JXB Advance Access originally published online on August 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 391, pp. 2351-2361,
October 1, 2003
© 2003 Oxford University Press
Ethylene and phosphorus availability have interacting yet distinct effects on root hair development
Received 10 March 2003; Accepted 20 June 2003
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Department of Horticulture, Penn State University, University Park, PA 16802, USA
* Present address: The Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA. To whom correspondence should be addressed. Fax: +1 814 863 6139. E-mail: kbe{at}psu.edu
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Abstract |
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The hypothesis that ethylene participates in the regulation of root hair development by phosphorus availability in Arabidopsis thaliana was tested by chemically manipulating ethylene synthesis and response and with ethylene-insensitive mutants. Low phosphorus-induced root hair development could be mimicked by adding the ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC), to high phosphorus media, and inhibited by adding ethylene inhibitors to low phosphorus media. Ethylene-insensitive mutants showed a reduced response to low phosphorus, indicating ethylene involvement in root hair responses to phosphorus deficiency. To dissect the nature of this involvement, the morphological and anatomical changes associated with increased root hair density were investigated. Growth in low phosphorus resulted in smaller, more numerous cortical cells, resulting in a larger number of root hair-bearing epidermal cell files. Cortical cell number was not affected by ethylene inhibitors, ACC, or mutations reducing ethylene sensitivity in roots grown with low phosphorus, indicating that ethylene does not participate in this response. The exception was the eir1 mutation, which strongly reduced this change in radial anatomy, supporting a role for polar auxin transport in this process. Trichoblast cell length was reduced by low phosphorus availability in all genotypes, but even more so for ein2-1 and ein4. The proportion of epidermal cells forming hairs and root hair length were reduced in ethylene-insensitive mutants, especially in the presence of low phosphorus. These results demonstrate multiple effects of low phosphorus from the earliest stages of root hair development, and cross-talk between ethylene and phosphorus in the control of a subset of the low phosphorus effects, concentrating on those later in development.
Key words: Arabidopsis thaliana, ethylene, ethylene-insensitive mutants, phosphorus, root hair density, root hair length, trichoblast.
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Introduction |
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Root hairs are subcellular protrusions of epidermal cells that are important for the acquisition of immobile nutrients such as phosphorus (Clarkson, 1985; Jungk, 2001). Root hair production is stimulated by the deficiency of several nutrients, including iron, zinc, manganese, and phosphorus, but, at least in Arabidopsis, phosphorus has a greater effect than other nutrients on root hair density and length (Bates and Lynch, 1996; Foehse and Jungk, 1983; Gahoonia and Nielsen, 1997; Ma et al., 2001a; Schmidt and Schikora, 2001). The effects of low phosphorus availability on root hair density, length, and distribution act synergistically to increase the effective volume of soil from which the plant can acquire phosphorus, and therefore its growth and competitiveness when phosphorus availability is limiting (Bates and Lynch, 2000b, 2001; Ma et al., 2001b).
In Arabidopsis, root hairs are formed from specific epidermal cells located over the intercellular space between underlying cortical cells, i.e. cells in the H position, while cells located directly over a single cortical cell (in the N position) do not form hairs (Dolan et al., 1994). Not all epidermal cells in the H position actually form hairs, i.e. become trichoblasts, and ectopic hairs may occur on cells in the N position with ethylene and other treatments (Dolan et al., 1994; Ma et al., 2001a; Schmidt and Schikora, 2001; Tanimoto et al., 1995).
Trichoblast files are typically separated by one to three files of atrichoblasts (non-hair-bearing cells) (Dolan and Costa, 2001; Pemberton et al., 2001). Most reports indicate that there are eight cortical cells in cross-section and correspondingly eight trichoblast files (Dolan and Costa, 2001). Ma et al. (2001a) found that Arabidopsis grown under phosphorus deficiency had 12 cortical cell files, and therefore 12 trichoblast files. This increase in the number of potential hair-bearing cells, along with an increase in the proportion of H cells forming root hairs, accounted for most of the increased root hair density observed under phosphorus deficiency (Ma et al., 2001a).
