Carlos Lucena

Ethylene and nitric oxide involvement in the up-regulation of key genes related to iron acquisition and homeostasis in Arabidopsis

In a previous work it was shown that ethylene participates in the up-regulation of several Fe acquisition genes of Arabidopsis, such as AtFIT, AtFRO2, and AtIRT1. In this work the relationship between ethylene and Fe-related genes in Arabidopsis has been looked at in more depth. Genes induced by Fe deficiency regulated by ethylene were searched for. For this, studies were conducted, using microarray analysis and reverse transcription-PCR (RT-PCR), to determine which of the genes up-regulated by Fe deficiency are simultaneously suppressed by two different ethylene inhibitors (cobalt and silver thiosulphate), assessing their regulation by ethylene in additional experiments. In a complementary experiment, it was determined that the Fe-related genes up-regulated by ethylene were also responsive to nitric oxide (NO). Further studies were performed to analyse whether Fe deficiency up-regulates the expression of genes involved in ethylene biosynthesis [S-adenosylmethionine synthetase, 1-aminocyclopropane-1-carboxylate (ACC) synthase, and ACC oxidase genes] and signalling (AtETR1, AtCTR1, AtEIN2, AtEIN3, AtEIL1, and AtEIL3). The results obtained show that both ethylene and NO are involved in the up-regulation of many important Fe-regulated genes of Arabidopsis, such as AtFIT, AtbHLH38, AtbHLH39, AtFRO2, AtIRT1, AtNAS1, AtNAS2, AtFRD3, AtMYB72, and others. In addition, the results show that Fe deficiency up-regulates genes involved in both ethylene synthesis (AtSAM1, AtSAM2, AtACS4, AtACS6, AtACS9, AtACO1, and AtACO2) and signalling (AtETR1, AtCTR1, AtEIN2, AtEIN3, AtEIL1, and AtEIL3) in the roots.

Bicarbonate blocks the expression of several genes involved in the physiological responses to Fe deficiency of Strategy I plants

Bicarbonate is considered one of the most important factors causing Fe chlorosis in Strategy I plants, mainly on calcareous soils. Most of its negative effects have been attributed to its capacity to buffer a high pH in soils, which can diminish both Fe solubility and root ferric reductase activity. Besides its pH-mediated effects, previous work has shown that bicarbonate can inhibit the induction of enhanced ferric reductase activity in Fe-deficient Strategy I plants. However, to date it is not known whether bicarbonate affects the upregulation of the ferric reductase gene and other genes involved in Fe acquisition. The objective of this work has been to study the effect of bicarbonate on the expression of several Fe acquisition genes in Arabidopsis (Arabidopsis thaliana L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.) and cucumber (Cucumis sativus L.) plants. Genes for ferric reductases AtFRO2, PsFRO1, LeFRO1 and CsFRO1; iron transporters AtITR1, PsRIT1, LeIRT1 and CsIRT1; H+-ATPases CsHA1 and CsHA2; and transcription factors AtFIT and LeFER have been examined. The results showed that bicarbonate could induce Fe chlorosis by inhibiting the expression of the ferric reductase, the iron transporter and the H+-ATPase genes, probably through alteration of the expression of Fe efficiency reactions (FER) (or FER-like) transcription factors.

Ethylene and the Regulation of Physiological and Morphological Responses to Nutrient Deficiencies

Physiological and morphological responses to increase the mobilization and uptake of nutrients from the soil are subject to regulation by ethylene. To cope with nutrient deficiencies, plants develop both morphological and physiological responses. The regulation of these responses is not totally understood, but some hormones and signaling substances have been implicated. It was suggested several years ago that ethylene participates in the regulation of responses to iron and phosphorous deficiency. More recently, its role has been extended to other deficiencies, such as potassium, sulfur, and others. The role of ethylene in so many deficiencies suggests that, to confer specificity to the different responses, it should act through different transduction pathways and/or in conjunction with other signals. In this update, the data supporting a role for ethylene in the regulation of responses to different nutrient deficiencies will be reviewed. In addition, the results suggesting the action of ethylene through different transduction pathways and its interaction with other hormones and signaling substances will be discussed.

