Unravelling the molecular and physiological components that contribute to iron deficiency chlorosis

Student thesis: Doctoral Thesis


Iron (Fe) deficiency chlorosis (IDC) is a serious condition affecting plants which are grown under calcareous or water logged soils. Under such conditions,Fe forms insoluble oxides and becomes unavailable for plant uptake, leading to stunted growth and severe yield reduction, causing aggravated agricultural losses. In the past years,efforts have been made to increase plant Fe content(so-called plant biofortification), in order to reduce the incidence of iron deficiency anaemia (IDA) prevalent around the world. To this end, legume grains and cereals, due to their rich nutritional profile and high worldwide intake by the population,have gained an important role in biofortification studies, which depend on the available molecular and physiological data for their successful implementation. The aim of this thes is was to contribute to the understanding of the molecular, physiological and biochemical mechanisms associated to Fe uptake and transport in Fe-stressed plants and to test a new class of Fe chelates as an efficient tool to prevent IDC. With the purpose of understanding the transcriptomic response to Fe deficiency in a set of different legume species, a non-targeted analysis was performed using Illumina technology. Transcriptome analysis was performed in the roots of soybean (Glycine max), common bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula) grown in Fe deficiency and Fe sufficiency, and 114,723 annotated genes were obtained for all samples. Four IDC-related gene families were up-regulated in common by the three species and can be considered key players involved in the IDC response, namely, metal ligands, transferases, zinc ion binding and metal ion binding genes. Also, amongst the most highly expressed genes were genes of theisoflavonoid pathway and, on the other hand, oxidoreductases were the most down-regulated genes.Still on the search for IDC molecular players, two targetedgenetic analyses were performed,one on G.maxand M. truncatula and another on rice (Oryza sativa). Both studies involved the growth of plants under Fe sufficiency and Fe deficiency in order to compare the regulation of IDC related genes. Soybean and barrel medicare strategy I-crops,which means that, before uptake, they need to reduce Fe(III) to Fe(II) via an enzyme encoded by the FRO2 gene and, afterwards, Fe(II) is transported to the roots via ametal transporter encoded by the IRT1 gene. The expression of these two genes was analysed and both behaved similarly between species, appearingto be co-regulated.Moreover, the Fe transportersYSL1 andVIT1and the main Fe storage protein-encoding gene –ferritin–were up-regulated in the presence of Fe. The NRAMP3 gene, responsible for Fe remobilization from the vacuoles, was up-regulated under Fe deficiency,as was theGCN2gene, indicating a putative role of the latterin Fe metabolism and homeostasis.The targeted study performed in rice, a strategy II cereal that releases phytosiderophores in order to chelate and absorb Fe, involved the analysis of two rice cultivars with distinct susceptibilities to IDC –cv. Nipponbare and cv. Bico Branco. This different susceptibility was confirmed by their contrasting leaf chlorosis development and tissue nutrient accumulation patterns. Thecv. Nipponbare, that showed lower IDC susceptibility, was able to induce higher levels of the key reduction enzyme activity(Fe reductase)and showed higher levels of expression of the strategy I-OsFRO2 gene in roots.In contrast, cv. Bico Branco induced more genes involved in strategy II, specially, the transcription factor OsIRO2 and the phytosiderophore precursor OsTOM1.The screening for tolerant genotypes to IDC is an important tool in plant breeding programs. The most common IDC indicator is the degree of chlorosis development, which is quantified using a numerical scale. Therefore, after gathering the molecular data, the physiological mechanisms triggered by IDC were studied. The model crop G. max was selected,as it comprises lines well characterized according to their IDC-susceptibilities. To this end, two studies were performed. In the first study we aimed at understanding if the ability to partition Fe could be related to Fe-efficiency. We concluded that IDC susceptible lines, when compared to efficient lines,have lower ability to translocate Fe to the shoots, having about two fold higher Fe content at the root level, and they have lower capacity to induce the ferric reductase enzyme, having about three fold lower enzyme activity. In the second study the regulation of the antioxidant and tetrapyrrole systems under Fe deficiency was analysedfor the first time and we inferred that higher levels of oxidative stress might induce the oxidation of the tetrapyrrole heme into hemin, which leads to the induction of the heme-containing catalase enzyme and the reduction of ferric reductase activity. Taken together, the previous results indicate that low ferric reductase activity and Fe accumulation in the root tissue could be added as new IDC-related physiological markers.The application of fertilizers and Fe chelating agentsis one of the most frequently used tools to manage IDC. However, most of them are ineffective,too expensive or recalcitrant in the environment. Hence, the search for new Fe chelates is of utmost importance. In the last step of this thesis, we investigated the potential of a tris(3-hydroxy-4-pyridinonate) Fe(III) complex(Fe(mpp)3, which has never be enutilized in agricultural context)as na Fe fertilizer. Soybean plants were grown hydroponically under Fe deficiency and with Fe(mpp)3or Fe EDDHA supplementation. Results of both physiological and molecular markers showed that the new Fe complex led to healthier plants with increased growthby 24%,42% higher SPAD units and lower Fe retention in the roots.In general, the results presented in this thesis have contributed to a better understanding of the IDC-associated mechanisms and elucidated the key factors to be considered when analysing Fe deficient plants and their defence responses.
Date of Award12 Jul 2017
Original languageEnglish
Awarding Institution
  • Universidade Católica Portuguesa
SupervisorMarta Vasconcelos (Supervisor), António O. S. S. Rangel (Co-Supervisor) & Susana Maria Pinto de Carvalho (Co-Supervisor)


  • Doutoramento em Biotecnologia

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