It has been proposed that the position-dependent differentiation of trichoblasts may be regulated by ethylene transported from the inner root through the apoplast (Michael, 2001; Tanimoto et al., 1995). This was based on the enhancement of root hair production by ethylene, ACC, and the ctr1 (constitutive ethylene response) mutation, and the inhibition of root hair production by the ethylene inhibitors aminoethoxyvinylglycine (AVG) and Ag+ (Dolan et al., 1994; Kieber et al., 1993; Masucci and Schiefelbein, 1996; Michael, 2001; Tanimoto et al., 1995). The fact that ctr1 and ACC could cause the development of ectopic hairs supported the idea that ethylene was responsible for the differentiation of epidermal cells to form hairs. However, later work showed that neither 1-methylcyclopropene (1-MCP), an ethylene response inhibitor, nor mutants with impaired ethylene response showed a reduction in root hair density unless root hair formation was stimulated by hormonal or environmental stimuli (Cho and Cosgrove, 2002).
The similarities in root hair response to low phosphorus and ethylene suggest a role for ethylene in mediating this nutrient deficiency response. Nutrient stress, either deficiency or toxicity, can change ethylene biosynthesis and/or responsiveness (Lynch and Brown, 1997). Borch et al. (1999) found that phosphorus-deficient bean roots produced twice as much ethylene per unit dry weight as roots supplied with adequate phosphorus. It is therefore reasonable to suggest that ethylene might mediate root hair production in response to phosphorus deficiency. However, Schmidt and Schikora (Schmidt and Schikora, 2001) found that phosphorus deficiency increased root hair density of Arabidopsis even in the presence of the ethylene antagonists AVG, amino-oxyacetic acid (AOA), Co2+, and STS. Based on this result, the authors proposed a hypothetical model in which phosphorus deficiency may not affect ethylene synthesis and signal transduction pathways, but interacts directly with ethylene-responsive genes associated with root hair formation (Schmidt, 2001; Schmidt and Schikora, 2001).
In this paper, the hypothesis was tested that ethylene and phosphorus interact to control root hair density and length. The root hair responses to phosphorus deficiency and ethylene manipulation were compared and the ability of ethylene-insensitive mutants to respond to phosphorus was investigated. Since root hair formation involves many developmental stages (Parker et al., 2000; Schiefelbein, 2000) and low phosphorus availability affects several of these (Ma et al., 2001a), the effects of phosphorus and ethylene manipulation on specific aspects of root anatomy known to contribute to increased root hair density were examined.
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Materials and methods |
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Seeds of Arabidopsis thaliana L. (Heynh) Columbia and isogenic ethylene-insensitive mutants ein2-1, ein3-1, ein4, ein5-1, ein7, and eir1-1, as well as ein6, which is in the Landsberg erecta background, were obtained from the Arabidopsis Biological Resource Center, of the Ohio State University. Seeds propagated in the greenhouse were used in all experiments reported here.
Growth media composition and preparation, seed germination, seedling culture, and root hair observation were carried out according to Ma et al. (2001a), except for the use of a dark period, in a 16/8 h light/dark alternation. A completely randomized design was used with at least six replicates per treatment. NH4H2PO4 was added to give targeted phosphorus concentrations of 1, 10, 100 or 1000 µM, and (NH4)2SO4 was used to balance the N among phosphorus treatments. Treatments with 1 or 1000 µM phosphate are referred to as low phosphorus or high phosphorus, respectively. To manipulate ethylene production and sensitivity, fresh media containing the ethylene precursor ACC, the ethylene production inhibitor AVG, or the ethylene action inhibitor STS (silver thiosulphate), were added to the existing media after seedlings had grown 12 d, as described by Bates and Lynch (1996). For treatment with the gaseous ethylene action inhibitor 1-MCP (EthylBloc®: 0.43% 1-methylcyclopropene, Floralife Inc. 751 Thunderbolt Drive, Walterboro, SC), open Petri dishes with 12-d-old plants were placed in an 8.0 l, air-tight container, which also contained a 5 ml vial containing 2 mg EthylBloc l–1 of chamber volume. The container was surface-disinfected with 70% alcohol before use. The chamber was sealed, and 0.04 ml distilled water was added to the vial through a septum to release 1-MCP gas. The treatment lasted 2 d until root hair measurement. At 14 d of growth (2 d after adding ethylene inhibitors or ACC, or after the initiation of 1-MCP treatment), six plants of each treatment were evaluated under a stereomicroscope for root hair length and density. The experiment was repeated four times.
For the root anatomical observations, roots of at least five plants were sampled from plants of the same age as used for root hair measurement, and root segments (2–3 mm long) were taken from the same region for root hair observation. Root fixation, embedding, cross-sectioning, and staining were done according to Ma et al. (2001a).
