Rubisco Activase Activity in Spinach Leaf Extracts

Rubisco activase is a chloroplast stromal protein that catalyzesthe activation of ribulose-1,5- bisphosphate carboxylase/oxygenase(rubisco) in vivo. Activation must occur before rubisco cancatalyze the photosynthetic assimilation of CO₂. In leaves,photosynthesis and rubisco activation increase with increasinglight intensity. Techniques are described that allow the activityof rubisco activase to be measured in extracts of spinach (Spinaceaoleracea L.) leaf tissue. In this context, rubisco activaseactivity is defined as the ability to promote activation ofthe inactive ribulose-1,5- bisphosphate-bound rubisco in anATP-dependent reaction. Determination of rubisco activase activityin extracts of dark and light treated leaf tissue revealed thatthe activation state of rubisco activase was independent oflight intensity. ¹Present address: Department of Biological Sciences, 213 Carson-TaylorHall, Louisiana Tech University, Ruston, Louisiana 71272, U.S.A.     

小麦Rubisco 活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶, 我们利用PCR技术, 从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段, 该片段长度为850 bp, 编码201个氨基酸。Northern blot表明, 小麦叶片在暗诱导衰老的条件下, 叶片中活化酶基因表达水平逐渐下降; 同时, 小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势。这些结果表明, 衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关。     

小麦Rubisco活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶,我们利用PCR技术,从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段,该片段长度为850 bp,编码201个氨基酸.Northern blot表明,小麦叶片在暗诱导衰老的条件下,叶片中活化酶基因表达水平逐渐下降;同时,小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势.这些结果表明,衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关.     

水稻叶片Rubisco 活化酶表达的昼夜变化

在 48h的自然光照、连续光照和连续黑暗处理下 ,水稻幼苗rcamRNA的含量均表现出昼夜节奏特性 ,其中以自然光周期中变化最为明显。在光暗交替的条件下 ,RCA含量虽也表现出明显的昼夜变化 ,但连续黑暗处理其含量持续下降 ,连续光照则其含量先上升然后下降 ,说明它不发生节昼现象。这些结果表明RCA表达在转录和翻译水平上的调控机制不同 ,转录既由光暗交替控制又受内生节奏调节 ,而翻译则更大程度上由光调节     

Rubisco活化酶的分子生物学

Rubisco活化酶是广泛存在于光合生物中、调节Rubisco活性的酶,Rubisco活化酶同时具有活化Rubisco和催化ATP水解的作用.它依赖ATP水解,促使RuBP或其它磷酸糖类从Rubisco上解离下来,以恢复Rubisco的活性.该文介绍Rubisco活化酶的分子特性、作用机制、光合作用调节及基因工程的最新研究进展.     

大麦和玉米叶片叶绿体中Rubisco及其活化酶的免疫金标记定位

运用免疫金标记电镜技术研究了禾本科C3植物大麦(Hordeum vulgare L.)和C4植物玉米(Zea mays L.)叶片中Rubisoo及其活化酶(RCA)的细胞定位,结果表明:两种植物叶片解剖结构及叶绿体超微结构差别明显.在大麦叶细胞中,只有一种叶肉细胞叶绿体,Rubisoo和RCA主要分布于叶绿体的间质中.在玉米叶细胞中,存在着维管束鞘细胞和叶肉细胞两种类型叶绿体,Rubisco主要分布于鞘细胞叶绿体的基质中,但在叶肉细胞叶绿体中亦有少量特异性标记;RCA在鞘细胞叶绿体和叶肉细胞叶绿体的基质中都有分布.两种植物叶绿体结构及光合作用关键酶定位的不同,体现了C3植物和C4植物在光合器结构与功能上的差异.     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

Rubisco Activase mRNA Expression in Spinach: Modulation by Nanoanatase Treatment

Characterized by a photocatalysis property, nanoanatase is closely related to the photosynthesis of spinach. It could not only improve light absorbance, transformation from light energy to electron energy, and active chemical energy, but also promote carbon dioxide (CO₂) assimilation of spinach. However, the molecular mechanism of carbon reaction promoted by nanoanatase remains largely unclear. In this study, we report that the amounts of Rubisco activase (rca) mRNA in the nanoanatase-treated spinach were increased by about 51%, whereas bulk-TiO₂ treatment produced an increase of only 5%. Accordingly, the protein level of Rubisco activase from the nanoanatase-treated spinach was increased by 42% compared with the control; however, bulk-TiO₂ treatment resulted in a 5% improvement. Further analysis indicated that the activity of Rubisco activase in the nanoanatase-treated spinach was significantly higher than the control by up to 2.75 times, and bulk-TiO₂ treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nanoanatase is that the nanoanatase treatment results in the enhancement of rca mRNA expressions, protein levels, and activities of Rubisco activase, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.     

