roots during lateral root development
Veth-Tello, L.M.
Citation
Veth-Tello, L. M. (2005, March 2). Analysis of gene expression in the outer cell layers of Arabidopsis roots during lateral root development. Retrieved from
https://hdl.handle.net/1887/2315
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Chapter 5
A role for the AIR3 subtilisin-like protease from
Arabidopsis in nitrate regulation of
root branching
Luz M. Veth-Tello, Rebecca Hewitt1, Brian G. Forde1, Paul J.J. Hooykaas and Bert J. van der Zaal*
Institute of Biology Leiden, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands; 1Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom
Abstract
AIR3 is an auxin-inducible gene from Arabidopsis thaliana that encodes a putative subtilisin-like serine protease. AIR3 has been shown to be expressed in the outer cell layers of the primary root specifically at sites of lateral root formation, but its function is unknown. We have generated Arabidopsis lines constitutively expressing AIR3 under the control of the CaMV 35S promoter. These lines have markedly altered root architecture: lateral root lengths were greatly increased, while primary root lengths and lateral root densities were unaffected. Histological analysis indicated that this phenotype was the result of enhanced meristematic activity in the lateral root tip. When the 35S::AIR3 lines were grown at a range of NO3- concentrations the lateral root
phenotype was found to be dependent on the NO3- supply, being strongest at the
highest [NO3-]. This indicates an effect of NO3- on lateral root growth. Using a
possible role of AIR3 in regulating lateral root growth and its relation with auxin and NO3- signaling is discussed.
Introduction
Lateral roots are an important means for the plant to increase its absorptive area and the volume of substrate explored. In Arabidopsis, lateral roots originate in the pericycle, the outermost layer of the vascular cylinder, from a subset of cells located adjacent to the two xylem poles named founder cells (Laskowski et al., 1995). Two adjacent founder cells within the same cell file undergo polarized asymmetric transverse divisions. Further radial expansion and subsequent periclinal divisions result in the formation of a lateral root primordium (Casimiro et al., 2001; Malamy and Benfey, 1997).
external and internal cues indicating that this family of genes plays important and varied roles in plant growth, development and defense.
Auxin plays a central role in lateral root formation and this has been extensively demonstrated by physiological and genetic studies. Besides auxin, environmental and nutritional signals have also a major influence on lateral root development. Plants have evolved a root system highly responsive to the availability of nutrients within the soil. Most plants can utilize NO3-, NH4+, urea and amino acids as nitrogen source with NO3
-being the most abundant N-containing substrate in well-aerated soils (Parson and Sunley, 2001). In non-symbiotic N metabolism the NO3- imported into the symplast is
reduced to NO2- by nitrate reductase. Because of its high toxicity, NO2- is rapidly
reduced to NH4+ by nitrite reductase and then further assimilated into organic
compounds (Parson and Sunley, 2001). In many plant species, exposure of the root to localized nitrate sources results in an increased rate of lateral root proliferation (Forde and Lorenzo, 2001). In Arabidopsis, this response consists primarily of an increase in the elongation rate, with little effect on lateral root number (Zhang and Forde, 1998; Zhang et al., 1999). However, at high concentrations, NO3- has an inhibitory effect on
lateral root elongation (Zhang and Forde, 1998; Zhang et al., 1999). Primary root growth is not affected by low or high [NO3-], indicating that the effect of NO3- is specific
for lateral roots (Zhang and Forde, 1998; Zhang et al., 1999).
Analyses of plants containing an AIR3::GUS construct have previously shown that the expression conferred by the AIR3 promoter is restricted to a distinct cluster of epidermal and cortical cells at the site of lateral root emergence (Neuteboom et al., 1999b). This expression pattern was first evident in the early stages of lateral root initiation, but the cells in which expression takes place do not directly contribute to the developing lateral root primordium. It was hypothesized that AIR3 digests structural proteins in the extracellular matrix of cells located above sites of lateral root formation, leading to weakened cell-to-cell connections and thereby facilitating lateral emergence (Neuteboom et al., 1999b).
evidence that AIR3 expression is down-regulated at high rates of NO3- supply. We
discuss the implications of these findings on the role of AIR3 in regulating lateral root growth and its relationship with the NO3- regulation of lateral root development.