Ethylene Participates in the Regulation of Fe Deficiency Responses in Strategy I Plants and in Rice

Iron (Fe) is very abundant in most soils but its availability for plants is low, especially in calcareous soils. Plants have been divided into Strategy I and Strategy II species to acquire Fe from soils. Strategy I species apply a reduction-based uptake system which includes all higher plants except the Poaceae. Strategy II species apply a chelation-based uptake system which includes the Poaceae. To cope with Fe deficiency both type of species activate several Fe deficiency responses, mainly in their roots. These responses need to be tightly regulated to avoid Fe toxicity and to conserve energy. Their regulation is not totally understood but some hormones and signaling substances have been implicated. Several years ago it was suggested that ethylene could participate in the regulation of Fe deficiency responses in Strategy I species. In Strategy II species, the role of hormones and signaling substances has been less studied. However, in rice, traditionally considered a Strategy II species but that possesses some characteristics of Strategy I species, it has been recently shown that ethylene can also play a role in the regulation of some of its Fe deficiency responses. Here, we will review and discuss the data supporting a role for ethylene in the regulation of Fe deficiency responses in both Strategy I species and rice. In addition, we will review the data about ethylene and Fe responses related to Strategy II species. We will also discuss the results supporting the action of ethylene through different transduction pathways and its interaction with other signals, such as certain Fe-related repressive signals occurring in the phloem sap. Finally, the possible implication of ethylene in the interactions among Fe deficiency responses and the responses to other nutrient deficiencies in the plant will be addressed.

Plant Hormones Influencing Iron Uptake in Plants

Different experimental results based on split-root and grafting experiments suggest the involvement of systemic signals in the regulation of Fe deficiency stress responses by Strategy I plants. Until now, the nature of this (or these) systemic signal(s) is unknown, but several authors have proposed some plant hormones, or their precursors, as participants in this signalling process. Among the hormones that have received more attention are auxin and ethylene. Both of them can induce morphological changes similar to the ones induced by Fe deficiency, such as subapical root hairs and transfer cells, when applied to Fe-sufficient Strategy I plants. Furthermore, the addition of either auxin or ethylene inhibitors to Fe-deficient Strategy I plants can block some of these morphological changes. These results, and others obtained by using ethylene mutants and other experimental approaches, suggest that auxin and/or ethylene could be involved in the regulation of some of the morphological changes developed by Fe-deficient Strategy I plants. Since auxin can increase the production of ethylene, some of the effects of auxin could be mediated through ethylene. Auxin and ethylene have also been involved in the regulation of some of the physiological responses to Fe deficiency developed by Strategy I plants, such as acidification and ferric reductase activity. However, their participation in such regulation is more controversial. There are no studies relating hormones to Fe uptake in Strategy II plants, although ethylene and phytosiderophores share common precursors. Auxin and ethylene have also been involved in other nutrient deficiencies, namely those of P and K. In this chapter, we review existing evidence suggesting a role for auxin, ethylene and other hormones and signaling substances in the regulation of Fe deficiency stress responses and other aspects of Fe nutrition, and discuss the involvement of hormones in the responses to other nutrient deficiencies.

Hypoxia and bicarbonate could limit the expression of iron acquisition genes in Strategy I plants by affecting ethylene synthesis and signaling in different ways

In a previous work, it was shown that bicarbonate (one of the most important factors causing Fe chlorosis in Strategy I plants) can limit the expression of several genes involved in Fe acquisition. Hypoxia is considered another important factor causing Fe chlorosis, mainly on calcareous soils. However, to date it is not known whether hypoxia aggravates Fe chlorosis by affecting bicarbonate concentration or by specific negative effects on Fe acquisition. Results found in this work show that hypoxia, generated by eliminating the aeration of the nutrient solution, can limit the expression of several Fe acquisition genes in Fe-deficient Arabidopsis, cucumber and pea plants, like the genes for ferric reductases AtFRO2, PsFRO1 and CsFRO1; iron transporters AtIRT1, PsRIT1 and CsIRT1; H(+) -ATPase CsHA1; and transcription factors AtFIT, AtbHLH38, and AtbHLH39. Interestingly, the limitation of the expression of Fe-acquisition genes by hypoxia did not occur in the Arabidopsis ethylene constitutive mutant ctr1, which suggests that the negative effect of hypoxia is related to ethylene, an hormone involved in the upregulation of Fe acquisition genes. As for hypoxia, results obtained by applying bicarbonate to the nutrient solution suggests that ethylene is also involved in its negative effect, since ACC (1-aminocyclopropane-1-carboxylic acid; ethylene precursor) partially reversed the negative effect of bicarbonate on the expression of Fe acquisition genes. Taken together, the results obtained show that hypoxia and bicarbonate could induce Fe chlorosis by limiting the expression of Fe acquisition genes, probably because each factor negatively affects different steps of ethylene synthesis and/or signaling.

