Combined Effects of CO2 and Light on the N2-Fixing Cyanobacterium Trichodesmium IMS101: Physiological Responses

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum (IMS101) showed that increasing CO₂ partial pressure (pCO₂) enhances N₂ fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO₂, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses of Trichodesmium IMS101 grown under a matrix of low and high levels of pCO₂ (150 and 900 μatm) and irradiance (50 and 200 μmol photons m⁻² s⁻¹). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO₂ and light levels in the cultures. The pCO₂-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO₂ stimulated rates of N₂ fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO₂. HCO₃⁻ was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO₂ uptake was enhanced under high pCO₂. A comparison between carbon fluxes in vivo and those derived from ¹³C fractionation indicates high internal carbon cycling, especially in the low-pCO₂ treatment under high light. Light-dependent oxygen uptake was only detected under low pCO₂ combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO₂ effect on N₂ fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO₂. These findings are supported by a complementary study looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.Human-induced climate change will significantly alter the marine environment within the next century and beyond. Future scenarios predict an increase from currently approximately 380 to about 750 to 1,000 μatm CO₂ partial pressure (pCO₂) in the atmosphere until the end of this century (Raven et al., 2005; Raupach et al., 2007). As the ocean takes up this anthropogenic CO₂, dissolved inorganic carbon (DIC) in the surface ocean increases while the pH decreases (Wolf-Gladrow et al., 1999). Rising global temperatures will increase surface ocean stratification, which may affect the light regime in the upper mixed layer as well as nutrient input from deeper waters (Doney, 2006). Uncertainties remain regarding both the magnitude of the physicochemical changes and the biological responses of organisms, including species and populations of the oceanic primary producers at the basis of the food webs.In view of potential ecological implications and feedbacks on climate, several studies have examined pCO₂ sensitivity in phytoplankton key species (Burkhardt and Riebesell, 1997; Riebesell et al., 2000; Rost et al., 2003; Tortell et al., 2008). Pronounced responses to elevated pCO₂ were observed in N₂-fixing cyanobacteria (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007; Fu et al., 2008; Kranz et al., 2009), which play a vital role in marine ecosystems by providing a new source of biologically available nitrogen species to otherwise nitrogen-limited regions. Recent studies focused on the impact of different environmental factors on the filamentous Trichodesmium species, which is known for high abundance and the formation of massive blooms in tropical and subtropical areas (Capone et al., 2005; Mahaffey et al., 2005). Higher pCO₂ levels stimulated growth rates, biomass production, and N₂ fixation (Hutchins et al., 2007; Levitan et al., 2007; Kranz et al., 2009) and affected inorganic carbon acquisition of the cells (Kranz et al., 2009). While elevated sea surface temperatures are predicted to shift the spatial distribution of Trichodesmium species toward higher latitudes (Breitbarth et al., 2007), the combined effects of pCO₂ and temperature may favor this species and extend its niche even farther (Hutchins et al., 2007; Levitan et al., 2010a). An increase in the average light intensity, caused by the predicted shoaling of the upper mixed layer, may further stimulate photosynthesis and thus growth and N₂ fixation of Trichodesmium (Breitbarth et al., 2008). To our knowledge, the combined effects of light and pCO₂ have not been studied yet, although these environmental factors are likely to influence photosynthesis and other key processes in Trichodesmium.To understand the responses of an organism to changes in environmental conditions, metabolic processes must be studied. In Trichodesmium, photosynthetically generated energy (ATP and NADPH) is primarily used for the fixation of CO₂ in the Calvin-Benson cycle. A large proportion of this energy, however, is also required for the process of N₂ fixation via nitrogenase and for the operation of a CO₂-concentrating mechanism (CCM). The latter involves active uptake of inorganic carbon, which functions to increase the rate of carboxylation reaction mediated by Rubisco. This ancient and highly conserved enzyme is characterized by low affinities for its substrate CO₂ and a susceptibility to a competing reaction with oxygen (O₂) as substrate (Badger et al., 1998); the latter initiates photorespiration. As cyanobacterial Rubisco possesses one of the lowest CO₂ affinities among phytoplankton (Badger et al., 1998), a considerable amount of resources have to be invested to achieve sufficient rates of carbon fixation and to avoid photorespiration. A first step toward a mechanistic understanding of responses in Trichodesmium has been taken by Levitan et al. (2007), focusing on pCO₂ dependency of nitrogenase activity and photosynthesis. Subsequently, Kranz et al. (2009) described variations in CCM efficiency with pCO₂ and suggested that the observed plasticity in CCM regulation allowed energy reallocation under high pCO₂, which may explain the observed pCO₂-dependent changes in nitrogenase activity, growth, and elemental composition (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007).In this study, we measured growth responses as well as metabolic key processes in Trichodesmium erythraeum (IMS101) under environmental conditions that likely alter the energy budget and/or energy allocation of the cell. Cultures were acclimated to a matrix of low and high pCO₂ (150 and 900 μatm) at two different light intensities (50 and 200 μmol photons m⁻² s⁻¹). For each of the four treatments, changes in growth rates, elemental ratios, and the accumulation of particulate carbon and nitrogen were measured. Metabolic processes (gross photosynthesis, CCM activity, and O₂ uptake) were obtained by means of membrane-inlet mass spectrometry (MIMS), while N₂ fixation was detected by gas chromatography. As these processes may vary over the diurnal cycle in Trichodesmium (Berman-Frank et al., 2001; Kranz et al., 2009), measurements were performed in the morning and around midday. The results on metabolic processes were accompanied by measurements of the fluorescence of PSII, ratios of the photosynthetic units (PSI:PSII), and pool sizes of key proteins involved in carbon and nitrogen fixation as well as assimilation (Levitan et al., 2010b).     