小麦RCA的cDNA片断克隆和反义表达载体的构建

目的:试图分离和克隆小麦Rubisco活化酶cDNA片断并构建反义表达载体。方法:采用RT-PCR技术克隆cDNA片断,对序列用Blast等软件进行分析,并将该片断反向连接于植物表达载体pROK2的CaMV35S启动子下游,构建反义表达载体。结果:获得了小麦Rubisco活化酶(RCA)的cDNA片断(GenBank注册号:DQ984669);小麦RCA的cDNA片断推导的氨基酸序列与其它植物的RCA氨基酸序列高度同源。序列比较表明,用本实验所得的cDNA序列推导出的氨基酸序列与GenBank登录的大麦(AAA63163)、南极银须草(AAP83928)、水稻(AAX95414)、玉米(AAC97932)、拟南芥(NP 850321)和烟草(CAA78703)等的RCA序列的同源性分别为97%、95%、88%、83%、82%和82%;分析表明,该序列为新的小麦RCAα的cDNA序列;通过选择和引入合适的酶切位点进行载体构建,构建了小麦RCA的cDNA反义表达载体pR-AntiRCA。结论:构建成了小麦RCA的cDNA反义表达载体。     

Heat Denaturation Profiles of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) and Rubisco Activase and the Inability of Rubisco Activase to Restore Activity of Heat-Denatured Rubisco

We compared the heat-denaturation profiles of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase and further examined the ability of Rubisco activase to restore the activity of heat-denatured Rubisco originally reported (E. Sanchez de Jimenez, L. Medrano, and E. Martinez-Barajas [1995] Biochemistry 34: 2826-2831). Rubisco was heat-treated in both the carbamylated and uncarbamylated forms and in the presence and absence of 10 mM dithiothreitol (DTT). Both forms were highly resistant to heat denaturation and further protection was gained in the presence of DTT. A 50% loss in total activity occurred after 1 h at 57.5 and 55.2[deg]C for uncarbamylated Rubisco and at 60.2 and 59.6[deg]C for carbamylated Rubisco, in each case with and without DTT, respectively. In contrast, Rubisco activase lost 50% activity after only 5 min at 33[deg]C and the loss in activity was not affected by the presence of Rubisco. When Rubisco, heat-denatured to various extents, was incubated at room temperature with Rubisco activase or bovine serum albumin as a control, Rubisco activase did not have a significant specific ability to restore Rubisco activity. We conclude that Rubisco activase alone does not have the ability to restore the activity of heat-denatured Rubisco and is unlikely to protect or restore Rubisco activity from heat denaturation in vivo because it is more heat-labile than Rubisco.     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。     

Rubisco活化酶的研究进展

Rubisco活化酶是近年中发现的一种可以调节Rubisco活性的酶,它能使Rubisco在植株体内条件下达到最大活化程度。Rubisco活化酶不仅具有活化Rubisco的活性,而且具有ATP水解酶活性。在ATP水解过程中,Rubisco活化酶促使各种磷酸糖抑制物从Rubisco上解离下来,恢复Rubisco活性。Rubisco活化酶的发现与研究使许多Rubisco体内活化中的疑难问题得到了阐明。本文还介绍了Rubisco活化酶的分子特性、酶作用机制以及环境因素对它活性影响等方面的最新研究进展。     

Rubisco活化酶的研究进展

Rubisco活化酶是近年中发现的一种可以调节Rubisco活性的酶 ,它能使Rubisco在植株体内条件下达到最大活化程度。Rubisco活化酶不仅具有活化Rubisco的活性 ,而且具有ATP水解酶活性。在ATP水解过程中 ,Rubisco活化酶促使各种磷酸糖抑制物从Rubisco上解离下来 ,恢复Rubisco活性。Rubisco活化酶的发现与研究使许多Rubisco体内活化中的疑难问题得到了阐明。本文还介绍了Rubisco活化酶的分子特性、酶作用机制以及环境因素对它活性影响等方面的最新研究进展。     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。     

Rubisco Activase Mediates ATP-Dependent Activation of Ribulose Bisphosphate Carboxylase

The activation level of ribulosebisphosphate carboxylase following preincubation with ribulose 1,5-bisphosphate was increased by ATP and ribulosebisphosphate carboxylase activase in the absence of thylakoids or illumination. Maximal activation was obtained with 0.5 millimolar ATP in the presence of an ATP regenerating system (phosphoenolpyruvate and pyruvate kinase). Without the ATP regenerating system, activation was lower, linearly dependent on ATP concentration up to 1.0 millimolar, and was strongly inhibited by ADP.     

