Plants Actively Avoid State Transitions upon Changes in Light Intensity: Role of Light-Harvesting Complex II Protein Dephosphorylation in High Light

Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, PROTON GRADIENT REGULATION5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery.Photosynthetic light reactions take place in the chloroplast thylakoid membrane. Primary energy conversion reactions are performed by synchronized function of the two light energy-driven enzymes PSII and PSI. PSII uses excitation energy to split water into electrons and protons. PSII feeds electrons to the intersystem electron transfer chain (ETC) consisting of plastoquinone, cytochrome b₆f, and plastocyanin. PSI oxidizes the ETC in a light-driven reduction of NADP to NADPH. Light energy is collected by the light-harvesting antenna systems in the thylakoid membrane composed of specific pigment-protein complexes (light-harvesting complex I [LHCI] and LHCII). The majority of the light-absorbing pigments are bound to LHCII trimers that can serve the light harvesting of both photosystems (Galka et al., 2012; Kouřil et al., 2013; Wientjes et al., 2013b). Energy distribution from LHCII is regulated by protein phosphorylation (Bennett, 1979; Bennett et al., 1980; Allen et al., 1981) under control of the STN7 and STN8 kinases (Depège et al., 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005) and the TAP38/PPH1 and Photosystem II Core Phosphatase (PBCP) phosphatases (Pribil et al., 2010; Shapiguzov et al., 2010; Samol et al., 2012). LHCII trimers are composed of LHCB1, LHCB2, and LHCB3 proteins, and in addition to reversible phosphorylation of LHCB1 and LHCB2, the protein composition of the LHCII trimers also affects the energy distribution from the light-harvesting system to photosystems (Damkjaer et al., 2009; Pietrzykowska et al., 2014). Most of the LHCII trimers are located in the PSII-rich grana membranes and PSII- and PSI-rich grana margins of the thylakoid membrane, and only a minor fraction resides in PSI- and ATP synthase-rich stroma lamellae (Tikkanen et al., 2008b; Suorsa et al., 2014). Both photosystems bind a small amount of LHCII trimers in biochemically isolatable PSII-LHCII and PSI-LHCII complexes (Pesaresi et al., 2009; Järvi et al., 2011; Caffarri et al., 2014). The large portion of the LHCII, however, does not form isolatable complexes with PSII or PSI, and therefore, it separates as free LHCII trimers upon biochemical fractionation of the thylakoid membrane by Suc gradient centrifugation or in native gel analyses (Caffarri et al., 2009; Järvi et al., 2011), the amount being dependent on the thylakoid isolation method. Nonetheless, in vivo, this major LHCII antenna fraction serves the light-harvesting function. This is based on the fact that fluorescence from free LHCII, peaking at 680 nm in 77-K fluorescence emission spectra, can only be detected when the energy transfer properties of the thylakoid membrane are disturbed by detergents (Grieco et al., 2015).Regulation of excitation energy distribution from LHCII to PSII and PSI has, for decades, been linked to LHCII phosphorylation and state transitions (Bennett, 1979; Bennett et al., 1980; Allen et al., 1981). It has been explained that a fraction of LHCII gets phosphorylated and migrates from PSII to PSI, which can be evidenced as increase in PSI cross section and was assigned as transition to state 2 (for review, see Allen, 2003; Rochaix et al., 2012). The LHCII proteins are, however, phosphorylated all over the thylakoid membrane (i.e. in the PSII- and LHCII-rich grana core) in grana margins containing PSII, LHCII, and PSI as well as in PSI-rich stroma lamellae also harboring PSII-LHCII, LHCII, and PSI-LHCII complexes in minor amounts (Tikkanen et al., 2008b; Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013a)—making the canonical-state transition theory inadequate to explain the physiological role of reversible LHCII phosphorylation (Tikkanen and Aro, 2014). Moreover, the traditional-state transition model is based on lateral segregation of PSII-LHCII and PSI-LHCI to different thylakoid domains. It, however, seems likely that PSII and PSI are energetically connected through a shared light-harvesting system composed of LHCII trimers (Grieco et al., 2015), and there is efficient excitation energy transfer between the two photosystems (Yokono et al., 2015). Nevertheless, it is clear that LHCII phosphorylation is a prerequisite to form an isolatable PSI-LHCII complex called the state transition complex (Pesaresi et al., 2009; Järvi et al., 2011). Existence of a minor state transition complex, however, does not explain why LHCII is phosphorylated all over the thylakoid membrane and how the energy transfer is regulated from the majority of LHCII antenna that is shared between PSII and PSI but does not form isolatable complexes with them (Grieco et al., 2015).Plants grown under any steady-state white light condition show the following characteristics of the thylakoid membrane: PSII core and LHCII phosphoproteins are moderately phosphorylated, phosphorylation takes place all over the thylakoid membrane, and the PSI-LHCII state transition complex is present (Järvi et al., 2011; Grieco et al., 2012; Wientjes et al., 2013b). Upon changes in the light intensity, the relative phosphorylation level between PSII core and LHCII phosphoproteins drastically changes (Rintamäki et al., 1997, 2000) in the timescale of 5 to 30 min. When light intensity increases, the PSII core protein phosphorylation increases, whereas the level of LHCII phosphorylation decreases. On the contrary, a decrease in light intensity decreases the phosphorylation level of PSII core proteins but strongly increases the phosphorylation of the LHCII proteins (Rintamäki et al., 1997, 2000). The presence and absence of the PSI-LHCII state transition complex correlate with LHCII phosphorylation (similar to the state transitions; Pesaresi et al., 2009; Wientjes et al., 2013b). Despite all of these changes in thylakoid protein phosphorylation, the relative excitation of PSII and PSI (i.e. the absorption cross section of PSII and PSI measured by 77-K fluorescence) remains nearly unchanged upon changes in white-light intensity (i.e. no state transitions can be observed despite massive differences in LHCII protein phosphorylation; Tikkanen et al., 2010).The existence of the opposing behaviors of PSII core and LHCII protein phosphorylation, as described above, has been known for more than 15 years (Rintamäki et al., 1997, 2000), but the physiological significance of this phenomenon has remained elusive. It is known that PSII core protein phosphorylation in high light (HL) facilitates the unpacking of PSII-LHCII complexes required for proper processing of the damaged PSII centers and thus, prevents oxidative damage of the photosynthetic machinery (Tikkanen et al., 2008a; Fristedt et al., 2009; Goral et al., 2010; Kirchhoff et al., 2011). It is also known that the damaged PSII core protein D1 needs to be dephosphorylated before its proteolytic degradation upon PSII turnover (Koivuniemi et al., 1995). There is, however, no coherent understanding available to explain why LHCII proteins are dephosphorylated upon exposure of plants to HL and PSII core proteins are dephosphorylated upon exposure to low light (LL).The above-described light quantity-dependent control of thylakoid protein phosphorylation drastically differs from the light quality-dependent protein phosphorylation (Tikkanen et al., 2010). State transitions are generally investigated by using different light qualities, preferentially exciting either PSI or PSII. State 1 light favors PSI excitation, leading to oxidation of the ETC and dephosphorylation of both the PSII core and LHCII proteins. State 2 light, in turn, preferentially excites PSII, leading to reduction of ETC and strong concomitant phosphorylation of both the PSII core and LHCII proteins (Haldrup