Stromal protein degradation is incomplete in Arabidopsis thaliana autophagy mutants undergoing natural senescence
© Lee et al.; licensee BioMed Central Ltd. 2013
Received: 21 December 2012
Accepted: 15 January 2013
Published: 17 January 2013
Degradation of highly abundant stromal proteins plays an important role in the nitrogen economy of the plant during senescence. Lines of evidence supporting proteolysis within the chloroplast and outside the chloroplast have been reported. Two extra-plastidic degradation pathways, chlorophagy and Rubisco Containing Bodies, rely on cytoplasmic autophagy.
In this work, levels of three stromal proteins (Rubisco large subunit, chloroplast glutamine synthetase and Rubisco activase) and one thylakoid protein (the major light harvesting complex protein of photosystem II) were measured during natural senescence in WT and in two autophagy T-DNA insertion mutants (atg5 and atg7). Thylakoid-localized protein decreased similarly in all genotypes, but stromal protein degradation was incomplete in the two atg mutants. In addition, degradation of two stromal proteins was observed in chloroplasts isolated from mid-senescence leaves.
These data suggest that autophagy does contribute to the complete proteolysis of stromal proteins, but does not play a major degenerative role. In addition, support for in organello degradation is provided.
KeywordsAutophagy Leaf senescence Stromal protein degradation
Stromal proteins in C3 mesophyll chloroplasts contain approximately 55% of total cellular nitrogen, mostly in the form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), while approximately 20% of total nitrogen is allocated to thylakoid proteins . During senescence most of the nitrogen from these two sources is exported from the aging leaf [2, 3], but the proteolytic process is not well understood [4–6]. Genetic approaches towards understanding senescence have focused on the isolation of stay-green mutants, and these studies have shown that stromal and thylakoid proteolysis can be uncoupled. One class of stay-green mutants, nonfunctional type C, retain thylakoid-localized light harvesting complex proteins while stromal proteins are degraded [7, 8].
The high nitrogen content of stromal proteins has led to extensive investigation of their proteolysis during leaf senescence. No chloroplast proteases specifically involved in Rubisco or other stromal protein degradation have been identified to date . A Zn-dependent EP1 protease activity was partially purified , but no corresponding gene or gene product has been reported. Chloroplast stromal Clp proteases are likely candidates for stromal protein degradation during senescence, however the protein levels of the catalytic ClpP subunit were observed to be greatly diminished in older leaves .
Active oxygen treatment led to Rubisco cleavage in isolated chloroplasts  and in chloroplast lysates [13, 14]. These findings suggested that stromal protein degradation could occur within chloroplasts with high levels of free radicals, a likely condition during the later stages of senescence. However, Rubisco degradation begins during the earliest stages of senescence  when photosynthesis is still occurring and free radicals are actively scavenged. For this reason, purified, intact chloroplasts were incubated in the dark to determine if stromal protein degradation could occur in the absence of free radical formation. These chloroplasts were re-purified to be certain they remained intact during the incubation period  and four stromal proteins were found to be degraded within intact plastids . Thus, numerous lines of evidence suggest that stromal protein degradation can occur within chloroplasts. However, a cysteine protease inhibitor (cystatin) predominantly expressed in tobacco cytosol inhibited Rubisco degradation in older leaves suggesting that stromal protein degradation is occurring outside of the plastid as well .
Although chloroplast numbers only decrease slightly during natural senescence , whole chloroplast engulfment via autophagy (chlorophagy) has been observed in individually darkened leaves . The dependence on autophagosome formation was demonstrated by the lack of chlorophagy in the Arabidopsis atg4a4b double mutant, however, Rubisco protein levels were found to decrease similarly to wild type in individually darkened leaves of atg4a4b mutants . Thus the contribution of chlorophagy to total stromal protein degradation is likely minimal. As most chloroplasts remain intact until the final stages of senescence, extra-plastidic pathways specific to the disposal of stroma proteins have been identified. There have been numerous reports of plastid protuberances that contain Rubisco [20–22], and two distinct entities, Rubisco Containing Bodies (RCBs) and Senescence Associated Vacuoles (SAVs), have been identified.
