Response of sugarcane plants with modified cytokinin homeostasis under water deficit conditions

Cytokinins (CK) are one of the main phytohormones involved in plant growth and development and play a role in plant adaptation to stress. In this study, the response to water deficit stress was investigated in sugarcane (Saccharum spp. hybrids) plants after exogenous application of CK and partial silencing of endogenous cytokinin oxidase/dehydrogenase (CKX) gene expression in transgenic plants. The CKX enzyme is responsible for CK breakdown in plant cells. A foliar drench of N6-benzyladenin on leaves (100 mu M) resulted in plants that survived more than 10 days of water deficit stress compared with the untreated control plants, which displayed senescence and leaf wilting after 10 days without water. Furthermore, after 10 days without water, the treated plants maintained a high efficiency of the photosystem II (PSII) and displayed higher levels of chlorophyll and relative water content, and after 20 days without water, high catalase activity and proline content when compared with the untreated controls. In addition, sugarcane calli were transformed with a recombinant vector containing a 237 bp sugarcane ShCKX target sequence which included a conserved CK binding domain cloned in sense and antisense orientations. Partial silencing of CKX expression in the transgenic plants resulted in significantly lower salicylic acid levels after 21 days of deficit stress compared with untransformed controls. Stressed transgenic plants accumulated less H2O2, more antioxidants (superoxide dismutase and catalase) and proline when compared with the wild type (WT) plants. Moreover, the transgenic plants were able to retain high levels of chlorophyll and most maintained the efficiency of PSII after watering was stopped. The transgenic genotype with the highest level of CKX silencing displayed delayed leaf senescence and maintained high leaf water content under water deficit stress compared with the WT plants. Both the exogenous application of CK and the partial silencing of CKX expression enabled the plants to resprout after re-watering following the stress period. The results indicate that the manipulation of CK levels, either by increasing CK exogenously or deceasing their break down by partial silencing of the CKX gene, in sugarcane positively affects the response to water deficit stress through a variety of physiological mechanisms.