated in hormone-freecontrol buffer for 24 h to allow the expression ofHVA22 protein. These samples were then transferredto a new buffer with or without 1 mM GA for another48 h before observation by confocal microscopy. Lowand high magnifications of representative aleuronecells are shown in Figure 2, B and C, respectively.Under the control conditions, most cells transformedwith vector only or expressing HVA22 contained many

small PSVs (Fig. 2, B and C), and there was nosignificant difference in the degree of vacuolation(Fig. 2D). After treatment with 1 mM GA for 48 h, cellstransformed with vector only developed extensivevacuolation (Fig. 2, B and C), and more than 80% ofobserved cells were vacuolated (Fig. 2D). In contrast,most cells overexpressing HVA22 proteins still con-tained small PSVs (Fig. 2, B and C), with only around30% of observed cells being vacuolated (Fig. 2D).

To exclude the possibility that the inhibition ofvacuolation by HVA22 is due to a general effect ofoverexpressing a toxic protein or any ABA-inducedprotein, constructs to overexpress GFP or barley HVA1were also cotransformed with DesRed (Fig. 2A). BarleyHVA1 was also isolated from ABA-induced cDNAclones of aleurone tissues and has been proposed toprotect cells from dehydration during seed develop-ment (Hong et al., 1988; Xu et al., 1996). However,overexpression of HVA1 or GFP resulted in a similarlevel of vacuolated cells to those transformed withvector only under no-hormone (control) or GA-treatedconditions (Fig. 2D). These results indicate that HVA22specifically inhibits the GA-induced PCD-associatedvacuolation of PSVs.

HVA22 Functions Downstream of GAMyb

In the GA signaling pathway in cereal aleurone cells,SLN1 is an upstream repressor of the GAMyb tran-scription factor, which is a crucial positive regulator ofGA induction events (see Fig. 10 below; Gubler et al.,2002; Zentella et al., 2002; Kaneko et al., 2004). It hasbeen reported that SLN1 also negatively regulates GA-induced PCD in aleurone cells (Zentella et al., 2002).Transient expression of SLN1RNAi was able to triggerGA signaling to induce PSV vacuolation more than thevector-alone control by 35%. Whether PCD of aleuronecells is also regulated by GAMyb transcription has notbeen shown. To further determine where in the GAsignaling pathway HVA22 interferes with the GA-mediated PCD, we analyzed if GAMyb was alsorequired for GA-induced PCD and how overexpress-ing HVA22 affects GAMyb action.

To express GAMyb transiently, the ORF for GAMybwas cloned into the same vector as HVA22, where itsexpression was under the control of the maize ubiq-uitin promoter UBi1 (Fig. 3A). Without GA treatment,overexpression of GAMyb resulted in approximately80% vacuolated cells (Fig. 3B), which is similar to thevacuolation level induced by GA treatment (Fig. 1C).Overexpression of HVA22 together with GAMybcould still inhibit GAMyb-induced vacuolation by40%. These results suggest that GAMyb is also in-volved in GA-mediated PCD and HVA22 functiondownstream of GAMyb action.

Whether GAMyb is absolutely necessary to inducePCD in aleurone cells was further resolved via theRNA interference (RNAi) approach. Introduction of anRNAi-generating construct with particle bombard-

Figure 1. Vacuolation of barley aleurone cells is regulated by GA andABA treatment. A, Scheme of the gene construct. The gray arrowrepresents the ORF of DesRed (not drawn to scale), the expression ofwhich is driven by the maize ubiquitin promoter, Ubi1. B, Confocalmicrographs of transformed aleurone cells. Embryoless half-seeds weretransformed with DesRed to observe the morphology of PSVs. Cellswere examined 48 h after incubation in buffers containing no hormone(Control), 1 mM GA (GA), or 1 mM GA plus 20 mM ABA (GA1ABA). C,Percentage of vacuolated cells in transformed aleurone tissues. Aleu-rone tissues were bombarded as described for B. Cells vacuolated werescored, and the percentage of vacuolated cells was calculated relativeto total cells observed (see ‘‘Materials and Methods’’). The resultsrepresent averages 6 SE (n 5 6).

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1712 Plant Physiol. Vol. 147, 2008



ment has been shown to be an effective method toperform transient loss-of-function studies of targetgenes in the aleurone tissue (Zentella et al., 2002).Thus, an RNAi effector construct, GAMybRNAi, wasgenerated using a region of the GAMyb gene (351 bp)that encodes the transactivation domain of this tran-scription factor (Fig. 3A). When GAMybRNAi wascobombarded with GAMyb into the aleurone cells, ex-pression of GAMybRNAi reduced GAMyb-mediatedPSV vacuolation by 40% under control conditions(without GA treatment) after 48 h of transformation(Fig. 3B). Consistently, in aleurone cells treated with1 mM GA for 48 h, GAMybRNAi inhibited GA-inducedvacuolation by 35% compared with the vector-alonecontrol (Fig. 3C). Cotransformation of GAMybRNAiwith HVA22 could only lower another 15% of vacuola-tion cells compared with GAMybRNAi only, similar tothe level seen with HVA22 alone. This result indicatesthat little additive effect exits between HVA22 andGAMybRNAi and that the actions of HVA22 andGAMyb on PCD are likely in the same regulatorypathway in aleurone cells.

