Sucrose promotes stem branching through cytokinin

Abstract

Shoot branching is an important aspect of plant architecture because it substantially affects plant biology and agricultural performance. Sugars play an important role in the induction of shoot branching in several species, including potato (Solanum tuberosum L.). However, the mechanism by which sugars affect shoot branching remains mostly unknown. In the present study, we addressed this question using sugar-mediated induction of bud outgrowth in potato stems under etiolated conditions. Our results indicate that sucrose feeding to detached stems promotes the accumulation of cytokinin (CK), as well as the expression of vacuolar invertase (VInv), an enzyme that contributes to sugar sink strength. These effects of sucrose were suppressed by CK synthesis and perception inhibitors, while CK supplied to detached stems induced bud outgrowth and VInv activity in the absence of sucrose. CK-induced bud outgrowth was suppressed in vinv mutants, which we generated by genome editing. Altogether, our results identify a branching-promoting module, and suggest that sugar-induced lateral bud outgrowth is in part promoted by the induction of CK-mediated VInv activity.

Figure 1

Exogenous sucrose or hexoses induce lateral bud burst and elongation in etiolated stems. Sprouts were detached from the tubers and supplemented with sugars (sucrose, glucose + fructose, sorbitol, each at 300 mM) or water for 9 d in the dark. A, Number of branches. B, Lateral bud length. C, Images showing the lateral node after 7 d of treatment. Bars = 100 μm. Results are means of 10 biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05).

Figure 2

Sucrose and hexoses translocate from the stem into the lateral bud. Detached etiolated stems were fed with a water solution containing A, 1 µCi [U-¹⁴C]sucrose or B, 1 µCi [U-¹⁴C]glucose + [U-¹⁴C]fructose for 0, 2, and 4 h in the dark. Results are means of five biological replicates. Error bars represent se. Different letters indicate significant differences between time points for each treatment (one-way ANOVA, P < 0.05).

Figure 3

Sucrose feeding of stems induces higher expression and activity of VInv in the lateral bud. Detached etiolated stems were fed with 300-mM sucrose, hexoses, or sorbitol for 24 h in the dark. A, VInv transcript level at the lateral bud was determined by real-time quantitative polymerase chain reaction (PCR) using gene-specific primers. Gene transcript levels are expressed relative to controls (0 h), which were set to 1 and normalized with the elongation factor 1-α gene (ef1α) transcript level. B, C, VInv and CWInv activity at the stem node, respectively. Results are means of three biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05). FW, fresh weight.

Figure 4

Silencing VInv reduces the effect of sucrose on stem branching. Detached etiolated stems of “Desiree” (WT) and VInv-knockout lines (vinv-7 and vinv-8) were fed with 300-mM sucrose for 20 d at 14°C, 95% relative humidity, in the dark. A, Number of branches. B, Lateral bud length. C, VInv activity at the stem node of the lateral bud. Results are means of eight biological replicates. Error bars represent se. Different letters represent significant differences between treatments at each time point (one-way ANOVA, P < 0.05). FW, fresh weight.

Figure 5

Feeding etiolated stems with sucrose induces higher content of endogenous CK in the node of the lateral bud. Levels of A, zeatin riboside and B, zeatin in untreated sprouts (0 h) or sprouts supplemented with sugars (sucrose, glucose + fructose, sorbitol at 300 mM), at different time intervals at 14°C, 95% relative humidity, in the dark. Results are means of three biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05). FW, fresh weight.

Figure 6.

Sucrose and CK have an additive effect on etiolated stem branching and lateral bud elongation. Sprouts were detached from the tubers, incubated at 14°C, 95% relative humidity, in the dark, and fed for 20 d with A, B, 0-, 100-, 200-, or 300-mM sucrose; C, D, 0-, 100-, 200-, or 300-µM BAP; E, F, 200-mM sucrose with or without 200-µM BAP, BAP alone, or water. The number of branches developed (A, C, E), and lateral bud length (B, D, F) were measured. G, Typical lateral bud after 15 d of feeding. Bars = 1,000 μm. Results are means of 10 biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05).

Figure 7

CK inhibitors eliminate branching induction by sucrose. Etiolated stems were detached from the tubers and fed with 300-mM sucrose, or 300-mM sucrose with CK-synthesis inhibitor (lovastatin, 200 µM) or with CK-perception inhibitors (LGR-991, PI-55, 200 µM), or water for 20 d at 14°C, 95% relative humidity, in the dark. A, Number of branches. B, Lateral bud length. C, Typical lateral bud after 15 d. Bars = 100 μm. Results are means of 10 biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05).

Figure 8

CK inhibitors reduce VInv activity induced by sucrose or BAP. Detached etiolated stems were incubated for 6 h, in the dark and were fed with A, 300-mM sucrose, or 300-mM sucrose with CK-synthesis inhibitor (200-µM lovastatin) or with CK-perception inhibitor (200-µM LGR-991), or water, (B) 200-µM BAP, BAP with CK-synthesis inhibitor (200-µM lovastatin), BAP with CK-perception inhibitor (200-µM LGR-991), or water. Results are means of five biological replicates. Error bars represent se. Different letters indicate significant differences between treatments at each time point (one-way ANOVA, P < 0.05). FW, fresh weight.

Figure 9

Silencing VInv activity reduces CK effect on stem branching. Etiolated stems were detached from tubers of WT (Désirée) and vinv mutants (vinv-7 and vinv-8). Stems were supplied with 100- or 200-μM BAP or water for 15 d at 14°C in 95% relative humidity, in the dark. A, B, Number of branches. C, D, Lateral bud length. Results are means of eight biological replicates. Error bars represent se. * Indicates significant differences between WT and each vinv mutant at each time point (Student’s t test, P < 0.05).