Leaf Senescence Signaling: The Ca2+-Conducting Arabidopsis Cyclic Nucleotide Gated Channel2 Acts through Nitric Oxide to Repress Senescence Programming

Ca²⁺ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca²⁺. The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca²⁺ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca²⁺ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca²⁺ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca²⁺ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H₂O₂ generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca²⁺ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca²⁺ signaling provides evidence consistent with genetic studies of the relationship between Ca²⁺ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca²⁺ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.Senescence can be considered as the final stage of a plant’s development. During this process, nutrients will be reallocated from older to younger parts of the plant, such as developing leaves and seeds. Leaf senescence has been characterized as a type of programmed cell death (PCD; Gan and Amasino, 1997; Quirino et al., 2000; Lim et al., 2003). During senescence, organelles such as chloroplasts will break down first. Biochemical changes will also occur in the peroxisome during this process. When the chloroplast disassembles, it is easily observed as a loss of chlorophyll. Mitochondria, the source of energy for cells, will be the last cell organelles to undergo changes during the senescence process (Quirino et al., 2000). At the same time, other catabolic events (e.g. protein and lipid breakdown, etc.) are occurring (Quirino et al., 2000). Hormones may also contribute to this process (Gepstein, 2004). From this information we can infer that leaf senescence is regulated by many signals.Darkness treatment can induce senescence in detached leaves (Poovaiah and Leopold, 1973; Chou and Kao, 1992; Weaver and Amasino, 2001; Chrost et al., 2004; Guo and Crawford, 2005; Ülker et al., 2007). Ca²⁺ can delay the senescence of detached leaves (Poovaiah and Leopold, 1973) and leaf senescence induced by methyl jasmonate (Chou and Kao, 1992); the molecular events that mediate this effect of Ca²⁺ are not well characterized at present.Nitric oxide (NO) is a critical signaling molecule involved in many plant physiological processes. Recently, published evidence supports NO acting as a negative regulator during leaf senescence (Guo and Crawford, 2005; Mishina et al., 2007). Abolishing NO generation in either loss-of-function mutants (Guo and Crawford, 2005) or transgenic Arabidopsis (Arabidopsis thaliana) plants expressing NO degrading dioxygenase (NOD; Mishina et al., 2007) leads to an early senescence phenotype in these plants compared to the wild type. Corpas et al. (2004) showed that endogenous NO is mainly accumulated in vascular tissues of pea (Pisum sativum) leaves. This accumulation is significantly reduced in senescing leaves (Corpas et al., 2004). Corpas et al. (2004) also provided evidence that NO synthase (NOS)-like activity (i.e. generation of NO from l-Arg) is greatly reduced in senescing leaves. Plant NOS activity is regulated by Ca²⁺/calmodulin (CaM; Delledonne et al., 1998; Corpas et al., 2004, 2009; del Río et al., 2004; Valderrama et al., 2007; Ma et al., 2008). These studies suggest a link between Ca²⁺ and NO that could be operating during senescence.In animal cells, all three NOS isoforms require Ca²⁺/CaM as a cofactor (Nathan and Xie, 1994; Stuehr, 1999; Alderton et al., 2001). Notably, animal NOS contains a CaM binding domain (Stuehr, 1999). It is unclear whether Ca²⁺/CaM can directly modulate plant NOS or if Ca²⁺/CaM impacts plant leaf development/senescence through (either direct or indirect) effects on NO generation. However, recent studies from our lab suggest that Ca²⁺/CaM acts as an activator of NOS activity in plant innate immune response signaling (Ali et al., 2007; Ma et al., 2008).Although Arabidopsis NO ASSOCIATED PROTEIN1 (AtNOA1; formerly named AtNOS1) was thought to encode a NOS enzyme, no NOS-encoding gene has yet been identified in plants (Guo et al., 2003; Crawford et al., 2006; Zemojtel et al., 2006). However, the AtNOA1 loss-of-function mutant does display reduced levels of NO generation, and several groups have used the NO donor sodium nitroprusside (SNP) to reverse some low-NO related phenotypes in Atnoa1 plants (Guo et al., 2003; Bright et al., 2006; Zhao et al., 2007). Importantly, plant endogenous NO deficiency (Guo and Crawford, 2005; Mishina et al., 2007) or abscisic acid/methyl jasmonate (Hung and Kao, 2003, 2004) induced early senescence can be successfully rescued by application of exogenous NO. Addition of NO donor can delay GA-elicited PCD in barley (Hordeum vulgare) aleurone layers as well (Beligni et al., 2002).It has been suggested that salicylic acid (SA), a critical pathogen defense metabolite, can be increased in natural (Morris et al., 2000; Mishina et al., 2007) and transgenic NOD-induced senescent Arabidopsis leaves (Mishina et al., 2007). Pathogenesis related gene1 (PR1) expression is up-regulated in transgenic Arabidopsis expressing NOD (Mishina et al., 2007) and in leaves of an early senescence mutant (Ülker et al., 2007).Plant cyclic nucleotide gated channels (CNGCs) have been proposed as candidates to conduct extracellular Ca²⁺ into the cytosol (Sunkar et al., 2000; Talke et al., 2003; Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007; Demidchik and Maathuis, 2007; Frietsch et al., 2007; Kaplan et al., 2007; Ma and Berkowitz, 2007; Urquhart et al., 2007; Ma et al., 2009a, 2009b). Arabidopsis “defense, no death” (dnd1) mutant plants have a null mutation in the gene encoding the plasma membrane-localized Ca²⁺-conducting CNGC2 channel. This mutant also displays no hypersensitive response to infection by some pathogens (Clough et al., 2000; Ali et al., 2007). In addition to involvement in pathogen-mediated Ca²⁺ signaling, CNGC2 has been suggested to participate in the process of leaf development/senescence (Köhler et al., 2001). dnd1 mutant plants have high levels of SA and expression of PR1 (Yu et al., 1998), and spontaneous necrotic lesions appear conditionally in dnd1 leaves (Clough et al., 2000; Jirage et al., 2001). Endogenous H₂O₂ levels in dnd1 mutants are increased from wild-type levels (Mateo et al., 2006). Reactive oxygen species molecules, such as H₂O₂, are critical to the PCD/senescence processes of plants (Navabpour et al., 2003; Overmyer et al., 2003; Hung and Kao, 2004; Guo and Crawford, 2005; Zimmermann et al., 2006). Here, we use the dnd1 mutant to evaluate the relationship between leaf Ca²⁺ uptake during plant growth and leaf senescence. Our results identify NO, as affected by leaf Ca²⁺ level, to be an important negative regulator of leaf senescence initiation. Ca²⁺-mediated NO production during leaf development could control senescence-associated gene (SAG) expression and the production of molecules (such as SA and H₂O₂) that act as signals during the initiation of leaf senescence programs.     