To measure trichoblast cell length, Columbia, ein2-1 and ein4 were used, each of which was transformed with ß-glucuronidase (GUS) reporter gene under the control of the promoter of AtEXP7, an 
-type cell wall expansin specifically expressed in hair-bearing epidermal cells (Cho and Cosgrove, 2002). For AtEXP7::GUS-transformed materials, treatments included low phosphorus, low phosphorus plus 3 µM AVG, high phosphorus, and high phosphorus plus 1 µM ACC. Living roots of transformed plants were stained at 37 °C for 1 h with 1.0 mM X-glucuronide in the buffer (0.1 M NaH2PO4, 10 mM EDTA, 0.5 mM K-ferricyanide, 0.1% Triton X-100, pH adjusted to 7.0 with 1 N NaOH), washed three times with the same buffer, then stained with 0.05% aqueous solution of Neutral red for 5 min at room temperature. For non-transformed plants, living roots were stained with 0.05% Toluidine Blue O in 0.1 M Na2HPO4/NaH2PO4 buffer (pH 6.8) for 5 min at room temperature. Stained root images were recorded with a digital camera connected with a microscope, and the cell length was then measured with Photoshop (Adobe Photoshop® 6.0, Adobe System Incorporated). Trichoblast cell lengths were measured on six cells from each of six plants.
Root growth rate of untransformed plants grown with low or high phosphorus was recorded daily by marking the root tip position on the bottom of the Petri dish, starting from day 9 of culture. Daily root growth was estimated by scanning the Petri dish and analysing the digital image with WinRHIZO (Regent Instruments Inc., 4040 Rue Blain, Quebec, G2B 5C3 Canada, Version 3.10b). Each treatment was repeated six times. Preliminary experiments showed a steady growth rate from day 9 to day 13; values taken from day 9 to day 10 are presented.
ANOVA of root hair data was conducted using statistical software MiniTab (Minitab Inc.).
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Results |
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Root hair growth in response to low phosphorus availability and ethylene
Ethylene production and action were manipulated using the ethylene production inhibitor AVG, the ethylene action inhibitors STS or 1-MCP, or the ethylene precursor ACC. The inhibitors reduced root hair density and length of Columbia plants grown in low phosphorus, while ACC increased root hair density and length of plants grown in high phosphorus (Table 1). Ethylene inhibitors reduced root hair density and length as much or more than high phosphorus availability. Root hair growth could be manipulated in the growing plant by adding ACC or AVG to the medium with similar results (Fig. 1).
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Root hair density and length of the ethylene response mutants were evaluated under low (1 µM) or high phosphorus (1000 µM) (Fig. 2). For all genotypes, root hair density and length in low phosphorus media were significantly greater than in high phosphorus media. In high phosphorus, ethylene-insensitive genotypes had root hair lengths and densities varying from greater than to less than those of Columbia. All mutant genotypes had shorter and less dense root hairs than Columbia with low phosphorus, except ein6, which had root hair length equal to Columbia. The responsiveness to low phosphorus was defined as the ratio of means between low and high phosphorus treatments, which was the greatest for Columbia (Fig. 2). All the mutants were less responsive to phosphorus availability than Columbia.
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Columbia and two ethylene-insensitive mutants, eir1-1 and ein7, displayed increasing root hair density and length as phosphorus availability declined (Fig. 3). The root hair density of these genotypes was the same with 1000 µM phosphorus, but Columbia increased its hair density more sharply than the mutants as phosphorus concentration decreased to 1 µM (Fig. 3A). Similarly, the mutants were less sensitive than Columbia to the effects of reduced phosphorus availability on root hair length (Fig. 3B). As a result, both mutants had shorter hairs than Columbia when plants were grown in 1 µM phosphorus, even though at 1000 µM phosphorus, ein7 had the longest hairs of the three genotypes. The two mutants had similar responses to phosphorus for density but not for length.
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Low phosphorus and ethylene have different effects on radial anatomy
When Columbia plants were grown with high phosphorus, the typical eight files of cortical cells were always observed, and root hairs formed only on cells in the H position (Table 2; Fig. 4A). Low phosphorus stress resulted in the production of more and smaller cortical cells (Table 2; Fig. 4C). As a result, more epidermal cells were located in the H position, as previously found by Ma et al. (2001a). Sometimes this resulted in no atrichoblast cell files between trichoblast files, so that two adjacent epidermal cells were hair-bearing cells (Fig. 4C). Root diameters were slightly (5%) larger in low phosphorus plants (Table 3), as previously reported (Ma et al., 2001a).
