Regulation Of Iron-Deficiency Responses By Carbon Monoxide And Identification Of Iron Deficiency-Responsive Micrornas In Arabidopsis Thaliana

Carbon monoxide (CO) is an endogenous gaseous molecule and regulates a variety of biological processes in animals. However, whether CO regulates biochemical and stressful responses in plants is largely unknown. In this study, we described a novel role of CO in regulating iron-homeostasis in iron-starved Arabidopsis.50μM CO aqueous solution was able to prevent iron-deficiency induced chlorosis and improve chlorophyll accumulation in Arabidopsis. CO-mediated increase in chlorophyll content was dose-dependent. Also,50μM CO aqueous solution could revert chlorosis of monocot maize under iron deficiency. Further, expressions of FIT1, AtFRO2and AtIRTl were up-regulated by CO exposure in iron-starved seedlings.50μM CO aqueous solution not only increased level of FRO2transcripts, but also promoted significantly the activity of ferric-chelate reductase as compared to the treatment of iron-deficiency alone, which indicated higher ability of reduction of Fe(Ⅲ) to Fe(Ⅱ) in roots, and this change may be convenient for iron uptake in Arabidopsis. In addition, CO caused morphological changes of iron-deficient roots and increased root hair density and length. After analysis of iron content in roots and shoots of Arabidopsis, we found that iron content was also significantly higher in50μM CO aqueous solution-treated seedlings than those of the controls (iron-deficiency alone).Endogenous CO is a product of heme catalyzed by heme oxygenase1(HO1). Transcription of HOI in roots and shoots of Arabidopsis was induced under iron deficiency, which suggest that endogenous CO respond to iron deficiency in Arabidopsis. In order to study the possible role of HO1gene or endogenous CO in iron nutrition, we used Arabidopsis hyl and hy2mutants in our experiments. Endogenous CO by HO1was hardly produced in hyl mutants because of mutation of HOI gene. In contrast, production of endo-genous CO may not be affected in hy2mutants because of presence of HOI gene. Treatment of hy2mutants with50μM CO aqueous solution could revert chlorosis of hy2mutants and improve chlorophyll content in leaves under iron deficiency. While50μM CO aqueous solution was not able to revert chlorosis of hyl mutants under iron deficiency. The above results indicated HO1gene or endogenous CO might play a role in exogenous CO-mediated chlorophyll increase in Arabidopsis.To evaluate the capacity of CO to avoid the iron deficiency phenotype when iron uptake mechanisms are altered, ysl and ys3maize plants were treated with50μM CO. CO was able to completely revert interveinal chlorosis of iron-inefficient maize mutants yellow stripel (ysl) and yellow stripe3(ys3) and improve chlorophyll content.In order to investigate the possible role of endogenous NO among CO-mediated chlorophyll increase under iron deficiency, we detected NO production in roots using DAF-2DA fluorescent emission. NO production and root hairs increased after50μM CO treatment, when cPTIO, the NO specific scavenger, was used to treat iron-deficiency seedlings in the presence of CO, the results showed that cPTIO was able to prevent the CO-improved chlorophyll accumulation and reduced the transcription of iron uptake-related genes and root hair development. The above results suggest that endogenous NO involve in CO-regulated chlorophyll increase in Arabidopsis. Additionally, we also investigated the possible role of HO1gene or endogenous CO in exogenous NO-mediated chlorophyll increase using Arabidopsis hyl and hy2mutants. The NO donor, SNP (5μM) improved the chlorophyll content of Arabidopsis hy2mutants under iron deficiency. While treatment with SNP (5μM) could not revert chlorosis of hyl mutants under same conditions. These results suggest that HOI gene or endogenous CO play a role in exogenous NO-mediated chlorophyll increase in Arabidopsis.MicroRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that have base pair with their target genes to repress their translation or induce their degradation. Recent studies have shown that several miRNAs regulate plant adaptation to sulfate, phosphate, nitrogen and copper deficiency, respectively. However, whether miRNAs are involved in regulation of stressful response to iron deficiency is unknown. In this study, we carried out a survey of Arabidopsis microRNA genes in response to iron-deficiency and identified IDE1/IDE2(/ron-Deficiency-responsive cis-Element1and2) in their promoter regions. We constructed a small RNA library from Arabidopsis seedlings under iron-deficiency. Sequencing analysis revealed8known miRNA genes in5families, all of which were up-regulated under iron-deficiency. Further, we analyzed c/s-regulatory elements upstream of187of all known miRNA genes in Arabidopsis and found24miRNA genes containing IDE1/IDE2-Like motifs in their promoter regions. Transcriptional analysis using RT-PCR have shown that70.8%(17/24) of the IDE-containing miRNA genes were up-expressed in response to iron-deficiency. Our analysis approach for this study is useful and efficient based on our results, so it is applicable to cis-element finding for miRNAs responding to any other abiotic stresses. Also, the data obtained in this study may aid our understanding of the role of iron-deficiency responsive specific sequences upstream of miRNA genes and the functional implications of miRNA genes in response to iron stress in plants.