A Non-Classical Member of the Protein Disulfide Isomerase Family, PDI7 of Arabidopsis thaliana, Localizes to the cis-Golgi and Endoplasmic Reticulum Membranes.

Members of the protein disulfide isomerase (PDI)-C subfamily are chimeric proteins containing the thioredoxin (Trx) domain of PDIs, and the conserved N- and C-terminal Pfam domains of Erv41p/Erv46p-type cargo receptors. They are unique to plants and chromalveolates. The Arabidopsis genome encodes three PDI-C isoforms: PDI7, PDI12 and PDI13. Here we demonstrate that PDI7 is a 65 kDa integral membrane glycoprotein expressed throughout many Arabidopsis tissues. Using a PDI7-specific antibody, we show through immunoelectron microscopy that PDI7 localizes to the endoplasmic reticulum (ER) and Golgi membranes in wild-type root tip cells, and was also detected in vesicles. Tomographic modeling of the Golgi revealed that PDI7 was confined to the cis-Golgi, and accumulated primarily at the cis-most cisterna. Shoot apical meristem cells from transgenic plants overexpressing PDI7 exhibited a dramatic increase in anti-PDI7 labeling at the cis-Golgi. When N- or C-terminal fusions between PDI7 and the green fluorescent protein variant, GFP(S65T), were expressed in mesophyll protoplasts, the fusions co-localized with the ER marker, ER-mCherry. However, when GFP(S65T) was positioned internally within PDI7 (PDI7-GFPint), the fusion strongly co-localized with the cis-Golgi marker, mCherry-SYP31, and faintly labeled the ER. In contrast to the Golgi-resident fusion protein (Man49-mCherry), PDI7-GFPint did not redistribute to the ER after brefeldin A treatment. Protease protection experiments indicated that the Trx domain of PDI7 is located within the ER/Golgi lumen. We propose a model where PDI-C isoforms function as cargo receptors for proteins containing exposed cysteine residues, cycling them from the Golgi back to the ER.

Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity.

BACKGROUND: In eukaryotes, classical protein disulfide isomerases (PDIs) facilitate the oxidative folding of nascent secretory proteins in the endoplasmic reticulum by catalyzing the formation, breakage, and rearrangement of disulfide bonds. Terrestrial plants encode six structurally distinct subfamilies of PDIs. The novel PDI-B subfamily is unique to terrestrial plants, and in Arabidopsis is represented by a single member, PDI8. Unlike classical PDIs, which lack transmembrane domains (TMDs), PDI8 is unique in that it has a C-terminal TMD and a single N-terminal thioredoxin domain (instead of two). No PDI8 isoforms have been experimentally characterized to date. Here we describe the characterization of the membrane orientation, expression, sub-cellular localization, and biochemical function of this novel member of the PDI family. RESULTS: Histochemical staining of plants harboring a PDI8 promoter:ß-glucuronidase (GUS) fusion revealed that the PDI8 promoter is highly active in young, expanding leaves, the guard cells of cotyledons, and in the vasculature of several organs, including roots, leaves, cotyledons, and flowers. Immunoelectron microscopy studies using a PDI8-specific antibody on root and shoot apical cells revealed that PDI8 localizes to the endoplasmic reticulum (ER). Transient expression of two PDI8 fusions to green fluorescent protein (spGFP-PDI8 and PDI8-GFP-KKED) in leaf mesophyll protoplasts also resulted in labeling of the ER. Protease-protection immunoblot analysis indicated that PDI8 is a type I membrane protein, with its catalytic domain facing the ER lumen. The lumenal portion of PDI8 was able to functionally complement the loss of the prokaryotic protein foldase, disulfide oxidase (DsbA), as demonstrated by the reconstitution of periplasmic alkaline phosphatase in Escherichia coli. CONCLUSION: The results indicate that PDI8 is a type I transmembrane protein with its catalytic domain facing the lumen of the ER and functions in the oxidation of cysteines to produce disulfide bonds. It likely plays a role in folding newly-synthesized secretory proteins as they translocate across the ER membrane into the lumen. These foundational results open the door to identifying the substrates of PDI8 to enable a more thorough understanding of its function in plants.

Phylogenetic characterization and promoter expression analysis of a novel hybrid protein disulfide isomerase/cargo receptor subfamily unique to plants and chromalveolates.

