Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida

Abstract

Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.

Keywords: Auxin; Rosa sp.; bud burst; cytokinins; shoot branching; strigolactones; sugar; sugar signalling..

Fig. 1.

(A) Bud elongation. (B) Estimated lag times before elongation and relative elongation rates of buds cultivated in vitro. Experiments were conducted with 100mM mannitol or 10, 50, 100, or 250mM sucrose. Data are mean ± SE of 10 replicates. Letters indicate significant differences between means.

Fig. 2.

Elongation of buds grown with (A) 80mM mannitol, sucrose or non-metabolizable sucrose analogues (lactulose, melibiose, turanose, and palatinose) for R. hybrida, or with 30mM sucrose or 30mM non-metabolizable sucrose analogues for (B) Arabidopsis and (C) P. sativum. Data are mean ± SE of 8–10 replicates.

Fig. 3.

GUS staining in bud-bearing stem sections of the DR5::GUS-expressing pea line (A) before treatment (T0); or grown for 48h with 100mM (B) mannitol or (C) sucrose; or grown for 96h with 100mM (D) mannitol or (E) sucrose or 80mM (F) lactulose or (G) palatinose. (H) Elongation of buds grown with 30mM sucrose and treated with a drop of gelose on buds containing 1% PEG, 0.01% Tween-20, and 0.2% DMSO supplemented or not with 1mM NPA. (A–G) Images are representative of five replicates; (H) mean ± SE of eight replicates. Asterisks indicate significant differences between the treatment conditions. White bars represent 1mm.

Fig. 4.

(A) Relative IAA content (relative to T0 value) in buds of R. hybrida grown in vitro with 100mM sucrose or 100mM mannitol for 96h. (B, C) Relative IAA content (relative to mannitol value) in buds grown for 48h with (B) 100mM mannitol or a range of sucrose concentrations (10, 50, 100, or 250mM), or (C) 100mM mannitol (Mtl) or 80mM sucrose analogues: palatinose (Pal), turanose (Tur), or lactulose (Lac). (D, E) Relative expression of (D) RhTAR1 and (E) RhYUC1. Data are mean ± SE of three measurements on a pool of 60 buds. Asterisks and letters indicate significant differences between the different treatments for each time-point.

Fig. 5.

Relative expression of (A) RhPIN1, (B) RhPID, and (C) RhPP2A in buds grown in vitro for 96h with 100mM mannitol or sucrose. (D) Relative fluorescence of the GFP signal at the polarized pole of the cells in bud stems of the pPIN1::PIN1-GFP-expressing tomato line grown for 24h or 96h with 100mM mannitol or sucrose. Data are mean ± SE of three measurements on a pool of 60 buds (A–C) and four replicates (D). Asterisks indicate significant differences between the different treatments for each time point.

Fig. 6.

(A) Nodal stem content in isopentenyl adenine riboside 5′-monophosphate (iPRMP), zeatin riboside 5′-monophosphate (ZRMP), isopentenyl adenine riboside (iPR), zeatin riboside (ZR), isopentenyl adenine (iP), zeatin (Z), Z O-glucoside (ZOG), and ZR O-glucoside (ZROG) for intact plants at the FBV stage (T0) or grown for 24h on 100mM mannitol, 100mM sucrose, or 30mM palatinose; contents are expressed relative to T0. Relative expression is also shown of (B) RhIPT3 and (C) RhIPT5 in nodal stem sections grown in vitro with 100mM mannitol or 100mM sucrose up to 96h after their excision. Also elongation of buds grown with (D) 30mM sucrose alone or with 10 µM lovastatine, PI-55, or LGR-991; and (E) with 30mM mannitol alone or with 10 µM 6-benzylaminopurine (BAP). Data are mean ± SE of three measurements on a pool of 60 buds (A–C) and 10 replicates (D, E). Asterisks and letters indicate significant differences between the different treatments for each time point.

Fig. 7.

Relative expression of (A) RwMAX3, (B) RwMAX4, and (C) RwMAX2 in nodal stems grown for 96h with 100mM mannitol or sucrose; relative expression of (D) RwMAX2, and (E) RhBRC1 in buds grown for 96h with 100mM mannitol or sucrose; and (F) relative expression of RwMAX2 and RhBRC1 in buds grown for 24h with 100mM mannitol or 10, 50, 100, or 250mM sucrose. Data are mean ± SE of three replicates. Asterisks indicate significant differences between sucrose and mannitol for each time point. Letters indicate significant differences between means for each gene.