Fe Deficiency-responsive Genes Research Articles

A global transcriptional activator involved in the iron homeostasis in cyanobacteria.

Cyanobacteria use a series of adaptation strategies and a complicated regulatory network to maintain intracellular iron (Fe) homeostasis. Here, a global activator named IutR has been identified through three-dimensional chromosome organization and transcriptome analysis in a model cyanobacterium Synechocystis sp. PCC 6803. Inactivation of all three homologous IutR-encoding genes resulted in an impaired tolerance of Synechocystis to Fe deficiency and loss of the responses of Fe uptake-related genes to Fe-deplete conditions. Protein-promoter interaction assays confirmed the direct binding of IutR with the promoters of genes related to Fe uptake, and chromatin immunoprecipitation sequencing analysis further revealed that in addition to Fe uptake, IutR could regulate many other physiological processes involved in intracellular Fe homeostasis. These results proved that IutR is an important transcriptional activator, which is essential for cyanobacteria to induce Fe-deficiency response genes. This study provides in-depth insights into the complicated Fe-deficient signaling network and the molecular mechanism of cyanobacteria adaptation to Fe-deficient environments.

Multilayered regulation of iron homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient for plant growth and development due to its role in crucial processes such as photosynthesis and modulation of the redox state as an electron donor. While Fe is one of the five most abundant metals in the Earth's crust, it is poorly accessible to plants in alkaline soils due to the formation of insoluble complexes. To limit Fe deficiency symptoms, plant have developed a highly sophisticated regulation network including Fe sensing, transcriptional regulation of Fe-deficiency responsive genes, and post-translational modifications of Fe transporters. In this mini-review, we detail how plants perceive intracellular Fe status and how they regulate transporters involved in Fe uptake through a complex cascade of transcription factors. We also describe the current knowledge about intracellular trafficking, including secretion to the plasma membrane, endocytosis, recycling, and degradation of the two main Fe transporters, IRON-REGULATED TRANSPORTER 1 (IRT1) and NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1). Regulation of these transporters by their non-Fe substrates is discussed in relation to their functional role to avoid accumulation of these toxic metals during Fe limitation.

The transcription factor POPEYE negatively regulates the expression of bHLH Ib genes to maintain iron homeostasis.

Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency-responsive genes to promote Fe uptake. POPEYE (PYE) is a key bHLH (basic helix-loop-helix) transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive in Arabidopsis. We found that the overexpression of PYE attenuates the expression of Fe deficiency-responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an ethylene response factor-associated amphiphilic repression (EAR) motif, it does not interact with the transcriptional co-repressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Sub-cellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a nuclear export signal (NES) which is required for the cytoplasmic localization of PYE. Mutation of the NES amplifies the repression function of PYE, resulting in down-regulation of Fe deficiency-responsive genes. Co-expression assays indicated that three bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses the transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides new insights into the Fe deficiency response signalling pathway and enhances the understanding of PYE functions in Arabidopsis.

The bHLH Transcription Factor OsbHLH057 Regulates Iron Homeostasis in Rice

Many basic Helix-Loop-Helix (bHLH) transcription factors precisely regulate the expression of Fe uptake and translocation genes to control iron (Fe) homeostasis, as both Fe deficiency and toxicity impair plant growth and development. In rice, three clade IVc bHLH transcription factors have been characterised as positively regulating Fe-deficiency response genes. However, the function of OsbHLH057, another clade IVc bHLH transcription factor, in regulating Fe homeostasis is unknown. Here, we report that OsbHLH057 is involved in regulating Fe homeostasis in rice. OsbHLH057 was highly expressed in the leaf blades and lowly expressed in the roots; it was mainly expressed in the stele and highly expressed in the lateral roots. In addition, OsbHLH057 was slightly induced by Fe deficiency in the shoots on the first day but was not affected by Fe availability in the roots. OsbHLH057 localised in the nucleus exhibited transcriptional activation activity. Under Fe-sufficient conditions, OsbHLH057 knockout or overexpression lines increased or decreased the shoot Fe concentration and the expression of several Fe homeostasis-related genes, respectively. Under Fe-deficient conditions, plants with an OsbHLH057 mutation showed susceptibility to Fe deficiency and accumulated lower Fe concentrations in the shoot compared with the wild type. Unexpectedly, the OsbHLH057-overexpressing lines had reduced tolerance to Fe deficiency. These results indicate that OsbHLH057 plays a positive role in regulating Fe homeostasis, at least under Fe-sufficient conditions.

