Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein-protein interactions.

Fibrillins (FBNs) are plastidial proteins found in photosynthetic organisms from cyanobacteria to higher plants. The function of most FBNs remains unknown. Here, we focused on members of the FBN subgroup comprising FBN1a, FBN1b, and FBN2. We show that these three polypeptides interact between each other, potentially forming a network around the plastoglobule surface. Both FBN2 and FBN1s interact with allene oxide synthase, and the elimination of any of these FBNs results in a delay in jasmonate-mediated anthocyanin accumulation in response to a combination of moderate high light and low temperature. Mutations in the genes encoding FBN1s or FBN2 also affect the protection of PSII under the combination of these stresses. Fully developed leaves of these mutants have lower maximum quantum efficiency of PSII (Fv/Fm) and higher oxidative stress than wild-type plants. These effects are additive, and the fbn1a-1b-2 triple mutant shows a stronger decrease in Fv/Fm and a greater increase in oxidative stress than fbn1a-1b or fbn2 mutants. Co-immunoprecipitation analysis indicated that FBN2 also interacts with other proteins involved in different metabolic processes. We propose that these fibrillins facilitate accurate positioning of different proteins involved in distinct metabolic processes, and that their elimination leads to dysfunction of those proteins.

CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis.

KEY MESSAGE: Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery. The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61-71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8-12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum.

Thioredoxins (TRXs) f and m are key components in the light regulation of photosynthetic metabolism via thiol-dithiol modulation in chloroplasts of leaves; however, little is known about the factors modulating the expression of these proteins. To investigate the effect of sugars as photosynthetic products on the expression of PsTRX f and m1 genes, sucrose and glucose were externally supplied to pea plants during the day. There was an increase in the mRNA levels of PsTRX f and m1 genes in response mainly to glucose. When leaf discs were incubated for up to 4h in the dark, glucose also led to an increase in both mRNA and protein levels of TRXs f and m, while sucrose had no substantial effect. Expression of PsDOF7, a carbon metabolism-related transcription factor gene, was also induced by glucose. Protein-DNA interaction showed that PsDOF7 binds specifically to the DOF core located in PsTRX f and m1 gene promoters. Transient expression in agroinfiltrated pea leaves demonstrated that PsDOF7 activated transcription of both promoters. The incubation of leaf discs in dithiotreitol (DTT) to increase the redox status led to a marked increase in the mRNA and protein levels of both TRXs within 4h. The increase in TRX protein levels occurred after 1h DTT feeding, implying a rapid effect of the thiol status on TRX f and m1 protein turnover rates, while transcriptional regulation took 3h to proceed. These results show that the protein levels of both TRXs are under short-term control of the sugar and thiol status in plants.

Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor.

Chloroplastic thioredoxins f and m (TRX f and TRX m) mediate light regulation of carbon metabolism through the activation of Calvin cycle enzymes. The role of TRX f and m in the activation of Calvin cycle enzymes is best known among the TRX family. However, the discoveries of new potential targets extend the functions of chloroplastic TRXs to other processes in non-photosynthetic tissues. As occurs with numerous chloroplast proteins, their expression comes under light regulation. Here, the focus is on the light regulation of TRX f and TRX m in pea and Arabidopsis during the day/night cycle that is maintained during the subjective night. In pea (Pisum sativum), TRX f and TRX m1 expression is shown to be governed by a circadian oscillation exerted at both the transcriptional and protein levels. Binding shift assays indicate that this control probably involves the interaction of the CCA1 transcription factor and an evening element (EE) located in the PsTRX f and PsTRX m1 promoters. In Arabidopsis, among the multigene family of TRX f and TRX m, AtTRX f2 and AtTRX m2 mRNA showed similar circadian oscillatory regulation, suggesting that such regulation is conserved in plants. However, this oscillation was disrupted in plants overexpressing CCA1 (cca1-ox) or repressing CCA1 and LHY (cca1-lhy). The physiological role of the oscillatory regulation of chloroplastic TRX f and TRX m in plants during the day/night cycle is discussed.

Current Knowledge on Mechanisms Preventing Photosynthesis Redox Imbalance in Plants.

