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First published online January 16, 2009; 10.1104/pp.108.131490 Plant Physiology 149:1679-1689 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Autophagy Is Enhanced and Floral Development Is Impaired in AtHVA22d RNA Interference Arabidopsis[C],[W],[OA]Department of Biology, Washington University, St. Louis, Missouri 63130 (C.-N.N.C., G.V., T.-H.D.H.); Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan (C.-N.N.C., T.-H.D.H.); Institute of Biomedical Science, National Chung Cheng University, Chia-Yi 621, Taiwan (H.-R.C.); and Department of Biological Sciences, Missouri University of Science and Technology, Rolla, Missouri 65409 (C.-N.N.C., S.-Y.Y.)
Autophagy is an intracellular process in which a portion of cytoplasm is transported into vacuoles for recycling. Physiological roles of autophagy in plants include recycling nutrients during senescence, sustaining life during starvation, and the formation of central digestive vacuoles. The regulation of autophagy and the formation of autophagosomes, spherical double membrane structures containing cytoplasm moving toward vacuoles, are poorly understood. HVA22 is a gene originally cloned from barley (Hordeum vulgare), which is highly induced by abscisic acid and environmental stress. Homologs of HVA22 include Yop1 in yeast, TB2/DP1 in human, and AtHVA22a to -e in Arabidopsis (Arabidopsis thaliana). Reverse genetics followed by a cell biology approach were employed to study the function of HVA22 homologs. The AtHVA22d RNA interference (RNAi) Arabidopsis plants produced small siliques with reduced seed yield. This phenotype cosegregated with the RNAi transgene. Causes of the reduced seed yield include short filaments, defective carpels, and dysfunctional pollen grains. Enhanced autophagy was observed in the filament cells. The number of autophagosomes in root tips of RNAi plants was also increased dramatically. The yop1 deletion mutant of Saccharomyces cerevisiae was used to verify our hypothesis that HVA22 homologs are suppressors of autophagy. Autophagy activity of this mutant during nitrogen starvation increased in 5 min and reached a plateau after 2 h, with about 80% of cells showing autophagy, while the wild-type cells exhibited low levels of autophagy following 8 h of nitrogen starvation. We conclude that HVA22 homologs function as suppressors of autophagy in both plants and yeast. Potential mechanisms of this suppression and the roles of abscisic acid-induced HVA22 expression in vegetative and reproductive tissues are discussed.
Abscisic acid (ABA) regulates the expression of hundreds of genes in a plant (Seki et al., 2002  ). The specific functions of many of these genes are still not clear, but they are assumed to be related to the physiological roles of ABA, such as promoting seed maturation, inhibiting seed germination, and conferring tolerance to environmental stress. HVA22, a gene isolated from barley (Hordeum vulgare) aleurone cells treated with ABA, encodes a small peptide of 130 amino acids without an obvious functional motif (Shen et al., 1993). Expression of HVA22 is dramatically up-regulated by ABA not only in aleurone cells but also in vegetative tissues (Shen et al., 2001). Homologs of HVA22 have been identified in diverse eukaryotes, including plants, fungi (named Yop1 in Saccharomyces cerevisiae), mammals (named TB2 or DP1 in human), flies, and worms, but not in any prokaryotes. Expression of yeast Yop1 is also up-regulated by stress (Shen et al., 2001). Studies of the physiological role of Yop1 protein revealed that Yop1p is a Yip1p-interacting protein that is an essential protein regulating a critical step in the Rab GTPase-mediated membrane transport in S. cerevisiae (Calero et al., 2001). In another study, synthetic enhancement mutant screening was performed and SEY1 was identified as a Yop1-dependent complementation gene (Brands and Ho, 2002). The yeast gene SEY1 is a homolog of the Arabidopsis (Arabidopsis thaliana) gene RHD3, whose function is to facilitate vesicle transport in root hairs. Mutation of RHD3 causes vesicle accumulation at the tips of root hairs and the formation of short and wavy root hairs (Schiefelbein and Somerville, 1990; Galway et al., 1997). Due to the apparent vesicle accumulation in the yeast yop1/sey1 double mutant, it was suggested that Yop1 protein is involved in vesicular trafficking.
