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SUMMARY: We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH‐isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress‐related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non‐stressed plants contained twofold elevated fructose‐2,6‐bisphosphate (F2,6P2) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose‐to‐sucrose ratios, but also more lipids. Above‐ground biomass lay in between susceptible Xanthi and resistant SNN, with photo‐assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid‐to‐carbohydrate ratios, resulting in increased harvest yields ‐ also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non‐stressed plants. In leaves of salt‐watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non‐stressed plants showed enhanced PSII‐induction kinetics (upon dark–light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids – especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi‐cP2 lines. Apparently, metabolic priming by F2,6P2—combined with sustained NADP(H) turnover—bypasses the genetically fixed growth–defence trade‐off, rendering tobacco plants more stress‐resilient and productive. [ABSTRACT FROM AUTHOR]

Summary: The oligosaccharyltransferase (OT) complex catalyzes N‐glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity‐tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission‐electron microscopy showed that STT3a becomes incorporated into OT–ribosome super‐complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a. [ABSTRACT FROM AUTHOR]

We analyzed plant-derived α1,4-fucosyltransferase ( FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc-green fluorescent protein ( GFP) or tomato LeFucTc-GFP restored Lewis-a formation in a fuctc mutant, confirming functionality in the trans-Golgi. AtFucTc-GFP partly accumulated at the nuclear envelope ( NE) not observed for other homologs or truncated AtFucTc lacking the N-terminus or catalytic domain. Analysis of At/ LeFucTc-GFP swap constructs with exchanged cytosolic, transmembrane and stalk ( CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino-acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N-terminal GFP copies did, indicating localization at the inner nuclear membrane ( INM). Tunicamycin treatment of AtFucTc-GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N-glycosylation. Yet neither expression in protoplasts of Arabidopsis N-glycosylation mutants nor elimination of the N-glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum ( ER)-to-Golgi transport by co-expression of Sar1( H74L) trapped tunicamycin-released AtFucTc-GFP in the ER, however, without NE localization. Since recovery after tunicamycin-washout required de novo-protein synthesis, our analyses suggest that AtFucTc localizes to the NE/ INM due to interaction with an unknown (glyco)protein. [ABSTRACT FROM AUTHOR]

Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx-mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxCS exchange in Trx. Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes. [ABSTRACT FROM AUTHOR]

Summary We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features. [ABSTRACT FROM AUTHOR]