Exogenous abscisic acid and sodium nitroprusside regulate flavonoid biosynthesis and photosynthesis of Nitraria tangutorum Bobr in alkali stress

Abstract

Abscisic acid (ABA) and nitric oxide (NO) are involved in mediating abiotic stress-induced plant physiological responses. Nitraria tangutorum Bobr is a typical salinized desert plant growing in an arid environment. In this study, we investigated the effects of ABA and NO on N.tangutorum seedlings under alkaline stress. Alkali stress treatment caused cell membrane damage, increased electrolyte leakage, and induced higher production of reactive oxygen species (ROS), which caused growth inhibition and oxidative stress in N.tangutorum seedlings. Exogenous application of ABA (15μm) and Sodium nitroprusside (50μm) significantly increased the plant height, fresh weight, relative water content, and degree of succulency in N.tangutorum seedlings under alkali stress. Meanwhile, the contents of ABA and NO in plant leaves were significantly increased. ABA and SNP can promote stomatal closure, decrease the water loss rate, increase leaf surface temperature and the contents of osmotic regulator proline, soluble protein, and betaine under alkali stress. Meanwhile, SNP more significantly promoted the accumulation of chlorophyll a/b and carotenoids, increased quantum yield of photosystem II (φPSII) and electron transport rate (ETRII) than ABA, and decreased photochemical quenching (qP), which improved photosynthetic efficiency and accelerated the accumulation of soluble sugar, glucose, fructose, sucrose, starch, and total sugar. However, compared with exogenous application of SNP in the alkaline stress, ABA significantly promoted the transcription of NtFLS/NtF3H/NtF3H/NtANR genes and the accumulation of naringin, quercetin, isorhamnetin, kaempferol, and catechin in the synthesis pathway of flavonoid metabolites, and isorhamnetin content was the highest. These results indicate that both ABA and SNP can reduce the growth inhibition and physiological damage caused by alkali stress. Among them, SNP has a better effect on the improvement of photosynthetic efficiency and the regulation of carbohydrate accumulation than ABA, while ABA has a more significant effect on the regulation of flavonoid and anthocyanin secondary metabolite accumulation. Exogenous application of ABA and SNP also improved the antioxidant capacity and the ability to maintain Na⁺/K⁺ balance of N. tangutorum seedlings under alkali stress. These results demonstrate the beneficial effects of ABA and NO as stress hormones and signaling molecules that positively regulate the defensive response of N. tangutorum to alkaline stress.

Keywords: ABA and NO; Nitraria tangutorum Bobr; alkali stress; anthocyanin accumulation; flavone pathway; photosynthetic efficiency.

Figure 1

Phenotypes and indicators of N. tangutorum treated with different concentrations of alkaline salt(AS). (A): Phenotype under different alkali treatments(AS: NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5); (B): Fresh weight and plant height; (C): Relative water content and degree of succulent; (D): Eletrical leakage and MDAcontent; (E): Total flavonoids and total anthocyanin content; (F): Chlorophyll content. Selected 40-day-old plants were treated with different concentrations of alkaline salt(AS)(10/30/50/100/150 mM). Plants grew in Hoagland solution served as controls(CK). Each group was treated with 25 plants and repeated 3 times. Data presented are the mean ± SDs (n=3). Different letters next to the number indicate significant difference (Duncan multiple range test; P<0.05).

Figure 2

ABA and SNP alleviate ROS accumulation and accelerates antioxidant defenses under 100mM alkali stress in leaves (AS: NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5). Growth status of N. tangutorum 40-day-old seedlings treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5 μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days. (A): DAB and NBT staining of the leaves; (B): Electrical leakage (EL); (C): MDA; (D): Hydrogen peroxide content (H₂O₂); (E): Superoxide anion content (O₂ ^-); (F): GSH content; (G): GR activity; (H):CAT activity; (I): GSSG content;(J): Total antioxidant capacity; (K): SOD activity; (L): GSH/GSSG ratio; (M): GST activity; (N): APX activity. Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 3

Ion content and quantification of related genes in N. tangutorum seedlings under different treament. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5 μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days(AS : NaHCO₃: Na₂CO_{3 =} 9:1, PH=9.5). Plants grew in Hoagland solution served as controls (CK). (A) Na⁺ content, (B) K⁺ content, (C) the Na⁺/K⁺ ratios, (D, E) The expression level of ion transporter genes in leaf and root, including NtSOS1, NtNHX1/2/3, NtKUP4, NtKCO, NtHAK6/12, NtKEA2/3/5 and NtHKT1. Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 4