Effects of Several Metals on Both Fe(III)‐ and Cu(II)‐Reduction by Roots of Fe‐Deficient Cucumber Plants

Abstract The ferric‐chelate reductase induced by Fe deficiency is also able to reduce other ions such as Cu2+. This Cu(II)‐reduction has been less studied and it has been suggested that Cu2+ ion rather than Cu2+‐chelate serves as the substrate. Ferric‐chelate reductase activity is inhibited by some metals, but the mechanisms implicated are not known. In the present work we use Fe‐deficient cucumber seedlings to study the interactions of Cu2+, Ni2+, Mn4+, and Fe3+ on both Fe(III)‐reduction and Cu(II)‐reduction activities. The response of Cu(II)‐reduction activity to Cu concentration, in the presence or absence of citrate, was also studied. Results showed that inhibition of the ferric‐chelate reductase activity by Cu2+ or Ni2+ could be partially reversed by increasing the concentration of Fe‐EDTA. The Cu(II)‐reduction activity was even stimulated by Fe‐EDTA or Ni2+; it was inhibited by a high concentration of Cu2+ itself; and it was not affected by the absence of citrate. Mn4+ caused a moderate inhibition of both Fe(III)‐reduction and Cu(II)‐reduction activities. Results agree with the hypothesis that free Cu2+ ion is the substrate for Cu(II)‐reduction and suggest that the mechanisms involved in Fe(III)‐reduction and Cu(II)‐reduction could have some differences and be affected by metals in different ways. The mode of action of metals on the reductase activity are discussed, but they are still not well known.

A Shoot Fe Signaling Pathway Requiring the OPT3 Transporter Controls GSNO Reductase and Ethylene in Arabidopsis thaliana Roots

Ethylene, nitric oxide (NO) and glutathione (GSH) increase in Fe-deficient roots of Strategy I species where they participate in the up-regulation of Fe acquisition genes. However, S-nitrosoglutathione (GSNO), derived from NO and GSH, decreases in Fe-deficient roots. GSNO content is regulated by the GSNO-degrading enzyme S-nitrosoglutathione reductase (GSNOR). On the other hand, there are several results showing that the regulation of Fe acquisition genes does not solely depend on hormones and signaling molecules (such as ethylene or NO), which would act as activators, but also on the internal Fe content of plants, which would act as a repressor. Moreover, different results suggest that total Fe in roots is not the repressor of Fe acquisition genes, but rather the repressor is a Fe signal that moves from shoots to roots through the phloem [hereafter named LOng Distance Iron Signal (LODIS)]. To look further in the possible interactions between LODIS, ethylene and GSNOR, we compared Arabidopsis WT Columbia and LODIS-deficient mutant opt3-2 plants subjected to different Fe treatments that alter LODIS content. The opt3-2 mutant is impaired in the loading of shoot Fe into the phloem and presents constitutive expression of Fe acquisition genes. In roots of both Columbia and opt3-2 plants we determined 1-aminocyclopropane-1-carboxylic acid (ACC, ethylene precursor), expression of ethylene synthesis and signaling genes, and GSNOR expression and activity. The results obtained showed that both ‘ethylene’ (ACC and the expression of ethylene synthesis and signaling genes) and ‘GSNOR’ (expression and activity) increased in Fe-deficient WT Columbia roots. Additionally, Fe-sufficient opt3-2 roots had higher ‘ethylene’ and ‘GSNOR’ than Fe-sufficient WT Columbia roots. The increase of both ‘ethylene’ and ‘GSNOR’ was not related to the total root Fe content but to the absence of a Fe shoot signal (LODIS), and was associated with the up-regulation of Fe acquisition genes. The possible relationship between GSNOR(GSNO) and ethylene is discussed.

The Role of Ethylene and Other Signals in the Regulation of Fe Deficiency Responses by Dicot Plants

Iron (Fe) is abundant but its availability for plants is low specially on calcareous soils. To facilitate its acquisition, dicot (Strategy I) plants switch on several morphological and physiological changes in their roots, known as Fe responses. Once plants acquire enough Fe, the responses are switched off. Their regulation is not totally known but since the 1990s different results have supported a role for the plant hormone ethylene in such a process. Most of those results have been based on morphological and physiological studies and have been previously reviewed. Although the role of ethylene in the regulation of morphological Fe responses has been generally accepted, its role in the regulation of physiological Fe responses has been more controversial. In this review, we discuss the most recent results supporting a role for ethylene in the regulation of physiological Fe responses, most of them based on transcriptomic, proteomic, molecular and genetic analyses. In addition, we review results suggesting a role, either as activators or suppressors of physiological Fe responses, of other hormones and non-hormonal substances, such as auxin, nitric oxide, glutathione, and phloem Fe. As conclusion, we propose a Working Model that integrates both positive and negative signals in the regulation of physiological Fe responses. As positive signals, ethylene and nitric oxide would act at the end of the signalling cascade leading to the activation of physiological Fe responses while phloem Fe could play an important role as repressor.