To obtain more insight into the role of HVA22, a functional analysis of HVA22 homologs was carried out in Arabidopsis. Five HVA22 homologs were identified in Arabidopsis, named AtHVA22a to -e. Expression of these five genes is differentially up-regulated by ABA and environmental stress, except for AtHVA22c, which is nearly unaffected by these treatments. Expression levels of AtHVA22 genes vary in different organs; generally, fast-growing organs such as flowers and inflorescence stems have higher expression levels than slow-growing organs such as mature rosette and cauline leaves. Among the five, expression of AtHVA22d is most tightly regulated by ABA in vegetative tissues (Chen et al., 2002
Autophagy is a cytoplasmic recycling mechanism by which a portion of cytoplasm is transported into vacuoles (lysosomes in animal cells) and degraded therein. The regained nutrients are either mobilized to different parts of the organism or used in the same cell. Aspects of autophagy, including physiological and molecular, have been described in eukaryotes (Yorimitsu and Klionsky, 2005
Autophagosome formation is the central part of macroautophagy. Similar to microautophagy, which engulfs cytoplasm into vacuoles nonselectively, macroautophagy nonselectively enwraps cytoplasm using a double membrane, thereby creating autophagosomes. When autophagosomes fuse with vacuoles, the outer membrane integrates into the vacuole membrane and the cytoplasm surrounded by the inner membrane is released into the vacuole lumen. Although many proteins have been identified that are associated with the preautophagosomal structures (Suzuki et al., 2001 Here, we report the results from studies on AtHVA22 genes in Arabidopsis and Yop1 in S. cerevisiae. Our results reveal that these HVA22 homologs negatively regulate autophagy in both Arabidopsis and yeast.
Selection and Phenotyping of AtHVA22d RNAi Transgenic Plants AtHVA22d cDNA, including parts of its 5' and 3' untranslated regions, was used to make the RNAi construct for Arabidopsis transformation (Fig. 1A ). After transformation, T1 seeds were harvested and germinated on agar plates containing kanamycin for transgenic plant selection. One hundred ninety-four lines were selected and grown in soil for phenotyping and allowed to set T2 seeds. Among these plants, 16 lines displayed a promising phenotype of aberrant siliques in which no or few seeds were produced. The selection scheme is shown in Figure 1B. Among the 16 lines, some were able to produce seeds by self-pollination. Those that could not set seeds by self-pollination either perished or were rescued by backcrossing with wild-type ecotype Columbia (Col-0) pollen. Among the surviving lines, four showed stably heritable phenotypes, and these lines were used in this study. Among the four lines, di 23 bc F2 was derived from a backcross as follows. This line was generated by backcrossing di 23 T1 with Col-0 pollen to produce di 23 bc F1. The F1 seeds were germinated on agar plates containing kanamycin, and the surviving seedlings (about 10% survived) were transferred to soil. The di 23 bc F2 seeds were harvested from one of these di 23 bc F1 plants that exhibited a heritable phenotype. Because homozygous di 23 bc F3 could not be identified (see below for explanation), this line was retained as a segregating F2 population for this study. Another line, di 69 T2, resulting from self-pollination, was also retained as T2 segregates because homozygous T3 could not be identified. The transgenic-nontransgenic ratios of the F2 and the T2 populations were both about 1:1 as genotyped by PCR (see below) and based on kanamycin selection. This non-Mendelian segregation of di 23 bc F2 and di 69 T2 and the low kanamycin resistance ratio of di 23 bc F1 seeds suggest that lethality was associated with the action of the AtHVA22d RNAi transgene. Two other lines, di S3-2 and di 64-2, were T3 homozygous, and their phenotype was weaker than that of the aforementioned F2 and T2 lines. Their nontransformed T3 segregates were di S3-5 and di 64-5, respectively.
