Combined effects of girdling and leaf removal on fluorescence characteristic of Alhagi sparsifolia leaf senescence.

Plant Biology ISSN 1435-8603

RESEARCH PAPER

Combined effects of girdling and leaf removal on fluorescence characteristic of Alhagi sparsifolia leaf senescence G. Tang1,2,3,4, X. Li1,2,3, L. Lin1,2,3, H. Guo1,2,3,4 & L. Li1,2,3 1 2 3 4

State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China Cele National Station of Observation and Research for Desert-Grassland Ecosystems, Cele, Xinjiang, China Key Laboratory of Biogeography and Bioresource in Arid Zone, Chinese Academy of Sciences, Urumqi, China University of the Chinese Academy of Sciences, Beijing, China

Keywords Alhagi sparsifolia; carbohydrate content; chlorophyll fluorescence; leaf removal; phloem girdling; senescence. Correspondence X. Li, Xinjiang Institute of Ecology and Geography, CAS, 818 South Beijing Rd., Urumqi, Xinjiang 830011, China. E-mail: [email protected] Received: 14 January 2015; Accepted: 27 January 2015 doi:10.1111/plb.12309

ABSTRACT Plant senescence is largely influenced by carbohydrate content. In order to investigate the impact of carbohydrate content on leaf senescence and photosystem II (PSII) during the senescence process, phloem girdling (PG), leaf removal (LR) and a combination of phloem girdling and leaf removal (GR) were performed on Alhagi sparsifolia (Fabaceae) at the end of the growing season. The results showed that during senescence, leaf soluble sugar content, starch content, the energy absorbed by the unit reaction centre (ABS/RC) increased; whereas, leaf photosynthetic rate, photosynthetic pigment content, maximum photochemical efficiency (φPo) and energy used by the acceptor site in electron transfer (ETo/RC) decreased. The degree of change was PG> GR> CK (control)> LR. The results of the present work implied that phloem girdling (PG) significantly accelerated leaf senescence, and that single leaf removal (LR) slightly delayed leaf senescence; although leaf removal significantly delayed the senescence process on the girdled leaf (GR). Natural or delayed senescence only slightly inhibited the acceptor site of PSII and did not damage the donor site of PSII. On the other hand, induced senescence not only damaged the donor site of PSII (e.g. oxygenevolving complex), but also significantly inhibited the acceptor site of PSII. In addition, leaf senescence led to an increase in the energy absorbed by the unit reaction centre (ABS/RC), which subsequently resulted in increasing excitation pressure in the reaction centre (DIo/RC), as well as additional saved Car for absorbing residual light energy and quenching reactive oxygen species during senescence.

INTRODUCTION Constituting the last stage of leaf development in plants (Fukao et al. 2012; Sakuraba et al. 2012), leaf senescence proceeds through a highly regulated process in order to redistribute nutrients from the senescing leaves to other plant organs (Buchanan-Wollaston 1997; Quirino et al. 2000; Shan et al. 2011; Besseau et al. 2012). Senescence may contribute to the survival of a plant in the next season or its next generation (Breeze et al. 2011), as nutrients such as nitrogen, phosphorus, potassium and sulphur will flow to the sink (Robinson et al. 2012; Sakuraba et al. 2012). Leaf senescence starts from chloroplast decomposition, followed by catabolism of macromolecules, such as chlorophyll (Chl), proteins, lipids and RNA (Hopkins et al. 2007; Lim et al. 2007; Besseau et al. 2012). Although the most significant feature of plant senescence is that it repeats annually in deciduous plants of temperate regions (Bhalerao et al. 2003; Brouwer et al. 2012), senescence can also be induced by a number of endogenous factors, developmental cues and reproductive growth (Gan & Amasino 1995; Pic et al. 2002; Riefler et al. 2006; Besseau et al. 2012). Among these factors, plant hormones play a particularly important role in the regulation of leaf senescence. These hormones include auxin, abscisic acid, cytokinin, salicylic acid, 980

jasmonic acid and ethylene (Jung et al. 2007; Shan et al. 2011; Besseau et al. 2012). The effects of hormones on leaf senescence may work through the induction of senescence-associated genes (Besseau et al. 2012). Genes normally expressed during photosynthesis and other anabolic processes in senescent leaves are down-regulated, while genes responsible for catabolism and transport of macromolecules are up-regulated (Fukao et al. 2012). In addition to regulation of endogenous factors, senescence may also be subject to induction of some environmental stressors, such as a lack of resources, darkness, excess energy, drought, salinity, nutrient limitation and injury (Schippers et al. 2007; Balazadeh et al. 2008; Shan et al. 2011). Emergence and development of senescence are controlled via a complex signal transduction channel mediated through the degree of plant development and through environmental factors (Besseau et al. 2012). Carbohydrate concentration also plays a vital role in plant senescence, in which both ‘carbon feast’ (Wingler et al. 2006; Parrott et al. 2007, 2010) or ‘carbon starvation’ (Matile et al. 1992; Pourtau et al. 2006) may result in increased plant senescence. Phloem girdling, which leads to the destruction of sieve tubes, consequently blocks the flow of phloem-transported metabolites beyond the girdle point. Previous work has demonstrated that girdling leads to the accumulation of

Plant Biology 17 (2015) 980–989 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands

Tang, Li, Lin, Guo, Li

carbohydrates in leaves and subsequent acceleration of leaf senescence (Krapp & Stitt 1995; Parrott et al. 2007). The most obvious change of senescence is leaf yellowing, which is associated with the loss of Chl content, protein and RNA degradation and decreased photosynthetic activity (Im Kim et al. 2011; Sakuraba et al. 2012). In fact, the most common cause of this phenomenon is that Chl appears to be more ‘vulnerable’ during the senescence process and degrades ahead of carotenoids (Ag€ uera et al. 2012). In addition, in the senescence process, new compounds will be synthesised, such as anthocyanins and phenolic compounds, which in turn accelerate leaf yellowing (Hendry et al. 1987; Hendry 1988; Matile 1992; Smart 1994; Rajcan et al. 1999). Recent studies have elucidated one of the approaches of Chl breakdown: conversion of Chl in the senescence process to colourless linear tetrapyrroles, the socalled non-fluorescent Chl catabolites (NCCs) as end products of Chl breakdown (H€ ortensteiner 2006; Kr€autler 2008; H€ ortensteiner & Kr€autler 2011; Sakuraba et al. 2012). There has, however, been little research conducted on Chl fluorescence in either the natural or induced senescence process, especially on photosystem II (PSII) energy transfer, and structural and functional changes in the acceptor and donor site. The literature on changes in PSII that demonstrate fast kinetic parameters of Chl fluorescence is small (Strasser et al. 2004). However, the functioning of PSII is essential to leaf photosynthesis during all types of senescence, and it is necessary and important to observe changes in PSII using Chl fluorescence in both the natural and induced senescence process. We examined Alhagi sparsifolia (Fabaceae) in the senescence phase in the Cele Oasis, located on the southern edge of the Taklimakan Desert, China, through a combination of phloem girdling and/or leaf removal. We chose this species because A. sparsifolia is a typical eremophyte (a plant that goese in desert conditions), and little research has been conducted on this kind of growth form. Moreover, as a deep-rooted plant, A. sparsifolia may have a special response when experiencing girdling or leaf removal. The purpose of this study is to understand the effect of phloem girdling and leaf removal on leaf senescence in A. sparsifolia. We are particularly interested in PSII energy transfer, structural and functional changes in the acceptor and donor site during leaf senescence, as well as elucidating the response mechanism of plant photosystems.

