the autophagic degradation of chloroplasts via rubisco ......the majority of plant nitrogen and...

The Autophagic Degradation of Chloroplasts viaRubisco-Containing Bodies Is Specifically Linked toLeaf Carbon Status But Not Nitrogen Statusin Arabidopsis1[W][OA]

Masanori Izumi, Shinya Wada, Amane Makino, and Hiroyuki Ishida*

Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University,Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981–8555, Japan

Autophagy is an intracellular process facilitating the vacuolar degradation of cytoplasmic components and is important fornutrient recycling during starvation. We previously demonstrated that chloroplasts can be partially mobilized to the vacuoleby autophagy via spherical bodies named Rubisco-containing bodies (RCBs). Although chloroplasts contain approximately80% of total leaf nitrogen and represent a major carbon and nitrogen source for new growth, the relationship between leafnutrient status and RCB production remains unclear. We examined the effects of nutrient factors on the appearance of RCBs inleaves of transgenic Arabidopsis (Arabidopsis thaliana) expressing stroma-targeted fluorescent proteins. In excised leaves, theappearance of RCBs was suppressed by the presence of metabolic sugars, which were added externally or were producedduring photosynthesis in the light. The light-mediated suppression was relieved by the inhibition of photosynthesis. During adiurnal cycle, RCB production was suppressed in leaves excised at the end of the day with high starch content. Starchlessmutants phosphoglucomutase and ADP-Glc pyrophosphorylase1 produced a large number of RCBs, while starch-excess mutantsstarch-excess1 and maltose-excess1 produced fewer RCBs. In nitrogen-limited plants, as leaf carbohydrates were accumulated,RCB production was suppressed. We propose that there exists a close relationship between the degradation of chloroplastproteins via RCBs and leaf carbon but not nitrogen status in autophagy. We also found that the appearance of non-RCB-typeautophagic bodies was not suppressed in the light and somewhat responded to nitrogen in excised leaves, unlike RCBs. Theseresults imply that the degradation of chloroplast proteins via RCBs is specifically controlled in autophagy.

Autophagy is the major pathway by which proteinsand organelles are transported for degradation inthe vacuoles of yeast and plants or the lysosomes ofanimals (for detailed mechanisms, see reviews byOhsumi, 2001; Levine and Klionsky, 2004; Thompsonand Vierstra, 2005; Bassham et al., 2006; Bassham,2009). In these systems, a portion of the cytoplasm,including entire organelles, is engulfed in a double-membrane vesicle called an autophagosome and de-livered to the vacuole/lysosome. The outer membraneof the autophagosome then fuses with the vacuolar/lysosomal membrane, and the inner membrane struc-ture called the autophagic body is degraded within thevacuole/lysosome by resident hydrolytic enzymes. A

recent genome-wide search confirmed that Autophagy-related genes (ATGs) are well conserved across yeast,plants, and animals (Meijer et al., 2007). Using ATGknockout mutants and a monitoring system withan autophagy marker, GFP-ATG8, numerous studieshave demonstrated the presence of the autophagysystem in plants and its importance in several biolog-ical processes (Yoshimoto et al., 2004, 2009; Liu et al.,2005; Suzuki et al., 2005; Thompson et al., 2005; Xionget al., 2005, 2007; Fujiki et al., 2007; Patel and Dinesh-Kumar, 2008; Phillips et al., 2008; Hofius et al., 2009).Plant autophagy is thought to play an important rolein nutrient recycling under starvation, similar to itsrole in starvation previously noted in yeast and ani-mals (Thompson and Vierstra, 2005; Bassham et al.,2006). For example, the creation of autophagosomesis induced by nitrogen, carbon, or both combinedstarvation in plant heterotrophic tissues such as roots(Yoshimoto et al., 2004; Xiong et al., 2005), hypocotyls(Thompson et al., 2005; Phillips et al., 2008), andsuspension-cultured cells (Chen et al., 1994; Aubertet al., 1996; Moriyasu and Ohsumi, 1996; Rose et al.,2006). Arabidopsis (Arabidopsis thaliana) atg mutantsshow accelerated leaf senescence and cannot surviveand respond to nutrient resupply after severe carbonor nitrogen starvation (Doelling et al., 2002; Hanaokaet al., 2002; Thompson et al., 2005; Xiong et al., 2005;Phillips et al., 2008).

1 This work was supported by KAKENHI (grant nos. 20200061,20780044, and 22780054 for H.I., grant no. 21114006 for A.M., andgrant no. 21–6184 for M.I.), by Genomics for Agricultural Innovation(grant no. GPN–0007 for A.M.), and by Research Fellowships of theJapan Society for the Promotion of Science for Young Scientists toM.I.

* 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:Hiroyuki Ishida ([email protected]).

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

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The majority of plant nitrogen and other nutrientsare distributed to leaves in the vegetative growthstage (Schulze et al., 1994; Makino et al., 1997). In C3plants, 75% to 80% of total leaf nitrogen is distributedto chloroplasts, primarily as photosynthetic proteinssuch as Rubisco (Makino and Osmond, 1991; Makinoet al., 2003). During senescence and suboptimal en-vironmental conditions, Rubisco and most stromalproteins are degraded, and the released nitrogen isremobilized to growing organs and finally stored inseeds (Friedrich and Huffaker, 1980; Mae et al., 1983).Chloroplast proteins can be degraded under carbon-limited conditions caused by darkness (Wittenbach,1978), with their carbon used as a substrate for respi-ration. Therefore, considering the role of autophagy innutrient recycling, much attention should be paid tothe study of chloroplast degradation. ATG transcriptabundances are elevated during starvation-inducedleaf senescence (Yoshimoto et al., 2004; van der Graaffet al., 2006; Chung et al., 2009). However, previousreports have not analyzed the effects of nutrient statuson the appearance of autophagosomes or autophagicbodies in leaves. Recently, we observed the accumu-lation of autophagic bodies and Rubisco-containingbodies (RCBs), a kind of autophagic body containingchloroplast stroma, using fluorescent markers in thevacuole of excised leaves treated with concanamycinA, which suppresses vacuolar lytic activity (Ishidaet al., 2008). Autophagy of chloroplast componentscould be observed during senescence of individuallydarkened leaves (Wada et al., 2009). However, it is notclear how the production of autophagic bodies andRCBs is affected by nutrient status in leaves.In this study, we aimed to demonstrate the relation-

ship between the nutrient status of Arabidopsis leavesand chloroplast degradation via RCBs. We examinedthe effects of nutrient conditions during leaf incuba-tion, leaf carbohydrate contents over a diurnal cycle,mutations affecting starch metabolism, and nitrogenlimitation on the appearance of RCBs. All analysesshowed that carbon status is a major factor controllingthe production of RCBs, while nitrogen status is lessimportant in the nutrient response of autophagy inleaves. We also found that the production response ofRCBs and non-RCB-type autophagic bodies containingcytoplasmic components other than chloroplasts tonutrient conditions was not always the same in excisedleaves. This suggests that there is a mechanism specif-ically controlling RCB production in plant autophagy.