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ACC added to high phosphorus media failed to induce a change in cortical cell number like that found with the low phosphorus treatment (Table 2), but promoted hair formation on both H and N cells (Fig. 4B). AVG added to low phosphorus media did not significantly affect cortical cell number (Table 2; Fig. 4D).
The ethylene-insensitive mutants ein2-1 and ein4 grown in low phosphorus had an anatomy similar to Columbia, since they produced more cortical cell files, and therefore more trichoblast cell files, than plants grown in high phosphorus (Table 2; Fig. 5A–D). Ectopic hairs were occasionally observed on the roots of ein4 grown in low phosphorus medium (Fig. 5D). The eir1-1 mutant showed very little increase in cortical cell number under phosphorus deficiency (Table 2; Fig. 5E, F).
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To exclude the possibility that the difference in cortical cell size and number was caused by differences in root development associated with low phosphorus stress, roots of both phosphorus treatments were sectioned from the middle of the hair-free zone near the root tip to the first lateral root, and found that the cortical cell number was constant throughout the root hair differentiation zone (data not shown).
Phosphorus and ethylene affect trichoblast cell length
Arabidopsis genotypes Columbia, ein2, and ein4 transformed with the GUS gene attached to an expansin gene (AtEXP7) promoter, were used for trichoblast identification and cell length measurements. This promoter was previously demonstrated to be specific for cells forming root hairs (Cho and Cosgrove, 2002) and allows easy visualization of the cross walls. Trichoblast cells were much shorter under low phosphorus than under high phosphorus for all genotypes (Table 3). ACC added to high phosphorus medium reduced trichoblast length of Columbia roots by 65%, while AVG added to low phosphorus medium increased trichoblast length by 45% (Table 3). ACC also significantly increased root diameter (Table 3), consistent with the known effects of ethylene. Columbia and ein4 had similar trichoblast lengths and root diameters at both phosphorus levels (Table 3). However, ein2 seemed to be more responsive than Columbia to phosphorus availability, since trichoblast length increased with high phosphorus and decreased with low phosphorus (Table 3). Root diameter was reduced by ein2 at both phosphorus levels, but more so at low phosphorus (Table 3).
To examine whether the AtEXP7::GUS insert affected trichoblast length, the same genotypes were examined without transformation (data not shown). Although the absolute values varied slightly from the previous experiment, the patterns of response were identical, i.e. low phosphorus reduced trichoblast length to a similar extent in Columbia and ein4, but ein2 had a more exaggerated response to phosphorus.
Percentage of H cells forming hairs
The percentage of H cells forming hairs was calculated from the other variables, trichoblast length, number of trichoblast cell files, and root hair density, and also measured directly. Both methods showed that most H cells formed hairs in Columbia, regardless of phosphorus availability (Table 4). ACC increased the percentage of H cells forming hairs to almost 100%, while AVG reduced it by half. The percentage of H cells forming hairs was reduced in ein2-1 and ein4, especially with the low phosphorus treatment (Table 4).
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Discussion |
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Gross effects of low phosphorus and ethylene are similar
Ethylene and low phosphorus have similar effects on root hair development in Arabidopsis. Ethylene (ACC) and low phosphorus significantly increase hair density and length (Fig. 1), and significantly reduce trichoblast cell length (Table 3). Inhibiting ethylene synthesis and action prevents the low phosphorus response (Table 1; Fig. 1), and inhibiting ethylene action via ethylene-insensitive mutants reduces the low phosphorus response (Figs 2, 3). These results are consistent with the hypothesis that ethylene mediates the responses of root hair growth to low phosphorus.