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.     

Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant''s endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.Many legumes establish nitrogen-fixing root nodules following reciprocal signal exchange between the plant and rhizobia (Hayashi et al., 2000; Hirsch et al., 2003). The host plant produces chemical compounds, frequently flavonoids, which induce rhizobial nod genes, whose products are involved in the synthesis and secretion of Nod factor. Perception of this chitolipooligosaccharide by the host plant results in the triggering of a signal transduction cascade that leads to root hair deformation and curling and subsequent cortical cell divisions, which establish the nodule primordium. The rhizobia enter the curled root hair cell and nodule primordial cells through an infection thread. Eventually, the rhizobia are released into nodule cells, enclosed within a membrane, and differentiate into nitrogen-fixing bacteroids that reduce atmospheric nitrogen into ammonia. In return, the host plant supplies photosynthetic products, to be used as carbon sources, to the rhizobia (Zuanazzi et al., 1998; Hayashi et al., 2000).The host plant is known to be important for regulating the number of nodules established on its roots. For example, hypernodulating mutants such as nitrate-tolerant symbiotic1 (nts1; Glycine max), hypernodulation aberrant root formation1 (har1; Lotus japonicus), super numeric nodules (sunn; Medicago truncatula), and symbiosis29 (sym29; Pisum sativum) disrupt the balance between supply and demand by developing excessive root nodules (Oka-Kira and Kawaguchi, 2006). Grafting experiments demonstrated that leaf tissue is a principal source of the systemic signals contributing to the autoregulation of nodulation (Pierce and Bauer, 1983; Kosslak and Bohlool, 1984; Krusell et al., 2002; Nishimura et al., 2002b; van Brussel et al., 2002; Searle et al., 2003; Schnabel et al., 2005). The Nts1, Har1, Sunn, and Sym29 genes encode a receptor-like kinase similar to CLAVATA1, which regulates meristem cell number and differentiation (Krusell et al., 2002; Nishimura et al., 2002a; Searle et al., 2003; Schnabel et al., 2005).Phytohormones are also known to regulate nodulation (Hirsch and Fang, 1994). For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads (Nukui et al., 2000; Oldroyd et al., 2001; Ma et al., 2003). The ethylene-insensitive mutant sickle1 (skl1) of M. truncatula has a hypernodulating phenotype (Penmetsa and Cook, 1997). Skl1 is homologous to Ethylene insensitive2 of Arabidopsis (Arabidopsis thaliana), which is part of the ethylene-signaling pathway (Alonso et al., 1999; Penmetsa et al., 2008). In contrast, cytokinin is a positive regulator of nodulation. The cytokinin-insensitive mutant hyperinfected1 (loss of function) of L. japonicus and the spontaneous nodule formation2 (gain of function) mutants of M. truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007).Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes (Phillips, 1971; Cho and Harper, 1993; Bano et al., 2002; Bano and Harper, 2002; Suzuki et al., 2004; Nakatsukasa-Akune et al., 2005; Liang et al., 2007). Recently, M. truncatula overexpressing abscisic acid insensitive1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA-signaling pathway (Leung et al., 1994; Hagenbeek et al., 2000; Gampala et al., 2001; Wu et al., 2003), was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype (Ding et al., 2008).In this study, we isolated a L. japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and proceeded to carry out its characterization. This mutant, named enhanced nitrogen fixation1 (enf1), exhibits enhanced symbiotic nitrogen fixation activity. Most legume nitrogen fixation activity mutants, such as ineffective greenish nodules1 (ign1), stationary endosymbiont nodule1, and symbiotic sulfate transporter1 (sst1), are Fix⁻ (Suganuma et al., 2003; Krusell et al., 2005; Kumagai et al., 2007).     

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.     

Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense.Nitric oxide (NO) is a small ubiquitous molecule derived from nitrogen-containing precursors that is one of the earliest and most widespread signaling molecules in living organisms from metazoans to mammals (Torreilles, 2001). The regulatory functions of NO have been extensively studied in mammals, where it is synthesized from Arg through the activity of NO synthases (Knowles and Moncada, 1994). By contrast, the biosynthesis and function of this molecule in plants are largely unknown. During the last 10 years, NO biosynthesis in plants has been one of the most controversial topics in plant biology (Durner and Klessig, 1999; Wendehenne et al., 2001; del Río et al., 2004; Zeier et al., 2004; Lamotte et al., 2005; Meyer et al., 2005; Modolo et al., 2005; Crawford, 2006; Crawford et al., 2006; Zemojtel et al., 2006a). Despite the controversy about its biosynthesis, it is now clear that NO regulates many physiological processes of plants, including seed germination, cell death, defense responses against pathogens, stomata function, senescence, and flowering (Beligni and Lamattina, 2000; Pedroso et al., 2000; Neill et al., 2002; Lamattina et al., 2003; He et al., 2004; Romero-Puertas et al., 2004; Wendehenne et al., 2004; Delledonne, 2005; Guo and Crawford, 2005; Simpson, 2005; Grün et al., 2006; Melotto et al., 2006; Planchet et al., 2006; Ali et al., 2007; Mishina et al., 2007).The molecular mechanisms underlying the control of seed dormancy and germination are still poorly characterized. Genetic data support a central role of abscisic acid (ABA) in regulating seed dormancy, whereas gibberellins promote germination (Finkelstein et al., 2008; Holdsworth et al., 2008). In addition, NO has been lately characterized as a new component in the signaling pathway leading to dormancy breakage. NO-releasing compounds reduce dormancy in a NO-dependent manner in Arabidopsis (Arabidopsis thaliana), some warm-season grasses, and certain barley (Hordeum vulgare) cultivars (Bethke et al., 2004; Sarath et al., 2006). More recently, the aleurone layer cells have been characterized as responsive to NO, gibberellins, and ABA, thus becoming a primary determinant of seed dormancy in Arabidopsis (Bethke et al., 2007).Two main enzyme-based pathways have been proposed to be functional for NO biosynthesis in plants. One is based on the activity of nitrate reductases (Meyer et al., 2005; Modolo et al., 2005), and another one, yet undefined, is based on the direct or indirect function of the Nitric Oxide-Associated1/Resistant to Inhibition by Fosfidomycin1 (AtNOA1/RIF1) protein. It has been also reported that NO synthesis from nitrite occurs in mitochondria associated with mitochondrial electron transport (Planchet et al., 2005) and also that this pathway is mainly functioning in roots under anoxia (Gupta et al., 2005). Moreover, the balance between mitochondrial nitrite reduction and superoxide-dependent NO degradation seems to be derived from factors controlling NO levels in Arabidopsis (Wulff et al., 2009). It has been recently reported that the synthesis of NO in floral organs requires nitrate reductase activity (Seligman et al., 2008) and also that homologues of AtNOA1 participate in NO biosynthesis in diatoms (Vardi et al., 2008), mammals (Zemojtel et al., 2006b; Parihar et al., 2008a, 2008b), and Nicotiana benthamiana (Kato et al., 2008). Recently, the identification of the rif1 mutant, carrying a null mutation in the AtNOA1 locus (At3g47450), allowed uncovering of a function for AtNOA1/RIF1 in the expression of plastome-encoded proteins (Flores-Pérez et al., 2008). Moreover, another recent report claims that AtNOA1 is not a NO synthase but a cGTPase (Moreau et al., 2008), likely playing a role in ribosome assembly and subsequent mRNA translation to proteins in the chloroplasts.To date, it is not clear if both pathways coexist in plants and, if so, the corresponding contributions of each pathway to NO biosynthesis. In this work, we have addressed the functions of both pathways in Arabidopsis by generating a triple mutant in both nitrate reductases and AtNOA1 that is severely impaired in NO production. Further characterization of NO-deficient plants allowed us to identify a functional cross talk between NO and ABA in controlling seed germination and dormancy as well as plant resistance to water deficit.     

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins

Spatial segregation of metabolism, such as cellular-localized CO₂ fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.A key enzyme in photosynthesis is the CO₂ fixation enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco not only fixes CO₂, resulting in carbon assimilation, but it can also fix O₂, leading to photorespiration. Suppressing the unwanted oxygenase activity of Rubisco by sequestering Rubisco with a source of CO₂ is Nature’s solution to this substrate discrimination problem. While C4 plants compartmentalize CO₂ fixation in specific cells (Hibberd et al., 2008; Parry et al., 2011), cyanobacteria have evolved a specialized organelle composed entirely of protein to encapsulate Rubisco—the carboxysome.The carboxysome is just one type of bacterial microcompartment (BMC), widespread, functionally diverse bacterial organelles (Axen et al., 2014). All BMCs consist of an enzymatic core surrounded by a selectively permeable protein shell (Kerfeld et al., 2005; Tanaka et al., 2008; Chowdhury et al., 2014; Kerfeld and Erbilgin, 2015). While the encapsulated enzymes differ among functionally distinct BMCs, they share an architecturally similar shell composed of three types of proteins: BMC-H, BMC-T, and BMC-P forming hexamers, pseudohexamers, and pentamers, respectively (Kerfeld and Erbilgin, 2015). These constitute the building blocks of a self-assembling, apparently icosahedral shell with a diameter ranging from 40 to 400 nm (Shively et al., 1973a,b, 1998; Price and Badger, 1991; Bobik et al., 1999; Iancu et al., 2007, 2010; Petit et al., 2013; Erbilgin et al., 2014). Recent studies have also shown that in the biogenesis of BMCs an encapsulation peptide (EP) (Fan and Bobik, 2011; Kinney et al., 2012; Aussignargues et al., 2015; Jakobson et al., 2015), a short (approximately 18 residues) amphipathic α-helix mediates interactions between a subset of core protein and the shell (Fan and Bobik, 2011; Choudhary et al., 2012; Kinney et al., 2012; Lawrence et al., 2014; Lin et al., 2014; Aussignargues et al., 2015). Indeed, because they are self-assembling organelles composed entirely of protein, BMCs hold great promise for diverse applications in bioengineering and development of bionanomaterials (Frank et al., 2013; Chowdhury et al., 2014; Chessher et al., 2015; Kerfeld and Erbilgin, 2015); the key features of BMCs include selective permeability, spatial colocalization of enzymes, the establishment of private cofactor pools, and the potentially beneficial effects of confinement on protein stability. For example, introducing carboxysomes into plants could provide a saltational enhancement of crop photosynthesis (Price et al., 2013; Zarzycki et al., 2013; Lin et al., 2014; McGrath and Long, 2014).The β-carboxysome, which sequesters form 1B Rubisco, has been an important model system for the study of the structural basis of carboxysome function, assembly, and engineering (Kerfeld et al., 2005; Tanaka et al., 2008; Cameron et al., 2013; Aussignargues et al., 2015; Cai et al., 2015). Beta-carboxysomes assemble from the inside out (Cameron et al., 2013; Gonzalez-Esquer et al., 2015). Two proteins that are absolutely conserved and unique to β-carboxysomes, CcmM and CcmN, play essential roles in this process: CcmM crosslinks Rubisco through its C-terminal Rubisco small subunit-like domains (SSLDs; pfam00101); CcmM and CcmN interact through their N-terminal domains; and C-terminal EP of CcmN interacts with the carboxysome shell.Here we describe a system for producing synthetic β-carboxysome shells and encapsulating nonnative cargo. We constructed a synthetic operon composed of ccmK1, ccmK2, ccmL, and ccmO, genes encoding, respectively, two BMC-H proteins, a BMC-P protein, and a BMC-T protein of the carboxysome shell of the halotolerant cyanobacterium, Halothece sp. PCC 7418 (Halo hereafter). Recombinant shells composed of all four proteins were produced and purified. We also demonstrated that the terminal α-helices of CcmK1 and CcmK2 are not, as had been proposed (Samborska and Kimber, 2012), required for the shell formation, and that the synthetic shell is a single-layered protein membrane. Cargo could be targeted to the interior of the synthetic shells using either the EP of CcmN or the N-terminal domain of CcmM; the latter observation provides new insight into the organization of the β-carboxysome. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly but also provide a platform to study carboxysome shell permeability. These results will be useful in guiding the design and optimization of carboxysomes and other BMCs for introduction into plants.     

