Plant Physiology and Biochemistry xxx (2014) 1e6

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Research article

Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon Shahniyar Bayramov*, Novruz Guliyev Institute of Botany, Azerbaijan National Academy of Sciences, 40 Patamdar Shosse, AZ-1073 Baku, Azerbaijan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2013 Accepted 20 January 2014 Available online xxx

Regulation of Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) gene expression and polypeptide content were determined in Brachypodium distachyon leaves, stems and ear elements at different developmental stages under optimal growth conditions as well as under drought and salt stress conditions. B. distachyon leaf contains a much greater amount of Rubisco activase small (RCAS) isoform than the large one (RCAL) under optimal growth conditions. Increased levels of the RCAL isoform compared with the RCAS isoform were found in leaves and in green stems under salt and drought stress, respectively. Transcriptional levels of RCA are almost identical in different leaf positions. Short-term drought and salt stresses did not cause the impairment of RCA gene expression in early seedlings. But gradually increasing drought stress significantly decreased gene expression in early seedling samples. Amounts of the RCAS isoform were found to be more in different leaves of the plant compared with the RCAL isoform and their ratio was constant under normal condition. In green stems gene expression of RCA decreased under salt and drought stresses, although as it was in green leaves protein amounts of RCAL isoform increased compared with the RCAS isoform. All of the above described results clearly indicate that the accumulation of each RCA isoform is differentially regulated by developmental and environmental cues. Ó 2014 Published by Elsevier Masson SAS.

Keywords: Brachypodium distachyon Drought stress Gene expression Protein content Rubisco activase Salt stress

1. Introduction Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme catalyzing carbon assimilation in plants, exhibits maximal activity when all its active sites are in the proper conformation and are available for catalysis. In plant Rubisco, the lysine residue at position 201 (numbering based on residue positions in tobacco Rubisco) in the active site pocket must be carbamylated and bind a Mg2þ ion as cofactor for the enzyme to become catalytically active. Binding of ribulose-1,5-bisphosphate to uncarbamylated Rubisco, or of 2-carboxy-D-arabinitol 1-phosphate to the carbamylated enzyme at night, results in the trapping of sugar phosphate and in the inhibition of the enzyme. However, Rubisco sites can be deactivated by various mechanisms, and the rate of deactivation increases with temperature (Spreitzer and Salvucci,

Abbreviations: EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonylfluoride; PVP, polyvinylpolypyrrolidone; Rubisco, Ribulose-1,5bisphosphate carboxylase/oxygenase; Rubisco LS, Rubisco large subunit; RCA, Rubisco activase; RCAL, Rubisco activase large isoform; RCAS, Rubisco activase small isoform; RT-PCR, reverse transcription polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: þ994 504273377; fax: þ994 125102433. E-mail address: [email protected] (S. Bayramov).

2002). Rubisco activity during photosynthesis is regulated by the Rubisco activase (RCA), which facilitates the dissociation of ribulose-1,5-bisphosphate and other inhibitory sugar phosphates from the active site of Rubisco in an ATP-dependent reaction. Activase is a relatively abundant nuclear-encoded AAAþ protein located in the chloroplasts of higher plants and algae. Like other AAAþ proteins, activase functions as a mechanical motor, remodeling the conformation of its target protein, Rubisco. RCA rescues Rubisco sites from deadend inhibition by promoting ATP-dependent conformational changes that open closed sites, making them more accessible to solvent and facilitating the dissociation of inhibitory sugar phosphates (Portis et al., 2008). In this way, RCA is a molecular chaperone, controlling the switching of Rubisco conformation from inactive to active (Spreitzer and Salvucci, 2002). In several plant species, RCA consists of two polypeptides, which differ in length by the presence of an extra 30 amino acids at the C-terminus of one of the forms (Salvucci et al., 1987; Werneke et al., 1988). Even under non-stressed conditions, control of RCA expression is complicated and is regulated at various levels in different species. Most species studied contain two isoforms of RCA, an a-isoform of 46e48 kDa and a b-isoform of 41e43 kDa. The Rubisco activase large isoform (RCAL) differs from the Rubisco activase small isoform (RCAS) by the presence of a carboxyterminus extension, which contains the redox-sensitive disulfides.