Cross-Suppression of the Expression of the AtHVA22 Gene Family by the RNAi Transgene
AtHVA22a and AtHVA22d RNAi transgenic plants were generated initially to compare their phenotypes. The phenotype of AtHVA22a T1 RNAi transgenics was similar to that of AtHVA22d T1 RNAi plants, although weaker. To examine the cosuppression effectiveness of the two RNAi transgenes, two T1 plants were arbitrarily selected from AtHVA22a and AtHVA22d RNAi plants for northern-blot analysis. As shown in Figure 2
, expression of AtHVA22a was suppressed significantly but not for the other three homologs (AtHVA22c, -d, and -e) in AtHVA22a RNAi plants. In AtHVA22d RNAi plants, however, apparent suppression was observed for the four genes (AtHVA22a, c, d, and e) whose transcripts could be detected by northern-blot analysis. Expression of AtHVA22b was too low to detect (Chen et al., 2002
The Seed Reduction Phenotype Cosegregated with the RNAi Transgene In addition to slower growth rate (Fig. 3A ), these AtHVA22d RNAi plants produced smaller siliques containing no or fewer mature seeds compared with the wild-type plants, as shown in Figure 3B. To examine whether this phenotype was due to artificial selection or caused by the action of the RNAi transgene, a statistical analysis was carried out with the F2, T2, and T3 populations to determine whether this phenotype cosegregated with the RNAi transgene. Plants from these populations were genotyped by either amplifying the GFP DNA fragment in the transgene (for di 23 bc F2 and di 69 T2; Fig. 1) using PCR or by examining their kanamycin resistance (for di S3-2, di S3-5, di 64-2, and di 64-5). The fourth to sixth siliques, numbering from the base to the apex on the primary inflorescence, were dissected to count the number of successful and unsuccessful seeds. The results show that the AtHVA22d RNAi transgene cosegregates with the seed reduction phenotype of these plants (Fig. 4 ). This indicates that the seed reduction phenotype is indeed caused by the RNAi transgene.
Stamen Development of the AtHVA22d RNAi Arabidopsis Was Impaired To determine the cause of seed reduction in AtHVA22d RNAi siliques, flowers of these RNAi plants that were grown in long-day growth conditions were dissected. Intriguingly, the stamens of the RNAi plants were not long enough to pollinate. As shown in Figure 5A , growth of the stamens one night before flower opening was documented every 4 h from 5 PM on day 1 to 9 AM on day 2. The stamens of the nontransformed segregate (di 23 bc F2 NT) elongated substantially from 5 PM to 9 PM on day 1, allowing the length of these stamens to match that of the carpel and permit self-pollination (Fig. 5A, bottom row). The growth of the stamens in the RNAi transgenic plants (di 23 bc F2 T), however, was impaired, and they were still shorter than the carpel when the flower was open (Fig. 5A, top row). To further investigate the stamen length defect, the latest open flowers of di 23 bc F2 T and NT at the midreproductive stage of the plants were dissected and the lengths of the carpels and stamens were measured. The carpels of di 23 bc F2 T were about 0.2 to 0.25 mm longer than the stamens of the same flower on average, while carpels of di 23 bc F2 NT were about the same length as their corresponding stamens (Fig. 5B). To be sure that the length difference between the carpels and stamens in the di 23 bc F2 T flowers was caused by short stamens rather than by long carpels, the lengths of carpels of di 23 bc F2 T and NT were compared, and those of those stamens were compared as well. The carpel length of the RNAi and control plants was 2.49 ± 0.031 mm (n = 23; mean ± SE) and 2.74 ± 0.027 mm (n = 17), respectively; the stamen length of the RNAi and the control plants was 2.25 ± 0.036 mm (n = 23) and 2.75 ± 0.025 mm (n = 17), respectively. This indicates that the presence of the AtHVA22d RNAi transgene results in a length decrease in both stamens and carpels, but the effect on the stamens is greater, causing reduction in self-pollination in RNAi transgenic plants.
Pollen Development Was Also Affected by the AtHVA22d RNAi Transgene We were unable to rescue some T1 transgenic plants using their pollen to fertilize wild-type plants. This led us to examine the pollen grains of these perishing T1 plants and the surviving transgenics. The majority of the pollen grains of these perishing T1 plants were deformed. Pollen grains of di 23 bc F2 T, di 69 T2 T, and wild-type Col-0 were stained with propidium iodide to examine their viability. Propidium iodide is a dead cell stain that emits light in the yellow-red region when it binds to nucleic acid and is excited by blue light. As shown in Figure 6A , about 10% to 20% of pollen grains from the RNAi plants examined by confocal laser scanning microscopy absorbed the stain, indicating that they were dead. Since di 23 bc F2 T and di 69 T2 T are not homozygous lines, the actual percentage of dead pollen grains among those containing the RNAi transgene is expected to be higher. Many abnormal pollen grains were observed in the di 69 T2 T line (Fig. 6A), suggesting that proper expression of the AtHVA22 gene family is required for pollen development. Scanning electron microscopy revealed that many pollen grains from di 69 T2 T plants were collapsed (Fig. 6B).