Senescence response of girdling and leaf removal

MATERIAL AND METHODS Study site The study was performed at the Desert Experimental Area in the Cele National Field Research Station for Desert Steppe Ecosystems, Chinese Academy of Sciences. The research area is located in the Taklimakan Desert at an oasis–desert transitional zone on the southern rim of the Taklimakan Desert (35°170 55″–39°300 00″ N, 80°030 24″–82°100 34″ E). Plant material and experimental design Alhagi sparsifolia Shap. plants were used for this study. On 25 August 2013, 12 quadrats, each 4 m 9 4 m, with ten to 12 plants, were set up on flat land. Each quadrat was randomly selected for four different treatments: CK, control (no girdling or leaf removal); PG, phloem girdling (girdle at the base of the stem); LR, leaf removal (removal of about half of the total leaves); and GR, combined girdling and leaf removal (we first girdled phloem at the base of the stem and then removed about half of the total leaves on branches), see Fig. 1. Each treatment (control, PG, LR and GR) consisted of three quadrats (each 4 m 9 4 m and with ten to 12 plants). Girdling consisted of removing a 10–12-mm wide band of bark at the base of the main stem of each branch. Leaf removal was defined as the removal of every other leaf from the base to the top of each branch; thus, there was the possibility that each leaf type (young, mature and old) remained or was removed. Soluble sugar content, starch content, net photosynthetic rate (Pn), Chl content and Chl fluorescence parameters of A. sparsifolia were measured on day 1, 11, 21 and 31 after the above treatments. Measurement of photosynthesis Net photosynthesis (Pn) was measured in the field according to Mittler et al. (2001) using a portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Beginning on 26 August 2013, we measured photosynthetic rates of A. sparsifolia once every 10 days within the quadrat, for a total of four times. All 4 days were cloudless when we measured Pn of A. sparsifolia. We conducted measurements at 10:00 h (GMT+6), when the

Fig. 1. Sketch of treatment of Alhagi sparsifolia girdling and leaf removal. CK: control (remain intact); PG: phloem girdling (girdle phloem at the bottom of stem); LR: leaf removal (remove about half of the total leaves); and GR: combine girdling and removal (we first girdled phloem at the bottom of stem and then removed about half of the total leaves in branches). The stems with yellow mean that they were girdled, and the leaves with yellow mean that they will be removed. From the bottom of the stem to the top, every other leaf was removed. The width of girdling was 10 mm. The growth directions of leaves are random, but not shown in the sketch.


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H€ ortensteiner S. (2006) Chlorophyll degradation during senescence. Annual Review of Plant Biology, 57, 55–77. H€ ortensteiner S., Kr€autler B. (2011) Chlorophyll breakdown in higher plants. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1807, 977–988. Im Kim J., Murphy A.S., Baek D., Lee S.-W., Yun D.-J., Bressan R.A., Narasimhan M.L. (2011) YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana. Journal of Experimental Botany, 62, 3981–3992. Jung C., Lyou S.H., Yeu S., Kim M.A., Rhee S., Kim M., Lee J.S., Do Choi Y., Cheong J.-J. (2007) Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana. Plant Cell Reports, 26, 1053– 1063. Krapp A., Stitt M. (1995) An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves. Planta, 195, 313–323. Krause G., Weis E. (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annual Review of Plant Biology, 42, 313–349. Kr€autler B. (2008) Chlorophyll breakdown and chlorophyll catabolites in leaves and fruit. Photochemical & Photobiological Sciences, 7, 1114–1120. Li N., Zhang S., Zhao Y., Li B., Zhang J. (2011) Overexpression of AGPase genes enhances seed weight and starch content in transgenic maize. Planta, 233, 241–250. Lichtenthaler H.K. (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148, 350–382. Lichtenthaler H.K., Babani F. (2004) Light adaptation and senescence of the photosynthetic apparatus. Changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity. In: Papageorgiou G.C., Govindjee (Eds), Chlorophyll a fluorescence. advances in photosynthesis and respiration, Vol. 19. Springer, Dordrecht, The Netherlands, pp 713–736. Lim P.O., Kim H.J., Gil Nam H. (2007) Leaf senescence. Annual Review of Plant Biology, 58, 115–136. Lu Q., Lu C., Zhang J., Kuang T. (2002) Photosynthesis and chlorophyll a fluorescence during flag leaf senescence of field-grown wheat plants. Journal of Plant Physiology, 159, 1173–1178. Masclaux C., Valadier M.-H., Brugiere N., Morot-Gaudry J.-F., Hirel B. (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta, 211, 510–518. Matile P. (1992) Chloroplast senescence. Topics in Photosynthesis, 12, 413–440. Matile P., Schellenberg M., Peisker C. (1992) Production and release of a chlorophyll catabolite in isolated senescent chloroplasts. Planta, 187, 230–235. Merzlyak M.N., Solovchenko A.E. (2002) Photostability of pigments in ripening apple fruit: a possible photoprotective role of carotenoids during plant senescence. Plant Science, 163, 881–888. Mikkelsen T.N., Heide-Jørgensen H.S. (1996) Acceleration of leaf senescence in Fagus sylvatica L. by low levels of tropospheric ozone demonstrated by leaf


colour, chlorophyll fluorescence and chloroplast ultrastructure. Trees, 10, 145–156. Mittler R., Merquiol E., Hallak-Herr E., Rachmilevitch S., Kaplan A., Cohen M. (2001) Living under a ‘dormant’canopy: a molecular acclimation mechanism of the desert plant Retama raetam. The Plant Journal, 25, 407–416. Parrott D., Yang L., Shama L., Fischer A. (2005) Senescence is accelerated, and several proteases are induced by carbon “feast” conditions in barley (Hordeum vulgare L.) leaves. Planta, 222, 989–1000. Parrott D.L., McInnerney K., Feller U., Fischer A.M. (2007) Steam-girdling of barley (Hordeum vulgare) leaves leads to carbohydrate accumulation and accelerated leaf senescence, facilitating transcriptomic analysis of senescence-associated genes. New Phytologist, 176, 56–69. Parrott D.L., Martin J.M., Fischer A.M. (2010) Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression: a family C1A cysteine protease is specifically induced under conditions characterized by high carbohydrate, but low to moderate nitrogen levels. New Phytologist, 187, 313–331. Petrie P.R., Trought M.C., Howell G.S., Buchan G.D. (2003) The effect of leaf removal and canopy height on whole-vine gas exchange and fruit development of Vitis vinifera L. Sauvignon Blanc. Functional Plant Biology, 30, 711–717. Pic E., de La Serve B.T., Tardieu F., Turc O. (2002) Leaf senescence induced by mild water deficit follows the same sequence of macroscopic, biochemical, and molecular events as monocarpic senescence in pea. Plant Physiology, 128, 236–246. Polívka T.S., Frank H.A. (2010) Molecular factors controlling photosynthetic light harvesting by carotenoids. Accounts of Chemical Research, 43, 1125–1134. Pourtau N., Jennings R., Pelzer E., Pallas J., Wingler A. (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta, 224, 556–568. Quentin A., Close D., Hennen L., Pinkard E. (2013) Down-regulation of photosynthesis following girdling, but contrasting effects on fruit set and retention, in two sweet cherry cultivars. Plant Physiology and Biochemistry, 73, 359–367. Quirino B.F., Noh Y.-S., Himelblau E., Amasino R.M. (2000) Molecular aspects of leaf senescence. Trends in Plant Science, 5, 278–282. Rajcan I., Dwyer L.M., Tollenaar M. (1999) Note on relationship between leaf soluble carbohydrate and chlorophyll concentrations in maize during leaf senescence. Field Crops Research, 63, 13–17. Riefler M., Novak O., Strnad M., Schm€ ulling T. (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. The Plant Cell, 18, 40–54. Robinson W.D., Carson I., Ying S., Ellis K., Plaxton W.C. (2012) Eliminating the purple acid phosphatase AtPAP26 in Arabidopsis thaliana delays leaf senescence and impairs phosphorus remobilization. New Phytologist, 196, 1024–1029. Sakuraba Y., Schelbert S., Park S.-Y., Han S.-H., Lee B.-D., Andres C.B., Kessler F., H€ ortensteiner S., Paek N.-C. (2012) Stay-Green and chlorophyll catabolic enzymes interact at light-harvesting complex II for