RESULTS

Responses of RCB Production to Carbon But NotNitrogen Supply in Excised Leaves

RCBs could be visualized in the vacuolar lumenwith laser-scanning confocal microscopy (LSCM)when leaves of chloroplast stroma-targeted GFP-expressing Arabidopsis (named CT-GFP) were excisedand incubated in darkness in “incubation buffer,”

containing concanamycin A, an inhibitor of vacuolarH+-ATPase that suppresses vacuolar lytic activity (Fig.1A; Ishida et al., 2008). RCBs were rarely seen whenattached leaves were excised and immediately ob-served during natural senescence (Ishida et al., 2008).RCBs were also rarely observed even if excised leaveswere incubated in darkness without concanamycinA for 20 h, suggesting that they are rapidly degradedin the vacuolar lumen under natural cell conditions(Ishida et al., 2008). Thus, inhibitor treatments forsuppressing vacuolar lytic activity are essential forquantitative examination of RCB production. Whenexcised leaves were incubated in incubation buffer,RCBs were detected more in mature and early senes-cent leaves than in young leaves, but RCBs were rarelyseen by the presence of Murashige and Skoog (MS)medium containing Suc, even in early senescent leaves(Ishida et al., 2008). These previous data suggestedthat the production of RCBs was influenced by nutri-ent conditions during incubation in excised leaves.First, we quantitatively analyzed the effects of externalnutrient supply and light conditions during incuba-tion on the appearance of RCBs (Fig. 1E). Around 50RCBs were detected in the field of view (188 mm 3188 mm each) in leaves incubated in nutrient-free me-dium in darkness (dark MES; Fig. 1A). The addition ofMS medium containing Suc (dark MS + Suc) greatlysuppressed the appearance of RCBs. This suppressionof RCBs was not relieved by the depletion of nitrogen(dark MS-N + Suc) but by the depletion of Suc (darkMS-N; dark MS). Furthermore, RCB appearance wasalso suppressed by the sole addition of Suc (dark +Suc; Fig. 1B), Glc (dark + Glc), or Fru (dark + Fru).Mannitol did not affect the appearance of RCBs(dark + Mann); thus, it is unlikely that osmotic stressaffects RCB appearance.

The appearance of RCBs was suppressed by irradi-ation (light MES; Fig. 1C). The light-mediated suppres-sion of RCB appearancewas relieved by the addition of3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), aninhibitor of photosynthetic electron transport (light +DCMU; Fig. 1D), indicating the possibility that theappearance of RCBs was suppressed by photosynthe-sis, or the products of photosynthesis, rather than bylight-mediated signals. Changes in starch and meta-bolic sugar contents were determined in excised leavesincubated either in darkness or in the light (Fig. 2).Although starch and metabolic sugars were decreasedand almost consumed after 20 h of incubation in dark-ness (dark MES) or in the light with DCMU (light +DCMU), carbohydrate levels were increased duringincubation in the light (light MES). These resultssuggested that RCB production was affected by photo-synthetically accumulated internal carbohydrates inthe light aswell as externally added sugars in darkness.

Differential RCB accumulation might be due to dif-ferences in the stability of RCBs or the maker, GFP,in the vacuolar lumen rather than differential RCBproduction between the various treatments usedhere. To clarify this possibility, we used transgenic

Nutrient Response of Autophagy in Leaves

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Arabidopsis expressing vacuole-targeted GFP, whichis a GFP fusion of N-terminal signal peptide andC-terminal signal propeptide of pumpkin (Cucurbitasp.) 2S albumin (SP-GFP-2SC; Tamura et al., 2003), asan indicator for the stability of the GFP signal in thevacuole (Supplemental Fig. S1). When leaves of SP-GFP-2SC were excised and immediately observedwith LSCM, the GFP signal was not observed in thevacuole but in the endoplasmic reticulum and Golgicomplex, as reported previously (Supplemental Fig.S1A; Tamura et al., 2003). It was also reported that GFPwas rapidly degraded by vacuolar proteases in thelight but that the degradation was suppressed by theaddition of concanamycin A or E-64d (Tamura et al.,2003). When leaves of SP-GFP-2SC plants were incu-bated in the presence of concanamycin A and E-64d,GFP was observed in the vacuole irrespective of thetreatment conditions used, darkness, darkness + MSmedium containing Suc, or in the light, where thedegree of RCB appearance was greatly different (Fig. 1;Supplemental Fig. S1, B–D). This suggests that the

treatments used did not have significant effects onvacuolar lytic activity. If RCBs were degraded in leavesof CT-GFP plants under conditions where RCB ap-pearance was significantly suppressed (Fig. 1), stroma-targeted GFP, which was mobilized from chloroplastsvia RCBs into the vacuole, should be distributeduniformly as SP-GFP-2SC in the vacuolar lumen, sinceGFP would not be degraded in the presence of prote-ase inhibitors (Supplemental Fig. S1, B–D). However,we could not observe uniformly distributed GFP invacuolar lumen in the leaves of CT-GFP plants underthose conditions (Fig. 1). These results suggest that theappearance of RCBs in various incubation conditionswas mainly due to differences in RCB production.

RCB Production Is Specifically Controlled duringAutophagy of Mesophyll Cells

Previous studies have shown that autophagic bodiesaccumulate under nitrogen, carbon, or nitrogen andcarbon combined starvation in heterotrophic tissues

Figure 1. Effects of incubation conditionson the appearance of Rubisco-containingbodies. A to D, Visualization of RCBs bystroma-targeted GFP in various incuba-tion conditions. Fourth leaves of stroma-targeted GFP expressing Arabidopsis at30 d after sowing (5 d after bolting) wereexcised and incubated in 10 mM MES-NaOH (pH 5.5) with 1 mM concanamycinA and 100 mM E-64d at 23�C for 20 h indarkness (A), with 3% (w/v) Suc in dark-ness (B), in the light (C), or with DCMU inthe light (D). GFP appears green, andchlorophyll fluorescence appears red. Inmerged images, overlap of GFP and chlo-rophyll fluorescence appears yellow. InB, C, and D, merged images are shown.Bars = 50 mm. E, The number of RCBsin various incubation conditions. Excisedleaves were incubated as described in A toD in darkness (blue columns) or in thelight (yellow columns) and with variousconstituents: MES, no other constituent;MS, MS medium; MS-N, nitrogen-free MSmedium; +Suc, 3% (w/v) Suc; +Glc, 3%(w/v) Glc; +Fru, 3% (w/v) Fru; +Mann, 3%(w/v) mannitol; +DCMU, 10 mM DCMU.Leaves from four independent plants wereincubated, eight quadrangular regions(188 mm 3 188 mm each) per leaf weremonitored by LSCM, and the number ofaccumulated RCBs was counted (RCBnumber). Each image was considered asan independent data point and subjectedto statistical analysis by Tukey’s test. Datarepresent means 6 SE (n = 32). Columnswith the same letter were not significantlydifferent (P # 0.01).