Ethylene-insensitive mutants reveal complex ethylene/phosphorus interaction
When the effects of low phosphorus on ethylene-insensitive mutants were investigated, it was found that all mutants responded to low phosphorus with increased root hair length and density, but that the ratio of values for low phosphorus/high phosphorus was reduced to an extent that varied among genotypes (Fig. 2). This is consistent with a partial involvement of ethylene in low phosphorus responses. There was no relationship between the root hair development of the mutants in response to low phosphorus and their responsiveness to ethylene as determined by the extent of inhibition of the triple response (Roman et al., 1995). For example, ein7 was the mutant most responsive to reduced phosphorus levels (Fig. 2), but was one of the weaker mutants in terms of the lesion in the ability of ethylene to initiate the triple response, while ein2-1 and ein4 were strongly inhibited in the triple responses (Roman et al., 1995), but not in phosphorus responsiveness (Fig. 2). Not only were differences in phosphorus responsiveness among mutants not correlated with lesions in the triple response, but the extent of reduction in phosphorus responses for the two root hair traits, root hair density and root hair length, were not consistent among mutants (Fig. 2). The ein2-1 mutant had the weakest inhibition of low phosphorus response for root hair density, but the second largest inhibition of low phosphorus response for root hair length. This lack of correlation among responses may indicate the extent to which ethylene is important for each response as well as the specific action of each mutant gene in signal transduction for ethylene responses related to seedling growth versus root hair development.
Some of the strongest inhibitions of the low phosphorus response were observed in mutants that have yet to be cloned and identified. The ein6 and ein7 mutants strongly reduced the low phosphorus/high phosphorus ratio for both root hair density and root hair length (Fig. 2). Compared with Columbia, the ein6 mutant increased root hair density under high phosphorus and reduced it under low phosphorus (Fig. 2). EIN6 and EIN7 have been placed after CTR1 in ethylene signal transduction based on genetic analysis (Roman et al., 1995). The ein6 mutant is the only ethylene-response mutant affected by the microtubule-destabilizing drug taxol (Johnson and Ecker, 1998) and the only ethylene-response mutant in which mechanical stimulation of TCH3 gene expression is inhibited (Wright et al., 2002). EIN6 may, therefore, be involved in responses to ethylene and other factors that affect the direction of growth, which could include phosphorus effects on trichoblast elongation.
There was also a strong reduction in the low phosphorus/high phosphorus ratio for root hair traits in eir1-1 (Fig. 2), which was originally isolated as an ethylene-response mutant displaying interactions with the auxin-response pathway (Roman et al., 1995), and is now identified as carrying a mutated auxin-efflux carrier (Chen et al., 1998; Luschnig et al., 1998). This mutation reduces root hair density and length at both phosphorus concentrations, but more so at low phosphorus (Fig. 2). In high phosphorus, eir1-1 seedlings had longer roots than Columbia, indicating that increased root hair density could not have resulted from an inhibition of root elongation (Roman et al., 1995). Interestingly, eir1-1 showed about 33% inhibition of response to 2 µM ACC for increased root hair density, but no inhibition of ACC enhancement of root hair length (data not shown), i.e. it retains ethylene sensitivity for that particular response. The fact that eir1 had such a strong effect suggests that its effect on auxin transport is probably important for low phosphorus responses.
Schmidt and Schikora (2001) reported a lack of interaction between ethylene and phosphorus in controlling root hair density. They used two Arabidopsis ethylene-insensitive mutants, etr1-3, which has a mutated receptor protein (Schaller and Bleecker, 1995), and ein2-1, which has a mutation in the gene believed to transduce the signal across the nuclear membrane (Alonso et al., 1999). They found that these two mutants exhibited a slightly reduced number of hairs when transferred to phosphorus-free medium, compared with Columbia. Based on the fact that these ethylene-response mutations did not depress the enhancement of root hair density by phosphorus deficiency, and the lack of effect of the ethylene synthesis inhibitors AVG and AOA and the ethylene action inhibitor STS, they proposed that a phosphorus-deficiency stress signal was not dependent on genes involved in ethylene signalling (Schmidt and Schikora, 2001). In these experiments, ein2-1 also had only a weak effect on low phosphorus enhancement of root hair density, and etr1 was not used. However, the other mutants had a more pronounced effect (Fig. 2), and in this study, ethylene synthesis and action inhibitors also had a strong effect (Table 1; Fig. 1). To explain these contradictory observations, it is necessary to dissect the root hair density phenotype into its components.
Phenotypic components of root hair density
Three components contribute to root hair density (if ectopic hairs are not considered): trichoblast length, number of trichoblasts per cross-section (trichoblast file number), and percentage of H cells forming hairs. Ectopic hairs in Columbia plants grown with either phosphorus treatment were sought, but were not observed. Schmidt and Shikora (2001) found only a small number of ectopic hairs in Arabidopsis plants transferred from a very high phosphorus (2.5 mM) to a no-phosphorus medium; in that case the ectopic hairs could have resulted from the stress of transfer. Therefore, the increase in root hair density in plants grown with low phosphorus should be explained by alterations in trichoblast length, trichoblast file number, and/or percentage of H cells forming hairs.