chlorophyll detoxification during leaf senescence in Arabidopsis. The Plant Cell, 24, 507–518. Schippers J.H., Jing H.-C., Hille J., Dijkwel P.P. (2007) Developmental and hormonal control of leaf senescence. In: Gan S., (Ed.), Senescence processes in plants. Blackwell, Oxford, UK, pp 145–170. Shan X., Wang J., Chua L., Jiang D., Peng W., Xie D. (2011) The role of Arabidopsis Rubisco activase in jasmonate-induced leaf senescence. Plant Physiology, 155, 751–764. Smart C.M. (1994) Gene expression during leaf senescence. New Phytologist, 126, 419–448. Song J., Deng W., Beaudry R.M., Armstrong P.R. (1997) Changes in chlorophyll fluorescence of apple fruit during maturation, ripening, and senescence. HortScience, 32, 891–896. Srivastava A., Guisse B., Greppin H., Strasser R.J. (1997) Regulation of antenna structure and electron transport in Photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1320, 95–106. Strasser R., Govindjee (1992) On the O-J-I-P fluorescence transient in leaves and D1 mutants of Chlamydomonas reinhardtii. In: Murata N. (Ed.), Research in photosynthesis, 2nd edition, vol. 2. Kluwer Academic, Dordrecht, the Netherlands, pp 529–532. Strasser B., Strasser R. (1995) Measuring fast fluorescence transients to address environmental questions: the JIP test. In: Mathis P. (Ed.), Photosynthesis: from light to biosphere, Vol V. Kluwer Academic, Dordrecht, the Netherlands, pp 977–980. Strasser R., Srivastava A., Tsimilli-Michael M. (2000) The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus M., Pathre U., Mohanty P. (Eds), Probing photosynthesis: mechanisms, regulation and adaptation, Ch. 25. Taylor & Francis, London, UK, pp 445–483. Strasser R.J., Tsimilli-Michael M., Srivastava A. (2004) Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G., Govindjee (Eds), Chlorophyll fluorescence a signature of photosynthesis. advances in photosynthesis and respiration. Kluwer, Dordrecht, the Netherlands, pp 321–362. Suzuki Y., Shioi Y. (2004) Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence. Physiologia Plantarum, 122, 291–296. Van Heerden P.D., Tsimilli-Michael M., Kr€ uger G.H., Strasser R.J. (2003) Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiologia Plantarum, 117, 476–491. Van Heerden P.D., Strasser R.J., Kr€ uger G.H. (2004) Reduction of dark chilling stress in N2-fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics. Physiologia Plantarum, 121, 239–249. Wingler A., Purdy S., MacLean J.A., Pourtau N. (2006) The role of sugars in integrating environmental signals during the regulation of leaf senescence. Journal of Experimental Botany, 57, 391–399.

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Fig. 2. Soluble sugar and starch content of A. sparsifolia for the control (CK), phloem girdling (PG), leaf removal (LR) and combined girdling and removal (GR) treatments after 1, 11, 21 and 31 days. (a) Soluble sugar. (b) Starch content. *Significant difference at the 0.05 probability level according to LSD test (n = 5) estimated with twotailed ANOVA between control and other treatments (PG, LR, GR); Data points represent the means of five biological replicates SE.

decline. On day 31, compared with controls, Pn of A. sparsifolia subjected to PG, LR and GR decreased 82.0% (P < 0.05), 9.9% (P > 0.05) and 43.3% (P < 0.05; Fig. 3), respectively. For controls, Pn decreased 23.5% (P < 0.05) from day 1 to 31 (Fig. 3). For PG, Pn decreased 85.3% (P < 0.05) from day 1 to 31; for LR, Pn decreased 18.4% (P < 0.05) from day 1 to 31; for GR, Pn decreased 56.0% (P < 0.05) from day 1 to 31 (Fig. 3). Response of photosynthetic pigments to phloem girdling and leaf removal Our study found that under all treatments, Chl a, Chl b, total Chl, Car, Chl a/b and Chl/Car reduced from day 1 to 31. On day 1, there was little difference (P > 0.05) between treatments (control, PG, LR and GR; Fig. 4). During senescence, treatments (control, PG, LR and GR) showed a decline in Chl a, Chl b, total Chl, Car, Chl a/b and Chl/Car, and the rate of decline was PG> GR> CK> LR. On day 31, compared with controls, Chl a, Chl b, total Chl, Car, Chl a/b and Chl/Car in A. sparsifolia subjected to PG decreased 60.0% (P < 0.05), 39.6% (P < 0.05), 54.5% (P < 0.05), 29.9% (P < 0.05), 34.3% (P < 0.05) and 34.9% (P < 0.05), respectively (Fig. 4). Chl a, Chl b, total Chl, Car, Chl a/b and Chl/Car in A. sparsifolia subjected to GR decreased 29.5% (P < 0.05), 20.2% (P < 0.05), 27.0% (P < 0.05), 13.3% (P < 0.05), 12.2% (P < 0.05) and 15.9% (P < 0.05), respectively (Fig. 4); Chl a, Chl b, total Chl, Car, Chl a/b and Chl/Car in A. sparsifolia subjected to LR increased 4.7% (P > 0.05), 4.0% (P > 0.05), 4.5% (P > 0.05), 1.7% (P > 0.05), 0.9% (P > 0.05) and 3.6% (P > 0.05), respectively (Fig. 4). Response of chlorophyll fluorescence to phloem girdling and leaf removal We examined changes in the donor side of PSII. The Chl fluorescence kinetics of A. sparsifolia showed a typical O-J-I-P phase on day 1; regardless of treatment, there was no K-phase (Fig. 5a). On day 11, Chl kinetics for PG showed a K-phase;

whereas there was no K-phase in other treatments (Fig. 5b). On day 21, only PG treatment exhibited an obvious K-phase (Fig. 5c), while on day 31, a K-phase appeared in PG and GR treatments, but there was no K-phase in the control or LR treatment (Fig. 5d). On day 1, there was no significant difference (P > 0.05) in initial slope of the fluorescence transient (Mo); normalised total complementary area above the O-J-I-P transient [reflecting single-turnover primary bound plastoquinone (QA) reduction events] (Sm); quantum yield for electron transport (at t = 0) (φEo); probability that a trapped exciton moves an electron into the electron transport chain beyond QA (at t = 0) (wo); maximum quantum yield for primary photochemistry

Fig. 3. Photosynthesis of A. sparsifolia for control (CK), phloem girdling (PG), leaf removal (LR) and combined girdling and removal (GR) treatments after 1, 11, 21 and 31 days. Measurement of photosynthesis was at 10:00 h, when photosynthesis reached a day-time peak. *Significant difference at the 0.05 probability level according to LSD test (n = 5) estimated with two-tailed ANOVA between control and other treatments (PG, LR, GR); Data points represent the means of five biological replicates SE.


