Journal of Integrative Agriculture
Advanced Online Publication: 2013
Doi: 10.1016/S2095-3119(13)60670-X
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Low light stress down-regulated Rubisco gene expression and photosynthetic
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capacity during cucumber (Cucumis sativus L.) leaf development1
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SUN Jian-lei1*, SUI Xiao-lei1*, HUANG Hong-yu1, WANG Shao-hui2, WEI Yu-xia1, ZHANG Zhen-xian1
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1
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protected vegetable crops, China Agricultural University, Beijing 100193, People’s Republic of China.
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2
College of Agriculture and Biotechnology, Beijing key laboratory of growth and development regulation for
Plant Science Department, Beijing University of Agriculture, Beijing 102206, People’s Republic of China.
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9
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Abstract
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Low light stress is one of the most important factors affecting photosynthesis and growth in
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cucumber (Cucumis sativus L.) winter production in solar greenhouses in northern China. Here,
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two genotypes of cucumber (Deltastar and Jinyan No.2) are used to determine the effect of low
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light stress on Rubisco expression and photosynthesis of leaves from emergence to senescence.
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During leaf development, the net photosynthetic rate (PN), stomatal conductance (gs), Rubisco
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initial activity and activation state, transcript levels of rbcL and rbcS, and the abundance of rbcL
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and rbcS DNA in these two genotypes increase rapidly to reach maximum in 10-20 days, and
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then decrease gradually. Meanwhile, the actual photosystem II efficiency (ФPS II) of cucumber
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leaves slowly increased in the early leaf developing stages, but it declined quickly in leaf
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senescent stages, accompanied by an increased non-photochemical quenching (NPQ). Moreover,
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PN, gs, initial Rubisco activity, and abundance of protein, mRNA and DNA of Rubisco subunits
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of leaves grown under 100 μmol·m-2·
s-1 are lower, and require more time to reach their maxima
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than those grown under 600 μmol·m-2·
s-1 during leaf development. All these results suggest that
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lower photosynthetic capacity of cucumber leaves from emergence to senescence under low light
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stress is probably due to down-regulated Rubisco gene expression in transcript and protein levels,
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and decreased initial and total activity as well as activation state of Rubisco. Deltastar performs
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better than Jinyan No.2 under low light stress.
1
*These authors contributed equally to this work.
Correspondence
Zhang
[email protected] ;
Zhenxian
g,
Tel:
+86-10-62731952 ， Fax:
+86-10-62731952 ，
E-mail:
Journal of Integrative Agriculture
Advanced Online Publication: 2013
Doi: 10.1016/S2095-3119(13)60670-X
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Key words: Cucumber, photosynthetic capacity, Rubisco, rbcL, rbcS, low light
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INTRODUCTION
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Cucumber (Cucumis sativus L.) is an important vegetable crop grown under single-slope solar
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greenhouses in winter and spring seasons in northern China. Low light availability is one of the
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most important limiting factors affecting cucumber production in solar greenhouses (Gao et al.
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2010). Leaves of plants grown under low light are generally thinner, larger in surface area and
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have a higher ratio of palisade/spongy tissues compared to leaves of plants grown under high light
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(Murchie et al. 2005). Low light inhibits photosynthetic performance, which leads to reductions in
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net photosynthetic rate (PN), linear whole-chain electron transport rate and partitioning proportion
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for photochemical reaction of light energy absorbed by PS II of leaves. At the same time, low light
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can induce an imbalance of partitioning of excitation energy between PS I and PS II (Zhou et al.
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2004). Low light intensity or darkness results in reduced expression of light-dependent genes and
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disappearance of photosynthetic proteins and chlorophylls (Wingler et al. 1998). Recently, we
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have shown that greenhouse ecotypes of cucumbers are more resistant to inhibition of
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photosynthesis by low light stress than field ecotypes (Li et al. 2008). We have also shown that
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low light results in the loss and inactivation of ribulose-1, 5-bisphosphate carboxylase-oxygenase
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(Rubisco) (Sui et al. 2011).