The ferroxidases LPR1 and LPR2 control iron translocation in the xylem of Arabidopsis plants

Iron (Fe) deficiency is common in agricultural crops and affects millions of people worldwide. Translocation of Fe in the xylem is a key step for Fe distribution in plants. The mechanism controlling this process remains largely unknown. Here, we report that two Arabidopsis ferroxidases, LPR1 and LPR2, play a crucial and redundant role in controlling Fe translocation in the xylem. LPR1 and LPR2 are mainly localized in the cell walls of xylem vessels and the surrounding cells in roots, leaves, and stems. Knockout of both LPR1 and LPR2 increased the proportion of Fe(II) in the xylem sap, and caused Fe deposition along the vascular bundles especially in the petioles and main veins of leaves, which was alleviated by blocking blue light. The lpr1 lpr2 double mutant displayed constitutive expression of Fe deficiency response genes and overaccumulation of Fe in the roots and mature leaves under Fe-sufficient supply, but Fe deficiency chlorosis in the new leaves and inflorescences under low Fe supply. Moreover, the lpr1 lpr2 double mutant showed lower Fe concentrations in the xylem and phloem saps, and impaired 57Fe translocation along the xylem. In vitro assays showed that Fe(III)-citrate, the main form of Fe in xylem sap, is easily photoreduced to Fe(II)-citrate, which is unstable and prone to adsorption by cell walls. Taken together, these results indicate that LPR1 and LPR2 are required to oxidize Fe(II) and maintain Fe(III)-citrate stability and mobility during xylem translocation against photoreduction. Our study not only uncovers an essential physiological role of LPR1 and LPR2 but also reveals a new mechanism by which plants maintain Fe mobility during long-distance translocation in the xylem.

FE UPTAKE-INDUCING PEPTIDE1 maintains Fe translocation by controlling Fe deficiency response genes in the vascular tissue of Arabidopsis.

FE UPTAKE-INDUCING PEPTIDE1 (FEP1), also named IRON MAN3 (IMA3) is a short peptide involved in the iron deficiency response in Arabidopsis thaliana. Recent studies uncovered its molecular function, but its physiological function in the systemic Fe response is not fully understood. To explore the physiological function of FEP1 in iron homoeostasis, we performed a transcriptome analysis using the FEP1 loss-of-function mutant fep1-1 and a transgenic line with oestrogen-inducible expression of FEP1. We determined that FEP1 specifically regulates several iron deficiency-responsive genes, indicating that FEP1 participates in iron translocation rather than iron uptake in roots. The iron concentration in xylem sap under iron-deficient conditions was lower in the fep1-1 mutant and higher in FEP1-induced transgenic plants compared with the wild type (WT). Perls staining revealed a greater accumulation of iron in the cortex of fep1-1 roots than in the WT root cortex, although total iron levels in roots were comparable in the two genotypes. Moreover, the fep1-1 mutation partially suppressed the iron overaccumulation phenotype in the leaves of the oligopeptide transporter3-2 (opt3-2) mutant. These data suggest that FEP1 plays a pivotal role in iron movement and in maintaining the iron quota in vascular tissues in Arabidopsis.

The miR157-SPL-CNR module acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato.