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(Yoshimoto et al., 2004; Thompson et al., 2005; Xionget al., 2005; Phillips et al., 2008). RCBs are a kind ofautophagic body that specifically contain the stromalportion of chloroplasts. However, the pattern of RCBaccumulation was not similar to autophagic bodyaccumulation shown in these previous studies (Fig.1). Therefore, we next compared RCB production withautophagic bodies containing cytoplasmic compo-nents other than the stroma (non-RCB-type autopha-gic bodies) in excised leaves under various nutrientconditions.RCBs and entire autophagic bodies can be visual-

ized at the same time using transgenic plants express-ing stroma-targeted DsRed and the GFP-ATG8 fusion(Fig. 3, A and B; Ishida et al., 2008). In these transgenicplants (ecotype Wassilewskija), the number of RCBsand non-RCB-type autophagic bodies detected in thefield of view (188 mm3 188 mm each) was 416 2.7 and20 6 1.7 (n = 64) under control conditions (dark MES;Fig. 3C), respectively. The appearance of RCBs wassignificantly suppressed by the presence of Suc or inthe light, although this was relieved by the presence ofDCMU (Fig. 3C), similar to the CT-GFP plants (ecotypeColumbia; Fig. 1). The pattern of appearance of non-RCB-type autophagic bodies was not always the sameas the pattern of RCB appearance (Fig. 3C). In dark-ness, the appearance of non-RCB-type autophagicbodies was suppressed by the presence of Suc, similarto RCBs (dark MS + Suc, dark MS-N + Suc, dark + Suc,and light + Suc). This suggested that exogenous excesssugars suppress entire autophagy. In Suc-free condi-tions, the appearance of non-RCB-type autophagicbodies was also suppressed by the presence of MSmedium (dark MS) or nitrogen (dark + N), while RCB

production was unaffected. These data indicated thatnitrogen also affects the appearance of non-RCB-typeautophagic bodies to some extent in leaves, as reportedpreviously (Yoshimoto et al., 2004; Thompson et al.,2005; Xiong et al., 2005; Phillips et al., 2008), but theeffect of nitrogen was small compared with the effectof Suc. The appearance of non-RCB-type autophagicbodies was not suppressed in the light (light MES),while RCB production was consistently and markedlysuppressed. This specific suppression of RCB pro-duction related to photosynthesis suggests that RCBproduction was more sensitive to increases in leafcarbohydrate contents than autophagosome creation.

Effects of Timing of Leaf Excision in a Diurnal Cycle onthe Appearance of RCBs

To examine the effects of physiological changes inleaf carbohydrate contents on the appearance of RCBs,we investigated RCB production during a diurnalcycle. Fourth rosette leaves of CT-GFP plants wereexcised at three time points: the end of the regularnight cycle, the end of a prolonged night, and the endof the day, and carbohydrate contents and RCB num-ber were determined (Fig. 4A). As expected, starchlevels were significantly higher at the end of the daycompared with the end of the night, with most of thestarch used up at the end of prolonged night (Fig. 4B).Among soluble sugars, Suc was significantly lower atthe end of prolonged night, although no significantchanges were detected in Glc or Fru levels. Recordedsugar levels were similar to those in previous reports(Gibon et al., 2004; Blasing et al., 2005; Smith and Stitt,2007). Leaves were excised and incubated in incuba-tion buffer for 10 h in darkness. The number of RCBswas low in leaves excised at the end of the day, whencarbohydrate levels were highest, and high in leavesexcised at the end of prolonged night, when carbohy-drate levels were at a minimum. Particularly, starchcontents seemed to be a primary factor affecting RCBproduction.

Effects of Mutations Affecting Leaf Starch Content on theAppearance of RCBs

To provide further evidence of the close relationshipbetween RCB production and leaf carbohydrate con-tents, we examined RCB production in starch contentgenetic mutants, specifically starchless phosphogluco-mutase (pgm-1) and ADP-Glc pyrophosphorylase1 (adg1-1)and starch-excess starch-excess1 (sex1-1) and maltose-excess1 (mex1-3). The mutant pgm-1 lacks chloroplastphosphoglucomutase (Caspar et al., 1985), and adg1-1lacks the activity of ADP-Glc pyrophosphorylase (Linet al., 1988). Neither mutant can accumulate starchduring the day. The mutant sex1-1 lacks glucanwater dikinase 1 and is reduced in its capacity todegrade starch (Caspar et al., 1991; Yu et al., 2001),while mex1-3 lacks the chloroplast maltose transporterand contains both high levels of starch and very high

Figure 2. Changes in starch, Suc, Glc, and Fru contents in incubatedleaves. Excised leaves from transgenic Arabidopsis at 30 d after sowingwere incubated at 23�C for 8 h or for 20 h in 10 mM MES-NaOH (pH5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness (blacksquares), in the light (white circles), or with 10 mM DCMU in the light(white triangles), and carbohydrate content was determined. Datarepresent means 6 SE (n = 3).





levels of maltose (Niittyla et al., 2004). These starch-related mutants grow like wild-type plants in contin-uous light; however, their growth is progressivelyimpaired as the duration of the night period is in-creased (Caspar et al., 1985, 1991; Lin et al., 1988). Wefirst set up starch-related mutants expressing stroma-targeted GFP and visualized the chloroplast stroma ofeach mutant (Supplemental Fig. S2). In mex1-3, weobserved both normal and abnormally shaped chloro-plasts, which exhibited swollen chloroplasts, as re-vealed by the fluorescence signals of stroma-targetedGFP (Supplemental Fig. S2, D and E). This “swollenchloroplast” phenotype has been noted previously inmex1 mutant cells observed by transmission electronmicroscopy (Stettler et al., 2009).

