Tolerance of citrus plants to the combination of high temperatures and drought is associated to the increase in transpiration modulated by a reduction in abscisic acid levels

Abstract

Background: In natural environments, several adverse environmental conditions occur simultaneously constituting a unique stress factor. In this work, physiological parameters and the hormonal regulation of Carrizo citrange and Cleopatra mandarin, two citrus genotypes, in response to the combined action of high temperatures and water deprivation were studied. The objective was to characterize particular responses to the stress combination.

Results: Experiments indicated that Carrizo citrange is more tolerant to the stress combination than Cleopatra mandarin. Furthermore, an experimental design spanning 24 h stress duration, heat stress applied alone induced higher stomatal conductance and transpiration in both genotypes whereas combined water deprivation partially counteracted this response. Comparing both genotypes, Carrizo citrange showed higher phostosystem-II efficiency and lower oxidative damage than Cleopatra mandarin. Hormonal profiling in leaves revealed that salicylic acid (SA) accumulated in response to individual stresses but to a higher extent in samples subjected to the combination of heat and drought (showing an additive response). SA accumulation correlated with the up-regulation of pathogenesis-related gene 2 (CsPR2), as a downstream response. On the contrary, abscisic acid (ABA) accumulation was higher in water-stressed plants followed by that observed in plants under stress combination. ABA signaling in these plants was confirmed by the expression of responsive to ABA-related gene 18 (CsRAB18). Modulation of ABA levels was likely carried out by the induction of 9-neoxanthin cis-epoxicarotenoid dioxygenase (CsNCED) and ABA 8'-hydroxylase (CsCYP707A) while conversion to ABA-glycosyl ester (ABAGE) was a less prominent process despite the strong induction of ABA O-glycosyl transferase (CsAOG).

Conclusions: Cleopatra mandarin is more susceptible to the combination of high temperatures and water deprivation than Carrizo citrange. This is likely a result of a higher transpiration rate in Carrizo that could allow a more efficient cooling of leaf surface ensuring optimal CO2 intake. Hence, SA induction in Cleopatra was not sufficient to protect PSII from photoinhibition, resulting in higher malondialdehyde (MDA) build-up. Inhibition of ABA accumulation during heat stress and combined stresses was achieved primarily through the up-regulation of CsCYP707A leading to phaseic acid (PA) and dehydrophaseic acid (DPA) production. To sum up, data indicate that specific physiological responses to the combination of heat and drought exist in citrus. In addition, these responses are differently modulated depending on the particular stress tolerance of citrus genotypes.

Keywords: Carrizo citrange; Cleopatra mandarin; Combined stress conditions; Heat; Hormone regulation; Salicylic acid.

Fig. 1

Phenotypic traits of citrus plants in response to a combination of drought and heat stress. Intact sprouts (%) of Carrizo and Cleopatra seedlings subjected to drought and heat stress (40 °C) in combination for 10 days. For each genotype, asterisks denote statistical significance with respect to initial values at p ≤ 0.05

Fig. 2

Relative water content (RWC) (a) and proline concentration (b) in Carrizo and Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences

Fig. 3

Gas exchange parameters in citrus plants subjected to different stress treatments. Leaf photosynthetic rate, A (a), transpiration, E (b), ratio of substomatal-to-ambient CO₂, C_i/C_a (c), stomatal conductance, g_s (d) in Carrizo and Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences

Fig. 4

Chlorophyll fluorescence parameters in citrus plants subjected to different stress treatments. Quantum efficiency (Φ_PSII) (a) and maximum efficiency of PSII photochemistry (F_v/F_m ratio) (b) in Carrizo and Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences

Fig. 5

Effect of the different stress treatments on metabolism and signaling of SA. CsPAL (a) and CsICS (b) relative expression, SA concentration (c) and CsPR2 (d) relative expression in Carrizo and Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences

Fig. 6

ABA, ABAGE, PA and DPA levels in citrus plants subjected to different stress treatments. ABA (a), ABAGE (c), PA (b) and DPA (d) levels in Carrizo and Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences

Fig. 7

Expression of genes involved in ABA biosynthesis, catabolism, conjugation and signaling in citrus plants subjected to different stress treatments. Relative expression of ABA-biosynthetic gene CsNCED1 (a), ABA-related catabolism gene CsCYP707A1 (b), ABA-related conjugation genes CsAOG and CsBG18 (c-d) and ABA-signaling gene CsRAB18 (e) in leaves of Carrizo and Cleopatra plants in response to drought (WS), heat (HS) and their combination (WS + HS). Different letters denote statistical significance at p ≤ 0.05. G: genotypes; T: stress treatment; GxT: interaction genotype x stress treatment. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no statistical differences