Since the GAMybRNAi construct was not able tocompletely block GAMyb- or GA-induced PCD-associated PSV vacuolation, we wondered whetherthe GAMybRNAi construct was sufficiently effectiveat inactivating GA signaling in vivo. A previous studyhas shown that GAMyb is not only sufficient but alsonecessary for GA-upregulated a-amylase transcrip-tion (Zentella et al., 2002). We further examined theeffectiveness of GAMybRNAi on the GA induction ofa-amylase expression. The reporter construct usedwould express GUS driven by the a-amylase32 pro-moter (AmyTGUS; Fig. 4A; Lanahan et al., 1992).Another reporter construct, which expressed lucifer-ase (UbiTLUC) under the control of a constitutivemaize Ubi1 promoter, was used to provide an internalcontrol of transformation. The embryoless barley half-seeds were cobombarded with the effector constructstogether with both of these reporter constructs. Therelative GUS activity in vivo was analyzed after 24 hof incubation. Without GA treatment, expression ofGAMyb significantly induced GUS activity (97-foldcompared with vector alone) and GAMybRNAi al-most completely blocked GAMyb-induced GUS activ-ity (Fig. 4B). When treated with 1 mM GA for 24 h,expression of the AmyTGUS reporter construct wasinduced 27-fold compared with that in the no-GAcontrol (Fig. 4C). However, expression of GAMybRNAidramatically diminished the expression of GUS to thelevel seen in samples treated without GA (Fig. 4C).These observations indicated that the GAMybRNAiconstruct used was effective at inhibiting GAMyb-induced a-amylase expression.

Figure 2. Barley HVA22 inhibits PCD/vacuolation in aleurone cells inthe presence of GA. A, Schemes of gene constructs. Gray arrowsrepresent the ORF (not drawn to scale), the expression of which isdriven by the maize ubiquitin promoter, Ubi1. B, Confocal micrographsof aleurone cells cobombarded with DesRed and a construct express-ing HVA22 or vector. Samples were preincubated in control buffer for24 h, then transferred to buffer containing no hormone (Control) or

1 mM GA (GA). After another 48 h, aleurone cells were observed with aconfocal microscope. C, Magnified images of representative cells fromB. D, Percentage of vacuolated cells was calculated as described forFigure 1C. The results represent averages 6 SE (n 5 6).





Taken together, the results from overexpression ofGAMyb and GAMyb plus HVA22 show that GAMybis required for the GA-mediated PCD, which is in-hibited by HVA22 acting downstream from GAMyb.

HVA22 Is Not the Only Regulatory Protein for ABA

Inhibition of GA-Induced PCD/Vacuolation

Since ABA inhibits GA-induced PCD and theexpression of HVA22 is highly induced by ABA, wehypothesize that ABA induces HVA22 accumulationto inhibit PCD during seed development. To investi-gate whether HVA22 is absolutely necessary for ABAinhibition of PCD, the RNAi effector constructHVA22RNAi was generated to inactivate endogenousHVA22 mRNA (Fig. 5A) by inserting inverted repeatsof a conserved TB2/DP1 domain of HVA22 (306 bp)into the GFP ORF (750 bp). When cells were cotrans-formed with HVA22 and HVA22RNAi effectorconstructs, the inhibitory effect of HVA22 on GA-induced vacuolation was completely abolished (Fig.5B). This result confirmed that HVA22RNAi waseffective at blocking HVA22 inhibition on PCD. How-ever, when compared with the vector-only control,HVA22RNAi did not result in a significantly highervacuolation level in aleurone cells treated with GAplus ABA (Fig. 5C), indicating that HVA22RNAi couldnot block the effect of ABA on GA-induced PCD.

Figure 4. GAMybRNAi is effective at blocking GA-induced a-amylaseexpression in aleurone cells. A, Schemes of gene constructs. Grayarrows represent the orientation of genes or gene fragments (not drawnto scale). Except that GUS expression is driven by the a-amylase32promoter, expression of other genes is under the control of the maizeubiquitin promoter, Ubi1. B and C, GUS activity produced by trans-formed aleurone tissues under control conditions or GA treatment. Thereporter constructs, AmyTGUS and UbiTLUC, were cobombardedwith empty vector, constructs expressing GAMyb or GAMybRNAi, orvarious combinations as indicated. Bars indicate relative GUS activity 6

SE (see ‘‘Materials and Methods’’) after 24 h of incubation in buffercontaining no hormone or 1 mM GA.

Figure 3. Barley HVA22 inhibits PCD/vacuolation downstream ofGAMyb in aleurone cells. A, Schemes of gene constructs. Gray arrowsrepresent the orientation of genes or gene fragments (not drawn toscale). The expression of these constructs is driven by the maizeubiquitin promoter. B, Percentage of vacuolated cells in transformedaleurone tissues under hormone-free conditions. The reporter DesRedwas cobombarded with empty vector, or GAMyb, or GAMyb andGAMybRNAi, or GAMyb and HVA22 as indicated. After 48 h ofincubation in control buffer, percentage of vacuolated cells wascalculated as described for Figure 1C. C, Percentage of vacuolatedcells in transformed aleurone tissues with GA treatment. The reporterDesRed was cobombarded with constructs expressing HVA22 orGAMybRNAi, vector only, or various combinations as indicated. Thealeurone tissues were preincubated in the control buffer for 48 h, thentransferred to buffer containing no hormone or 1 mM GA. After another48 h of incubation, percentage of vacuolated cells was calculated asdescribed for Figure 1C. The results represent averages 6 SE (n 5 6).