0981-9428/$ e see front matter Ó 2014 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.plaphy.2014.01.013

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013

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The activity of longer isoform was regulated by redox state via thioredoxin f and this redox regulation was due to an interaction between carboxyl extension and nucleotide-binding pocket in RCAL. The C-terminal extension of the a-isoform contains two redox-regulated Cys residues that are modulated by thioredoxin f. When these residues are oxidized to a disulfide, the affinity for ATP decreases and enzyme activity is more sensitive to inhibition by ADP. Physiological ratios of ADP to ATP significantly inhibit the activity of the Arabidopsis a-isoform when in the oxidized state, but inhibition is much less when this isoform has been reduced by thioredoxin. In contrast, the shorter Arabidopsis b-isoform is not redox regulated and is less sensitive to inhibition by ADP (Zhang and Portis, 1999). In most of the species that have been examined, the two isoforms are products of a single, alternatively spliced pre-mRNA. In plants such as Arabidopsis, spinach, and rice, which have two RCA isoforms, the two forms are encoded by mRNAs produced from alternative splicing of the transcribed pre-mRNA from a single RCA gene (Zielinski et al., 1989; Zhang and Komatsu, 2000). However, in cotton, the two isoforms are encoded by separate genes (Salvucci et al., 2003). In barley there are two genes, one that is alternatively spliced and a second that encodes only the smaller isoform. Two RCA polypeptides are encoded by two separate genes and the transcription rate of the two RCA genes differed during leaf development in barley leaves (Rundle and Zielinski, 1991). Ayala-Ochoa et al. (2004) reported that the accumulation of two maize RCA polypeptides, encoded by two separate genes, was regulated during leaf development. RCA gene expression seems to be tissue specific in all higher plants examined. It occurs almost only in green parts of the plant and is developmentally regulated by leaf age and light. The number of RCA -encoding genes varies depending on the plant species. Certain plant species like Arabidopsis, camelina, and spinach express equal amounts of a-isoform and b-isoform, while others like rice (Oryza sativa) and wheat (Triticum aestivum) accumulate much more b-isoform than a-isoform (Fukayama et al., 2012). RCA is also considered to be a heat-labile protein and a key regulation point for photosynthesis, especially under moderately high temperature stress (Portis, 2003). Previously, a temperaturedependent dual function has been proposed for RCA at optimal temperatures. It works in releasing inhibitory sugar phosphates from the Rubisco active site, but during heat stress, RCA might function as a chaperone, protecting the protein synthesis machinery against heat inactivation (Rokka et al., 2001). Over-expression of sedoheptulose-1,7-bisphosphatase in rice suggested that yields were also improved under drought and heat stress by protecting the RCA (Feng et al., 2007). Research with the model plant, Arabidopsis, has already demonstrated that the thermotolerance of photosynthesis can be improved by increasing the thermal stability of RCA (Kumar et al., 2009). Currently available X-ray crystallographic data of RCA provide for a spiral subunit packing arrangement within the crystal with unknown physiological relevance (Stotz et al., 2011). RCA structure, activity and protein expression have been the focus of studies examining the effect of heat stress on this enzyme, but less is known about the response of RCA gene expression and protein content under salt and drought stress conditions. The genome of B. distachyon, a wild annual grass endemic to the Mediterranean and Middle East, has been sequenced. B. distachyon represents a wild, undomesticated grass species, in contrast to cultured crops such as rice, wheat, barley, sorghum, and maize. Comparison of the Brachypodium, rice and sorghum genomes shows a precise history of genome evolution across a broad diversity of the grasses, and establishes a template for analysis of the large genomes of economically important pooid grasses such as wheat. The high-quality genome sequence, coupled with ease of cultivation and transformation, small size and rapid life cycle, will help Brachypodium

reach its potential as an important model system for developing new energy and food crops (Vogel et al., 2010). The aim our research is to study RCA gene expression and protein content in B. distachyon grown under normal and also salt and drought stress conditions. 2. Results 2.1. Western blot analysis of Rubisco activase Western blot analysis indicated that there are two RCA proteins in B. distachyon. The molecular mass of these polypeptides were approximately 46 and 42 kDa. Expression of RCA was detected in leaves, green stems and ear elements. RCA protein amount changed differently in leaves and stems of the early seedlings of plants subjected to drought and watered with 50 mM NaCl solution (Fig. 1). RCAL amount increased in initial leaves of seedlings exposed to drought as well as to salt stress, but this increase was more pronounced under salt stress. In stems of the early seedlings amount of the RCAL was sharply induced under drought, whereas under salt stress only the expression of the RCAS occurred. The results showed that content of RCAS was 10-fold more than that of RCAL content in different mature leaf positions and the RCAL/RCAS ratio reached maximum in the second and third leaf under optimal growth conditions (Fig. 2). RCAS and RCAL protein abundance levels decreased, in B. distachyon mature leaves in response to drought stress. Significant decrease of RCAL abundant was observed after 5 days of drought treatment (Fig. 2). But RCAL content increased after 24 h of rewatering. RCAL content increased in stem after 7 days of drought stress. RCAL/RCAS ratio increased under salt stress and this increase became more pronounced with increasing duration of the stress (Fig. 3). Contrary to the plants grown under normal conditions at the stages of ear forming and flowering, protein contents of the RCAS and RCAL isoforms were close and their total amount was less compared with that in leaves. At the beginning of the grain filling stage amount of the RCAL was more than that of the RCAS in awns (Fig. 4). Amounts of both isoforms appeared to be less in glumes compared with awns. Amounts of both isoforms increased parallely in awns under drought stress compared with watered plants, while in glumes more pronounced increase was observed for the RCAS. Immunoblot analysis did not reveal RCA protein expression in developing grains (Fig. 4). In contrast, expression of Rubisco large subunit (Rubisco LS) observed at the initial stages of grain development sharply decreased at the final stages and disappeared in fully mature grains. Rubisco protein content was found to be higher in awns and glumes compared with developing grains. Both RCA isoforms and Rubisco LS protein contents appeared to be high in glumes. However, unlike to RCA, Rubisco LS quantity decreased in glumes and awns in response to drought stress (Fig. 5). 2.2. Expression patterns of B. distachyon RCA RCA transcript level was assessed by reverse transcriptionpolymerase chain reaction (RT-PCR). The expression of RCA gene

Fig. 1. Western-blot analysis of Rubisco activase in Brachypodium distachyon early leaf (L) and stem (ST) samples under control (C), salt stress (S) and drought stress (D) conditions.

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013

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Fig. 2. Western-blot analysis of Rubisco activase in Brachypodium distachyon different leaf position (L) and stem (ST) samples under normal watering (C) and drought stress (DS) conditions.

Fig. 3. Western-blot analysis of Rubisco activase in Brachypodium distachyon Leaf (L), head (H) and stem (ST) samples under control (C) and salt stress (S) conditions after 4d, 5-d, 7-d, 10-d, 12-d.

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Fig. 6. Rubisco activase gene expression in Brachypodium distachyon different leaf (L) positions and stem (ST) samples under control conditions (C), after 4-d, 5-d, 7d drought stress (D), and after rewatering (Rew). NAD-GADPH was used as a reference gene.

seedlings remained unchanged for a short period under drought stress. Whereas 100 mM NaCl caused a decrease in the gene expression compared with control variants. However, close expression patterns were observed under the influence of 200 mM NaCl for drought stressed and control variants (Fig. 7). During progressively imposed drought stress the abundance of RCA transcript levels significantly decreased in the early seedling leaves, but under 100 mM NaCl concentration RCA transcript level did not change compared to optimal growth conditions (Fig. 8). Also during progressively imposed drought stress, RCA transcript levels weakly decreased in the mature leaves and ear elements. A gradual decrease of the RCA transcript level was observed in B. distachyon mature leaves, stems and spike during water stress. RCA gene expression did not occur in developing grains. Watering of B. distachyon with 100 mM NaCl solution caused an increase in Rubisco activase gene expression in leaves in the tillering phase but RCA gene expression decreased in green stems under salt stress (Fig. 9). Increased messenger RNA levels in salt stressed plants compared to optimal growth conditions were observed for RCA. Drought had opposite to salt effects, leading to a decrease of transcript quantities of RCA in leaf and ear elements.

Fig. 4. Western-blot analysis of Rubisco activase in Brachypodium distachyon Leaf (L), head (H) and stem (ST) samples under control (C) and salt stress (S) conditions after 4d, 5-d, 7-d, 10-d, 12-d.

in stem, leaves and ear elements was investigated. RCA were ubiquitously expressed in the early seedling green parts, mature leaves, stem and ear elements. RCA expression was strongest in leaves being 1.0e1.5 times higher than the level found in other tissues. Transcriptional levels of RCA are almost identical in different leaf positions (Fig. 6). RCA gene show differential expression in B. distachyon organs and in response to environmental stress. Expression of the RCA gene in leaves of the early

Fig. 5. Western-blot analysis of Rubisco LS in Brachypodium distachyon development grain (DG) and ear elements (awnes and glumes) grown under normal watering (C) and drought stress (DS) conditions.

Fig. 7. Rubisco activase gene expression in the Brachypodium distachyon seedling leaf (L) samples under control conditions (C), after 5-h, 8-h and 12-h drought stress (D) and salt stress (S). NAD-GADPH was used as a reference gene.

Fig. 8. Rubisco activase gene expression in the Brachypodium distachyon seedling leaf (L) samples under control conditions (C), after 1-d, 2-d and 3-d drought (D) and salt (S) stress conditions. NAD-GADPH was used as a reference gene.

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013

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Fig. 9. Rubisco activase gene expression in the Brachypodium distachyon leaf (L), head (H) and stem (ST) samples under control condition (C) and salt stress (S) condition after 2-d, 6-d, 7-d, 8-d and 10-d. NAD-GADPH was used as a reference gene.

3. Discussion Western blot analysis of RCA revealed two isoforms in leaf extracts from early seedlings and mature leaves. In spite of the difference in accumulation of two RCA polypeptides in different leaf positions, the changes of their RCA gene expression patterns were close. We may conclude that despite the close amounts of the gene transcription in different leaf positions their isoform contents and ratio were not attributed to transcriptional regulation. Higher RCAL/RCAS ratio was recently observed in rice leaves with higher photosynthetic capacity and the decrease in photosynthetic rate and Rubisco activation state highly correlated with the decline of RCAL/RCAS ratio during leaf aging. It suggests that there is a posttranscriptional mechanism regulating the RCAL/RCAS ratio, which may play a role of the regulator, modulating photosynthetic capacity during leaf aging in rice plant (Wang et al., 2009). RCA gene expression patterns have previously been determined in barley leaf, coleoptile, seed, and root organs and were found to be only expressed in leaf (Zielinski et al., 1989). Recently obtained results indicated that in sweet potato RCA expression occurred only in leaf tissue (Xu et al., 2010). Our results showed the Rubisco activase gene was expressed in leaf, green stem and ear elements in B. distachyon, where photosynthesis takes place. Previous studies on maize showed that water restriction caused the 43/41 kDa RCA ratio to decrease by up to half of the control levels, particularly in maize leaves from stages five and six (Sanchez de Jimenez and Martınez-Barajas, 1995). The gradual application of drought stress in this experiment allowed us to detect significant changes in gene expression of RCA in early seedlings, mature leaves, stems and spike in B. distachyon. Recent studies showed that transcript abundance levels of RCA gene did not change in response to drought or post drought rewatering of Kentucky bluegrass and there were no detectable differences between two cultivars. They suggest that RCA transcripts were not sensitive to changes in water status in Kentucky bluegrass (Xu et al., 2013). However, decreases (Pelloux et al., 2001) and increases (Ji et al., 2011) in transcript abundance of RCA have been observed in other plant species and/or relative stress intensity. Our previous study found that depending on the duration and severity of drought protein content of RCA changed differently in various wheat genotypes and decreased significantly under severe water stress (Bayramov et al., 2010). High expression levels of Rubisco and RCA protein contents in awns and glumes indicated the active photosynthetic assimilation of CO2 in ear green parts. Previous investigations showed that ear organs made a substantial contribution to grain yield in wheat (Araus et al., 1993), especially during drought (Abbad et al., 2004). The importance of the ear as a source of assimilates seems to be related to its better photosynthetic performance compared to the flag leaf under stress, including a higher water use efficiency, delayed senescence, higher drought or heat tolerance, the refixation of the CO2 respired by developing grains and some degree of

