Plant materials and growth conditions
The LYP9 rice cultivar was cultivated in experimental fields of Nanjing Normal University. Regular management was performed according to Yu et al. (2012). Sampling was performed in the mornings (09:30 − 10:30 a.m.) on clear days at approximately 7-day intervals from September 11 (premature senescence) to October 10 (near grain harvesting time). A leaf was carefully detached from the petiole with fine forceps and for in vitro experiments, the mid-vein was removed from the leaf, and where necessary any contaminating leaf tissue was stripped removed, following procedures described by Brown et al. (2010). Several plant samples were pooled to obtain sufficient material, frozen in liquid nitrogen and stored at − 80 °C.
Detection of photosynthetic parameters
Measurements of the assimilation rate (A), transpiration rate (E), internal CO2 concentration (Ci) and water use efficiency (WUE) of the leaf lamina and mid-vein were carried out in the field using a portable photosynthesis system (CIRAS-3, PP-Systems Hitchin, UK). The conditions were: ambient CO2 concentration was 390±10 mmol mol-1, PAR intensity was at 1, 200 ± 50 μmol m-2s-1, flow rate was 300 ml min-1, leaf temperature was 25±1°C, and relative air humidity was 65-70% (Zhang et al. 2006). In order to accurately measure the photosynthetic rate of the leaf lamina and mid-vein, a 3 mm × 30 mm rectangular area was used to cover other tissues and fitted between clips of the CIRAS-3 and the mid-vein or leaf lamina during measurements (Pavlovic et al. 2009). The mid-vein was enclosed in a leaf cuvette and measurements were started in the morning between 09:00 and 10:00 a.m.with 10 repeats for each leaf analyzed, between the period from September 11 (premature senescence) to October 10 (near grain harvesting time).
Measurement of chlorophyll a fluorescence
Chlorophyll fluorescence parameters in the leaf lamina and mid-vein were estimated simultaneously with a portable fluorometer (Handy PEA, Hansatech, UK) as previously described (Strasser et al. 1995). Samples of leaf lamina and mid-vein still attached to the plants were collected from the midsection of the same leaf during the period from September 11 (premature senescence) to October 10 (near grain harvesting time). To make sure that photon exchange between the instrument and the mid-vein or leaf lamina did not interfere with each other when measuring light intensity, a 3 mm × 15 mm rectangular area and a non-fluorescing piece of black tape, were used as in Manetas (2004) and Panda et al. (2013). Prior to each measurement, leaf clips for dark adaptation were placed on the leaves for 30 min and the leaves were then illuminated with continuous red LED light (peak at 650 nm) at an excitation irradiance of 3,000 μmol m–2 s–1 with a duration of 800 ms. We repeated and recorded these measurements 10 times for each leaf, and the data analysis was performed using the professional PEA Plus and Biolyzer HP3 software (Hansatech, UK). Parameters are described in Table 1.
Measurement of photosynthetic pigments
For measurements of total chlorophyll (Chl) and carotenoid (Car) concentrations, pigments were extracted with acetone: ethyl alcohol (1:1 v/v) as described by Arnon (1949) and Lichtenthaler (2001). The concentrations of Chl and Car were measured with a spectrophotometer (Genesys 10UV, Thermo, USA) (Lichtenthaler and Wellburn 1983).
The extraction and analysis of xanthophyll by high performance liquid chromatography (HPLC) was performed as previously described (Wright et al. 2011). Under dimmed room lighting, a sample was extracted separately with 85% acetone and 100% acetone, before centrifugation for 4 min at 12,000 g at 4°C. The supernatant was further cleared by passing through a 0.22 μm nylon filter of organic phase (Nylon 66, Jinteng, China). The xanthophyll was eluted using 100% of solution A (acetonitrile: methanol, 85:15 v/v) for the first 14.5 min followed by a 2 min linear gradient with 100% solution B (methanol: ethyl acetate, 68:32 v/v) for an additional 28 min. The xanthophylls were then separated using a non-endcapped Zorbax Elipse (250 mm × 4.6 mm ID, XPB-C18, 5 μm) analytical column (Agilent 1100, USA). Pigments were detected by measuring absorbance at 445 nm.