Iron (Fe) homeostasis is critical for plant growth, development, and stress responses. Fe levels are tightly controlled by intricate regulatory networks in which transcription factors (TFs) play a central role. A series of basic helix-loop-helix (bHLH) TFs have been shown to contribute to Fe homeostasis, but the regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF SlSPL-CNR negatively regulates Fe-deficiency responses in tomato (Solanum lycopersicum) roots. Fe deficiency rapidly repressed the expression of SlSPL-CNR, and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-generated SlSPL-CNR knock-out lines compared to the wild-type. Comparative transcriptome analysis identified 47 Fe deficiency-responsive genes the expression of which is negatively regulated by SlSPL-CNR, one of which, SlbHLH101, helps regulate Fe uptake genes. SlSPL-CNR localizes the nucleus and interacts with the GTAC and BOX 4 (ATTAAT) motifs in the SlbHLH101 promoter to repress its expression. Inhibition of SlSPL-CNR expression in response to Fe deficiency was well correlated with the expression of the microRNA SlymiR157. SlymiR157-overexpressing tomato lines displayed enhanced Fe deficiency responses, as did SlSPL-CNR loss-of-function mutants. We propose that the SlymiR157-SlSPL-CNR module represents a novel pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.

Comparative Physiological and Transcriptomic Analyses Reveal Altered Fe-Deficiency Responses in Tomato Epimutant Colorless Non-ripening.

The mechanisms associated with the regulation of iron (Fe) homeostasis have been extensively examined, however, epigenetic regulation of these processes remains largely unknown. Here, we report that a naturally occurring epigenetic mutant, Colorless non-ripening (Cnr), displayed increased Fe-deficiency responses compared to its wild-type Ailsa Craig (AC). RNA-sequencing revealed that a total of 947 and 1,432 genes were up-regulated by Fe deficiency in AC and Cnr roots, respectively, while 923 and 1,432 genes were, respectively, down-regulated. Gene ontology analysis of differentially expressed genes showed that genes encoding enzymes, transporters, and transcription factors were preferentially affected by Fe deficiency. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed differential metabolic responses to Fe deficiency between AC and Cnr. Based on comparative transcriptomic analyses, 24 genes were identified as potential targets of Cnr epimutation, and many of them were found to be implicated in Fe homeostasis. By developing CRISPR/Cas9 genome editing SlSPL-CNR knockout (KO) lines, we found that some Cnr-mediated Fe-deficiency responsive genes showed similar expression patterns between SlSPL-CNR KO plants and the Cnr epimutant. Moreover, both two KO lines displayed Fe-deficiency-induced chlorosis more severe than AC plants. Additionally, the Cnr mutant displayed hypermethylation in the 286-bp epi-mutated region on the SlSPL-CNR promoter, which contributes to repressed expression of SlSPL-CNR when compared with AC plants. However, Fe-deficiency induced no change in DNA methylation both at the 286-bp epi-allele region and the entire region of SlSPL-CNR gene. Taken together, using RNA-sequencing and genetic approaches, we identified Fe-deficiency responsive genes in tomato roots, and demonstrated that SlSPL-CNR is a novel regulator of Fe-deficiency responses in tomato, thereby, paving the way for further functional characterization and regulatory network dissection.

Transcriptome Profiling of Cu Stressed Petunia Petals Reveals Candidate Genes Involved in Fe and Cu Crosstalk.

Copper (Cu) is an essential element for most living plants, but it is toxic for plants when present in excess. To better understand the response mechanism under excess Cu in plants, especially in flowers, transcriptome sequencing on petunia buds and opened flowers under excess Cu was performed. Interestingly, the transcript level of FIT-independent Fe deficiency response genes was significantly affected in Cu stressed petals, probably regulated by basic-helix-loop-helix 121 (bHLH121), while no difference was found in Fe content. Notably, the expression level of bHLH121 was significantly down-regulated in petals under excess Cu. In addition, the expression level of genes related to photosystem II (PSII), photosystem I (PSI), cytochrome b6/f complex, the light-harvesting chlorophyll II complex and electron carriers showed disordered expression profiles in petals under excess Cu, thus photosynthesis parameters, including the maximum PSII efficiency (FV/FM), nonphotochemical quenching (NPQ), quantum yield of the PSII (ΦPS(II)) and photochemical quenching coefficient (qP), were reduced in Cu stressed petals. Moreover, the chlorophyll a content was significantly reduced, while the chlorophyll b content was not affected, probably caused by the increased expression of chlorophyllide a oxygenase (CAO). Together, we provide new insight into excess Cu response and the Cu–Fe crosstalk in flowers.