Protein disulfide isomerases (PDIs) play critical roles in protein folding by catalyzing the formation and rearrangement of disulfide bonds in nascent secretory proteins. There are six distinct PDI subfamilies in terrestrial plants. A unique feature of PDI-C subfamily members is their homology to the yeast retrograde (Golgi-to-endoplasmic reticulum) cargo receptor proteins, Erv41p and Erv46p. Here, we demonstrate that plant Erv41p/Erv46p-like proteins are divided into three subfamilies: ERV-A, ERV-B and PDI-C, which all possess the N-proximal and C-proximal conserved domains of yeast Erv41p and Erv46p. However, in PDI-C isoforms, these domains are separated by a thioredoxin domain. The distribution of PDI-C isoforms among eukaryotes indicates that the PDI-C subfamily likely arose through an ancient exon-shuffling event that occurred before the divergence of plants from stramenopiles and rhizarians. Arabidopsis has three PDI-C genes: PDI7, PDI12, and PDI13. PDI12- and PDI13-promoter: ß-glucuronidase (GUS) gene fusions are co-expressed in pollen and stipules, while PDI7 is distinctly expressed in the style, hydathodes, and leaf vasculature. The PDI-C thioredoxin domain active site motif CxxS is evolutionarily conserved among land plants. Whereas PDI12 and PDI13 retain the CxxS motif, PDI7 has a CxxC motif similar to classical PDIs. We hypothesize that PDI12 and PDI13 maintain the ancestral roles of PDI-C in Arabidopsis, while PDI7 has undergone neofunctionalization. The unusual PDI/cargo receptor hybrid arrangement in PDI-C isoforms has no counterpart in animals or yeast, and predicts the need for pairing redox functions with cargo receptor processes during protein trafficking in plants and other PDI-C containing organisms.

Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in Arabidopsis.

Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the Arabidopsis PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. In vitro and in vivo, PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the pdi9 mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the ire1a-ire1b mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.

Development of a GFP biosensor reporter for the unfolded protein response-signaling pathway in plants: incorporation of the bZIP60 intron into the GFP gene.

The ability to measure the activation of the unfolded protein response (UPR) in plants is important when they are exposed to stressful environments. To this end, we developed a unique and versatile biosensor-reporter system to indicate the activation of UPR in living plant cells. The small cytoplasmically spliced intron from the bZIP60 locus was incorporated into the 5' end of the GFP gene, creating the 35S::bZIP60 intron:GFP construct. When this construct is transiently expressed in Arabidopsis protoplasts, the presence of the bZIP60 intron prevents GFP mRNA from being translated under non-UPR conditions. However, when UPR is activated, the IRE1 kinase/ribonuclease splices this intron from the GFP mRNA and its translation proceeds, generating GFP fluorescence. We demonstrated the utility of the system in Arabidopsis leaf protoplasts treated with DTT, which is a chemical inducer of UPR, followed by visualization and quantification using confocal microscopy. The 35S::bZIP60 intron:GFP construct was also expressed in protoplasts from an overexpressor line containing the coding sequence for the UPR-induced, protein folding chaperone, protein disulfide isomerase-9 (PDI9). PDI9 also influences the strength of the UPR signaling pathway. Protoplasts from WT and PDI9 overexpressor plants treated with DTT exhibited significantly higher GFP fluorescence relative to untreated protoplasts, indicating that the bZIP60 intron was spliced from the GFP mRNA in response to activation of UPR. RT-PCR further confirmed the higher induction of PDI9 and bZIP60 (total and spliced) mRNA levels in DTT-treated protoplasts relative to controls. This system can be adapted for monitoring crop stress and for basic studies dissecting the UPR signaling pathway.

The Use of Fluorescent Protein Fusions to Monitor the Unfolded Protein Response and Protein Foldase-Substrate Interactions in Plant Protoplasts.

Endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR) are critical stress response pathways in eukaryotes. To study these types of interactions in plants, a wide range of methods have been used, including generation of transgenic plants, subcellular immunolocalization of protein foldases, and co-immunoprecipitation (co-IP) assays. Although these more time-consuming methods have been successfully implemented, there is a need for a versatile and rapid in vivo system to investigate ER stress and UPR. Here, we describe a transient expression system that uses plant protoplasts to define in vivo subcellular localizations and protein-protein interactions of protein foldases and their substrates fused to fluorescent protein reporters. This accurate and robust assay utilizes a variety of analyses, such as subcellular localization, FLIM-FRET, co-IP, mutagenesis, and RT-PCR in the genetically amenable Arabidopsis model system. We demonstrate the methodology by using the representative protein foldase, protein disulfide isomerase-9 (PDI9), as well as subcellular markers, secretory proteins, and dithiothreitol (DTT)-mediated induction of the UPR as monitored by RT-PCR. Together, these methods yield reliable high output results for investigating subcellular localization and protein-protein interactions in plants to decipher the UPR pathways.

The cyclic nucleotide-gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis.

The suppression of the cyclic nucleotide-gated channel (CNGC) AtCNGC10 alters K(+) transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca(2+), K(+), Li(+), Cs(+) and Rb(+) across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K(+) in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non-invasive ion-specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H(+), Ca(2+) and Mg(2+) in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca(2+) influx from the external media corresponded to a lower intracellular Ca(2+) activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H(+) influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K(+) and lower Ca(2+) and Mg(2+) content in the shoots, indicating disturbed long-distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K(+), Ca(2+) and Mg(2+) concentrations in the solution led to altered K(+), Ca(2+) and Mg(2+) content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca(2+) (and probably Mg(2+)) transport in plants, which in turn regulates K(+) transporters' activities.

The Arabidopsis Protein Disulfide Isomerase Subfamily M Isoform, PDI9, Localizes to the Endoplasmic Reticulum and Influences Pollen Viability and Proper Formation of the Pollen Exine During Heat Stress.

Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.

Novel thigmomorphogenetic responses in Carica papaya: touch decreases anthocyanin levels and stimulates petiole cork outgrowths.

BACKGROUND AND AIMS: Because of its rapid growth rate, relative ease of transformation, sequenced genome and low gene number relative to Arabidopsis, the tropical fruit tree, Carica papaya, can serve as a complementary genetic model for complex traits. Here, new phenotypes and touch-regulated gene homologues have been identified that can be used to advance the understanding of thigmomorphogenesis, a multigenic response involving mechanoreception and morphological change. METHODS: Morphological alterations were quantified, and microscopy of tissue was conducted. Assays for hypocotyl anthocyanins, lignin and chlorophyll were performed, and predicted genes from C. papaya were compared with Arabidopsis touch-inducible (TCH) and Mechanosensitive channel of Small conductance-like genes (MscS-like or MSL). In addition, the expression of two papaya TCH1 homologues was characterized. KEY RESULTS: On the abaxial side of petioles, treated plants were found to have novel, hypertrophic outgrowths associated with periderm and suberin. Touched plants also had higher lignin, dramatically less hypocotyl anthocyanins and chlorophyll, increased hypocotyl diameter, and decreased leaf width, stem length and root fresh weight. Papaya was found to have fewer MSL genes than Arabidopsis, and four touch-regulated genes in Arabidopsis had no counterparts in papaya. Water-spray treatment was found to enhance the expression of two papaya TCH1 homologues whereas induction following touch was only slightly correlated. CONCLUSIONS: The novel petiole outgrowths caused by non-wounding, mechanical perturbation may be the result of hardening mechanisms, including added lignin, providing resistance against petiole movement. Inhibition of anthocyanin accumulation following touch, a new phenotypic association, may be caused by diversion of p-coumaroyl CoA away from chalcone synthase for lignin synthesis. The absence of MSL and touch-gene homologues indicates that papaya may have a smaller set of touch-regulated genes. The genes and novel touch-regulated phenotypes identified here will contribute to a more comprehensive view of thigmomorphogenesis in plants.

Light enhances the unfolded protein response as measured by BiP2 gene expression and the secretory GFP-2SC marker in Arabidopsis.