In continuous-light growth conditions, both thewild type and the mutants all took about 21 d to startbolting. Mutants pgm-1, adg1-1, and sex1-1were almostthe same size and had the same leaf number as thewild type at 5 d after bolting (Fig. 5A). Only mex1-3was smaller than the wild type; however, leaf numberwas almost the same as in the wild type (Fig. 5A).Carbohydrate contents were determined in the fourthleaves at 5 d after bolting (Fig. 5B). As expected, starchlevels were significantly higher in starch-excess mu-tants than in the wild type. No starch was detected instarchless mutants. Soluble sugar levels were similarin wild-type and mutant plants. Leaves from each setof plants were excised and incubated in incubationbuffer. RCBs were also observed in leaves of each of

Figure 3. Effects of incubation conditionson the appearance of RCBs and autopha-gic bodies. A and B, Visualization of bothRCBs and autophagic bodies. Excisedleaves from both GFP-ATG8- and stroma-targeted DsRed-expressing plants at 25 dafter sowing (5 d after bolting) were incu-bated at 23�C for 20 h in 10 mM MES-NaOH (pH 5.5) with 1 mM concanamycinA and 100 mM E-64d in darkness (A) orin the light (B). GFP appears green andDsRed appears red. In merged images,overlap of GFP and DsRed appears yel-low. Bars = 10 mm. C, The appearance ofRCBs and non-RCB-type autophagicbodies in various incubation conditions.Excised leaves from GFP-ATG8- andstroma-targeted DsRed-expressing plantswere incubated as described in A and Bin darkness or in the light with various con-stituents: MES, no other constituent; MS,MS medium; MS-N, nitrogen-free MS me-dium; +Suc, 3% (w/v) Suc; +N, nitrogennutrition of MS medium; +DCMU, 10 mM

DCMU. Leaves from six independentplants were incubated, eight quadrangularregions (188 mm 3 188 mm each) per leafwere monitored by LSCM, and the numberof accumulated RCBs (red columns) andnon-RCB-type autophagic bodies notcontaining DsRed (green columns) werecounted. Each image was considered as anindependent data point and subjected tostatistical analysis by Tukey’s test. Eachvalue is shown as a percentage of the levelin “dark MES.” Data represent means 6 SE

(n = 64). Values with the same letter werenot significantly different in RCBs (redvalues) and non-RCB-type autophagicbodies (green values; P # 0.05).

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the starchless and starch-excess mutants, although thenumber of RCBs was significantly higher in leaves ofstarchless mutants and lower in starch-excess mutantsthan in the wild type (Fig. 6).Next, we analyzed RCB production throughout

the leaf life span in the wild type and starch-relatedmutants under long-day growth conditions with a14-h photoperiod, because the growth rates of eachmutant differed under long-day conditions. Here,wild-type plants took 25 d to bolt. A further 2 to 3 dwere required in mex1-3, 3 to 4 d in sex1-1, and 5 to 6 din pgm1-1 and adg1-1. In wild-type and mutant plants,leaf area of the fourth leaf rapidly increased beforebolting, and leaf expansion had almost finished asbolting commenced (Supplemental Fig. S3). We quan-tified the appearance of RCBs in fourth leaves from 5 dbefore bolting, during active leaf expansion, to 10 dafter bolting, the senescent stage, in each plant (Fig.7A). In the wild type, the number of RCBs increased inexpanding, mature and early senescent leaves, asreported previously (Ishida et al., 2008), but decreasedat the later senescent stage. In starchless mutants, theappearance of RCBs was significantly higher than inthe wild type in expanding and mature leaves but

declined during leaf senescence faster than in the wildtype. In leaves of starchless mutants, the transcriptionlevels of senescence-associated genes, SAG12 andSAG13, were higher than in the wild type (Fig. 7B).This implied rapid leaf senescence in starchless mu-tants, leading to a rapid decline of RCB production.In starch-excess mutants, RCB appearance was al-ways lower than in wild-type plants during the pe-riod examined. In senescent leaves, the appearance ofRCBs was significantly lower in starch-excess mutantsthan in the wild type. The maximum number of RCBsduring the leaf life span was significantly higher instarchless mutants and lower in starch-excess mutantscompared with the wild type (Fig. 8A). Starch couldnot be detected in the leaves of starchless mutants, andsignificantly higher starch contents were detected inleaves of starch-excess mutants than in the wild typewhen the number of RCBs after incubation was max-imum in each plant (Fig. 8B), while soluble sugarlevels were higher in starch-related mutants than inthe wild type (Fig. 8C). The starch-mediated suppres-sion of RCB production in starch-related mutants wasalso found in the long-day growth condition usedhere.

Figure 4. Effects of timing of leaf excision in a diurnal cycle on the appearance of RCBs. A, Schematic representation of thisexperiment. Fourth leaves of stroma-targeted GFP transgenic plants at 30 d after sowing were excised at the end of the regularnight cycle (EN) or after 4 h of prolonged night (PN) or the end of the day (ED) in a day cycle. B, The leaf carbohydrate contentsand the appearance of RCBs. The contents of starch (left) and Suc, Glc, and Fru (center) in leaves at the end of the night (graycolumns), after the prolonged night (black columns), and the end of the day (white columns) were determined. Excised leaves ateach point were incubated at 23�C for 10 h in 10 mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM E-64d indarkness. Leaves from four independent plants were incubated, eight quadrangular regions (188 mm 3 188 mm each) per leafwere monitored by LSCM, and the number of accumulated RCBs was counted (right). Data represent means 6 SE (n = 3–4[carbohydrate contents] or 32 [RCB number]). Statistical analysis was performed by Tukey’s test. Columns with the same letterwere not significantly different (P # 0.05).





RCB Production Is Attenuated duringNitrogen-Limited Senescence

Carbon or nitrogen limitation accelerates leaf senes-cence. We previously reported that RCBs can be ob-served in the vacuole without inhibitor treatment inindividually darkened leaves, where senescence wasinduced by carbon limitation (Wada et al., 2009). Here,we examined the appearance of RCBs during leafsenescence accelerated by nitrogen limitation at thewhole plant level.

Nitrogen-limited plants were smaller than nitrogen-sufficient plants at 5 d after the imposition of treat-ments (Fig. 9A). In nitrogen-limited plant leaves, thecontents of chlorophyll, nitrogen, soluble protein, and

Rubisco rapidly decreased (Fig. 9B). In spite of theaccelerated degradation of Rubisco and soluble pro-tein, RCBs were not observed without concanamycin Atreatment in leaves of nitrogen-limited plants (data notshown). Even if excised leaves were incubated withincubation buffer containing concanamycin A, signifi-cantly fewer RCBs were noted in the leaves of nitrogen-limited plants compared with nitrogen-sufficientplants from 1 d after the start of the treatment (Fig.10A). Five days after the start of the treatment, fewRCBs could be noted in leaves of nitrogen-limitedplants even in the presence of concanamycin A (Fig.10A). In nitrogen-limited plants, carbohydrates, starch,Glc, and Fru were rapidly accumulated, and Suc wasalso increased (Fig. 10B). When attached leaves ofnitrogen-limited plants were individually darkened toimpede the accumulation of carbohydrates, few RCBsand faint GFP signals were observed in the vacuole,similar to individually darkened leaves of nitrogen-sufficient plants (data not shown). This result supportedour conclusion that RCB production is primarily medi-ated by carbohydrate accumulation rather than bynitrogen limitation in individual leaves.