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Senescence is a phase of leaf development marked by a decline in photosynthetic activity,
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disintegration of organelle structures, intensive loss of chlorophylls and proteins, and finally leaf
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death and abscission (Ananieva et al. 2008; Guo et al. 2004; Smart 1994). The process involves
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degradation of proteins and chlorophylls, and remobilization of limiting nutrients to growing
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and/or storage organs (Brouwer et al. 2012). One of the most conspicuous features of senescence
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is the decline in photosynthetic capacity, which has usually been divided into three phases during
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leaf ontogeny in dicotyledons: an early phase of increased photosynthetic rate when the leaf is
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actively expanding, a phase of maximal rate at full leaf expansion, and finally, a prolonged
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senescence phase of steady decline in photosynthetic rate (Jiang et al. 1993). A variety of factors
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are involved in controlling photosynthetic rate during leaf development. One of the major
Journal of Integrative Agriculture
Advanced Online Publication: 2013
Doi: 10.1016/S2095-3119(13)60670-X
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characteristics of late leaf development and senescence is loss of proteins, especially the loss of
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Rubisco, which accounts for a major portion of the loss of C3 species (He et al. 1997).
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Ribulose-1, 5-bisphosphate carboxylase /oxygenase (Rubisco, EC 4.1.1.39) is a key enzyme in
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photosynthesis and the most abundant protein in leaves. It accounts for 15–30% of total leaf N
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content in C3 species (Evans and Malmberg 1989; Makino et al. 1992) and constitutes >50% of
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all soluble proteins in mature leaves of C3 plants (Makino et al. 1984). Rubisco catalyzes two
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competing reactions: CO2 fixation in photosynthesis and the production of 2-phosphoglycolate in
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the photorespiratory pathway. Furthermore, Rubisco is a rate-limiting factor for both
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photosynthesis and photorespiration under conditions of saturating light at atmospheric CO2 and
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O2 levels (Hudson et al. 1992; Quick et al. 1991). In higher plants, Rubisco is composed of eight
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small subunits encoded by a nuclear multigene family (rbcS) (Dean et al. 1989), and eight large
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subunits encoded by a single gene (rbcL) in the chloroplast genome (Rodermel et al. 1996).
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Rubisco content increases rapidly during leaf expansion, reaching its maximum around maturation,
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and then declines gradually during senescence (Imai et al. 2008; Imai et al. 2005; Suzuki et al.
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2009b). A decrease in Rubisco activity is a hallmark of senescence (Craftsbrandner 1992; Ford
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and Shibles 1988; Secor et al. 1984). Rubisco is degraded, and its degradation products are
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utilized as sources of N for developing tissues during leaves senescence (Suzuki et al. 2001).
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Changes in the amount of Rubisco in leaves are thus directly related to carbon and N economy in
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plants. Rubisco content in a leaf is dictated by a balance between its synthesis and degradation.
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Changes of the levels of rbcL and rbcS mRNAs and their relationship to the synthesis of Rubisco
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from emergence to senescence have been studied in bean (Phaseolus vulgaris L.) (Bate et al.
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1991), amaranth (Nikolau and Klessig 1987), and rice leaves (Suzuki et al. 2001). Although
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severa1 reports have shown altered rates of Rubisco subunit synthesis at the transcriptional and
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translational levels during senescence (Bate et al. 1991; Jiang et al. 1993; Suzuki et al. 2001;
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Suzuki et al. 2009a), the regulation of Rubisco synthesis may be different among species. The
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question arises whether the accumulation of this protein also is regulated differently among
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various leaves of a given species.
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The main aims of the present study are: 1) to analyze the changes of rbcL and rbcS expressions,
Journal of Integrative Agriculture
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protein content, activity and activation state of Rubisco, and photosynthetic characteristics during
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leaf development; 2) to explore the effects of growth light intensity on photosynthetic
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characteristics and leaf senescence; 3) to compare the capacity of different genotypes of cucumber
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plants to respond to low light stress during leaf development.
