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The accumulation of endogenous proline induces changes in gene expression of several antioxidant enzymes in leaves of transgenic Swingle citrumelo

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Plant exposure to abiotic stresses leads to an accumulation of reactive oxygen species with the concomitant increase in antioxidant defense mechanisms. Previous studies showed that exogenous application of proline mitigate the deleterious effects caused by oxidative stress due to its ability to increase the activity of antioxidant enzymes. However, there are no reports of the effects of high endogenous accumulation of proline in the transcriptional pattern of antioxidant enzymes genes under normal conditions of water supply or in response to water deficit. Here, we show that isoforms of four antioxidant enzymes genes (Ascorbate peroxidase—APX, Catalase—CAT, Superoxide dismutase—SOD and Glutathione reductase—GR) were differentially regulated in leaves of Swingle citrumelo transgenic plants with high endogenous proline accumulation submitted to water deficits and also under normal water supply condition. Proline per se caused a two-fold change in the transcription activity of APX1, APXcl, CAT2 and Cu/ZnSOD2, while during water deficit proline influenced mRNAs levels in APXs and Cu/ZnSODs isoforms, MnSODmit and GRcl. This study adds new information on the role of proline during drought conditions and, more important, without the potential confounding effects imposed by water deficiency. We showed that, in addition to its known effects on diverse plant physiological and biochemical processes, high endogenous proline can also acts as a regulatory/signalling molecule capable of altering the transcript levels of stress-related genes.

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We thank Celso J. Marur and Fábio S. Souza for plant water status evaluation. This work was supported by a grant from EMBRAPA/OEPAS. KC gratefully acknowledges CAPES for scholarship. LGEV and LFPP are CNPq research fellows.

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Correspondence to Luiz Gonzaga Esteves Vieira.

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Fig. S1 UPGMA dendrogram of the deduced amino acid sequence of APXs. Bootstrap values are indicated for each branch divergence. Sequences are GenBank accession numbers: Citrus spp. (CsAPX1, CsAPX2, CsAPXcl); Arabidopsis thaliana (NP_172267 – AtAPX1; NP_177873 - AtAPXcl); Gossypium hirsutum (ABR18607 - GhAPX1); Oryza sativa (NP_001049769 - OsAPX1; NP_001047111); Populus trichocarpa (ABK93180 - PtAPX2); Solanum lycopersicum (AXX84654 - SlAPX1); Vitis vinifera (ABX79340 - VvAPX2; CA061885 – VvAPXcl) (JPG 41 kb)

Fig. S2 UPGMA dendrogram of the deduced amino acid sequence of CATs. Bootstrap values are indicated for each branch divergence. Sequences are GenBank accession numbers: Citrus spp. (CsCAT1; CsCAT2); Arabidopsis thaliana (NP_564120 – AtCAT2; CAA_64220 - AtCAT1); Gossypium hirsutum (P17598 - GhCAT2); Oryza sativa (NP_001048861 - OsCAT1); Prunus persica (CAD42908 – PpCAT2; CAD42909 - PpCAT1) Populus trichocarpa (CAI43948 - PtCAT2); Solanum lycopersicum (Q9XHH3 - SlCAT1); Vitis vinifera (CA066235 - VvCAT2; AAL83720 – VvCAT1) (JPG 32 kb)

Fig. S3 UPGMA dendrogram of the deduced amino acid sequence of GRs. Bootstrap values are indicated for each branch divergence. Sequences are GenBank accession numbers: Citrus spp. (CsGR; CsGRcl); Arabidopsis thaliana (AAK25938 – AtGR; NP_191026 - AtGRcl); Oryza sativa (NP_001048485 - OsGR; NP_001049057 - OsGRcl); Populus trichocarpa (EEE84081 – PtGR; EEF05642 - PtGRcl); Vitis vinifera (CAO71585 - VvGR; CAN66042 – VvGRcl) (JPG 33 kb)

Fig. S4 UPGMA dendrogram of the deduced amino acid sequence of SODs. Bootstrap values are indicated for each branch divergence. Sequences are GenBank accession numbers: Citrus spp. (CsCu/ZnSOD2; CsCu/ZnSODcl; CsCu/ZnSOD1; CsMnSODmit; CsFeSODcl); Arabidopsis thaliana (NP_172360 – AtCu/ZnSOD2 ; NP_565666 - AtCu/ZnSODcl; NP_199923 – AtFeSODcl; AAM62550 - AtMnSODmit); Citrus limon (AAQ14591 - ClCu/ZnSOD2); Gossypium hirsutum (ABA00453 - GhCu/ZnSOD2; ABA00454 - GhCu/ZnSODcl; ABA00456 – GhFeSODcl; ABA00455 - GhMnSODmit); Oryza sativa (NP_001060564 - OsCu/ZnSOD2; ABF95937 - OsCu/ZnSOD2; EAZ43665 - OsCu/ZnSODcl; NP_001056612 – OsFeSODcl; NP_001055195 - OsMnSODmit); Prunus persica (Q9SM64 – PpMnSODmit); Populus trichocarpa (ABK95956 PtCu/ZnSOD2; ABK96672 - PtCu/ZnSODcl; EEF06442 – PtFeSODcl; EEE95255 - PtMnSODmit); Solanum lycopersicum (AAQ09007 - SlCu/ZnSODcl; AAQ18699 - SlFeSODcl); Vitis vinifera (CAO15111 - VvCu/ZnSOD2; CAN59834 – VvCu/ZnSODcl; CAO62292 – VvFeSODcl; CAN61687 - VvMnSODmit) (JPG 85 kb)

Fig. S5 Differences in leaf rolling symptoms between non-transformed (A) and transgenic (B) “Swingle” citrumelo plants after twenty days without irrigation (JPG 29 kb)

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de Carvalho, K., de Campos, M.K.F., Domingues, D.S. et al. The accumulation of endogenous proline induces changes in gene expression of several antioxidant enzymes in leaves of transgenic Swingle citrumelo. Mol Biol Rep 40, 3269–3279 (2013). https://doi.org/10.1007/s11033-012-2402-5

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  • Proline
  • Reactive oxygen species
  • Antioxidant enzymes
  • mRNA
  • Expression levels