New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida.

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called "darkly-veined" petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All "darkly-veined" genotypes have the identical mutation in the AN2 gene, but express a different functional paralog - ANTHOCYANIN-4 (AN4) - abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis - CHALCONE ISOMERASE-A (CHI-A) - the plant responded directly by upregulating of another paralogs - DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI-B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the "darkly-veined" phenotypes as a result of the breeding process under human selection, and CHI-B is a backup for CHI-A acitvity in some floral tissues.

Mechanical Stimulation Decreases Auxin and Gibberellic Acid Synthesis but Does Not Affect Auxin Transport in Axillary Buds; It Also Stimulates Peroxidase Activity in Petunia × atkinsiana.

Thigmomorphogenesis (or mechanical stimulation-MS) is a term created by Jaffe and means plant response to natural stimuli such as the blow of the wind, strong rain, or touch, resulting in a decrease in length and an increase of branching as well as an increase in the activity of axillary buds. MS is very well known in plant morphology, but physiological processes controlling plant growth are not well discovered yet. In the current study, we tried to find an answer to the question if MS truly may affect auxin synthesis or transport in the early stage of plant growth, and which physiological factors may be responsible for growth arrest in petunia. According to the results of current research, we noticed that MS affects plant growth but does not block auxin transport from the apical bud. MS arrests IAA and GA3 synthesis in MS-treated plants over the longer term. The main factor responsible for the thickening of cell walls and the same strengthening of vascular tissues and growth arrestment, in this case, is peroxidase (POX) activity, but special attention should be also paid to AGPs as signaling molecules which also are directly involved in growth regulation as well as in cell wall modifications.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

KEY MESSAGE: Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

Molybdenum and iron mutually impact their homeostasis in cucumber (Cucumis sativus) plants.

Molybdenum (Mo) and iron (Fe) are essential micronutrients required for crucial enzyme activities in plant metabolism. Here we investigated the existence of a mutual control of Mo and Fe homeostasis in cucumber (Cucumis sativus). Plants were grown under single or combined Mo and Fe starvation. Physiological parameters were measured, the ionomes of tissues and the ionomes and proteomes of root mitochondria were profiled, and the activities of molybdo-enzymes and the synthesis of molybdenum cofactor (Moco) were evaluated. Fe and Mo were found to affect each other's total uptake and distribution within tissues and at the mitochondrial level, with Fe nutritional status dominating over Mo homeostasis and affecting Mo availability for molybdo-enzymes in the form of Moco. Fe starvation triggered Moco biosynthesis and affected the molybdo-enzymes, with its main impact on nitrate reductase and xanthine dehydrogenase, both being involved in nitrogen assimilation and mobilization, and on the mitochondrial amidoxime reducing component. These results, together with the identification of > 100 proteins differentially expressed in root mitochondria, highlight the central role of mitochondria in the coordination of Fe and Mo homeostasis and allow us to propose the first model of the molecular interactions connecting Mo and Fe homeostasis.

Visualization and quantification of protein interactions in the biosynthetic pathway of molybdenum cofactor in Arabidopsis thaliana.

The molybdenum cofactor (Moco) is the active compound at the catalytic site of molybdenum enzymes. Moco is synthesized by a conserved four-step pathway involving six proteins in Arabidopsis thaliana. Bimolecular fluorescence complementation was used to study the subcellular localization and interaction of those proteins catalysing Moco biosynthesis. In addition, the independent split-luciferase approach permitted quantification of the strength of these protein-protein interactions in vivo. Moco biosynthesis starts in mitochondria where two proteins undergo tight interaction. All subsequent steps were found to proceed in the cytosol. Here, the heterotetrameric enzyme molybdopterin synthase (catalysing step two of Moco biosynthesis) and the enzyme molybdenum insertase, which finalizes Moco formation, were found to undergo tight protein interaction as well. This cytosolic multimeric protein complex is dynamic as the small subunits of molybdopterin synthase are known to go on and off in order to become recharged with sulphur. These small subunits undergo a tighter protein contact within the enzyme molybdopterin synthase as compared with their interaction with the sulphurating enzyme. The forces of each of these protein contacts were quantified and provided interaction factors. To confirm the results, in vitro experiments using a technique combining cross-linking and label transfer were conducted. The data presented allowed the outline of the first draft of an interaction matrix for proteins within the pathway of Moco biosynthesis where product-substrate flow is facilitated through micro-compartmentalization in a cytosolic protein complex. The protected sequestering of fragile intermediates and formation of the final product are achieved through a series of direct protein interactions of variable strength.

Sulfite oxidase controls sulfur metabolism under SO2 exposure in Arabidopsis thaliana.

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).

Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors.

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.

Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana.

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K(D) values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.

New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation.

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.