The effect of sodium bicarbonate on plant performance and iron acquisition system of FA-5 (Forner-Alcaide 5) citrus seedlings

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO₃. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-⁵⁷FeEDDHA experiments indicated that ⁵⁷Fe uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more ⁵⁷Fe in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H⁺-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe²⁺ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.     

Bicarbonate blocks iron translocation from cotyledons inducing iron stress responses in Citrus roots

The effect of bicarbonate ion (HCO₃⁻) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated “in vitro” on two iron-deprived media, supplemented or not with 10 mM HCO₃⁻ (−Fe+Bic and −Fe, respectively). After 21 d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO₃⁻ (+Fe+Bic or +Fe, respectively). −Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than −Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO₃⁻ upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H⁺-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When −Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15 d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO₃⁻ prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities.     

Endophytic yeast protect plants against metal toxicity by inhibiting plant metal uptake through an ethylene-dependent mechanism

Toxic metal pollution requires significant adjustments in plant metabolism. Here, we show that the plant microbiota plays an important role in this process. The endophytic Sporobolomyces ruberrimus isolated from a serpentine population of Arabidopsis arenosa protected plants against excess metals. Coculture with its native host and Arabidopsis thaliana inhibited Fe and Ni uptake. It had no effect on host Zn and Cd uptake. Fe uptake inhibition was confirmed in wheat and rape. Our investigations show that, for the metal inhibitory effect, the interference of microorganisms in plant ethylene homeostasis is necessary. Application of an ethylene synthesis inhibitor, as well as loss-of-function mutations in canonical ethylene signalling genes, prevented metal uptake inhibition by the fungus. Coculture with S. ruberrimus significantly changed the expression of Fe homeostasis genes: IRT1, OPT3, OPT6, bHLH38 and bHLH39 in wild-type (WT) A. thaliana. The expression pattern of these genes in WT plants and in the ethylene signalling defective mutants significantly differed and coincided with the plant accumulation phenotype. Most notably, down-regulation of the expression of IRT1 solely in WT was necessary for the inhibition of metal uptake in plants. This study shows that microorganisms optimize plant Fe and Ni uptake by fine-tuning plant metal homeostasis.     

Glutathione plays an essential role in nitric oxide‐mediated iron‐deficiency signaling and iron‐deficiency tolerance in Arabidopsis

Iron (Fe) deficiency is a common agricultural problem that affects both the productivity and nutritional quality of plants. Thus, identifying the key factors involved in the tolerance of Fe deficiency is important. In the present study, the zir1 mutant, which is glutathione deficient, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Other glutathione‐deficient mutants also showed various degrees of sensitivity to Fe‐limited conditions. Interestingly, we found that the glutathione level was increased under Fe deficiency in the wild type. By contrast, blocking glutathione biosynthesis led to increased physiological sensitivity to Fe deficiency. On the other hand, overexpressing glutathione enhanced the tolerance to Fe deficiency. Under Fe‐limited conditions, glutathione‐deficient mutants, zir1, pad2 and cad2 accumulated lower levels of Fe than the wild type. The key genes involved in Fe uptake, including IRT1, FRO2 and FIT, are expressed at low levels in zir1; however, a split‐root experiment suggested that the systemic signals that govern the expression of Fe uptake‐related genes are still active in zir1. Furthermore, we found that zir1 had a lower accumulation of nitric oxide (NO) and NO reservoir S‐nitrosoglutathione (GSNO). Although NO is a signaling molecule involved in the induction of Fe uptake‐related genes during Fe deficiency, the NO‐mediated induction of Fe‐uptake genes is dependent on glutathione supply in the zir1 mutant. These results provide direct evidence that glutathione plays an essential role in Fe‐deficiency tolerance and NO‐mediated Fe‐deficiency signaling in Arabidopsis.     

Unraveling the iron deficiency responsive proteome in <i>Arabidopsis?</i>shoot by iTRAQ-OFFGEL approach

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.     

Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition

To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> IRT2 cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells

Iron is an essential nutrient for all organisms but toxic when present in excess. Consequently, plants carefully regulate their iron uptake, dependent on the FRO2 ferric reductase and the IRT1 transporter, to control its homeostasis. Arabidopsis IRT2 gene, whose expression is induced in root epidermis upon iron deprivation, was shown to encode a functional iron/zinc transporter in yeast, and proposed to function in iron acquisition from the soil. In this study, we demonstrate that, unlike its close homolog IRT1, IRT2 is not involved in iron absorption from the soil since overexpression of IRT2 does not rescue the iron uptake defect of irt1-1 mutant and since a null irt2 mutant shows no chlorosis in low iron. Consistently, an IRT2-green fluorescent fusion protein, transiently expressed in culture cells, localizes to intracellular vesicles. However, IRT2 appears strictly co-regulated with FRO2 and IRT1, supporting the view that IRT2 is an integral component of the root response to iron deficiency in root epidermal cells. We propose a model where IRT2 likely prevents toxicity from IRT1-dependent iron fluxes in epidermal cells, through compartmentalization.     

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains

Background

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe + and Fe − medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe − conditions.

Results

HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe − condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna.

Conclusion

HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.     

Flooding Impairs Fe Uptake and Distribution in Citrus Due to the Strong Down-Regulation of Genes Involved in Strategy I Responses to Fe Deficiency in Roots

This work determines the ffects of long-term anoxia conditions—21 days—on Strategy I responses to iron (Fe) deficiency in Citrus and its impact on Fe uptake and distribution. The study was carried out in Citrus aurantium L. seedlings grown under flooding conditions (S) and in both the presence (+Fe) and absence of Fe (-Fe) in nutritive solution. The results revealed a strong down-regulation (more than 65%) of genes HA1 and FRO2 coding for enzymes proton-ATPase and Ferric-Chelate Reductase (FC-R), respectively, in –FeS plants when compared with –Fe ones. H+-extrusion and FC-R activity analyses confirmed the genetic results, indicating that flooding stress markedly repressed acidification and reduction responses to Fe deficiency (3.1- and 2.0-fold, respectively). Waterlogging reduced by half Fe concentration in +FeS roots, which led to 30% up-regulation of Fe transporter IRT1, although this effect was unable to improve Fe absorption. Consequently, flooding inhibited 57Fe uptake in +Fe and –Fe seedlings (29.8 and 66.2%, respectively) and ⁵⁷Fe distribution to aerial part (30.6 and 72.3%, respectively). This evidences that the synergistic action of both enzymes H+-ATPase and FC-R is the preferential regulator of the Fe acquisition system under flooding conditions and, hence, their inactivation implies a limiting factor of citrus in their Fe-deficiency tolerance in waterlogged soils.     

Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper

The Arabidopsis FRO2 gene encodes the iron deficiency-inducible ferric chelate reductase responsible for reduction of iron at the root surface; subsequent transport of iron across the plasma membrane is carried out by a ferrous iron transporter (IRT1). Genome annotation has identified seven additional FRO family members in the Arabidopsis genome. We used real-time RT-PCR to examine the expression of each FRO gene in different tissues and in response to iron and copper limitation. FRO2 and FRO5 are primarily expressed in roots while FRO8 is primarily expressed in shoots. FRO6 and FRO7 show high expression in all the green parts of the plant. FRO3 is expressed at high levels in roots and shoots, and expression of FRO3 is elevated in roots and shoots of iron-deficient plants. Interestingly, when plants are Cu-limited, the expression of FRO6 in shoot tissues is reduced. Expression of FRO3 is induced in roots and shoots by Cu-limitation. While it is known that FRO2 is expressed at high levels in the outer layers of iron-deficient roots, histochemical staining of FRO3-GUS plants revealed that FRO3 is predominantly expressed in the vascular cylinder of roots. Together our results suggest that FRO family members function in metal ion homeostasis in a variety of locations in the plant.