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Fig. 4. Chl a, Chl b, Chl a+b, Car, Chl a/b and Chl/Car content of A. sparsifolia under control (CK), phloem girdling (PG), leaf removal (LR) and combined girdling and removal (GR) after 1, 11, 21 and 31 days. (a) Chl a. (b) Chl b. (c) Chl a+b. (d) Car. (e) Chl a/b. (f) Chl/Car. Chl: chlorophyll; Car: carotenoids. *Significant difference at the 0.05 probability level according to LSD test (n = 5) estimated with two-tailed ANOVA between control and other treatments (PG, LR, GR); Data points represent the means of five biological replicates SE.

(φPo); performance index on absorption basis (PIabs; Fig. 6); absorption flux (of antenna Chl) per reaction centre (RC) (also a measure of PSII apparent antenna size) (ABS/RC); trapped energy flux (leading to QA reduction) per RC (TRo/RC); dissipated flux per RC (DIo/RC); electron transport flux (beyond QA) per RC (ETo/RC); density of RCs (QA reducing PSII reaction centres) (RC/CS) (Table 1). In each treatment (control, PG, LR and GR), Mo (Fig. 6a), ABS/RC (Table 1) and DIo/RC (Table 1) increased during senescence; whereas Sm (Fig. 6b), φEo (Fig. 6c), wo (Fig. 6d), φPo (Fig. 6e), PIabs (Fig. 6f), TRo/RC (Table 1), ETo/RC (Table 1) and RC/CS (Table 1) decreased during senescence. On day 31, compared to controls, Mo (Fig. 6a), ABS/RC (Table 1) and DIo/RC (Table 1) in A. sparsifolia subjected to PG increased 99.9% (P < 0.05), 498.9% (P < 0.05) and 994.2% (P < 0.05), respectively; Sm (Fig. 6b), φEo (Fig. 6c), wo (Fig. 6d), φPo (Fig. 6e), PIabs (Fig. 6f), TRo/RC (Table 1), ETo/ RC (Table 1) and RC/CS (Table 1) decreased 46.9% (P < 0.05), 63.7% (P < 0.05), 40.6% (P < 0.05), 71.8% (P < 0.05), 88.2% (P < 0.05), 47.0% (P < 0.05), 24.8% (P < 0.05) and 94.2% (P < 0.05), respectively. On day 31, compared to controls, Mo (Fig. 6a), ABS/RC (Table 1), DIo/RC (Table 1) and ETo/RC (Table 1) in A. sparsifolia subjected to LR decreased 8.2% (P < 0.05), 8.2% (P < 0.05), 28.2% (P < 0.05) and 4.6% (P > 0.05), respectively; and Sm (Fig. 6b), φEo (Fig. 6c), wo (Fig. 6d), φPo (Fig. 6e), PIabs (Fig. 6f), TRo/RC (Table 1) and RC/CS (Table 1) increased 8.5% (P < 0.05), 4.8% (P > 0.05), 0.4% (P > 0.05), 2.0% (P > 0.05), 6.3% (P > 0.05) (Fig. 6), 984

13.8% (P < 0.05) and 11.5% (P < 0.05; Table 1), respectively. On day 31, compared to controls, Mo (Fig. 6a), ABS/RC (Table 1) and DIo/RC (Table 1) in A. sparsifolia subjected to GR increased 45.9% (P < 0.05), 179.8% (P < 0.05) and 368.4% (P < 0.05), respectively; and Sm (Fig. 6b), φEo (Fig. 6c), wo (Fig. 6d), φPo (Fig. 6e), PIabs (Fig. 6f), TRo/RC (Table 1), ETo/ RC (Table 1, and RC/CS (Table 1) decreased 25.4% (P < 0.05), 30.9% (P < 0.05), 26.5% (P < 0.05), 34.3% (P < 0.05), 62.7% (P < 0.05), 28.1% (P < 0.05), 12.8% (P < 0.05) and 84.0% (P < 0.05), respectively. DISCUSSION Carbohydrate content has a strong relationship with leaf senescence (Parrott et al. 2005, 2007), and the most direct effect of girdling is accumulation of carbohydrates above the girdle (Eltom et al. 2013). We therefore measured the changes in soluble sugar and starch content in our experiment to elucidate possible reasons for leaf senescence. In the present study, both soluble sugars and starch increased under the treatments PG and GR (Fig. 2), with PG exhibiting a more obvious change compared with GR. Girdling leading to increases in carbohydrate content has been found in many previous studies (e.g. Parrott et al. 2005, 2007). The current study suggested that when girdling and leaf removal existed together, the plant showed similar changes to girdling, while leaf removal partially offset increases in carbohydrates induced by phloem girdling. Leaf removal decreased carbohydrate content (Caspari et al.




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Fig. 5. The O-J-I-P Chl a fluorescence transient curves (log time scale) in A. sparsifolia during senescence in the four treatments (CK, PG, LR, GR) after 1(a), 11 (b), 21(c) and 31(d) days.

1998; Petrie et al. 2003), which was also demonstrated in our study, where both soluble sugar and starch content of LR leaves declined compared to controls (Fig. 2). A previous study showed that in the natural senescence process, carbohydrate content in the plant first increased and then decreased (Masclaux et al. 2000), which was also found in the control treatment (Fig. 2). However, neither induced senescence (PG, GR) nor delayed senescence (LR) showed a similar change to natural senescence (control); carbohydrate content in the PG, GR and LR treatments increased continuously over 31 days (Fig. 2). Carbohydrate increases in PG and GR may be due to destruction of phloem, so that carbohydrates cannot be transported to other organs, which can be accomplished in non-girdled plants. In the LR treatment, carbohydrate content did not decrease, possibly because senescence was delayed until day 31; thus, the plant did not need to recycle carbohydrates in leaves. In addition, photosynthesis always increases when leaves are removed, and this may compensate for the reduced leaf area (Petrie et al. 2003). As an essential indicator of senescence, we also observed a change in net photosynthesis during the study. In the present study, leaf removal increased net photosynthesis (Fig. 3), which may be due to a decrease in carbohydrates because source–sink relations exhibit a strong relationship with net photosynthesis (Arp 1991). Hence, a decrease of net photosynthesis in the PG and GR treatments (Fig. 3) may be a result of the accumulation of carbohydrates or high source–sink ratio, as found in previous studies (Krapp & Stitt 1995; Quentin et al. 2013). Yellowing of leaves and a decline in photosynthetic pigments is considered as an important symptom of leaf senescence