DISCUSSION

Recent works have demonstrated the importance ofautophagy in nutrient recycling during both nitrogenand carbon starvation in plants (Doelling et al., 2002;Hanaoka et al., 2002; Yoshimoto et al., 2004; Thompsonet al., 2005; Xiong et al., 2005; Phillips et al., 2008).However, it has not been revealed how autophagy ofchloroplast proteins, which are a major source ofcarbon and nitrogen for new growth, is regulated bynutrient status. In this study, we examined the ap-pearance of RCBs, autophagic bodies containing chlo-roplast proteins, under various conditions that affectthe nutrient status of leaves. Our data indicate that theautophagy of chloroplasts via RCBs is closely andspecifically linked to leaf carbon but not to nitrogenstatus in individual leaves.

Physiological Roles of RCB and Autophagy in Leaves

The data presented here suggest one possible role ofRCBs, which is energy supply under temporal carbonstarvation. In the light, chloroplasts provide energy forcellular processes and growth via photosynthesis. Indarkness or insufficient light conditions, which causeenergy limitation, photosynthetic carbon fixation isreduced and the proteins may be used as an energysource via autophagic recycling. In unicellular organ-isms, degradation of unnecessary cellular compo-nents is known to be an important role of autophagy.For example, when methylotrophic yeasts such asPichia pastoris and Hansenula polymorpha are grown inthe presence of methanol as their sole carbon source,they increased their peroxisome numbers, as thisorganelle is essential for metabolizing methanol.

Figure 5. The leaf carbohydrate contents in starch-related mutantsunder continuous illumination. A, Photograph of wild-type, starchlessmutant (pgm-1, adg1-1), and starch-excess mutant (sex1-1, mex1-3)plants expressing stroma-targeted GFP grown for 26 d after sowing (5 dafter bolting) in continuous illumination (60 mmol quanta m22 s21). B,The leaf carbohydrate contents in the wild type and starch-relatedmutants. The contents of starch (left) and Suc, Glc, and Fru (right) weredetermined in leaves of stroma-targeted GFP transgenic plants ofbackground wild type, pgm-1, adg1-1, sex1-1, or mex1-3 at 26 d aftersowing. Data represent means 6 SE (n = 3). Statistical analysis wasperformed by Tukey’s test. Columns with the same letter were notsignificantly different (P # 0.05).

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When they are transferred to a Glc- or ethanol-richenvironment, the excess peroxisomes are selectivelydegraded via autophagy for adaptation to the envi-ronmental change (Dunn et al., 2005; Sakai et al., 2006).This mechanism is called pexophagy. RCBs may be asimilarly selective autophagy system for the degra-dation of the chloroplast component. In pexophagysystems, whole peroxisomes are transported to thevacuole. Chloroplasts are usually essential for carbonassimilation in plants, while many peroxisomes arenot required for growth on most carbon sources otherthan methanol in yeast. Whole chloroplast degrada-tion would impair the resumption of photosynthesiswhen light conditions improve. Partial degradationvia RCBs is possibly effective for maintaining somebasal functions of chloroplasts with a supply ofneeded energy. Therefore, one possible role of RCBsis supplying stromal proteins and chloroplast enve-lope as a carbon source under carbon-limited condi-tions without the loss of whole chloroplasts. In fact,free amino acid levels increase during extended dark-ness in Arabidopsis leaves, possibly by protein deg-radation (Usadel et al., 2008). Several Arabidopsis atgmutants exhibit accelerated leaf senescence and couldnot survive under severe carbon limitation with ex-

tended darkness (Doelling et al., 2002; Hanaoka et al.,2002; Thompson et al., 2005; Xiong et al., 2005; Phillipset al., 2008). RCBs may contribute to adaptation andsurvival under extended darkness by supplying res-piratory carbon.

In plants, carbon starvation by darkness acceleratesleaf senescence in some cases. Weaver and Amasino(2001) provided an experimental model of leaf senes-cence in Arabidopsis; senescence was induced whenindividual leaves were darkened but not when wholeplants were darkened. Dark-induced senescence washighly localized (i.e. senescence was locally acceler-ated in darkened but not in illuminated areas in thesame leaf). Our recent work demonstrates that chlo-roplast autophagy occurs in the senescence of indi-vidually darkened leaves (Wada et al., 2009). Togetherwith this report, our data here strongly indicate thatRCBs also have an important role in chloroplast deg-radation during accelerated senescence by darkness inArabidopsis. During senescence of individually dark-ened leaves, transportation of whole chloroplasts byATG4 gene-dependent autophagy was also observed(Wada et al., 2009). This implied that maintenance ofbasal chloroplast functions was no longer necessary indarkened sectors, and it may be more advantageous

Figure 6. Effects of mutations affecting leaf starch content on the appearance of RCBs. A to E, Visualization of RCBs in leaves ofstarch-related mutants. Transgenic plants expressing stroma-targeted GFP of background wild type (A), pgm-1 (B), adg1-1 (C),sex1-1 (D), ormex1-3 (E) were incubated at 23�C for 20 h in 10mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM

E-64d in darkness and observed with LSCM. GFP appears green and chlorophyll fluorescence appears red. Merged images areshown. Bars = 50mm. F, The appearance of RCBs in leaves of starch-related mutants. Leaves excised from five independent plantsof the wild type and each mutant were incubated, eight quadrangular regions (188 mm3 188 mm each) per leaf were monitoredby LSCM, and the number of accumulated RCBs was counted. Data represent means 6 SE (n = 40). Statistical analysis wasperformed by Tukey’s test. Columns with the same letter were not significantly different (P # 0.05).





for plant growth that nutrients were remobilized toorgans under more direct illumination. In fact, inindividual leaves, dark-induced senescence was notreversed by a return to light; conversely, whole dark-ened plants exhibited delayed senescence relative tonontreated plants (Weaver and Amasino, 2001). Thus,two roles of RCBs, supply of temporal energy sourceunder plant carbon limitation and effective degrada-tion of chloroplasts under leaf carbon limitation, mayexist. RCBs that possibly contribute to the effectivedegradation of chloroplast in darkened leaves coop-erated with whole chloroplast degradation. Alterna-tively, because autophagic machinery may not havesufficient size capacity for the transportation of normalchloroplasts, shrinkage of chloroplast size by RCBsmay be a prerequisite for the degradation of wholechloroplasts.