et al., 2001). Shifts between states 1 and 2 lights induce state transitions, mechanisms that change the excitation between PSII and PSI (Murata and Sugahara, 1969; Murata, 2009). Similar to shifts between state lights, the shifts between LL and HL intensity also change the phosphorylation of the PSII core and LHCII proteins (Rintamäki et al., 1997, 2000). Importantly, the white-light intensity-induced changes in thylakoid protein phosphorylation do not change the excitation energy distribution between the two photosystems (Tikkanen et al., 2010). Despite this fundamental difference between the light quantity- and light quality-induced thylakoid protein phosphorylations, a common feature for both mechanisms is a strict requirement of LHCII phosphorylation for formation of the PSI-LHCII complex. However, it is worth noting that LHCII phosphorylation under state 2 light is not enough to induce the state 2 transition but that the P-LHCII docking proteins in the PSI complex are required (Lunde et al., 2000; Jensen et al., 2004; Zhang and Scheller, 2004; Leoni et al., 2013).Thylakoid protein phosphorylation is a dynamic redox-regulated process dependent on the interplay between two kinases (STN7 and STN8; Depège et al., 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005) and two phosphatases (TAP38/PPH1 and PBCP; Pribil et al., 2010; Shapiguzov et al., 2010; Samol et al., 2012). Concerning the redox regulation mechanisms in vivo, only the LHCII kinase (STN7) has so far been thoroughly studied (Vener et al., 1997; Rintamäki et al., 2000; Lemeille et al., 2009). The STN7 kinase is considered as the LHCII kinase, and indeed, it phosphorylates the LHCB1 and LHCB2 proteins (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). In addition to this, STN7 takes part in the phosphorylation of PSII core proteins (Vainonen et al., 2005), especially in LL (Tikkanen et al., 2008b, 2010). The STN8 kinase is required for phosphorylation of PSII core proteins in HL but does not significantly participate in phosphorylation of LHCII (Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005; Tikkanen et al., 2010). It has been shown that, in traditional state 1 condition, which oxidizes the ETC, the dephosphorylation of LHCII is dependent on TAP38/PPH1 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010), whereas the PSII core protein dephosphorylation is dependent on the PBCP phosphatase (Samol et al., 2012). However, it remains unresolved whether and how the TAP38/PPH1 and PBCP phosphatases are involved in the light intensity-dependent regulation of thylakoid protein phosphorylation typical for natural environments.Here, we have used the two kinase (stn7 and stn8) and the two phosphatase (tap38/pph1and pbcp) mutants of Arabidopsis (Arabidopsis thaliana) to elucidate the individual roles of these enzymes in reversible thylakoid protein phosphorylation and distribution of excitation energy between PSII and PSI upon changes in light intensity. It is shown that the TAP38/PPH1-dependent, redox-regulated LHCII dephosphorylation is the key component to maintain excitation balance between PSII and PSI upon increase in light intensity, which at the same time, induces strong phosphorylation of the PSII core proteins. Collectively, reversible but opposite phosphorylation and dephosphorylation of the PSII core and LHCII proteins upon increase or decrease in light intensity are shown to be crucial for maintenance of even distribution of excitation energy to both photosystems, thus preventing state transitions. Moreover, evidence is provided indicating that the pH gradient across the thylakoid membrane is yet another important component in regulation of the distribution of excitation energy to PSII and PSI, possibly by affecting the regulation of thylakoid kinases and phosphatases.     

The Stromal Chloroplast Deg7 Protease Participates in the Repair of Photosystem II after Photoinhibition in Arabidopsis

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).Chloroplasts of higher plants carry out one of the most important biochemical reactions: the capture of light energy and its conversion into chemical energy. Although light is the ultimate source of energy for photosynthesis, it can also be harmful to plants. Light-induced loss of photosynthetic efficiency, which is generally termed as photoinhibition, limits plant growth and lowers productivity, especially when combined with other abiotic stresses.The main target of photoinhibition is PSII, which catalyzes the light-dependent water oxidation concomitantly with oxygen production (for review, see Prasil et al., 1992; Aro et al., 1993; Adir et al., 2003). In higher plants, PSII consists of more than 20 subunits, including the reaction center D1 and D2 proteins, cytochrome (Cyt) b₅₅₉, the light-harvesting chlorophyll a-binding proteins CP47 and CP43, the oxygen-evolving 33-kD protein (PsbO), and several low molecular mass proteins (Nelson and Yocum, 2006). The PSII reaction center D1 protein has been identified among PSII proteins as the primary target of light-induced damage (Kyle et al., 1984; Mattoo et al., 1984; Ohad et al., 1984; Adir et al., 1990), but several studies have shown that the D2, CP47, and CP43 proteins are degraded under photoinhibitory conditions (Schuster et al., 1988; Yamamoto and Akasaka, 1995; Jansen et al., 1999; Adir et al., 2003). Moreover, several small PSII subunits, such as PsbH, PsbW, and Cyt b₅₅₉, were also found to be frequently replaced within PSII (Hagman et al., 1997; Ortega et al., 1999; Bergantino et al., 2003). Evidence for the involvement of two families of proteases, FtsH and Deg, in the degradation of the D1 protein in thylakoids of higher plants has been recently described (Lindahl et al., 1996, 2000; Bailey et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; Kapri-Pardes et al., 2007; Sun et al., 2007a, 2007b). However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases.DegP (or HtrA) proteases were initially identified based on the fact that they are required for the survival of Escherichia coli at high temperatures and for the degradation of abnormal periplasmic proteins (Lipinska et al., 1988; Strauch and Beckwith, 1988). DegP is an ATP-independent Ser endopeptidase, and it contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (Gottesman, 1996; Pallen and Wren, 1997; Clausen et al., 2002). PDZ domains appear to be important for complex assembly and substrate binding through three or four residues in the C terminus of their target proteins (Doyle et al., 1996; Harris and Lim, 2001). DegP switches between chaperone and protease functions in a temperature-dependent manner. The chaperone function dominates at low temperatures, and DegP becomes proteolytically active at elevated temperatures (Spiess et al., 1999). Crystal structures of different members of the DegP protein family (Krojer et al., 2002; Li et al., 2002; Kim et al., 2003; Wilken et al., 2004) have revealed the structure-function relationship of these PDZ-containing proteases. Trimeric DegP is the functional unit, and the hexameric DegP is formed via the staggered association of trimers (Clausen et al., 2002; Kim and Kim, 2005). At normal growth temperatures, the active site of the protease is located within the chamber of hexameric DegP, which is not accessible to the substrates. However, at high temperatures, conformational changes induce the activation of the protease function (Krojer et al., 2002). Recent studies have shed light on the substrate binding-induced formation of larger oligomeric complexes of DegP (Jiang et al., 2008; Krojer et al., 2008).In Arabidopsis (Arabidopsis thaliana), 16 genes coding for DegP-like proteases have been identified, and at least seven gene products are predicted to be located in chloroplasts (Kieselbach and Funk, 2003; Huesgen et al., 2005; Adam et al., 2006; Sakamoto, 2006; Kato and Sakamoto, 2009). Based on proteomic data, four Deg