Different pathways are involved in phosphate and iron stress-induced alterations of root epidermal cell development

Low bioavailability of phosphorus (P) and iron (Fe) induces morphogenetic changes in roots that lead to a higher surface-to-volume ratio. In Arabidopsis, an enlargement in the absorptive surface area is achieved by an increase in the length and frequency of hairs in roots of Fe- and P-deficient plants. The extra root hairs are often located in positions that are occupied with non-hair cells under normal conditions, i.e. over a tangential wall of underlying cortical cells. An involvement of auxin and ethylene in root epidermis cell development of Fe- and P-deficient plants was inferred from phenotypical analysis of hormone-related Arabidopsis mutants and from the application of substances that interfere with either synthesis, transport, or perception of the hormones. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-D caused a marked increase in root hair density in plants of all growth types and confers a phenotype characteristic of ethylene-overproducing mutants. Hormone insensitivity and application of hormone antagonists inhibited the initiation of extranumerary root hairs induced by Fe deficiency, but did not counteract the formation of extra hairs in response to P deprivation. A model is presented summarizing putative pathways for alterations in root epidermal cell patterning induced by environmental stress.     

Acclimative changes in root epidermal cell fate in response to Fe and P deficiency: a specific role for auxin?

Summary Root hair formation and the development of transfer cells in the rhizodermis was investigated in various existing auxinrelated mutants ofArabidopsis thaliana and in the tomato mutantdiageotropica. Wild-type Arabidopsis plants showed increased formation of root hairs when the seedlings were cultivated in Fe- or P-free medium. These extranumerary hairs were located in normal positions and in positions normally occupied by nonhair cells, e.g., over periclinal walls of underlying cortical cells. Defects in auxin transport or reduced auxin sensitivity inhibited the formation of root hairs in response to Fe deficiency completely but did only partly affect initiation and elongation of hairs in P-deficient roots. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-dichlorophenoxyacetic acid did not rescue the phenotype of the auxin-resistantaxr2 mutant under control and Fe-deficient conditions, indicating that functionalAXR2 product is required for translating the Fe deficiency signal into the formation of extra hairs. The development of extra hairs inaxr2 roots under P-replete conditions was not affected by auxin antagonists, suggesting that this process is independent of auxin signaling. In roots of tomato, growth under Fe-deficient conditions induced the formation of transfer cells in the root epidermis. Transfer cell frequency was enhanced by application of 2,4-dichlorophenoxyacetic acid but was not inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid. In thediageotropica mutant, which displays reduced sensitivity to auxin, transfer cells appeared to develop in both Fe-sufficient and Fe-deficient roots. Similar to the wild type, no reduction in transfer cell frequency was observed after application of the above auxin transport inhibitor. These data suggest that auxin has no primary function in inducing transfer cell development; the formation of transfer cells, however, appears to be affected by the hormonal balance of the plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - TIBA triiodobenzoic acid - NPA N-1-naphthylphthalamic acid - STS silver thiosulfate     

Modulation of the root epidermal phenotype by hormones,inhibitors and iron regime

Patterning of epidermal cells is subject to genetic regulation but also influenced by environmental stimuli. To adapt to unfavorable environmental conditions plants have developed various mechanisms to increase the plasma membrane's surface area of epidermal root cells, for example through the formation of root hairs and differentiation of rhizodermal transfer cells. Mechanisms controlling cell fate speciation in the rhizodermis were investigated by application of hormones and hormone antagonists. In addition, the effect of Fe deficiency on root epidermal patterning and Fe(III)-reduction activity was examined. In the iron-hyperaccumulating pea mutants dgl and brz and in the Arabidopsis mutant man1 Fe(III)-reduction activity was found to be up-regulated under both high and low iron supply. In contrast, morphological responses such as the development of transfer cells and extranumerary root hairs was repressed by a high iron concentration in the external medium. All morphological responses can be mimicked by exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D). Conversely, Fe(III)-reduction rates were not influenced or only slightly affected by the hormone treatment. Application of inhibitors of ethylene synthesis, ethylene action or auxin transport was effective only in inhibiting the formation of extra root hairs, indicating that these hormones are not required for transfer cell formation or expression of Fe(III) reduction. These data suggest that the Fe reductase induced by iron stress does not depend on the formation of transfer cells and further imply separate regulatory pathways for the two responses. The data are compatible with a model in which root reduction activity is modulated by a shoot-borne signal coordinating iron uptake with the shoot demand, while the epidermal phenotype is primarily dependent on the intracellular iron concentration of root cells.     