Photosynthesis includes a set of redox reactions that are the source of reducing power and energy for the assimilation of inorganic carbon, nitrogen and sulphur, thus generating organic compounds, and oxygen, which supports life on Earth. As sessile organisms, plants have to face continuous changes in environmental conditions and need to adjust the photosynthetic electron transport to prevent the accumulation of damaging oxygen by-products. The balance between photosynthetic cyclic and linear electron flows allows for the maintenance of a proper NADPH/ATP ratio that is adapted to the plant's needs. In addition, different mechanisms to dissipate excess energy operate in plants to protect and optimise photosynthesis under adverse conditions. Recent reports show an important role of redox-based dithiol-disulphide interchanges, mediated both by classical and atypical chloroplast thioredoxins (TRXs), in the control of these photoprotective mechanisms. Moreover, membrane-anchored TRX-like proteins, such as HCF164, which transfer electrons from stromal TRXs to the thylakoid lumen, play a key role in the regulation of lumenal targets depending on the stromal redox poise. Interestingly, not all photoprotective players were reported to be under the control of TRXs. In this review, we discuss recent findings regarding the mechanisms that allow an appropriate electron flux to avoid the detrimental consequences of photosynthesis redox imbalances.

cpFBPaseII, a novel redox-independent chloroplastic isoform of fructose-1,6-bisphosphatase.

A full-length FBPase cDNA has been isolated from Fragaria x ananassa (strawberry) corresponding to a novel putative chloroplastic FBPase but lacking the regulatory redox domain, a characteristic of the plastidial isoenzyme (cpFBPaseI). Another outstanding feature of this novel isoform, called cpFBPaseII, is the absence of the canonical active site. Enzymatic assays with cpFBPaseII evidenced clear Mg(2+)-dependent FBPase activity and a K(m) for fructose-1,6-bisphosphate (FBP) of 1.3 mM. Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma. Nevertheless, unlike cpFBPaseI, which is redox activated, cpFBPaseII did not increase its activity in the presence of either DTT or thioredoxin f (TRX f) and is resistant to H(2)O(2) inactivation. Additionally, the novel isoform was able to complement the growth deficiency of the yeast FBP1 deletion fed with a non-fermentable carbon source. Furthermore, orthologues are restricted to land plants, suggesting that cpFBPaseII is a novel and an intriguing chloroplastic FBPase that emerged late in the evolution of photosynthetic organisms, possibly because of a pressing need of land plants.

Changing sugar partitioning in FBPase-manipulated plants.

This review offers an overview of the current state of our knowledge concerning the role of fructose-1,6-bisphosphatase (FBPase) in sugar partitioning and biosynthesis, through the analysis of genetically manipulated plants. The existence of two well-characterized isoforms is a consequence of the subcellular compartmentalization of photosynthetic eukaryotes, conditioning their respective regulatory mechanisms and their influence over plant metabolism and photosynthesis. Both isoforms are important, as has been deduced from previous work with different plant species, although there is still much to be done in order to gain a definitive vision of this issue. Despite that, alteration of the FBPase content follows a general pattern, there are some differences that could be considered species-specific. Modifications lead to profound changes in the carbohydrate content and carbon allocation, raising questions as to whether flux of some sugars or sugar precursors from one side to the other of the chloroplast envelope occurs to rebalance carbohydrate metabolism or whether other compensatory, though not fully efficient, enzymatic activities come into play. Due to the pleiotropic nature of modifying the core carbon metabolism, an answer to the above questions would require an exhaustive study involving diverse aspects of plant physiology.

Proteomic Analyses of Thioredoxins f and m Arabidopsis thaliana Mutants Indicate Specific Functions for These Proteins in Plants.

A large number of plastidial thioredoxins (TRX) are present in chloroplast and the specificity versus the redundancy of their functions is currently under discussion. Several results have highlighted the fact that each TRX has a specific target protein and thus a specific function. In this study we have found that in vitro activation of the fructose-1,6-bisphosphatase (FBPase) enzyme is more efficient when f1 and f2 type thioredoxins (TRXs) are used, whilst the m3 type TRX did not have any effect. In addition, we have carried out a two-dimensional electrophoresis-gel to obtain the protein profiling analyses of the trxf1, f2, m1, m2, m3 and m4 Arabidopsis mutants. The results revealed quantitative alteration of 86 proteins and demonstrated that the lack of both the f and m type thioredoxins have diverse effects on the proteome. Interestingly, 68% of the differentially expressed proteins in trxf1 and trxf2 mutants were downregulated, whilst 75% were upregulated in trxm1, trxm2, trxm3 and trxm4 lines. The lack of TRX f1 provoked a higher number of down regulated proteins. The contrary occurred when TRX m4 was absent. Most of the differentially expressed proteins fell into the categories of metabolic processes, the Calvin⁻Benson cycle, photosynthesis, response to stress, hormone signalling and protein turnover. Photosynthesis, the Calvin⁻Benson cycle and carbon metabolism are the most affected processes. Notably, a significant set of proteins related to the answer to stress situations and hormone signalling were affected. Despite some studies being necessary to find specific target proteins, these results show signs that are suggest that the f and m type plastidial TRXs most likely have some additional specific functions.

AtCXXS: atypical members of the Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide isomerase activity.