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MATERIALS AND METHOD
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Plant material and growth conditions
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The seeds of two cucumber (Cucumis sativus L.) genotypes, Deltastar (a greenhouse cultivar
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from Rijk Zwaan Co. Holland) and Jinyan No. 2 (a field cultivar from Tianjin Cucumber Institute,
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China), were sowed in 12 cm-diameter plastic pots and the seedlings were cultivated with
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Hoagland solution in phytotron under a 10 h light/14 h dark regime, 25/18°C day/night
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temperature, 65-85% relative humidity (RH) and a photosynthetic photon flux density (PFD) of
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550-600 μmol·m-2·
s-1. At 3-leaf stage, the seedlings of each genotype were divided into two
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groups. One group was exposed to 75-100 μmol·m-2·
s-1 (low light, LL), whereas another group
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was kept at 550-600 μmol·m-2·
s-1 (control light, CT), under the same photoperiod (10 h),
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temperature regime (25/18°C day/night) and RH (65%-85%), we used artificial light controlling
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the different light intensity. There were four combinations between genotypes and growth light
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conditions: Deltastar control (CT-D), Deltastar low light treatment (LL-D), Jinyan No.2 control
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(CT-J), and Jinyan No.2 low light treatment (LL-J). The date of emergence of all new leaves was
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noted, and the fourth leaves (from bottom of the plant) of both growth light conditions were
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sampled at 1, 5, 10, 15, 20, 30, 40, 50 days respectively after the treatment, and used to determine
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parameters of photosynthesis and chlorophyll fluorescence and to analyze gene expression and
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enzyme activity. Each treatment was replicated four times; and each measurement of index was
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replicated three times, respectively.
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Plant growth and leaf characteristics
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Leaf area, specific leaf weight (SLW), soluble protein content and total chlorophyll content of
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the fourth leaves of two varieties emergence through to senescence were determined at 1, 5, 10, 15,
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20, 30, 40, 50 days respectively after the treatment. Leaf area was traced onto paper, and the area
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was measured by weighing the paper (Pandey and Singh, 2011). To determine specific leaf weight
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(SLW), leaf discs were taken from the middle of the leaf lamella and fresh weight was measured.
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SLW was calculated as fresh weight of leaf discs (g) divided by their area (m2). Total chlorophylls
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were extracted with 95% ethanol and chlorophyll concentrations were then determined by
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measuring absorbance at A663 and A645 nm using a spectrophotometer according to Wintermans
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and Demots (Winterma and Demots 1965).
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Photosynthesis and chlorophyll a fluorescence
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Gas exchange parameters of the leaves tested were measured with a LI-6400 portable
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photosynthesis system (Li-Cor, Lincoln, NE, USA). The actual CO2 assimilation rate (PN),
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stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) were
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recorded under 600 μmol·m-2·
s-1 or 100 μmol·m-2·
s-1 with a constant ambient CO2 concentration
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400±10 μmol·
mol-1, air humidity 50-60% and leaf temperature 25±1°C throughout all
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measurements. Air flow rate through the assimilation chamber was maintained at 500 μmol·
s-1.
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Water use efficiency (WUE) was calculated as PN /Tr.
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Chlorophyll fluorescence parameters were measured using a chlorophyll fluorometer
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(PAM-2100, Walz, Effeltrich, Germany). Prior to measurements, leaf blades were dark adapted in
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the leaf disk chamber for 20 min. The fluorescence parameters, including the maximum PSII
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quantum yield in dark-adapted state (Fv/Fm), the steady-state PSII quantum yield in light (ΦPSII)
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and steady-state non-photochemical quenching in light (NPQ), were calculated as follows: (1)
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Fv/Fm =( Fm-Fo)/ Fm; (2) ФPSII = (Fm＇－ Fs)/Fm＇; and (3) NPQ= Fm/Fm＇－ 1 (Bilger and
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Bjorkman 1990; Genty et al. 1989).
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Enzyme extraction and measurement of total soluble protein
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Frozen leaf samples (0.1 g) were homogenized using chilled mortar and pestle with cold
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extraction buffer (1 mL) containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM MgCl2,
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12.5% (v/v) glycerol, 1% polyvinylpolypyrrolidone (PVPP) and 10 mM β-mercaptoethanol. The
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homogenate was centrifuged for 15 min at 15,000 g and 4°C. The supernatant was used for
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determination of enzyme activity and quantity.
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Total soluble protein was determined according to the method of Bradford (Bradford 1976) at
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595 nm, with bovine serum albumin as standard.
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Measurement of Rubisco activity
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Initial and total activities of Rubisco were measured spectrophotometrically by coupling
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3-phosphoglyceric acid formation with NADH oxidation at 25°C as described by Zheng (Zheng
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2006). Initial Rubisco activity was assayed immediately after extraction, whereas total Rubisco
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activity was assayed after the extract was activated in a 0.1 mL activation mixture containing 33
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mM Tris-HCl (pH 7.5), 0.67 mM EDTA, 33 mM MgCl2, 10 mM NaHCO3 at 30°C for 10 min.