CPK13, a Noncanonical Ca2+-Dependent Protein Kinase,Specifically Inhibits KAT2 and KAT1 Shaker K+ Channels and Reduces Stomatal Opening

Ca²⁺-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca²⁺, CPKs can be sorted into three types: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K⁺ Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels.Stomata are microscopic organs at the leaf surface, each made of two so-called guard cells forming a pore. Opening or closing these pores is the way through which plants control their gas exchanges with the atmosphere (i.e. carbon dioxide uptake to feed the photosynthetic process and transpirational loss of water vapor). Stomatal movements result from osmotically driven fluxes of water, which follow massive exchanges of solutes, including K⁺ ions, between the guard cells and the surrounding tissues (Hetherington, 2001; Nilson and Assmann, 2007).Both Ca²⁺-dependent and Ca²⁺-independent signaling pathways are known to control stomatal movements (MacRobbie, 1993, 1998; Blatt, 2000; Webb et al., 2001; Mustilli et al., 2002; Israelsson et al., 2006; Marten et al., 2007; Laanemets et al., 2013). In particular, Ca²⁺ signals have been reported to promote stomatal closure through the inhibition of inward K⁺ channels and the activation of anion channels (Blatt, 1991, 1992, 2000; Thiel et al., 1992; Grabov and Blatt, 1999; Schroeder et al., 2001; Hetherington and Brownlee, 2004; Mori et al., 2006; Marten et al., 2007; Geiger et al., 2010; Brandt et al., 2012; Scherzer et al., 2012). However, little is known about the molecular identity of the links between Ca²⁺ events and Shaker K⁺ channel activity. Several kinases and phosphatases are believed to be involved in both the Ca²⁺-dependent and Ca²⁺-independent signaling pathways. Plants express two large kinase families whose activity is related to Ca²⁺ signaling. Firstly, CBL-interacting protein kinases (CIPKs; 25 genes in Arabidopsis [Arabidopsis thaliana]) are indirectly controlled by their interaction with a set of calcium sensors, the calcineurin B-like proteins (CBLs; 10 genes in Arabidopsis). This complex forms a fascinating network of potential Ca²⁺ signaling decoders (Luan, 2009; Weinl and Kudla, 2009), which have been addressed in numerous reports (Xu et al., 2006; Hu et al., 2009; Batistic et al., 2010; Held et al., 2011; Chen et al., 2013). In particular, some CBL-CIPK pairs have been shown to regulate Shaker channels such as Arabidopsis K⁺ Transporter1 (AKT1; Xu et al., 2006; Lan et al., 2011) or AKT2 (Held et al., 2011). Second, Ca²⁺-dependent protein kinases (CPKs) form an even larger family (34 genes in Arabidopsis) of proteins combining a kinase domain with the ability to bind Ca²⁺, thanks to the so-called EF hands (Harmon et al., 2000; Harper et al., 2004). CPKs, which, interestingly, are not found in animal cells, exhibit different calcium dependencies (Boudsocq et al., 2012). With respect to this, three types of CPKs can be considered: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially Ca²⁺-insensitive CPKs (however, structurally close to kinases of groups 1 and 2).Pioneering work by Luan et al. (1993) demonstrated in Vicia faba guard cells that inward K⁺ channels were regulated by some Ca²⁺-dependent kinases. Then, such a Ca²⁺-dependent kinase was purified from guard cell protoplasts of V. faba and shown to actually phosphorylate the in vitro-translated KAT1 protein, a Shaker channel subunit natively expressed in Arabidopsis guard cells (Li et al., 1998). KAT1 regulation by CPK was shown by the inhibition of KAT1 currents after the coexpression of KAT1 and CDPK from soybean (Glycine max) in oocytes (Berkowitz et al., 2000). Since then, several cpk mutant lines of Arabidopsis have been shown to be impaired in stomatal movements, for example cpk10 (Ca²⁺ insensitive), cpk4/cpk11 (Ca²⁺ dependent), and cpk3/cpk6/cpk23 (Ca²⁺ dependent; Mori et al., 2006; Geiger et al., 2010; Munemasa et al., 2011; Hubbard et al., 2012).Of the nine genes encoding voltage-dependent K⁺ channels (Shaker) in Arabidopsis (Véry and Sentenac, 2002, 2003; Lebaudy et al., 2007; Hedrich, 2012), six are expressed in guard cells and play a role in stomatal movements: the Gated Outwardly-Rectifying K⁺ (GORK) gene, encoding an outward K⁺ channel subunit, and the AKT1, AKT2, Arabidopsis K⁺ Rectifying Channel1 (AtKC1), KAT1, and KAT2 genes, encoding inward K⁺ channel subunits (Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Pandey et al., 2007; Lebaudy et al., 2008a). Shaker channels result from the assembly of four subunits, and it has been shown that inward subunits tend to heterotetramerize, thus potentially widening the functional and regulatory scope of inward K⁺ conductance in guard cells (Xicluna et al., 2007; Jeanguenin et al., 2008; Lebaudy et al., 2008a, 2010). Inhibition of inward K⁺ channels has been shown to reduce stomatal opening (Liu et al., 2000; Kwak et al., 2001). This has grounded a strategy for disrupting inward K⁺ channel conductance in guard cells by expressing a nonfunctional KAT2 subunit (dominant negative mutation) in a kat2 knockout Arabidopsis line. The resulting Arabidopsis lines, named kincless, have no functional inward K⁺ channels and exhibit delayed stomatal opening (Lebaudy et al., 2008b) with, in the long term, a biomass reduction compared with the Arabidopsis wild-type line.Among the CPKs presumably expressed in Arabidopsis guard cells (Leonhardt et al., 2004), we looked for CPK13, which belongs to the atypical Ca²⁺-insensitive type of CPKs (Kanchiswamy et al., 2010; Boudsocq et al., 2012; Liese and Romeis, 2013) and whose role remains unknown in stomatal movements. Here, we confirm first that CPK13 kinase activity is independent of Ca²⁺ and show that CPK13 expression is predominant in Arabidopsis guard cells using CPK13-GUS lines. We then report that overexpression of CPK13 in Arabidopsis induces a dramatic default in stomatal aperture. Based on the previously reported kincless phenotype (Lebaudy et al., 2008b), we propose that CPK13 could reduce the activity of inward K⁺ channels in guard cells, particularly that of KAT2. We confirm this hypothesis by voltage-clamp experiments and show an inhibition of KAT2 and KAT1 activity by CPK13 (but not that of AKT2). In addition, we present peptide array phosphorylation assays showing that CPK13 targets, with some specificity, several KAT2 and KAT1 polypeptides. Finally, we demonstrate that KAT2 and CPK13 interact in planta using Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM).     