Different Rhizospheric pH Conditions Affect Nutrient Accumulations in Rice under Salinity Stress

This study was conducted to determine the responses to saline-alkaline (SA) stress with regard to nutrient accumulation in two rice varieties having different tolerances to salt-stress. A salinity-tolerant landrace, Pokkali, and a salinity-sensitive variety, PTT1, were exposed to three levels of SA conditions, pH 7.0 (mild), pH 8.0 (moderate), and pH 9.0 (severe), under 50 mM Na stress. The results indicated that Pokkali had comparably greater SA tolerance than PTT1 owing to its higher biomass production. The maintenance of the lower Na/K ratio in Pokkali shoots was achieved by the higher expression of OsHKT1;5 encoding a Na+ transporter in the shoots, OsNHX1 encoding a tonoplast-localized Na+/H+ antiporter in the roots, and OsHAK16 encoding a K+ transporter in the roots under SA conditions. We propose that the high expression of Fe deficiency-responsive genes, OsIRT1, OsIRO2, OsYSL15, OsNAS1, and OsNAS2, in both rice varieties under all SA conditions should contribute to Fe homeostasis in the shoots. In addition, SA treatment increased the concentrations of Ca, Mn, Zn, and Cu in the roots but decreased their concentrations in the shoots of both varieties. Overall, the results indicated that high rhizospheric pH influenced nutrient uptake and translocation from the roots to the shoots in rice.

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis.

Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3'-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2, in comparison to other pathways such as TCA cycle-, amino acid- metabolisms and photosynthesis. The cell wall pectin metabolism, cell wall proteins and degradation process were highly modulated. Intriguingly Fe-deficiency responsive genes and drought stress-induced genes were also up-regulated, suggesting the importance of thermospermi'ne flux on regulation of gene network. Histological observation showed that the vascular system of the joint part between stem and leaves was structurally dissociated, indicating its involvement in vascular maintenance. Endogenous increase in T-Spm and reduction in H2O2 contents were found in mutant grown in T-Spm containing media. The results indicate that T-Spm homeostasis by a fine tuned balance of its synthesis and catabolism is important for maintaining gene regulation network and the vascular system in plants.

The bHLH protein OsIRO3 is critical for plant survival and iron (Fe) homeostasis in rice (Oryza sativa L.) under Fe-deficient conditions

ABSTRACT Iron (Fe) is an essential element for most organisms since it is involved in various biological reactions. Fe deficiency is a major constraint for plant growth and leads to low crop yields and poor nutritional quality. Fe uptake and translocation in plants require the coordinated expression of multiple genes, which is regulated by various transcription factors (TFs), especially members of the basic helix-loop-helix (bHLH) family. OsIRO3 was first reported as a repressor related to Fe homeostasis gene regulation in roots based on evidence in overexpressing rice. However, its functions in regulating rice growth and Fe homeostasis between roots and shoots during Fe deficiency is poorly understood. Here, we developed osiro3 knockout mutants by the CRISPR/Cas9 system. The leaves of osiro3 knockout mutants showed abnormal growth and development after Fe-deficiency treatment. Strong lesions occurred on the young leaves of osiro3 knockout mutants, which even led to plant death during severe Fe deficiency. Furthermore, accumulation of reactive oxygen species (ROS), Fe and manganese concentrations were all elevated in the shoots of osiro3 knockout mutants under the Fe deficiency stress. Quantitative RT-PCR showed that OsNAC4 for ROS induced plant hypersensitive cell death and OsFerritins for Fe storage and overload were all significantly activated in osiro3 knockout shoots after Fe deficiency treatment. In osiro3 knockout mutants, Fe-deficiency response genes were induced in roots, but suppressed in shoots, and furthermore Fe sufficiency/overload genes were induced in shoots under Fe-deficient conditions according to DNA microarray. Yeast two-hybrid analysis revealed a positive interaction between OsIRO3 and the homologous protein OsbHLH062, which was also transcriptionally increased in Fe-deficient roots. OsIRO3 is essential for maintaining plant survival in rice under Fe-deficient conditions and plays an important role in signal transmission from shoots to roots which is critical for plants to prevent Fe overload.

