A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh

Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast biogenesis, differentiation, and senescence in sweet orange flesh.Chromoplasts are special organelles with superior ability to synthesize and store massive amounts of carotenoids, bringing vivid red, orange, and yellow colors to many flowers, fruits, and vegetables (Li and Yuan, 2013). Chromoplasts exhibit various morphologies, such as crystalline, globular, tubular, and membranous structures (Egea et al., 2010). The relationship between the architecture and carotenoid composition has been well stated in diverse pepper (Capsicum annuum) and tomato (Solanum lycopersicum) fruits (Kilcrease et al., 2013; Nogueira et al., 2013). Crystalline bodies have been observed in carrot (Daucus carota; Frey-Wyssling and Schwegler, 1965) and tomato (Harris and Spurr, 1969), which predominantly consist of β-carotene and lycopene, respectively. Globular and/or tubular-globular chromoplasts, in which numerous lipid droplets (also named plastoglobules), which act as passive storage compartments for triglycerides, sterol ester, and some pigments, are accumulated, were described for yellow fruits from kiwi (Actinidia deliciosa), papaya (Carica papaya), and mango (Mangifera indica), which contain lutein, β-cryptoxanthin, and β-carotene as the major pigments, respectively (Vasquez-Caicedo et al., 2006; Montefiori et al., 2009; Schweiggert et al., 2011). Carotenoid composition has been reported to be regulated by the expression of carotenogenic genes in the flesh of various citrus fruits differing in their internal colors (Fanciullino et al., 2006, 2008). Chromoplasts are frequently derived from fully developed chloroplasts, as seen during fruit ripening from green to red or yellow fruits in tomato and pepper (Egea et al., 2010). In some cases, chromoplasts also arise from nonphotosynthetic plastids, such as colorless proplastids, leucoplasts, or amyloplasts (Knoth et al., 1986; Schweiggert et al., 2011). To date, most studies on chromoplast differentiation have been focused on the synthesis of carotenoids by combining biochemical and molecular analyses (Cazzonelli and Pogson, 2010; Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013), and little is known about the molecular mechanisms underlying chromoplast biogenesis (Li and Yuan, 2013).Recently, proteomics has become an efficient tool to study the protein composition of subcellular organelles such as chromoplasts and their dynamic changes during the development of a particular plant organ/tissue. The majority of chromoplast-related studies are concerned with the functions of these organelles in various crops, such as pepper, tomato, watermelon (Citrulis lanatus), carrot, cauliflower (Brassica oleracea), and papaya (Siddique et al., 2006; Wang et al., 2013). However, only a few of such studies addressed the mechanisms underlying plastid differentiation, such as the transition from proplastid to chloroplast in maize (Zea mays; Majeran et al., 2010), from etioplast to chloroplast in pea (Pisum sativum; Kanervo et al., 2008) and rice (Oryza sativa; Kleffmann et al., 2007), and from chloroplast to chromoplast in tomato (Barsan et al., 2012). In tomato, chromoplastogenesis appears to be associated with major metabolic shifts, including a strong decrease in abundance of the proteins involved in light reaction and an increase in terpenoid biosynthesis and stress-response proteins (Barsan et al., 2012). These changes in proteins are in agreement with the structural changes occurring in tomato during fruit ripening, which is characterized by the loss of chlorophyll and the synthesis of colored compounds. Chromoplast differentiation from nonphotosynthetic plastids occurs frequently in a number of plant tissues, such as watermelon flesh and carrot root (Kim et al., 2010; Wang et al., 2013). However, to the best of our knowledge, no large-scale proteomic study for understanding this developmental process has been reported.Citrus is one of the most economically important fruit crops in the world. Different from the model fruit tomato, which represents climacteric fruits, citrus shows nonclimacteric characteristics during fruit maturation. Additionally, citrus fruits exhibit a unique anatomical fruit structure consisting of two major sections, the pericarp and the edible flesh. Considerable progress has been made in the understanding of chromoplast differentiation in the pericarp of citrus fruits (Eilati et al., 1969; Iglesias et al., 2007), which is a process similar to that of tomato and pepper (Egea et al., 2010). However, little is known about the molecular basis of chromoplast differentiation in the edible flesh, even though there is increasing evidence suggesting an essential role of carotenoid synthesis in inducing chromoplast differentiation (Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013). Recently, we successfully isolated and purified intact chromoplasts containing a large number of plastoglobules from the flesh of sweet orange (Citrus sinensis) fruits at the maturation stage (Zeng et al., 2011). The same method has also been used successfully to isolate plastids from sweet orange flesh in earlier maturation stages (Zeng et al., 2014), thus making comparative and quantitative proteomic analyses of plastid differentiation possible. In this study, we investigated how ultrastructural changes of plastids/chromoplasts during sweet orange fruit maturation might be associated with changes in the composition of carotenoids and the expression of carotenogenic genes in red and yellow flesh of the fruits. Furthermore, we employed the isobaric tag for relative and absolute quantitation (iTRAQ)-based technology to investigate how protein compositional changes might be correlated with metabolic and structural changes in the plastids of sweet orange flesh during their transformation from amyloplasts to chromoplasts.     