Guo and Ho




These results suggest that HVA22 is not absolutelyrequired for ABA-mediated inhibition on PSV vacuo-lation and PCD.

Localizations of HVA22 in ER and the Golgi Apparatus

What is the mechanism of HVA22 in regulatingPCD? To address this question, the subcellular local-izations of HVA22 in aleurone cells were examined.The coding region of barley HVA22 cDNA wasfused in frame with GFP ORF at the N terminus(HVA22:GFP), and the resulting construct was cotrans-formed into aleurone cells with DesRed (Fig. 6A). Toconfirm that the HVA22:GFP fusion protein is func-tional, the effect of expressing HVA22:GFP on GA-induced PCD was analyzed. When compared withcells transformed with GFP, overexpression of HVA22:GFP significantly reduced the percentage of vacuolatedcells by 20% and 40% under control and GA-treatedconditions (Fig. 6B), respectively. This result demon-strates that the fusion protein HVA22:GFP is able toperform similar functions as native HVA22 proteinsin vivo.

In the aleurone cells cotransformed with HVA22:GFP and DesRed, the HVA22:GFP fusion proteinsproduced punctate patterns and some network-likestrings in both nonvacuolated (Fig. 6C, a and c) andvacuolated (Fig. 6C, g and i) cells, whereas DesRedalone produced the expected diffuse pattern (Fig. 6C, band h). When GFP and DesRed were cotransformed incells, both proteins generated exactly the same diffusepattern in both nonvacuolated (Fig. 6C, d–f) andvacuolated (Fig. 6C, j–l) cells.

The punctate and network patterns were reminis-cent of the localization of the ER and Golgi apparatus(Kim et al., 2001), as observed previously with HVA22homologs in yeast and animal cells (Calero et al., 2001;Voeltz et al., 2006). In plant cells, the BiP:RFP fusionprotein between red fluorescent protein (RFP) andportions of the chaperone binding protein (BiP) hasbeen widely used as an ER marker (Kim et al., 2001;Song et al., 2006), and ST:mRFP, a chimeric proteinbetween rat sialyltransferase and monomeric red fluo-rescent protein (mRFP), has been shown to be local-ized in the Golgi stacks (Kim et al., 2001). In order tofurther define the subcellular localizations of HVA22protein, HVA22:GFP was cotransformed with BiP:RFPor ST:mRFP for comparison.

When transformed with both HVA22:GFP andBiP:RFP, the aleurone cells displayed the BiP:RFPfluorescence in filamentoid and patchy ER patterns(Fig. 7B), which is consistent with previous observa-tions in Arabidopsis cells (Kim et al., 2001). Someportions of the HVA22:GFP-positive dim filamentsand bright punctate stains were colocalized with fluo-rescence from BiP:RFP (Fig. 7, A–C, arrowheads). Theintensity profile of the green and red fluorescencesignals along the transect in Figure 7D further showedthat the arrowhead-highlighted peaks of HVA22:GFPsignals corresponded to the BiP:RFP signals located atthe ER.

In aleurone cells transformed with HVA22:GFP andST:mRFP, the red fluorescence of ST:mRFP was ob-served in the punctuate Golgi complexes and the spacebetween the plasma membrane and cell walls (Fig. 7F).Most of the dot-like green fluorescent signals ofHVA22:GFP overlapped with the red fluorescent sig-nals of ST:mRFP to yield yellow signals (Fig. 7, E–G,arrowheads). The intensity profile of GFP and RFPsignals along the transect in Figure 7H further dem-onstrated that those peaks of HVA22:GFP signalscorresponded to the positions of ST:mRFP signals.These results suggest that barley HVA22 proteins arelikely localized in the ER and Golgi apparatus.

Roles of Transmembrane Domains in HVA22 Function

According to Pfam prediction (http://pfam.sanger.ac.uk/family?id5TB2_DP1_HVA22&tab5speciesBlock),the barley HVA22 protein contains three potentialtransmembrane domains located in the TB2/DP1 re-gion (Fig. 8A). To examine the function of these trans-membrane domains in vivo, we generated effector

Figure 5. HVA22RNAi does not block ABA-inhibited PCD/vacuolationin aleurone cells. A, Schemes of gene constructs. Gray arrows representthe orientation of genes or gene fragments (not drawn to scale). B and C,Percentage of vacuolated cells in transformed aleurone tissues. Thereporter DesRed was cobombarded with empty vector, constructsexpressing HVA22 or HVA22RNAi, or various combinations as indi-cated. Samples were preincubated in control buffer for 48 h, thentransferred to buffer containing no hormone (Control), 1 mM GA, or1 mM GA and 20 mM ABA. After another 48 h, aleurone cells wereobserved by confocal microscopy. Percentage of vacuolated cells wascalculated as described for Figure 1C. The results represent averages 6

SE (n 5 6).





constructs expressing truncated forms of HVA22 pro-teins, DTM1, DTM2, and DTM3 (Fig. 8A). The individ-ual transmembrane domains (TM1, TM2, and TM3)were deleted such that the remaining amino acidsformed in-frame protein with the other two domains.When these truncated HVA22 proteins (DTM1, DTM2,and DTM3) were overexpressed with DesRed, none ofthese truncated proteins could inhibit GA-inducedPCD and PSV vacuolation; in the same experiment,however, intact HVA22 protein reduced the vacuola-tion by around 60% compared with the vector-alonecontrol (Fig. 8B).

