Proteomic Responses to Alkali Stress in Oats and the Alleviatory Effects of Exogenous Spermine Application

Abstract

Alkali stress limits plant growth and yield more strongly than salt stress and can lead to the appearance of yellow leaves; however, the reasons remain unclear. In this study, we found that (1) the down-regulation of coproporphyrinogen III oxidase, protoporphyrinogen oxidase, and Pheophorbide a oxygenase in oats under alkali stress contributes to the appearance of yellow leaves (as assessed by proteome and western blot analyses). (2) Some oat proteins that are involved in the antioxidant system, root growth, and jasmonic acid (JA) and indole-3-acetic acid (IAA) synthesis are up-regulated in response to alkalinity and help increase alkali tolerance. (3) We added exogenous spermine to oat plants to improve their alkali tolerance, which resulted in higher chlorophyll contents and plant dry weights than in plants subjected to alkaline stress alone. This was due to up-regulation of chitinase and proteins related to chloroplast structure, root growth, and the antioxidant system. Spermine addition increased sucrose utilization efficiency, and promoted carbohydrate export from leaves to roots to increase energy storage in roots. Spermine addition also increased the IAA and JA contents required for root growth.

Keywords: alkali stress; carbohydrate; hormone; oat; proteome; spermine.

FIGURE 1

Effects of exogenous spermine on oat growth under alkali stress at booting stage. (A) The cone-tainer planting method. (B) Effects of alkali stress on chlorophyll contents at varied stages. (C) Image related to the effects of exogenous spermine on shoot growth under alkali stress at booting stage. (D) Image related to the effects of exogenous spermine on root growth under alkali stress at booting stage. (E) Effects of spermine on chlorophyll contents under alkali stress at booting stage. (F) Effects of spermine on shoot dry weight per plant under alkali stress at booting stage. (G) Effects of spermine on root dry weight per plant under alkali stress at booting stage. CK, control; AS, alkali stress (35 mmol.L^–1 Na₂CO₃:NaHCO₃ = 1:1); AS + Spm, 35 mmol.L^–1 Na₂CO₃:NaHCO₃ + 0.01 mmol.L^–1 spermine.

FIGURE 2

Principal component analysis among all the replicates and treatments. CK, control; AS, alkali stress (35 mmol.L^–1 Na₂CO₃:NaHCO₃ = 1:1); AS + Spm, alkali stress + spermine (35 mmol.L^–1 Na₂CO₃:NaHCO₃ + 0.01 mmol.L^–1 spermine); R, root; L, leaf, 1, replicate 1; 2, replicate 2; 3, replicate 3.

FIGURE 3

(A) GO and (B) KEGG analysis of differentially expressed proteins in leaves at CK vs. AS. CK, control; AS, alkali stress (35 mmol.L^–1 Na₂CO₃:NaHCO₃ = 1:1). The color gradient indicated significance, *** indicated P < 0.001, ** indicated P < 0.01, * indicated P < 0.05.

FIGURE 4

Western blot analysis. CK, control; AS, alkali stress (35 mmol.L^–1 Na₂CO₃:NaHCO₃ = 1:1); AS + Spm, alkali stress + spermine (35 mmol.L^–1 Na₂CO₃:NaHCO₃ + 0.01 mmol.L^–1 spermine); R, roots; L, leaves. The coproporphyrinogen III oxidase, protoporphyrinogen oxidase, ATP-dependent zinc metalloprotease FTSH 8 and cytochrome f expressions of leaves at CK vs. AS were detected by western blot assay, and representative bands were shown in panel (A), the cytochrome f and photosystem II 10 kDa polypeptide expressions of leaves at AS vs. AS + Spm were detected by western blot assay, and representative bands were shown in panel (B), the profilin expressions of roots at AS vs. CK was detected by western blot assay, and representative bands was shown in panel (C), the glucan endo-1,3-beta-glucosidase, polyphenol oxidase expressions of roots at AS vs. AS + Spm were detected by western blot assay, and representative bands were shown in panel (D).

FIGURE 5

Effects of spermine on ABA, ACC, IAA, JA contents under alkali stress. CK, control; AS, alkali stress (35 mmol.L^–1 Na₂CO₃:NaHCO₃ = 1:1); AS + Spm, alkali stress + spermine (35 mmol.L^–1 Na₂CO₃:NaHCO₃ + 0.01 mmol.L^–1 spermine).