proteases have been shown to be localized to the chloroplast (Peltier et al., 2002; Schubert et al., 2002) and functionally characterized. Deg1, Deg5, and Deg8 are located in thylakoid lumen, and Deg2 is peripherally associated with the stromal side of thylakoid membranes (Itzhaki et al., 1998; Haußühl et al., 2001; Sun et al., 2007a). Recombinant DegP1, now renamed Deg1, has been shown to be proteolytically active toward thylakoid lumen proteins such as plastocyanin and PsbO of PSII in vitro (Chassin et al., 2002). A 5.2-kD C-terminal fragment of the D1 protein was detected in vitro after incubation of recombinant Deg1 with inside-out thylakoid membranes. In transgenic plants with reduced levels of Deg1, fewer of its 16- and 5.2-kD degradation products were observed (Kapri-Pardes et al., 2007). Deg5 and Deg8 form a dodecameric complex in the thylakoid lumen, and recombinant Deg8 is able to degrade the photodamaged D1 protein of PSII in an in vitro assay (Sun et al., 2007a). The 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in a deg5 deg8 double mutant after high-light treatment. The deg5 deg8 double mutant showed increased sensitivity to high light and high temperature in terms of growth and PSII activity compared with the single mutants deg5 and deg8, suggesting that Deg5 and Deg8 have overlapping functions in the primary cleavage of the CD loop of the D1 protein (Sun et al., 2007a, 2007b). In vitro analysis has demonstrated that recombinant stroma-localized Deg2 was also shown to be involved in the primary cleavage of the DE loop of the D1 protein (Haußühl et al., 2001). However, analysis of a mutant lacking Deg2 suggested that Deg2 may not be involved in D1 degradation in vivo (Huesgen et al., 2006).Here, we have expressed and purified a recombinant DegP protease, His-Deg7. In vitro experiments showed that His-Deg7 is proteolytically active toward the PSII proteins D1, D2, CP43, and CP47. In vivo analyses of a deg7 mutant revealed that the mutant is more sensitive to high light stress than the wild-type plants. We demonstrated that Deg7 is a chloroplast stroma protein associated with the thylakoid membranes and that it interacts with PSII, which suggests that it can cleave the stroma-exposed region of substrate proteins. Our results also provide evidence that Deg7 is important for maintaining PSII function.     

Differential Mobility of Pigment-Protein Complexes in Granal and Agranal Thylakoid Membranes of C3 and C4 Plants

The photosynthetic performance of plants is crucially dependent on the mobility of the molecular complexes that catalyze the conversion of sunlight to metabolic energy equivalents in the thylakoid membrane network inside chloroplasts. The role of the extensive folding of thylakoid membranes leading to structural differentiation into stacked grana regions and unstacked stroma lamellae for diffusion-based processes of the photosynthetic machinery is poorly understood. This study examines, to our knowledge for the first time, the mobility of photosynthetic pigment-protein complexes in unstacked thylakoid regions in the C₃ plant Arabidopsis (Arabidopsis thaliana) and agranal bundle sheath chloroplasts of the C₄ plants sorghum (Sorghum bicolor) and maize (Zea mays) by the fluorescence recovery after photobleaching technique. In unstacked thylakoid membranes, more than 50% of the protein complexes are mobile, whereas this number drops to about 20% in stacked grana regions. The higher molecular mobility in unstacked thylakoid regions is explained by a lower protein-packing density compared with stacked grana regions. It is postulated that thylakoid membrane stacking to form grana leads to protein crowding that impedes lateral diffusion processes but is required for efficient light harvesting of the modularly organized photosystem II and its light-harvesting antenna system. In contrast, the arrangement of the photosystem I light-harvesting complex I in separate units in unstacked thylakoid membranes does not require dense protein packing, which is advantageous for protein diffusion.In higher plants, the photosynthetic apparatus is compartmentalized in the specialized chloroplast organelle. The molecular machinery for the primary photosynthetic processes, the sunlight-driven generation of metabolic energy equivalents, is harbored in an intricate thylakoid membrane system within the chloroplasts. Recent improvements in electron tomography have led to three-dimensional models of the complex architecture of thylakoid membranes (Mustárdy and Garab, 2003; Nevo et al., 2009; Austin and Staehelin, 2011; Daum et al., 2010; Kouřil et al., 2011). Although important details about the thylakoid structure are still highly controversial, consensus exists about the overall design of this membrane system. The thylakoid membrane consists of two morphologically distinct domains: strictly stacked cylindrical grana regions with a diameter of 300 to 600 nm are interconnected by unstacked stroma lamellae, thus forming a continuous membrane system. The molecular complexes that catalyze energy transformation are distributed heterogeneously between the stacked and unstacked membrane regions. The majority of the PSII complex and light-harvesting complex II (LHCII) are localized in stacked thylakoid regions, whereas PSI and the ATP-synthase complex are lacking from stacked grana (Staehelin and van der Staay, 1996; Albertsson, 2001; Dekker and Boekema, 2005). It is assumed that the fifth photosynthetic protein complex (cytochrome b₆f complex) is homogenously distributed.An essential feature of the thylakoid membrane system is its high flexibility, which is required for adaptability and maintenance of the photosynthetic machinery in plants. Highly responsive to environmental conditions, both the overall thylakoid architecture (e.g. number of grana discs) and the molecular membrane composition can change remarkably to optimize, protect, and maintain the photosynthetic apparatus (Walters, 2005; Anderson et al., 2008; Chuartzman et al., 2008; Dietzel et al., 2008; Betterle et al., 2009; Johnson et al., 2011). The underlying molecular processes require brisk protein traffic between stacked and unstacked thylakoid domains (Kirchhoff, 2008). The role of grana in these transport-based processes is poorly understood.Although photosynthetic energy conversion is possible without grana (Anderson et al., 2008), the fact that stacked thylakoids are ubiquitous in almost all land plants (with the exception of chloroplasts in bundle sheath [BS] cells in some C₄ plants; see below) highlights the evolutionary pressure to preserve this complex structural feature. Recently, the importance of grana formation was highlighted in Arabidopsis (Arabidopsis thaliana) mutants that lack the GRANA-DEFICIENT CHLOROPLAST1 gene; they grow much slower than the wild type and exhibit seed lethargy due to missing grana formation (Cui et al., 2011). The functional advantages of grana formation have been discussed extensively (Trissl and Wilhelm, 1993; Mullineaux, 2005; Anderson et al., 2008). It was hypothesized that grana could (1) increase the thylakoid membrane area, and the pigment concentration, in chloroplasts, (2) avoid energy spillover from PSII to PSI, (3) regulate the balance of energy distribution between PSII and PSI by state transition, and (4) enable transversal exciton energy transfer between adjacent grana discs. Although there are good arguments that these possibilities are important for photosynthetic energy conversion, the basis for the evolutionary development of grana has not been determined (Mullineaux, 2005; Anderson et al., 2008).A less considered aspect of grana formation is that it leads to a concentration of protein complexes (Murphy, 1986; Kirchhoff, 2008). The membrane area fraction that belongs to integral photosynthetic protein complexes is about 70%, making grana discs one of the most crowded biomembranes (Kirchhoff, 2008). Light