It is possible that the truncated forms of HVA22proteins (DTM1, DTM2, and DTM3) are not stable invivo or are misexpressed in other subcellular com-partments. To clarify these possibilities, the localiza-tions of these truncated proteins were furtherinvestigated. The truncated forms of HVA22 werefused in frame with GFP at the N terminus(DTM1:GFP, DTM2:GFP, and DTM3:GFP) in the samemanner as HVA22:GFP used in Figure 6A. As ex-pected, the expression of HVA22:GFP resulted in dis-tinct punctate and filamentoid patterns (Fig. 8C, a andc), in contrast to the diffused pattern of DesRed (Fig.8Cb). The localizations of DTM1:GFP (Fig. 8Ce) andDTM3:GFP (Fig. 8Cm) were similar to those of the full-

length protein, HVA22:GFP; however, 95% of cells(from a total of 40 cells observed) transformed withDTM2:GFP displayed weak green fluorescence anddiffuse patterns as DesRed (Fig. 8C, i–k). The distri-butions of the signal intensities of green and red fluo-rescence of these representative cells further showedlow intensity and diffuse patterns of DTM2:GFP sig-nals (Fig. 8Cl) when compared with HVA22:GFP (Fig.8Cd), DTM1:GFP (Fig. 8Ch), and DTM3:GFP (Fig.8Cp). These observations highlight the importance ofTM2 for its protein stability and correct subcellularlocalization in vivo. However, we cannot exclude thepossibility that other sequences are also required forits function. Therefore, further studies will be requiredto examine the effects of other hydrophilic domainson PCD.

Arabidopsis HVA22 Also Inhibits PCD/Vacuolation

Electron microscopy showed that the ultrastructureof Arabidopsis aleurone cells is similar to that of cerealaleurone cells (Bethke et al., 2007). The same GA-induced PCD and PSV vacuolation were also observedin the Arabidopsis aleurone cells. It is possible thatArabidopsis HVA22 homologs perform similar func-tions to barley HVA22. In Arabidopsis, 16 HVA22

Figure 6. Localization of HVA22:GFP in barley aleurone cells. A, Schemes of gene constructs. Gray arrows indicate ORF ofHVA22 (not drawn to scale), fused in frame with GFP protein at the N terminus. B, Percentage of vacuolated cells in transformedaleurone cells. The reporter DesRed was cobombarded with a construct expressing HVA22:GFP or GFP alone. Samples werepreincubated in control buffer for 24 h, then transferred to buffer containing no hormone (Control) or 1 mM GA. After another48 h, aleurone cells were observed by confocal microscopy. Percentage of vacuolated cells was calculated as described forFigure 1C. The results represent averages 6 SE (n 5 6). C, Confocal micrographs of aleurone cells observed in B. Representativecells 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.

Guo and Ho




homologs were identified through blasting the TB2/DP1 domain of barley HVA22 (Supplemental Fig. S1).Among these HVA22 homologs, only AtHVA22A,AtHVA22C, AtHVA22D, and AtHVA22E are predictedto contain similar transmembrane domains as barleyHVA22 (Supplemental Fig. S2). To investigate if theseArabidopsis homologs function similarly to barleyHVA22, the reporter DesRed was cobombarded witheffector constructs expressing AtHVA22A, AtHVA22B,AtHVA22C, AtHVA22D, and AtHVA22E driven by themaize Ubi1 promoter (Fig. 9A). In the presence of GA,overexpressing AtHVA22D proteins was most effec-tive at reducing GA-induced PSV vacuolation, with its

effect similar to that caused by barley HVA22 (Figs. 2Dand 9B). AtHVA22B, AtHVA22C, and AtHVA22E pro-teins only provided some degree of inhibition on PCD,and AtHVA22A had no effect on vacuolation in aleu-rone cells.

DISCUSSION

The cereal aleurone layer is a secretory tissue inwhich hydrolytic enzymes are de novo synthesizedand secreted for breaking down the reserves stored instarchy endosperm for the early stages of seedling

Figure 7. Colocalization of HVA22:GFP with Golgi and ER markersin barley aleurone cells. Confocalmicrographs of aleurone cells co-bombarded with HVA22:GFP andBiP:RFP (A–D) or ST-mRFP (E–H) areshown. Fluorescence from green (Aand E), red (B and F), and both (C andG) channels was observed after 48 hof transformation under control con-ditions. D and H show pixel intensi-ties of green (green trace) and red (redtrace) fluorescence along the tran-sects in the left panels. The bluearrowheads indicate the coalignmentof intensity peaks. The green stars andorange circles represent the start andend positions of the transacts.





growth (Filner and Varner, 1967; Eastmond and Jones,2005). The hydrolases are synthesized from aminoacids previously incorporated in storage proteins,which originate from the ER and Golgi pathway andaccumulate primarily in specific differentiated vacu-oles, PSVs (Herman and Larkins, 1999; Vitale andHinz, 2005). When the aleurone layer has completedits digestive function, PCD of aleurone cells occursconcurrently with extensive fusion of PSVs to generatelarge central vacuoles (vacuolation; Jones and Price,1970; Bethke et al., 1999). GA is an enhancer, whileABA is a negative regulator, of this PCD process inaleurone cells (Bethke et al., 1999, 2007).

