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An Arabidopsis MADS Box Gene That Controls Nutrient-Induced Changes in Root Architecture

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Science  16 Jan 1998:
Vol. 279, Issue 5349, pp. 407-409
DOI: 10.1126/science.279.5349.407

Abstract

The development of plant root systems is sensitive to the availability and distribution of nutrients within the soil. For example, lateral roots proliferate preferentially within nitrate (NO3 )-rich soil patches. A NO3 -inducible Arabidopsis gene (ANR1), was identified that encodes a member of the MADS box family of transcription factors. Transgenic plants in whichANR1 was repressed had an altered sensitivity to NO3 and no longer responded to NO3 -rich zones by lateral root proliferation, indicating that ANR1 is a key determinant of developmental plasticity in Arabidopsis roots.

The structure of a root system is determined by an interplay between the intrinsic developmental program and external biotic and abiotic stimuli (1). One important role of such plasticity is to enable the plant to forage for mineral nutrients (2), which are usually distributed unevenly within the soil. Nitrate is the major source of the mineral N for many higher plant species, and its supply often limits plant growth and crop yields. Plants respond to its presence in a variety of ways that enhance their ability to absorb and metabolize it (3). Most dramatic of these is the stimulation of lateral root development that occurs specifically within NO3 -enriched patches of soil (4).

In the course of screening Arabidopsis roots for NO3 -inducible genes we identified a cDNA clone, pANR1, that has homology to the MADS (MCM1, AGAMOUS, DEFICIENS, and serum response factor) box family of transcription factors (5). Products of MADS box genes, found in a variety of eukaryotes, share a conserved motif within the DNA binding domain (6). In plants, most of the MADS box genes so far identified (more than 24 in Arabidopsis alone) are expressed in flowers; many of these affect floral organ identity (7). TheANR1 gene belongs to the same subfamily (6) as two MADS box genes of unknown function: AGL17, anArabidopsis root-specific gene (55% amino acid identity) (8), and DEFH125, an Antirrhinumpollen-specific gene (59% amino acid identity) (9). Hybridization of pANR1 to Southern blots of Arabidopsisgenomic DNA showed that no other members of the MADS box family are sufficiently similar to cross-hybridize with it at high stringency (10).

Northern (RNA) blots showed that ANR1 expression, undetectable in N-starved roots, is rapidly induced when NO3 is supplied (Fig.1A) (11). We were unable to detect ANR1 mRNA in stems or leaves of mature plants (Fig.1B), suggesting that ANR1, like AGL17(8), is expressed preferentially or specifically in roots. We investigated whether ANR1 is induced as part of a general response to nutrient starvation and resupply (Fig. 1C) and found that expression of the gene was maintained in NO3 -grown roots regardless of changes in the supply of either K+ or inorganic PO4 3–.

Figure 1

Developmental and nutritional regulation ofANR1 expression. (A) ANR1 is rapidly induced by NO3 . Nitrogen-starved seedlings were treated with KNO3 at time 0. Lanes contain total RNA from roots harvested at intervals after NO3 treatment, and the blot was hybridized with an ANR1 cDNA probe (11). A β-tubulin cDNA was hybridized to the same filter as a loading control. (B) ANR1 is expressed mainly or exclusively in roots. Lanes contain total RNA extracted from the aerial parts of greenhouse-grown plants and from roots of seedlings grown in liquid culture (11). (C) ANR1 is not under general nutritional control. Seedlings were starved of K+or inorganic PO4 3– (Pi) and at time 0 were resupplied with the appropriate nutrient (11). Lanes contain total RNA from roots harvested at intervals after nutrient resupply.

To investigate the function of ANR1, we generated transgenicArabidopsis lines in which expression of ANR1 was down-regulated to various degrees by antisense or cosuppression effects (12) (Fig. 2A). The response of the transgenic root systems to either ubiquitous or locally concentrated supplies of NO3 was then analyzed.

Figure 2

Sensitivity of lateral root development in ANR1-repressed lines to inhibition by NO3 . (A) TransgenicArabidopsis lines with various degrees of ANR1repression. Lanes contain total RNA from NO3 -induced roots of the parental line (C24), four ANR1 antisense lines (A1, A11, A13, and A15), and one sense line (S10). Roots were induced for 3 hours with 2 mM KNO3 (11). The Northern blot was hybridized with the ANR1 and β-tubulin probes as in Fig. 1. (B) NO3 response curves for roots of C24 (O), a transgenic control line 43-3(Δ), A1 (•), A11 (▪), A13 (▴), and A15 (▾). Seedlings were grown on agar plates containing a range of [NO3 ] and the lengths of the primary (upper panel) and lateral (lower panel) roots were measured after 14 days (13). Each point represents the mean of data from four to eight seedlings; those marked with an asterisk differed significantly from the 43-3 and C24 controls (P < 0.05 in a t test).

First we examined the effect on root growth of a range of external NO3 concentrations (Fig. 2B) (13). Previous studies have demonstrated that lateral root development is sensitive to changes in the NO3 supply (4, 14). In the parental line (C24) and a control transgenic line (43-3), increasing the concentration of NO3 ([NO3 ]) from 10 μM to 1 mM had no significant effect on the growth of lateral roots, whereas at 10 mM and particularly at 50 mM their development was inhibited (Fig. 2B). When we compared the numbers of emerged and unemerged laterals in roots grown on 1 mM and 50 mM KNO3 we found little difference (15), showing that the suppression of lateral root development that occurs at concentrations ≥10 mM is due to an effect on lateral root elongation rather than on initiation. As has been found for other species (4), primary root elongation was insensitive to [NO3 ] (Fig.2B), demonstrating that the inhibition of lateral root growth by [NO3 ] ≥10 mM is not part of a general inhibitory effect on plant growth.