The Arabidopsis thaliana thioredoxin subgroup h III is composed of four members and includes the two monocysteinic (CXXS) thioredoxins encoded by the genome. We show that AtCXXS1 is the ortholog of monocysteinic thioredoxins present in all higher plants. In contrast, unicellular algae and the moss Physcomitrella patens do not encode monocysteinic thioredoxin. AtCXXS2, the second monocysteinic thioredoxin of Arabidopsis has no ortholog in any other higher plants. It probably appeared recently by duplications of a dicysteinic thioredoxin of the same subgroup h III. Both monocysteinic thioredoxins show a low disulfide reductase activity in vitro but are very efficient as disulfide isomerases in RNAse refolding tests. The possible interactions of these proteins with the glutathione glutaredoxin pathway are discussed on the basis of recent papers.

Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum.

Redox regulation is of great importance in chloroplasts. Many chloroplast enzymes, such as those belonging to the Calvin-Benson cycle (CBC), have conserved regulatory cysteines which form inhibitory disulphide bridges when physiological conditions become unfavourable. Amongst these enzymes, cFBP1, the CBC fructose-1,6-bisphosphatase (FBPase) isoform, is well known to be redox activated by thioredoxin f through the reduction of a disulphide bridge involving Cys153 and Cys173. Moreover, data obtained during recent years point to S-nitrosylation as another redox post-translational modification putatively regulating an increasing number of plant enzymes, including cFBP1. In this study we have shown that the Pisum sativum cFBP1 can be efficiently S-nitrosylated by GSNO and SNAP, triggering the formation of the regulatory disulphide. Using in vivo experiments with P. sativum we have established that cFBP1 S-nitrosylation only occurs during the light period and we have elucidated by activity assays with Cys-to-Ser mutants that this enzyme may be inactivated through the S-nitrosylation of Cys153. Finally, in the light of the new data, we have proposed an extended redox-regulation model by integrating the S-nitrosylation and the TRX f-mediated regulation of cFBP1.

Plastid thioredoxins f and m are related to the developing and salinity response of post-germinating seeds of Pisum sativum.

Plastid thioredoxins (TRXs) f and m have long been considered to regulate almost exclusively photosynthesis-related processes. Nonetheless, some years ago, we found that type-f and m TRXs were also present in non-photosynthetic organs such as roots and flowers of adult pea plants. In the present work, using pea seedlings 2-5 days old, we have determined the mRNA expression profile of the plastid PsTRX f, m1, and m2, together with the ferredoxin NADP reductase (FNR). Our results show that these TRX isoforms are expressed in cotyledons, underlying similar expression levels in roots for PsTRX m2. We have also noted plastid TRX expression in cotyledons of etiolated seedlings of Arabidopsis thaliana lines carrying constructs corresponding to PsTRX f and m1 promoters fused to the reporter gene GUS, pointing to a role in reserve mobilization. Furthermore, the response of plastid TRXs to NaCl and their capacity in restoring the growth of a TRX-deficient yeast under saline conditions suggest a role in the tolerance to salinity. We propose that these redox enzymes take part of the reserve mobilization in seedling cotyledons and we suggest additional physiological functions of PsTRX m2 in roots and PsTRX m1 in the salinity-stress response during germination.

Localization in roots and flowers of pea chloroplastic thioredoxin f and thioredoxin m proteins reveals new roles in nonphotosynthetic organs.

Plant thioredoxins (TRXs) are involved in redox regulation of a wide variety processes and usually exhibit organ specificity. We report strong evidence that chloroplastic TRXs are localized in heterotrophic tissues and suggest some ways in which they might participate in several metabolic and developmental processes. The promoter regions of the chloroplastic f and m1 TRX genes were isolated from a pea (Pisum sativum) plant genomic bank. Histochemical staining for beta-glucuronidase (GUS) in transgenic homozygous Arabidopsis (Arabidopsis thaliana) plants showed preferential expression of the 444-bp PsTRXf1 promoter in early seedlings, stems, leaves, and roots, as well as in flowers, stigma, pollen grains, and filaments. GUS activity under the control of the 1,874-bp PsTRXm1 promoter was restricted to the leaves, roots, seeds, and flowers. To gain insight into the translational regulation of these genes, a series of deletions of 5' elements in both TRX promoters were analyzed. The results revealed that a 126-bp construct of the PsTRXf2 promoter was unable to reproduce the expression pattern observed with the full promoter. The differences in expression and tissue specificity between PsTRXm1 and the deleted promoters PsTRXm2 and PsTRXm3 suggest the existence of upstream positive or negative regulatory regions that affect tissue specificity, sucrose metabolism, and light regulation. PsTRXm1 expression is finely regulated by light and possibly by other metabolic factors. In situ hybridization experiments confirmed new localizations of these chloroplastic TRX transcripts in vascular tissues and flowers, and therefore suggest possible new functions in heterotrophic tissues related to cell division, germination, and plant reproduction.

A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana.

Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.