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.

Carica papaya L. is an economically significant crop in tropical and subtropical regions, with a gross production value of 6.2 × 109 in 2020. However, various biotic and abiotic stresses threaten crop productivity. To enhance stress resistance, genetic engineering and traditional breeding have been employed. Unfortunately, these methods are limited by the scarcity of innate disease resistance genes in the genome and the poor fertility of interspecific hybrids. Therefore, to circumvent these limitations, we developed a papaya protoplast-based gene editing system. By optimizing protoplast isolation, 28

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 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.

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.

This paper provides an overview on the self-adaptation considered as a human skill that we can train and develop in order to balance our organizations. We try here to explain what kind of organizational structures and behaviours can be used to facilitate and accelerate transformations and changes in our economic and social contexts. We are facing fast changes and new problems in economy and society. this current complexity requires new perspectives in designing and managing organizations. We strive to create organizational models and techniques that are as robust and versatile as biological organisms are and evolve in nature. Self-adaptation is an attribute of several systems in nature: it allows the reliability and scalability of recursive processes based on adjustment of collective behaviour of elements and without a central control of structure. Observing adaptive organisms biology focuses on processes at the sub-organism level. Using the latest molecular and physiological tools the adaptations of organisms to environmental stresses are studied and developed. The regulatory mechanisms are understood, including the genetic constraints, the physiological plasticity and the evolutionary history of the responses of organisms. Leafing through a biology book you will be fascinated by the multitude of self-adaptation solutions implemented by natural organisms: plants, animals and micro-organisms convey crucial information for an understanding of the effects of an environmental change on the organisms and the effects of organisms on the environment change. We suggest an approach able to catalyse transformation in people and organisations exposing behaviours and structures able to optimise themselves. These catalysers sometimes are people (change agents), sometimes are things (digital technologies) and sometimes are pieces of culture (knowledge and experience) and more often are combinations of these three factors. This approach has been used in this research for collecting and analysing results achieved in more than 5 years of projects evolution in the change management field.

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.

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: β-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.

The induction of systemic acquired resistance (SAR) in pineapples (Ananas comosus) was studied as shown by the up-regulation of the PR-1 gene (the SAR marker) and examination of the SAR effect on the reniform nematode, Rotylenchulus reniformis. Real-time polymerase chain reaction assay was performed using degenerate primers designed from the PR-1 genes of several monocotyledonous (monocots) and dicotyledonous (dicots) plants. A 266 bp cDNA band was evident only in plants treated with the SAR inducer, acibenzolar-s-methyl. This 266 bp cDNA was sequenced and found to be highly homologous to a number of PR-1 genes from monocots. In addition, the amino acid sequence deduced from the 266 bp cDNA showed a high identity to PR-1 proteins from both monocots and dicots. Therefore, it was highly likely that this cloned fragment was part of the A. comosus PR-1 gene, indicating that A. comosus has an SAR pathway. The time course of PR-1 expression was studied. The results showed that PR-1 induction was initiated as early as 1 d after acibenzolar application and continued through 3 wk thereafter. The effect of SAR on the nematodes, R. reniformis, in pineapples was also elucidated. The results showed that the reproduction of nematodes on the pineapples treated with 100 mg/L or 200 mg/L was 55% lower than that on pineapples treated with 0 mg/L or 50 mg/L. Nematode reproduction on pineapples treated with the same concentration but inoculated at different times was not significantly different (p > 0.05).

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:β-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.

The adoption of Agrobacterium-mediated transformation to agronomically important Carica papaya cultivars and genotypes, such as "Kapoho', has been challenging. To address this problem, an alternative transformation protocol was developed for papaya using embryogenic suspension-derived cultures. The ability of the tissues (cultivar 'Kapoho') to regenerate plantlets from these cultures was demonstrated for both transgenic lines and untransformed controls. Suspension-derived cultures at the young globular calli stage contained embryos highly receptive to Agrobacterium infection and did not require the problematic wounding treatments inherent in other protocols. The optimum parameters selected to obtain transgenic calli were as follows: 1-d co-cultivation with Agrobacterium, the cell density used for transformation was A(600) of 0.005, and elimination of Agrobacterium post-transformation was done using 250 mg/L carbenicillin and cefotaxime. Putative transgenic calli were confirmed positive for the presence of the eGFP transgene, Cp45 promoter sequence, and hygromycin resistance gene (hptII) using PCR. The presence of eGFP messenger RNA (mRNA) and protein were detected using reverse transcription PCR (RT-PCR) and Western blot analysis, respectively. Visualization of qualitative eGFP fluorescence in roots, stems, and leaves further confirmed the expression of Cp45:eGFP fusion in the transformed papaya plantlets. This technique serves as an alternative and efficient method to generate transgenic plants in a simple laboratory setup that facilitates Agrobacterium-mediated transformation of previously difficult papaya cultivars and genotypes.

