Rossitza Atanassova

A Grape ASR Protein Involved in Sugar and Abscisic Acid Signaling

The function of ASR (ABA [abscisic acid]-, stress-, and ripening-induced) proteins remains unknown. A grape ASR, VvMSA, was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putative monosaccharide transporter (VvHT1). This promoter contains two sugar boxes, and its activity is induced by sucrose and glucose. VvMSA and VvHT1 share similar patterns of expression during the ripening of grape. Both genes are inducible by sucrose in grape berry cell culture, and sugar induction of VvMSA is enhanced strongly by ABA. These data suggest that VvMSA is involved in a common transduction pathway of sugar and ABA signaling. Gel-shift assays demonstrate a specific binding of VvMSA to the 160-bp fragment of the VvHT1 promoter and more precisely to two sugar-responsive elements present in this target. The positive regulation of VvHT1 promoter activity by VvMSA also is shown in planta by coexpression experiments. The nuclear localization of the yellow fluorescent protein–VvMSA fusion protein and the functionality of the VvMSA nuclear localization signal are demonstrated. Thus, a biological function is ascribed to an ASR protein. VvMSA acts as part of a transcription-regulating complex involved in sugar and ABA signaling.

Modular organization and developmental activity of an Arabidopsis thaliana EF-1α gene promoter

The activity of the Arabidopsis thalana A1 EF-1α gene promoter was analyzed in transgenic Arabidopsis plants. The 5′ upstream sequence of the A1 gene and several promoter deletions were fused to the β-glucuronidase (GUS) coding region. Promoter activity was monitored by quantitative and histochemical assays of GUS activity. The results show that the A1 promoter exhibits a modular organization. Sequences both upstream and downstream relative to the transcription initiation site are involved in quantitative and tissue-specific expression during vegetative growth. One upstream element may be involved in the activation of expression in meristematic tissues; the downstream region, corresponding to an intron within the 5′ non-coding region (5′IVS), is important for expression in roots; both upstream and downstream sequences are required for expression in leaves, suggesting combinatorial properties of EF-1α cis-regulatory elements. This notion of specific combinatorial regulation is reinforced by the results of transient expression experiments in transfected Arabidopsis protoplasts. The deletion of the 5′IVS has much more effect on expression when the promoter activity is under the control of A1 EF-1α upstream sequences than when these upstream sequences were replaced by the 35S enhancer. Similarly, a synthetic oligonucleotide corresponding to an A1 EF-1α upstream cis-acting element (the TEF1 box), is able to restore partially the original activity when fused to a TEF1-less EF1-α promoter but has no significant effect when fused to an enhancer-less 35S promoter.

Sugar-regulated expression of a putative hexose transport gene in grape.

Different lengths of the promoter of grape (Vitis vinifera) VvHT1 (Hexose Transporter 1) gene, which encodes a putative hexose transporter expressed during the ripening of grape, have been transcriptionally fused to the β-glucuronidase reporter gene. In transgenic tobacco (Nicotiana tabacum) transformed with these constructs,VvHT1 promoters were clearly responsible for the sink organ preferential expression. The potential sugar effectors ofVvHT1 promoter were studied in tobacco cv Bright-Yellow 2 cells transformed with chimeric constructs. Glucose (56 mm), sucrose (Suc; 58 mm), and the non-transported Suc isomer palatinose doubled the β-glucuronidase activity conferred by the VvHT1 promoter, whereas fructose did not affect it. These effects were the strongest with the 2.4-kb promoter, which contains all putative sugar-responsive elements (activating and repressing), but they were also significant with the 0.3-kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both Suc and palatinose was confirmed in the homologous grape berry cell culture. The data provide the first example of a putative sugar transporter, which is induced by both glucose and Suc in higher plants. Although induction ofVvHT1 expression by Suc does not require transport, the presence of glucosyl moiety is necessary for Suc sensing. These results provide new insights into sugar sensing and signaling in plants.

