Altered membrane lipase expression delays leaf senescence

A cDNA clone encoding a lipase that is up-regulated in senescing leaves and flower petals has been isolated by screening an expression library. The abundance of the lipase mRNA increases as flowers and leaves begin to senesce, and expression of the gene is also induced by treatment with ethylene. Transgenic Arabidopsis plants in which levels of the senescence-induced lipase protein have been reduced show delayed leaf senescence.     

arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts

A novel mutant of Arabidopsis thaliana, arc6 (accumulation and replication of chloroplasts), has been isolated from a transfer DNA-mutagenized population of Arabidopsis seedlings. arc6 has the most extreme arc mutant phenotype we have yet described, with only one to three chloroplasts per leaf mesophyll cell compared to a mean of 83 in cells of the wild-type var Wassilewskija. The chloroplasts of arc6 are 20-fold larger than wild-type chloroplasts.Chloroplast division is almost certainly precluded in arc6 mesophyll cells, since chloroplast number per cell does not increase during mesophyll cell expansion. arc6 chloroplasts are long and thin in cross-section and only one-half the width of wild-type chloroplasts and the arrangement of thylakoid membranes is largely unaltered. arc6 segregates as a monogenic recessive nuclear mutation in a normal Mendelian manner and the arc6 phenotype is stably inherited for at least four generations. arc6 plants grow normally and are fertile, although the rosette leaves appear curled and twisted. arc6 plants accumulate 70 to 75% of the biomass of wild type. The phenotype of this novel mutant is discussed in relation to the nature of the control of chloroplast division in leaf cells.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts.     

The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis

This report describes the characterisation of ATHB16, a novel Arabidopsis thaliana homeobox gene, which encodes a homeodomain-leucine zipper class I (HDZip I) protein. We demonstrate that ATHB16 functions as a growth regulator, potentially as a component in the light-sensing mechanism of the plant. Endogenous ATHB16 mRNA was detected in all organs of Arabidopsis, at highest abundance in rosette leaves. Reduced levels of ATHB16 expression in transgenic Arabidopsis plants caused an increase in leaf cell expansion and consequently an increased size of the leaves, whereas leaf shape was unaffected. Transgenic plants with increased ATHB16 mRNA levels developed leaves that were smaller than wild-type leaves. Therefore, we suggest ATHB16 to act as a negative regulator of leaf cell expansion. Furthermore, the flowering time response to photoperiod was increased in plants with reduced ATHB16 levels but reduced in plants with elevated ATHB16 levels, indicating that ATHB16 has an additional role as a suppressor of the flowering time sensitivity to photoperiod in wild-type Arabidopsis. As deduced from the response of transgenic plants with altered levels of ATHB16 expression in hypocotyl elongation assays, the gene may act to regulate plant development as a mediator of a blue light response.     

Characterization of an ultraviolet B-induced lipase in Arabidopsis

An Arabidopsis expressed sequence tag clone, 221D24, encoding a lipase has been characterized using an antisense approach. The lipase gene is expressed during normal growth and development of Arabidopsis rosette leaves but is down-regulated as the leaves senesce. When plants are exposed to sublethal levels of UV-B radiation, expression of the lipase is strongly up-regulated. The lipase protein is localized in the cell cytosol and is present in all organs of Arabidopsis plants. Recombinant lipase protein produced in Escherichia coli preferentially hydrolyzed phospholipids, indicating that the gene encodes a phospholipase. Transgenic plants in which lipase expression is suppressed showed enhanced tolerance to UV-B stress but not osmotic stress and were unable to up-regulate PR-1 expression when irradiated with UV-B. The observations collectively indicate that the lipase is capable of deesterifying membrane phospholipids and is up-regulated in response to UV-B irradiation.     

Control of starch granule numbers in Arabidopsis chloroplasts

The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of clp genes expressed in senescing Arabidopsis leaves.

Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola

Deoxyhypusine synthase (DHS; EC 2.5.1.46) mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-5A (eIF-5A) to an activated form, thought to facilitate translation. A full-length cDNA clone encoding canola ( Brassica napus cv. Westar) DHS was isolated from a cDNA-expression library prepared from senescing leaves. Transgenic canola lines with suppressed DHS expression were obtained by introducing a transgene expressing antisense 3'-UTR canola DHS cDNA under the regulation of the constitutive cauliflower mosaic virus 35S (CaMV-35S) promoter. Transformed seed was obtained by vacuum infiltration of canola inflorescences using the protocol developed for Arabidopsis with modifications. The resultant transgenic plants had reduced levels of leaf DHS protein and exhibited delayed natural leaf senescence. Suppression of DHS also increased leaf size by 1.5- to two-fold and resulted in increases in seed yield of up to 65%. Moreover, the enhanced performance of transgenic plants reflected increased tolerance to chronic sublethal stress. When wild-type and transgenic plants were grown in 6-inch pots, the increase in seed yield accruing from suppression of DHS was approximately 4.5-fold greater than when the plants were grown in 12-inch pots. Thus, suppression of DHS appears to ameliorate the effects of sublethal stress engendered by growth in small containers.     