Results
The phenotype of Arabidopsis lines overexpressing AIR3
To investigate the effects of constitutive overexpression of the AIR3 gene we transformed Arabidopsis with a construct in which the AIR3 coding sequence (plus the first intron) was fused downstream of the CaMV 35S promoter (see Materials and Methods). Five independent 35S::AIR3 lines were generated (S1, S8, S9, S23 and S29), each carrying a single T-DNA insertion, as well as a transgenic control (C1) carrying the empty vector.
For phenotypic analysis, seeds were germinated on vertical agar plates containing ½ Murashige and Skoog (MS) medium. Up to 9 d after germination no clear phenotypic differences were apparent between 35S::AIR3 and control plants (not shown). By 12 d, however, lateral roots from 35S::AIR3 plants were visibly longer than those from the control and by 14 d the differences in lateral root length were even more evident (Figure 1A). In lines S1, S9, S23 (Figure 1A) and S8 (not shown), any effect on primary root growth was minor compared to the effect on lateral roots. A fifth line (S29) had a relatively short primary root, but still showed the characteristic ‘long-lateral-roots’ phenotype observed for the other 35S::AIR3 lines (Figure 1A). The aerial part of 35S::AIR3 plants appeared to be normal, again with the exception of line S29, which had a short hypocotyl.
The long-lateral-roots phenotype was thus associated with five independent transgenic lines. Furthermore, the line with the weakest root phenotype (S23) was also the line in which the 35S::AIR3 transgene was least strongly expressed, as judged by northern blot (Figure 1E). Note that even after auxin treatment, the expression of the endogenous AIR3 gene is very low by comparison to that driven by the 35S promoter (Figure 1F).
the most pronounced change in root morphology was an increase in lateral root length, with almost no effect on lateral root density or primary root growth (Figure 1B-D).
We conclude that a high constitutive level of AIR3 mRNA primarily affects lateral root growth, without altering lateral root initiation or causing any gross morphological defects.
Histological analysis of 35S::AIR3 roots
An increase in root growth can occur either through an increase in cell production rate in the root tip or an increase in the length of mature root cells, or a combination of the two. To distinguish between these possibilities, we performed a histological analysis of roots from lines S1, S29 and the transgenic control (C1). This showed only minor differences in cell length that could not account for differences in growth rate. The length of lateral root epidermal cells in lines C, S1 and S29 were: 138.3 (+/- 4.8) µm, 151.7 (+/- 4.0) µm and 141.7 (+/-11.1) µm (+/- standard error; n = 6). Similarly, cross-sections of primary and lateral roots showed no differences in cell size or cellular structure between the two 35S::AIR3 lines and the control (data not shown). We conclude that the increased rate of lateral root growth in the 35S::AIR3 lines was primarily due to enhanced meristematic activity in the lateral root tips.
Effects of an AIR3 knock-out mutation
A mutant line carrying a T-DNA insertion in the fourth intron of the AIR3 gene has been isolated and shown to be lacking in AIR3 mRNA (see Materials and Methods). Root growth studies of the air3-T mutant line revealed the seedlings to have an essentially normal phenotype, apart from a slightly increased rate of lateral root growth compared to the wild-type at 10 µM NO3- (data not shown). There were no
Len gth (mm) 0 20 40 60 80 C
A
S1 S9 S23 S29B
C
S1 S29 S9 S23 S8 AIR3 rRNA LR de nsity (mm -1 ) 0 1 2 3 4C
Le ng th ( m m ) 0 2 4 6 8 10 12D
E
1NAA H2O1NAA H20 WT CF
C S1 S9 C S1 S9 C S1 S9 Len gth (mm) 0 20 40 60 80 CA
S1 S9 S23 S29B
C
S1 S29 S9 S23 S8 AIR3 rRNA S1 S29 S9 S23 S8 AIR3 rRNA LR de nsity (mm -1 ) 0 1 2 3 4C
Le ng th ( m m ) 0 2 4 6 8 10 12D
E
1NAA H2O1NAA H20 WT C 1NAA H20 1NAA H2O WT C 1NAA H2O WT CF
C S1 S9 C S1 S9 C S1 S9Figure 1. Phenotypic and molecular analysis of AIR3 overexpressing lines. A, Images showing the root
morphology of seedlings transformed with the CaMV 35S::AIR3 construct (S1, S9, S23 and S29) and a transgenic control line (C1), which had been grown at 21oC for 15 d on vertical agar plates containing ½ MS medium. Primary root length (B), lateral root density (C) and individual lateral root lengths (D) were measured for seedlings of lines C1, S1 and S9. The seedlings were germinated on vertical agar plates containing a dilute B5 medium (Zhang and Forde, 1998) with 10 µM NH4NO3 as N source. After 4 d at
25oC, seedlings of homogenous size were transferred to plates containing 1 mM KNO3 (3 seedlings per
plate). Root measurements were made on 15 d-old seedlings. Bars represent standard errors (n = 6-9).