RCBs are 0.5 to 1.5 μm in diameter, cross-react with antibodies to Rubisco LSU, SSU and chloroplast glutamine synthase (GS2), and have multiple membranes . Stromal-targeted GFP lines have been used to detect RCBs within vacuoles of concanamycin-A treated cells in which vacuolar proteolysis has been prevented due to inhibition of vacuolar-H+ ATPases . RCBs appear as Rubisco levels decline in the primary leaves of wheat and are not formed in Arabidopsis atg5 mutants . ATG5 is required for ATG8 lipidation, and atg5 mutants cannot form autophagosomes [26, 27]. A further connection between autophagy and RCBs is the colocalization of stromal-targeted DsRed and GFP-ATG8, the molecule that coats the autophagosome [28, 29]. The presence of RCBs is inversely correlated to starch levels , but how this correlates to Rubisco levels is not clear. The decline in Rubisco during natural senescence was measured with RBCS-mRFP fusions, and 10% of the transgenic fusion protein degradation was estimated to be autophagy-dependent .
SAVs are 0.5 to 0.8 μm in diameter and were first detected by R-6502, a cysteine protease substrate that becomes fluorescent upon cleavage . Senescent-specific SAVs are acidic compartments that stain with Lysotracker Red and harbor SAG12, a senescence-specific cysteine protease. SAV membranes contain vacuolar H+-ATPases, and thus SAVs are considered to be vacuolar compartments. SAVs have also been detected in the atg7 mutant (which is inhibited at a similar phase of autophagosome formation as atg5) indicating SAV formation is not dependent on functional autophagy. SAVs purified on sucrose gradients contained stromal proteins, but not thylakoid proteins, and slow degradation of Rubisco LSU was observed in the isolated SAVs .
Nitrogen remobilization efficiency (NRE) was measured in three different Arabidopsis autophagy mutants (atg5, atg9 and atg18 RNAi) by a 15N pulse treatment of leaves and then subsequent transfer of 15N into seeds during plant growth . NRE was significantly lower in all autophagy mutants suggesting that autophagy does contribute to nitrogen remobilization. In this study, levels of three native stromal proteins were measured during natural senescence in two autophagy mutants, atg5 and atg7, in order to directly assess the contribution of autophagy towards stromal protein degradation. In addition, degradation of stromal proteins was evaluated in chloroplasts isolated from fully-expanded mid-senescent leaves. Our data provide supporting evidence that autophagy does contribute to stromal, but not to thylakoid, protein degradation, and that stromal proteins might be degraded in organello.
Results and discussion
Stromal protein degradation is incomplete in autophagy mutants
Concern exists that the higher stromal protein levels in the younger atg tissue resulted from less time for stromal protein degradation and were not related to the loss of autophagy. Double mutants have been constructed between atg5 and NahG as well as sid2 that decrease SA levels and thus reverse the early senescence phenotype . However the prevention of SA accumulation by NahG and sid2 does increase leaf longevity [41, 42] and thus can over-compensate for the early activation of the SA signaling pathway since SA can never accumulate, even at the proper developmental time. Thus an autophagy mutant in a background with normal timing of natural senescence does not yet exist. In addition, if the retention of the three stromal proteins was a result of faster senescence, and not the loss of autophagy, this would indicate that autophagy plays no role in stromal protein degradation, which would be inconsistent with previously published results .
Proteolysis in isolated Arabidopsis chloroplasts
In an effort to identify chloroplast proteases that might contribute to stromal protein degradation, we isolated T-DNA insertions that disrupted At5g11650, a gene encoding a serine protease that is strongly up-regulated in senescent leaves . At5g11650 is distantly related to pheophytinase , but At5g11650 mutants display normal loss of chlorophyll in older leaves. Stromal protein degradation was identical in chloroplasts isolated from WT and At5g11650 mutant chloroplasts demonstrating that this chloroplast-localized serine protease is unlikely to play a major role in stromal protein degradation (data not shown).