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Rubisco activity was measured in a 0.5 mL reaction mixture containing 50 mM Hepes-KOH (pH
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8.0), 1 mM EDTA, 20 mM MgCl2, 2.5 mM dithiothreitol (DTT), 10 mM NaHCO3, 5 mM
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ATP-Na2, 5 mM Phosphocreatine disodium hydrate, 0.15 mM NADH2, 5U of creatine
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phosphokinase, 5U of 3-phosphoglyceric phosphokinase, 5U of glyceraldehydes 3-phosphate
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dehydrogenase, 0.6 mM RuBP and 50 μL of extract. The change in absorbance at 340 nm was
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monitored for 60 s. The ratio of initial to total activity, indicating a good estimate of Rubisco
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carbamylation when the leaf was frozen, was taken as the activation state of Rubisco (Butz and
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Sharkey 1989).
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Western blot analysis
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The relative amounts of Rubisco large subunit protein (LSU) and Rubisco small subunit
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protein (SSU) were determined by Western blotting. Samples were made to 1:1 (v/v) in a buffer
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containing 2% (w/v) sodium dodecyl sulphate (SDS), 100 mM Tris, 10% (v/v) glycerol, 5% (v/v)
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β-mercaptoethanol and 0.2% bromophenol blue. Proteins were denatured by boiling at 100°C for 3
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min. Equal amounts of total protein (20 μg) were used for SDS-polyacrylamide gel electrophoresis
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(PAGE) in an Amersham Biosciences MiniVE (Amersham Biosciences, Freiburg, Germany).
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Migration was performed at 30 mA per mini gel for 2 h using 12.5% separating and 4.5% stacking
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gels. After electrophoretic separation, proteins were electro-blotted onto a nitrocellulose
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membrane (Amersham, les Ulis, France) at 25 V using a semidry system (Trans Blot SD;
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Amersham, Freiburg, Germany) for 2 h. The membrane was blocked with 3% (w/v) BSA in TBS
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(10 mM Tris, pH 7.5, 150 mM NaCl). The immunochemical detection of Rubisco LSU and SSU
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was carried out with Rubisco LSU antibody, form I and form II (Agrisera, Vännäs, Sweden), and
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Rubisco SSU antibody (Agrisera, Vännäs, Sweden). A horseradish peroxidase conjugated
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goat-anti-rabbit
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chemiluminescence. The visualization was performed with NBT and BCIP. Films were scanned
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using an Alpha Imager EP (NatureGene Corp, USA).
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RNA extraction and Northern blot
secondary
antibody
(Sigma-Aldrich,
USA)
was
then
detected
by
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Total RNA extraction was performed from 0.1-0.2 g leaf tissues using the Column Plant
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RNAout Kit (Tiandz, Beijing, China). DNA was digested with the RNase-Free DNase I (Tiangen
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Biotech, Beijing CO., LTD. China), RNA was reverse-transcribed with oligo (dT) using reverse
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transcriptase (Promega, Madison, WI, USA), and cDNA was then amplified by PCR. Specific
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primers for rbcL and rbcS sequence amplification were designed in their conserved sequences:
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rbcL: forward primer, 5′ -ACAACTGTGTGGACCGATGGGCTTA -3′
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reverse primer, 5′- CAAGGGTGCCCCAAAGTTCCTCCGC -3′
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rbcS: forward primer, 5′- CCATTCTCTCATCCGCCGCTGTTGC -3′
reverse primer, 5′- AACATAGTGGGATTCAACAGACAAA -3′
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Amplification was achieved under the following conditions: 2 min at 94°C, 30 cycles of
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denaturation at 94°C for 15 s, annealing at 60°C (rbcL) or 58°C (rbcS) for 30 s, and elongation at
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68°C for 1 min, followed by elongation at 68°C for 10 min. PCR products were analyzed by
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agarose gel electrophoresis, cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA) and
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used for sequencing or DIG-labelled probe synthesis.