Trichoblast cell length and root elongation reduced by low phosphorus and ethylene
Trichoblast cell length is an important factor contributing to root hair density, since shorter trichoblast cells would increase the number of H cells per unit length of root, and therefore the number of potential trichoblasts. Trichoblast cell length was reduced with low P, so that the number of trichoblasts per unit length of root is increased 1.6-fold (Table 3), accounting for about half the observed increase in root hair density. ACC reduced trichoblast cell length of low phosphorus plants, while AVG increased it in high phosphorus plants (Table 3). These results show that both phosphorus and ethylene influence trichoblast cell length, which would be expected to correlate with overall root elongation. Indeed, low phosphorus reduced root growth rate to 40–45% that of high phosphorus controls (Table 3). The ein4 mutant had very little effect on trichoblast cell length or root growth rate (Table 3). The ein2 mutant, on the other hand, had longer trichoblasts and a higher root growth rate when grown in high phosphorus, but shorter trichoblasts and slower growth under low phosphorus compared with Columbia (Table 3). This indicates that endogenous ethylene limits extension growth of roots under high phosphorus, but is below optimal for root extension under low phosphorus. Similar results were found for the main root growth of common bean roots treated with AVG (Borch et al., 1999) and for tap root growth of 1-MCP-treated Arabidopsis roots (Ma et al., 2003). In both mutants grown with low phosphorus, the root hair density was reduced, despite the increase in the number of trichoblasts per unit length, compared with Columbia (Fig. 2; Table 3). The effects on trichoblast cell length and root elongation must be counteracted by another variable to produce lower root hair densities. That variable is percent of H cells forming hairs (see below).
Auxin, but not ethylene, required for increased trichoblast cell file number
Low phosphorus increases trichoblast cell file number partially by increasing the number of cortical cells (Ma et al., 2001a). These results show that low phosphorus and ethylene have distinct effects on cortical cell organization (Fig. 4; Table 2). Low phosphorus has impacts on tissue organization that ethylene cannot mimic, i.e. an increased number of files of smaller cortical cells. Ethylene (from ACC) induces swelling of cortical cells, but has no effect on cortical cell number, and likewise, AVG and ethylene-insensitive mutants have no effect on cortical cell number (Fig. 4; Table 2). The only exception was the eir1-1 mutant, which showed only a slight increase in cortical cell number with low phosphorus (Table 2). Since the behaviour of eir1-1 was inconsistent with AVG-treated Columbia or the other ethylene-insensitive mutants, its failure to alter cortical cell organization in response to low phosphorus must result from the primary lesion in eir1-1, the impairment of auxin efflux (Luschnig et al., 1998), implicating auxin in the alterations of root anatomy resulting from phosphorus stress. Since low phosphorus increases cortical cell numbers but ethylene does not, low phosphorus increases root hair density through at least one mechanism that is completely independent of ethylene.
Ethylene is important for root hair formation on H cells
The final factor that could be responsible for increased root hair density is the percentage of H cells forming hairs. No significant effect of low phosphorus on this variable were observed, either when it was calculated from the other two variables or when it was observed directly (Table 4). However, ACC increased the percentage of H cells forming hairs to nearly 100%, while AVG and ethylene-response mutations reduced it. Although percentage of H cells forming hairs was not affected by phosphorus in Columbia, the ein2 and ein4 mutants had even greater reductions in the percentage of H cells forming hairs under low phosphorus than they did under high phosphorus. This confirms the importance of ethylene for root hair development (Masucci and Schiefelbein, 1994, 1996; Tanimoto et al., 1995) and also indicates that ethylene is more important for root hair development in plants grown with low phosphorus stress than in unstressed plants.
In another study, phosphorus had a significant effect on the percentage of H cells forming hairs (Ma et al., 2001a). High phosphorus treatment reduced the percentage of H cells forming hairs to only 24%, while in low phosphorus plants, 90% of H cells formed hairs, as in this paper. In that study, the percentage of H cells forming hairs was not measured directly, but calculated from the other components of root hair density (Ma et al., 2001a). In addition, the length of trichoblasts with low phosphorus was only slightly less than that from high phosphorus, so the increase in root hair density with low phosphorus was due to the increased number of trichoblast cell files and the increased percentage of H cells forming hairs (Ma et al., 2001a). In this study, reduced trichoblast length and the increased number of trichoblast cell files account for the root hair density increase. The reduction in trichoblast cell length was about the same as the reduction in overall root growth (Table 3). Each of these three components may contribute to changes in root hair density, depending on the experimental conditions and the extent of phosphorus deficiency. Only two of them, trichoblast cell length and percentage of H cells forming hairs, are influenced by ethylene.
