The impact of light and thioredoxins on the plant thiol-disulfide proteome.

Thiol-based redox regulation is a crucial posttranslational mechanism to acclimate plants to changing light availability. Here, we conducted a biotin switch-based redox proteomics study in Arabidopsis (Arabidopsis thaliana) to systematically investigate dynamics of thiol-redox networks in response to temporal changes in light availability and across genotypes lacking parts of the thioredoxin (Trx) or NADPH-Trx-reductase C (NTRC) systems in the chloroplast. Time-resolved dynamics revealed light led to marked decreases in the oxidation states of many chloroplast proteins with photosynthetic functions during the first 10 min, followed by their partial reoxidation after 2 to 6 h into the photoperiod. This involved f, m, and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys peroxiredoxins, and Trx y2 showed an opposing pattern, being more oxidized in light than dark. In Arabidopsis trxf1f2, trxm1m2, or ntrc mutants, most proteins showed increased oxidation states in the light compared to wild type, suggesting their light-dependent dynamics were related to NTRC/Trx networks. While NTRC deficiency had a strong influence in all light conditions, deficiencies in f- or m-type Trxs showed differential impacts on the thiol-redox proteome depending on the light environment, being higher in constant or fluctuating light, respectively. The results indicate plant redox proteomes are subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. The importance of the individual elements of the NTRC/Trx networks mediating these responses depend on the extent of light variability, with NTRC playing a crucial role to balance protein-redox states in rapidly fluctuating light.

Thioredoxins o1 and h2 jointly adjust mitochondrial dihydrolipoamide dehydrogenase-dependent pathways towards changing environments.

Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX-based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild-type and the glycine decarboxylase T-protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)-dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C-labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine-tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions.

Plant NADPH-dependent thioredoxin reductases are crucial for the metabolism of sink leaves and plant acclimation to elevated CO2.

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.

NTRC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism.

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O2 conditions. However, an increase in the stoichiometry of photorespiratory CO2 release was found during O2 -dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD+ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.

Expression of a gene encoding ß-ureidopropionase is critical for pollen germination in tomatoes.

Global warming has seriously decreased world crop yield. High temperatures affect development, growth and, particularly, reproductive tissues in plants. A gene encoding ß-ureidopropionase (SlUPB1, EC 3.5.1.6) was isolated from the stamens of a heat-tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of ß-alanine, the only ß-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing at 35/30°C (day/night), was 2.16 and 2.93 times greater than that in a heat-sensitive tomato (L4783) cultivated at 30/25°C or 25/20°C, respectively. Transgenic tomatoes, upregulating SlUPB1 in L4783 and downregulating SlUPB1 in CL5915, were constructed, and the amount of ß-alanine measured by liquid chromatography-electrospray ionization-mass spectrometry in the transgenic overexpression of SlUPB1 was higher than that of L4783. However, the ß-alanine in the transgenics downregulating SlUPB1 was significantly lower than the ß-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUPB1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics downregulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of ß-alanine further indicated that ß-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together, these results showed that the expression of SlUPB1 is important for pollen germination, and ß-alanine may play a role in pollen germination under both optimal and high temperatures.

Thioredoxin h2 and o1 Show Different Subcellular Localizations and Redox-Active Functions, and Are Extrachloroplastic Factors Influencing Photosynthetic Performance in Fluctuating Light.

Arabidopsis contains eight different h-type thioredoxins (Trx) being distributed in different cell organelles. Although Trx h2 is deemed to be confined to mitochondria, its subcellular localization and function are discussed controversially. Here, cell fractionation studies were used to clarify this question, showing Trx h2 protein to be exclusively localized in microsomes rather than mitochondria. Furthermore, Arabidopsis trxo1, trxh2 and trxo1h2 mutants were analyzed to compare the role of Trx h2 with mitochondrial Trx o1. Under medium light, trxo1 and trxo1h2 showed impaired growth, while trxh2 was similar to wild type. In line with this, trxo1 and trxo1h2 clustered differently from wild type with respect to nocturnal metabolite profiles, revealing a decrease in ascorbate and glutathione redox states. Under fluctuating light, these genotypic differences were attenuated. Instead, the trxo1h2 double mutant showed an improved NADPH redox balance, compared to wild type, accompanied by increased photosynthetic efficiency, specifically in the high-light phases. Conclusively, Trx h2 and Trx o1 are differentially localized in microsomes and mitochondria, respectively, which is associated with different redox-active functions and effects on plant growth in constant light, while there is a joint role of both Trxs in regulating NADPH redox balance and photosynthetic performance in fluctuating light.

Characterization of the complete chloroplast genome sequence of Daphne retusa Hemsl. (Thymelaeaceae), a rare alpine plant species in northwestern China.

Daphne retusa Hemsl. (Thymelaeaceae) is an evergreen shrub plant. First, we characterized the complete nucleotide sequence of chloroplast (cp) genome of D. retusa. The total length of cp genome was found to be 170,553 bp, including a large single copy (LSC) region of 84,886 bp, a small single copy (SSC) region of 2,437 bp, and a pair of inverted repeats (IRs) of 41,617 bp. The cp genome of Daphne retusa Hemsl. contains 134 genes, including 90 protein-coding genes (75 PCG species), 37 transfer RNA genes (29 tRNA species), and 6 rRNA genes (3 RNA species). A total of 13 genes (trnK-UUU, trnS-CGA, atpF, rpoC1, trnL-UAA, trnC-ACA, petD, rpl16, rpl2, ndhB, trnE-UUC, ndhA, and trnA-UGC) contain a single intron, and one gene (ycf3) contains two introns. The GC content in whole cp genome, LSC region, SSC region, and IR region was 36.75%, 34.83%, 28.19%, and 38.96% respectively, like other Thymelaeaceae plants. Phylogenetic analysis suggested that D. retusa has a close relationship with congeneric Daphne tangutica.

Dual resistance of transgenic plants against Cymbidium mosaic virus and Odontoglossum ringspot virus.

Taxonomically distinct Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are two of the most prevalent viruses worldwide; when co-infecting orchids, they cause synergistic symptoms. Because of the huge economic loss in quality and quantity in the orchid industry with virus-infected orchids, virus-resistant orchids are urgently needed. To date, no transgenic resistant lines against these two viruses have been reported. In this study, we generated transgenic Nicotiana benthamiana expressing various constructs of partial CymMV and ORSV genomes. Several transgenic lines grew normally and remained symptomless after mixed inoculation with CymMV and ORSV. The replication of CymMV and ORSV was approximately 70-90% lower in protoplasts of transgenic lines than wild-type (WT) plants. Of note, we detected extremely low or no viral RNA or capsid protein of CymMV and ORSV in systemic leaves of transgenic lines after co-infection. Grafting experiments further revealed that CymMV and ORSV trafficked extremely inefficiently from co-infected WT stocks to transgenic scions, presumably due to RNA-mediated interference. This study reports the first successful creation of dual resistant transgenic lines against CymMV and ORSV. Our studies shed light on the commercial development of transgenic orchid production to combat the global viral threat.