Hydrogen Peroxide Increases during Endodormancy and Decreases during Budbreak in Grapevine (Vitis vinifera L.) Buds

Abstract

Changes in the level of hydrogen peroxide (H₂O₂) is a good indicator to monitor fluctuations in cellular metabolism and in the stress responses. In this study, the changes in H₂O₂ content during bud endodormancy (ED) and budbreak were analysed in grapevine (Vitis vinifera L.). The results showed a gradual increase in the H₂O₂ content during the development of bud ED, which was mainly due to an increase in the activity of peroxidases (PODs). The maximum H₂O₂ content reached in the grapevine buds coincided with the maximum depth of bud ED. In contrast, during budbreak, the H₂O₂ content decreased. As the plant hormones cytokinin (CK) and auxin play an important role in budbreak and growth resumption in grapevine, the effect of exogenous applications of H₂O₂ on the expression of genes involved in CK and auxin metabolism was analysed. The results showed that H₂O₂ represses the expression of the CK biosynthesis genes VvIPT3a and VvLOG1 and induces the expression of the CK-inactivating gene VvCKX3, thus reducing potentially the CK content in the grapevine bud. On the other hand, H₂O₂ induced the expression of the auxin biosynthesis genes VvAMI1 and VvYUC3 and of the auxin transporter gene VvPIN3, thus increasing potentially the auxin content and auxin transport in grapevine buds. In general, the results suggest that H₂O₂ in grapevine buds is associated with the depth of ED and negatively regulates its budbreak.

Keywords: auxin; budbreak; cytokinin; dormancy; grapevine buds; hydrogen peroxide; peroxidases.

Figure 1

Relationship between the degree of endodormancy and H₂O₂ levels in Thompson seedless grapevine buds grown in Santiago, Chile. The degree of dormancy was determined by means of a log-rank test between the probabilistic function KM of the buds collected before (reference) and after the onset of the ED [26]. H₂O₂ was determined by a chemiluminescence method [29].

Figure 2

Expression of peroxidase genes (VvPODs), peroxidase activity and H₂O₂ levels during grape bud endodormancy. (a) Relationship between H₂O₂ levels and peroxidase activity (VvPOD) during endodormancy in Thompson seedless grapevine buds grown in Santiago, Chile. (b) Changes in the expression of peroxidase genes (VvPOD) throughout bud growth in Tempranillo grapevines grown in Alcalá de Henares, Madrid [21] and adapted to the Southern Hemisphere conditions.

Figure 3

Expression profile of RESPIRATORY BURST OXIDASE HOMOLOGS (VvRBOHs) genes in grapevine buds during endodormancy. The expression profile of VvRBOH genes in Tempranillo grapevine buds grown in Alcalá de Henares, Madrid [21] and adapted to the Southern Hemisphere conditions.

Figure 4

Catalase activity and expression of catalase genes in grapevine buds during endodormancy. (a) Catalase activity during endodormancy in Thompson seedless grapevine buds grown in Santiago, Chile. (b) Expression of catalase genes (VvCAT1 and VvCAT2) in buds of Tempranillo grapevines grown in Alcalá de Henares, Madrid [21] and adapted to the Southern Hemisphere conditions.

Figure 5

Aminotriazole effect on H₂O₂ content and budbreak in grapevine buds. The effect of aminotriazole (a) on the level of H₂O₂ and (b) on the budbreak of Thompson seedless grapevine buds was analysed. Buds collected at June 11 were excised into single-bud cuttings and sprayed with 2.5% HC, 2% Aminotriazole and water as control. The treated single-bud cuttings were placed in the growth chamber and H₂O₂ was determined at the desired time by a chemiluminescence method [29]. Values are the average of three biological replicates ± SD; and (*) indicates statistically significance differences (Dunnett’s multiple comparison test α = 0.05). Buds collected at July 18 were excised into single-bud cuttings (30 per treatment) and sprayed with 2.5% HC, 2% Aminotriazole and water as control. The treated single-bud cuttings were placed in the growth chamber and budbreak was assessed by the presence of green tips.

Figure 6

Rate of H₂O₂ reduction in dormant and quiescent grapevine buds under forcing budbreak. Decreases in H₂O₂ content in (a) endodormant and (b) quiescent buds of Thompson seedless grapevines under forced growth conditions. H₂O₂ was determined by a chemiluminescence method [29]. Values are the average of three biological replicates ± SD; (*) indicates statistically significance differences. Student’s test (α = 0.05).

Figure 7

Transcriptional effect of H₂O₂ on the homeostasis of CK and auxin related genes. Effect of H₂O₂ applications on the expression of (a) cytokinin (CK) VvIPT3a, VvLOG1, and VvCKX3 and (b) auxin VvAMI1, VvYUC3 and VvPIN3 genes. Gene expression analysis was performed by RT-qPCR and normalised against VvUBIQITIN. Values are the average of three biological replicates with three technical repetitions ± SD; (*) indicates statistically significance differences p ≤ 0.05; (**) p ≤ 0.01 (Dunnett’s multiple comparison test).