A maltose transporter from apple is expressed in source and sink tissues and complements the Arabidopsis maltose export-defective mutant

Prior to the cytosolic synthesis of transport sugars during transitory starch utilization, intermediate products of starch breakdown, such as maltose, must be exported from chloroplasts. Recent work in Arabidopsis indicates that a novel transporter mediates maltose transfer across the chloroplast inner envelope membrane. We cloned a gene from an apple cDNA library that is highly homologous with the Arabidopsis maltose transporter, MEX1. Expression levels of MdMEX determined by real-time PCR were low in the tips of growing shoots, higher in expanding leaves and maximal in mature leaves. Expression was also detected in fruits and roots, indicating a role for MdMEX in starch mobilization in sink tissues. The cDNA from apple was subcloned into an expression cassette between the cauliflower mosaic virus 35S promoter and the sGFP (green fluorescent protein) coding sequence. Plants of the Arabidopsis maltose excess1-1 mutant, which is homozygous for a defective MEX1 allele, were transformed with the 35S:MdMEX:GFP construct. Fluorescence of GFP was localized to chloroplasts, indicating that Arabidopsis recognized the predicted 55 amino acid chloroplast transit peptide in the apple protein. The phenotypes of several independently transformed lines were analyzed. The complemented plants were relieved of the severe stunting and chlorosis characteristic of mex1-1 plants. Furthermore, starch levels and concentrations of soluble sugars, leaf chlorophyll content and maximum quantum efficiency of PSII were restored to wild-type levels. MdMEX (Malus domestica maltose transporter) is the second member of the unique maltose transporter gene family.     

The impact of alteration of polyunsaturated fatty acid levels on C6-aldehyde formation of Arabidopsis thaliana leaves.

C6-aldehydes are synthesized via lipoxygenase/hydroperoxide lyase action on polyunsaturated fatty acid (PUFA) substrates in plant leaves. The source pools and subcellular location of the processes are unknown. A close relationship is found between the composition of PUFA and the composition of C6-aldehydes. In the current study, this relationship was tested using the Arabidopsis PUFA mutant lines act1, fad2, fad3, fad5, fad6, and fad7. The results indicate that C6-aldehyde formation is influenced by the alteration of C18 PUFA levels. Mutants act1 and fad5, which are deficient in C16 unsaturated fatty acids, had wild-type levels of C6-aldehyde production. Mutants deficient in the chloroplast hexadecenoic acid/oleic acid desaturase (fad6) or hexadecadienoic acid/linoleic acid desaturase (fad7) had altered C6-aldehyde formation in a pattern similar to the changes in the PUFA. Mutations that impair phosphatidylcholine desaturase activity, such as fad2 and fad3, however, resulted in increased E-2-hexenal formation. The enzymes involved in C6-aldehyde production were partially characterized, including measurement of pH optima. The differences in C6-aldehyde formation among the fatty acid mutants of Arabidopsis appeared not to result from alteration of lipoxygenase/hydroperoxide lyase pathway enzymes. Investigation of the fatty acid composition in leaf phospholipids, glycolipids, and neutral lipids and analysis of the fatty acid composition of chloroplast and extrachloroplast lipids indicate that chloroplasts and glycolipids of chloroplasts may be the source or major source of C6-aldehyde formation in Arabidopsis leaves.     

A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts

We generated transgenic tobacco (Nicotiana tabacum cv Xanthi) plants that contained only one to three enlarged chloroplasts per leaf mesophyll cell by introducing NtFtsZ1-2, a cDNA for plastid division. These plants were used to investigate the advantages of having a large population of small chloroplasts rather than a few enlarged chloroplasts in a leaf mesophyll cell. Despite the similarities in photosynthetic components and ultrastructure of photosynthetic machinery between wild-type and transgenic plants, the overall growth of transgenic plants under low- and high-light conditions was retarded. In wild-type plants, the chloroplasts moved toward the face position under low light and toward the profile position under high-light conditions. However, chloroplast rearrangement in transgenic plants in response to light conditions was not evident. In addition, transgenic plant leaves showed greatly diminished changes in leaf transmittance values under both light conditions, indicating that chloroplast rearrangement was severely retarded. Therefore, under low-light conditions the incomplete face position of the enlarged chloroplasts results in decreased absorbance of light energy. This, in turn, reduces plant growth. Under high-light conditions, the amount of absorbed light exceeds the photosynthetic utilization capacity due to the incomplete profile position of the enlarged chloroplasts, resulting in photodamage to the photosynthetic machinery, and decreased growth. The presence of a large number of small and/or rapidly moving chloroplasts in the cells of higher land plants permits more effective chloroplast phototaxis and, hence, allows more efficient utilization of low-incident photon flux densities. The photosynthetic apparatus is, consequently, protected from damage under high-incident photon flux densities.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.     