Ethylene and phosphorus control root hair length
Root hair elongation (length) is promoted by low phosphorus or ACC and reduced by ethylene synthesis and action inhibitors (Fig. 1; Table 1). This is consistent with previous reports showing that low phosphorus increases both growth rate and duration of growth to produce longer root hairs (Bates and Lynch, 1996) and with the well-known effects of ethylene on root hair elongation (Abeles et al., 1992; Pitts et al., 1998; Schmidt and Schikora, 2001). The effects of ethylene response mutations on root hair length depend on phosphorus availability, indicating an interaction between ethylene and phosphorus (Figs 2, 3). All mutants except ein6 had shorter root hairs than Columbia under low phosphorus, but the mutations had variable effects with high phosphorus. Likewise, Cho and Cosgrove (2002) found that root hair length was reduced in some, but not all, ethylene-insensitive genotypes at high phosphorus, while 1-MCP usually reduced root hair length. These results indicate that the role of ethylene in root hair elongation is not as simple as it may appear from work with ACC, AVG, and the first few mutants examined (Pitts et al., 1998). The fact that ethylene response mutations are more inhibitory at low phosphorus suggests that, as with root hair density, the action of ethylene appears to be more crucial when phosphorus is limiting.
Cross-talk between ethylene and phosphorus in root hair development
Phosphorus regulates root hair length and density by modifying some subset of the enormously complex set of processes regulating these traits, including root growth rate, trichoblast cell specification and growth, and all of the many events governing the development and elongation of the root hairs themselves (Grierson et al., 2001). Some of these processes may be responsive to environmental factors regulating root hair development, among which phosphorus is one of the most important (Bates and Lynch, 1996; Foehse and Jungk, 1983; Ma et al., 2001a). In its effect on cortical cell number, phosphorus affects the very earliest stages of root hair development by changing the patterns of meristematic cell division. Sections very close to the meristem show similar cortical cell numbers to those in the root hair zone shown in Fig. 4, indicating that these changes do, in fact, occur in or near the meristem and not later in root development (data not shown). This profound effect on radial organization does not involve ethylene action, but does require polar auxin transport, since this response was strongly suppressed in the eir1-1 mutant (Table 2). The effect of phosphorus on radial organization affects cell specification (trichoblast versus atrichoblast) by influencing the number of epidermal cells in the H position.
Ethylene has effects on root hair development that are distinct from phosphorus effects. Ethylene, but not phosphorus, is important for the initiation of root hairs at the proper site on the trichoblast (Masucci and Schiefelbein, 1994). Abnormalities were not observed in root hair position in plants grown with a wide range of phosphorus concentrations, though alterations were clearly visible with AVG treatment. Excess ethylene also causes root hair development from N cells, i.e. ectopic hairs (Dolan et al., 1994; Tanimoto et al., 1995), while phosphorus (at high or low availability) does not.
Low phosphorus has additional effects which result in an even greater increase in root hair density than would be achieved with the increase in trichoblast file number alone, including reduced root growth and trichoblast elongation and, under some conditions, an increase in the proportion of H cells forming hairs. Each of these responses appears to involve ethylene action. Once hairs form, low phosphorus increases their length by increasing both the growth rate and the duration of elongation (Bates and Lynch, 1996). Inhibition of ethylene under phosphorus deficiency results in reduced root growth, fewer H cells forming hairs, and reduced root hair length, all part of the programme to increase root hair density and length. These morphological changes are synergistic in their effects on phosphorus acquisition and increase performance and competitiveness when phosphorus is limiting (Bates and Lynch, 2000a, 2000b, 2001; Ma et al., 2001b). Ethylene is required for the plant to fully engage these adaptive responses to phosphorus deficiency.
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Acknowledgements |
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The authors gratefully acknowledge the pre-publication gift of the AtEXP7 promoter lines used for trichoblast cell length measurements from DC Cosgrove and H-T Cho, Penn State University. This work was supported by BARD grant IS-2949-97R and USDA-NRI grant 99-35100-7596.
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