The sensitivity of lateral root elongation to [NO3 ] was altered in theANR1-repressed lines (Fig. 2B). At 10 μM KNO3, lateral root growth in the antisense lines did not differ from that in the control lines. However, concentrations of 100 μM or 1 mM, which had no effect on the control lines, significantly inhibited lateral root growth in A1 and A13, the most strongly down-regulated lines (Fig.2B). S10, a line carrying the ANR1 sense construct (12), had the lowest amounts of ANR1 mRNA (Fig.2A), probably because of cosuppression (16), and this line had a phenotype similar to A1 and A13 (10). Two lines with intermediate amounts of ANR1 mRNA (A11 and A15) showed an intermediate sensitivity to NO3 inhibition (Fig. 2B). As in the control lines, primary root growth in theANR1-repressed lines was unaffected by [NO3 ] (Fig. 2B), showing that the consequences of down-regulating ANR1 are specific to lateral roots.

To investigate further the phenotype of theANR1-repressed lines, we tested the effect of locally concentrated supplies of NO3 on root development (Fig. 3) (17). In the control lines (C24 and 43-3), lateral root growth in the NO3 -enriched segment was stimulated two- to threefold compared with KCl-treated controls, whereas growth of the lateral roots in the top (low [NO3 ]) segment was slightly (30%) inhibited (Fig. 3B). The localized NO3 treatment had no significant effect on the number of lateral roots in C24 (18), and more detailed analysis has shown that the increased proliferation of lateral roots is due to a twofold increase in the average rate of lateral root elongation in the treated zone (10).

Figure 3

Insensitivity of lateral root development in ANR1-repressed lines to a localized supply of NO3 . (A) Experimental set-up for applying localized NO3 treatments to Arabidopsisroots (17). The agar plates were divided into three segments so that different concentrations of NO3 could be maintained in different parts of the plate. At the start of the experiment, seedlings were placed on the plate as shown on the left; lateral root lengths were measured when the seedlings reached the stage shown on the right. (B) Effect of a localized NO3 treatment on lateral root development in the control lines (C24 and 43-3), twoANR1-repressed lines (A13 and S10), and a NR-deficient mutant (nia1 nia2). All three agar segments contained 10 μM NH4NO3, and the middle segment also contained 1 mM KCl (designated C) or 1 mM KNO3 (+N). Twelve days after transfer of the seedlings to the segmented plates (or 13 days for nia1 nia2 because of its slower growth rate) lateral root lengths were measured in the top (open bars) and middle segments (closed bars) (17). (The bottom segment was not included in the analysis because it did not yet contain many laterals). Each bar represents the mean of data from 8 to 12 seedlings; those marked with an asterisk differ significantly (P < 0.05 in a t test) from the control treatment to the same line. (C) Root morphology of seedlings of C24 and S10 that received (+NO3 ) or did not receive (C) a localized NO3 treatment. Representative seedlings from the experiment in (B) were stained with toluidine blue and photographed.

The localized NO3 treatment was also applied to an Arabidopsis mutant (nia1 nia2) that has just 0.5% of wild-type nitrate reductase (NR) activity and is therefore ineffective at using NO3 as a source of N (19). This mutant showed a similar response to the localized NO3 treatment as the control lines (Fig. 3B). This result argues against previous hypotheses that it is the assimilation of NO3 locally at its site of uptake, and the consequent increased flux of photosynthate to that region of the root, that leads to localized lateral root proliferation (20) and supports an earlier suggestion (21) that the effect is due to the signaling properties of the NO3 ion itself.

When the localized NO3 treatment was applied to the three strongly ANR1-repressed lines, there was no significant effect on lateral root growth in the treated zone (shown for A13 and S10 in Fig. 3); the two intermediately repressed lines (A11 and A15) did show some response, but one which was diminished by about 70% compared with the response by C24 (10). Thus, the stimulation of lateral root elongation by localized applications of NO3 is dependent on expression of theANR1 gene (22).

To explain the manner in which down-regulation of ANR1alters the root's responses to localized and ubiquitous supplies of NO3 , we suggest a model in which the NO3 supply has two opposing effects on lateral root elongation: a localized stimulatory effect that requiresANR1 expression and depends on the external [NO3 ] at the lateral root tip, and a systemic inhibitory effect that results from its influence on the N status of the shoot, which in turn depends on how much NO3 is absorbed by the whole root system. The latter effect would not involve ANR1 and would require some shoot-derived signal that suppresses lateral root elongation (23). This model can explain how a localized NO3 treatment stimulates lateral root elongation specifically within the treated segment. In this situation, the inhibitory effect should be felt by all the lateral roots, both outside and inside the NO3 -rich zone, whereas the stimulatory effect should be sensed only by those lateral roots present within the NO3 -rich zone.

The model is also consistent with both aspects of the phenotype of theANR1-repressed lines. The absence of a positive response to the localized NO3 treatment (Fig. 3) would be due to the impairment of the signal transduction pathway that normally mediates the stimulatory effect of external NO3 . The increased sensitivity of the lateral roots to NO3 inhibition when NO3 is ubiquitously supplied (Fig. 2) would be the phenotype expected if the stimulatory effect of NO3 had been blocked, but the inhibitory effect had not.

What role might ANR1 play in converting a NO3 stimulus at the lateral root tip into an increased rate of elongation? One possibility is suggested by analogy with another MADS box transcription factor, the human serum response factor. This protein is responsible for the rapid and coordinate activation of a set of “immediate-early” genes when quiescent human cell lines receive an extracellular mitogenic stimulus (24). In a similar way the ANR1 gene product might be the transcriptional regulator of a set of genes that modulate the rate of lateral root elongation.

  • * To whom correspondence should be addressed. E-mail: brian.forde{at}bbsrc.ac.uk

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