Ascorbate Alleviates Fe Deficiency-Induced Stress in Cotton (Gossypium hirsutum) by Modulating ABA Levels

Abstract

Fe deficiency causes significant losses to crop productivity and quality. To understand better the mechanisms of plant responses to Fe deficiency, we used an in vitro cotton ovule culture system. We found that Fe deficiency suppressed the development of ovules and fibers, and led to tissue browning. RNA-seq analysis showed that the myo-inositol and galacturonic acid pathways were activated and cytosolic APX (ascorbate peroxidase) was suppressed in Fe-deficient treated fibers, which increased ASC (ascorbate) concentrations to prevent tissue browning. Suppression of cytosolic APX by RNAi in cotton increased ASC contents and delayed tissue browning by maintaining ferric reduction activity under Fe-deficient conditions. Meanwhile, APX RNAi line also exhibited the activation of expression of iron-regulated transporter (IRT1) and ferric reductase-oxidase2 (FRO2) to adapt to Fe deficiency. Abscisic acid (ABA) levels were significantly decreased in Fe-deficient treated ovules and fibers, while the upregulated expression of ABA biosynthesis genes and suppression of ABA degradation genes in Fe-deficient ovules slowed down the decreased of ABA in cytosolic APX suppressed lines to delay the tissue browning. Moreover, the application of ABA in Fe-deficient medium suppressed the development of tissue browning and completely restored the ferric reduction activity. In addition, ABA 8'-hydroxylase gene (GhABAH1) overexpressed cotton has a decreased level of ABA and shows more sensitivity to Fe deficiency. Based on the results, we speculate that ASC could improve the tolerance to Fe deficiency through activating Fe uptake and maintaining ABA levels in cotton ovules and fibers, which in turn reduces symptom formation.

Keywords: Fe deficiency; abscisic acid; ascorbate; cotton fiber; cytosolic APX.

FIGURE 1

The phenotype and ferric reduction activity of fiber-bearing ovules cultured with different concentrations of Fe. (A) 0.5 DPA ovules of YZ1 were cultured for 12 days in medium with various concentrations of Fe (0, 15, 30, 60, 120, and 240 μM). Fe deficiency (0 μM) suppressed the development of ovule and fiber and resulted in tissue browning. Scale bar: 5 mm. Phenotype (B), fresh weight per ovule (C) and ferric reduction activities (D) of YZ1 0.5-DPA-ovules cultured for 6 days under Fe deficient (-Fe, 0 μM Fe) or Fe sufficient (+Fe, 60 μM Fe) conditions. Scale bar: 2 mm (B). (E) Ferric reduction activities of ovules cultured under Fe deficient or Fe sufficient condition for 10 days. Ferric reduction activities (F) and phenotype (G) of xuzhou142 (xu142) and xuzhou142 fuzzless-lintless (xu142-fl) mutant cultured under Fe deficient (-Fe, 0 μM Fe) or Fe sufficient (+Fe, 60 μM Fe) conditions for 6 days. Error bars represent SD. Every sample includes four biological replicates. Scale bar: 2 mm (G). Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05).

FIGURE 2

Screening and analysis of differentially expressed genes (DEGs) in fibers cultured with three concentrations of Fe for 10 days. (A) Phenotype of ovules and fibers cultured for 10 days in medium with three concentrations of Fe. Scale bar: 5 mm. -Fe, 0 μM Fe; +Fe, 60 μM Fe; ++Fe, 240 μM Fe. (B) DEGs in fibers between each two treatments with Fe. +Fe/-Fe indicates the gene expression levels in +Fe compared to the gene expression levels in -Fe. ++Fe/-Fe indicates the gene expression levels in ++Fe compared to the gene expression levels in -Fe. ++Fe/+Fe indicates the gene expression levels in ++Fe compared to the gene expression levels in +Fe. (C) Venn diagram of DEGs in the response to three levels of Fe. (D) PCA plots of six simples treated with three concentrations of Fe (each treatment has two biological repeats) based on RNA-seq data.

FIGURE 3

The expression level changes of Fe deficiency response marker genes and the contents of ions in 10 days fibers or ovules treated with three concentrations of Fe. (A) Cluster of marker genes responding to Fe deficiency (-Fe) based on the expression levels in 10 days fibers treated with three concentrations of Fe. -Fe, 0 μM Fe; +Fe, 60 μM Fe; ++Fe, 240 μM Fe. Contents of Fe (B), K and Ca (C), Zn, Mn, Mo, and Co (D) in ovules treated with three concentrations of Fe for 10 days. Each treatment includes three biological replicates. Error bars represent SD. Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05).

