Interplay of sugar, light and gibberellins in expression of Rosa hybrida vacuolar invertase 1 regulation.

Our previous findings showed that the expression of the Rosa hybrida vacuolar invertase 1 gene (RhVI1) was tightly correlated with the ability of buds to grow out and was under sugar, gibberellin and light control. Here, we aimed to provide an insight into the mechanistic basis of this regulation. In situ hybridization showed that RhVI1 expression was localized in epidermal cells of young leaves of bursting buds. We then isolated a 895 bp fragment of the promoter of RhVI1. In silico analysis identified putative cis-elements involved in the response to sugars, light and gibberellins on its proximal part (595 bp). To carry out functional analysis of the RhVI1 promoter in a homologous system, we developed a direct method for stable transformation of rose cells. 5' deletions of the proximal promoter fused to the uidA reporter gene were inserted into the rose cell genome to study the cell's response to exogenous and endogenous stimuli. Deletion analysis revealed that the 468 bp promoter fragment is sufficient to trigger reporter gene activity in response to light, sugars and gibberellins. This region confers sucrose- and fructose-, but not glucose-, responsive activation in the dark. Inversely, the -595 to -468 bp region that carries the sugar-repressive element (SRE) is required to down-regulate the RhVI1 promoter in response to sucrose and fructose in the dark. We also demonstrate that sugar/light and gibberellin/light act synergistically to up-regulate ß-glucuronidase (GUS) activity sharply under the control of the 595 bp pRhVI1 region. These results reveal that the 127 bp promoter fragment located between -595 and -468 bp is critical for light and sugar and light and gibberellins to act synergistically.

Photocontrol of bud burst involves gibberellin biosynthesis in Rosa sp.

Light is a critical determinant of plant shape by controlling branching patterns and bud burst in many species. To gain insight into how light induces bud burst, we investigated whether its inductive effect in rose was related to gibberellin (GA) biosynthesis. In axillary buds of beheaded plants subject to light, the expression of two GA biosynthesis genes (RoGA20ox and RoGA3ox) was promptly and strongly induced, while that of a GA-catabolism genes (RoGA2ox) was reduced. By contrast, lower expression levels of these two GA biosynthesis genes were found in darkness, and correlated with a total inhibition of bud burst. This effect was dependent on both light intensity and quality. In in vitro cultured buds, the inductive effect of light on the growth of preformed leaves and SAM organogenic activity was inhibited by ancymidol and paclobutrazol, two effectors of GA biosynthesis. This effect was concentration-dependent, and negated by GA(3). However, GA(3) alone could not rescue bud burst in the dark. GA biosynthesis was also required for the expression and activity of a vacuolar invertase, and therefore for light-induced sugar metabolism within buds. These findings are evidence that GA biosynthesis contributes to the light effect on bud burst and lay the foundations of a better understanding of its exact role in plant branching.

Insight into the role of sugars in bud burst under light in the rose.

Bud burst is a decisive process in plant architecture that requires light in Rosa sp. This light effect was correlated with stimulation of sugar transport and metabolism in favor of bud outgrowth. We investigated whether sugars could act as signaling entities in the light-mediated regulation of vacuolar invertases and bud burst. Full-length cDNAs encoding two vacuolar invertases (RhVI1 and RhVI2) were isolated from buds. Unlike RhVI2, RhVI1 was preferentially expressed in bursting buds, and was up-regulated in buds of beheaded plants exposed to light. To assess the importance of sugars in this process, the expression of RhVI1 and RhVI2 and the total vacuolar invertase activity were further characterized in buds cultured in vitro on 100 mM sucrose or mannitol under light or in darkness for 48 h. Unlike mannitol, sucrose promoted the stimulatory effect of light on both RhVI1 expression and vacuolar invertase activity. This up-regulation of RhVI1 was rapid (after 6 h incubation) and was induced by as little as 10 mM sucrose or fructose. No effect of glucose was found. Interestingly, both 30 mM palatinose (a non-metabolizable sucrose analog) and 5 mM psicose (a non-metabolizable fructose analog) promoted the light-induced expression of RhVI1 and total vacuolar invertase activity. Sucrose, fructose, palatinose and psicose all promoted bursting of in vitro cultured buds under light. These findings indicate that soluble sugars contribute to the light effect on bud burst and vacuolar invertases, and can function as signaling entities.

Role of the sucrose synthase encoding PrSus1 gene in the development of the parasitic plant Phelipanche ramosa L. (Pomel).

Phelipanche ramosa L. (Pomel) is a major root-parasitic weed attacking many important crops. Success in controlling this parasite is rare and a better understanding of its unique biology is needed to develop new specific control strategies. In the present study, quantitative polymerase chain reaction experiments showed that sucrose synthase encoding PrSus1 transcripts accumulate at their highest level once the parasite is connected to the host (tomato) vascular system, mainly in the parasite tubercles, which bear numerous adventitious roots. In situ hybridization experiments revealed strong PrSus1 expression in both shoot and root apices, especially in shoot apical meristems and in the vascular tissues of scale leaves and stems, and in the apical meristems and developing xylem in roots. In addition, immunolocalization experiments showed that a sucrose synthase protein co-localized with cell-wall thickening in xylem elements. These findings highlight the role of PrSus1 in the utilization of host-derived sucrose in meristematic areas and in cellulose biosynthesis in differentiating vascular elements. We also demonstrate that PrSus1 is downregulated in response to 2,3,5-triiodobenzoic acid-induced inhibition of polar auxin transport in the host stem, suggesting that PrSus1 activity in xylem maturation is controlled by host-derived auxin.

Regulation of RhSUC2, a sucrose transporter, is correlated with the light control of bud burst in Rosa sp.

In roses, light is a central environmental factor controlling bud break and involves a stimulation of sugar metabolism. Very little is known about the role of sucrose transporters in the bud break process and its regulation by light. In this study, we show that sugar promotes rose bud break and that bud break is accompanied by an import of sucrose. Radio-labelled sucrose accumulation is higher in buds exposed to light than to darkness and involves an active component. Several sucrose transporter (RhSUC1, 2, 3 and 4) transcripts are expressed in rose tissues, but RhSUC2 transcript level is the only one induced in buds exposed to light after removing the apical dominance. RhSUC2 is preferentially expressed in bursting buds and stems. Functional analyses in baker's yeast demonstrate that RhSUC2 encodes a sucrose/proton co-transporter with a K(m) value of 2.99 mm at pH 4.5 and shows typical features of sucrose symporters. We therefore propose that bud break photocontrol partly depends upon the modulation of sucrose import into buds by RhSUC2.