Overall, our data suggest that complete degradation of stromal proteins requires autophagy-dependent processes, but much of stromal protein degradation relies on autophagy-independent pathways which may include proteolysis within the chloroplasts or SAVs.
Plant material and growth conditions
Arabidopsis plants were grown under continuous white light (70 μmoles photons m-2 sec-1) at 24°C in Sunshine Mix #1/LC1 (Sun Gro Horticulture, Inc.) and watered weekly with diluted Gro-Power Liquid (Gro Power, Inc.). SAIL_128_B07 (atg5-1, Col-0 ecotype, same allele used in ) and SAIL_11_H07 (atg7, Col-0 ecotype) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH), and lines homozygous for T-DNA were selected by PCR amplification of genomic DNA.
Chlorophyll, protein isolation and immunoblots
Two leaf disks (1/4 inch diameter) were incubated in 1.5 ml dimethylformamide for 4–24 hours in the dark at room temperature and total chlorophyll was quantified according to . Protein was extracted from two leaf disks in 133 μL of buffer E . Ten microliters of protein extract were subject to SDS-PAGE (13% acrylamide) and immunoblot analysis . The anti-LSU antibody was generated by Antibodies, Inc. and used at a titer of 1:1,000. Anti-GS2 and anti-Lhcb1 were obtained from Agrisera, Inc. and used at titers of 1:5,000 and 1:10,000, respectively. The anti-RCA antibody was a gift of Dr. Michael Salvucci and used at a titer of 1:5,000. The secondary antibody was goat anti-rabbit coupled to alkaline phosphatase (Millipore, Inc.). Alkaline phosphatase activity was detected by nitroblue tetrazolium and 5-bromo-4-chloro-3’-indolyl phosphate. Blots were scanned and pixels quantified by NIH Image J.
The chloroplast isolation protocol was adapted from techniques used in Arabidopsis  which was modified from a protocol developed in Hordeum vulgare. Additional modifications were adopted from a protocol developed in pea [15, 16]. 2.5-5.0 g of mature leaf tissue was minced with a scissors prior to homogenization with a Omni TH tissue homogenizer (Omni, Inc.) in increments of 1.0 to 2.0 g in 30.0 mL Grinding Buffer at 4°C (50.0 mM HEPES-KOH, 2.0 mM EDTA-NaOH, 1.0 mM MnCl2, 1.0 mM MgCl2, 165.0 mM sorbitol, 5.7 mM ascorbic acid, 0.25% BSA (w/v), final pH 7.5). Non-homogenized tissue was allowed to float to the top while the sample stayed on ice, then only the top 10.0 -15.0 mL was re-homogenized to avoid disturbing existing contents. Homogenate was then filtered through one layer of Miracloth in increments of 5.0 mL, clearing debris from the Miracloth in between addition of more homogenate. Filtered homogenate was then centrifuged at 1000 × g for 8 minutes at 4°C.
The resulting pellet was resuspended in 4.0 mL of Grinding Buffer, and loaded onto a 40-85% Percoll step gradient in a 15.0 mL centrifuge tube loaded with 4.0 mL 85% solution and 3.0 mL 40% solution [40% solution: 40.0% Percoll (GE Healthcare Bio-Sciences), 330 mM sorbitol, 2.1 mM MgCl2, 1.6 mM MgCl2, 50 mM HEPES-KOH pH 7.6, 2.0 mM EDTA-NaOH pH 8.0, 0.1% (w/v) BSA); 85% solution: 85.0% Percoll, 50 mM HEPES-KOH pH 7.6, 330 mM sorbitol]. 40-85% Percoll step gradients containing the resuspended chloroplasts were centrifuged at 6,000 × g for 15 minutes at 4°C. Intact chloroplasts were collected from the 85% solution surface, washed with 30.0 mL of Incubation Buffer [50.0 mM HEPES-KOH, 1.0 mM MgCl2, 1.0 mM MgCl2, 165.0 mM sorbitol, 5.7 mM ascorbic acid, 0.25% BSA( w/v), final pH 7.5], and centrifuged at 1,000 × g for 6 minutes.
The chloroplast pellet was resuspended in 1.0 mL of Incubation Buffer, and chlorophyll concentration was adjusted to 200 μg/mL. Chloroplasts were incubated in a foil-wrapped Oakridge tube to prevent light exposure, and stored in a closed drawer at room temperature. Harvested samples were immediately loaded onto a 40-85% Percoll gradient and centrifuged for 15 minutes at 6,000 × g. Intact chloroplasts were collected from the 85% solution surface, washed in 30.0 mL of Incubation Buffer and centrifuged at 1000 × g for 6 minutes. The resulting pellet was then resuspended in Incubation Buffer and stored at −80°C for immunoblot analysis.
Coomassie Brilliant Blue
Chloroplast glutamine synthetase
Major light harvesting complex proteins of photosystem II
Nitrogen remobilization efficiency
Rubisco Containing Body
Senescence associated vacuole.
Dr. Michael Salvucci provided the RCA antibody and Dr. Ana Rus-Canterbury provided critical review of the manuscript. Initial work was funded by the National Science Foundation, grant IBN 0415108.
- Makino A, Osmond B: Effects of nitrogen nutrition on nitrogen partitioning between chloroplasts and mitochondria in pea and wheat. Plant Physiol. 1991, 96: 355-362. 10.1104/pp.96.2.355.PubMedPubMed CentralView Article
- Himelblau E, Amasino RM: Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence. J Plant Physiol. 2001, 158: 1317-1323. 10.1078/0176-1617-00608.View Article
- Mae T, Makino A, Ohira K: Changes in the amounts of ribulose bisphosphate carboxylase synthesized and degraded during the life span of rice leaf (Oryza sativa L.). Plant and Cell Physiology. 1983, 24 (6): 1079-1086.
- Feller U, Anders I, Mae T: Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. J Exp Bot. 2008, 59 (7): 1615-1624.PubMedView Article
- Krupinska K: Fate and activities of plastids during leaf senescence. The Structure and Function of Plastids. Edited by: Wise RR, Hoober JK. 2006, The Netherlands: Springer, 433-449.View Article
- Lim P, Kim H, Nam H: Leaf Senescence. Annu Rev Plant Biol. 2007, 58: 115-136. 10.1146/annurev.arplant.57.032905.105316.PubMedView Article
- Park S-Y, Yu J-W, Park J-S, Li J, Yoo S-C, Lee N-Y, Lee S-K, Jeong S-W, Seo HS, Koh H-J, et al: The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell. 2007, 19: 1649-1664. 10.1105/tpc.106.044891.PubMedPubMed CentralView Article
- Thomas H, Ougham H, Canter P, Donnison I: What stay-green mutants tell us about nitrogen remobilization in leaf senescence. J Exp Bot. 2002, 53: 801-808. 10.1093/jexbot/53.370.801.PubMedView Article
- Gregersen PL, Holm PB, Krupinska K: Leaf senescence and nutrient remobilization in barley and wheat. Plant Biology. 2008, 10: 37-49.PubMedView Article
- Bushnell TP, Bushnell D, Jagendorf AT: A purified zinc protease of pea chloroplasts, EP1, degrades the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Physiol. 1993, 103: 585-591.PubMedPubMed Central
- Weaver ML, Froehilich JE, Amasino RM: Chloroplast-targeted ERD1 protein declines but its mRNA increases during senescence in Arabidopsis. Plant Physiol. 1999, 119: 1209-1216. 10.1104/pp.119.4.1209.PubMedPubMed CentralView Article
- Desimone M, Henke A, Wagner E: Oxidative stress induces partial degradation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in isolated chloroplasts of barley. Plant Physiol. 1996, 111: 789-796.PubMedPubMed Central
- Desimone M, Wagner E, Johanningmeier U: Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplast proteases requires ATP-hydrolysis. Planta. 1998, 205: 459-466. 10.1007/s004250050344.View Article
- Ishida H, Nishimori Y, Sugisawa M, Makino A, Mae T: The large subunit of ribulose-1,5-bisophosphate carboxylase/oxygenase is fragmented into 37-kDa and 16-kDa polypeptides by active oxygen in the lysates of chloroplasts from primary leaves of wheat. Plant Cell Physiol. 1997, 38 (4): 471-479. 10.1093/oxfordjournals.pcp.a029191.PubMedView Article