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Total RNA (rbcL 2 μg or rbcS 5 μg) was separated in a denaturing 1.2% (w/v) agarose gel
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containing 2% formaldehyde and transferred to a positively charged nylon membrane (Hybond-N+
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Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to standard protocol. The
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membrane was oven-baked at 80°C for 2 h. Hybridization and washing of the membrane was
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carried out according to the digoxlgenin (DIG) system user’s guide (My lab, Indianapolis, IN,
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China). The DIG-labelled DNA probes for rbcL and rbcS were prepared from fragments of the
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cucumber rbcL and rbcS genes, respectively, according to the PCR DIG probe synthesis kit (My
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lab, Indianapolis, IN, China). Signals were detected with a DIG luminescent detection kit. Films
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were then scanned (Alpha Imager EP).
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DNA extraction and Southern blot
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Total DNA was extracted from leaves of cucumber using cetyltrimethyl ammonium bromide
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(CTAB). For DNA blot analysis, 25 μg total DNA was digested with EcoRV (LSU) or BamHI
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(SSU), phenol-extracted, and then ethanol-precipitated and dissolved in ddH2O. The DNA fraction
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was separated in a 0.8% agarose gel, and blotted onto cellulose acetate membrane. The membrane
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was then dried at 80°C. Methods for the hybridization, detection and determination of signal
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intensities were the same as those described above for the Northern blot analysis.
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Statistics
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Data and the graphs were processed using Microsoft Excel 2003. Origin 7.5 Statistical analysis
205
of variance (ANOVA) was done using SPSS software Version 16.0, and each value of means and
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standard errors in figures represents four replicates.
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RESULTS
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The leaf characteristics in the fourth leaf
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After the emergence of the fourth leaf, leaf area increased rapidly in the first 30 days, then
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continued to slowly increase until reaching its maximum (Fig. 1-A). SLW was only slightly
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changed during the time of full expansion, especially in the first 15 days (Fig. 1-B). Total soluble
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protein increased rapidly during leaf expansion, reaching maximum at 10 days, and then rapidly
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declined (Fig. 1-C). Chlorophyll (Chl) contents reached maximum at tenth day in plants grown
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under 100 μmol·m-2·
s-1and fifth day in plants grown under 600 μmol·m-2·
s-1 respectively, then
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declined gradually (Fig. 1-D).
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Comparing with control light, leaf areas of plants grown under PPFD 100 μmol·m-2·
s-1 were
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larger, but soluble protein content and SLW were lower (Fig.1-A-C). In addition, total Chl content
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was significantly increased in Deltastar during the whole progress of leaf development under low
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light stress, but not in Jinyan No. 2 (Fig. 1-D).
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The parameters of photosynthesis and chlorophyll a fluorescence
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Light intensity had a great effect on photosynthesis parameters in cucumber leaves from
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emergence to senescence. PN, gs and WUE increased rapidly after leaf emergence, and reached
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maxima in 10 to 20 d, then declined gradually (Fig. 2-A, B and D). However, Ci, showing the
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opposite trend, decreased rapidly after leaf emergence, reached minimum in 10-15 d, then
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increased gradually (Fig. 2-C).
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The steady state PN, gs and WUE of leaves grown under the 100 μmol·m-2·
s-1 were obviously
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lower than those grown under 600 μmol·m-2·
s-1, However, the time it took to reach maximum PN,
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gs and WUE under low light (twenty days) longer than under control light (ten days) (Fig. 2-A, B
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and D). When grown under 100 μmol·m-2·
s-1, Deltastar had slightly higher steady state PN, gs and
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WUE than Jinyan No.2. These implied that Deltastar was more tolerant to low light than Jinyan
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No.2.
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Changes in photosystem II (PS II) efficiency were measured via chlorophyll fluorescence
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(Fv/Fm) kinetics (Fig. 3A). Fv/Fm ratio of the fourth leaf stayed at maximum (0.8) until 10-15d
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after emergence, then sharply decreased under control light (Fig. 3-A). However, Fv/Fm started to
235
decline slowly from 30 days after the beginning of the treatment in low light-grown leaves. The
236
quantum yield of PS II (ФPS II) (Fig. 3-B) increased gradually, reaching maximum in the tenth
237
day after leaf emergence, then declined rapidly. On the contrary, NPQ (Fig. 3-C) had an opposite
238
variation trend to ФPS II.
