Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

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.     

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.     

Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Steady-State Phosphorylation of Light-Harvesting Complex II Proteins Preserves Photosystem I under Fluctuating White Light

According to the “state transitions” theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.Plant acclimation to different quantities and qualities of light has been extensively investigated. The light quality experiments have usually concerned the red/blue and far-red light acclimation strategies, which have been closely related to the state transitions and the phosphorylation of the light-harvesting complex II (LHCII) proteins, Lhcb1 and Lhcb2, by the state transition7 (STN7) kinase (Allen, 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006; Rochaix, 2007). Such studies on acclimation to different qualities of light have uncovered key mechanisms required for the maintenance of photosynthetic efficiency in dense populations and canopies (Dietzel et al., 2008). However, the role of LHCII phosphorylation under fluctuations in the quantity of white light has been scarcely investigated. Light conditions in natural environments may be very complex with respect to the quantity of white light, which constantly fluctuates both in short- and long-term durations (Smith, 1982; Külheim et al., 2002). Thus, the acclimation strategies to natural environments must concomitantly meet the challenges of both high- and low-light acclimation. Changing cloudiness, for example, would initiate both the high-light and low-light acclimation signals in the time scale of minutes and hours, whereas the movements of leaves in the wind or the rapid movement of clouds would initiate even more frequent light acclimation signals. The kinetics of reversible LHCII phosphorylation is far too slow to cope with rapid environmental changes.The phosphorylation level of LHCII proteins in the thylakoid membrane is regulated by both the STN7 kinase and the counteracting PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010). No definite results are available about regulation of the PPH1/TAP38 phosphatase, but the STN7 kinase is strongly under redox regulation (Lemeille et al., 2009) and controls the phosphorylation level of LHCII proteins under varying white light intensities as well as according to chloroplast metabolic cues, as described already decades ago (Fernyhough et al., 1983; Rintamäki et al., 2000; Hou et al., 2003). So far, research on the role of the STN7 kinase and LHCII phosphorylation in the light acclimation of higher plants has heavily focused on reversible LHCII phosphorylation and concomitant state transitions. The state 1-to-state 2 transition, by definition, means the phosphorylation of LHCII proteins, their detachment from PSII in grana membranes, and migration to the stroma membranes to serve in the collection of excitation energy to PSI (Fork and Satoh, 1986; Williams and Allen, 1987; Wollman, 2001; Rochaix, 2007; Kargul and Barber, 2008; Murata, 2009; Lemeille et al., 2010; Minagawa, 2011). Concomitantly, the absorption cross section of PSII decreases and that of PSI increases (Canaani and Malkin, 1984; Malkin et al., 1986; Ruban and Johnson, 2009). Indeed, state transitions have been well documented when different qualities (blue/red and far red) of light, preferentially exciting either PSII or PSI, have been applied.Different from state transitions, the white light intensity-dependent reversible LHCII phosphorylation does not result in differential excitation of the two photosystems (Tikkanen et al., 2010). Instead, both photosystems remain nearly equally excited independently whether the LHCII proteins are heavily phosphorylated or strongly dephosphorylated. Moreover, it is worth noting that the different qualities of light generally used to induce reversible LHCII phosphorylation and state transitions (blue/red and far-red lights) have usually been of very low intensity (for review, see Haldrup et al., 2001), and apparently, minimal protonation of the lumen takes place under such illumination conditions. Yet another difference between induction of LHCII protein phosphorylation by different qualities of light or different quantities of white light concerns the concomitant induction of PSII core protein phosphorylation. In the former case, the level of PSII core protein phosphorylation follows the phosphorylation pattern of LHCII proteins, whereas under different quantities of white light, the phosphorylation behavior of PSII core and LHCII proteins is the opposite (Tikkanen et al., 2008b).To gain a more comprehensive understanding of the physiological role of white light-induced changes in LHCII protein phosphorylation, we have integrated Arabidopsis (Arabidopsis thaliana) LHCII phosphorylation with other light-dependent regulatory modifications of light harvesting and electron transfer in the thylakoid membrane, which include the nonphotochemical quenching of excitation energy (for review, see Niyogi, 1999; Horton and Ruban, 2005; Barros and Kühlbrandt, 2009; de Bianchi et al., 2010; Jahns and Holzwarth, 2012; Ruban et al., 2012) and the photosynthetic control of electron transfer by the cytochrome b₆f (Cytb6f) complex (Rumberg and Siggel, 1969; Witt, 1979; Tikhonov et al., 1981; Bendall, 1982; Nishio and Whitmarsh, 1993; Joliot and Johnson, 2011; Suorsa et al., 2012; for review, see Foyer et al., 1990, 2012), both strongly dependent on lumenal protonation.It is demonstrated that the steady-state LHCII phosphorylation is particularly important under rapidly fluctuating light (FL) conditions. This ensures equal energy distribution to both photosystems, prevents the accumulation of electrons in the intersystem electron transfer chain (ETC), eliminates perturbations in chloroplast redox balance, and maintains PSI functionality upon rapid fluctuations in white light intensity.     