Effects of different treatments on leaf transpiration and osmomodulatory substances of N. tangutorum seedlings. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5 μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days(AS : NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5), and subsequent experiments are the same. Plants grew in Hoagland solution served as controls(CK). (A, B, D): Infrared imaging, leaf water loss rate and leaf temperature of leaves of N. tangutorum seedlings under different stress treatments. (C) and (E): stomatal phenotype and stomatal aperture of N. tangutorum seedlings under different treatments. (F): Intercellular CO₂ concentration; (G): Betaine content; (H): Soluble protein content; (I): Proline content. Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 5

Effects of different treatments on chlorophyll content and photosynthesis in leaves of N. tangutorum seedlings. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5 μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days (AS : NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5), and subsequent experiments are the same. Plants grew in Hoagland solution served as controls(CK). (A): Phenotypes of old and new leaves under different stress treatments; (B): determination of chlorophyll content; (C): Fluorescence imaging of plant chlorophyll in vivo; (D–I): Determination of photosynthetic indexes under different treatments, including: Photochemical efficiency of photosystem II (Fv/Fm); photochemical quenching (qP); photosystem II apparent photosynthetic electron transfer efficiency (ETRII); non photochemical quenching (NPQ); quantum yield of photosystem II (φPSII); net photosynthetic rate(Pn). Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 6

Effects of different treatments on sugar accumulation in N. tangutorum seedlings. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days(AS: NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5). Plants grew in Hoagland solution served as controls (CK). (A–F): Changes in the accumulation of carbohydrates in leaves of N. tangutorum seedlings under different stress treatments, in order: glucose, fructose, sucrose, starch, soluble sugar and total sugar. Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 7

ABA and SNP promote the accumulation of flavonoid and anthocyanin secondary metabolites under alkaline stress. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA/5 μm Flu, 100 mM alkali +50 μm SNP/70 μm cPTIO) for 12 days (AS : NaHCO₃: Na₂CO_{3 =} 9:1, pH=9.5). Plants grew in Hoagland solution served as controls(CK). (A): Total flavonoid content; (B): Flavanol content; (C): Total anthocyanin content; (D): Proanthocyanidin content; (E): Catechin content; (F): Quercetin content; (G): Isoamnetin rhcontent; (H): Kaempferol content; (I): Naringenin content. Bars represent the means ± SDs of three replicates. Significant differences among treatments are indicated by different letters within a panel based on Duncan’s multiple range test (P < 0.05).

Figure 8

RT-PCR analysis of the effects of exogenous ABA and SNP on the expression of related genes in the synthesis pathways of phenylpropane, flavonoids and anthocyanins under alkaline stress. 40-day-old seedlings were treated with different treatments (100 mM alkali, 100 mM alkali +15 μm ABA, 100 mM alkali +50 μm SNP) for 12 days (AS : NaHCO3: Na₂CO_{3 =} 9:1, pH=9.5). Plants grew in Hoagland solution served as controls (CK). The detected genes include: NtPAL(DN22899/DN30845/DN32316/DN27795/DN28855),NtC4H(DN22630),Nt4CL(DN27635/DN26161/DN26165/DN26520/DN22466/DN24012), NtCHS(DN26129), NtCHI(DN25681/DN30553/DN26750/DN23554), NtFHT(DN24264), NtFLS(DN27463/DN25711/DN36475/DN26381/DN28412/DN31474/DN11103), NtF3’H (DN40094/DN33376/DN34723/DN11734/DN26220), NtF3H(DN12356), NtF3’5’H (DN26145), NtDFR(DN29583/DN20811/DN29793/DN2454 0), NtLDOX(DN27002), NtANS(DN28028), NtANR(DN27423), NtUFGT(DN30699/DN26126/DN29030/DN23964/), Nt3/5/7GT(DN22396/DN28282/DN25447/DN30437/DN22901),NtGST(DN30723/DN46257/DN18263/DN27066/DN30434/DN22619/DN23788/DN33175/DN8177/DN25640/DN25885/DN16942),NtOMT(DN 13998/DN37666).