Regulatory Factors Involved In Cellular Signal Transduction Upon Iron Deficiency In Arabidopsis

Iron (Fe) deficiency is one of the major limiting factors affecting crop production in calcareous soils worldwide. Fortunately, many plants have developed various strategies in the process of Fe solubilization, uptake, transport and reutilization to cope with Fe deficiency in those soils. Nongraminaceous monocots and dicots plants, so-called Strategy I plants, employ a range of responses to Fe deficiency to increase Fe acquisition from soil, including:(1) induction of both a plasmalemma ferric-chelate reductase (FCR) and plasmalemma Fe(II) transporter in root cells,(2) enhanced release of protons, chelators and reductants such as organic acids and phenolic compounds into the rhizosphere, and (3) changes in root architecture, including enhanced root branching and sub-apical root hair development. Among these responses, the activation of FCR is of great importance (Curie and Briat,2003) as Strategy I plants must enzymatically reduce Fe(III) before their root cells can take it up in the form of Fe(II). However, the signals involved in the regulatory cascade leading to these Fe-deficiency-induced responses are still not well understood. In this study, a Strategy I model plant, Arabidopsis(Arabidopsis thaliana) was employed to investigate the roles of plant hormones and other regulation factors involved in the induction of these reponses. These results are summarized as following:1. NO acts downstream of auxin in FCR inductionIn response to Fe deficiency, dicots employ a reduction-based mechanism by inducing ferric-chelate reductase (FCR) at the root plasma membrane to enhance Fe uptake. However, the signal pathway leading to FCR induction is still unclear. Here, we found that Fe-deficiency-induced increase of auxin and nitric oxide (NO) levels in the wild type Arabidopsis (WT) was accompanied by up-regulation of root FCR activity, the bHLH transcription factor FIT and the ferric reductase FRO2gene expression, which was further stimulated by application of auxin analog (NAA) or NO donor (GSNO), but suppressed by either polar auxin transport inhibition with NPA or NO scavenging with cPTIO, tungstate, or L-NAME. On the other hand, root FCR activity, NO level and gene expression of FIT and FRO2were even higher in auxin-overproducing mutant yucca under Fe deficiency, which were sharply restrained by cPTIO treatment. The opposite response was observed in a basipetal auxin transport impired mutant auxl-7, which was slightly rescued by exogenous GSNO application. Furthermore, Fe deficiency or NAA application failed to induce Fe deficiency responses in noal and nial nia2, two mutants with reduced NO synthesis, but root FCR activities in both mutants could be significantly elevated by GSNO. The inability to induce NO burst and FCR activity was also found in a double mutant yucca noal with elevated auxin production and reduced NO accumulation. Therefore, we presented a novel signaling pathway that NO acts downstream of auxin to activate root FCR activity under Fe deficiency in Arabidopsis.2. The function of14-3-3protein GRF11in term of Fe deficiency responseThe14-3-3proteins are a family functioning as phosphoserine-binding proteins that regulate a wide array of target proteins involved in signal transduction, cell cycle, metabolism, and stress response. We found that loss-of-function of GRF11resulted in the failure of acidification, FCR induction and the expression of genes mediating Fe uptake and acidification, namely IRTI, FR02, and AHA2both under Fe deficient and sufficient conditions, thus decreased Fe uptake, implying GRF11is required for the proper operation of Fe acquisition mechanisms at physiological and gene expression levels in Arabidopsis. Moreover, we found that the expression of GRF11and FIT was inter-related, on one hand, knockout of GRF11resulted in significant decrease of FIT transcription and over-expression of GRF11enhanced FIT transcription under Fe-sufficient conditions, implying a positive regulation of GRF11on FIT expression, but has no influence on those genes whose expression was involved in posttranscriptional regulation of FIT. On the other hand, knockout of FIT or over-expression of FIT caused significant decrease or increase of GRF11transcription under Fe-sufficient conditions, supporting FIT as a transcription regulator of GRF11. Taken these results together, a model can be proposed in which Fe deficiency results in GRF11accumulation, and GRF11transcriptional regulates the expression of FIT, which in turn promotes GRF11transcription. Furthermore, we demonstrated that NO-dependent regulation of FIT transcription under Fe deficiency requires the functioning of GRF11. First, in the WT plant, Fe-deficiency-induced expression of GRF11could be further enhanced by exogenous application of NO and eliminated by NO scavengers and inhibitors of NO biosynthesis. Secondly, in the endogenous NO levels reduced mutant plants, the transcriptional response of GRF11to Fe deficiency was eliminated. Finally, in GRF11knockout mutant, exogenous application of NO could not restore the Fe-deficiency-induced FCR activity. In conclusion, we demonstrated that a member from14-3-3protein family, GRF11, acts downstream of NO to trigger Fe-deficiency-induced response.in Arabidopsis, and the activity of FIT is necessary for the transcriptional activation of GRF11expression in the signaling pathway.