ABSTRACT
Iron chlorosis is one of the main abiotic stress factors in plants, and as such, is a widespread agricultural problem especially affecting those crops grown on calcareous soils, as in the case of citrus in the Valencia region.
The aim of this study was to characterize the iron uptake system in citrus plants and their response to different chlorotic situations that compromise availability of the element, causing Fe deficiency in the plant. The components of the Fe-absorption system -acidification of the medium, reduction of Fe(III), iron transport through the membrane and alteration of the organic acids synthesis- were analyzed at the physiological, biochemical and molecular level, in conditions of absence of external Fe, presence of bicarbonate (HCO3-) and other micronutrients such as zinc (Zn) and manganese (Mn).
To achieve these objectives, experiments were carried on various citrus genotypes with a different tolerance to iron chlorosis and inducing various chlorotic mediums, either by removing Fe or adding HCO3-, Zn2+ or Mn2+ ions. Depending on each experiment, we assessed the vegetative growth of the plants, total Fe content in various organs and in root apoplast and the state of the photosynthetic system of the leaves after induction of iron chlorosis. Likewise, we determined the ability of reduction and acidification of the roots, by analyzing the activity of the main enzymes involved, such as the Ferric-Chelate Reductase (FC-R), the Proton-ATPase (H+-ATPase), the phosphoenol pyruvate carboxylase (PEPCase) and other enzymes that participate in the biosynthesis of organic acids, such as malate dehydrogenase (MDH), pyruvate kinase (PK), malic enzyme (ME), citrate synthase (CS), aconitase (ACO) and fumarase (FUM). Additionally, we identified in the citrus genome, sequences homologous to genes FRO1, FRO2, HA1, HA2, IRT1 and IRT2 of Arabidopsis thaliana, which encode enzymes FC-R and H+-ATPase and the major iron transporters. Also, we homologated the sequences of PEPC1, cMDH, ME, CS, ACO, FUM, mMDH and DTC coding for the corresponding enzymes of the Krebs cycle and the mitochondrial dicarboxylate-tricarboxylate acids transporter. In all cases, we designed the sequences of oligonucleotides for analysing gene expression by RT-PCR. Finally, we determined the rate of iron uptake by the root and its transport in the plant by isotope dilution technique.
The expression analysis of genes showed that Fe-deficiency increases FRO2, HA1 and IRT1 transcripts abundance encoding for FC-R and H+-ATPase enzymes and the iron transporter. This was confirmed with the induction of the corresponding enzymatic activities and the increased 57Fe uptake. The iron chlorosis-tolerant genotype, Cleopatra mandarin, presented greater FC-R activity than the sensitive one, Carrizo citrange, as well as an increased capacity for accumulating Fe in its root apoplast. Citrus roots accumulated organic acids under iron chlorosis conditions by stimulating the activity of some enzymes of the Krebs cycle, such as PEPCase, MDH, CS and FUM and the di-tricarboxylate acids carrier DTC. Major acids accumulated are citrate and malate, which once synthesized in the roots are released to the xylem sap (to promote the long-distance transport of Fe) or to the exterior (to acidify the rhizosphere). The presence of bicarbonate in the medium induced iron chlorosis, causing the alteration of the photosynthetic system in the leaves and the reduction of plant growth, particularly pronounced in the roots. HCO3- ion inhibits iron absorption by the roots, which results in a decrease in Fe concentration in the organs of the plant. This effect appears to be due to blockage of medium acidification caused by HCO3- buffering power. Consequently with the decreased absorption of Fe by the bicarbonate effect, there is a reduction of Fe concentration in the roots, which in turn induces the responses to the deficiency of this element. In addition, bicarbonate hinders Fe transport from the roots to the shoots and also blocks the mobilization of Fe from the cotyledons to the roots of seedlings grown without external Fe. The presence of relatively high concentrations of Zn2+ or Mn2+ in the medium reduces Fe absorption. This seems to be due to a competition effect at the transport level across the plasma membrane, since responses to iron deficiency are activated when the entry of Fe in the root cells is reduced.
Overall results indicate that citrus ability for iron absorption increases in the absence of Fe in the medium, due to the activation of the element acquisition system components. In addition, all responses to Fe deficiency in roots are metabolically inter-correlated with each other and are involved in the absorption of this element by the plant, solubilising Fe by rhizosphere acidification, reducing the ion Fe3+ to Fe2+ available to plants and activating iron transporters from the root plasma membrane. The level of citrus tolerance to iron chlorosis is determined by the level of response of the components of the Fe acquisition system in a chlorotic situation, being especially important the role of the enzyme FC-R.