Effect of Rubisco Activase Deficiency on the Temperature Response of CO2 Assimilation Rate and Rubisco Activation State: Insights from Transgenic Tobacco with Reduced Amounts of Rubisco Activase

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. To elucidate its role in maintaining CO₂ assimilation rate at high temperature, we examined the temperature response of CO₂ assimilation rate at 380 μL L⁻¹ CO₂ concentration (A₃₈₀) and Rubisco activation state in wild-type and transgenic tobacco (Nicotiana tabacum) with reduced Rubisco activase content grown at either 20°C or 30°C. Analyses of gas exchange and chlorophyll fluorescence showed that in the wild type, A₃₈₀ was limited by ribulose 1,5-bisphosphate regeneration at lower temperatures, whereas at higher temperatures, A₃₈₀ was limited by ribulose 1,5-bisphosphate carboxylation irrespective of growth temperatures. Growth temperature induced modest differences in Rubisco activation state that declined with measuring temperature, from mean values of 76% at 15°C to 63% at 40°C in wild-type plants. At measuring temperatures of 25°C and below, an 80% reduction in Rubisco activase content was required before Rubisco activation state was decreased. Above 35°C, Rubisco activation state decreased slightly with more modest decreases in Rubisco activase content, but the extent of the reductions in Rubisco activation state were small, such that a 55% reduction in Rubisco activase content did not alter the temperature sensitivity of Rubisco activation and had no effect on in vivo catalytic turnover rates of Rubisco. There was a strong correlation between Rubisco activase content and Rubisco activation state once Rubisco activase content was less that 20% of wild type at all measuring temperatures. We conclude that reduction in Rubisco activase content does not lead to an increase in the temperature sensitivity of Rubisco activation state in tobacco.The catalytic sites of Rubisco must be activated for CO₂ fixation to take place. This requires the carbamylation of a Lys residue at the catalytic sites to allow the binding of Mg²⁺ and ribulose 1,5-bisphosphate (RuBP; Andrews and Lorimer, 1987). Rubisco activase facilitates carbamylation and the maintenance of Rubisco activity by removing inhibitors such as tight-binding sugar phosphates from Rubisco catalytic sites in an ATP-dependent manner (Andrews, 1996; Spreitzer and Salvucci, 2002; Portis, 2003; Parry et al., 2008). The activity of Rubisco activase is regulated by the ATP/ADP ratio and redox state in the chloroplast (Zhang and Portis, 1999; Zhang et al., 2002; Portis, 2003).In many plant species, Rubisco activation state decreases at high temperature in vivo (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004b; Cen and Sage, 2005; Yamori et al., 2006b; Makino and Sage, 2007). However, it is unclear what the primary mechanisms underlying the inhibition of Rubisco activation are and whether Rubisco deactivation limits CO₂ assimilation rate at high temperature. It has been proposed that Rubisco activation state decreases at high temperature, because the activity of Rubisco activase is insufficient to keep pace with the faster rates of Rubisco inactivation at high temperatures (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a, 2004c; Kim and Portis, 2006). In in vitro assays using purified Rubisco and Rubisco activase, the activity of Rubisco activase was sufficient for the activation of Rubisco at the optimum temperature but not at high temperatures (Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a, 2004c). ATP hydrolysis activity of Rubisco activase