Chloroplasts autophagy during senescence of individually darkened leaves

We recently reported that autophagy plays a role in chloroplasts degradation in individually-darkened senescing leaves. Chloroplasts contain approximately 80% of total leaf nitrogen, mainly as photosynthetic proteins, predominantly ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth. Concomitantly, while decreasing in size, chloroplasts undergo transformation to non-photosynthetic gerontoplasts. Likewise, over time the population of chloroplasts (gerontoplasts) in mesophyll cells also decreases. While bulk degradation of the cytosol and organelles is mediated by autophagy, the role of chloroplast degradation is still unclear. In our latest study, we darkened individual leaves to observe chloroplast autophagy during accelerated senescence. At the end of the treatment period chloroplasts were much smaller in wild-type than in the autophagy defective mutant, atg4a4b-1, with the number of chloroplasts decreasing only in wild-type. Visualizing the chloroplast fractions accumulated in the vacuole, we concluded that chloroplasts were degraded by two different pathways, one was partial degradation by small vesicles containing only stromal-component (Rubisco containing bodies; RCBs) and the other was whole chloroplast degradation. Together, these pathways may explain the morphological attenuation of chloroplasts during leaf senescence and describe the fate of chloroplasts.Key words: Arabidopsis, autophagy, chloroplast, dark treatment, leaf senescence, nutrients recyclingThe most abundant chloroplast protein is Rubisco, comprising approximately 50% of the soluble protein.¹ The amount of Rubisco decreases rapidly in the early phase of leaf senescence, and more slowly in the later phase. During senescence, chloroplasts gradually shrink and their numbers gradually decrease in mesophyll cells.^2,3 During leaf senescence, leaves lose approximately 75% of their Rubisco, while chloroplast numbers decrease by only about 15%.⁴ Previous studies showed chloroplasts localized within the central vacuole by electron microscopy, indicating chloroplast degradation in the highly hydrolytic vacuole.⁵ However, there was no direct evidence showing translocation of chloroplasts from the cytosol to the vacuole, and the mechanism of transportation was also unclear.Recent reverse genetic approaches are helping to elucidate the autophagy system in plants, which has a similar molecular mechanism as in yeast.^6–11 In Arabidopsis (Arabidopsis thaliana), atg mutants have phenotypically accelerated leaf senescence, insufficient root elongation in nutrient starvation condition and reduced seeds yields, therefore, autophagy is considered to be important for nutrient recycling especially nutrient starvation and senescence in plants.¹²In Arabidopsis, individually darkened rosette leaves (IDLs) exhibit enhanced senescence.¹³ Appling IDLs treatment as an experimental model of leaf senescence, we recently demonstrated that chloroplasts are degraded in two different pathways by autophagy, one for RCBs,^14,15 and one for whole chloroplast.¹⁶ Darkened leaves became pale in 3 to 5 days treatment, while illuminated parts normally grow in both wild-type and autophagy defective mutant, atg4a4b-1. Furthermore, genes specifically expressed during senescence, SAG12 and SEN1, were rapidly upregulated, meanwhile, photosynthetic genes, such as RBCS2B and CAB2B, were gradually downregulated. All analyzed ATG genes were also upregulated under IDL treatment, which suggests that autophagy is important in IDL senescence. It has been reported that approximately three quarter genes of upregulated in IDL were also upregulated in naturally senescing leaves, including the ATG genes.¹⁷ This suggests that the autophagy pathways used in IDLs are also used in naturally senescing leaves.Over the 5 day treatment period, chloroplasts of wild-type IDL shrink to approximately one third their original size. In atg4a4b-1, by contrast, chloroplasts shrinkage occurred immediately after the start of IDL treatment after which no further shrinkage was noted. While the shrunk chloroplasts in fixed cells of wild-type were still smooth and round, while wrinkly chloroplasts were observed in atg4a4b-1. At same time, in the living mesophyll cells of wild-type IDL, RCBs accumulated in the vacuole (Fig 1B). The shrinkage of chloroplasts may be due to the consumption of the chloroplast envelope by RCB formation. Immunological quantification of inner and outer envelope proteins might confirm this hypothesis. The chloroplast number was also gradually decreased in IDL of wild-type plants, but no decline in chloroplast number was noted in