HAPLESS13, the Arabidopsis μ1 Adaptin,Is Essential for Protein Sorting at the trans-Golgi Network/Early Endosome

In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The µ1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis µ1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.Plant cells contain sophisticated endomembrane compartments, including the endoplasmic reticulum, the Golgi, the trans-Golgi network (TGN)/early endosome (EE), the prevacuolar compartments/multivesicular bodies (PVC/MVB), various types of vesicles, and the plasma membrane (PM; Ebine and Ueda, 2009; Richter et al., 2009). Intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes (Richter et al., 2009; De Marcos Lousa et al., 2012). Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the TGN/EE, while vesicles in the endocytic route start from the PM before reaching the TGN/EE (Dhonukshe et al., 2007; Viotti et al., 2010). The TGN/EE in Arabidopsis (Arabidopsis thaliana) is an independent and highly dynamic organelle transiently associated with the Golgi (Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010), distinct from the animal TGN. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM (Ebine and Ueda, 2009; Richter et al., 2009). Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Fine-tuned control of intracellular protein sorting at the TGN/EE is essential for plant development (Geldner et al., 2003; Dhonukshe et al., 2007, 2008; Richter et al., 2007; Kitakura et al., 2011; Wang et al., 2013). An auxin gradient is crucial for pattern formation in plants, whose dynamic maintenance requires the polar localization of auxin efflux carrier PINs through endocytic recycling (Geldner et al., 2003; Blilou et al., 2005; Paciorek et al., 2005; Abas et al., 2006; Jaillais et al., 2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008). Receptor-like kinases (RLKs) have also been recognized as major cargos undergoing endocytic trafficking, which are either recycled back to the PM or sent for vacuolar degradation (Geldner and Robatzek, 2008; Irani and Russinova, 2009). RLKs are involved in most if not all developmental processes of plants (De Smet et al., 2009).Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals (Boehm and Bonifacino, 2001; Robinson, 2004; Dhonukshe et al., 2007). Adaptor proteins (AP) play a key role (Boehm and Bonifacino, 2001; Robinson, 2004) in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits (β and γ/α/δ/ε), a small subunit (σ), and a medium subunit (µ) that is crucial for cargo selection (Boehm and Bonifacino, 2001). APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers (Boehm and Bonifacino, 2001). Five APs have been identified so far, classified by their components, subcellular localization, and function (Boehm and Bonifacino, 2001; Robinson, 2004; Hirst et al., 2011). Of the five APs, AP-1 associates with the TGN or recycling endosomes (RE) in yeast and mammals (Huang et al., 2001; Robinson, 2004), mediating the sorting of cargo proteins to compartments of the endosomal-lysosomal system or to the basolateral PM of polarized epithelial cells (Gonzalez and Rodriguez-Boulan, 2009). Knockouts of AP-1 components in multicellular organisms resulted in embryonic lethality (Boehm and Bonifacino, 2001; Robinson, 2004).We show here that the recently identified Arabidopsis µ1 adaptin AP1M2 (Park et al., 2013; Teh et al., 2013) is a key component in the cellular machinery mediating intracellular protein sorting at the TGN/EE. AP1M2 was previously named HAPLESS13 (HAP13), whose mutant allele hap13 showed male gametophytic lethality (Johnson et al., 2004). In recent quests for AP-1 in plants, HAP13/AP1M2 was confirmed as the Arabidopsis µ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN (Park et al., 2013; Teh et al., 2013). A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis (Park et al., 2013; Teh et al., 2013). However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences. Using the same mutant allele, we found that functional loss of HAP13 (hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling. Cell morphogenesis was also disturbed in hap13-1. Importantly, hap13-1 was insensitive to brefeldin A (BFA) washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation factor (ArfGEF)-mediated post-Golgi trafficking. Furthermore, HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a µ1 adaptin. These results demonstrate the importance of the Arabidopsis µ1 adaptin for intracellular protein sorting centered on the TGN/EE.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.