The progression of leaf senescence is regulated byseveral interacting environmental signals, with nutri-ent status being one of the most important (Lim et al.,2003; Yoshida, 2003). Sugar is considered as an im-portant internal signal; it has been indicated thatleaf senescence is induced not only by sugar starva-tion caused by darkness but also by sugar accumula-tion (Weaver and Amasino, 2001; Pourtau et al., 2006;Wingler et al., 2006, 2009; van Doorn, 2008). Leaf car-bon limitation can be an important factor controllingsenescence in natural conditions. For example, in aplant canopy, upper leaves can shade older leaves,which can be a factor accelerating loss of nitrogen andshortening a leaf life span in shaded leaves (Oikawaet al., 2006). Our study suggests that RCBs can functionduring senescence under natural conditions depend-ing on leaf carbon status, particularly promoted bycarbon limitation with insufficient light.

RCB Production and Carbohydrate Metabolism inStarch-Related Mutants

The appearance of RCBs tended to be higher inleaves of starchless mutants and lower in starch-excessmutants compared with the wild type in both contin-uous light and day/night conditions (Figs. 5–8). Theseresults suggest that starch accumulation directly in-fluences RCB production. Leaf starch content alsoseemed to be a key factor of RCB production over adiurnal cycle (Fig. 4). Accumulation of starch granulesmay alter the tension of the chloroplast envelope. Twomechanosensory proteins, MSL2 and MSL3, whichhave been identified in the plastid envelope, controlplastid shape and size (Haswell and Meyerowitz,2006). Envelope tension seems to be a signal thatregulates RCB production. However, the appearanceof RCBs was also suppressed by light during leafincubation in starchless mutants similar to wild-typeplants (data not shown). Additionally, in nitrogen-limited plants, the suppression of RCB appearancecould be observed from 1 d after treatment, whilestarch accumulation was detected later (Fig. 10). Glcand Fru accumulated from 1 d after the start of thetreatment (Fig. 10). Thus, we cannot attribute thesuppression of RCB production simply as results ofleaf starch accumulation. Rather, we can understandthe changes of RCB production in starch-related mu-tants by referring to a diurnal metabolism of leafcarbohydrates. Carbon is assimilated during the day,with a part of the photoassimilate retained as starchin the chloroplast; however, at night, this starch isdegraded to support leaf respiration, allowing diurnalmaintenance of Suc levels in Arabidopsis leaves(Gibon et al., 2004; Smith and Stitt, 2007). In starchlessmutants, soluble sugars accumulated to higher levelsthan in the wild type during the day, but they rapidlydecreased and were almost completely consumed bythe middle of the night (Caspar et al., 1985; Gibonet al., 2004). Similarly, during incubation of excisedleaves in darkness, reduced starch levels appeared to

Figure 7. Effects of mutations affecting leaf starch content on theappearance of RCBs throughout leaf life span under a 14-h photope-riod. A, Changes of the appearance of RCBs in leaves of starch-relatedmutants under a 14-h photoperiod. Leaves of stroma-targeted GFPtransgenic plants of backgroundwild type (black squares), pgm-1 (blackcircles), adg1-1 (white circles), sex1-1 (black triangles), or mex1-3(white triangles) were excised 5 d before bolting and 0, 5, and 10 d afterbolting. Leaves were incubated at 23�C for 20 h in 10 mM MES-NaOH(pH 5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness.Leaves from six independent plants were incubated, eight quadrangularregions (188 mm 3 188 mm each) per leaf were monitored by LSCM,and the number of accumulated RCBs was counted at each stage. Datarepresent means 6 SE (n = 48). B, The transcription levels of SAG13,SAG12, and RBCS2B as marker genes for leaf senescence. Total RNAfrom third and fourth leaves of stroma-targeted GFP transgenic plants ofbackground wild type and each mutant during 10 d after bolting wasisolated and subjected to semiquantitative reverse transcription-PCRusing gene-specific primers. PCR products were separated by electro-phoresis, stained with SYBR Green I, and quantified by a fluorescenceimage analyzer. 18S ribosomal RNA was used as an internal control.

Izumi et al.




allow more rapid consumption of sugars in starchlessmutants, possibly resulting in increased production ofRCBs (Figs. 5–8). During a diurnal cycle in starch- andmaltose-excess mutants, soluble sugar levels weresimilar or slightly lower than wild-type levels duringthe night, although the supply of soluble sugars waspartially defective by lower starch degradation capac-ity (Caspar et al., 1991; Trethewey and ap Rees, 1994;Zeeman and ap Rees, 1999; Niittyla et al., 2004). Dur-ing the extended night, there was a more than 30%decrease in starch content in sex1 mutant leaves, whilemost of the starch was already consumed at the startof the extended night in the wild type (Caspar et al.,1991). During leaf incubation in darkness, solublesugars may have been supplied for a longer periodby degradation of starch in starch-excess mutants thanin wild-type plants, leading to lower production ofRCBs (Figs. 5–8). We propose that available carbohy-drate contents may be an important factor controllingRCB production in leaves, although its sensing mech-anisms are largely unknown. Starch contents are greatlydifferent from soluble sugar contents (Figs. 4, 5, and 8),indicating that starch is the main available carbohy-drate in darkness.Recently, Stettler et al. (2009) reported that the accu-

mulation ofmaltose andmalto-oligosaccharides inmex1mutants causes chloroplast dysfunction, which triggerschloroplast degradation, possibly by autophagy. In ourstudy, the appearance of RCBs was low in leaves of

mex1 plants (Figs. 6–8). However, we occasionally couldobserve aggregated or round structures exhibiting chlo-rophyll fluorescence in dark-incubated leaves of mex1plants expressing stroma-targeted GFP (data notshown). Vacuole-located chloroplasts transported byATG4 gene-dependent autophagy also exhibited chlo-rophyll fluorescence without stroma-targeted fluo-rescent proteins (Wada et al., 2009). In mex1 plants,only whole chloroplast autophagy, not RCBs, may beinduced. When naturally senescent leaves weresubjected to 20 h of darkness with concanamycin A,autophagy of only RCBs, but not whole chloroplasts,was observed (Fig. 1A; Ishida et al., 2008). Therefore,the results in mex1 plants also suggest that the partialmobilization of chloroplast components via RCBs andautophagy of whole chloroplasts may be differentiallyregulated depending upon the cellular status.