Carpels of the AtHVA22d RNAi Flowers Were Also Defective Since the RNAi plants produced fewer seeds than the controls and the average length of the transgenic carpels was shorter than that of nontransformed segregates, this raised the question of a potential developmental problem associated with the female parts of the flowers of the RNAi plants. To examine whether development of the female parts was impaired, reciprocal crosses were carried out between the RNAi plants and wild-type Col-0. As shown in Figure 7 , siliques from di 69 T2 T carpels fertilized with Col-0 pollen (C) were much smaller than the siliques from Col-0 self-pollination (A) or Col-0 carpels fertilized with di 69 T2 T pollen (B). Seventeen seeds were produced by six siliques resulting from Col-0 pollinating di 69 T2 T carpels, which was much fewer than the approximately 60 seeds produced by a normal Col-0 silique. This indicates that the AtHVA22d RNAi transgene affected not only the development of stamens and pollen but also the development of female parts of the transgenic plants. This conclusion is supported by the observation that Col-0 pollinating the RNAi lines produced a lower than expected number of seeds able to survive kanamycin selection: 10% of di 23 T1-derived seeds, 25% from di 23 bc F2 T, and 6% from di 69 T2 T plants. These ratios are markedly lower than the 50% (assuming that they were single-locus inserted transgenic plants) that would be expected from these hemizygous plants in the absence of any fertility defects.
Autophagy Was Enhanced in the AtHVA22d RNAi Plants
The shortened stamens of the RNAi Arabidopsis lines prompted us to investigate the subcellular structure of the filament cells. Images obtained with transmission electron microscopy showed vesicles and membranes accumulated in the vacuoles of many filament cells (Fig. 8A2
). Invagination of cytoplasm into vacuoles was also detected (Fig. 8A3). Abnormal electron-dense particles in the cytoplasm and thinning of cytoplasm were also observed (Fig. 8A, 3, 5 and 6). These events were not seen in the filament cells of the control plants (Fig. 8A, 1 and 4). These observations led to our hypothesis that autophagy is enhanced in the RNAi plants, resulting in impaired development of some cell types. To more directly examine levels of autophagy activity in these plants, roots of the RNAi plants and wild-type Col-0 were stained with the autophagosome-specific fluorescent dye monodansylcadaverine (Contento et al., 2005
Autophagy Was Enhanced in the yop1 Deletion Mutant of S. cerevisiae
If it was true that autophagy was enhanced in AtHVA22d RNAi plants, we expected that the yop1 deletion mutant in S. cerevisiae would have a similar phenotype, because Yop1 is the only HVA22 homolog in S. cerevisiae. In S. cerevisiae, the membrane protein Atg8p has been used as an autophagy molecular marker because it is associated with autophagosomes and transported into vacuoles when autophagy occurs (Kirisako et al., 1999
To verify the autophagy detected by western-blot analysis, a cytosolic GFP was overexpressed in yeast to label the distribution of its cytoplasm. Laser scanning confocal microscopy was used to monitor autophagy by monitoring the distribution of GFP-labeled cytoplasm. Little difference was discovered between the wild-type yeast and the yop1 deletion mutant under nonstarvation conditions. However, a difference emerged rapidly when the two strains were transferred to a nitrogen starvation medium. In the yop1 deletion mutant, GFP-labeled cytoplasm started to appear in the vacuoles in as soon as 5 min under nitrogen starvation (Fig. 10 ). Although this phenomenon was observed only in a low percentage of cells initially, cells displaying autophagy increased to over 80% after 2 h of the starvation. In contrast, autophagy in the wild-type cells was much less obvious through 8 h of nitrogen starvation. For more details of our monitoring of autophagy in yeast, see http://biology4.wustl.edu/autophagy.