Responses of Arabidopsis and Wheat to Rising CO2 Depend on Nitrogen Source and Nighttime CO2 Levels

A major contributor to the global carbon cycle is plant respiration. Elevated atmospheric CO₂ concentrations may either accelerate or decelerate plant respiration for reasons that have been uncertain. We recently established that elevated CO₂ during the daytime decreases plant mitochondrial respiration in the light and protein concentration because CO₂ slows the daytime conversion of nitrate (NO₃−) into protein. This derives in part from the inhibitory effect of CO₂ on photorespiration and the dependence of shoot NO₃− assimilation on photorespiration. Elevated CO₂ also inhibits the translocation of nitrite into the chloroplast, a response that influences shoot NO₃− assimilation during both day and night. Here, we exposed Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) plants to daytime or nighttime elevated CO₂ and supplied them with NO₃− or ammonium as a sole nitrogen (N) source. Six independent measures (plant biomass, shoot NO₃−, shoot organic N, ¹⁵N isotope fractionation, ¹⁵NO₃⁻ assimilation, and the ratio of shoot CO₂ evolution to O₂ consumption) indicated that elevated CO₂ at night slowed NO₃− assimilation and thus decreased dark respiration in the plants reliant on NO₃−. These results provide a straightforward explanation for the diverse responses of plants to elevated CO₂ at night and suggest that soil N source will have an increasing influence on the capacity of plants to mitigate human greenhouse gas emissions.The CO₂ concentration in Earth’s atmosphere has increased from about 270 to 400 µmol mol^–1 since 1800, and may double before the end of the century (Intergovernmental Panel on Climate Change, 2013). Plant responses to such increases are highly variable, but plant nitrogen (N) concentrations generally decline under elevated CO₂ (Cotrufo et al., 1998; Long et al., 2004). One explanation for this decline is that CO₂ inhibits nitrate (NO₃−) assimilation into protein in the shoots of C₃ plants during the daytime (Bloom et al., 2002, 2010, 2012, 2014; Cheng et al., 2012; Pleijel and Uddling, 2012; Myers et al., 2014; Easlon et al., 2015; Pleijel and Högy, 2015). This derives in part from the inhibitory effect of CO₂ on photorespiration (Foyer et al., 2009) and the dependence of shoot NO₃− assimilation on photorespiration (Rachmilevitch et al., 2004; Bloom, 2015).A key factor in global carbon budgets is plant respiration at night (Amthor, 1991; Farrar and Williams, 1991; Drake et al., 1999; Leakey et al., 2009). Nighttime elevated CO₂ may inhibit, have a negligible effect on, or stimulate dark respiration, depending on the plant species (Bunce, 2001, 2003; Wang and Curtis, 2002), plant development stage (Wang et al., 2001; Li et al., 2013), experimental approach (Griffin et al., 1999; Baker et al., 2000; Hamilton et al., 2001; Bruhn et al., 2002; Jahnke and Krewitt, 2002; Bunce, 2004), and total N supply (Markelz et al., 2014). The current study is, to our knowledge, the first to examine the influence of N source, NO₃− versus ammonium (NH₄+), on plant dark respiration at elevated CO₂ during the night.Plant organic N compounds account for less than 5% of the total dry weight of a plant, but conversion of NO₃− into organic N expends about 25% of the total energy in shoots (Bloom et al., 1989) and roots (Bloom et al., 1992). During the day, photorespiration supplies a portion of the energy (Rachmilevitch et al., 2004; Foyer et al., 2009), but at night, this energetic cost is borne entirely by the respiration of C substrates (Amthor, 1995) and may divert a substantial amount of reductant from the mitochondrial electron transport chain (Cousins and Bloom, 2004). The relative importance of NO₃− assimilation at night versus the day, however, is still a matter of intense debate (Nunes-Nesi et al., 2010). Here, we estimated NO₃− assimilation using several independent methods and show in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum), two diverse C₃ plants, that NO₃− assimilation at night can be substantial, and that elevated CO₂ at night inhibits this process.     