RCB Production Is Specifically Controlled in Autophagy

The effects of incubation conditions on the appear-ance of RCBs and non-RCB-type autophagic bodieswere not always the same (Fig. 3), indicating that thereexists a novel mechanism specifically controlling RCBproduction in the progression of autophagy. In yeastand animal cells, autophagy is induced by the starva-tion of either nitrogen or carbon sources (Takeshigeet al., 1992; Mizushima, 2005). Starvation-inducedautophagy has also been confirmed directly using a

Figure 8. The leaf carbohydrate contents in starch-related mutants under a 14-h photoperiod. A, The maximum number ofaccumulated RCBs in incubated leaves throughout leaf life span. The maximum number of accumulated RCBs in leaves ofstroma-targeted GFP transgenic plants of background wild type, pgm-1, adg1-1, sex1-1, or mex1-3 was excerpted from Figure7A. B and C, The leaf carbohydrate contents when the appearance of RCBs was maximum. The contents of starch (B) and Suc,Glc, and Fru (C) were determined in fourth leaves at 4 to 6 h into the photoperiod at 5 d after bolting in wild-type and mex1-3plants or at bolting in pgm-1, adg1-1, and sex1-1, when the appearance of RCBs after leaf incubation wasmaximum in Figure 7A.Data represent means6 SE (n = 48 [RCB number] or 4 [carbohydrate contents]). Statistical analysis was performed by Tukey’s test.Columns with the same letter were not significantly different (P # 0.05).





fluorescent marker, GFP-ATG8, in roots, hypocotyls,and cultured plant cells (Yoshimoto et al., 2004;Thompson et al., 2005; Xiong et al., 2005, Phillipset al., 2008). In our study, we grew plants autotrophi-cally and observed autophagy in leaves. Specific fea-tures of RCB induction may reflect differences inheterotrophs and autotrophs and also in heterotrophic

and autotrophic tissues in plants. In nature, plantshave to get carbohydrates by photosynthesis andassimilate inorganic nitrogen into organic compounds.Chloroplasts have a central role in both photosynthesisand nitrogen assimilation, and around 80% of totalleaf nitrogen is distributed to chloroplasts. Therefore,although the underpinning mechanisms are still

Figure 10. Effects of nitrogen limitation on the appearance of RCBs and leaf carbohydrate contents. A, Effects of nitrogenlimitation on the appearance of RCBs. Fourth leaves of stroma-targeted GFP-expressing plants under nitrogen-sufficient (blacksquares) and nitrogen-limited (white circles) conditions were excised over the treatment period and incubated at 23�C for 20 h in10 mM MES-NaOH (pH 5.5) with 1 mM concanamycin A and 100 mM E-64d in darkness. Leaves from four independent plantswere incubated, eight quadrangular regions (188 mm 3 188 mm each) per leaf were monitored by LSCM, and the number ofaccumulated RCBs was counted at each period. Data represent means 6 SE (n = 32). B, The leaf carbohydrate contents innitrogen-limited plants. Changes in the starch, Suc, Glc, and Fru contents were measured in fourth leaves of plants undernitrogen-sufficient (black squares) and nitrogen-limited (white circles) conditions over the treatment period. Data representmeans 6 SE (n = 3).

Figure 9. Nitrogen-limited senescence. A, Photographs of plants under nitrogen-sufficient condition as a control (top image) ornitrogen-limited condition (bottom image) at 5 d after treatment. Stroma-targeted GFP-expressing plants were hydroponicallygrown with nitrogen-rich solution. When plants started bolting (23 d after sowing), hydroponic solution was replaced with eitherthe same nitrogen-rich solution as the control or nitrogen-free solution, and plants were grown for 5 d. Bars = 10 mm. B, Changesin the chlorophyll, nitrogen, soluble protein, and Rubisco protein contents in leaves of plants under nitrogen-sufficient (blacksquares) and nitrogen-limited (white circles) conditions over the treatment period. Data represent means 6 SE (n = 3).

Izumi et al.




unknown, it seems reasonable to consider that au-tophagy of chloroplast proteins must be specificallycontrolled. In yeast, some selective autophagy path-ways have been identified (e.g. cytoplasm-to-vacuoletargeting pathway, mitophagy, pexophagy, and alsospecific ATG genes required for respective pathwayswere identified; for review, see Kraft et al., 2009;Nakatogawa et al., 2009). Our study is suggestive ofthe existence of novel plant ATG genes specificallyrequired for the autophagic degradation of chloro-plasts, and identification of such genes may be effec-tive for elucidating the regulatory mechanism of RCBproduction and specific features of plant autophagy.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Transgenic Arabidopsis (Arabidopsis thaliana) ecotype Columbia expressing

CT-GFP and ecotype Wassilewskija expressing both stroma-targeted DsRed

and the GFP-ATG8a fusion protein were described previously (Ishida et al.,

2008). Transgenic Arabidopsis ecotype Columbia expressing SP-GFP-2SC was

described previously (Tamura et al., 2003). The seeds of Arabidopsis mutant

allele pgm1-1 (Caspar et al., 1985), adg1-1 (Lin et al., 1988), sex1-1 (Caspar et al.,

1991), and the T-DNA insertion mutantmex1-3 (SAIL_574_D11) were obtained

from the Arabidopsis Biological Resource Center. mex1-3 was identified by

confirming homozygosity using PCR with gene-specific primers. Further-

more, the purportedmex1-3 plants exhibited the same phenotype as the report

that first identified mex1 mutants (Niittyla et al., 2004). These mutants were

crossed to transgenic plants expressing stroma-targeted GFP. Homozygosity

of pgm-1, adg1-1, and sex1-1 was confirmed by sequencing regions including

G-to-A substitutions of PGM1 (Periappuram et al., 2000), ADG1 (Wang et al.,

1998), and SEX1 (Yu et al., 2001) in each mutant, respectively. Homozygosity

ofmex1-3was confirmed by PCR using gene-specific primers. Under long-day

conditions, plants were grown on soil (Metro-Mix 350; Sun Gro Horticulture)

on a 14-h-light/10-h-dark photoperiod with fluorescent lamps (100 mmol

quanta m22 s21) at 23�C day/18�C night temperatures. Under continuous light

conditions, plants were grown on the same soil with fluorescent lamps

(60 mmol quanta m22 s21) at 23�C. For nitrogen-limitation treatment, CT-GFP

plants were grown hydroponically on horticultural rockwools as described

previously (Wada et al., 2009). The hydroponic solution contained 3mMKNO3,

2.5 mM potassium phosphate buffer (pH 5.5), 2.0 mM Ca(NO3)2, 2.0 mM MgSO4,

50 mM Fe-EDTA, 0.7 mM H3BO4, 170 mM MnCl2, 2.0 mM Na2MoO4, 100 mM NaCl,

5.0 mM CuSo4, 10 mM ZnSO4, and 0.1 mM CoCl2. When the plants started bolting,

the rockwools were washed with deionized water and the nutrient solution

was replaced with nitrogen-free solution. The nitrogen-free solution was

prepared by replacing KNO3 and Ca(NO3)2 with KCl and CaCl2, respectively.

Image Analysis with LSCM

LSCMwas performed with a Nikon C1si system equipped with a CFI Plan

Apo VC603water-immersion objective (numerical aperture = 1.20; Nikon) as

described previously in detail (Ishida et al., 2008). The signals of GFP and

DsRed were obtained using the unmixing program of Nikon C1si software

from fluorescence spectra between 500 and 650 nm.