HVA22 was one of the many stress-induced proteins in plants whose function had not been fully explored. The presence of HVA22 homologs among diverse eukaryotes provided us the opportunity to investigate its function in both Arabidopsis and yeast. Taking advantage of convenient features in genetics and cell biology in these organisms, we have been able to reveal that HVA22 functions as an autophagy suppressor in both systems. We have demonstrated that autophagy is enhanced in both AtHVA22d RNAi transgenic Arabidopsis lines and the yop1 deletion mutant of S. cerevisiae. This suggests that HVA22 homologs regulate autophagy in a negative manner. A recent study showed that Yop1p and TB2/DP1 (all are HVA22 homologs) are integral proteins in tubular ER membranes with three cytosolic domains (N terminus, central, and C terminus) separated by two hydrophobic hairpins inserted into the outer leaflet of the ER lipid bilayer and that the insertion per se is required to maintain the shape of tubular ER (Voeltz et al., 2006 ). Our findings coupled with this work suggest the following two possible cellular roles for HVA22 homologs in autophagy. First, ER membrane with HVA22 homologs inserted could be restrained from forming preautophagosomal structures, because the shape of the ER membrane is constrained by the presence of the protein, leading to negative autophagy regulation. Second, it is possible that HVA22 homologs prevent preautophagosomal structure formation by interacting with an unknown protein(s) that contributes to negative regulation of autophagy. The topology of Yop1p and TB2/DP1 in the ER membrane suggests that these proteins execute multiple functions in cells. In addition to maintaining the shape of tubular ER, the studies of Calero et al. (2001) and Brands and Ho (2002) showed that Yop1p is involved in vesicular trafficking and that the N terminus is responsible for this activity. However, the most conserved part of Yop1p homologs is located in the central cytosolic domain. It is reasonable to assume that this central cytosolic domain is responsible for a cellular mechanism that has yet to be discovered. As for the C-terminal domain, it is very diverse among these homologs; thus, presumably this domain is not responsible for the conserved autophagy. The initial attempt in this work was to use RNAi to study the role of the AtHVA22d gene particularly, because AtHVA22d was the most responsive to ABA and stress treatments. Although T-DNA insertion mutants are available for the AtHVA22d gene, phenotypes we have observed in the AtHVA22d mutants are much milder than the RNAi lines (Supplemental Data S2). As shown in Figure 2, the RNAi lines have reduced transcript levels for all members of the AtHVA22 gene family. Therefore, the phenotypes described in this paper are most likely related to knocking down of the expression of all members of AtHVA22 family.
HVA22 is barely detectable and highly inducible by ABA in barley aleurone cells and young vegetative tissues (Shen et al., 2001
Autophagy is less active in fast-growing cells (Stephan and Herman, 2006 Many of the T1 RNAi plants in this study showing severe phenotypes, including dwarf and bushy stature, perished because they could not set seeds by either self-pollination or crossing to wild-type plants. This suggests that strong suppression of the AtHVA22 gene family is lethal. This view is supported by the non-Mendelian segregation of di 23 bc F2 and di 69 T2 and the difficulties in identifying di 23 bc F3 and di 69 T3 homologous transgenic RNAi plants. These phenomena are well explained by our observation of overactive autophagy in these RNAi plants: strong suppression of the AtHVA22 gene family causes overactive autophagy that leads to self-destruction of the transgenic cells. The severity of phenotype is expected to be correlated with the degree of gene expression knockdown in transgenic plants if low degrees of gene knockdown cause an easily quantifiable phenotype and lethality is not associated with high levels of knockdown. In our study, we obtained 16 lines of transgenic plants with obvious and stable phenotypes among the original 194 T1 plants. This indicates that minor levels of knockdown did not lead to an apparent phenotype. This notion is supported by the fact that single-gene T-DNA insertion lines (Supplemental Data S2) also did not show any phenotype. If it takes a certain threshold level of knockdown in order to cause a phenotype, the correlation between phenotype severity and degree of knockdown may not be observed. Moreover, lethality in this study had caused the loss of many lines with severe phenotypes. This problem further cuts down the analyzable range of phenotype versus gene expression knockdown. Phenotyping based on bias had been the greatest concern since the beginning of this study. We eventually investigated four transgenic lines with inheritable and stable phenotypes, two strong ones and two weak ones. The best way in this case to exclude the possibility of artificial selection was to conduct a phenotype-versus-transgene cosegregation analysis. Although this approach may not be flawless, we believe it to be the most convincing way to correlate the presence of an RNAi-generating transgene with the appearance of a phenotype. In addition, our hypothesis obtained from Arabidopsis was confirmed in yeast. The negative regulation mechanism executed by AtHVA22 and Yop1p might be downstream of a starvation sensor, as shown by the production of autophagosomes in the root tips of the RNAi plants and the yop1 deletion yeast only in starvation conditions. However, the relation between AtHVA22/Yop1p and Tor1p, as well as the Atg1p-Atg13p complex, is not clear. We hypothesize that AtHVA22/Yop1p controls the lipid flow from the ER to the preautophagosomal structures by either stabilizing the ER membrane or by recruiting a negative autophagy regulator(s) that interacts with their central cytosolic domain. This would place their activity upstream of the Atg1p-Atg13p complex. Since Tor1p inactivation enhances autophagy in nonstarvation conditions and its substrate has yet to be identified, it is difficult to determine whether Tor1p may act in the same pathway as AtHVA22/Yop1p. Clearly, further studies are required to clarify the regulation of autophagy.