E, Northern blot analysis of AIR3 mRNA abundance in the 35S::AIR3 lines. The northern blots were
hybridized with an AIR3 RNA probe (rRNA= loading control). F, northern blot analysis of AIR3 mRNA expression in C24 (WT) and the transgenic control (C1) after a 24 h inductive treatment with the synthetic auxin 1-NAA (10-6 M) or without induction (H2O).
Does the overexpression of AIR3 alter root sensitivity to nitrate?
Of all the environmental factors that influence root architecture, NO3- is
Linkohr et al., 2002) Low external NO3- concentrations (<1 mM) stimulate lateral root
growth, while higher concentrations lead to a systemic inhibition of lateral root development, a phenomenon that has been explained by the ‘dual pathways’ model for NO3- regulation of root branching (Zhang et al., 1999). In view of the very specific way
in which AIR3 overexpression affects lateral root growth, we therefore decided to investigate whether lateral root development in the 35S::AIR3 lines displayed an altered sensitivity to NO3-. Three 35S::AIR3 lines (S1, S9 and S29), and the control
lines (C1 and wild-type), were grown on vertical agar plates containing a range of NO3-
concentrations: low (10 µM), medium (1mM) and high (25 mM). To simultaneously examine the effect of varying the C/N ratio, we used two different sucrose concentrations: 0.5% and 2% (w/v). The lengths of the main root and the two oldest lateral roots were measured in 9 d-old seedlings (Figure 2).
In agreement with Zhang et al., (1999), we observed in the control lines that there was no significant effect on primary root elongation of varying the [NO3-] at either
sucrose concentration (Figure 2A). On the other hand, lateral root growth on 0.5% sucrose was significantly inhibited by growth on 25 mM NO3- compared to 10 µM NO3
-(Figure 2B and C), an inhibitory effect that was alleviated by increasing the sucrose concentration to 2% (Figure 2B and C; Zhang et al., 1999). The response of the two 35S::AIR3 lines differed most noticeably from the controls at 25 mM NO3- and 0.5%
sucrose, the conditions under which the inhibitory effect ofNO3- is most pronounced in
the controls. Under these conditions, lateral root growth (but not primary root growth) in S1, S9 and S29 was stimulated compared to the 10 µM and 1 mM treatments (Figure 2A-C). At 2% sucrose, this stimulation of lateral root growth in the 35S::AIR3 lines at the highest NO3- concentration was not observed (Figure 2B and C).
The aerial part of the 35S::AIR3 plants showed wild-type responses to different NO3- concentrations. At low NO3- (10 µM KNO3), cotyledons and leaves were small
and bleached and at higher NO3- (1 or 25 mM KNO3) they were large and green (not
shown). These results demonstrate that despite ectopic overexpression of AIR3 mRNA in most cell types, as can be expected from the 35S promoter, its effect was only discernible during lateral root development.