harvesting by PSII benefits from a high protein-packing density for two reasons. First, a concentration of PSII and LHCII in grana ensures a high concentration of light-absorbing pigments that increase the probability of capturing sunlight, which is a “dilute” energy source on the molecular scale (Blankenship, 2002). Second, it has been demonstrated that a high protein-packing density in grana thylakoids is required for efficient intermolecular exciton energy transfer between LHCII and PSII (Haferkamp et al., 2010). Macromolecular crowding ensures that weakly interacting LHCII and PSII complexes come in close contact, allowing efficient Förster-type energy transfer.Besides these advantages, lateral protein traffic is challenged by macromolecular crowding (Mullineaux, 2005; Kirchhoff, 2008). The molecular mobility of proteins in grana thylakoids is reduced by numerous collisions of the diffusing object in the two-dimensional reaction space of the membrane with obstacles, integral membrane proteins, that increase the apparent diffusion path and, consequently, the diffusion time. The strong impairment of a high protein density in grana thylakoids on protein mobility was demonstrated by computer simulations (Tremmel et al., 2003; Kirchhoff et al., 2004) and by diffusion measurement on isolated grana membranes (Kirchhoff et al., 2008) and intact chloroplasts (Goral et al., 2010) using the fluorescence recovery after photobleaching (FRAP) technique. Processes that are expected to be affected by restricted protein mobility are a regulation of energy distributed between PSII and PSI by state transitions (Lemeille and Rochaix, 2010), the repair of photodamaged PSII (Mulo et al., 2008), membrane remodeling triggered by long-term environmental changes (Walters, 2005; Anderson et al., 2008), and the biogenesis of the thylakoid membrane network (Adam et al., 2011). Recently, evidence has accumulated that photoprotective high-energy quenching also requires large-scale diffusion-based structural reorganization within grana thylakoids (Betterle et al., 2009; Johnson et al., 2011).In contrast to our current understanding of diffusion-based processes in thylakoid membranes, knowledge about the factors that determine the mobility of photosynthetic protein complexes in different thylakoid domains is still fragmentary (Mullineaux, 2008). The protein-packing density is very likely a main element that determines protein mobility (Kirchhoff et al., 2008). However, other factors, like electrostatic interactions between proteins by membrane surface charges (Tremmel et al., 2005) or the size and molecular shape of protein complexes (Tremmel et al., 2003), can contribute significantly. However, data only exist about protein mobility for isolated grana thylakoids (Kirchhoff et al., 2008) and for chloroplasts from the grana-containing C₃ plant Arabidopsis (Goral et al., 2010). The diffusion characteristics of the latter are almost completely determined by granal proteins. Limiting information on protein diffusion exists for stroma lamellae of C₃ plants (Consoli et al., 2005; Vladimirou et al., 2009), and no data are available for agranal thylakoids, which occur in BS cells of some C₄ species.This study fills this gap in the knowledge base by studying lateral protein diffusion in unstacked thylakoid membranes in BS chloroplasts of two NADP-malate enzyme (ME)-type C₄ species, maize (Zea mays) and sorghum (Sorghum bicolor), in comparison with the grana-containing mesophyll (M) chloroplasts. The analysis was also complemented by studies on isolated thylakoid subfragments (grana core, grana, and stroma lamellae) from Arabidopsis. The protein mobility was measured by FRAP (Mullineaux and Kirchhoff, 2007), which has been shown to be a straightforward method to analyze protein diffusion in photosynthetic membranes by utilizing natural chlorophyll fluorescence (Kirchhoff et al., 2008; Goral et al., 2010). The comparison with diffusion characteristics in unstacked versus stacked membrane areas highlights the significance of grana formation on the lateral mobility of photosynthetic pigment-protein complexes.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.Light capture and its conversion to chemical energy occur in a set of transmembrane protein complexes of the thylakoid membrane. PSII, the cytochrome b₆f complex, and PSI drive photosynthetic electron flow and the creation of a proton gradient across the thylakoid membrane. ATP synthase couples the dissipation of this gradient to the synthesis of ATP. The light-harvesting antennae play an important role in collecting light and transferring energy to the photosystems. Light-Harvesting Complex I (LHCI) exclusively transfers light energy to PSI, with which it is tightly associated (Croce and van Amerongen, 2014). In contrast, LHCII, which is the most abundant complex of the thylakoid membrane, can transfer energy to PSI or PSII (Grieco et al., 2015). Light is highly variable in natural environments, and plants experience continuous changes in both the spectrum and intensity of light on timescales as short as seconds. Changes in light quality may unbalance the activity of the two photosystems since their absorption spectra differ, whereas high light intensity can lead to overexcitation and induce photodamage. At low or moderate light intensities, the LHCII complex differentially associates with PSII or PSI, in a phosphorylation-dependent process known as state transitions, to rapidly respond to changes in the spectrum of light. In brief, under light quality that activates PSII more than PSI (e.g. blue light), LHCII is phosphorylated, and as a consequence, its binding to PSI is favored (state 2). Conversely, under light that preferentially excites PSI (enriched in far-red), this association can be reverted by dephosphorylation of the LHCII antenna, which favors its binding to PSII (state 1; Goldschmidt-Clermont and Bassi, 2015; Kim et al., 2015). A protein kinase, STATE TRANSITION7 (STN7), and a protein phosphatase, PROTEIN PHOSPHATASE1 (PPH1)/THYLAKOID-ASSOCIATED PHOSPHATASE38 (TAP38), are essential for the rapid phosphorylation and dephosphorylation of the LHCII antenna that regulates its differential association to PSI or PSII (Bellafiore et al., 2005; Pribil et al., 2010; Shapiguzov et al., 2010). Only a relatively small fraction of the LHCII antenna (<20%) is estimated to participate in state transitions in Arabidopsis (Arabidopsis thaliana; Allen, 1992). However, the process is conserved across the green eukaryotes and is relevant to plant fitness (Frenkel et al., 2007). Under high light, energy-dependent quenching of LHCII predominates, and furthermore, this antenna can uncouple from PSII (Wientjes et al., 2013b).The differential association of photosystems, LHCII, and other components of the thylakoid membrane gives rise to a set of supercomplexes that are central in ensuring photosynthetic efficiency and a rapid response to environmental cues (Caffarri et al., 2009; Duffy et al., 2013; Pietrzykowska et al., 2014; Fristedt et al., 2015). Fine tuning the dynamic assembly of these supercomplexes involves the association of antennae containing specific sets of Lhcb proteins. The major LHCII antenna comprises homo- and heterotrimers of Lhcb1 to Lhcb3 (Jackowski et al., 2001), whereas the minor LHCII isoforms (Lhcb4–Lhcb6) are monomeric (de Bianchi et al., 2008). Lhcb1 and Lhcb2 share a very similar primary structure and associated pigments (Formaggio et al., 2001; Zhang et al., 2008), whereas Lhcb3 appears to have slightly different features (Standfuss and Kühlbrandt, 2004). In Arabidopsis, five genes encode Lhcb1 isoforms, three genes encode Lhcb2 isoforms, and a single gene encodes Lhcb3. The principal discriminant between these classes is a short stretch of residues at the N-terminal end, which is of particular importance since it contains the Thr that is reversibly phosphorylated during light-acclimation