Detection of RCBs and Autophagic Bodies

Fourth rosette leaves were excised from four to six independent plants

expressing CT-GFP 4 to 6 h into the photoperiod. Incubation buffer containing

10 mM MES-NaOH (pH 5.5), 1 mM concanamycin A, and 100 mM E-64d was

infiltrated into excised leaves, which were then incubated for 20 h at 23�C in

darkness. When effects of nutrient factors during leaf incubation on RCB

production were examined, full-strength MS medium and various constitu-

ents were added to the incubation buffer, or light (50 mmol quanta m22 s21)

was irradiated. The concentration of Suc, Glc, Fru, and mannitol added was

3% (w/v), and DCMU was 10 mM. In incubation with nitrogen-free MS

medium (MS-N), MS-N medium was prepared by removal of NH4NO3 and

replacing the KNO3 with KCl. When diurnal effects were examined, leaves

were excised at three time points representing the end of the regular night,

the end of the prolonged night, and the end of the day, and incubation time

was 10 h as described in Figure 4A. After incubation, each leaf was divided

into four parts, and two quadrangular regions (188 mm3 188 mmeach) in each

section were monitored by LSCM and images were obtained. The number

of accumulated RCBs in the images was counted. Each image was considered

an independent data point and subjected to statistical analysis.

The analysis of production of RCBs and autophagosomes in plants

expressing stroma-targeted DsRed and the GFP-ATG8 fusion was performed

in the same way, except that leaves from six independent plants at 25 d after

sowing (5 d after bolting) were used. The number of RCBs and non-RCB-type

autophagic bodies that do not exhibit DsRed was counted in each image.

Sugar Analysis

Leaves for carbohydrate quantification were rapidly frozen in liquid N2 and

then stored at 280�C until analysis. Frozen leaves were homogenized in a

liquid N2-chilled tube with a pestle. The resulting powder was immediately

extracted four times with 80% (v/v) ethanol at 80�C (Nakano et al., 1995), and

the extractant was pooled and concentrated by vacuum evaporation. The

residuewas dissolved in distilledwater, and the concentrations of Suc, Glc, and

Fru were determined using an F-kit for a-Glc/Suc/Fru (Roche Diagnostics).

The 80% ethanol-insoluble fraction was dried overnight at room temper-

ature. Starch in the ethanol-insoluble fraction was extracted with 0.6 mL of

dimethyl sulfoxide and 0.15 mL of 8 M HCl at 60�C for 30 min, and the pH

was neutralized to between 4.0 and 5.0 with 0.1 mL of 2 M Na-acetate buffer

(pH 4.5) and 8 M NaOH. The solution was made up to its final volume of

1.0 mLwith distilled water. The starch concentration was determined using an

F-kit for starch (Roche Diagnostics).

Quantification of Chlorophyll, Nitrogen, SolubleProteins, and Rubisco Protein

Frozen fourth rosette leaves were homogenized in an ice-chilled mortar

and pestle in 50 mM Na-phosphate buffer (pH 7.5) containing 2 mM iodole-

acetic acid, 0.8% (v/v) 2-mercaptoethanol, and 5% (v/v) glycerol (Makino

et al., 1994). Chlorophyll was determined by the method of Arnon (1949).

Soluble protein content was measured in supernatant of part of the homog-

enate according to Bradford (1976) using a Bio-Rad protein assay with bovine

serum albumin as the standard. Total leaf nitrogen was determined from part

of the homogenate with Nessler’s reagent after Kjeldahl digestion. Triton

X-100 (0.1% final concentration) was added to the remaining homogenate.

After centrifugation, the supernatant was mixed with an equal volume of

SDS sample buffer containing of 200 mM Tris-HCl (pH 8.5), 2% (w/v) SDS,

0.7 M 2-mercaptoethanol, and 20% (v/v) glycerol, boiled for 3 min, and then

subjected to SDS-PAGE. The Rubisco content was determined spectrophoto-

metrically by formamide extraction of the Coomasie Brilliant Blue R-250-stained

bands corresponding to the large and small subunits of Rubisco separated by

SDS-PAGE. Calibration curves were made with bovine serum albumin.

Semiquantitative Reverse Transcription-PCR Analysis

Total RNA was isolated from leaves using the RNeasy Plant Mini Kit

(Qiagen). Isolated RNAwas treated with DNase (DNA-free; Ambion) prior to

the synthesis of first-strand cDNAwith random hexamer primers (SuperScript

III first-strand synthesis system; Invitrogen). An aliquot of the synthesized

first-strand cDNA derived from 1.7 ng of total RNA was used for PCR

amplification (total volume of 6 mL) with Taq polymerase (PrimeSTAR;

TAKARA). The gene-specific primer pairs and the number of PCR cycles

were as follows: 5#-CAGCTTGCCCACCCATTGTTA-3# and 5#-GTCGTA-

CGCACCGCTTCTTTCTTA-3#, 28 cycles for SAG13 (Barth et al., 2004);

5#-GATGAAGGCAGTGGCACACCAA-3# and 5#-TCCCACACAAACA-

TACACAATTAAAAGC-3#, 24 cycles for SAG12 (Panchuk et al., 2005);

5#-ACCTTCTCCGCAACAAGTGG-3# and 5#-GAAGCTTGGTGGCTTGT-

AGG-3#, 18 cycles for RBCS2B (Acevedo-Hernandez et al., 2005); and Quan-

tumRNA 18S internal standard (Ambion), 14 cycles for 18S ribosomal RNA.

Supplemental Data

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





Supplemental Figure S1. Effects of incubation conditions on the accumu-

lation of vacuole-targeted GFP in vacuolar lumens.

Supplemental Figure S2.Visualization of stroma-targeted GFP in leaves of

starch-related mutants.

Supplemental Figure S3. Changes of leaf area in starch-related mutants

under a 14-h photoperiod.

ACKNOWLEDGMENTS

We thank Dr. Louis Irving for critical reading of the manuscript, Dr.

Maureen R. Hanson for the use of transgenic Arabidopsis expressing CT-

GFP and critical reading of the manuscript, Dr. Kohki Yoshimoto and Dr.

Yoshinori Ohsumi for the use of transgenic Arabidopsis expressing GFP-

ATG8, Dr. Ikuko Hara-Nishimura for providing seeds of transgenic Arabi-

dopsis expressing SP-GFP-2SC, and the Arabidopsis Biological Resource

Center and original donors for providing seeds of starch-related mutant

Arabidopsis.

Received April 27, 2010; accepted August 30, 2010; published August 31, 2010.

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the autophagic degradation of chloroplasts via rubisco ......the majority of plant nitrogen and...

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