The use of promoters for downstream metabolic engineering in plants requires efficiently screening their transcriptional strength and the resulting levels of the desired metabolite. Carica papaya is a complex slow-growing tropical tree, which is difficult to rapidly screen for promoter activity. Therefore, we used the simple model system, Arabidopsis thaliana (Columbia), to compare the effectiveness of two native papaya promoters (Cp9 and Cp29) with a positive control (CaMV35S promoter), in driving transcription of the stilbene synthase gene (Vst1) and production of the phytoalexin trans-resveratrol glucoside (piceid). Single transgene copy numbers were verified via Southern analysis of at least three independent transgenic lines for each promoter construct. The Cp29 and control CaMV35S promoters consistently produced high Vst1 mRNA levels, whereas Vst1 mRNA was 50–60 % less in the Cp9:Vst1 lines. The evolutionarily related chalcone synthase (CHS) gene produced 20 and 40 % less mRNAs relative to WT in the Cp29:Vst1 and 35S:Vst1 lines, respectively, but was unchanged in the Cp9:Vst1 lines. The transgenic lines accumulated piceid as identified through RP-HPLC and tandem mass spectrometry. Piceid levels were the highest in the 35S:Vst1 followed by Cp29:Vst1 and Cp9:Vst1 in the studied tissues. No overt deleterious phenotypes were observed in the Cp9:Vst1 and Cp29:Vst1 lines; however, 35S:Vst1 produced smaller plants. The levels of secondary metabolites (anthocyanins) and seed pigments (tannins) decreased in the CaMV35S:Vst1 and Cp29:Vst1 lines, likely due to competition between CHS and stilbene synthase for precursors p-coumaroyl- and malonyl-CoA. The Cp29:Vst1 expression in Arabidopsis produced adequate levels of piceid for future disease-resistance studies.

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. (C) 2015 Elsevier Ireland Ltd. Al! rights reserved.

Although Anthurium is an attractive and commercially popular ornamental plant, its genetic enhancement has lagged behind that of other ornamental crops. There are several agronomically important traits in need of improvement. These include novel flower colors and morphologies, increased shelf and vase lives, and resistance to bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae), burrowing nematodes and abiotic stresses. The production of transgenic Anthuriums is critical because the conventional breeding of a cultivar with beneficial traits typically requires 8–10 years. This review evaluates the problems, challenges and progress associated with developing molecular markers for Anthurium and in genetically transforming this ornamental. Recent improvements have hastened the tissue culture and regeneration of transgenic plants primarily using Agrobacterium-based methods. Promoter analyses have focused on constitutive and tissue-enhanced gene expression with the green fluorescent protein being a more reliable reporter than β-glucuronidase. The development of molecular markers assists with phylogenetic analyses and PCR-based markers such as RAPD, SSR, SPAR, ISSR and AFLP can be used to differentiate cultivars and for genetic fingerprinting. The marker-assisted breeding of Anthurium will become more feasible once available data are used for association to specific traits. Work on the identification of quantitative trait loci for disease resistance and other traits such as flower colour is required and should incorporate new approaches, such as next-generation sequencing technologies. By highlighting the aforementioned bottlenecks and successes in this review, it is expected that the pace of Anthurium genetic improvement will increase with the multifaceted incorporation of focused priorities and new technology advancements.