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The maximum Fv/Fm and ФPS II were higher in plants grown under 100 μmol·m-2·
s-1 than
240
those grown under 600 μmol·m-2·
s-1 in leaf fast growth stage (Figs. 3-A and 1-A). This indicated
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that the original activity of PS II reaction centre was increased, and the transformation efficiency
242
of primary light energy was improved in the low light-acclimated leaves of cucumber. However,
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differences between the two genotypes in changes of Fv/Fm, ФPS II and NPQ in response to low
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light were less apparent.
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Rubisco activity and activation state
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To assess the contributions of Rubisco activity and activation state to the control of
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photosynthesis capacity during leaf development, we measured Rubisco initial activity, total
248
activity and Rubisco activation state (Fig. 4). The results showed that initial activity (Fig. 4-A),
249
total activity (Fig. 4-B) and activation state (Fig. 4-C) of Rubisco increased rapidly to reach
250
maxima in 10-15 days of leaf development, and then gradually decreased during leaf senescence.
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Comparing with the control light, the initial activity (Fig. 4-A), total activity (Fig. 4-B) and
252
activation state (Fig. 4-C) of Rubisco in leaves grown under 100 μmol·m-2·
s-1 had lower maximum
253
values and delayed kinetics. Under control light, Jinyan No.2 had a higher maximum values of
254
initial (Fig. 4-A) and total (Fig. 4-B) Rubisco activities than Deltastar, but under 100 μmol·m-2·
s-1
255
low light, the values were higher for Deltastar than for Jinyan No.2, especially the Rubisco
256
activation state.
257
Changes in the protein levels of LSU and SSU
258
Western blotting was used to monitor the changes of the protein levels of LSU and SSU. A 53
259
kDa polypeptide of LSU was detected in cucumber leaves. The relative abundance of LSU
260
increased rapidly just after leaf emergence and reached their maximum after 10-15 days, and then
261
decreased gradually during leaf development (Fig. 5). LSU was clearly discernible for the first 15
262
days, but its presence in older leaves was more difficult to evaluate unequivocally because of the
263
appearance of another partially resolved band at about 50 kDa, which may be partially degraded
264
LSU. Lower LSU content was detected in leaves grown under 100 μmol·m-2·
s-1 than that grown
265
under 600 μmol·m-2·
s-1, especially in the leaves of Jinyan No.2. Moreover, LSU content was
266
higher in leaves of Deltastar than in leaves of Jinyan No.2 when both grown under 100
267
μmol·m-2·
s-1 (Fig. 5). A polypeptide of 14 KD of SSU detected in cucumber leaves had equivalent
268
trends to LSU, but its expression declined more slowly than LSU (Fig. 5).
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Changes in the levels of rbcL mRNA, rbcS mRNA, rbcL DNA and rbcS DNA
270
Northern blot analysis showed that the transcript level of rbcL increased rapidly just after leaf
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emergence, reached maximum on the 10th or 20th day, then declined gradually during leaf
272
development (Fig. 6-A). Comparing with the control light condition, rbcL transcript abundance of
273
cucumber leaves grown under 100 μmol·m-2·
s-1 was lower and had delayed kinetics. The
274
maximum expression was reached in twentieth days after leaf emergence in plants grown under
275
low light, as opposed to tenth days for those grown under control light. The levels of rbcS
276
expression had a similar trend as rbcL, but changed more slowly from leaf emergence to
277
senescence.
278
DNA template availability is an important factor influencing transcript abundance. To estimate
279
the availability of the rbcL and rbcS templates, Southern blot analysis was performed to examine
280
the DNA abundance of rbcL and rbcS. The result showed that the level of rbcL DNA increased
281
gradually after leaf emergence, reached its maximum just before full expansion, and then declined
282
significantly during leaf development (Fig. 6-B). The level of rbcS DNA also increased after leaf
283
emergence and either remained almost constant or slightly declined thereafter during senescence
284
(Fig. 6-B). DNA levels of rbcL and rbcS in leaves grown under 100 μmol·m-2·
s-1 were lower than
285
under 600 μmol·m-2·
s-1 (Fig. 6-B). Under 100 μmol·m-2·
s-1, the expression levels of rbcL and rbcS
286
were decreased more significantly in Jinyan No.2 than in Deltastar (Fig. 6-B).