Rhizobacteria-Mediated Activation of the Fe Deficiency Response in Arabidopsis Roots: Impact on Fe Status and Signaling.

The beneficial root-colonizing rhizobacterium Pseudomonas simiae WCS417 stimulates plant growth and induces systemic resistance against a broad spectrum of plant diseases. In Arabidopsis thaliana (Arabidopsis), the root transcriptional response to WCS417 shows significant overlap with the root response to iron (Fe) starvation, including activation of the marker genes MYB72 and IRT1. Here, we investigated how colonization of Arabidopsis roots by WCS417 impacts Fe homeostasis in roots and shoots. Under Fe-sufficient conditions, root colonization by WCS417 induced a transient Fe deficiency response in the root and elevated both the total amount of Fe in the shoot and the shoot fresh weight. When plants were grown under Fe-starvation conditions, WCS417 still promoted plant growth, but did not increase the total amount of Fe, resulting in chlorosis. Thus, increased Fe uptake in response to WCS417 is essential to maintain Fe homeostasis in the more rapidly growing plant. As the WCS417-induced Fe deficiency response is known to require a shoot-derived signal, we tested whether the Fe deficiency response is activated in response to an increased Fe demand in the more rapidly growing shoot. Exogenous application of Fe to the leaves to reduce a potential shoot Fe shortage did not prevent WCS417-mediated induction of the Fe deficiency response in the roots. Moreover, the leaf Fe status-dependent shoot-to-root signaling mutant opt3-2, which is impaired in the phloem-specific Fe transporter OPT3, still up-regulated the Fe deficiency response genes MYB72 and IRT1 in response to WCS417. Collectively, our results suggest that the WCS417-induced Fe deficiency response in the root is controlled by a shoot-to-root signaling system that functions independently of both leaf Fe status and OPT3.

BHLH104 confers tolerance to cadmium stress in Arabidopsis thaliana.

Cd is a non-essential heavy metal that is toxic to both plants and animals. Here, we reveal that the transcription factor bHLH104 positively regulates Cd tolerance in Arabidopsis thaliana. We show that Fe deficiency-responsive genes were induced by Cd treatment, and that their upregulation was suppressed in bhlh104 loss-of-function mutants, but enhanced upon overexpression of bHLH104. Correspondingly, the bhlh104 mutants displayed sensitivity to Cd stress, whereas plants overexpressing bHLH104 exhibited enhanced Cd tolerance. Further analysis suggested that bHLH104 positively regulates four heavy metal detoxification-associated genes, IREG2, MTP3, HMA3 and NAS4, which play roles in Cd sequestration and tolerance. The bHLH104 overexpression plants accumulated high levels of Cd in the root but low levels of Cd in the shoot, which might contribute to the Cd tolerance in those lines. The present study thus points to bHLH104 as a potentially useful tool for genetic engineering of plants with enhanced Cd tolerance.

Complementary Evaluation of Iron Deficiency Root Responses to Assess the Effectiveness of Different Iron Foliar Applications for Chlorosis Remediation.