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

Stomata control the exchange of CO₂ and water vapor in land plants. Thus, whereas a constant supply of CO₂ is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO₂ concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.Stomata are functionally specialized microscopic pores that control the essential exchange of CO₂ and H₂O with the environment in land plants. Stomata are found on the surfaces of the majority of the aerial parts of plants, rendering them as the main control point regulating the flow of gases between plants and their surrounding atmosphere. Accordingly, the majority of water loss from plants occurs through stomatal pores, allowing plant transpiration and CO₂ absorption for the photosynthetic process (Bergmann and Sack, 2007; Kim et al., 2010). The maintenance of an adequate water balance through stomatal control is crucial to plants because cell expansion and growth require tissues to remain turgid (Sablowski and Carnier Dornelas, 2014), and minor reductions in cell water volume and turgor pressure will therefore compromise both processes (Thompson, 2005). As a result, the high sensitivity of plant tissues to turgor has prompted the use of reverse genetic studies in attempt to engineer plants with improved performance (Cowan and Troughton, 1971; Xiong et al., 2009; Borland et al., 2014; Franks et al., 2015).In most land plants, not only redox signals invoked by shifts in light quality (Busch, 2014) but also the transport of inorganic ions (e.g. K⁺, Cl⁻, and NO⁻₃) as well as metabolites such as the phytohormone abscisic acid (ABA), Suc, and malate, are important players controlling stomatal movements (Hetherington, 2001; Roelfsema and Hedrich, 2005; Pandey et al., 2007; Blatt et al., 2014; Kollist et al., 2014). In this context, although organic acids in plants is known to support numerous and diverse functions both within and beyond cellular metabolism, only recently have we obtained genetic evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal movements (Nunes-Nesi et al., 2007; Araújo et al., 2011b; Penfield et al., 2012; Medeiros et al., 2015). Notably malate, in particular, has been considered as a key metabolite and one of the most important organic metabolites involved in guard cell movements (Hedrich and Marten, 1993; Fernie and Martinoia, 2009; Meyer et al., 2010). During stomatal aperture, the flux of malate into guard cells coupled with hexoses generated on starch breakdown lead to decreases in the water potential, and consequently, water uptake by the guard cells ultimately opens the stomata pore (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lee et al., 2008). On the other hand, during stomatal closure, malate is believed to be converted into starch, which has no osmotic activity (Penfield et al., 2012) or, alternatively, is released from the guard cells to the surrounding apoplastic space (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010).The role of organic acids on the stomatal movements has been largely demonstrated by studies related to malate transport (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In the last decade, two protein families were identified and functionally characterized to be directly involved with organic acid transport at the guard cell plasma membrane and to be required for stomatal functioning (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In summary, AtABCB14, a member of the ABC (ATP binding cassette) family, which is involved in malate transport from apoplast to guard cells, was described as a negative modulator of stomatal closure induced by high CO₂ concentration; notably, exogenous application of malate minimizes this response (Lee et al., 2008). In addition, members of a small gene family, which encode the anion channels SLAC1 (slow anion channel 1) and four SLAC1-homologs (SLAHs) in Arabidopsis (Arabidopsis thaliana), have been described to be involved in stomatal movements. SLAC1 is a well-documented S-type anion channel that preferentially transports chloride and nitrate as opposed to malate (Vahisalu et al., 2008, 2010; Geiger et al., 2010; Du et al., 2011; Brandt et al., 2012; Kusumi et al., 2012). Lack of SLAC1 in Arabidopsis and rice (Oryza sativa) culminated in a failure in stomatal closure in response to high CO₂ levels, low relative humidity, and dark conditions (Negi et al., 2008; Vahisalu et al., 2008; Kusumi et al., 2012). Although mutations in AtSLAC1 impair S-type anion channel functions as a whole, the R-type anion channel remained functional (Vahisalu et al., 2008). Indeed, a member of the aluminum-activated malate transporter (ALMT) family, AtALMT12, an R-type anion channel, has been demonstrated to be involved in malate transport, particularly at the plasma membrane of guard cells (Meyer et al., 2010; Sasaki et al., 2010). Although AtALMT12 is a member of ALMT family, it is not activated by aluminum, and therefore Meyer et al. (2010) proposed to rename it as AtQUAC1 (quick-activating anion channel 1; Imes et al., 2013; Mumm et al., 2013). Hereafter, we will follow this nomenclature. Deficiency of a functional AtQUAC1 has been documented to lead to changes in stomatal closure in response to high levels of CO₂, dark, and ABA (Meyer et al., 2010). Taken together, these studies have clearly demonstrated that both S- and R-type anion channels are key modulators of stomatal movements in response to several environmental factors.Despite a vast number of studies involving the above-mentioned anion channels, little information concerning the metabolic changes caused by their impairment is currently available. Such information is important to understand stomatal movements, mainly considering that organic acids, especially the levels of malate in apoplastic/mesophyll cells, have been highlighted as of key importance in leaf metabolism (Fernie and Martinoia, 2009; Araújo et al., 2011a, 2011b; Lawson et al., 2014; Medeiros et al., 2015). Here, we demonstrate that a disruption in the expression of AtQUAC1, which leads to impaired stomatal closure (Meyer et al., 2010), was accompanied by increases in mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c). By further characterization of atquac1 knockout plants, we demonstrated that reduced diffusive limitations resulted in higher photosynthetic rates and altered respiration that, in turn, led to enhanced biomass accumulation. Overall, the results obtained are discussed both in terms of the importance of organic acid transport in plant cell metabolism and with regard to the contribution that it plays in the regulation of both stomatal function and growth.     