Iron stress-induced changes in root epidermal cell fate are regulated independently from physiological responses to low iron availability

Iron-overaccumulating mutants were investigated with respect to changes in epidermal cell patterning and root reductase activity in response to iron starvation. In all mutants under investigation, ferric chelate reductase activity was up-regulated both in the presence and absence of iron in the growth medium. The induction of transfer cells in the rhizodermis appeared to be iron regulated in the pea (Pisum sativum L. cv Dippes Gelbe Viktoria and cv Sparkle) mutants bronze and degenerated leaflets, but not in roots of the tomato (Lycopersicon esculentum Mill. cv Bonner Beste) mutant chloronerva, suggesting that in chloronerva iron cannot be recognized by putative sensor proteins. Experiments with split-root plants supports the hypothesis that Fe(III) chelate reductase is regulated by a shoot-borne signal molecule, communicating the iron status of the shoot to the roots. In contrast, the formation of transfer cells was dependent on the local concentration of iron, implying that this shoot signal does not affect their formation. Different repression curves of the two responses imply that the induction of transfer cells occurs after the enhancement of electron transfer across the plasma membrane rather than being causally linked. Similar to transfer cells, the formation of extra root hairs in the Arabidopsis mutant man1 was regulated by the iron concentration of the growth medium and was unaffected by interorgan signaling.     

Does Ethylene Mediate Cluster Root Formation under Iron Deficiency?

Casuarina glauca develops proteoid (cluster) roots in response to Fe deficiency. This study set out to investigate the possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots (CR). For this purpose, the effect of Ag+ added as silver thiosulfate, an inhibitor of ethylene action has been studied in plants growing hydroponically. No CR formation was observed in these growth conditions. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine, 1- aminoisobutyric acid, aminoxyacetic acid or cobalt chloride also eliminated the positive effect of Fe deficiency on CR formation in C. glauca. CR were not formed in Fe- deficient roots in the presence of ethylene inhibitors, suggesting a role for ethylene in the morphological responses to Fe deficiency. Interestingly, treatment of Casuarina plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid stimulated significantly the formation of CR, even if plants are supplied with Fe. However, this stimulation did not reach the level of CR obtained in Fe-deficient plants. These results suggest that an ethylene-mediated signalling pathway is involved in CR formation process in C. glauca.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

Iron deficiency-induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover

Phenolic compounds are frequently reported to be the main components of root exudates in response to iron (Fe) deficiency in Strategy I plants, but relatively little is known about their function. Here, we show that removal of secreted phenolics from the root-bathing solution almost completely inhibited the reutilization of apoplastic Fe in roots of red clover (Trifolium pratense). This resulted in much lower levels of shoot Fe and significantly higher root Fe compared with control and also resulted in leaf chlorosis, suggesting this approach stimulated Fe deficiency. This was supported by the observation that phenolic removal significantly enhanced root ferric chelate reductase activity, which is normally induced by plant Fe deficiency. Furthermore, root proton extrusion, which also is normally increased during Fe deficiency, was found to be higher in plants exposed to the phenolic removal treatment too. These results indicate that Fe deficiency-induced phenolics secretion plays an important role in the reutilization of root apoplastic Fe, and this reutilization is not mediated by proton extrusion or the root ferric chelate reductase. In vitro studies with extracted root cell walls further demonstrate that excreted phenolics efficiently desorbed a significant amount of Fe from cell walls, indicating a direct involvement of phenolics in Fe remobilization. All of these results constitute the first direct experimental evidence, to our knowledge, that Fe deficiency-induced secretion of phenolics by the roots of a dicot species improves plant Fe nutrition by enhancing reutilization of apoplastic Fe, thereby improving Fe nutrition in the shoot.     