atg4a4b-1. Chloroplasts exhibiting chlorophyll auto-fluorescence were found in the vacuole of wild-type IDLs, but not in atg4a4b-1 IDLs. These results show that whole chloroplast degradation is also performed by autophagy. However, the transport pathway of whole chloroplasts into the vacuole remains unclear. The chloroplast, even in its shrunken state, is a large organelle, and the autophagosome, the carrier bodies of autophagy, which usually target small spherical organelles like mitochondria and peroxisomes, may be incapable of isolating large organelles. In the yeast autophagy system, specific cellular organelles and fractions are also transported via vacuolar membrane invagination using the microautophagy system.¹⁸ RCB uptake into the vacuole is termed macroautophagy, while larger organelles, such as chloroplasts, are engulfed in a process known as microautophagy. Whether there exists a molecular difference between these processes, or whether this is an arbitrary division based solely on the size of the consumed body is unclear.Open in a separate windowFigure 1Visualization of stroma-targeted DsRed and chlorophyll autofluorescence in living mesophyll cells of wild-type plants by laser-scanning confocal microscopy. A excised control leaf (A, Light) and an individually darkened leaf (B, IDL) from plants grown under 14 h-photoperiod condition and a leaf from whole-plant darkened condition (WD, C) for 5days were incubated with 1 µM concanamycin A in 10 mM MES-NaOH (pH 5.5) at 23C° for 20 h in darkness. Stroma-targeted DsRed appears green and chlorophyll fluorescence appears red. In merged images, overlap of DsRed and chlorophyll fluorescence appears yellow. Small vesicles with stromal-targeted DsRed, i.e. RCBs, can be found in the vacuole (A, B). In IDL (B), massive accumulation of stroma-targeted DsRed is entirely seen in the vacuolar lumen and chloroplasts losing DsRed fluorescence are found in some cells. Bars = 50 µm.Whole darkened plants exhibit retarded leaf aging, in contrast to the accelerated senescence in IDLs.¹³ Whole darkened plants suppress leaf senescence with the leaves retaining green color. After 5 days, in the mesophyll cells of whole darkened plants, any translocation of chloroplast components, stroma-targeted DsRed, RCBs, and whole chloroplasts, into the vacuole could hardly be detected (Fig. 1C). This suggests that autophagy is not induced by darkness alone, and is associated closely with senescence. ATG genes were downregulated in the whole darkened wild-type plants less than control plants during the treatment. Previous studies have shown that following about 5 day period of whole plant darkening, atg mutants lose their ability to protect themselves against photo-damage.⁷ Upon return to the light, these plant quickly undergo terminal photo-bleaching.Concentrations of chlorophyll, soluble protein, leaf nitrogen and Rubisco rapidly declined under IDL condition of both wild-type and atg4a4b-1. Considering the accumulated fluorescence of stroma-targeted Ds-Red in the vacuole and autophagy dependent size shrinkage of chloroplasts in IDL, in wild-type plants RCB autophagy appear to be responsible for a sizable proportion of chloroplast protein degradation. In atg4a4b-1 which cannot form RCBs, alternative degradation pathways must be upregulated, with chloroplast proteases the most likely candidates. Intriguingly, the decrease in Rubisco concentration proceeds at the almost identical rates in both wild-type and atg4a4b-1 plants, despite the different degradation pathways. It seems likely that the rate of Rubisco degradation may be regulated at an early step in the degradation pathway, by some, as yet unknown, factors.Chloroplasts appear to have the ability to control their volume during cell division, dividing and increasing their density up to the certain level,¹⁹ and transferring their cellular components between them via stromules.²⁰ How chloroplasts are able to regulate their volume remains unclear, but it seems likely that chloroplasts grow and divide, like any other bacteria, as long as sufficient resources remain in the environment, in this case the cell. Total chloroplast volume, therefore, may be limited by the availability of carbon, nitrogen, or other nutrients in the cell during leaf emergence. Chloroplasts may be also able to reduce and control their volumes during leaf senescence via multiple degradation pathways. Our next goal is to estimate the contribution of both RCBs and whole chloroplasts autophagy in chloroplast protein degradation during natural leaf senescence. Further investigations are required for understanding the specific molecular mechanisms of RCB production and whole chloroplast degradation.