To obtain a more detailed picture of the effect of AIR3 overexpression on root architecture, we repeated the NO3- experiment with two of the 35S::AIR3 lines (S1 and
lengths of all the lateral roots. The data in Figure 3A confirm for lines S1 and S9 that the effects seen in Figure 2 extend to the entire population of lateral roots.
lengths (mm) 20 15 10 5 0 lengths (mm) lengths (mm) 4 12 8 0 0.01 1 25 0.01 1 25 w.t C S1 S9 S29
mM NO
-30.5% sucrose
2% sucrose
50 40 30 20 10 0A
B
C
mM NO
-3 lengths (mm) 20 15 10 5 0 20 15 10 5 0 lengths (mm) lengths (mm) 4 12 8 0 4 12 8 0 0.01 1 25 0.01 1 25 w.t C S1 S9 S29 w.t C S1 S9 S29mM NO
-30.5% sucrose
2% sucrose
50 40 30 20 10 00.5% sucrose
2% sucrose
50 40 30 20 10 0 50 40 30 20 10 0A
B
C
mM NO
-3 Figure 2. The NO3responsiveness of root growth of the 35S::AIR3 lines at two different sucrose concentrations. Seedlings from lines S1, S9 and S29, the transgenic control (C1) and the wild-type were germinated on vertical agar plates containing dilute B5 medium (0.5% sucrose and 10 µM NH4NO3).
After 3 d seedlings were transferred to agar plates containing 0.01 mM, 1 mM or 25 mM KNO3 and
either 0.5% or 2% (w/v) sucrose. Measurements were done on 9 d-old seedlings. A, primary root lengths; B, length of the first lateral root; and C, length of the second lateral root. The differences in lateral root length between the control (C) and the sense lines were statistically significant in all treatments (t-test; p=0.05). Bars represent standard errors (n=12).
At 0.1 mM NO3-, the majority of lateral roots in C1 were <5 mm in length and
proportion of roots in the largest size class (>20 mm). At 25 mM NO3-, this size class
represented 27% and 17% of the population in S1 and S9 respectively, with a few roots extending to over 40 mm. As before, primary root lengths were unaffected by the overexpression of the AIR3 gene at all NO3- concentrations (Figure 3B).
Since one of the major effects of a high [NO3-] treatment is an inhibition of the
early stages of lateral root development, at around the time of emergence (Zhang et al., 1999; Signora et al., 2001), it was possible that a reduction in the NO3- sensitivity of
this phase of root branching could at least partly account for the altered NO3
-responsiveness of the 35S::AIR3 lines. However, measurements of the lateral root density (visible lateral roots per cm primary root) showed that overexpression of AIR3 had no effect on the number of emerged laterals: the 25 mM NO3- treatment had
similar inhibitory effect on the number of emerged laterals in lines S1 and S9 as in C1 (Figure 3C). Thus, to account for the difference between the NO3- responsiveness of
the C1 and the 35S::AIR3 lines, it appears that AIR3 overexpression has produced a phenotype in which growth of mature lateral roots is strongly stimulated by high [NO3-],
rather than inhibited as it is in the control line.
Is AIR3 expression subject to regulation by NO3-?
The finding that constitutive overexpression of AIR3 appears to overcome an inhibitory effect of high [NO3-] on lateral root growth suggests that NO3- could exert its
inhibitory effect by down-regulating AIR3 expression. To test this hypothesis, we investigated the effect of high rates of NO3- supply on AIR3 expression. The availability
of an AIR3::GUS reporter line (Neuteboom et al., 1999b) made it possible to study the effect of NO3- on AIR3 expression in its likely site of action, adjacent to the developing
lateral root primordium. The localization of AIR3::GUS expression in the epidermal and cortical cells overlying a lateral root primordium can be seen in Figure 4A. Figure 4B shows how the emerging lateral root penetrates the cluster of cells expressing AIR3::GUS.
The AIR3::GUS seedlings were grown on medium containing either 1 mM or 50 mM NO3- and 7 d after germination they were histochemically stained for GUS activity.
