Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems

Abstract

Background: Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems.

Results: Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H₂O₂) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H₂O₂ and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H₂O₂ scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H₂O₂ are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H₂O₂ and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H₂O₂ to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS.

Conclusions: CmCADs but CmCAD4 are positively regulated by ABA, H₂O₂ and JA under drought stress and participate in lignin synthesis.

Keywords: Abscisic acid; Cinnamyl alcohol dehydrogenase; Drought; Hydrogen peroxide; Jasmonic acid; Lignin; Oriental melon.

Fig. 1

Contents of ABA (a, d), H₂O₂ (b, e) and JA (c, f) in stems of melon seedlings. a-c, changes of ABA, H₂O₂ and JA contents under 8% PEG6000 treatment. d-f, contents of ABA, H₂O₂ and JA under PEG and/or inhibitor or scavenger treatments with respect to ABA, H₂O₂ and JA at peak-value time point according to a-c. Values are the means ± SE of three independent experiments. Different lowercase letters indicate significant differences from control using Duncan’s Multiple Range Test at P< 0.05

Fig. 2

qRT-PCR analysis the relative expressions of CmCAD genes in stems of melon seedlings regulated by ABA (a-e), H₂O₂ (f-j) and MeJA (k-o). Values are the means ± SE of three independent experiments. Different lowercase letters indicate significant differences from control using Duncan’s Multiple Range Test (P< 0.05)

Fig. 3

Heat map depicting expression fold changes of lignin biosynthesis genes in lignin monomer synthesis pathway. Data presented as Log₂[abundance in sample/abundance in control]. Data are collected from biological replicates (n = 3)

Fig. 4

Effects of pretreated with ABA inhibitor fluridone (a), H₂O₂ scavenger imidazol (b) and MeJA inhibitor ibuprofen (c) on CAD activity in stems of melon seedlings exposed to PEG. Values are the means ± SE of three independent experiments. Different lowercase letters indicate significant differences from control using Duncan’s Multiple Range Test (P< 0.05)

Fig. 5

Lignin deposition regulated by ABA, H₂O₂ and JA under PEG treatment. a, c, e, Effects of ABA, H₂O₂, JA and their corresponding inhibitor or scavenger on lignin contents under PEG treatments. b, d, f, histochemical staining of xylem tissue from the 3rd internode of stems from melon seedlings with phloroglucinol-HCl for lignin observation. mx: metaxylem; px: protoxylem. Different lowercase letters indicate significant differences from control using Duncan’s Multiple Range Test (P< 0.05). Bars=50 μm

Fig. 6

Histochemical staining (a) and activity detection (b) of GUS driven by promoters with respect to CmCAD1–3 treated with ABA, H₂O₂ and MeJA. Different lowercase letters indicate significant differences from control using Duncan’s Multiple Range Test (P< 0.05). ND: not detected; ns: no significance