In this study, we provide evidence to show thatexpression of barley HVA22 specifically inhibits GA-induced PCD/vacuolation of aleurone cells (Figs.

1 and 2). The action of HVA22 is downstream of thetranscription factor GAMyb (Fig. 3). Based on theseresults and previous signal transduction studies, wepropose a model of HVA22 function as shown inFigure 10. HVA22 is the downstream ABA-inducedmembrane protein negatively regulating GA-inducedPCD in barley aleurone cells. The synthesis of HVA22is downstream from transcription factors ABI5/VP1,and the action of HVA22 is downstream from the GA-mediated transcription factor GAMyb (Casaretto andHo, 2003).

GAMyb has been shown to be the principal tran-scription activator for hydrolase expression in aleu-rone cells (Gubler et al.,1995; Zentella et al., 2002;Kaneko et al., 2004). It is surprising that GAMyb issufficient but may not be absolutely necessary for GA-

Figure 8. Transmembrane domainsare necessary for HVA22 function.A, Schemes of gene constructs.White boxes indicate predictedtransmembrane domains of HVA22protein. Black boxes and arrows in-dicate the same N-terminal andC-terminal regions used in theseconstructs. B, Percentage of vacuo-lated cells in transformed aleuronetissues. The reporter DesRed wascobombarded with empty vector, aconstruct expressing HVA22, or con-structs expressing various HVA22truncated forms (DTM1, DTM2, andDTM3) into aleurone cells. Sampleswere preincubated in control bufferfor 24 h, then transferred to buffercontaining no hormone (Control) or1 mM GA. After another 48 h, aleu-rone cells were observed by confocalmicroscopy. Percentage of vacuo-lated cells was calculated as de-scribed for Figure 1C. The resultsrepresent averages 6 SE (n 5 6). C,Confocal micrographs of trans-formed cells expressing HVA22:GFP(a–c), DTM1:GFP (e–g), DTM2:GFP(i–k), and DTM3:GFP (m–o). Theintensity profiles of green and redfluorescence from representativecells expressing HVA22:GFP (d),DTM1:GFP (h), DTM2:GFP (l), andDTM3:GFP (p) are also shown.

Guo and Ho




induced PCD, since depletion of GAMyb expressionvia GAMybRNAi only decreased GA-induced vacuo-lation by 40% (Fig. 3). A similar result was alsoobserved for its upstream repressor, SLN1, which isan important intermediate of GA signaling (Fig. 10;Gubler et al., 2002; Zentella et al., 2002). However, wecannot exclude the possibility that there is anotherunknown factor to activate the GA signaling pathway.Also, while GAMybRNAi is able to effectively blockGA activation on a-amylase expression (Fig. 4C), arelatively small amount of a-amylase activity (7-foldcompared with vector only) could still be observedwhen GAMyb and GAMybRNAi were cotransformed(Fig. 4B). These data suggest that some residualamount of GAMyb remained in vivo under theseconditions, which could have triggered the down-stream PCD.

Loss of function of HVA22 via transformation withthe HVA22RNAi construct did not alleviate ABAinhibition of the GA effect (Fig. 5), suggesting thatHVA22 is sufficient but not necessary for ABA inhibi-tion of PCD. This could be explained by functionalcompensation by other HVA22-like genes or othersynergistic components. This notion is supported bythe fact that HVA22 genes constituted families of 51

and 16 genes in rice (Oryza sativa) and Arabidopsis,respectively (Supplemental Fig. S1). It is also worthnoting that HVA22/Yop1p/DP1-deficient yeast ornematode worms grow normally (Brands and Ho,2002; Voeltz et al., 2006; Audhya et al., 2007). Thedefective phenotypes were only observed in combina-tion with mutation/deletion of another coordinatedprotein, Rtn4 or Sey1p, which does not share sequencesimilarity.

Alternatively, ABA could directly inhibit the expres-sion of GAMyb that is important for downstream GA-mediated processes (Fig. 10). It has been shown thatABA could inhibit GAMyb transcription to preventa-amylase expression (Zentella et al., 2002). Why docells need two ABA regulatory pathways in control-ling PCD? When barley aleurone protoplasts werepreincubated with GA for 24 h, adding ABA was nolonger able to block a-amylase expression (Bethkeet al., 1999; Fath et al., 2001), demonstrating that ABAinhibition of the GA signaling pathway is dependenton timing and the fine balance between GA and ABAaction (Finch-Savage and Leubner-Metzger, 2006).This could be particularly important for seedlingsunder stress, in which the GA-mediated processesare already under way yet need to be suppressed inresponse to stressful conditions. It is conceivable thatthe stress-induced ABA in turn suppresses PCDdownstream from GAMyb, which is already inducedby GA. Therefore, the stressed seedlings could stillslow the nutrient mobilization processes that are notneeded during stress. During late stages of seed de-velopment, the accumulation of HVA22 transcripts inbarley embryo is correlated with endogenous ABAbiosynthesis (Shen et al., 2001). Highly accumulatedHVA22 mRNA is maintained in dry seeds until imbi-bition, whereas ABA is degraded to lower levels infully mature seeds (Karssen et al., 1983). Thus, accu-mulation of HVA22 proteins could provide anothercontrol mechanism in aleurone cells to prevent pre-mature GA-induced PCD to maintain dormancy whenABA is low in mature seeds. This hypothesis is furthersupported by the fact that the amount of HVA22transcripts is correlated with the status of seed dor-mancy (Shen et al., 2001), which has been suggested to