A
Length (mm) NO3-concentration NO3-concentration La teral roo t de nsi ty (cm -1)B
C
Fr eq ue ncy ( % ) 0 20 40 60 80 Freq ue n c y (%) 0 20 40 60 80 Fr eq ue ncy ( % ) 0 20 40 60 80 100 C1 S1 S9 0-5 5-10 10-15 15-20 >20Lateral root lengths (mm)
0.1 mM NO3 -1 mM NO3 -25 mM NO3 -Fr eq ue ncy ( % ) 0 20 40 60 80 Freq ue n c y (%) 0 20 40 60 80 Fr eq ue ncy ( % ) 0 20 40 60 80 100 C1 S1 S9 0-5 5-10 10-15 15-20 >20
Lateral root lengths (mm)
0.1 mM NO3 -1 mM NO3 -25 mM NO3
-A
Length (mm) NO3-concentration NO3-concentration La teral roo t de nsi ty (cm -1)B
C
Length (mm) NO3-concentration NO3-concentration La teral roo t de nsi ty (cm -1)B
C
Fr eq ue ncy ( % ) 0 20 40 60 80 Freq ue n c y (%) 0 20 40 60 80 Fr eq ue ncy ( % ) 0 20 40 60 80 100 C1 S1 S9 0-5 5-10 10-15 15-20 >20Lateral root lengths (mm)
0.1 mM NO3 -1 mM NO3 -25 mM NO3 -Fr eq ue ncy ( % ) 0 20 40 60 80 Freq ue n c y (%) 0 20 40 60 80 Fr eq ue ncy ( % ) 0 20 40 60 80 100 C1 S1 S9 0-5 5-10 10-15 15-20 >20
Lateral root lengths (mm)
0.1 mM NO3
-1 mM NO3
-25 mM NO3
-Figure 3. Effect of different NO3- concentrations on numbers of emerged lateral roots and the
distribution of lateral root lengths in 35S::AIR3 lines. A, Effect of the NO3
supply on the distribution of lateral root lengths in seedlings of the control (C1) and the 35S::AIR3 lines (S1 and S9). The seedlings were germinated on vertical agar plates containing dilute B5 medium (Zhang and Forde, 1998) with 10 µM NH4NO3 and 0.5% (w/v) sucrose. After 4 d, the seedlings were transferred to plates containing
different KNO3 concentrations (3 seedlings per plate) and lateral root measurements were made 11 d
later. B, Effect of the NO3- supply on primary root growth in the same experiment. Bars represent
standard errors (n = 6-9). C, Effect of the NO3
supply on lateral root densities in the same experiment. Lateral roots were scored by their visibility in 2 x enlarged images of the root systems, so only fully emerged lateral roots are included. Bars represent standard errors (n = 6-9).
lateral roots initiated was not affected (Figure 4D). Thus the effect of 50 mM NO3- on
the number of staining events in the AIR3::GUS line cannot be attributed to a reduction in the number of lateral roots. Since there were fewer staining events than there were lateral roots, we conclude that this was due to the low but variable level of expression conferred by the AIR3 promoter, such that in many instances expression of the reporter gene was at levels insufficient for histochemical detection. The high [NO3-]
treatment appears to have reduced the level of AIR3 expression still further, so that detectable GUS activity is associated with only a very small proportion of laterals.
Is a second auxin-regulated gene (AIR1), which is expressed in the same spatial pattern as AIR3, similarly affected by high [NO3-]?
Shoot-derived auxin is involved in the stimulation of lateral root emergence (Bhalerao et al., 2002), and it has been hypothesized that high rates of NO3- could act
on lateral root development by inhibiting auxin biosynthesis in the shoot, or its transport to the root (Forde, 2002). Since AIR3 is known to be auxin-inducible, this model could potentially account for its down-regulation under conditions of high [NO3-].
In this case we would expect other auxin-regulated genes that are associated with lateral root development to be down-regulated by high NO3-. To test this hypothesis,
we examined the effect of the high [NO3-] treatment on the expression of AIR1, another
auxin-regulated gene whose expression is associated with lateral root development (Neuteboom et al., 1999a). The AIR1 gene product is related to a family of proline-rich proteins of unknown function (Neuteboom et al., 1999a). Using an AIR1::GUS reporter gene construct it has been shown that AIR1 is expressed in roots in a very similar spatial pattern as AIR3, i.e. localized to the outer cell layers adjacent to the site of lateral root development (Neuteboom, 2000). The AIR1::GUS line was grown for 7 d on 1 mM or 50 mM NO3- and histochemically stained for GUS. As seen in Figure 4E,
based on the number of observed staining events, the expression of the AIR1::GUS construct was unaffected by the increase in external NO3- concentration. We also
observed no differences in the general intensity of staining at the two NO3
-concentrations (not shown). We confirmed that lateral root development in the AIR1::GUS line was inhibited by 50 mM NO3- in a similar way to the AIR3::GUS line (cf.