Formation of transfer cells and H(+)-ATPase expression in tomato roots under P and Fe deficiency

In roots of tomato ( Lycopersicon esculentum Mill.), extranumerary root hairs and transfer cell-like wall ingrowth depositions in the rhizodermis were developed in response to P and Fe deficiency. Immunocytolocalization of the plasma membrane H(+)-ATPase in roots of P-deficient plants revealed no appreciable increase in H(+)-ATPase density relative to control plants. In transfer cells, immunogold labeling was considerably higher than in ordinary rhizodermal cells. H(+)-ATPase sites were asymmetrically distributed in cells with and without wall ingrowths under P-deficient conditions. A split-root study revealed that the frequency of transfer cells was higher in the low-P half of the root system, but the density of H(+)-ATPase molecules was enhanced only in the high-P half of the split roots, suggesting that formation of transfer cells was controlled directly by the external Pi concentration, whereas ATPase expression was regulated indirectly by the internal nutrient status of the plant. The role of hormones in the induction of transfer cells was investigated by treating plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or various ethylene antagonists. Transfer cells were induced by ACC to an extent similar to that observed after P or Fe starvation, but inhibitors of either ethylene synthesis or action did not decrease their frequency. These results suggest that ethylene was not required for the induction of transfer cells but changes in ethylene levels appeared to modulate the number of cells forming wall ingrowths. In roots of ethylene-insensitive Never-ripe tomato plants the frequency of transfer cells was rather increased than decreased under most growth conditions relative to the wild type, indicating that ethylene responsiveness played no critical role in the differentiation of transfer cells and that the transduction of signals ultimately leading to their formation was independent of the ethylene signaling cascade.     

Natural variation for Fe-efficiency is associated with upregulation of Strategy I mechanisms and enhanced citrate and ethylene synthesis in Pisum sativum L.

Iron (Fe)-deficiency is a common abiotic stress in Pisum sativum L. grown in many parts of the world. The aim of the study was to investigate variation in tolerance to Fe deficiency in two pea genotypes, Santi (Fe-efficient) and Parafield (Fe-inefficient). Fe deficiency caused greater declines in chlorophyll score, leaf Fe concentration and root-shoot development in Parafield compared to Santi, suggesting greater Fe-efficiency in Santi. Fe chelate reductase activity and ethylene production were increased in the roots of Santi and to a lesser extent in Parafield under Fe deficiency, while proton extrusion was only occurred in Santi. Moreover, expression of the Fe chelate reductase gene, FRO1, and Fe transporter, RIT1 were upregulated in Fe-deficient roots of Santi. Expression of HA1 (proton extrusion) was also significantly higher in Santi when compared to Parafield grown in Fe-deficient conditions. Furthermore, the application of the ethylene biosynthesis inhibitor, 1-aminoisobutyric acid reduced the Fe chelate reductase activity, supporting a direct role for ethylene in its induction. A significant increase in root citrate was only observed in Santi under Fe deficiency indicating a role for citrate in the Fe-efficiency mechanism. Taken together, our physiological and molecular data indicate that genotypic variation in tolerance to Fe deficiency in Santi and Parafield plants is a result of variation in a number of Strategy I mechanisms and also suggest a direct role for ethylene in Fe reductase activity. The pea cultivar, Santi provides a new source of Fe-efficiency that can be exploited to breed more Fe-efficient peas.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

The Iron-Deficiency Induced Phenolics Accumulation May Involve in Regulation of Fe(III) Chelate Reductase in Red Clover

Although considerable researches have been conducted on the physiological responses to plant iron (Fe) deficiency stress in dicotyledonous plants, much still needs to be learned about the regulation of these processes. In the present research, red clover was used to investigate the role of root phenolics accumulation in regulating Fe-deficiency induced Fe(III) chelate reductase (FCR). The root FCR activity, IAA and phenolics accumulation, and also the phenolics secretion were greatly increased by the Fe deficiency treatment. The application of TIBA (2,3,5-triiodobenoic acid) to the stem, an IAA polar transport inhibitor, which could decrease IAA accumulation in root, significantly inhibited the FCR activity, but did not effect root phenolics accumulation and secretion, suggesting that IAA itself did not involve in root phenolics accumulation and secretion. In contrast, the Fe deficiency treatment significantly decreased the root IAA-oxidase activity. Interestingly the phenolics extracted from roots inhibited IAA-oxidase activity in vitro, and this inhibition was greater with phenolics extracted from roots of Fe deficient plants than that from Fe sufficient plants, indicating that the Fe deficiency-induced IAA-oxidase inhibition probably caused by the phenolics accumulation in Fe deficient roots. Based on these observations, we propose a model where under Fe deficiency stress in dicots, an increase in root phenolics concentrations plays a role in regulating root IAA levels through an inhibition of root IAA oxidase activity. This response, leads to, or at least partially leads to an increase in root IAA levels, which in turn help induce increased root FCR activity.Key Words: Fe deficiency, ferric chelate reductase, phenolics, Trifolium pretense     