C4 metabolism (Araus et al., 1993; Abbad et al., 2004). Increase in RCAL isoform protein in B. distachyon leaves under salt was similar to the increase observed in some plant leaves under heat stress. It suggests that similar to heat stress, salt stress also cause an increase in RCAL biosynthesis of RCA in B. distachyon. Being more resistant to adverse environmental factors, RCAL probably plays an important role in Rubisco activation. RCAL is likely more resistant to proteinase effects than the RCAS. In spite of the different changes in RCA gene expression under salt and water stress, amount of its RCAL increased under salt stress. These results indicate that salt and drought stresses alter RCA expression, most likely posttranscriptionally. Although rice leaves accumulate more RCAS than RCAL (Fukayama et al., 2012), it still keeps unknown whether or not the two isoforms change in leaf development and what relationship is between them and photosynthetic capacity. Recent studies of grapevine plants showed that, one of the earliest responses to water deficit was an increase in the transcript abundance of RCA (day 4), but this increase occurred much later in salt-stressed plants (day 12) (Cramer et al., 2007). However, we observed close timedependent increases in the RCAL isoform protein content in mature leaves of B. distachyon exposed to salt stress. Significant upregulation of the RCAL under salt stress suggests its involvement in the Rubisco- RCA complex during acclimation to this abiotic stress. These results indicate that the RCAL may function as a salt induced chaperone that contributes to the maintenance of Rubisco initial activity and photosynthetic rate at abiotic stress conditions. Recently, positive, linear correlation was found between the expression of wheat 45e46 kDa RCA and plant productivity under heat stress conditions. These results support the hypothesis that endogenous levels of RCA could play an important role in plant productivity under supraoptimal temperature conditions (Ristic et al., 2009). Also increased de novo synthesis of RCA and an altered isoenzyme pattern has been reported for heat-stressed wheat leaves (Law et al., 2001). The enhanced synthesis of RCA could compensate for the thermolability of RCA and contribute to the maintenance of the steady-state level of RCA. According to Crafts-Brandner et al. (1997), the thermal properties of RCA forms may differ both within and among species. Therefore it is difficult to compare our results concerning RCA with those reported with other plant species or cultivars. Investigations performed with rice plants showed that heat stress significantly induced the expression of RCAL as determined by both mRNA and protein levels. Also correlative analysis indicated that RCAS protein content was significantly related to Rubisco initial activity and net photosynthetic rate under both heat stress and normal conditions. Immunoblot analysis of the Rubiscoe RCA complex revealed that the ratio of RCAL to Rubisco increased markedly in heat-acclimated rice leaves (Wang et al., 2010). RCAL was upregulated in response to long- term salt stress. Increased RCAL may be required to tolerate long-term salt stress due to a direct reduction in stomatal conductance and subsequent low CO2 levels. Low stromal CO2 will result in increased rates of Rubisco inactivation through the binding of inhibitory sugars prior to carboxylation. Increases in stromal levels of the RCAL of the activase may directly allow carboxylation to occur at low CO2 levels (Parker et al., 2006). The RCAL is subject to redox regulation by disulphide bond formation in the C-terminal tail, which in turn increases the susceptibility to ADP inhibition. This suggests that RCA is only fully functional during active photosynthesis, when ADP levels are low and reduction equivalents are abundant (Zhang et al., 2002). Our results revealed that effects of salt and drought stresses on RCA were similar at the protein but not at the transcript level. Differential expression of two biochemically different forms of activase could provide a mechanism for optimizing Rubisco

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013

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activation at the development stage and prevailing environmental conditions. Our results demonstrated that in B. distachyonear elements drought induced a decrease in Rubisco LS protein quantities. Some reports indicated the absence of the correlation between Rubisco activase and Rubisco amount changes. The contents of Rubisco activase showed different pattern of change to that of Rubisco during leaf aging in rice (Fukayama et al., 1996). Antisense reduction of Rubisco activase leads to increase in Rubisco content in tobacco and rice (He et al., 1997; Jin et al., 2006). In addition, Rubisco was down-regulated under elevated CO2 where RCA was up-regulated (Fukayama et al., 2009). These observations suggest that the contents of two representative stromal proteins are regulated in a different manner in leaves. Our results also indicated that levels of the both RCA isoforms were upregulated under drought stress in ear elements, while Rubisco LS level was down-regulated. RT-PCR analysis indicates that salt and drought stresses regulate the expression of RCA in B. distachyon, suggesting that the RCA gene plays important roles in response of B. distachyon plant to salt and drought stresses. Transcriptional levels of RCA are almost identical in different leaf positions. 4. Conclusions Regulation of RCA polypeptide ratios was determined in plant leaves at different developmental stages and under stressing environmental conditions. Increased levels of 46/42 kDa RCA ratio were found in leaves under salt stress. Immunoblot analysis showed that salt stress increased and drought stress decreased the amount of an RCAL. In addition, our results from B. distachyon showed that RCAL was up- regulated under salt stress by increasing stress period. Therefore the level of RCA isoforms depends on the duration of drought and salt stresses. RT-PCR analysis revealed that RCA transcript expression levels were up-regulated by salt stress, but down-regulated by drought stress. A gradual decrease of the RCA transcript level was observed in B. distachyon early seedlings, mature leaves, stems and spike during water stress. We conclude that altered expression of RCA may be crucial for continued CO2 fixation under drought and salt stresses. 5. Materials and methods 5.1. Plant material and growth conditions Seeds of B. distachyon variety Bd21 were sterilized in 15% bleach for 15 min. After soaking in the bleach solution the seeds were rinsed with sterile distilled water. Sterilized seeds were sown on damp filter paper, cold treated at 4  C for 2 d in order to synchronize germination. Seeds were germinated at room temperature on sterile filter paper soaked with water. For drought treatment, 4-day-old seedlings were submerged in filter paper. For growing mature plants experiments were conducted in a glasshouse under natural light with day/night temperatures of 25/18  C and 16 h photoperiod with an additional illumination of 400 mmol photons m2 s1 and 60e70% relative humidity during the day. Samples were harvested after 4 h of illumination. For salt and drought treatments, 2-week-old plants were either watered with Hoagland medium supplemented with 100 mM NaCl or left without watering for periods of up to 10 days. The recovery of the drought stressed plants was determined 24 h after re-watering. 5.2. Immunocharacterization and electrophoresis Plant tissues were frozen in liquid nitrogen and ground with a mortar and pestle. Crude extracts were prepared from ground tissue with extraction buffer (50 mM TriseHCl pH 7.9, 1.0 mM EDTA, 0.5 mM PMSF). The homogenized mixture was centrifuged for

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20 min at 15 000  g. The resulting supernatant constituted the cellfree extract and was stored at 20  C. Extracted total soluble proteins were mixed with SDS-PAGE sample buffer (100 mM Tris- HCl, 4% (w/v) SDS, 20% glycerol, 0.1% (w/v) bromophenol blue and 0.2 M DDT) and heated at 95  C for 5 min. Denaturing (SDS-PAGE) polyacrylamide gel electrophoresis was conducted as described by Laemmli (Laemmli, 1970). In this work, 10% (w/v) resolving gel coupled to a 4.5% (w/v) stacking gel were utilized. Gel was loaded with equal amounts of protein samples. After electrophoresis, the gel was electroblotted onto a nitrocellulose membrane (N-8017; Sigma) at 10 V (2 mA cm2) for 2 h in a semidry transfer-blot apparatus (Bio- Rad). After transfer the blotted membrane was immersed into Ponceau S staining solution. This procedure allows for qualitative verification of the performed electro-transfer by reversible binding of the dye to the protein. The membrane was incubated for 2e5 min with the staining solution and subsequently washed in ddH2O until protein bands were clearly visible. Membranes were then soaked at room temperature for at least 1 h in 15 mM TriseHCl pH 7.4 containing 0.2 M NaCl (buffer A) and 5% w/v dry powdered milk. After overnight incubation at 4  C with polyclonal Arabidopsis anti-RCA antibodies (diluted 1:3000 in phosphate-buffered saline plus 1% nonfat milk), sheets were washed four times each for 15 min in buffer A containing 0.1% v/v Tween 20. Subsequent detection was with a peroxidase assay using affinity-purified goat anti-rabbit IgG horseradish peroxidase (diluted 1:10,000 in phosphate-buffered saline plus 5% nonfat milk conjugate) (Bio-Rad). The enhanced chemiluminescence western-blotting detection kit (Amersham) was used to develop the reaction. Immunoreacting bands were detected with Quantity One software. 5.3. RNA isolation and RT-PCR analysis Total RNA was extracted from B. distachyon leaves, stems and spikes using TRIZOL reagent and precipitated in 3 M LiCl. Total RNA was dissolved in diethyl pyrocarbonate treated water after extraction. RNA samples were treated with 2 units of DNase I for 1 h at 37  C, then DNase I was inactivated by treatment with DNase I Inactivating Reagent (Ambion, Austin, TX). The quantity and quality of total RNA were measured by spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific, MA) and 1.2% agarose/Ethidium bromide gel electrophoresis. One microgram of the isolated total RNA was used to synthesize first-strand cDNA using quantiscript reverse transcriptase. PCR amplification was carried out in a 20-ml reaction mixture containing a 200 mM concentration of each dNTP, a 20 mM concentration of each primer as follows: 1 ml cDNA, 4 ml 5xMyTaq reaction buffer, and 0.1 ml of MyTaqÔ DNA polymerase (Promega), 0.4 ml each primer and 14.5 ml water (ddH2O). PCR was carried out for 30 cycles of 1 min of initial denaturation 95  C, 15 s at 95  C, 15 s at 60  C, and 10 s at 72  C, followed by a final extension step for 4 min at 72  C using the primers (forward primer of 50 -GAG CTTTGAGATCCAGGACG-30 and reverse primer of 50 - CAGCGAGTCTCCTGATTCG -30 ) were designed for a region that includes the gene-specific region of RCA mRNA (Genbank accession XM_003580674). NAD-glyceraldehyde phosphate dehydrogenase (NAD-GAPDH) gene (Genbank accession XM_003573270) amplification was used to ensure that equal amounts of the template were added to each RT-PCR. The PCR products were loaded onto a 1.5% agarose gel containing ethidium bromide, and electrophoresis was performed. The gel was examined and photographed using a UV imager. ImageJ 1.38 program was used to analyse the intensity of the Western blot and RT-PCR products. 5.4. Protein determination Protein was measured by the method of Bradford (Bradford, 1976), using bovine serum albumin as the standard.

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013

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Acknowledgments This work was financially supported by the “About Post-doctoral European Scholarships for Young Scientific Researchers (to Sh. Bayramov) from Azerbaijan National Academy of Sciences” dated on February 09, 2010, No 719, signed by the President of the Republic of Azerbaijan. References Abbad, H., Jaafari, El S.A., Bort, J., Araus, J.L., 2004. Comparative relationship of the flag leaf and the ear photosynthesis with the biomass and grain yield of durum wheat under a range of water conditions and different genotypes. Agronomie 24, 19e28. Araus, J.L., Brown, H.R., Febrero, A., Bort, J., Serret, M.D., 1993. Ear photosynthesis, carbon isotope discrimination and the contribution of respiratory CO2 to differences in grain mass in durum wheat. Plant Cell. Environ. 16, 383e392. Ayala-Ochoa, A., Vargas-Suárez, M., Loza-Tavera, H., León, P., Jiménez-García, L.F., Sánchez-de-Jiménez, E., 2004. In maze, two distinct ribulose 1,5-bisphosphate carboxylase/oxygenase activase transcripts have different day/night patterns of expression. Biochimie 86, 439e449. Bayramov, S.M., Babayev, H.G., Khaligzade, M.N., Guliyev, N.M., Raines, Ch. A., 2010. Effect of water stress on protein content of some Calvin cycle enzymes in different wheat genotypes. Proc. NAS of Azerbaijan (Biol. Sci.) 65, 106e111. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Crafts-Brandner, S.J., van de Loo, F.J., Salvucci, M.E., 1997. The two forms of ribulose1,5-bisphosphate carboxylase/oxygenase activase differ in sensitivity to elevated temperature. Plant Physiol. 114, 439e444. Cramer, G.C., Ergül, A., Grimplet, J., et al., 2007. Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles. Funct. Integr. Genomics 7, 111e134. Feng, L., Wang, K., Li, Y., Tan, Y., Kong, J., Li, H., Li, Y., Zhu, Y., 2007. Overexpression of SBPase enhances photosynthesis against high temperature stress in transgenic rice plants. Plant Cell. Reports 26, 1635e1646. Fukayama, H., Uchida, N., Azuma, T., Yasuda, T., 1996. Relationships between photosynthetic activity and the amounts of Rubisco activase and Rubisco in rice leaves from emergence through senescence. Jpn. J. Crop. Sci. 65, 296e302. Fukayama, H., Fukuda, T., Masumoto, C., Taniguchi, Y., Sakai, H., Cheng, W., Hasegawa, T., Miyao, M., 2009. Rice plant response to long term CO2 enrichment: gene expression profiling. Plant Sci. 177, 203e210. Fukayama, H., Ueguchi, C., Nishikawa, K., Katoh, N., Ishikawa, C., Masumoto, C., Hatanaka, T., Misoo, S., 2012. Overexpression of Rubisco activase decreases the photosynthetic CO2 assimilation rate by reducing Rubisco content in rice leaves. Plant Cell Physiol. 53, 976e986. He, Z., von Caemmerer, S., Hudson, G.S., Price, G.D., Badger, M.R., Andrews, T.J., 1997. Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development. Plant Physiol. 115, 1569e1580. Ji, K., Wang, Y., Sun, W., Lou, Q., Mei, H., Shen, S., Chen, H., 2011. Drought-responsive mechanisms in rice genotypes with contrasting drought tolerance during reproductive stage. J. Plant Physiol. 169, 336e344. Jin, S.-H., Hong, J., Li, X.-Q., Jiang, D.-A., 2006. Antisense inhibition of Rubisco activase increases Rubisco content and alters the proportion of Rubisco activase in stroma and thylakoids in chloroplasts of rice leaves. Ann. Bot. 97, 739e744. Kumar, A., Li, C., Portis, A., 2009. Arabidopsis thaliana expressing a thermostable chimeric Rubisco activase exhibits enhanced growth and higher rates of photosynthesis at moderately high temperatures. Photosynth. Res. 100, 143e153. Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680e685.