Iron deficiency in plants is caused by a low availability of iron in the soil, and its main visual symptom is leaf yellowing due to a decrease in chlorophyll content, along with a reduction in plant growth and fruit quality. Foliar sprays with Fe compounds are an economic alternative to the treatment with expensive synthetic Fe-chelates applied to the soil, although the efficacy of foliar treatments is rather limited. Generally, plant response to Fe-foliar treatments is monitored by measuring chlorophyll content (or related parameters as SPAD index). However, different studies have shown that foliar Fe sprays cause a local regreening and that translocation of the applied Fe within the plant is quite low. In this context, the aim of this study was to assess the effects of foliar applications of different Fe compounds [FeSO4, Fe(III)-EDTA, and Fe(III)-heptagluconate] on Fe-deficient cucumber plants, by studying the main physiological plant root responses to Fe deficiency [root Fe(III) chelate reductase (FCR) activity; acidification of the nutrient solution; and expression of the Fe deficiency responsive genes encoding FCR, CsFRO1, Fe(II) root transporter CsIRT1, and two plasma membrane H+-ATPases, CsHA1 and CsHA2], along with SPAD index, plant growth and Fe content. The results showed that the overall assessment of Fe-deficiency root responses improved the evaluation of the efficacy of the Fe-foliar treatments compared to just monitoring SPAD indexes. Thus, FCR activity and expression of Fe-deficiency response genes, especially CsFRO1 and CsHA1, preceded the trend of SPAD index and acted as indicators of whether the plant was sensing or not metabolically active Fe due to the treatments. Principal component analysis of the data also provided a graphical tool to evaluate the evolution of plant responses to foliar Fe treatments with time.

H2O2 mediates nitrate‐induced iron chlorosis by regulating iron homeostasis in rice

The uptake of nitrate by plant roots causes a pH increment in rhizosphere and leads to iron (Fe) deficiency in rice. However, little is known about the mechanism how the nitrate uptake-induced high rhizosphere pH causes Fe deficiency. Here, we found that rice showed severe leaf chlorosis and large amounts of Fe plaque were aggregated on the root surface and intercellular space outside the exodermis in a form of ferrihydrite under alkaline conditions. In this case, there was significantly decreased Fe concentration in shoots, and the Fe deficiency responsive genes were strongly induced in the roots. The high rhizosphere pH induced excess hydrogen peroxide (H2 O2 ) production in the epidermis due to the increasing expression of NADPH-oxidase respiratory burst oxidase homolog 1, which enhanced root oxidation ability and improved the Fe plaque formation in rhizosphere. Further, the concentrated H2 O2 regulated the phenylpropanoid metabolism with increased lignin biosynthesis and decreased phenolics secretion, which blocked apoplast Fe mobilization efficiency. These factors coordinately repressed the Fe utilization in rhizosphere and led to Fe deficiency in rice under high pH. In conclusion, our results demonstrate that nitrate uptake-induced rhizosphere alkalization led to Fe deficiency in rice, through H2 O2 -dependent manners of root oxidation ability and phenylpropanoid metabolism.

Genome-wide analysis of gene expression profiling revealed that COP9 signalosome is essential for correct expression of Fe homeostasis genes in Arabidopsis.

In plant cells, either excess or insufficient iron (Fe) concentration triggers stress responses, therefore it is strictly controlled. Proteasome-mediated degradation through ubiquitination of Fe homeostasis proteins has just become the focus of research in recent years. Deactivating ubiquitin ligases, COP9 signalosome has a central importance in the translational control of various stress responses. The aim of the study was to investigate COP9 signalosome in Fe deficiency response of Strategy I plants. In silico analysis of a set of Fe-deficiency-responsive genes was conducted against the transcriptome of Arabidopsis csn mutant lines using Genevestigator software. Induced and suppressed genes were clustered in a hierarchical way and gene ontology enrichment categories were identified. In wild-type Arabidopsis, CSN genes did not respond to iron deficiency. In csn mutant lines, under Fe-sufficient conditions, hundreds of Fe-deficiency-responsive genes were misregulated. Among the ones previously characterized for their physiological roles under Fe deficiency IRT1, NAS4, BTS, NRAMP1 were down-regulated while AHA2, MTP8, FRD3 were up-regulated. Unexpectedly, from those which were regulated in opposite ways, some had been repeatedly shown to be tightly co-regulated by the same transcription factor, FIT. Two proteins from DELLA family, which were reported to interact with FIT to repress its downstream, were found to be strikingly repressed in csn mutants. Overall, the study underlined that the absence of a functional CSN greatly impacted the regulation of Fe homeostasis-related genes, in a manner which cannot be explained simply by the induction of the master transcription factor, FIT. Correct expression of Fe deficiency-responsive genes requires an intact COP9 signalosome in Arabidopsis.

