Drought-tolerant transgenic Swingle Citrumelo controls accumulation of proline modulating the expression of key genes of the proline metabolism
Diliane Barichello , Renato Farinacio , Camilla Carvalho Numes dos Santos , Giselly Aparecida Andrade , Getúlio Takashi Nagashima , Deoclécio Domingues Garbúglio , Inês Fumiko Ubukata Yada , Regiane de Fátima Travensolo2 , Beatriz Cutilak Bianchi , Luiz Gonzaga Esteves Vieira & Eduardo Fermino Carlos
The Brazilian citrus industry is primarily not irrigated, and growers often face dry spells that affects tree development, flowering, fruit set and juice quality, limiting the use of rootstocks and imposing considerable yield variation upon the industry. To mitigate water stress, cells increase osmoprotective compounds, such as the amino acid proline. The objectives of this study were to evaluate physiological parameters for transgenic Swingle citrumelo (Citrus paradisi Macfad. cv. Duncan x Poncirus trifoliata L. Raf.) rootstock able to accumulate proline, and to assess gene expression level of key enzymes in the proline biosynthesis and glutamate pathway. The transgenic plants resulted from the integration of the P5CS-F129A gene from Vigna aconitifolia, that codes for the mutant Δ1-pyrroline-5-carboxylate synthetase (P5CS), under the control of the constitutive CaMV35S promoter. The water deficit experiment was conducted under greenhouse conditions, and transcriptional analysis of leaf tissue using RNA-seq was performed. Transgenic plants had significant higher proline accumulation up to intermediated stress level, and higher Relative Water Content (RWC) and stomatal conductance from 26 up to 34 days without irrigation (d.w.i) compared to non-transgenic control. Analysis of the transcriptional data showed that the expression of the native Δ-pyrroline-5-carboxylate reductase (P5CR) was down-regulated while the expression of the proline dehydrogenase (ProDH) gene increased respectively in transgenic non irrigated and in both transgenic treatmens (irrigated and not irrigated), probably to counter balance proline accumulation under those circunstances. Based on this knowledge, new drought-tolerant transgenic rootstocks can be developed to tolerate drought stress for longer periods of time than drought sensitive genotypes.