(H€ ortensteiner 2006; Wingler et al. 2006). We also observed a change in photosynthetic pigments, i.e. Chl a, Chl b, Chl a+b, Car, Chl a/b and Chl/Car during our 31-day study. In the present study, Chl a, Chl b, Chl a+b and Car decreased with time (Fig. 4a, b, c, d); this was the result of senescence, as shown in previous studies (H€ ortensteiner 2006; Wingler et al. 2006). Compared with the control, Chl a, Chl b, Chl a+b and Car content in PG and GR leaves decreased after 31 days of treatment (Fig. 4a, b, c, d), similar to the reduced Chl content after girdling (Dai & Dong 2011). However, Chl a, Chl b, Chl a+b and Car increased in LR leaves compared with controls, and this may be why net photosynthesis increase in LR leaves (Fig. 3). The present study suggests that when phloem girdling and leaf removal exist together, A. sparsifolia showed a similar response as phloem girdling, i.e. both PG and GR accelerate leaf senescence, as previously demonstrated (Parrott et al. 2005, 2007). However, the extent of acceleration of senescence in PG was much higher than in GR; thus, leaf removal could significantly delay leaf senescence on the girdled leaves, while single leaf removal (LR) only slightly delayed leaf senescence. Changes in Chl a/b during senescence were complicated. The ratio of Chl a to Chl b in radish cotyledons increased slightly during senescence, which suggests that Chl b degraded faster than Chl a during senescence (Suzuki & Shioi 2004). It was also found that the Chl a/b ratio did not decline in Platanus occidentalis L. during autumnal leaf senescence (Adams et al. 1990). On the other hand, studies have consistently found a significant decrease in the Chl a/b ratio in leaves (Ag€ uera et al. 2012). Interestingly, both decline and no decline in the Chl a/b ratio in leaves were


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Table 1. Mean values SE for five JIP test parameters in A. sparsifolia under phloem girdling and leaf removal after 1, 11, 21 and 31 days. treatment parameters

time (days)

CK

PG

LR

GR

ABS/RC

1 11 21 31 1 11 21 31 1 11 21 31 1 11 21 31 1 11 21 31

2.01 0.08 2.26 0.08 2.67 0.11 3.00 0.11 1.70 0.08 1.69 0.07 1.63 0.10 1.43 0.09 0.31 0.01 0.58 0.02 1.04 0.05 1.57 0.04 1.04 0.04 1.07 0.06 0.97 0.04 0.90 0.03 943.30 39.18 768.82 43.94 655.20 29.99 564.2 28.48

2.27 0.08 5.83 0.25** 10.61 0.47** 17.94 0.75** 1.79 0.08 1.63 0.08 1.06 0.05** 0.76 0.03** 0.47 0.02** 4.20 0.20** 9.55 0.47** 17.18 0.75** 1.07 0.04 0.91 0.11 0.76 0.03** 0.68 0.03** 818.37 24.96* 421.3 19.03** 120.94 6.85** 32.95 1.44**

2.05 0.08 2.15 0.06** 2.54 0.09** 2.75 0.09** 1.69 0.07 1.63 0.07 1.71 0.10 1.62 0.05** 0.36 0.02 0.52 0.01** 0.83 0.01** 1.13 0.05** 0.96 0.03* 0.92 0.05 0.87 0.04 0.86 0.06 934.52 32.59 811.07 79.12** 700.19 24.14** 628.9 28.90**

2.03 0.08 2.68 0.05** 5.71 0.14** 8.38 0.20** 1.67 0.09 1.58 0.03 1.19 0.04 1.02 0.05 0.36 0.02** 1.10 0.07** 4.53 0.14** 7.36 0.23** 0.98 0.03 0.91 0.03 0.84 0.03* 0.79 0.02 895.42 45.60 678.58 22.39* 345.7 15.51** 90.04 3.40

TRo/RC

DIo/RC

ETo/RC

RC/CS

ABS/RC is photon absorbance rate per reaction centre (RC); TRo/RC is exciton trapping rate per RC; DIo/RC is exciton dissipation rate per RC; ETo/RC is electron transport rate per RC; RC/CS is density of PSII reaction centres per excited cross-section. CK, control; PG, phloem girdling; LR, leaf removal; GR, combined phloem girdling and leaf removal. *and **F-test of corresponding mean squares significant at the 0.05 or 0.01 probability levels. Values are means of five replicates, and significant differences in main effects and interactions were established using ANOVA and least significant difference (LSD; P < 0.05 or P < 0.01) are given.

found in our study during senescence (Fig. 4e). The control and LR leaves did not show a decline in Chl a/b ratio; however, a decline was found in the PG and GR treatment (Fig. 4e). Our study indicated that whether or not the ratio of Chl a/b changes depends on the particular type of senescence. Specifically, natural or delayed senescence did not influence the Chl a/b ratio, a finding that is consistent with previous studies that characterized leaf senescence as a controlled process (Adams et al. 1990). However, in induced senescence (e.g. girdling or high carbohydrate content), the senescence process may be over-controlled or disorganised so that the Chl a/b ratio shows a significant decline. Chlorophyll a fluorescence has been extensively utilised to evaluate changes in the photosynthetic apparatus in intact leaves (Srivastava et al. 1997), and these changes during senescence are very important to plants. Here, we used the fluorescence to measure the fast Chl a fluorescence transient, which offers additional possibilities to probe the nature of PSII characteristics during different kinds of senescence. Previous studies have shown that when the donor site of PSII is injured, the intensity of Chl a fluorescence increases after a very short period of time (before J point), and the K-phase appears (the characteristic loci of about 300 ls after illumination; Srivastava et al. 1997). The emergence of a K-phase was caused by inhibition of the water-splitting system and partial inhibition of the acceptor site before QA (Mikkelsen & Heide-Jørgensen 1996; Shan et al. 2011). In this inhibition process, the oxygen-evolving complex (OEC) is damaged, so the K-phase can indicate that the OEC is injured (Strasser et al. 2000, 2004). The present study showed that the K-phase first appeared on day 11 of the 986

PG treatment (Fig. 5b), while in GR leaves the K-phase only appeared on day 31 (Fig. 5d); whereas, no K-phase was found during the whole senescence process in control and LR leaves (Fig. 5). The present study suggested that the OEC was not damaged during either natural senescence or delayed senescence processes. This result is also consistent with previous studies, which found that leaf senescence was a controlled process, so that the photosynthetic apparatus would remain stable during senescence (Lu et al. 2002; Lichtenthaler & Babani 2004). However, the OEC will be damaged during induced leaf senescence under the girdling treatment (PG), and leaf removal will delay the time of the appearance of OEC damage in girdled leaves (GR). In the fast Chl fluorescence induction kinetic curve, parameters such as Mo, Sm, φEo and wo primarily reflect changes in the acceptor site of PSII, which mainly includes the primary bound plastoquinone (QA), secondary bound plastoquinone (QB) and plastoquinone (PQ) sink. Mo reflects the maximum rate of reduction of QA, i.e. the reduced rate of QA in the O-J process (Strasser & Strasser 1995; Strasser et al. 2000, 2004). Mo is relevant to the reaction centre pigments, light-harvesting pigments and QA status. Sm reflects the energy needed to reduce QA completely, i.e. the size of the acceptor site PQ sink in the PSII reaction centre. The more electrons enter the electron transport chain from QA, the longer it takes to arrive at maximum recorded fluorescence intensity (Fm), and the larger the value of Sm. When leaves are subjected to high light stress, degradation of protein D1 intensifies, causing electron transport complexity, especially QB exchange with plastoquinone from the plastoquinone pool. This results in reduced acceptor capacity,