Iron deficiency enhances the levels of ascorbate,glutathione, and related enzymes in sugar beet roots

Summary.  The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet (Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants. Received November 19, 2001; accepted September 30, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Apartado 202, 50080 Zaragoza, Spain.     

Radial expansion of root cells and elongation of root hairs of Arabidopsis thaliana induced by massive doses of gamma irradiation

Radial expansion of root cells and elongation of root hairs were induced within 3 d of a massive dose (3 kGy) of gamma irradiation to Arabidopsis thaliana. Because treatment with the antioxidant n-propyl gallate before irradiation suppressed these changes, gamma irradiation partially rescued the rhd2 mutant (defective in NADPH oxidase); the superoxide-generating reagent paraquat induced similar root morphogenesis. These responses appeared to be induced by the active oxygen species (AOS) generated by water radiolysis. Ethylene production was induced immediately after gamma irradiation and reached a steady level after about 2 h. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid partly induced a similar expansion of root cells and elongation of root hairs. Addition of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine, before gamma irradiation completely suppressed the formation of abnormal structures. These results suggest that the AOS is involved in the root morphological changes through the ethylene biosynthesis induced by gamma irradiation in Arabidopsis.     

Root Hair Initiation Is Coupled to a Highly Localized Increase of Xyloglucan Endotransglycosylase Action in Arabidopsis Roots

Root hairs are formed by two separate processes: initiation and subsequent tip growth. Root hair initiation is always accompanied by a highly localized increase in xyloglucan endotransglycosylase (XET) action at the site of future bulge formation, where the trichoblast locally loosens its cell wall. This suggests an important role of XET in the first stages of root hair initiation. The tip of growing root hairs is not marked by localized high XET action. Experiments in which root hair initiation was modulated and observations on root hair mutants support this view. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid shifts both root hair initiation and the local increase in XET action toward the root tip. On the other hand, roots treated with the ethylene inhibitor aminoethoxyvinyl-glycine, as well as roots of mutants affected in root hair initiation (rhl1, rhd6-1, and axr2-1) revealed no localized increases of XET action at all and consequently did not initiate root hairs. Disruption of actin and microtubules did not prevent the localized increase in XET action. Also, the temporal and spatial pattern of action as the specific pH dependence suggest that different isoforms of XET act in different processes of root development.     

The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin- and Ethylene-Associated Process

Root-hair initiation in Arabidopsis thaliana provides a model for studying cell polarity and its role in plant morphogenesis. Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized. We have identified a mutant, rhd6, that displays three defects: (a) a reduction in the number of root hairs, (b) an overall basal shift in the site of root-hair emergence, and (c) a relatively high frequency of epidermal cells with multiple root hairs. These defects implicate the RHD6 gene in root-hair initiation and indicate that RHD6 is normally associated with the establishment of, or response to, root epidermal cell polarity. Similar alterations in the site of root-hair emergence, although less extreme, were also discovered in roots of the auxin-, ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-resistant mutant etr1. All three rhd6 mutant phenotypes were rescued when either auxin (indoleacetic acid) or an ethylene precursor (1-aminocyclopropane-1-carboxylic acid) was included in the growth medium. The rhd6 root phenotypes could be phenocopied by treating wild-type seedlings with an inhibitor of the ethylene pathway (aminoethoxyvinylglycine). These results indicate that RHD6 is normally involved in directing the selection or assembly of the root-hair initiation site through a process involving auxin and ethylene.     

The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet)

The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase.