Overexpression of thioredoxin m in chloroplasts alters carbon and nitrogen partitioning in tobacco.

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.

Elevated CO2 has concurrent effects on leaf and grain metabolism but minimal effects on yield in wheat.

While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.

Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance.

The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.

Physiological performance of transplastomic tobacco plants overexpressing aquaporin AQP1 in chloroplast membranes.

The leaf mesophyll CO2 conductance and the concentration of CO2 within the chloroplast are major factors affecting photosynthetic performance. Previous studies have shown that the aquaporin NtAQP1 (which localizes to the plasma membrane and chloroplast inner envelope membrane) is involved in CO2 permeability in the chloroplast. Levels of NtAQP1 in plants genetically engineered to overexpress the protein correlated positively with leaf mesophyll CO2 conductance and photosynthetic rate. In these studies, the nuclear transformation method used led to changes in NtAQP1 levels in the plasma membrane and the chloroplast inner envelope membrane. In the present work, NtAQP1 levels were increased up to 16-fold in the chloroplast membranes alone by the overexpression of NtAQP1 from the plastid genome. Despite the high NtAQP1 levels achieved, transplastomic plants showed lower photosynthetic rates than wild-type plants. This result was associated with lower Rubisco maximum carboxylation rate and ribulose 1,5-bisphosphate regeneration. Transplastomic plants showed reduced mesophyll CO2 conductance but no changes in chloroplast CO2 concentration. The absence of differences in chloroplast CO2 concentration was associated with the lower CO2 fixation activity of the transplastomic plants. These findings suggest that non-functional pores of recombinant NtAQP1 may be produced in the chloroplast inner envelope membrane.

Differential effects of elevated CO2 on awn and glume metabolism in durum wheat (Triticum durum).

While the effect of CO2 enrichment on wheat (Triticum spp.) photosynthesis, nitrogen content or yield has been well-studied, the impact of elevated CO2 on metabolic pathways in organs other than leaves is poorly documented. In particular, glumes and awns, which may refix CO2 respired by developing grains and be naturally exposed to higher-than-ambient CO2 mole fraction, could show specific responses to elevated CO2 . Here, we took advantage of a free-air CO2 enrichment experiment and performed multilevel analyses, including metabolomics, ionomics, proteomics, major hormones and isotopes in Triticum durum . While in leaves, elevated CO2 tended to accelerate amino acid metabolism with many significantly affected metabolites, the effect on glumes and awns metabolites was modest. There was a lower content in compounds of the polyamine pathway (along with uracile and allantoin) under elevated CO2 , suggesting a change in secondary N metabolism. Also, cytokinin metabolism appeared to be significantly affected under elevated CO2 . Despite this, elevated CO2 did not affect the final composition of awn and glume organic matter, with the same content in carbon, nitrogen and other elements. We conclude that elevated CO2 mostly impacts on leaf metabolism but has little effect in awns and glumes, including their composition at maturity.

New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts.

Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.

Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-expression with Plastid Thioredoxin m.

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein's overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.

Assessing the evolution of wheat grain traits during the last 166 years using archived samples.

The current study focuses on yield and nutritional quality changes of wheat grain over the last 166 years. It is based on wheat grain quality analyses carried out on samples collected between 1850 and 2016. Samples were obtained from the Broadbalk Continuous Wheat Experiment (UK) and from herbaria from 16 different countries around the world. Our study showed that, together with an increase in carbohydrate content, an impoverishment of mineral composition and protein content occurred. The imbalance in carbohydrate/protein content was specially marked after the 1960's, coinciding with strong increases in ambient [CO2] and temperature and the introduction of progressively shorter straw varieties. The implications of altered crop physiology are discussed.

NTRC and Thioredoxin f Overexpression Differentially Induces Starch Accumulation in Tobacco Leaves.