processes (Goldschmidt-Clermont and Bassi, 2015). During evolution, land plants have maintained a major LHCII composed of different classes of Lhcb subunits. The phosphorylated N terminus of Lhcb2 was particularly well conserved (Alboresi et al., 2008; Zhang et al., 2008).PSII-LHCII supercomplexes have been isolated from Arabidopsis with up to four LHCII trimers bound to a PSII dimer, as well as the three minor monomeric antennae (Lhcb4–Lhcb6; Caffarri et al., 2009; Kouřil et al., 2012). In the LHCII trimers of these supercomplexes, different classes of Lhcb subunits are distributed differently, suggesting a specific role in light acclimation for each of them (Damkjaer et al., 2009; Pietrzykowska et al., 2014). In the stably bound S trimer, Lhcb1 and Lhcb2 are more abundant, whereas the moderately bound M trimer contains mostly Lhcb1 and Lhcb3 (Galka et al., 2012). PSII supercomplexes isolated from spinach (Spinacia oleracea) showed the presence of an extra LHCII trimer (L trimer); therefore, it is possible that, in Arabidopsis, other trimers are associated with the PSII dimer in a more labile supercomplex that cannot be isolated (Boekema et al., 1999). A single LHCII trimer, containing Lhcb1 and Lhcb2, stably associates with PSI to constitute the PSI-LHCII supercomplex, whose formation is dependent on LHCII phosphorylation by STN7 in state 2 (Kouřil et al., 2005; Galka et al., 2012).Previous reports have shown that the relative phosphorylation of Lhcb1 and Lhcb2 isoforms differs among thylakoid supercomplexes (Galka et al., 2012; Leoni et al., 2013). Here, we address the specific roles of Lhcb1 and Lhcb2 phosphorylation in photosynthetic acclimation. The improved protocol for SDS-PAGE in the presence of Phos-tag (Wako Chemicals) that we present allows quantification of the extent of phosphorylation for each class of antenna isoforms. We report that, in the PSI-LHCII supercomplex that is assembled in state 2, only the phosphorylated form of Lhcb2 is present, whereas the phosphorylated form of Lhcb1 is excluded. In contrast, both Lhcb1 and Lhcb2 are phosphorylated to different levels in other supercomplexes. This quantitative information on the level of phosphorylation of Lhcb1 and Lhcb2 offers new insights into the specific roles of the two classes of LHCII isoforms in light acclimation and supercomplex formation.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants

State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems.In oxygenic photosynthesis, PSII and PSI function in series to convert light energy into the chemical energy that fuels multiple metabolic processes. Most of this light energy is captured by the chlorophyll (Chl) and carotenoid pigments in the light-harvesting antenna complexes (LHCs) that are peripherally associated with the core complexes of both photosystems (Wobbe et al., 2016). However, since the two photosystems exhibit different absorption spectra (Nelson and Yocum, 2006; Nield and Barber, 2006; Qin et al., 2015), PSI or PSII is preferentially excited under naturally fluctuating light intensities and qualities. To optimize photosynthetic electron transfer, the excitation state of the two photosystems must be rebalanced in response to changes in lighting conditions. To achieve this, higher plants and green algae require rapid and precise acclimatory mechanisms to adjust the relative absorption cross-sections of the two photosystems.To date, the phenomenon of state transitions is one of the well-documented short-term acclimatory mechanisms. It allows a mobile portion of the light-harvesting antenna complex II (LHCII) to be allocated to either photosystem, depending on the spectral composition and intensity of the ambient light (Allen and Forsberg, 2001; Rochaix, 2011; Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). State transitions are driven by the redox state of the plastoquinone (PQ) pool (Vener et al., 1997; Zito et al., 1999). When PSI is preferentially excited (by far-red light), the PQ pool is oxidized and all the LHCII is associated with PSII. This allocation of antenna complexes is defined as state I. When light conditions (blue/red light or low light) favor exciton trapping of PSII, the transition from state I to state II occurs. The over-reduced PQ pool triggers the activation of the membrane-localized Ser-Thr kinase STN7, which phosphorylates an N-terminal Thr on each of two major LHCII proteins, LHCB1 and LHCB2 (Allen, 1992; Bellafiore et al., 2005; Shapiguzov et al., 2016). Phosphorylation of LHCII results in the dissociation of LHCII from PSII and triggers its reversible relocation to PSI (Allen, 1992; Rochaix, 2011). Conversely, when the PQ pool is reoxidized, STN7 is inactivated and the constitutively active, thylakoid-associated phosphatase TAP38/PPH1 dephosphorylates LHCII, which then reassociates with PSII (Pribil et al., 2010; Shapiguzov et al., 2010). The physiological significance of state transitions has been demonstrated by the reduction in growth rate seen in the stn7 knock-out mutant under fluctuating light conditions (Bellafiore et al., 2005; Tikkanen et al., 2010).The canonical state transitions model implies spatial and temporal regulation of the allocation of LHC between the two spatially segregated photosystems (Dekker and Boekema, 2005). PSII-LHCII supercomplexes are organized in a tightly packed form in the stacked grana regions of thylakoid membranes, while PSI-LHCI supercomplexes are mainly localized in the nonstacked stromal lamellae and grana margin regions (Dekker and Boekema, 2005; Haferkamp et al., 2010). It has been proposed that, in the grana margin regions, which harbor LHCII and both photosystems, LHCII can migrate rapidly between them (Albertsson et al., 1990; Albertsson, 2001). This idea is supported by the recent discovery of mega complexes containing both photosystems in the grana margin regions (Yokono et al., 2015). Furthermore, phosphorylation of LHCII was found to increase not only the amount of PSI found in the grana margin region of thylakoid membranes (Tikkanen et al., 2008a), but also to modulate the pattern of PSI-PSII megacomplexes under changing light conditions (Suorsa et al., 2015). Nonetheless, open questions remain in relation to the physiological significance of the detection of phosphorylated LHCII in all thylakoid regions, even under the constant light conditions (Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013), although LHCII phosphorylation has been shown to modify the stacking of thylakoid membranes (Chuartzman et al., 2008; Pietrzykowska et al., 2014).State I-to-state II transition is featured by the formation of LHCII-PSI-LHCI supercomplexes, in which LHCII favors the light-harvesting capacity of PSI. Recently, LHCII-PSI-LHCI supercomplexes have been successfully isolated and purified using various detergents (Galka et al., 2012; Drop et al., 2014; Crepin and Caffarri, 2015) or a styrene-maleic acid copolymer (Bell et al., 2015). These findings yielded further insights into the reorganization of supercomplexes associated with state transitions, and it was suggested that phosphorylation of LHCB2 rather than LHCB1 is the essential trigger for the formation of state transition supercomplexes (Leoni et al., 2013; Pietrzykowska et al., 2014; Crepin and Caffarri, 2015; Longoni et al., 2015). Furthermore, characterization of mutants deficient in individual PSI core subunits indicates that PsaH, L, and I are required for docking of LHCII at PSI (Lunde et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005; Plöchinger et al., 2016).Recently, the state transition capacity has been characterized in the Arabidopsis (Arabidopsis thaliana) mutants with missing LHCI components. Although the Arabidopsis knock-out mutants lacking one of the four LHCI proteins (LHCA1-4) showed enhanced accumulation of LHCII-PSI complexes, the absorption cross-section of PSI under state II conditions was still compromised in the lhca1-4 mutants, and it is suggested that LHCI mediates the detergent-sensitive interaction between ‘extra LHCII’ and PSI (Benson et al., 2015; Grieco et al., 2015). Furthermore, the Arabidopsis mutant ΔLhca lacking all LHCA1-4 proteins was shown to be compensated for the deficiency of LHCI by binding LHCII under state II conditions (Bressan et al., 2016). In spite of this finding, the significant reduction in the absorption cross-section of PSI was still observed in the ΔLhca mutant, suggesting a substantial role of LHCI in light absorption under canopy conditions (Bressan et al., 2016). However, these findings emphasize the acclimatory function of state transitions in balancing light absorption capacity between the two photosystems by modifying their relative antenna size and imply the dynamic and variable organization of PS-LHC supercomplexes.LHC proteins are encoded by the nuclear Lhc superfamily (Jansson, 1994). The biogenesis of LHCs includes the cytoplasmic synthesis of the LHC precursor proteins, their translocation into chloroplasts via the TOC/TIC complex, and their posttranslational targeting and integration into the thylakoid membranes by means of the chloroplast signal recognition particle (cpSRP) machinery (Jarvis and Lopez-Juez, 2013). The posttranslational cpSRP-dependent pathway for the final translocation of LHC proteins into the thylakoid membrane includes interaction of cpSRP43 with LHC apo-proteins and recruitment of cpSRP54 to form a transit complex. Then binding of this tripartite cpSRP transit complex to the SRP receptor cpFtsY follows, which supports docking of the transit complex to thylakoid membranes and its association with the LHC translocase ALB3. Ultimately, ALB3 inserts LHC apo-proteins into the thylakoid membrane (Richter et al., 2010). Importantly, stoichiometric amounts of newly synthesized Chl a and Chl b as well as carotenoid are inserted into the LHC apo-proteins by unknown mechanisms to form the functional LHCs that associate with the core complexes of both photosystems in the thylakoid membranes (Dall’Osto et al., 2015; Wang and Grimm, 2015).The first committed steps in Chl synthesis occur in the Mg branch of the tetrapyrrole biosynthesis pathway. 5-Aminolevulinic acid synthesis provides the precursor for the formation of protoporphyrin IX, which is directed into the Mg branch (Tanaka and Tanaka, 2007; Brzezowski et al., 2015). Chl synthesis ends with the conversion of Chl a to Chl b catalyzed by Chl a oxygenase (CAO; Tanaka et al., 1998; Tomitani et al., 1999). It has been hypothesized that coordination between Chl synthesis and the posttranslational cpSRP pathway is a prerequisite for the efficient integration of Chls into LHC apo-proteins.In this study, we intend to characterize the assembly of LHCs when the availability of Chl molecules or the integration of LHC apo-proteins into thylakoid membranes is limiting. To this end, we compared the assembly of LHCs and the organization of PS-LHC complexes in two different sets of Arabidopsis mutants. Firstly, we used the chlorina1-2 (ch1-2) mutant, which is defective in the CAO gene. The members of the second set of mutants carry knock-out mutations in genes involved in the chloroplast SRP pathway (Richter et al., 2010).Our studies revealed distinct accumulation of PS-LHC supercomplexes between the two sets of mutant relative to wild-type plants. In spite of the defect in synthesis of Chl b, ch1-2 retains predominantly intact PSI-LHCI supercomplexes but has strongly reduced amounts of LHCII. In contrast, the chaos (cpSRP43) mutant exhibits synchronously reduced contents of both LHCI and LHCII, which results in the accumulation of PS core complexes without accompanying LHCs. Thus, the distribution of LHCs in the thylakoid membranes of the two mutants, ch1-2 and chaos, were explored under varying light conditions with the aim of elucidating the influence of modified LHCI/LHCII antenna size on state transitions. Our results contribute to an expanding view on the variety of photosynthetic complexes, which can be observed in Arabidopsis plants with specified mutations in LHC biogenesis.     

Focus on Ethylene: Ethylene and the Regulation of Physiological and Morphological Responses to Nutrient Deficiencies

To cope with nutrient deficiencies, plants develop both morphological and physiological responses. The regulation of these responses is not totally understood, but some hormones and signaling substances have been implicated. It was suggested several years ago that ethylene participates in the regulation of responses to iron and phosphorous deficiency. More recently, its role has been extended to other deficiencies, such as potassium, sulfur, and others. The role of ethylene in so many deficiencies suggests that, to confer specificity to the different responses, it should act through different transduction pathways and/or in conjunction with other signals. In this update, the data supporting a role for ethylene in the regulation of responses to different nutrient deficiencies will be reviewed. In addition, the results suggesting the action of ethylene through different transduction pathways and its interaction with other hormones and signaling substances will be discussed.When plants suffer from a mineral nutrient deficiency, they develop morphological and physiological responses (mainly in their roots) aimed to facilitate the uptake and mobilization of the limiting nutrient. After the nutrient has been acquired in enough quantity, these responses need to be switched off to avoid toxicity and conserve energy. In recent years, different plant hormones (e.g. ethylene, auxin, cytokinins, jasmonic acid, abscisic acid, brassinosteroids, GAs, and strigolactones) have been implicated in the regulation of these responses (Romera et al., 2007, 2011, 2015; Liu et al., 2009; Rubio et al., 2009; Kapulnik et al., 2011; Kiba et al., 2011; Iqbal et al., 2013; Zhang et al., 2014).Before the 1990s, there were several publications relating ethylene and nutrient deficiencies (cited in Lynch and Brown [1997] and Romera et al. [1999]) without establishing a direct implication of ethylene in the regulation of nutrient deficiency responses. In 1994, Romera and Alcántara (1994) published an article in Plant Physiology suggesting a role for ethylene in the regulation of Fe deficiency responses. In 1999, Borch et al. (1999) showed the participation of ethylene in the regulation of P deficiency responses. Since then, evidence has been accumulating in support of a role for ethylene in the regulation of both Fe (Romera et al., 1999, 2015; Waters and Blevins, 2000; Lucena et al., 2006; Waters et al., 2007; García et al., 2010, 2011, 2013, 2014; Yang et al., 2014) and P deficiency responses (Kim et al., 2008; Lei et al., 2011; Li et al., 2011; Nagarajan and Smith, 2012; Wang et al., 2012, 2014c). Both Fe and P may be poorly available in most soils, and plants develop similar responses under their deficiencies (Romera and Alcántara, 2004; Zhang et al., 2014). More recently, a role for ethylene has been extended to other deficiencies, such as K (Shin and Schachtman, 2004; Jung et al., 2009; Kim et al., 2012), S (Maruyama-Nakashita et al., 2006; Wawrzyńska et al., 2010; Moniuszko et al., 2013), and B (Martín-Rejano et al., 2011). Ethylene has also been implicated in both N deficiency and excess (Tian et al., 2009; Mohd-Radzman et al., 2013; Zheng et al., 2013), and its participation in Mg deficiency has been suggested (Hermans et al., 2010).In this update, we will review the information supporting a role for ethylene in the regulation of different nutrient deficiency responses. For information relating ethylene to other aspects of plant mineral nutrition, such as N₂ fixation and responses to excess of nitrate or essential heavy metals, the reader is referred to other reviews (for review, see Maksymiec, 2007; Mohd-Radzman et al., 2013; Steffens, 2014).     