RNAi Construct The GFP cDNA fragment was amplified by PCR using primers 5'-ACGAATTCGTGGAGGAGGTATTCTAGATC-3' and 5'-ATGAGCTCATCCATGCCATGTGTAATCCC-3'. This cDNA fragment was inserted into the multicloning sites of the Arabidopsis (Arabidopsis thaliana) transformation vector pMON 10098. The AtHVA22d cDNA fragment was amplified by PCR using primers 5'-AAGAATTCGAGCTCTTTACACAGACTTGTCGCTCT-3' and 5'-TCTCTAGAGGATCCGAAATACGCGTGGAAGATTAG-3'. This amplified AtHVA22d cDNA fragment contained the coding region and parts of the 5' and 3' untranslated regions. There were two enzyme digestion sites at each end of this cDNA fragment, which were used for the sense or antisense orientation insertion between the GFP cDNA fragment and the terminator or the promoter, respectively.
Arabidopsis ecotype Col-0 was grown on a germination mix in a growth chamber as described by Chen et al. (2002)
Total RNA isolation and northern-blot analysis were carried out based on the procedures described by Chen et al. (2002)
Small-scale genomic DNA isolation was carried out with a modified mini-prep procedure (Cocciolone and Cone, 1993
Mature pollen was spread on glass slides and stained with 1 mg mL–1 propidium iodide (Sigma P4170) in distilled water. The absorption spectrum of propidium iodide bound to DNA is between 450 and 600 nm, and the emission is in the yellow-red region (550–700 nm). The samples were examined with 488-nm excitation wavelength using a Leica laser scanning confocal microscope. The emission spectrum was collected with two channels: one in the green region for the autofluorescence from the cell wall of pollen grains, the other in the yellow-red region for the propidium iodide emission.
Mature anthers were attached onto specimen mounts for scanning electron microscopy and allowed to dry on a slide warmer. The materials were then sputter coated with gold and imaged with a Hitachi S-450 scanning electron microscope. Stamens were fixed with 2.5% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7.4, containing a trace amount of Photo-Flo 200 (Kodak) as a wetting reagent. The samples were postfixed with 2% (w/v) osmium tetroxide in water, dehydrated, and embedded in Polybed 812 resin (Polysciences). Thin sections were stained with 2% uranyl acetate followed by 0.38 mM lead citrate. The samples were imaged with a Hitachi H-600 transmission electron microscope.
Autophagosome staining in root tips was carried out using a modified method of Contento et al. (2005)
Wild-type Saccharomyces cerevisiae (strain w303) and its yop1 deletion mutant (Brands and Ho, 2002
Wild-type yeast and the yop1 (HVA22 homolog in yeast) deletion mutant cells were transformed with cytosolic GFP driven by a Gal-inducible promoter to label the distribution of cytoplasm. These cells were grown in the synthetic dropout medium (–Trp) containing 2% Gal for 44 h to late log phase and then starved by transferring to nitrogen-depleted medium (without ammonium sulfate and amino acids) containing 2% Gal and 1 mM PMSF, and autophagy was monitored using laser scanning confocal microscopy. More details of this procedure are given in the Supplemental Data S1.
The following materials are available in the online version of this article.
We thank Drs. Craig Pikaard and Eric Richards for helpful discussions and Mr. Mike Veith and Dr. Tatsuhiko Noguchi for assistance with electron and fluorescence microscopy, respectively. Received November 14, 2008; accepted January 8, 2009; published January 16, 2009.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Tuan-Hua David Ho (tho{at}sinica.edu.tw).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.131490 * Corresponding author; e-mail tho{at}sinica.edu.tw.
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ABSTRACT
RESULTS
Rp and using antibodies to detect the dissociation of GFP and Atg8