(a)

(b)

(c)

(d)

(e)

(f)

Fig. 6. The Mo, Sm, φEo, wo, φPo and PIabs of A. sparsifolia under control (CK), phloem girdling (PG), leaf removal (LR) and combined girdling and removal (GR) after 1, 11, 21 and 31 days. (a) Mo. (b) Sm. (c) φEo. (d) wo. (e) φPo. (f) PIabs. Mo, approximated initial slope of the fluorescence transient; Sm, normalised total complementary area above the O-J-I-P transient (reflecting single turnover QA reduction events); φEo, quantum yield for electron transport (at t = 0); wo, probability that a trapped exciton moves an electron into the electron transport chain beyond QA (at t = 0); φPo, maximum quantum yield for primary photochemistry; PIabs, performance index on absorption basis. *Significant difference at the 0.05 probability level according to LSD test (n = 5) estimated with two-tailed ANOVA between control and other treatments (PG, LR, GR); Data points represent the means of five biological replicates SE.

manifested as decreased Sm. In the present study, both types of senescence complied with the same rules; during the process of senescence, Chl fluorescence parameters (Mo, Sm, φEo and wo) that reflect changes in the acceptor site of PSII showed a consistent trend among treatments (control, PG, LR and GR; Fig. 6a, b, c, d). In the leaf senescence process, regardless of whether it is induced or natural, activity of photosynthetic enzymes was inhibited, thereby degrading proteins related to photosynthesis. Degradation of protein D1 intensified, causing electron transport complexity, especially QB exchange with plastoquinone from the plastoquinone pool. This resulted in reduced acceptor capacity, manifest as decreased Sm (Fig. 6b). As the electron acceptor sink capacity (Sm) of the PSII acceptor site was inhibited, the probability that a trapped exciton moves an electron into the electron transport chain beyond QA (wo) (Fig. 6c) was increased, reducing the quantum yield for electron transport (φEo; Fig. 6d). More energy was used to restore QA, accelerating reduction of QA (Mo), shown as an increase in Mo (Fig. 6a). The present study indicates that in the induced senescence process (PG, GR), besides damage on the donor site of PSII (e.g. OEC), there was a significant (P < 0.05) inhibition on the acceptor site of PSII. On the other hand, in the natural (control) or delayed (LR) senescence, there was only a slight (P > 0.05) inhibition on the acceptor site of PSII and no damage on the donor site of PSII. So, in the natural/delayed senescence process of plants, although the overall performance of plant PSII was reduced to some degree, it still maintained a relatively good state to maintain photosynthesis.

The φPo reflects maximum photochemical efficiency after dark adaptation. It is the same as the parameter Fv/Fm determined by modulated pulse fluorescence. Change in φPo during senescence is also complicated, with some literature showing that φPo (Fv/Fm) changed very little during leaf senescence (Adams et al. 1990; Lu et al. 2002), and other studies demonstrating that φPo (Fv/Fm) declined with time during senescence (Mikkelsen & Heide-Jørgensen 1996; Song et al. 1997). Interestingly, both types of φPo change were found in our study. Specifically, in the natural (control) or delayed (LR) senescence, there was only a slight decline (P > 0.05) during senescence (Fig. 6e); whereas, in induced (PG, GR) senescence, φPo declined significantly (P < 0.05) during senescence (Fig. 6e). Hence, our study suggests that whether or not φPo changes during senescence depends on the type of senescence. As a highly controlled process, natural (or delayed) senescence did not change φPo substantially, while induced senescence (e.g. high levels of carbohydrates) severely inhibit PSII photochemical efficiency. Similar to φPo, the performance index PIabs is also a parameter reflecting the performance and state of the photosynthetic apparatus, except that it contains three parameters (RC/ABS, φPo and wo). These three parameters are interdependent, and thus the performance index can more accurately reflect the state of the photosynthetic apparatus (Appenroth et al. 2001). PIabs is more sensitive than φPo, and can better reflect the impact of stress on the photosynthetic apparatus (Appenroth et al. 2001; Van Heerden et al. 2003, 2004). The present study


987



also found that PIabs is more sensitive than φPo because, although the trend of PIabs is similar to that of φPo (decline during senescence), the degree of change in PIabs was much larger than that in φPo (Fig. 6f). Therefore, according to our study, compared with φPo (Fv/Fm), PIabs may be a better parameter to study the change of PSII during senescence. Specific activity of the photosynthetic apparatus could be analysed effectively through JIP determination, i.e. various quantum efficiencies (ABS/RC, TRo/RC, ETo/RC, DIo/RC, etc.) in the active reaction centre (RC) and the number of reaction centres per unit area (RC/CS; Table 1). The present study showed that leaf senescence (natural, delayed or induced) led to a decrease in active reaction centres (RC/CS) per unit area (Table 1), which subsequently resulted in an increase in the energy absorbed by the unit reaction centre (ABS/RC; Table 1). The increased light energy, however, could not be fully utilised by the plants. The absorbed energy, energy that had been fully captured by the reaction centre (TRo/RC; Table 1), energy used by the acceptor site in electron transfer (ETo/RC; Table 1) and photochemical reaction ability φPo (Fig. 6e) did not increase, but declined with senescence. Instead, excess absorbed light energy became a burden, leading to increased excitation pressure in the reaction centre (DIo/RC; Table 1). However, non-photochemical quenching will therefore rise, since the initiation of non-photochemical quenching takes longer than photosynthetic electron transfer (Krause & Weis 1991). Therefore, there will be a higher probability that senescent leaves will produce excess reactive oxygen in PSII. In order to mitigate the stress of reactive oxygen, plants save more Car compared with Chl during leaf senescence, shown as a decline in Chl/Car (Fig. 4f). This is because one of most important functions of Car is to absorb residual light energy and quench reactive oxygen species, thus preventing membrane lipid peroxidation (Polívka & Frank 2010). That Chl/Car decline and Car play a photoprotective role during senescence was also found in previous studies (Merzlyak & Solovchenko 2002). In summary, phloem girdling (PG) significantly accelerated leaf senescence and single leaf removal (LR) slightly REFERENCES Adams W.W., Winter K., Schreiber U., Schramel P. (1990) Photosynthesis and chlorophyll fluorescence characteristics in relationship to changes in pigment and element composition of leaves of Platanus occidentalis L. during autumnal leaf senescence. Plant Physiology, 92, 1184–1190. Ag€ uera E., Cabello P., de la Mata L., Molina E., de la Haba P. (2012) Metabolic regulation of leaf senescence in sunflower (Helianthus annuus L.) plants. Senescence. InTech Open Access Publisher, Rijeka, Croatia. Appenroth K.-J., St€ ockel J., Srivastava A., Strasser R. (2001) Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements. Environmental Pollution, 115, 49–64. Arp W. (1991) Effects of source–sink relations on photosynthetic acclimation to elevated CO2. Plant, Cell & Environment, 14, 869–875. Balazadeh S., Ria~ no-Pach on D., Mueller-Roeber B. (2008) Transcription factors regulating leaf senescence in Arabidopsis thaliana. Plant Biology, 10, 63– 75.