Disruption of the protein-folding capacity in the ER induces the accumulation of unfolded proteins and ER stress, which activate the unfolded protein response (UPR). Although UPR has been extensively studied in yeast and mammals, much less is known about UPR and its relationship with light in plants. Here, we examined the effects of chemically induced UPR and light on a molecular marker of UPR (binding protein, BiP2, gene expression) and a secretory green fluorescent protein marker (GFP-2SC) that is trafficked from the ER to vacuole in Arabidopsis thaliana (L). UPR, which was induced by DTT and tunicamycin (TM), increased Bip2 mRNA levels and decreased the levels of microsomal and vacuolar forms of GFP-2SC. Treatment with protease inhibitors lessened the effects of DTT and TM on GFP-2SC, indicating the decrease in GFP levels partially involved protein degradation. Light treatments synergistically enhanced the decrease in GFP levels in both the ER and vacuole and induced the expression of UPR marker genes for BiP2 and protein disulfide isomerase (PDI, EC 5.3.4.1). DTT and TM treatments required light for maximal induction of the UPR. Light-induced UPR occurred during the daily dark to light cycle and when dark-adapted plants were exposed to light. We propose that light activates the UPR to increase the protein folding capacity in the ER to accommodate an increase in translation during dark to light transitions.

The cyclic nucleotide-gated channel, AtCNGC10, influences salt tolerance in Arabidopsis.

Cyclic nucleotide-gated channels (CNGCs) in the plasma membrane transport K+ and other cations; however, their roles in the response and adaptation of plants to environmental salinity are unclear. Growth, cation contents, salt tolerance and K+ fluxes were assessed in wild-type and two AtCNGC10 antisense lines (A2 and A3) of Arabidopsis thaliana (L.) Heynh. Compared with the wild-type, mature plants of both antisense lines had altered K+ and Na+ concentrations in shoots and were more sensitive to salt stress, as assessed by biomass and Chl fluorescence. The shoots of A2 and A3 plants contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared with wild-type, whereas roots contained higher K+ concentrations and lower Na+/K+ ratios. Four-day-old seedlings of both antisense lines exposed to salt stress had smaller Na+/K+ ratios and longer roots than the wild-type. Under sudden salt treatment, the Na+ efflux was higher and the K+ efflux was smaller in the antisense lines, indicating that AtCNGC10 might function as a channel providing Na+ influx and K+ efflux at the root/soil interface. We conclude that the AtCNGC10 channel is involved in Na+ and K+ transport during cation uptake in roots and in long-distance transport, such as phloem loading and/or xylem retrieval. Mature A2 and A3 plants became more salt sensitive than wild-type plants because of impaired photosynthesis induced by a higher Na+ concentration in the leaves.

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells.

BACKGROUND: The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. RESULTS: AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50-150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. CONCLUSION: AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.

Dual protein trafficking to secretory and non-secretory cell compartments: clear or double vision?

Approximately 18% of Arabidopsis thaliana proteins encode a signal peptide for translocation to the endoplasmic reticulum (ER), the gateway of the eukaryotic secretory pathway. However, it was recently discovered that some ER proteins can undergo both co-translational import into the ER/secretory pathway and trafficking to compartments outside of the secretory pathway. This phenomenon is observed among members of the protein disulfide isomerase (PDI) family, which are traditionally regarded as ER enzymes involved in protein folding. Although classical PDIs possess an N-terminal signal peptide and a C-terminal ER retention signal, some also dual localize to secretory and non-secretory compartments, including mammalian PDI ERp57, Chlamydomonas reinhardtii PDI RB60, and A. thaliana AtPDI2. ERp57 is present in both the ER and nucleus where it influences gene transcription. RB60 localizes to the ER and chloroplast where it modulates the redox state of polyadenylate-binding protein RB47. AtPDI2, which interacts with transcription factor MEE8, localizes to the ER-secretory pathway and the nucleus. A model proposing secretory trafficking of AtPDI2 and nuclear co-translocation of an AtPDI2-MEE8 complex illustrates the diversity of dual targeting mechanisms, the multifunctional roles of some PDIs, and the potential co-translocation of other proteins to multiple subcellular compartments.

Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase.

Protein disulfide isomerases (PDIs) catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER). Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a') and two non-catalytic domains (b, b'), in the order a-b-b'-a'. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6). Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11) possess two thioredoxin domains, but without intervening b-b' domains. C-terminal green fluorescent protein (GFP) fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA-. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6) functionally complemented dsbA-. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA-. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

Protein disulfide isomerase-2 of Arabidopsis mediates protein folding and localizes to both the secretory pathway and nucleus, where it interacts with maternal effect embryo arrest factor.

Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXC-containing thioredoxin catalytic sites (a,a'), two noncatalytic thioredoxin fold domains (b,b'), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein-folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2-GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP- and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus.

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.

Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana.

Proteins entering the secretory pathway of eukaryotic cells are folded into their native structures in the endoplasmic reticulum (ER). Disruption of protein folding causes ER stress and activates signaling cascades, designated the unfolded protein response (UPR), that restore folding capacity. In mammals and yeast, the protein disulfide isomerases (PDIs) are key protein folding catalysts activated during UPR. However, little is known about the response of PDI genes to UPR in plants. In Arabidopsis thaliana, we identified 12 PDI genes that differed in polypeptide length, presence of signal peptide and ER retention signal, and the number and positions of thioredoxin and transmembrane domains. AtPDI gene expression was investigated in different tissues, in response to chemically induced UPR, and in null mutants of UPR signaling mediators (AtIRE1-2 and AtbZIP60). The expression of six AtPDI genes was significantly up-regulated by UPR and sharply attenuated by the transcription inhibitor, actinomycin D, indicating UPR induced AtPDI gene transcription. AtPDI and BIP2 (Binding protein) gene expression was not affected in the Atire1-2 mutant exposed to UPR, however, the expression of four AtPDI genes was decreased in the Atbzip60 mutant. We proposed that additional UPR signaling factors complement AtbZIP60 in the activation of AtPDI gene expression during ER stress in plants.

Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds.

Protein disulfide isomerase (PDI) oxidizes, reduces, and isomerizes disulfide bonds, modulates redox responses, and chaperones proteins. The Arabidopsis thaliana genome contains 12 PDI genes, but little is known about their subcellular locations and functions. We demonstrate that PDI5 is expressed in endothelial cells about to undergo programmed cell death (PCD) in developing seeds. PDI5 interacts with three different Cys proteases in yeast two-hybrid screens. One of these traffics together with PDI5 from the endoplasmic reticulum through the Golgi to vacuoles, and its recombinant form is functionally inhibited by recombinant PDI5 in vitro. Peak PDI5 expression in endothelial cells precedes PCD, whereas decreasing PDI5 levels coincide with the onset of PCD-related cellular changes, such as enlargement and subsequent collapse of protein storage vacuoles, lytic vacuole shrinkage and degradation, and nuclear condensation and fragmentation. Loss of PDI5 function leads to premature initiation of PCD during embryogenesis and to fewer, often nonviable, seeds. We propose that PDI5 is required for proper seed development and regulates the timing of PCD by chaperoning and inhibiting Cys proteases during their trafficking to vacuoles before PCD of the endothelial cells. During this transitional phase of endothelial cell development, the protein storage vacuoles become the de facto lytic vacuoles that mediate PCD.

The cyclic nucleotide-gated calmodulin-binding channel AtCNGC10 localizes to the plasma membrane and influences numerous growth responses and starch accumulation in Arabidopsis thaliana.

Cyclic nucleotide gated channels (CNGCs) that are regulated by calmodulin (CaM) have been shown to play essential roles in signal transduction, metabolism, and growth in animals. By contrast, very little is known about the subcellular location and the function of these channels in plants. Here we report on the effects of antisense suppression of the expression of AtCNGC10, a putative K+ channel, and the immunolocalization of the protein using an AtCNGC10-specific antiserum. In Arabidopsis thaliana leaves, AtCNGC10 was localized to the plasma membrane of mesophyll and parenchyma cells. Antisense AtCNGC10 plants had 40% of the AtCNGC10 mRNA levels and virtually undetectable protein levels relative to wild type plants. Antisense expression of AtCNGC10 did not affect the mRNA levels of AtCNGC13, the most closely related CNGC family member in the genome. Relative to wild type Columbia, antisense AtCNGC10 plants flowered 10 days earlier, and had a 25% reduction in leaf surface area, thickness and palisade parenchyma cell length. Their roots responded more slowly to gravitropic changes and the chloroplasts accumulated more starch. We propose that AtCNGC10, through interactions with CaM and cGMP, modulates cellular K+ balance across the plasma membrane, and that perturbations of this K+ gradient affect numerous growth and developmental processes.