Effect of modification of the O-methyltransferase activity on cell wall composition, ultrastructure and degradability of transgenic tobacco

The effect of O-methyltransferase (OMT) cDNA modulation on cell wall composition, ultrastructure and rumen degradability was measured on transgenic tobacco (Nicotiana tabacum). The expression of OMT cDNA in antisense orientation (AS) inhibited OMT activity by 92% whereas expression of sense constructs led to plants either co-suppressed (CS, 98% inhibition) or overexpressing OMT activity. The cell wall residues of stems were analysed for lignin content, products of nitrobenzene oxidation (NBO) and polysaccharide content. Degradability was determined by a cellulase method. Sections of stem were stained by acid phloroglucinol and Maule reactant. Stem samples were incubated in the rumen for 8, 24 and 48 h and observed by scanning electron microscopy (SEM). Compared to controls, OMT-depleted stems showed decreased hemicellulose content but unchanged lignin content. In contrast, syringyl units decreased by 40 and 90% in AS and CS samples respectively and NBO content followed a similar trend. Dry matter cellulase degradability was significantly improved by 3.5 and 5.6 percentage units in AS and CS samples respectively. SEM showed a greater bacterial colonisation in these samples and indicated a higher rate of rumen degradability in CS tissues than in controls. Overexpressing plants had a composition and a degradability similar to that of controls. For all the plants studied, the improvements in dry matter degradability were closely linked to the syringyl to guaiacyl ratio or to the NBO content. The modifications observed in down-regulated tobacco were similar to those produced by bm3 maize mutation, but without lignin decrease. Genetic modifications should therefore be considered for improving forage digestibility.

Plasma membrane transporters: a machinery for uptake of organic solutes and stress resistance

The paper gives an overview of the data concerning the plasma membrane transporters mediating the uptake of sucrose, monosaccharides, amino acids, small peptides and glutathione. Current problems and gaps are underlined, and a special focus is made on the importance of these transporters during biotic and abiotic stresses.

Replication-independent cis-acting element of a maize histone gene promoter

Abstract A chimeric gene containing the β-glucoronidase (GUS) coding sequence fused to the maize histone H4C7 gene promoter was expressed in transient assays in tobacco mesophyll protoplasts revealing the replication-independent activity of the plant histone gene promoter. Functional analysis of 5′ deletions of the H4C7 promoter suggested the presence of antagonistic activities in a region encompassing the plant histone-specific octamer and a hybrid hexameric motif, and revealed an essential cis-acting positive element between nucleotides -82 and -36 relative to the TATA box. Furthermore, point mutations introduced in the CCATCGAAC motif present in this region drastically reduced the activity of the H4C7 promoter. We conclude this nonameric sequence, present in the 5′ flanking DNA sequence of most plant histone genes, to be a positive cis-acting element controlling the basal replication-independent activity of the H4C7 promoter.

Les gènes d'histones: un modèle pour l'étude de la régulation de l'expression génique dans les méristèmes

Summary During germination, the amounts of histone H3 and H4 mRNAs increase in parallel to DNA synthesis and, in the organs of adult plants, they are directly related to the mitotic activity of the tissues. In transgenic plants, an Arabidopsis H4 promoter (H4A748) induces preferential expression of the s-glucuronidase (GUS) reporter gene in meristems. The elements necessary to confer the specificity are contained in a 126 bp fragment of the promoter. Studying the kinetics of expression of the H4A748 chimaeric gene in protoplasts isolated from transgenic tobacco leaves revealed the existence of two activities of the promoter: a basal replication—independent activity and a strong replication—dependent activity in parallel with DNA synthesis.

The Sugar-Signaling Hub: Overview of Regulators and Interaction with the Hormonal and Metabolic Network

Plant growth and development has to be continuously adjusted to the available resources. Their optimization requires the integration of signals conveying the plant metabolic status, its hormonal balance, and its developmental stage. Many investigations have recently been conducted to provide insights into sugar signaling and its interplay with hormones and nitrogen in the fine-tuning of plant growth, development, and survival. The present review emphasizes the diversity of sugar signaling integrators, the main molecular and biochemical mechanisms related to the sugar-signaling dependent regulations, and to the regulatory hubs acting in the interplay of the sugar-hormone and sugar-nitrogen networks. It also contributes to compiling evidence likely to fill a few knowledge gaps, and raises new questions for the future.

Comparative metabolomics and glycolysis enzyme profiling of embryogenic and nonembryogenic grape cells

A novel biological model was created for the comparison of grapevine embryogenic cells (EC) and nonembryogenic cells (NEC) sharing a common genetic background but distinct phenotypes, when cultured on their respective most appropriate media. Cytological characterization, 1H‐NMR analysis of intracellular metabolites, and glycolytic enzyme activities provided evidence for the marked metabolic differences between EC and NEC. The EC were characterized by a moderate and organized cell proliferation, coupled with a low flux through glycolysis, high capacity of phosphoenolpyruvate carboxylase and glucokinase, and high oxygen consumption. The NEC displayed strong anarchic growth, and their high rate of glycolysis due to the low energetic efficiency of the fermentative metabolism is confirmed by increased enolase capacity and low oxygen consumption.