- Mitsuhashi W, Crafts-Brandner SJ, Feller U: Ribulose-1,5-bis-phosphate carboxylase/oxygenase degradation in isolated pea chloroplasts incubated in the light or in the dark. J Plant Physiol. 1992, 139: 653-658. 10.1016/S0176-1617(11)81706-2.View Article
- Roulin S, Feller U: Light-independent degradaton of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations. Planta. 1998, 205: 297-304. 10.1007/s004250050324.View Article
- Prins A, van Heerden PDR, Olmos E, Kunert KJ, Foyer CH: Cysteine proteases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. J Exp Bot. 2008, 59 (7): 1935-1950.PubMedView Article
- Evans IM, Rus AM, Belanger EM, Kimoto M, Brusslan JA: Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence. Plant Biology. 2010, 12: 1-12.PubMedPubMed CentralView Article
- Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A: Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol. 2009, 149: 885-893.PubMedPubMed CentralView Article
- Bourett TM, Czymmek KJ, Howard RJ: Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta. 1999, 208: 472-479. 10.1007/s004250050584.View Article
- Park H, EL L, Roberson RW, Hoober JK: Transfer of proteins from the chloroplast to the vacuoles in Chlamydamonas reinhardtii (Chlorophyta): a pathway for degradation. Journal of Phycology. 1999, 35: 528-538. 10.1046/j.1529-8817.1999.3530528.x.View Article
- Yamane K, Mitsuya S, Taniguchi M, Miyake H: Salt-induced chloroplast protrusion is the process of exculsion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts into cytoplasm in leaves of rice. 2012, Cell & Environment: Plant
- Chiba A, Ishida H, Nishizawa NK, Makino A, Mae T: Exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol. 2003, 44 (9): 914-921. 10.1093/pcp/pcg118.PubMedView Article
- Ishida H, Yoshimoto K, Reisen D, Makino A, Ohsumi Y, Hanson MR, Mae T: Visualization of rubisco-containing bodies derived from chloroplasts in living cells of arabidopsis. Photosynthesis: Energy from the Sun: 14th International Congress on Photosynthesis. Edited by: JF A, Gantt E, Golbeck J, Osmond B. 2008, Dordrecht, The Netherlands: Springer, 1213-1216.
- Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T: Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiol. 2008, 148: 142-155. 10.1104/pp.108.122770.PubMedPubMed CentralView Article
- Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ, Yoshimoto K: Autophagy in development and stress responses of plants. Autophagy. 2006, 2 (1): 2-11.PubMedView Article
- Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD: Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. 2005, 138: 2097-2110. 10.1104/pp.105.060673.PubMedPubMed CentralView Article
- Li F, Vierstra RD: Autophagy: a multifaceted intracellular system for bulk and selective recycling. Trends Plant Sci. 2012, 17: 526-537. 10.1016/j.tplants.2012.05.006.PubMedView Article
- Xie Z, Klionsky DJ: Autophagasome formation: core machinery and adaptations. Nat Cell Biol. 2007, 9 (10): 1102-1109. 10.1038/ncb1007-1102.PubMedView Article
- Izumi M, Wada S, Makino A, Ishida H: The autophagic degradation of chloroplasts via Rubisco-containing bodies is specifically linked to leaf carbon status but not nitrogen status in Arabidopsis. Plant Physiol. 2010, 154: 1196-1209. 10.1104/pp.110.158519.PubMedPubMed CentralView Article
- Ono Y, Wada S, Izumi M, Makino A, Ishida H: Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana. 2012, Cell & Environment: Plant
- Otegui MS, Noh Y-S, Martinez DE, Vila Petroff MG, Staehelin LA, Amasino RM, Guiamet JJ: Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant Journal. 2005, 41: 831-844. 10.1111/j.1365-313X.2005.02346.x.PubMedView Article
- Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD: The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem. 2002, 277: 33105-33114. 10.1074/jbc.M204630200.PubMedView Article
- Martinez DE, Costa ML, Gomez FM, Otegui MS, Guiamet JJ: ‘Senescence-associated vacuoles’ are involved in the degradation of chloroplast proteins in tobacco leaves. Plant J. 2008, 41: 831-844.