in vitro has varying temperature optima among species (e.g. 25°C in Antarctic hairgrass [Deschampsia antarctica] and spinach [Spinacia oleracea] but 35°C in tobacco [Nicotiana tabacum] and cotton [Gossypium hirsutum]), and Rubisco activase more readily dissociates into inactive forms at high temperature, causing a loss of Rubisco activase capacity (Crafts-Brandner and Law, 2000; Salvucci and Crafts-Brandner, 2004b). Moreover, the rates of inhibitor formation by misprotonation of RuBP during catalysis increased at higher temperatures (Salvucci and Crafts-Brandner, 2004c; Kim and Portis, 2006). CO₂ assimilation rates and plant growth were improved under heat stress in transgenic Arabidopsis expressing thermotolerant Rubisco activase isoforms generated by either gene-shuffling technology (Kurek et al., 2007) or chimeric Rubisco activase constructs (Kumar et al., 2009). These results support the view that the reduction of Rubisco activase activity limits the Rubisco activation and, therefore, the CO₂ assimilation rates at high temperatures.It has also been suggested that the decrease in CO₂ assimilation rate at high temperatures is caused by a limitation of RuBP regeneration capacity (e.g. electron transport capacity) rather than by Rubisco deactivation per se (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007; Kubien and Sage, 2008). These groups suggest that Rubisco deactivation at high temperature may be a regulatory response to the limitation of one of the processes contributing to electron transport capacities. For example, at high temperature, protons can leak through the thylakoid membrane, impairing the coupling of ATP synthesis to electron transport (Pastenes and Horton, 1996; Bukhov et al., 1999, 2000). As the electron transport capacity becomes limiting, ATP/ADP ratios and the redox potential of the chloroplast decline, causing a loss of Rubisco activase activity and, in turn, a reduction in the Rubisco activation state (Zhang and Portis, 1999; Zhang et al., 2002; Sage and Kubien, 2007). Based on this understanding, the decline in the Rubisco activation state at high temperature may be a regulated response to a limitation in electron transport capacity rather than a consequence of a direct effect of heat on the integrity of Rubisco activase.Temperature dependence of CO₂ assimilation rate shows a considerable variation with growth temperature (Berry and Björkman, 1980; Hikosaka et al., 2006; Sage and Kubien, 2007). Plants grown at low temperature generally exhibit higher CO₂ assimilation rates at low temperatures compared with plants grown at high temperature, but they exhibit lower rates at high temperature. Furthermore, both the temperature response of Rubisco activation state and the limiting step of CO₂ assimilation rate (a Rubisco versus RuBP regeneration limitation) have been shown to differ depending on growth temperature (Hikosaka et al., 1999; Onoda et al., 2005; Yamori et al., 2005, 2006a, 2006b, 2008). This suggests that the regulation of Rubisco activation state could also differ in plants grown at different growth temperatures. Here, we analyzed the effects of Rubisco activase content on Rubisco activation state and CO₂ assimilation rate at leaf temperatures ranging from 15°C to 40°C in tobacco grown under two different temperature regimes (day/night temperatures of 20°C/15°C or 30°C/25°C). We used wild-type and transgenic tobacco with a range of reductions in Rubisco activase content to examine the dependence of Rubisco activation on Rubisco activase content over the range of leaf temperatures (Mate et al., 1993, 1996).     