An Extended AE-Rich N-Terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed during Ripening

Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation.Phytocystatins are Cys protease inhibitors from plants that reside in the cystatin superfamily and contain a distinctive α-helix-forming sequence, [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N, in the main body (Margis et al., 1998). The most investigated phytocystatin is rice (Oryza sativa) oryzacystatin I (OC-I; Abe et al., 1987). Its three-dimensional structure (Nagata et al., 2000) resembles the structure of chicken egg white cystatin (Bode et al., 1988). These structural features of OC-I include a five-stranded antiparallel β-pleated sheet, which is wrapped around the α-helix. Two regions are predicted to reversibly bind to the active site of papain-like Cys proteases. They are the highly conserved QxVxG motif that is situated on a loop between the second and third β-strand and a conserved W on a loop between the fourth and fifth β-strand (Arai et al., 1991; Urwin et al., 1995). A conserved G immediately precedes the main body at the N terminus. The region preceding the conserved G is referred to as the N-terminal trunk (NTT) and has been shown to interact with Cys protease (Machleidt et al., 1989; Björk et al., 1995; Girard et al., 2007), but the role of the NTT in phytocystatins is less clear.Although the NTT of OC-I did not affect the inhibition of papain (Abe et al., 1988; Chen et al., 1992), the NTTs of other phytocystatins were subsequently shown to modulate the binding affinities to various enzymes (Urwin et al., 1995; Kiggundu et al., 2006). Some phytocystatins were predicted to possess an N-terminal signal peptide for transport into the lumen of the endoplasmic reticulum and/or a C-terminal extension, which may be involved in binding legumain-type Cys proteases (Lim et al., 1996; Womack et al., 2000; Martínez et al., 2005a, 2007; Abraham et al., 2006; Gianotti et al., 2006). Other phytocystatins, designated multicystatins, contain multiple copies of the main body (Kouzuma et al., 2000; Diop et al., 2004; Christova et al., 2006; Girard et al., 2007).Phytocystatins function in diverse biological processes, such as protein turnover during seed development and germination (Kuroda et al., 2001; Martínez et al., 2005c; Abraham et al., 2006; Kiyosaki et al., 2007; Valdés-Rodríguez et al., 2007), organogenesis (Corre-Menguy et al., 2002; Massonneau et al., 2005; Rivard et al., 2007), programmed cell death (Beers et al., 2000; Belenghi et al., 2003), fruit development (Ryan et al., 1998), and defense against a variety of pests and pathogens (Koiwa et al., 2000; Gholizadeh et al., 2005; Christova et al., 2006; Girard et al., 2007). Thus, phytocystatins inhibit both endogenous and exogenous Cys proteases. It is expected that cystatins have a high affinity for their endogenous cognate targets because they have coevolved functionally in the same cellular environment and the cystatin could control potentially damaging proteolytic activity (Otlewski et al., 2005). Similarly, exogenous targets require effective inhibitor-enzyme binding to confer resistance upon pathogen/herbivore attack (Kiggundu et al., 2006). The identification of natural targets of phytocystatins and the elucidation of their regulatory mechanisms are critical to improve our understanding of their roles in plants and for the development of practical applications (Urwin et al., 1997; Arai et al., 1998; Lilley et al., 2004).In pineapple (Ananas comosus), four major Cys proteases have been identified. They are the stem (Ritonja et al., 1989) and fruit bromelains (Yamada et al., 1976; Rowan et al., 1990) and unique ananain (Lee et al., 1997) and comosain (Rowan et al., 1990). Stem and fruit bromelains are encoded by distinct genes (Harrach et al., 1998; Jung et al., 2008) and share 68% sequence identity. They both contain signal peptides for entering the secretory pathway and propeptides for intramolecular inhibition and assisting protein folding. However, the primary species of bromelains that accumulate in plant cells have the propeptide removed (Yamada et al., 1976; Ritonja et al., 1989). Due to their broad substrate specificity and strong proteolytic activity, pineapple Cys proteases have become of considerable economical importance in the food and pharmaceutical industry (Rowan et al., 1990; Maurer, 2001). Fruit and stem bromelains are highly abundant and have been extensively studied (Vanhoof and Cooreman, 1997). Only kiwifruit (Actinidia deliciosa) cystatin has some inhibitory effect on stem bromelain (Rasaam and Laing, 2004). Here, we analyzed a ubiquitously expressed pineapple cystatin, AcCYS1, that we found to be secreted to the apoplast. AcCYS1 is unusual in that it contains an extended NTT of 63 residues that is rich in Ala and Glu. We showed that the NTT is important for complete inhibition of fruit and stem bromelain in the picomolar range and it is cleaved upon fruit ripening. Based on in vitro inhibition analysis against fruit and stem bromelain of three different species of AcCYS1, differing in the length of their NTT, we hypothesize that the cleavage of the NTT enhances the proteolytic activity of fruit bromelain during fruit ripening and senescence.     

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.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.