- Guiboileau A, Yoshimoto K, Soulay F, Bataille M-P, Avice J-C, Masclaux-Daubresse C: Autophagy machinery controls nitrogen remobilization at the whole plant level under both limiting and ample nitrate conditions in Arabidopsis. New Phytol. 2012, 194 (3): 732-740. 10.1111/j.1469-8137.2012.04084.x.PubMedView Article
- Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consinni C, Panstruga R, Ohsumi Y, Shirasu K: Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune repsonse in Arabidopsis. Plant Cell. 2009, 21: 2914-2927. 10.1105/tpc.109.068635.PubMedPubMed CentralView Article
- Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata K, Tabata S, Ohsumi Y: Leaf senescence and starvation-induced chlorosis are accelerated by disruption of an Arabidopsis autophagy gene. Plant Physiol. 2002, 129: 1181-1193. 10.1104/pp.011024.PubMedPubMed CentralView Article
- Leong T-Y, Anderson JM: Adaptation of the thylakoid membranes of pea chloroplasts to light intensities. I. Study on the distribution of chlorophyll-protein complexes. Photosynthesis Research. 1984, 5: 105-115. 10.1007/BF00028524.PubMedView Article
- Sakuraba Y, Schelbert S, Park S-Y, Han S-H, Lee B-D, Andres CB, Kessler F, Hortensteiner S, Paek N-C: STAY-GREEN and chlorophyll catabolic enzymes interact at light harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell. 2012, 24: 507-518. 10.1105/tpc.111.089474.PubMedPubMed CentralView Article
- Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M: Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J. 2009, 57: 120-131. 10.1111/j.1365-313X.2008.03670.x.PubMedView Article
- Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim P, Nam H, Lin J-F, Wu S-H, Swidzinski J, Ishizaki K, et al: Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J. 2005, 42: 567-585. 10.1111/j.1365-313X.2005.02399.x.PubMedView Article
- Abreu ME, Munne-Bosch S: Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant of Arabidopsis thaliana. J Exp Bot. 2009, 60: 1261-1271. 10.1093/jxb/ern363.PubMedPubMed CentralView Article
- Breeze E, Harrison E, McHattie S, Hughes L, Hickman R, Hill C, Kiddle S, Kim Y-S, Penfold CA, Jenkins D, et al: High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. Plant Cell. 2011, 23: 873-894. 10.1105/tpc.111.083345.PubMedPubMed CentralView Article
- Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K, Hortensteiner S: Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell. 2009, 21: 767-785. 10.1105/tpc.108.064089.PubMedPubMed CentralView Article
- Porra RJ, Thompson WA, Kriedemann PE: Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta. 1989, 975: 384-394. 10.1016/S0005-2728(89)80347-0.View Article
- Martinez-Garcia JF, Monte E, Quail PH: A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant J. 1999, 20 (2): 251-257. 10.1046/j.1365-313x.1999.00579.x.PubMedView Article
- Schulz A, Knoetzel J, Scheller HV, Mant A: Uptake of a fluorescent dye as a swift and simple indicator of organelle intactness: import-competent chloroplasts from soil-grown Arabidopsis. J Histochem Cytochem. 2004, 52 (5): 701-704. 10.1177/002215540405200514.PubMedView Article
- Brock IW, Hazell L, Michl D, Nielsen VS, Moller BL, Herrmann RG, Klosgen RB, Robinson C: Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimization of in vitro assays. Plant Molecular Biololgy. 1993, 23: 717-725. 10.1007/BF00021527.View Article
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