The Regulatory Properties of Rubisco Activase Differ among Species and Affect Photosynthetic Induction during Light Transitions

Rubisco’s catalytic chaperone, Rubisco activase (Rca), uses the energy from ATP hydrolysis to restore catalytic competence to Rubisco. In Arabidopsis (Arabidopsis thaliana), inhibition of Rca activity by ADP is fine tuned by redox regulation of the α-isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive β-isoform, the response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was significantly inhibited by physiological ratios of ADP to ATP in species containing both α-Rca and β-Rca (Arabidopsis and camelina [Camelina sativa]) or just the β-Rca (tobacco [Nicotiana tabacum]). However, Rca activity was insensitive to ADP inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis β-Rca, although not in a transformant of Arabidopsis that expresses a tobacco-like β-Rca. ATP hydrolysis by recombinant Arabidopsis β-Rca was much less sensitive to inhibition by ADP than recombinant tobacco β-Rca. Mutation of 17 amino acids in the tobacco β-Rca to the corresponding Arabidopsis residues reduced ADP sensitivity. In planta, Rubisco deactivated at low irradiance except in the Arabidopsis rwt43 transformant containing an ADP-insensitive Rca. Induction of CO₂ assimilation after transition from low to high irradiance was much more rapid in the rwt43 transformant compared with plants containing ADP-sensitive Rca forms. The faster rate of photosynthetic induction and a greater enhancement of growth under a fluctuating light regime by the rwt43 transformant compared with wild-type Arabidopsis suggests that manipulation of Rca regulation might provide a strategy for enhancing photosynthetic performance in certain variable light environments.The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca), a specific catalytic chaperone (Spreitzer and Salvucci, 2002; Portis, 2003). Like other AAA+ ATPases (Snider et al., 2008), Rca uses the energy from ATP hydrolysis to remodel the conformation of its target protein, Rubisco. The conformational changes induced by Rca restore catalytic competence to Rubisco active sites that have been inactivated by the unproductive binding of sugar phosphates, including the substrate ribulose 1,5-bisphosphate (RuBP; Wang and Portis, 1992). Because of the requirement for ATP hydrolysis and the inhibition of activity by ADP (Robinson and Portis, 1988, 1989), Rca adjusts the rate of CO₂ fixation to the rates of electron transport activity via changes in the activation state of Rubisco (Salvucci et al., 1985). As a result of this coordinate regulation, the light response of Rubisco activation closely resembles the light response of CO₂ assimilation, and the levels of RuBP under steady-state conditions are relatively constant over a wide range of irradiance levels (Perchorowicz et al., 1981; Dietz and Heber, 1984).Many plant species express two isoforms of Rca, α and β, that are both active in ATP hydrolysis and Rubisco activation (Shen et al., 1991; Salvucci et al., 2003). In some plant species, these isoforms are the products of an alternative splicing event that generates two polypeptides, which are identical except for a 20- to 30-amino acid extension at the C terminus of the longer α-isoform (Werneke et al., 1989). In cotton (Gossypium hirsutum), soybean (Glycine max), and presumably other plant species, separate genes encode the two isoforms of Rca (Salvucci et al., 2003; Yin et al., 2010). In these species, the amino acid sequences of the overlapping regions of the α- and β-polypeptides are very similar, and the C-terminal extension of the longer α-isoform is similar to the extension produced by alternative splicing (Supplemental Fig. S1).Our current understanding of the role of the two Rca isoforms is based primarily on investigations with Arabidopsis (Arabidopsis thaliana; Zhang and Portis, 1999; Zhang et al., 2001; Wang and Portis, 2006). The C-terminal extension of the α-Rca contains two redox-regulated Cys residues that are modulated by thioredoxin f (Zhang and Portis, 1999). 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 α-Rca when in the oxidized state, but inhibition is much less when this isoform has been reduced by thioredoxin. In contrast, the shorter Arabidopsis β-Rca is not redox regulated and is less sensitive to inhibition by ADP (Zhang and Portis, 1999). Mixing experiments with recombinant Rca have shown that the properties of α-Rca are conferred to the heterooligomer, providing a mechanism for redox regulating the Rca holoenzyme (Zhang et al., 2001). In this way, Rca is similar to the chloroplastic glyceraldehyde 3-P dehydrogenase (GAPDH), which also has both redox-regulated (GAP-B) and non-redox-regulated (GAP-A) forms that differ by a C-terminal extension (Baalmann et al., 1996). Like Rca (Zhang and Portis, 1999; Zhang et al., 2001), redox regulation of two Cys residues in the extension exerts master control over the mixed GAPDH oligomer.Some plant species, including members of the Solanaceae family, as well as maize (Zea mays) and green algae, express only the shorter β-Rca (Salvucci et al., 1987). The β-Rca in these species is not responsive to redox regulation, even though the activation state of Rubisco in these plants is modulated by irradiance (Salvucci and Anderson, 1987) and seems to be associated with the redox status of the chloroplast (Ruuska et al., 2000). With GAPDH, all higher plants appear to have both chloroplastic isoforms, but the non-redox-sensitive form, Gap-A, can be regulated indirectly by thioredoxin through the binding of the small chloroplast protein CP12 (Trost et al., 2006). By analogy, a similar association with CP12 or a CP12-like protein could provide a means of conferring redox sensitivity to β-Rca in species that have only this Rca isoform. However, no association of Rca was observed when the native CP12 complex and other high-molecular-mass species were isolated from tobacco (Nicotiana tabacum) chloroplasts (Carmo-Silva et al., 2011b).In this study, the regulation of β-Rca activity was examined both in vivo and in vitro in plant species that contain only one (i.e. β-) or both (α- and β-) Rca isoforms. The response of enzyme activity to physiological ratios of ADP to ATP was measured for the native Rca in leaf extracts, as well as for recombinant Arabidopsis and tobacco enzymes, to determine the sensitivity of β-Rca to ADP in different species. In addition, experiments were conducted with transgenic Arabidopsis plants containing variants of β-Rca to determine the link between Rca regulation and photosynthetic induction. The results suggest a new strategy for enhancing photosynthetic performance under variable light environments based on altering the regulatory properties of Rca to increase the rate of photosynthetic induction.     

Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.