Law, R.D., Crafts-Brandner, S.J., Salvucci, M.E., 2001. Heat stress induces the synthesis of a new form of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in cotton leaves. Planta 214, 117e125. Parker, R., Timothy, J., Flowers, T.J., Moore, A.L., Nicholas, H.V.J., 2006. An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina. J. Exp. Bot. 57, 1109e1118. Pelloux, J., Jolivet, Y., Fontaine, V., Banvoy, J., Dizengremel, P., 2001. Changes in Rubisco and Rubisco activase gene expression and polypeptide content in Pinus halepensis M. subjected to ozone and drought. Plant Cell Environ. 24, 123e131. Portis Jr., A.R., 2003. Rubisco activase: Rubisco’s catalytic chaperone. Photosynth. Res. 75, 11e27. Portis Jr., A.R., Li, C., Wang, D., Salvucci, M.E., 2008. Regulation of Rubisco activase and its interaction with Rubisco. J. Exp. Bot. 59, 1597e1604. Ristic, Z., Momcilovic, I., Bukovnik, U., Prasad, P.V.V., Fu, J., DeRidder, B., Elthon, T.E., Mladenov, N., 2009. Rubisco activase and wheat productivity under heat-stress conditions. J. Exp. Bot. 60, 4003e4014. Rokka, A., Zhang, L., Aro, E.M., 2001. Rubisco activase: an enzyme with a temperature-dependent dual function? Plant J. 25, 463e471. Rundle, S.J., Zielinski, R.E., 1991. Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley. J. Biol. Chem. 266, 4677e4685. Salvucci, M.E., Werneke, J.M., Ogren, W.L., Portis Jr., A.R., 1987. Purification and species distribution of Rubisco activase. Plant Physiol. 84, 930e936. Salvucci, M.E., van de Loo, F.J., Stecher, D., 2003. Two isoforms of Rubisco activase in cotton, the products of separate genes not alternative splicing. Planta 216, 736e744. Sanchez de Jimenez, L., Martınez-Barajas, Medrano E., 1995. Rubisco activase, a possible new member of the molecular chaperone family. Biochemistry 34, 2826e2831. Spreitzer, R.J., Salvucci, M.E., 2002. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Ann. Rev. Plant Biol. 53, 449e475. Stotz, M., Mueller-Cajar, O., Ciniawsky, S., Wendler, P., Hartl, F.U., Bracher, A., HayerHartl, M., 2011. Structure of green-type Rubisco activase from tobacco. Nat. Struct. Mol. Biol. 18 (12), 1366e1378. Vogel, J.P., Garvin, D.F., Mockler, T.C., Schmutz, J., Rokhsar, D., Bevan, M.W., et al., 2010. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463, 763e768. Wang, D., Lu, Q., Li, X.F., Jiang, Q.S., Wu, J.X., Jiang, D.A., 2009. Relationship between Rubisco activase isoform levels and photosynthetic rate in different leaf positions of rice plant. Photosynthetica 47, 621e629. Wang, D., Li, X.-F., Zhou, Z.-J., Feng, X.-P., Yang, W.-J., Jiang, D.-A., 2010. Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant. Physiol. Plant. 139, 55e67. Werneke, J.M., Zielinski, R.E., Ogren, W.L., 1988. Structure and expression of spinach leaf cDNA encoding ribulose bisphosphate carboxylase/oxygenase activase. Proc. Natl. Acad. Sci. USA 85, 787e791. Xu, K., Bowen, H., Shuang, Z., Zhou, L., Zhang, Y., 2010. Cloning and characterization of the Rubisco activase gene from Ipomoea batatas (L.) Lam. Mol. Biol. Rep. 37, 661e668. Xu, L., Yu, L., Han, J., Huang, B., 2013. Photosynthesis enzyme activities and gene expression associated with drought tolerance and post-drought recovery in Kentucky bluegrass. Environ. Exp. Bot. 89, 28e35. Zhang, Z.L., Komatsu, S., 2000. Molecular cloning and characterization of cDNAsencoding two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenaseactivase in rice (Oryza sativa L.). J. Biochem 128, 383e389. Zhang, N., Portis Jr., A.R., 1999. Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f. Proc. Natl. Acad. Sci. USA 96, 9438e9443. Zhang, N., Kallis, R.P., Ewy, R.G., Portis Jr., A.R., 2002. Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform. Proc. Natl. Acad. Sci. USA 99, 3330e3334. Zielinski, R.E., Werneke, J.M., Jenkins, M.E., 1989. Coordinate expression of Rubisco activase and Rubisco during barley leaf cell development. Plant Physiol. 90, 516e521.

Please cite this article in press as: Bayramov, S., Guliyev, N., Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.013