Hormones induce an Fe-deficiency-like root epidermal cell pattern in the Fe-inefficient tomato mutantfer

Summary The tomato mutantfer (Lycopersion esculentum L. T3238fer) displayed a chlorotic phenotype at normal external Fe levels. Root cells of the mutant are incompetent to take up iron in adequate amounts and are incapable to induce any of the known responses to Fe deficiency stress. We report here that the ethylene precursor 1-aminocyclopropane-l-carboxylic acid and the auxin analog 2,4-dichlorophenoxyacetic acid induce the formation of extra root hairs and transfer cells in the epidermis, thus mimicking the root-morphological Fe stress responses. In contrast, the physiological reactions involved in iron acquisition are not affected by the hormone treatment. These results indicate that ethylene is essential for transducing environmental signals into adaptive changes in root morphology. The data further suggest that the mutation does not affect necessary steps in the differentiation processes of epidermal cells. TheFER gene appears to control sensing of iron levels and/or the regulation of mechanisms involved in iron uptake.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BPDS bathophenanthrolinedisulfonate - FeHEDTA Fe hydroxyethylethylenediaminetriacetic acid     

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     

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.     

Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

Role of hormones in the induction of iron deficiency responses in Arabidopsis roots

In "strategy I" plants, several alterations in root physiology and morphology are induced by Fe deficiency, although the mechanisms by which low Fe levels are translated into reactions aimed at alleviating Fe shortage are largely unknown. To prove whether changes in hormone concentration or sensitivity are involved in the adaptation to suboptimal Fe availability, we tested 45 mutants of Arabidopsis defective in hormone metabolism and/or root hair formation for their ability to increase Fe(III) chelate reductase activity and to initiate the formation and enlargement of root hairs. Activity staining for ferric chelate reductase revealed that all mutants were responsive to Fe deficiency, suggesting that hormones are not necessary for the induction. Treatment of wild-type plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid caused the development of root hairs in locations normally occupied by non-hair cells, but did not stimulate ferric reductase activity. Ectopic root hairs were also formed in -Fe roots, suggesting a role for ethylene in the morphological responses to Fe deficiency. Ultrastructural analysis of rhizodermal cells indicated that neither Fe deficiency nor 1-aminocyclopropane-1-carboxylic acid treatment caused transfer-cell-like alterations in Arabidopsis roots. Our data indicate that the morphological and physiological components of the Fe stress syndrome are regulated separately.     

Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H+-ATPase in rhizodermal cells

The immunocytochemical localization of the plasma membrane H⁺‐ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P‐type H⁺‐ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H⁺‐ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non‐acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe‐deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H⁺‐ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H⁺‐ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non‐active segments. Split‐root experiments indicated that both the steady‐state level of plasma membrane H⁺‐ATPase and proton extrusion activity are systemically regulated, indicating inter‐organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4‐dichlorophenoxyacetic acid and the ethylene precursor 1‐aminocyclopropane‐1‐carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe‐deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.     

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.     

Stage-specific crosstalk between light, auxin, and ethylene during low-pH-induced root hair formation in lettuce (Lactuca sativa L.) seedlings

In the light, transfer of lettuce seedlings precultured on liquid medium at pH 6.0 to fresh medium at pH 4.0 induces root hair formation. However, no root hairs form in the dark. Here, we investigated how light induces root hair formation. Randomization of the transverse cortical microtubule (CMT) arrays which occurs in root epidermal cells in the light prior to root hair initiation was not observed in the dark. However, addition of indole-3-acetic acid (IAA) or 1-aminocyclopropane-1-carboxylic acid (ACC) induced CMT randomization and root hair formation. In these cases, CMT randomization occurred in almost the same time-dependent manner as under light. However, root hair initiation was delayed for several hours in the dark. These results suggest that light promotes CMT randomization and root hair initiation via auxin and ethylene signaling but light additionally influences root hair initiation independently of these signaling mechanisms. Furthermore, addition of a microtubule-depolymerizing drug in the dark disrupted the transverse CMT arrays and initiated root hair formation; however, root hair elongation was still suppressed. Root hairs elongated when IAA or ACC was applied with the drug. These results suggest that light also promotes root hair elongation via auxin and ethylene signaling.     

Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation

Summary Sunflowers are known to respond to Fe deficiency (-Fe) with a typical root tip swelling and the formation of root hairs and transfer cells in the rhizodermis. The possible regulation of this process was examined by a comparative study of root morphology and cytology of intact seedlings (Helianthus annuus L. cv. Giganteus) under -Fe and hormonal treatment in nutrient solution. Longitudinal sections of -Fe roots showed root tip swelling is due to cessation of cell elongation and isodiarnetric volume increase of the cortical cells. Enhanced cell division in the pericycle leads to the formation of lateral root primordia in the swollen zone. Xylem vessel differentiation is markedly accelerated and accompanied by early differentiation of the casparian band in the endodermis. Exogenous application of IAA (10^–8-10^–7 M) via the nutrient solution to Fe sufficient plants causes symptoms which closely mimick the characteristics of Fe deficiency including root hair development. Moreover, rhizodermal cells produce peripheral protuberances reminiscent of -Fe transfer cells. Ethylene-releasing ethephon (10^–4M) also causes subapical swelling and root hair formation. However, wall protuberance development is less pronounced. ABA (10^–5 M) leads to similar root thickening and root hair formation but without any comparable transfer cell differentiation. From the striking similarities between -Fe and IAA treatment it is concluded that this hormone (possibly in cooperation with ethylene) is involved in the Fe stress response of sunflower roots. The importance of a continuous polar IAA transport for this process is discussed.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - Ethephone 2-chloro-ethylphosphonic acid - Fe(III)-EDTA ethylenediaminetetraacetic ferric-sodium salt - IAA indole-acetic acid - TIBA triiodobenzoic acid     

The Arabidopsis root hair mutants der2-der9 are affected at different stages of root hair development

Root hairs are an excellent model system to study cell developmental processes as they are easily accessible, single-celled, long tubular extensions of root epidermal cells. In a genetic approach to identify loci important for root hair development, we have isolated eight der (deformed root hairs) mutants from an ethylmethanesulfonate (EMS)-mutagenized Arabidopsis population. The der lines represent five new loci involved in root hair development and show a variety of abnormalities in root hair morphology, indicating that different root hair developmental stages are affected. A double mutant analysis with the short root hair actin2 mutant der1-2 confirmed that the der mutants are disturbed at different time points of root hair formation. Auxin and ethylene are known to be important for trichoblast cell fate determination and root hair elongation. Here, we show that they are able to suppress the phenotype of two der mutants. As the auxin- and ethylene-responsive der mutants are affected at different stages of root hair formation, our results demonstrate that the function of auxin and ethylene is not limited to cell differentiation and root hair elongation but that the two hormones are effective throughout the whole root hair developmental process.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.     

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.     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe(III)-chelate reductase in roots of strategy I species

Stimulation of root Fe(III) reductase activity by iron additions to iron-deficient growth media may be the result of iron activation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase required for ethylene biosynthesis. Two different ethylene inhibitors, aminooxyacetic acid (AOA) (20 m; ACC synthase inhibitor) and cobalt (3 m CoCl₂; ACC oxidase inhibitor), were used to study the effects of iron supply and cobalt inhibition on ethylene action in controlling the activity of Fe(III)-chelate reductase in pea (Pisum sativum L.) roots. Supplying 20 gm m Fe(III)-N,N-ethylenebis[2-(2-hydroxypheyl)-glycine [Fe(III)-EDDHA] to either cobalt-treated, iron-deficient Sparkle (normal parent) or E107 (brz mutant genotype) pea seedlings reversed the negative effects of cobalt on root Fe(III)-reductase activity. Re-supplying 20 m Fe(III)-EDDHA to iron-deficient, AOA-treated seedlings did not enhance root Fe(III)-reductase. Apparently, cobalt competes with iron for the active site in ACC oxidase during ethylene synthesis. Inhibition of root reductase activity by cobalt treatment lowered manganese, zinc, magnesium and potassium content of mutant E107 pea seedlings. In contrast, iron enhancement of root reductase activity in iron-deficient, cobalt-treated E107 seedlings resulted in higher seedling accumulations of manganese, zinc, magnesium and potassium. These results support the hypothesis that root cell plasma membrane reductase activity plays a role in cation uptake by root cells.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.