287
DISCUSSION
288
Rubisco is a key enzyme in crop photosynthesis. Senescence-associated loss in photosynthetic
289
capacity is usually correlated to alterations in Rubisco activity (Gepstein 1998). Our data shows
290
that photosynthesis is closely related to initial Rubisco activity (Figs. 2-A and 4-A), which may be
291
a consequence of alterations in either the activation state or abundance of the enzyme. Given the
292
likely importance of Rubisco content in determining photosynthetic capacity, it was of interest to
293
elucidate the mechanisms that regulate Rubisco content during cucumber leaf senescence. Our
294
data indicate that differences in photosynthetic capacities of leaves grown under 600 μmol·m-2·
s-1
295
and 100 μmol·m-2·
s-1 during senescence are correlated with alterations in Rubisco activity (Fig. 4)
296
and content (Fig. 5). Moreover, Western blot analysis (Fig. 5) shows that the level of LSU
297
declines faster than SSU during leaf senescence. We also observe that overall reductions in the
298
abundance of LSU and SSU are accompanied by reductions in the levels of rbcL and rbcS mRNAs
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during leaf senescence (Figs. 5 and 6-A). Furthermore, the level of rbcS changes more slowly than
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rbcL from leaf emergence to senescence. Transcriptional regulations of rbcS and rbcL mRNAs
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seem to be primarily responsible for the level of the holoenzyme during the senescence process in
302
cucumber leaves. Therefore, it appears that the decrease of expression levels of both nuclear and
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chloroplast-encoded photosynthetic proteins during senescence is primarily caused by a decrease
304
of mRNA levels (Bate et al. 1991), and the decline of translational efficiencies of rbcS and rbcL
305
mRNAs (Imai et al. 2008). Moreover, Rubisco content has been shown to be more highly
306
associated with the mRNA level of rbcL than with that of rbcS (Suzuki et al. 2007). These data
307
suggest that the synthesis of the large subunit strongly limits the extent Rubisco production
308
(Suzuki et al., 2007).
309
(Fig. 6-A) occurred almost in parallel with the reduction in the level of rbcL DNA (Fig. 6-B)
310
during leaf development. This observation indicates that the level of rbcL DNA is a major
311
determinant of the level of rbcL mRNA. It has been suggested that the decline in the level of rbcL
312
DNA can limit the level of rbcL mRNA during leaf senescence. For example, the level of rbcL
313
mRNA declines almost in parallel with the level of rbcL DNA in tobacco (Jiang and Rodermel
314
1995; Miller et al. 2000), and declines slightly ahead of rbcL DNA during senescence in soybean
315
(Jiang et al. 1993) and in rice (Suzuki et al. 2001).
From the results we can see that the decline in the level of rbcL mRNA
316
It has been observed in this study that changes in Chlorophyll (Chl) content (Fig. 2-A)
317
correspond approximately to alterations in photosynthetic capacity during leaf senescence. Chl
318
content starts to decrease (from 5-10 days after the beginning of the treatment) before the fourth
319
leaves reach their full surface area, and almost 50% of Chl and 50% of proteins are lost (Fig. 1-A
320
and D). In spite of a steady reduction in Chl content, PSII efficiency (measured as Fv/Fm, Fig. 3-A)
321
in the fourth leaves stays at a higher value until 30 days of treatments, especially in the low
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light-grown leaves. The functional organization of the remaining PSII units seems to not be
323
affected up to this time point. Accordingly, the photosynthetic apparatus still maintained high
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efficiency of PSII photochemistry in spite of a loss of Chl in the senescent leaves, which has been
325
observed in wheat leaves (Lu and Zhang 1998). NPQ is linearly related to excited energy
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dissipation, and can be regarded as a sensitive indicator of thermal dissipation. The decrease in
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ФPSII (Fig. 3-B) was accompanied by a corresponding increase in NPQ (Fig. 3-C) during leaf
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senescence, especially in the leaves grown under low light. It is suggested that heat dissipation
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increased to a greater extent to presumably protect the PSII photosystem from photoinhibition in
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the senescent leaves grown under low light (Yoo et al. 2003; Weng et al. 2005). Our study agreed
331
with the view that the decrease in PSII quantum yield (Fig. 3-B) may represent a mechanism of
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down-regulated phoyosynthetic electron transport to match the decreased CO2 assimilation
333
capacity (Fig. 2-A) (Sui et al. 2012).