Thioredoxin (Trx) f and NADPH-dependent Trx reductase C (NTRC) have both been proposed as major redox regulators of starch metabolism in chloroplasts. However, little is known regarding the specific role of each protein in this complex mechanism. To shed light on this point, tobacco plants that were genetically engineered to overexpress the NTRC protein from the chloroplast genome were obtained and compared to previously generated Trx f-overexpressing transplastomic plants. Likewise, we investigated the impact of NTRC and Trx f deficiency on starch metabolism by generating Nicotiana benthamiana plants that were silenced for each gene. Our results demonstrated that NTRC overexpression induced enhanced starch accumulation in tobacco leaves, as occurred with Trx f. However, only Trx f silencing leads to a significant decrease in the leaf starch content. Quantitative analysis of enzyme activities related to starch synthesis and degradation were determined in all of the genotypes. Zymographic analyses were additionally performed to compare the amylolytic enzyme profiles of both transplastomic tobacco plants. Our findings indicated that NTRC overexpression promotes the accumulation of transitory leaf starch as a consequence of a diminished starch turnover during the dark period, which seems to be related to a significant reductive activation of ADP-glucose pyrophosphorylase and/or a deactivation of a putative debranching enzyme. On the other hand, increased starch content in Trx f-overexpressing plants was connected to an increase in the capacity of soluble starch synthases during the light period. Taken together, these results suggest that NTRC and the ferredoxin/Trx system play distinct roles in starch turnover.

Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers.

Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato.

Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis.

The switch from budding to filamentous growth is a key aspect of invasive growth and virulence for the fungal phytopathogen Ustilago maydis. The cyclic AMP (cAMP) signaling pathway regulates dimorphism in U. maydis, as demonstrated by the phenotypes of mutants with defects in protein kinase A (PKA). Specifically, a mutant lacking the regulatory subunit of PKA encoded by the ubc1 gene displays a multiple-budded phenotype and fails to incite disease symptoms, although proliferation does occur in the plant host. A mutant with a defect in a catalytic subunit of PKA, encoded by adr1, has a constitutively filamentous phenotype and is nonpathogenic. We employed serial analysis of gene expression to examine the transcriptomes of a wild-type strain and the ubc1 and adr1 mutants to further define the role of PKA in U. maydis. The mutants displayed changes in the transcript levels for genes encoding ribosomal proteins, genes regulated by the b mating-type proteins, and genes for metabolic functions. Importantly, the ubc1 mutant displayed elevated transcript levels for genes involved in phosphate acquisition and storage, thus revealing a connection between cAMP and phosphate metabolism. Further experimentation indicated a phosphate storage defect and elevated acid phosphatase activity for the ubc1 mutant. Elevated phosphate levels in culture media also enhanced the filamentous growth of wild-type cells in response to lipids, a finding consistent with PKA regulation of morphogenesis in U. maydis. Overall, these findings extend our understanding of cAMP signaling in U. maydis and reveal a link between phosphate metabolism and morphogenesis.

Mapping of genomic regions (quantitative trait loci) controlling production and quality in industrial cultures of the edible basidiomycete Pleurotus ostreatus.

Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.

Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus.

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.

Differentially regulated, vegetative-mycelium-specific hydrophobins of the edible basidiomycete Pleurotus ostreatus.

Three different hydrophobins (Vmh1, Vmh2, and Vmh3) were isolated from monokaryotic and dikaryotic vegetative cultures of the edible fungus Pleurotus ostreatus. Their corresponding genes have a number of introns different from those of other P. ostreatus hydrophobins previously described. Two genes (vmh1 and vmh2) were expressed only at the vegetative stage, whereas vmh3 expression was also found in the fruit bodies. Furthermore, the expression of the three hydrophobins varied significantly with culture time and nutritional conditions. The three genes were mapped in the genomic linkage map of P. ostreatus, and evidence is presented for the allelic nature of vmh2 and POH3 and for the different locations of the genes coding for the glycosylated hydrophobins Vmh3 and POH2. The glycosylated nature of Vmh3 and its expression during vegetative growth and in fruit bodies suggest that it should play a role in development similar to that proposed for SC3 in Schizophyllum commune.