FIGURE 4

Fe deficiency leads to an increase in ASC in ovules and fibers after 10 days culture. (A) Cluster analysis of the expression levels for ASC synthesis and metabolism related genes responding to Fe deficiency. -Fe, 0 μM Fe; +Fe, 60 μM Fe; ++Fe, 240 μM Fe. (B) The contents of ASC increased in ovules and fibers treated with Fe deficiency for 10 days. Each treatment includes six biological replicates. Error bars represent SD. The concentrations of glucose (C), fructose (D), and sucrose (E) in ovules and fibers after treatment with three concentrations of Fe for 10 days. Each treatment includes six biological replicates. Error bars represent SD. Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05).

FIGURE 5

Cytosolic APX RNAi lines (IAO) showed higher tolerance than the control (Null) to Fe deficiency stress. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of cytosolic APX genes (GhAPX1, GhAPX8, GhAPX9, and GhAPX10) in ovules of transgenic cottons and the control cultured for 10 days under Fe-deficient or Fe-sufficient conditions. (B) Phenotype of GhAPX1 overexpressor (OA15/OA17), the control (Null) and cytosolic APXs suppressed (IAO24/IAO167) fiber-bearing ovules treated for 10 days under Fe-deficient and Fe-sufficient conditions. Scale bar: 5 mm. The dynamic phenotypes of GhAPX1 overexpressor (OA), control (Null) and cytosolic APXs suppressed (IAO) fiber-bearing ovules treated for 6 days (C), 8 days (D), 10 days (E) and 12 days (F) under Fe-deficient condition. Scale bar: 5 mm (C–F). Fresh weight per ovule of transgenic cotton ovules cultured in Fe-deficient or -sufficient medium for 6 days (G) or 10 days (H). Ferric reduction activities of transgenic cotton cultured for 6 days (I) or 10 days (J) under Fe-deficient or -sufficient conditions. -Fe, 0 μM Fe; +Fe, 60 μM Fe. Error bars represent SD. Every sample includes four biological replicates. Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05).

FIGURE 6

Optimal ascorbate (ASC) increases the tolerance of ovules to Fe deficiency. (A) The concentrations of ASC in ovules and fibers of APX transgenic lines and the control cultured in Fe-deficient (-Fe) or Fe-sufficient medium (+Fe) after 10 days culture. -Fe, 0 μM Fe; +Fe, 60 μM Fe. The phenotype (B), Ferric reduction activities (C) and fresh weight per ovule (D) of ovules cultured in Fe deficient (-Fe) or Fe sufficient (+Fe) medium applied with four levels of ASC. Scale bar: 5 mm (B). Error bars represent SD. Every sample includes four biological replicates. Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05).

FIGURE 7

Reduced decrease in ABA content in cytosolic APX RNAi (IAO) tissues improves tolerance to Fe deficiency stress. Quantification of ABA concentrations in fibers (A) and ovules (B) of APX transgenic lines and the control after 6, 10, or 12 days culture. -Fe, 0 μM Fe; +Fe, 60 μM Fe; ++Fe, 240 μM Fe. OA, GhAPX1 overexpression line; Null, the control; IAO, cytosolic APXs suppression line. (C) Concentrations of ABA in ovules and fibers cultured with three concentrations of Fe for 10 days. Each treatment includes four biological replicates. Error bars represent SD. Values with different letters above bars in the histogram indicate significant differences (one-way ANOVA and Duncan’s multiple comparisons, P < 0.05). (D,E) a serious of ABA application suppresses symptoms induced by Fe deficiency after 10 days culture. Scale bars: 5 mm (D). (F) The exogenous application of 10 μM ABA could not restore the fresh weight per ovule inhibited by Fe deficiency. (G) The exogenous application of 10 μM ABA completely recover the ferric reduction of ovules treated with Fe deficiency.

FIGURE 8

Reverse transcription polymerase chain reaction (qRT-PCR) analysis of ABA biosynthesis and metabolism genes in ovules of transgenic cotton and control, cultured for 10 days under Fe-deficient or Fe-sufficient conditions. The relative expression levels of genes were normalized against the expression of the cotton GhUB7 gene. Error bars represent SD of three technological repeats.