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Low light stress has an important effect on the photosynthetic capacity of cucumbers during
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leaf development. Steady state PN and gs of Leaves grown at 100 μmol·m-2·
s-1 was lower than
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those grown at 600 μmol·m-2·
s-1 for both genotypes (Fig. 2), especially in leaves of Jinyan No.2
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grown under low light. Moreover, the time reaching the maximum PN was longer for plants grown
338
under 100 μmol·m-2·
s-1 than for plants grown under 600 μmol·m-2·
s-1. Although the changes in
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Rubisco activity, protein contents and abundance of Rubisco transcripts and plastid DNA during
340
development of low light-grown leaves of cucumber were very similar to that changes under
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control light, the time reaching peak values under low light was also delayed and the maximum of
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the values decreased, especially in the low light-grown leaves of Jinyan No.2 (Figs. 1-D, 4, 5 and
343
6). In other words, it was probably that low light stress down-regulated photosynthesis through
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declining Rubisco gene expression, including transcript and translation, Rubisco activity, and
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activation state. Which was in agreement with the photosynthetic capacity was regulated by
346
Rubisco in Arabidopsis (Zhang et al. 2001), tobacco (Tholen et al. 2007) and potato (Cen and
347
Sage 2005). In addition, two cucumber varieties had different adaptability to low light stress. By
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comprasion, Rubisco expression, initial activity and activation state, as well as photosynthetic rate
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in Deltastar were larger than those in Jinyan No.2 under low light stress. That is to say, all these
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characteristics of Deltastar have contributed to better low light stress tolerance.
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CONCLUSION
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Our results demonstrated that low light had a significantly effect on the characteristics of
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photosynthesis and Rubisco of leaves from emergence to senescence. Under low light growth
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condition, PN, Rubisco activity, and abundance of protein, mRNA and DNA of Rubisco subunits
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of leaves increased gradually and decreased after reaching the maximum levels. The time reaching
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peak values under low light was delayed and the maximum of the values decreased, especially in
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the low light-grown leaves of Jinyan No.2 during leaf development. Meanwhile, the actual ФPS II
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of cucumber leaves increased slowly in the early leaf developing stages, but it declined quickly in
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leaf senescent stages, accompanied by an increased non-photochemical quenching (NPQ). In a
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word, low light stress down-regulated Rubisco gene expression in transcript and protein levels
362
result in lower photosynthetic capacity of cucumber leaves from emergence to senescence.
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Compared with low light-sensitive genotype (Jinyan No.2), Deltastar performs more low light
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tolerance.
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Acknowledgements
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This work was supported by China Agriculture Research System (CARS-25), the National Basic
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Research Program of China (973 Program, 2009CB11900) and the National Key Technologies
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Research and Development (R & D) Program of China (2011BAD12B01).
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Legends
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Fig. 1 Changes of leaf area (A), specific leaf weight (B), total soluble protein content (C) and
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chlorophyll content (D) in the fourth leaves from emergence to senescence. CT-D and CT-J:
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Deltastar and JY2 plants grown at 600 μmol·m-2·
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JY2 plants grown at 100 μmol·m-2·
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Fig. 2 Changes of PN (A), gs (B), Ci (C) and WUE (D) in cucumbr leaves from emergence to
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senescence. CT-D, CT-J, LL-D and LL-J are the same as in Fig. 1. Gas exchange parameters
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were determined at ambient CO2 (400 μmol·mol-1) and a temperature of 25°C.
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Fig. 3 Changes of Fv/Fm (A), ФPSⅡ (B) and NPQ (C) in cucumber leaves from emergence to
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senescence. CT-D, CT-J, LL-D and LL-J are the same as in Fig. 1. Each point is mean ± SE (n =
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4).
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Fig. 4 Changes of Rubisco inital (A) and total (B) activity and activation rate (C) in cucumber
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leaves from emergence to senescence. CT-D, CT-J, LL-D and LL-J are the same as in Fig. 1.
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Each point is mean ±SE (n = 4).
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Fig. 5 Western blot analysis of LSU and SSU in cucumber leaves from emergence to senescence.
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CT-D, CT-J, LL-D and LL-J are the same as in Fig. 1.
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Fig. 6 Northern blot analysis (A) and Southern blot analysis (B) of rbcL and rbcS in cucumber
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leaves from emergence to senescence. In the Northern blot analysis, Ethidium bromide staining
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of rRNA serves as the internal control of gene expression for equal loading. CT-D, CT-J, LLD
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and LL-J are the same as in Fig. 1.
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