Figure 9. Effects of overexpressing Arabidopsis AtHVA22 homologs onGA-induced PCD/vacuolation in barley aleurone cells. A, Schemes ofgene 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. Thereporter DesRed was cobombarded with empty vector or constructsexpressing Arabidopsis AtHVA11A, AtHVA11B, AtHVA11C, AtHVA11D,or AtHVA11E. Samples were preincubated in control buffer for 24 h, thentransferred to buffer containing no hormone (Control) or 1 mM GA. Afteranother 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 6 SE (n 5 6).

Figure 10. Scheme of factors and pathways involved in GA/ABA-regulated PCD/vacuolation in aleurone cells. A repressor, SLN1, and anactivator, GAMyb, are important regulators in the GA-mediated PCDand the associated PSV vacuolation. ABA negatively regulates PCDboth upstream and downstream of GAMyb. The ABA-induced HVA22is involved in the inhibition downstream of GAMyb.





be determined by the existence of aleurone tissues(Bethke et al., 2007).

Exclusive localizations of HVA22:GFP in the ER andGolgi stacks (Figs. 6 and 7) and the requirement oftransmembrane domains for its function (Fig. 8) implythat barley HVA22 exerts its function in ER and Golgimembranes. These results are consistent with previousobservations with yeast and animal HVA22 homologs,Yop1p and DP1. The Yop1p/DP1 proteins are pre-dominantly localized in the ER and have been shownto interact with reticulons to shape peripheral ERtubules (Voeltz et al., 2006; Audhya et al., 2007; Huet al., 2008). A subpopulation of Yop1p in S. cerevisiae isalso localized in the Golgi and interacts with Yip1p(Calero et al., 2001), a component of the Yip1p/Yif1p/Yos1p complex that appears to be involved in vesicleformation (Heidtman et al., 2005). It has been pro-posed that Yop1p, in conjunction with Yip1p, facili-tates Rab GTPase-mediated protein transport betweenthe ER and the Golgi network (Calero et al., 2001).With its localization similar to that of yeast Yop1p, it islikely that HVA22 participates in the ER-to-Golgisecretory pathway in planta.

The vacuolation of PSVs is similar to homotypicvacuole fusion in yeast. Studies in yeast have shownthat homotypic vacuole fusion requires cytosolic eno-lase, ATP, and several endomembrane secretory-related proteins (Wickner and Haas, 2000; Deckerand Wickner, 2006). In particular, a GTPase, Ypt7p, isrequired to concentrate SNAREs and other proteins toactivate the vacuole fusion machinery (Haas et al.,1995; Starai et al., 2007). Disrupting protein transportduring the secretory pathway will result in nonfusedfragmented vacuoles (Haas et al., 1995; Decker andWickner, 2006). Interestingly, the yeast HVA22 homo-log Yop1p also physically interacts with Ypt7p in vivo(Calero et al., 2001), and overexpressing Yop1p in yeastcells results in negative dominant defects in vacuoleprotein transport (Haas et al., 1995). Therefore, plantHVA22 protein may regulate homotypic vacuole fu-sion of PSVs by mediating vesicle trafficking betweenthe ER, Golgi, and PSVs. This notion is further sup-ported by the observation that in GA-treated aleuronecells, the ER and Golgi stacks lying adjacent to thePSVs proliferate vesicles that appear to fuse with thePSVs via autophagic internalization (Jones, 1969a,1969b, 1987; Jones and Price, 1970; Herman andLarkins, 1999).

Combining these studies, we propose that ABAinduces the biosynthesis of HVA22 to negatively reg-ulate vesicle transport between the ER and Golgi or theGolgi to PSVs, to prevent premature degradation ofPSVs and nutrient mobilization. When GA triggersgermination, HVA22 proteins are rapidly degraded tofacilitate the necessary protein trafficking for PSVmetabolism and fusion.

Phylogenetic analyses show that AtHVA22D andAtHVA22E are closer to barley HVA22 than toAtHVA22A, AtHVA22B, and AtHVA22C (Chen et al.,2002). Consistent with this finding, AtHVA22D is

more effective than other Arabidopsis HVA22 proteinsat inhibiting the GA-induced PCD (Fig. 9). Althoughthe level of expression and stability of ArabidopsisHVA22 proteins could influence these results, thesignificantly higher ability of AtHVA22D than otherAtHVA22s at inhibiting PCD suggests that AtHVA22Dmay also function in ER/Golgi secretory transport, asproposed for barley HVA22. Interestingly, among allAtHVA22s, AtHVA22D is also the most induced byABA and abiotic stresses in vegetative tissues (Chenet al., 2002). How the role of HVA22 in regulatingvesicle trafficking contributes to plant responses toabiotic stresses warrants further investigation.

MATERIALS AND METHODS

Plant Materials

Barley (Hordeum vulgare ‘Himalaya’) seeds from 1998 and 2002 harvested at

Washington State University in Pullman, Washington, were used in all

experiments. Embryoless half-seeds were prepared as described previously

(Shen et al., 1993).