Fe-deficiency-induced expression of bHLH104 enhances Fe-deficiency tolerance of Arabidopsis thaliana.

Expression of bHLH104 - GFP driven by the MYB72 promoter improves plants' tolerance to Fe deficiency and increases seed Fe concentrations. Iron (Fe) deficiency causes reduced crop yield and quality. In humans, Fe deficiency is directly associated with Fe-deficiency anemia. Therefore, breeding Fe-deficiency tolerant and Fe-enriched plants are an ideal approach to deal with these problems. Here, different strategies were explored to generate Fe-deficiency tolerant and Fe-enriched plants. Unexpectedly, the overexpression of Fe-deficiency responsive genes (IRT1, MYB72, and bHLH100) resulted in enhanced sensitivity to Fe deficiency, including leaf chlorosis and short roots under Fe-deficiency conditions. Next, three different types of Fe-deficiency responsive promoters (Pro IRT1 , Pro MYB72, and Pro bHLH100 ) were used to drive the expression of bHLH104-GFP fusion gene in Arabidopsis. Pro IRT1 :bHLH104-GFP plants showed the enhanced sensitivity to Fe deficiency on Fe-deficient media and the reduced fertility in alkaline soil. In contrast, Pro bHLH100 :bHLH104-GFP plants displayed a slight tolerance to Fe deficiency and Pro MYB72 :bHLH104-GFP plants had a significant advantage in growth in alkaline soil, including increased root length, chlorophyll, and biomass. Further analysis revealed that the expression of Fe-deficiency responsive genes was dramatically upregulated in both Pro MYB72 :bHLH104-GFP and Pro bHLH100 :bHLH104-GFP plants under Fe-deficiency conditions. When grown in alkaline soil, Pro MYB72 :bHLH104-GFP plants greatly improved the seed yield and Fe concentration. These results are fundamental for plant manipulation approaches to modify tolerance to Fe deficiency and Fe accumulation through alterations of bHLH104 gene expression.

Two bHLH Transcription Factors, bHLH34 and bHLH104, Regulate Iron Homeostasis in Arabidopsis thaliana.

The regulation of iron (Fe) homeostasis is critical for plant survival. Although the systems responsible for the reduction, uptake, and translocation of Fe have been described, the molecular mechanism by which plants sense Fe status and coordinate the expression of Fe deficiency-responsive genes is largely unknown. Here, we report that two basic helix-loop-helix-type transcription factors, bHLH34 and bHLH104, positively regulate Fe homeostasis in Arabidopsis (Arabidopsis thaliana). Loss of function of bHLH34 and bHLH104 causes disruption of the Fe deficiency response and the reduction of Fe content, whereas overexpression plants constitutively promote the expression of Fe deficiency-responsive genes and Fe accumulation. Further analysis indicates that bHLH34 and bHLH104 directly activate the transcription of the Ib subgroup bHLH genes, bHLH38/39/100/101 Moreover, overexpression of bHLH101 partially rescues the Fe deficiency phenotypes of bhlh34bhlh104 double mutants. Further investigation suggests that bHLH34, bHLH104, and bHLH105 (IAA-LEUCINE RESISTANT3) function as homodimers or heterodimers to nonredundantly regulate Fe homeostasis. This work reveals that plants have evolved complex molecular mechanisms to regulate Fe deficiency response genes to adapt to Fe deficiency conditions.

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

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.