The Roles And Mechanisms Of Sucrose And Nitric Oxide In Regulating Plants Iron-deficiency-induced Responses

Iron (Fe) is an essential microelement for plants, although Fe is the fourth abundant element in the earth's crust, however, in alkaline or calcareous soils with aerobic condition, the bioavailability of Fe is far below the level that required for normal plant growth. It's estimated that about 40% of world's arable lands were suffered from Fe deficiency to various degrees. Therefore, Fe deficiency became as one of the most limiting factors affecting crop production and quality worldwide. Plants have developed a series of adaptive strategies in response to Fe deficiency, and the regulation of Fe-deficiency-induced responses is a complex process regulated at different levels by the interaction of various signal molecules and transcription factors. Although the molecular and physiological mechanisms of Fe acquisition has been well clarified, the signal process underlying Fe-deficiency-induced responses still remain largely unkonwn. Therefore, in this study, we first investigated the gene expression profile in response to Fe deficiency in Arabidopsis(Arabidopsis thaliand) on transcript level by using RNA-Seq (RNA-sequencing), and proposed factors which may be involved in the regulation of Fe-deficiency-induced responses, and then we investigated the roles of these factors in the regulation of Fe-deficiency-induced responses by using the physiological, genetic and molecular biological approaches. The main results are as follows:1. The gene expression profile of Arabidopsis (Arabidopsis thaliana) under Fe deficiency was investigated by using RNA-Seq. The results showed that there are 316 genes differentially expressed in Fe-deficient roots as compared with Fe-sufficient ones. Among them 232 genes were significantly induced by Fe deficiency, while 84 genes were suppressed. GO (Gene Ontology) biological processes analysis indicated that these significantly affected genes mainly related to the physiological processes including phyto-hormones signaling, ions homeostasis, carbohydrate metabolism, secondary metabolism, cell wall synthesis and modification, defense responses., we found that genes related to Fe acquisition, including IRT1, FRO2, OPT3, NAS1 and transcription factors bHLHs that regulate Fe-uptake-related genes were significantly induced by Fe deficiency. The modulation of these genes under Fe deficiency should play an important role in enhancing plant's Fe acquisition capacity. Additionally, Fe deficiency also significantly up-regulate many genes involved in ethylene biosynthesis and signaling, auxin signaling which have previously been demonstrated to be involeved in the regulation of Fe-deficiency responses. These results implying that these signal molecules may play critical roles in regulating Fe-deficiency-induced responses. Interestingly, the genes being increased belong to the metabolism and transportation of carbohydrate and cell wall synthesis and modifications were also observed under Fe-deficient condition. Among them, the Fe-deficiency-induced enhanced sucrose translocation may affect auxin signaling, thus plays a role in the regulation of Fe acquisition, whereas modulation of cell wall synthesis and modification may have an impact on the reutilization of root apoplastic Fe.2. The regulatary roles and mechanisms of sucrose (Sue) on Fe-deficiency-induced responses was investigated by using Arabidopsis (Arabidopsis thaliana) of wild-type, Sue transport mutant suc2-5, Sue transport over-expression line 35S::SUC2 and auxin transport mutants auxl-7 and pinl-1.The results showed that Fe-deficiency increased sucrose level in roots of Arabidopsis. Exogenous application of sucrose further stimulated Fe-deficiency-induced ferric-chelate-reductase (FCR) activity and expression of Fe-acquisition-related genes FRO2, IRT1, and FIT in roots. The opposite patterns were observed in the dark treatment. In addition, FCR activity and expression of Fe-acquisition-related genes were higher in the sucrose-high-accumulating transgenic plant 35S::SUC2, but were lower in the sucrose-low-accumulating mutant suc2-5, compared to wild-type plants under Fe-deficient conditions. Consequently, Fe-deficiency tolerance was enhanced in 35S::SUC2, but was compromised in suc2-5. Furthermore, exogenous sucrose also increased root GUS activity in auxin-inducible reporter DR5-GUS transgenic plants under Fe-deficiency. However, exogenous sucrose failed to increase FCR activity and expression of Fe-acquisition-related genes in the auxin transport-impaired mutants aux1-7 and pin1-1, as well as in the wild type plants treated with an auxin transport inhibitor, under Fe-deficiency. Therefore, Fe-deficiency-induced accumulation of Sue in roots acts upstream of auxin in regulating Fe deficiency responses by increasing FCR activity and up-regulating genes involved in Fe acquisition, and consequently enhances plants tolerance to Fe deficiency.3. The RNA-Seq results indicated that the regulation of cell wall synthesis and modification may affect the reutilization of Fe stored in root apoplast. Our group previously reported that Fe deficiency can increase nitric oxide (NO) production in roots. However, the regulatory role of NO on the synthesis cell walls and its association with the reutilization of root apoplatic Fe remain unclear. Hence, the effect of NO production in response to Fe deficiency on the composition and chemical properties of root cell walls and its association with the reutilization of root apoplatic Fe were investigated by using tomato(Solarium lycopersicum). The results showed that Fe deficiency elevated NO level in tomato roots. However, application of S-nitrosoglutathione, a NO donor, significantly enhanced Fe retention in root apoplast of iron-deficient plants, accompanied with a decrease of Fe level in xylem sap. Consequently, S-nitrosoglutathione treatment increased Fe concentration in roots, but decreased it in shoots. The opposite was true for the NO scavenging treatment with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide (cPTIO). Interestingly, S-nitrosoglutathione treatment increased pectin methylesterase activity and decreased degree of pectin methylation in root cell wall of both Fe-deficient and Fe-sufficient plants, which led to an increased Fe retention in pectin fraction, thus increasing the binding capacity of Fe to the extracted cell wall. Altogether, these results suggested that Fe-deficiency-induced elevation of NO increases Fe immobilization in root apoplast by decreasing pectin methylation in cell wall, and ultimately inhibited the translocation of Fe from roots to shoots.