Plasmid Construction

The reporter and effector constructs were generated as follows. (1) GFP,

DesRed, GAMyb, Amy-GUS, and LUC plasmids were constructed as de-

scribed previously (Cercos et al., 1999; Zentella et al., 2002). The same

backbone plasmid (CYH8) was used, in which the maize (Zea mays) ubiquitin

promoter and its first intron (Ubi1) were cloned into the PstI site of pBluescript

II SK1 with the 3# Nos terminator. (2) For HVA22 plasmid, the complete

barley HVA22 ORF was amplified from the barley embryo cDNA using a pair

of primers (5#-AGAAACCCGGGTCATGGGCAAATCATG-3# and 5#-ATA-

TACTAGTTCAATGC ACACGACCATG-3#). The resulting 393-bp fragment

was subcloned into SmaI and SpeI sites of CYH8. (3) For HVA1 plasmid,

complete barley HVA1 ORF was released from a cDNA clone using NotI

digestion, then was subcloned into the NotI site of CYH8. (4) For the

HVA22RNAi construct, Gateway technology was used. A PCR product of

300 bp of HVA22 ORF (19–324 bp), including the conserved TB2/DP1 domain,

was amplified using primers 5#-CACCCTCCTCACCCACCTCCACT-3# and

5#-GTTCCTGCCGCGGTACTTCCT-3#. The amplified fragments were cloned

into the pENTR/D-TOPO vector (Invitrogen). The resulting HVA22-pENTR

construct was recombined with the Gateway destination vector pANDA-mini

(Miki and Shimamoto, 2004) by LR (recombination between attL and attR

sites) reaction using LR Clonase II (Invitrogen) following the manufacturer’s

instructions. (5) For GAMybRNAi, a PCR product of 351 bp of the partial

GAMyb ORF from barley was amplified using primers 5#-CACCTCCAC-

GAAGCCAACTCTAGC-3# and 5#-TCACGGAGAGCTGCTGATTCTTC-3#.

The region amplified shared the least similarity with other Myb genes and

was located in the transactivation domain (842–1,192 bp). The fragment was

subcloned into pANDA-mini vector using the same Gateway technology as

for the HVA22RNAi construct.

(6) For the DTM1 plasmid, a PCR product of 300 bp containing predicted

transmembrane domains TM2 and TM3 of HVA22 was amplified us-

ing primers 5#-AACCCGGGATGGGCAGCCCGTCCAAGGTGGAC-3# and

5#-ATATACTAGTTCAATGCACACGACCATG-3#. Then, the DTM1 fragment

was cloned into XmaI and SpeI sites of CYH8 plasmid. (7) For the DTM3

plasmid, a PCR product of 336 bp including predicted TM1 and TM2 of

HVA22 was amplified using primers 5#-AGAAACCCGGGTCATGGGC-

AAATCATG-3# and 5#-ATACTAGTTCAATGTGGGTACCACACCGGTAT-3#,

then was cloned into CYH8. (8) For the DTM2 construct, TM3 of HVA22 was

first amplified using primers 5#-ATACTGGATCCCGGTGTGGTACCCAG-

TGA-3# and 5#-ATACTAGTTCAATGTGGGTACCACACCGGTAT-3#. The

190-bp PCR product was then cloned into BamHI and SpeI sites of the DTM3

plasmid to replace TM2, so that TM1 and TM3 were fused in frame. (9) For

HVA22:GFP, DTM1:GFP, DTM2:GFP, and DTM3:GFP constructs, the complete

HVA22 ORF, or corresponding gene fragments for DTM1, DTM2, and DTM3

were amplified using HVA22, DTM1, DTM2, and DTM3 plasmids as tem-

Guo and Ho




plates, respectively. The primer pairs used were as follows: HVA22:GFP,

5#-AGAAACCCGGGTCATGGGCAAATCATG-3# and 5#-TATCCCGGGTCC-

ATGCACACGACCATGG-3#; DTM1:GFP, 5#-AACCCGGGATGGGCAGCC-

CGTCCAAGGTGGAC-3# and 5-TATCCCGGGTCCATGCACACGACCATGG-3#;DTM2:GFP, 5#-AGAAACCCGGGTCATGGGCAAATCATG-3# and 5#-TAT-

CCCGGGTCCATGCACACGACCATGG-3#; DTM3:GFP, 5#-AGAAACCCGGG-

TCATGGGCAAATCATG-3# and 5#-TAGACCCGGGTCCATGTGGGTACCAC-

ACC-3#. These PCR fragments were then cloned into the SmaI site of GFP

plasmid in order to fuse in frame with the N terminus of GFP ORF.