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delayed leaf senescence, while leaf removal significantly delayed the senescence process on girdled leaves (GR). The level of carbohydrate in leaves may be the key factor that leads to these phenomena. However, leaf senescence is a highly controlled process in which both natural and delayed senescence has little influence on Chl fluorescence. Natural or delayed senescence only slightly inhibits the acceptor site of PSII and does not damage the donor site of PSII. In natural or delayed senescence process in plants, although the overall performance of plant PSII was reduced to some degree, it still retained a relatively good state to maintain photosynthesis. However, in induced senescence, besides damage to the donor site of PSII (e.g. OEC), there was a significant inhibition on the acceptor site of PSII. In addition, leaf senescence led to a decrease in active reaction centres per unit area (RC/CS), which subsequently resulted in an increase in the energy absorbed by the unit reaction centre (ABS/RC). However, most of the energy cannot be captured by the reaction centre, so the excess energy caused a stress in terms of reactive oxygen, which led to more Car being saved for absorbing residual light energy and quenching reactive oxygen species during senescence. This study is crucial for improving our ability to understand the changes in PSII energy transfer, structure and function in the acceptor and donor sites during leaf senescence. ACKNOWLEDGEMENTS We thank Zichun Guo, Changjun Li and Zhuyu Gu for invaluable help with the experiments, and the farmers for their important field assistance. We also express gratitude to Jake Carpenter for polishing the English in this manuscript and an anonymous reviewer for comments and suggestions on this study. This research was supported by the National Natural Science Foundation of China (41271494) and Key Project in the National Science and Technology Pillar Program (2009BAC54B03).

Besseau S., Li J., Palva E.T. (2012) WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana. Journal of Experimental Botany, 63, 2667–2679. Bhalerao R., Keskitalo J., Sterky F., Erlandsson R., Bj€ orkbacka H., Birve S.J., Karlsson J., Gardestr€ om P., Gustafsson P., Lundeberg J. (2003) Gene expression in autumn leaves. Plant Physiology, 131, 430–442. Breeze E., Harrison E., McHattie S., Hughes L., Hickman R., Hill C., Kiddle S., Kim Y.-S., Penfold C.A., Jenkins D. (2011) High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. The Plant Cell, 23, 873–894. Brouwer B., Ziolkowska A., Bagard M., Keech O., Gardestr€ om P. (2012) The impact of light intensity on shade-induced leaf senescence. Plant, Cell & Environment, 35, 1084–1098. Buchanan-Wollaston V. (1997) The molecular biology of leaf senescence. Journal of Experimental Botany, 48, 181–199. Caspari H.W., Lang A., Alspach P. (1998) Effects of girdling and leaf removal on fruit set and vegetative growth in grape. American Journal of Enology and Viticulture, 49, 359–366.

Dai J., Dong H. (2011) Stem girdling influences concentrations of endogenous cytokinins and abscisic acid in relation to leaf senescence in cotton. Acta Physiologiae Plantarum, 33, 1697–1705. Eltom M., Trought M., Winefield C. (2013) The effects of cane girdling before budbreak on shoot growth, leaf area and carbohydrate content of Vitis vinifera L. Sauvignon Blanc grapevines. Functional Plant Biology, 40, 749–757. Fukao T., Yeung E., Bailey-Serres J. (2012) The submergence tolerance gene SUB1A delays leaf senescence under prolonged darkness through hormonal regulation in rice. Plant Physiology, 160, 1795–1807. Gan S., Amasino R.M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science, 270, 1986–1988. Hendry G. (1988) Where does all the green go? New Scientist, 5, 38–42. Hendry G.A., Houghton J.D., Brown S.B. (1987) The degradation of chlorophyll – a biological enigma. New Phytologist, 107, 255–302. Hopkins M., Taylor C., Liu Z., Ma F., McNamara L., Wang T.W., Thompson J.E. (2007) Regulation and execution of molecular disassembly and catabolism during senescence. New Phytologist, 175, 201–214.



H€ ortensteiner S. (2006) Chlorophyll degradation during senescence. Annual Review of Plant Biology, 57, 55–77. H€ ortensteiner S., Kr€autler B. (2011) Chlorophyll breakdown in higher plants. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1807, 977–988. Im Kim J., Murphy A.S., Baek D., Lee S.-W., Yun D.-J., Bressan R.A., Narasimhan M.L. (2011) YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana. Journal of Experimental Botany, 62, 3981–3992. Jung C., Lyou S.H., Yeu S., Kim M.A., Rhee S., Kim M., Lee J.S., Do Choi Y., Cheong J.-J. (2007) Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana. Plant Cell Reports, 26, 1053– 1063. Krapp A., Stitt M. (1995) An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves. Planta, 195, 313–323. Krause G., Weis E. (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annual Review of Plant Biology, 42, 313–349. Kr€autler B. (2008) Chlorophyll breakdown and chlorophyll catabolites in leaves and fruit. Photochemical & Photobiological Sciences, 7, 1114–1120. Li N., Zhang S., Zhao Y., Li B., Zhang J. (2011) Overexpression of AGPase genes enhances seed weight and starch content in transgenic maize. Planta, 233, 241–250. Lichtenthaler H.K. (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology, 148, 350–382. Lichtenthaler H.K., Babani F. (2004) Light adaptation and senescence of the photosynthetic apparatus. Changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity. In: Papageorgiou G.C., Govindjee (Eds), Chlorophyll a fluorescence. advances in photosynthesis and respiration, Vol. 19. Springer, Dordrecht, The Netherlands, pp 713–736. Lim P.O., Kim H.J., Gil Nam H. (2007) Leaf senescence. Annual Review of Plant Biology, 58, 115–136. Lu Q., Lu C., Zhang J., Kuang T. (2002) Photosynthesis and chlorophyll a fluorescence during flag leaf senescence of field-grown wheat plants. Journal of Plant Physiology, 159, 1173–1178. Masclaux C., Valadier M.-H., Brugiere N., Morot-Gaudry J.-F., Hirel B. (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta, 211, 510–518. Matile P. (1992) Chloroplast senescence. Topics in Photosynthesis, 12, 413–440. Matile P., Schellenberg M., Peisker C. (1992) Production and release of a chlorophyll catabolite in isolated senescent chloroplasts. Planta, 187, 230–235. Merzlyak M.N., Solovchenko A.E. (2002) Photostability of pigments in ripening apple fruit: a possible photoprotective role of carotenoids during plant senescence. Plant Science, 163, 881–888. Mikkelsen T.N., Heide-Jørgensen H.S. (1996) Acceleration of leaf senescence in Fagus sylvatica L. by low levels of tropospheric ozone demonstrated by leaf