Promoters are essential upstream genetic switches that activate and repress gene expression. Promoter characterization is a key prerequisite before using in downstream biotechnology applications. The model system, Arabidopsis thaliana, was utilized here to efficiently hasten the time to verify and characterize the functionality and strength of promoters from the more complex tropical tree, Carica papaya. Four putative promoter regions and their 5’UTRs were isolated from the genes encoding peroxidase (Cp9), β-1,3-glucanase (Cp29), ferulate-5-hydroxylase (Cp35) and hypersensitive-induced response protein (Cp45) and fused to eGFP. In silico analysis predicted the presence of several cis-elements associated with regulatory functions in stress and defense responses. The Arabidopsis transcriptional machinery readily recognized the promoters, as determined by qualitative and quantitative measurements of eGFP expression (fluorescence, mRNA and protein levels). The eGFP was expressed in a variety of tissues of the transgenic plants (vasculature, shoot apex, cotyledon, cotyledon petioles, hypocotyls, and root). The Cp29 and Cp45 promoters showed the highest and most promising overall expression. Comparison of eGFP mRNA and protein levels indicated post-transcriptional regulation. Identifying the precise transcription start sites (TSSs) demonstrated transcription fidelity and mapped the length of the 5′-UTR. Predicted mRNA 5′-UTR secondary structures potentially affected the translational efficiency of the mRNAs during development, most notably for Cp9 and Cp35. This work demonstrates the utility of using Arabidopsis to quickly evaluate and identify useful promoters (Cp29, Cp45) from complex tropical plants for future biotechnology goals, and to analyze 5′UTR regulation, cis-elements, and trans-acting factors.

Promoters are essential upstream genetic switches that activate and repress gene expression. Promoter characterization is a key prerequisite before using in downstream biotechnology applications. The model system, was utilized here to efficiently hasten the time to verify and characterize the functionality and strength of promoters from the more complex tropical tree, . Four putative promoter regions and their 5’UTRs were isolated from the genes encoding peroxidase (), β-1,3-glucanase (), ferulate-5-hydroxylase () and hypersensitive-induced response protein () and fused to eGFP. analysis predicted the presence of several -elements associated with regulatory functions in stress and defense responses. The transcriptional machinery readily recognized the promoters, as determined by qualitative and quantitative measurements of eGFP expression (fluorescence, mRNA and protein levels). The eGFP was expressed in a variety of tissues of the transgenic plants (vasculature, shoot apex, cotyledon, cotyledon petioles, hypocotyls, and root). The and promoters showed the highest and most promising overall expression. Comparison of eGFP mRNA and protein levels indicated post-transcriptional regulation. Identifying the precise transcription start sites (TSSs) demonstrated transcription fidelity and mapped the length of the 5′-UTR. Predicted mRNA 5′-UTR secondary structures potentially affected the translational efficiency of the mRNAs during development, most notably for and . This work demonstrates the utility of using to quickly evaluate and identify useful promoters () from complex tropical plants for future biotechnology goals, and to analyze 5′UTR regulation, -elements, and trans-acting factors.

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.

Plant cyclic nucleotide-gated channels (CNGCs) are implicated in the uptake of both essential and toxic cations, Ca2+ signalling, and responses to biotic and abiotic stress. The 20 CNGC paralogues of Arabidopsis are divided into five evolutionary groups. Group IV-A is highly isolated and consists only of two closely spaced genes, CNGC19 and CNGC20. Prior studies have shown that both genes are induced by salinity and biotic stress. A unique feature of CNGC19 and CNGC20 is their long hydrophilic N-termini. To determine the subcellular locations of CNGC19 and CNGC20, partial and full-length fusions to GFP(S65T) were generated. Translational fusions of the N-termini of CNGC19 (residues 1-171) and CNGC20 (residues 1-200) to GFP(S65T) were targeted to punctate structures when transiently expressed in leaf protoplasts. In the case of CNGC20, but not CNGC19, the punctate structures were co-labelled with a marker for the Golgi. The full-length CNGC19-GFP fusion co-localized with markers for the vacuole membrane (alpha TIP- and gamma TIP-mCherry). Vacuole membrane labelling by the full-length CNGC20-GFP fusion was also observed, but the signal was weak and accompanied by numerous punctate signals that did not co-localize with alpha TIP- or gamma TIP-mCherry. These punctate structures diminished, and localization of full-length CNGC20-GFP to the vacuole increased, when it was co-expressed with the full-length CNGC19-mCherry. Vacuole membrane labelling was also detected in planta via immunoelectron microscopy using a CNGC20-antiserum on cryopreserved ultrathin sections of roots. We hypothesize that the role of group IV-A CNGCs is to mediate the movement of cations between the central vacuole and the cytosol in response to certain types of abiotic and biotic stress.