(10) For the AtHVA22A, AtHVA22B, AtHVA22C, AtHVA22D, and AtHVA22E

constructs, the entire ORFs were amplified by PCR from Arabidopsis (Arabidopsis

thaliana) seedling cDNA using gene-specific primers for AtHVA22A

(At1g74520, 5#-TCAACCCGGGGATGGGATCTGGAGCTGGCA-3# and 5#-ATA-

TACTAGTTTAGTACTGATAACCCTCACCAT-3#), AtHVA22B (At5g62490,

5#-TCAGCCCGGGAATGAGTTCCGGAATCGGA-3# and 5#-TAAGGCGG-

CCGCCTAGTAGATATAGGCGTCATCA-3#), AtHVA22C (At1g69700, 5#-TGG-

ACCCGGGAATGCCTTCAAATTCAGGAGA-3# and 5#-ATATACTAGTTTA-

GTACCTATAGTCATCATCA-3#), AtHVA22D (At4g24960, 5#-CTGACCCGG-

GAATGGACAAATTTTGGACT-3# and 5#-ATATACTAGTTCAGTGACTGT-

GAGCCTCGTGTC-3#), and AtHVA22E (At5g50720, 5#-CTGACCCGGGAAT-

GACAAAACTATGGACTTC-3# and 5#-ATATACTAGTTCACTCAGCCTGAT-

GCTCTACCT-3#). Except for Ubi-AtHVA22B, these PCR fragments were

cloned into XmaI and SpeI sites of CYH8, whereas the AtHVA22B PCR product

was cloned into XmaI and NotI sites of CYH8. All of the plasmids were further

confirmed by sequencing.

Transient Expression via Particle Bombardment

Barley embryoless half-seeds were prepared and transformed transiently by

particle bombardment as described previously (Shen et al., 1993). In general,

2.5 mg of reporter construct was cobombarded with an effector construct as

indicated in the figures in a molar ratio of 1:1 except for GAMyb. The GAMyb

effector construct was cotransformed with reporter or other effector constructs

in a molar ratio of 1:10. After bombardment, the half-seeds were preincubated

in control shooting buffer for 0, 24, or 48 h depending on the experiment.

Then samples were transferred to buffer in the presence or absence of 1 mM GA

or 20 mM ABA for another 48 h before observation with a confocal microscope.

More than 20 cells from each aleurone tissue and six to eight barley half-seeds

were observed by confocal microscopy for each construct combination.

Cells containing only one to three large vacuoles were classified as vacuolated

cells, and the percentage of vacuolated cells was calculated in each aleurone

tissue (vacuolated cells/total observed cells 3 100%). The values shown in

the figures represent average percentages 6 SE of vacuolated cells of six to

eight barley aleurone tissues from independent half-seeds.

Confocal Microscopy

Aleurone layers were isolated by removing the starchy endosperm of the

embryoless half-seeds and observed with a Zeiss CLSM510 confocal micro-

scope. DesRed images were captured in the 560- to 615-nm range after

excitation at 543 nm with a HeNe laser beam. The GFP images were captured

in the 505- to 530-nm range after excitation at 488 nm with an argon laser beam.

The analysis of colocalization was performed using LSM 5 Image Examiner

software.

LUC and GUS Enzyme Assays

The half-seeds were homogenized in 1 mL of grinding buffer after 24 h of

transformation (Shen et al., 1993). After centrifugation at 12,000 rpm for 10

min, 100 mL of supernatant was used for LUC assays using a luminometer

(Lumat LB9507; Berthold Technologies). For GUS assays, 50 mL of supernatant

was mixed with 150 mL of GUS assay buffer (Shen et al., 1993) and incubated

for 20 h at 37�C in the dark; then, 50 mL of reaction mixture was diluted in 2 mL

of 200 mM sodium carbonate stop buffer. GUS activity was measured with a

fluorometer (TBS-380; Turner BioSystems) calibrated to 1,000 units with a

standard made by diluting 50 mL of 1 mM 4-methylumbelliferone in 2 mL of

200 mM sodium carbonate. Relative GUS activity was calculated by dividing

GUS activity by LUC activity of the respective samples and multiplying by a

constant of 100,000. All experiments consisted of three replicates, and the

entire experiment was repeated at least two times with similar results.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Taxonomic coverage of HVA22 homologs

containing the TB2/DP1 domain.

Supplemental Figure S2. Predicted structures of Arabidopsis HVA22

proteins (AtHVA22) available in the Pfam database.

ACKNOWLEDGMENTS

We thank Dr. Inhwan Hwang (Division of Molecular and Life Sciences,

Pohang University of Science and Technology, Pohang, South Korea) for

providing us with BiP-RFP and ST-mRFP constructs and for his valuable

suggestions and Dr. Chwan-Yang Hung (Department of Agricultural Chem-

istry, National Taiwan University, Taipei, Taiwan) for providing CYH8, GFP,

and DesRed plasmids. We also thank Ms. Mei-Jan Fang (Institute of Plant and

Microbial Biology, Academia Sinica) for her expert help with confocal

microscopic analyses. Thanks also go to Mr. Kuan-Te Lee for his contributions

in the early part of this work.

Received March 31, 2008; accepted June 19, 2008; published June 26, 2008.

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Guo and Ho




CORRECTIONS

Vol. 147: 1710–1722, 2008

Guo W.-J. and Ho T.-H.D. An Abscisic Acid-Induced Protein, HVA22, InhibitsGibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells.

This article was published with the name of corresponding author Tuan-Hua David Homisspelled. The name has been corrected in the online version of the article.

www.plantphysiol.org/cgi/doi/10.1104/pp.104.900271

1182 Plant Physiology, October 2008, Vol. 148, p. 1182, www.plantphysiol.org � 2008 American Society of Plant Biologists

an abscisic acid-induced protein, hva22, inhibits ......1991; xu et al., 1996; sivamani et al.,...

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