Integrating Image-Based Phenomics and Association Analysis to Dissect the Genetic Architecture of Temporal Salinity Responses in Rice

Salinity affects a significant portion of arable land and is particularly detrimental for irrigated agriculture, which provides one-third of the global food supply. Rice (Oryza sativa), the most important food crop, is salt sensitive. The genetic resources for salt tolerance in rice germplasm exist but are underutilized due to the difficulty in capturing the dynamic nature of physiological responses to salt stress. The genetic basis of these physiological responses is predicted to be polygenic. In an effort to address this challenge, we generated temporal imaging data from 378 diverse rice genotypes across 14 d of 90 mm NaCl stress and developed a statistical model to assess the genetic architecture of dynamic salinity-induced growth responses in rice germplasm. A genomic region on chromosome 3 was strongly associated with the early growth response and was captured using visible range imaging. Fluorescence imaging identified four genomic regions linked to salinity-induced fluorescence responses. A region on chromosome 1 regulates both the fluorescence shift indicative of the longer term ionic stress and the early growth rate decline during salinity stress. We present, to our knowledge, a new approach to capture the dynamic plant responses to its environment and elucidate the genetic basis of these responses using a longitudinal genome-wide association model.Nearly one-third of the 54 million ha of the highly saline soils in the world are located in South and Southeast Asia. Rice (Oryza sativa), which is the primary source of calories and protein for these two regions, is very sensitive to salinity stress, with even moderate salinity levels known to decrease yields by 50% (Zeng et al., 2002). Projected sea level rise due to climate change is expected to increase saltwater ingress in coastal rice-growing regions of South and Southeast Asia. Therefore, development of salt-tolerant rice cultivars is essential to maintain rice productivity in the salinity-affected regions globally.Salt tolerance, defined as the ability to maintain growth and productivity in saline conditions, is a complex polygenic trait that may be influenced by distinct physiological mechanisms (Munns et al., 1982; Munns and Termaat, 1986; Cheeseman, 1988; Munns and Tester, 2008; Horie et al., 2012; for a comprehensive review of genes involved in salinity tolerance in rice, see Negrão et al., 2011) At the cellular level, plants respond to saline conditions in two phases, namely an osmotic (shoot ion independent) and an ionic stress phase, which can occur in an overlapping manner with varying intensity during the course of salinity stress (Munns and Termaat, 1986; Munns, 2002; Munns and James, 2003; Munns and Tester, 2008; Horie et al., 2012). During the osmotic stress phase, which occurs soon after the onset of salinity, the reduction in external osmotic potential disrupts water uptake and impedes cell expansion, which, at the whole plant level, leads to reduced growth rate (Matsuda and Riazi, 1981; Munns and Passioura, 1984; Rawson and Munns, 1984; Azaizeh and Steudle, 1991; Fricke and Peters, 2002; Fricke, 2004; Boursiac et al., 2005). As salinity stress persists over several days and weeks, sodium ions (Na⁺) accumulate to toxic levels, resulting in cell death and precocious leaf senescence (Lutts and Bouharmont, 1996; Munns, 2002; Munns and James, 2003; Ghanem et al., 2008). This is typically observed during the ionic phase of the salinity response (Munns, 2002; Munns and James, 2003; Munns and Tester, 2008). Plants possess distinct mechanisms to adapt to these osmotic and ionic stresses that are controlled by a suite of genes (Apse et al., 1999; Carvajal et al., 1999; Halfter et al., 2000; Ishitani et al., 2000; Shi et al., 2000; Zeng and Shannon, 2000; Rus et al., 2001; Berthomieu et al., 2003; Martínez-Ballesta et al., 2003; Boursiac et al., 2005, 2008; Ren et al., 2005; Huang et al., 2006; Davenport et al., 2007; Obata et al., 2007; Székely et al., 2008; Horie et al., 2011; Rivandi et al., 2011; Asano et al., 2012; Munns et al., 2012; Latz et al., 2013; Schmidt et al., 2013; Campo et al., 2014; Choi et al., 2014; Liu et al., 2014). The genetic basis of temporal adaptive responses to salinity stress remains to be explored in rice and other crops. This is primarily due to challenges in capturing the dynamic physiological responses to salinity for a large number of genotypes in a nondestructive manner. Manual phenotyping to detect incremental changes in growth rate during the osmotic stress and slight shifts in leaf color due to ionic stress is difficult to quantify for a large number of genotypes.In rice, at least one major quantitative trait loci (QTL; saltol) for salinity tolerance has been characterized based on end point measurements of biomass, senescence/injury, and Na⁺ and K⁺ concentrations (Bonilla et al., 2002; Lin et al., 2004; Thomson et al., 2010). SHOOT K⁺ CONTENT1 (SKC1) is the causative gene underlying the saltol region. SKC1 encodes a Na⁺-selective high-affinity potassium transporter that regulates Na⁺/K⁺ homeostasis during salinity stress (Ren et al., 2005). High levels of Na⁺ displace cellular K⁺, an essential element for several enzymatic reactions and physiological processes (Gierth and Mäser, 2007). The ability to maintain cellular K⁺ levels during salinity through the action of Na⁺-selective potassium transporters or Na⁺/H⁺ antiporters is a well-characterized tolerance mechanism in cereals including rice (Ren et al., 2005; Sunarpi et al., 2005; Huang et al., 2006; Møller et al., 2009; Mian et al., 2011; Munns et al., 2012).Numerous studies have utilized conventional linkage mapping to identify QTL for morphological and physiological responses to salinity in rice using discrete end point measurements (Bonilla et al., 2002; Lin et al., 2004; Ren et al., 2005; Negrão et al., 2011; Wang et al., 2012). However, the physiological adaptation to saline conditions is a complex continuous process that develops over time. While some accessions will exhibit similar end point phenotypic values, the genetic and physiological mechanisms giving rise to the similar phenotypes may be very different and the growth trajectories throughout the experiment may be distinct. Although single time point studies have yielded important information regarding the genetic basis of salinity tolerance, such approaches are too simple to reveal the genetic architecture of stress adaptation. With the advent of high-throughput image-based phenotyping platforms, it is now feasible to quantify dynamic responses during the stress treatment for a large number of genotypes (Berger et al., 2010; Golzarian et al., 2011; Chen et al., 2014; Honsdorf et al., 2014).Image-based phenotyping has been combined with genome-wide association studies (GWAS) and linkage mapping to examine the genetic basis of complex developmental processes (Busemeyer et al., 2013; Moore et al., 2013; Topp et al., 2013; Slovak et