Determination of adenosine triphosphate (ATP) content, and calcium-ATPase (Ca2+-ATPase) and magnesium-ATPase (Mg2+-ATPase) activities
ATP content was measured using the bioluminescence method described by Zhu et al. (2001) and expressed as μmol (ATP) mg–1 (Chl). The assay for Ca2+-ATPase and Mg2+-ATPase activities was performed as described by Vallejos et al. (1983) and Ma et al. (2016).
Two-dimensional electrophoresis (2-DE) and image analysis
Rice leaves grown for 120 days were collected as material for 2-DE. Protein samples were isolated separately from 1 g of the leaf lamina and mid-vein using a trichloroacetic acid (TCA)-acetone/phenol extraction method (Wang et al. 2006). Samples were extracted with 10 % (w/v) trichloromethane in acetone and centrifuged at 16,000 g for 5 min at 4ºC. The pellet was washed with 0.1M ammonium acetate and 80% acetone, incubated at -20ºC for 1 h, and centrifuged at 16,000 g for 20 min at 4ºC. The pellet was air dried at room temperature. Approximately 0.1 g pellet was added to 0.6 mL of a Tris-saturated phenol solution (pH 8.0) and 0.6 mL of sodium dodecyl sulfate (SDS) buffer (30% sucrose, 2% SDS, 0.1M Tris-HCl, pH 8.0, 5% mercaptoethanol), incubated for 5 min, and centrifuged at 16,000 g for 20 min at 4ºC. The phenol phase was transferred to a new tube containing four to five times the volume of 0.1M ammonium acetate in 80% (v/v) methanol (100 mL), and the samples were incubated overnight at -20ºC. After centrifugation, the pellet was air dried at room temperature and resuspended in an immobilized aqueous solution (8M Urea, 20% CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue, 10 mL). Three independent samples were extracted as biological replicates.
2-DE and image analysis were performed as described by Carpentier et al. (2005). The first dimension isoelectric focusing (IEF) was performed with IPG strips (Bio-Rad, USA, pH 4-7, 24 cm) with an Ettan IPGphor 3 system (GE Healthcare, USA). The second-dimension electrophoresis was performed on 24 × 9 × 24 cm SDS-PAGE gels (12.5% acrylamide) without a stacking gel using an Ettan DALT six Large Vertical System (GE Healthcare, USA). A total of six 2-DE gels were loaded with equal amounts of protein (1.25 mg) dissolved in the aqueous solution to a final volume of 425 mL. The gels were stained by 0.1% (w/v) Coomassie brilliant blue R-250 for 2-3h, then the gel images (Figure S1) were analyzed using the method described by Carpentier et al. (2005), using the Image-Master 2-D Elite software version 4.01 (Amersham Biosciences). Protein spots showed differences in size that were observed in all the replicates were selected as targets.
In-gel digestion and MALDI-TOF/TOF MS analysis
In-gel tryptic digestion of proteins in the selected spots was performed as in Guha et al. (2013). Samples were analyzed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) with a proteomics analyzer (4800 Plus, Applied Biosystems, USA), and were internally calibrated using tryptic peptides from auto-digestion. Database searching and PMF (peptide mass fingerprinting) was performed using the in-house Mascot server (http://www.matrix science.com) for matching against to the National Center for Biotechnology non-redundant (NCBI nr) database.
Protein functions were assigned using the protein functional database UniProt (http://www.uniprot.org) and Inter-Pro (http://www.ebi.ac.uk/interpro/) (Apweiler et al. 2002). Proteins identified were then categorized according to their assigned biological functions as described by Bevan et al. (1998). The subcellular locations of the unique proteins identified in this study were predicted using WolfPsort (http://wolfpsort.org) (Wu et al. 2013).
Statistical analysis
Values are presented as mean ± standard deviation from at least three individual experiments. Data were assessed by independent samples t test analysis of variance using GraphPad Prism 6 and SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences between mid-vein and leaf lamina samples were considered significant at P<0.05.