MPK3/MPK6 are involved in iron deficiency-induced ethylene production in Arabidopsis

Abstract

Iron (Fe) is an essential micronutrient that participates in various biological processes important for plant growth. Ethylene production induced by Fe deficiency plays important roles in plant tolerance to stress induced by Fe deficiency. However, the activation and regulatory mechanisms of 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) genes in this response are not clear. In this study, we demonstrated that Fe deficiency increased the abundance of ACS2, ACS6, ACS7, and ACS11 transcripts in both leaves and roots as well as the abundance of ACS8 transcripts in leaves and ACS9 transcripts in roots. Furthermore, we investigated the role of mitogen-activated protein kinase 3 and 6 (MPK3/MPK6)-regulated ACS2/6 activation in Fe deficiency-induced ethylene production. Our results showed that MPK3/MPK6 transcript abundance and MPK3/MPK6 phosphorylation are elevated under conditions of Fe deficiency. Furthermore, mpk3 and mpk6 mutants show a lesser induction of ethylene production under Fe deficiency and a greater sensitivity to Fe deficiency. Finally, in mpk3, mpk6, and acs2 mutants under conditions of Fe deficiency, induction of transcript expression of the Fe-deficiency response genes FRO2, IRT1, and FIT is partially compromised. Taken together, our results suggest that the MPK3/MPK6 and ACS2 are part of the Fe starvation-induced ethylene production signaling pathway.

Keywords: 1-Aminocyclopropane-1-carboxylic acid synthase (ACS); Arabidopsis; Fe deficiency; ethylene; mitogen-activated protein kinase (MPK).

Figure 1

Time-course analysis of ethylene production in WT plants under Fe-sufficient (+Fe) and -deficient (−Fe) conditions. Ten-day-old seedlings were transferred to Fe-sufficient (50 μM EDTA-Fe) or Fe-deficient (0 μM EDTA-Fe) nutrient solutions. For each time point shown, ethylene accumulated over a 24 h period from 10 seedlings in the headspace of GC vials was measured. Data are shown as the mean ±SEM (n = 3). Columns marked with “^*” indicate a significant difference (P < 0.05), and “^**” indicate a highly significantly difference (P < 0.01).

Figure 2

Transcription levels of ACS genes in the leaves and roots of WT seedlings under Fe-sufficient (+Fe) and -deficient (−Fe) conditions. One-month-old seedlings were transferred to +Fe (50 μM EDTA-Fe) or −Fe (0 μM EDTA-Fe) nutrient solutions for 7 days. Leaves and roots were collected for RNA extraction. All values are expressed relative to the expression level under Fe-sufficient conditions (control—set to 1.0) as appropriate. Data are shown as the mean ±SEM (n = 3). Column marked with “**” indicate a highly significantly difference (P < 0.01). n.d. indicates “not detectable.”

Figure 3

Ethylene production of WT, acs mutant plants under Fe-sufficient (+Fe) and -deficient (−Fe) conditions. Ten-day-old seedlings were transferred to Fe-sufficient (50 μM EDTA-Fe) or -deficient (0 μM EDTA-Fe) nutrient solutions. Ethylene accumulated over a 24 h period from 10 seedlings in the headspace of GC vials was measured at 4 day after the treatment. Data are shown as the mean ±SEM (n = 3). Columns marked with “^*” indicate a significant difference (P < 0.05), and “^**” indicate a highly significantly difference (P < 0.01).

Figure 4

Transcription levels of Fe-deficiency responsive genes in acs2 and acs6 mutant plants. Ten day old seedlings cultured in swimming medium were transferred to Fe-deficient medium for 7 days and sampled for RNA extraction. All values are expressed relative to the expression level under Fe-sufficient conditions (control—set to 1.0) as appropriate. Data are shown as the mean ±SEM (n = 3). Column marked with “*” indicate a significant difference (P < 0.05).

Figure 5

Fe deficiency activates MPK3 and MPK6. (A) Transcription levels of MPK3 and MPK6 in the leaf and root of WT seedlings under Fe-sufficient (50 μM EDTA-Fe) and -deficient (0 μM EDTA-Fe) conditions. All values are expressed relative to the expression level under Fe-sufficient conditions (control—set to 1.0) as appropriate. Data are shown as the mean ±SEM (n = 3). (B) Immunoprecipitation kinase assay of MPK3 and MPK6 activity using Anti-p44/42 MAPK antibody. Ten-day-old seedlings were transferred to Fe-sufficient or -deficient nutrient solutions. Column marked with “**” indicate a highly significantly difference (P < 0.01).

Figure 6

Ethylene production and expression of ACS2 and ACS6 in mpk3 and mpk6 mutants. (A) Ethylene production in mpk3 and mpk6 mutants. (B) Transcript abundances of ACS2 and ACS6 genes in mpk3 and mpk6 mutants. Ten-day-old seedlings were transferred to Fe-sufficient (50 μM EDTA-Fe) or -deficient (0 μM EDTA-Fe) nutrient solutions. Ethylene accumulated over a 24 h period from 10 seedlings in the headspace of GC vials was measured at 4 days after the treatment. qRT-PCR was conducted using RNA extracted from whole seedlings at 7 days after –Fe treatment. Data are shown as the mean ±SEM (n = 3). Columns marked with “^*” indicate a significant difference (P < 0.05), and “^**” indicate a highly significantly difference (P < 0.01).

Figure 7

Phenotypes of mpk3 and mpk6 mutants grown in Fe-sufficient or Fe-deficient conditions. (A) Growth performance of WT, mpk3, and mpk6 mutant plants. 30 day-old seedlings were grown in Fe -sufficient (50 μM) or -deficient (0 μM) nutrient solution for 7 days. (B) Young leaves (from the third to the eighth) were detached to display their phenotypes. (C) SPAD values of WT, mpk3, and mpk6 mutants. (D) Soluble Fe concentration of WT, mpk3, and mpk6 mutants. Scale bar = 3 cm. Data are shown as the mean ±SEM (n = 3). Column marked with “*” indicate a significant difference (P < 0.05).

Figure 8

Transcription levels of Fe-deficiency responsive genes in mpk3 and mpk6 mutants. Ten day old seedlings cultured in swimming medium were transferred to Fe-deficient medium for 7 days and sampled for RNA extraction. All values are expressed relative to the expression level under Fe-sufficient conditions (control—set to 1.0) as appropriate. Data are shown as the mean ±SEM (n = 3). Columns marked with “^*” indicate a significant difference (P < 0.05), and “^**” indicate a highly significantly difference (P < 0.01).