al., 2014; Würschum et al., 2014; Yang et al., 2014; Bac-Molenaar et al., 2015). Moreover, the introduction of the time axis provides a better understanding of the physiological processes underlying complex stress and developmental responses compared with single end point measurements (Zhang et al., 2012; Moore et al., 2013; Brown et al., 2014; Chen et al., 2014; Slovak et al., 2014; Bac-Molenaar et al., 2015). However, to date, no studies have implemented an association mapping approach using image-derived phenotypes to address the genetic basis of dynamic stress responses in plants. Image-based phenotyping offers several advantages over conventional phenotyping: (1) quantitative measurements can be recorded over discrete time points to capture morphological and physiological responses in a nondestructive manner, and (2) the use of various types of spectral imaging address phenotypes that are not detectable to the human eye such as chlorophyll fluorescence and leaf water content. Integrating dynamic phenotypic data and association mapping has the potential to query genetic diversity across hundreds of accessions for complex traits and provides much higher resolution compared with conventional linkage mapping. Here, we explored the dynamic growth and chlorophyll responses to salinity of a diverse set of rice accessions using high-throughput visible and fluorescence imaging. To assess the genetic basis of plant growth in saline conditions, a logistic model was used to describe the temporal growth responses and was incorporated into the statistical framework necessary for association mapping. Coupled with temporal fluorescence imaging, we present, to our knowledge, new insights into the genetic architecture of osmotic and ionic responses during salinity stress in rice.     

Surveying Rubisco Diversity and Temperature Response to Improve Crop Photosynthetic Efficiency

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally “better” compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.In a changing climate and under pressure from a population set to hit nine billion by 2050, global food security will require massive changes to the way food is produced, distributed, and consumed (Ort et al., 2015). To match rising demand, agricultural production must increase by 50 to 70% in the next 35 years, and yet the gains in crop yields initiated by the green revolution are slowing, and in some cases, stagnating (Long and Ort, 2010; Ray et al., 2012). Among a number of areas being pursued to increase crop productivity and food production, improving photosynthetic efficiency is a clear target, offering great promise (Parry et al., 2007; von Caemmerer et al., 2012; Price et al., 2013; Ort et al., 2015). As the gatekeeper of carbon entry into the biosphere and often acting as the rate-limiting step of photosynthesis, Rubisco, the most abundant enzyme on the planet (Ellis, 1979), is an obvious and important target for improving crop photosynthetic efficiency.Rubisco is considered to exhibit comparatively poor catalysis, in terms of catalytic rate, specificity, and CO₂ affinity (Tcherkez et al., 2006; Andersson, 2008), leading to the suggestion that even small increases in catalytic efficiency may result in substantial improvements to carbon assimilation across a growing season (Zhu et al., 2004; Parry et al., 2013; Galmés et al., 2014a; Carmo-Silva et al., 2015). If combined with complimentary changes such as optimizing other components of the Calvin Benson or photorespiratory cycles (Raines, 2011; Peterhansel et al., 2013; Simkin et al., 2015), optimized canopy architecture (Drewry et al., 2014), or introducing elements of a carbon concentrating mechanism (Furbank et al., 2009; Lin et al., 2014a; Hanson et al., 2016; Long et al., 2016), Rubisco improvement presents an opportunity to dramatically increase the photosynthetic efficiency of crop plants (McGrath and Long, 2014; Long et al., 2015; Betti et al., 2016). A combination of the available strategies is essential for devising tailored solutions to meet the varied requirements of different crops and the diverse conditions under which they are typically grown around the world.Efforts to engineer an improved Rubisco have not yet produced a “super Rubisco” (Parry et al., 2007; Ort et al., 2015). However, advances in engineering precise changes in model systems continue to provide important developments that are increasing our understanding of Rubisco catalysis (Spreitzer et al., 2005; Whitney et al., 2011a, 2011b; Morita et al., 2014; Wilson et al., 2016), regulation (Andralojc et al., 2012; Carmo-Silva and Salvucci, 2013; Bracher et al., 2015), and biogenesis (Saschenbrecker et al., 2007; Whitney and Sharwood, 2008; Lin et al., 2014b; Hauser et al., 2015; Whitney et al., 2015).A complementary approach is to understand and exploit Rubisco natural diversity. Previous characterization of Rubisco from a limited number of species has not only demonstrated significant differences in the underlying catalytic parameters, but also suggests that further undiscovered diversity exists in nature and that the properties of some of these enzymes could be beneficial if present in crop plants (Carmo-Silva et al., 2015). Recent studies clearly illustrate the variation possible among even closely related species (Galmés et al., 2005, 2014b, 2014c; Kubien et al., 2008; Andralojc et al., 2014; Prins et al., 2016).Until recently, there have been relatively few attempts to characterize the consistency, or lack thereof, of temperature effects on in vitro Rubisco catalysis (Sharwood and Whitney, 2014), and often studies only consider a subset of Rubisco catalytic properties. This type of characterization is particularly important for future engineering efforts, enabling specific temperature effects to be factored into any attempts to modify crops for a future climate. In addition, the ability to coanalyze catalytic properties and DNA or amino acid sequence provides the opportunity to correlate sequence and biochemistry to inform engineering studies (Christin et al., 2008; Kapralov et al., 2011; Rosnow et al., 2015). While the amount of gene sequence information available grows rapidly with improving technology, knowledge of the corresponding biochemical variation resulting has yet to be determined (Cousins et al., 2010; Carmo-Silva et al., 2015; Sharwood and Whitney, 2014; Nunes-Nesi et al., 2016).This study aimed to characterize the catalytic properties of Rubisco from diverse species, comprising a broad range of monocots and dicots from diverse environments. The temperature dependence of Rubisco catalysis was evaluated to tailor Rubisco engineering for crop improvement in specific environments. Catalytic diversity was analyzed alongside the sequence of the Rubisco large subunit gene, rbcL, to identify potential catalytic switches for improving photosynthesis and productivity. In vitro results were compared to the average temperature of the warmest quarter in the regions where each species grows to investigate the role of temperature in modulating Rubisco catalysis.