The Tomato odorless-2 Mutant Is Defective in Trichome-Based Production of Diverse Specialized Metabolites and Broad-Spectrum Resistance to Insect Herbivores

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) produce a wide array of volatile and nonvolatile specialized metabolites. Many of these compounds contribute to the characteristic aroma of tomato foliage and constitute a key part of the language by which plants communicate with other organisms in natural environments. Here, we describe a novel recessive mutation called odorless-2 (od-2) that was identified on the basis of an altered leaf-aroma phenotype. od-2 plants exhibit pleiotrophic phenotypes, including alterations in the morphology, density, and chemical composition of glandular trichomes. Type VI glandular trichomes isolated from od-2 leaves accumulate only trace levels of monoterpenes, sesquiterpenes, and flavonoids. Other foliar defensive compounds, including acyl sugars, glycoalkaloids, and jasmonate-regulated proteinase inhibitors, are produced in od-2 leaves. Growth of od-2 plants under natural field conditions showed that the mutant is highly susceptible to attack by an indigenous flea beetle, Epitrix cucumeris, and the Colorado potato beetle, Leptinotarsa decemlineata. The increased susceptibility of od-2 plants to Colorado potato beetle larvae and to the solanaceous specialist Manduca sexta was verified in no-choice bioassays. These findings indicate that Od-2 is essential for the synthesis of diverse trichome-borne compounds and further suggest that these compounds influence host plant selection and herbivore community composition under natural conditions.The plant epidermal surface provides a formidable protective barrier to invasion by pathogens and arthropod herbivores. Hair-like protuberances, called trichomes, are among the most conspicuous defense-related structures on the aerial epidermis of leaves, stems, and floral organs. Trichomes are typically classified morphologically as being either nonglandular or glandular. Nonglandular trichomes physically impede the movement of small arthropod herbivores on the plant surface. Molecular and ecological studies indicate that trichome density is both a highly adaptive and a functionally important trait for resistance to herbivory (Kennedy, 2003; Kivimaki et al., 2007). In-depth knowledge of the molecular mechanisms that control trichome development in Arabidopsis (Arabidopsis thaliana), which produces unicellular nonglandular trichomes, has provided significant insight into the genetic basis of variation in trichome habit (Marks, 1997; Karkkainen and Agren, 2002; Yoshida et al., 2009).In contrast to our understanding of nonglandular trichomes, much less is known about the development and ecological function of glandular trichomes, many of which are multicellular. These epidermal structures synthesize a diverse array of specialized (i.e. secondary) metabolites that exert toxic or repellent effects on myriad phytophagous animals (Kennedy, 2003; Shepherd et al., 2005; Schilmiller et al., 2008). Rupture of the cuticle upon insect contact releases gland contents, which can rapidly oxidize to form a sticky exudate that physically entraps small insects. Among the major classes of compounds involved in trichome-mediated resistance are terpenoids, alkaloids, flavonoids, and defensive proteins (Shepherd and Wagner, 2007; Schilmiller et al., 2008). Large-scale sequencing of ESTs isolated from purified glands has provided unprecedented insight into the biochemical pathways that operate in glandular trichomes (Lange et al., 2000; Aziz et al., 2005; Wang et al., 2008, 2009; Xie et al., 2008; Schilmiller et al., 2009a; Dai et al., 2010). Many key biosynthetic genes in these pathways have been identified and characterized (Iijima et al., 2004; Falara et al., 2008; Slocombe et al., 2008; Ben-Israel et al., 2009; Marks et al., 2009; Schilmiller et al., 2009a).Cultivated tomato (Solanum lycopersicum) and its wild relatives produce several different types of nonglandular and glandular trichomes on aerial tissues (Luckwill, 1943; Kang et al., 2010). The chemical composition of glandular trichomes varies significantly within and between tomato species (Antonious, 2001; Schilmiller et al., 2008; Besser et al., 2009). Acyl sugars secreted by Solanum pennellii type IV trichomes provide effective resistance to a wide range of insects (Goffreda et al., 1990; Rodriguez et al., 1993; Juvik et al., 1994). Methyl ketone and sesquiterpene derivatives produced in type VI glands of Solanum habrochaites also exert powerful toxic and repellent effects on numerous insect pests (Williams et al., 1980; Maluf et al., 2001; Antonious and Snyder, 2006). Recent studies indicate that trichomes are also an important component of induced anti-insect defenses that are regulated by the plant hormone jasmonate (JA). For example, the density of type VI trichomes on tomato leaves is regulated by the JA pathway (Li et al., 2004; Boughton et al., 2005; Peiffer et al., 2009). JA also plays a role in controlling the accumulation of defense-related terpenoids in type VI glands (Li et al., 2004; van Schie et al., 2007). Recent studies provide evidence that type VI trichomes accumulate JA and may function as sensors for detecting insect movement on the leaf surface (Peiffer et al., 2009). These collective observations highlight the importance of glandular trichomes in shaping plant-insect relations.Our current understanding of the role of trichomes in mediating S. lycopersicum interaction with arthropod herbivores comes mainly from insect bioassays performed under controlled laboratory conditions (Kennedy, 2003; Li et al., 2004; Bleeker et al., 2009; Peiffer et al., 2009; Kang et al., 2010). Much less is known about the ecological relevance of trichomes in tomato plants grown under more natural conditions in the field. Here, we report the characterization of a tomato mutant, odorless-2 (od-2), that was identified on the basis of an altered leaf-aroma phenotype. This mutant exhibits defects in the development and density of glandular trichomes. Detailed chemical analysis of isolated type VI glands showed that od-2 disrupts the production of diverse specialized metabolites, including volatile terpenes and flavonoids. Consistent with important ecological roles for these compounds in host plant selection and defense, we show that od-2 plants are highly susceptible to natural populations of insect herbivores. Our results suggest that trichome-based chemical defenses play a major role in the resistance of cultivated tomato to opportunistic herbivores and also influence herbivore community composition under natural conditions.