colour, chlorophyll fluorescence and chloroplast ultrastructure. Trees, 10, 145–156. Mittler R., Merquiol E., Hallak-Herr E., Rachmilevitch S., Kaplan A., Cohen M. (2001) Living under a ‘dormant’canopy: a molecular acclimation mechanism of the desert plant Retama raetam. The Plant Journal, 25, 407–416. Parrott D., Yang L., Shama L., Fischer A. (2005) Senescence is accelerated, and several proteases are induced by carbon “feast” conditions in barley (Hordeum vulgare L.) leaves. Planta, 222, 989–1000. Parrott D.L., McInnerney K., Feller U., Fischer A.M. (2007) Steam-girdling of barley (Hordeum vulgare) leaves leads to carbohydrate accumulation and accelerated leaf senescence, facilitating transcriptomic analysis of senescence-associated genes. New Phytologist, 176, 56–69. Parrott D.L., Martin J.M., Fischer A.M. (2010) Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression: a family C1A cysteine protease is specifically induced under conditions characterized by high carbohydrate, but low to moderate nitrogen levels. New Phytologist, 187, 313–331. Petrie P.R., Trought M.C., Howell G.S., Buchan G.D. (2003) The effect of leaf removal and canopy height on whole-vine gas exchange and fruit development of Vitis vinifera L. Sauvignon Blanc. Functional Plant Biology, 30, 711–717. Pic E., de La Serve B.T., Tardieu F., Turc O. (2002) Leaf senescence induced by mild water deficit follows the same sequence of macroscopic, biochemical, and molecular events as monocarpic senescence in pea. Plant Physiology, 128, 236–246. Polívka T.S., Frank H.A. (2010) Molecular factors controlling photosynthetic light harvesting by carotenoids. Accounts of Chemical Research, 43, 1125–1134. Pourtau N., Jennings R., Pelzer E., Pallas J., Wingler A. (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta, 224, 556–568. Quentin A., Close D., Hennen L., Pinkard E. (2013) Down-regulation of photosynthesis following girdling, but contrasting effects on fruit set and retention, in two sweet cherry cultivars. Plant Physiology and Biochemistry, 73, 359–367. Quirino B.F., Noh Y.-S., Himelblau E., Amasino R.M. (2000) Molecular aspects of leaf senescence. Trends in Plant Science, 5, 278–282. Rajcan I., Dwyer L.M., Tollenaar M. (1999) Note on relationship between leaf soluble carbohydrate and chlorophyll concentrations in maize during leaf senescence. Field Crops Research, 63, 13–17. Riefler M., Novak O., Strnad M., Schm€ ulling T. (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. The Plant Cell, 18, 40–54. Robinson W.D., Carson I., Ying S., Ellis K., Plaxton W.C. (2012) Eliminating the purple acid phosphatase AtPAP26 in Arabidopsis thaliana delays leaf senescence and impairs phosphorus remobilization. New Phytologist, 196, 1024–1029. Sakuraba Y., Schelbert S., Park S.-Y., Han S.-H., Lee B.-D., Andres C.B., Kessler F., H€ ortensteiner S., Paek N.-C. (2012) Stay-Green and chlorophyll catabolic enzymes interact at light-harvesting complex II for


chlorophyll detoxification during leaf senescence in Arabidopsis. The Plant Cell, 24, 507–518. Schippers J.H., Jing H.-C., Hille J., Dijkwel P.P. (2007) Developmental and hormonal control of leaf senescence. In: Gan S., (Ed.), Senescence processes in plants. Blackwell, Oxford, UK, pp 145–170. Shan X., Wang J., Chua L., Jiang D., Peng W., Xie D. (2011) The role of Arabidopsis Rubisco activase in jasmonate-induced leaf senescence. Plant Physiology, 155, 751–764. Smart C.M. (1994) Gene expression during leaf senescence. New Phytologist, 126, 419–448. Song J., Deng W., Beaudry R.M., Armstrong P.R. (1997) Changes in chlorophyll fluorescence of apple fruit during maturation, ripening, and senescence. HortScience, 32, 891–896. Srivastava A., Guisse B., Greppin H., Strasser R.J. (1997) Regulation of antenna structure and electron transport in Photosystem II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1320, 95–106. Strasser R., Govindjee (1992) On the O-J-I-P fluorescence transient in leaves and D1 mutants of Chlamydomonas reinhardtii. In: Murata N. (Ed.), Research in photosynthesis, 2nd edition, vol. 2. Kluwer Academic, Dordrecht, the Netherlands, pp 529–532. Strasser B., Strasser R. (1995) Measuring fast fluorescence transients to address environmental questions: the JIP test. In: Mathis P. (Ed.), Photosynthesis: from light to biosphere, Vol V. Kluwer Academic, Dordrecht, the Netherlands, pp 977–980. Strasser R., Srivastava A., Tsimilli-Michael M. (2000) The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus M., Pathre U., Mohanty P. (Eds), Probing photosynthesis: mechanisms, regulation and adaptation, Ch. 25. Taylor & Francis, London, UK, pp 445–483. Strasser R.J., Tsimilli-Michael M., Srivastava A. (2004) Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G., Govindjee (Eds), Chlorophyll fluorescence a signature of photosynthesis. advances in photosynthesis and respiration. Kluwer, Dordrecht, the Netherlands, pp 321–362. Suzuki Y., Shioi Y. (2004) Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence. Physiologia Plantarum, 122, 291–296. Van Heerden P.D., Tsimilli-Michael M., Kr€ uger G.H., Strasser R.J. (2003) Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiologia Plantarum, 117, 476–491. Van Heerden P.D., Strasser R.J., Kr€ uger G.H. (2004) Reduction of dark chilling stress in N2-fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics. Physiologia Plantarum, 121, 239–249. Wingler A., Purdy S., MacLean J.A., Pourtau N. (2006) The role of sugars in integrating environmental signals during the regulation of leaf senescence. Journal of Experimental Botany, 57, 391–399.

989

Ameliorative effects of melatonin on dark-induced leaf senescence in gardenia (Gardenia jasminoides Ellis): leaf morphology, anatomy, physiology and transcriptome.

Transcriptional networks in leaf senescence.

Towards understanding abscisic acid-mediated leaf senescence.

Leaf senescence in a non-yellowing mutant of Festuca pratensis : III. Total acyl lipids of leaf tissue during senescence.

The effects of abscisic acid on senescence in leaf discs of radish, Raphanus sativus L.

Modelling transcriptional networks in leaf senescence.

[Changes of glyco- and phospholipid contents during leaf senescence].

Effects of combined ozone and cadmium stresses on leaf traits in two poplar clones.

ABA receptor PYL9 promotes drought resistance and leaf senescence.

Isolation and characterization of a spotted leaf 32 mutant with early leaf senescence and enhanced defense response in rice.

Transcriptional analyses of natural leaf senescence in maize.

LSD 2.0: an update of the leaf senescence database.

Mitochondria change dynamics and morphology during grapevine leaf senescence.

Alteration of the phenology of leaf senescence and fall in winter deciduous species by climate change: effects on nutrient proficiency.

Switchgrass (Panicum virgatum L) flag leaf transcriptomes reveal molecular signatures of leaf development, senescence, and mineral dynamics.

Hsc70 signaling complex dynamically regulates leaf senescence.

C1A cysteine protease-cystatin interactions in leaf senescence.

Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis.

Photosynthesis and stomatal conductance of potato leaves-effects of leaf age, irradiance, and leaf water potential.

Transcriptomic analysis of the primary roots of Alhagi sparsifolia in response to water stress.

Strigolactone Regulates Leaf Senescence in Concert with Ethylene in Arabidopsis.

Proteome data associated with the leaf senescence in Glycine max.

Effects of temperature on leaf hydraulic architecture of tobacco plants.

The effects of leaf roughness, surface free energy and work of adhesion on leaf water drop adhesion.