Discussion
Constitutive overexpression of AIR3 stimulates lateral root growth
In this study we have demonstrated that constitutive overexpression of the AIR3 gene has a very specific effect on the root architecture of Arabidopsis seedlings. The 35S::AIR3 lines were characterized by their long lateral roots, while lateral root density was unaffected and in most experiments there was no significant change in primary root growth. By measuring mature cell size in the lateral roots we showed that the increased rate of lateral root growth is mainly attributable to increased meristematic activity in the lateral root tips. The 35S::AIR3 lines are fully fertile and have no apparent morphological abnormalities in the shoot. Only one 35S::AIR3 line (S29), which was one of the strongest AIR3 expressers showed pleiotropic effects (a shorter primary root and hypocotyl), but we cannot rule out the possibility that this was due to an insertional mutation caused by T-DNA.
A null mutation in AIR3 produced no major alteration of growth and development of Arabidopsis plants. Under standard culture conditions air3-T knockout plants were similar to wild type plants, including a wild-type appearance of the root system. This result would be most easily explained by the existence of one or more additional subtilisin-like genes with similar properties to AIR3 and whose expression is able to mask the effect of the air3-T mutation. Functional redundancy amongst the Arabidopsis subtilases would not be surprising given the presence of at least 54 subtilisin-like genes in the Arabidopsis genome (Beers et al., 2004).
Mode of action of AIR3 in regulating lateral root growth
Figure 4. Effect of a high [NO3
-] treatment on the expression of AIR3::GUS and AIR1::GUS constructs in
Arabidopsis roots. A, Histochemical localization of GUS activity in the primary root of an AIR3::GUS line
in the vicinity of a lateral root primordium (arrow). B, GUS staining at the site of an emerging lateral root. Roots of the AIR3::GUS line (Neuteboom et al., 1999b) were stained with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) (Jefferson et al., 1987). A color picture of A and B is shown in the cover of this thesis. C, Effect of the NO3
supply on the number of GUS staining events seen in roots of the
AIR3::GUS line. AIR3::GUS seedlings were germinated on dilute B5 medium containing 10 µM NH4NO3
and transferred after 4 d at 25oC to vertical nutrient agar plates containing either 1 mM or 50 mM KNO3
and 0.5% (w/v) sucrose. After 7 d of further growth, the roots were histochemically stained for GUS activity by incubating overnight at 37oC in X-gluc. The number of staining events (stained cell clusters) per primary root was counted under a Leica MZ FLIII binocular microscope (bars represent standard errors; n = 50). D, Effect of the NO3
supply on the numbers of emerged and unemerged lateral roots per seedling in the AIR3::GUS line. Seedlings used for GUS staining were examined at x100 magnification to determine the number of lateral root primordia and emerged lateral roots (bars represent standard errors; n = 15). E, Effect of the NO3
-supply on the numbers of emerged and unemerged lateral roots per seedling in the AIR1::GUS line (bars represent standard errors; n = 15).
expected to be strongly expressed in most tissues, does not appear consistent with the notion that AIR3 has a non-specific role in degrading cell wall proteins.
An analogous result was obtained in studies of Arabidopsis plants overexpressing the SDD1 subtilisin-like protease which is involved in the regulation of stomatal patterning (von Groll et al., 2002). Transgenic lines carrying a 35S::SDD1 construct showed a reduced number of stomata and also arrested stomata. The effect of 35S::SDD1 expression was restricted to stomatal precursor cells, and again there were no large-scale effects on plant development.
In mammals, subtilases function as pro-protein convertases (PCs), whose role is to process a variety of inactive precursor proteins and peptides (including polypeptide hormones and other components of signalling pathways) into their active forms (Seidah and Chretien, 1999). It has been suggested that plant subtilases could have a similar processing function (Janzik et al., 2000; Berger and Altmann, 2000; Tanaka et al., 2001). Serna and Fenoll (2002) have hypothesized that SDD1 could generate a diffusible extracellular peptide that serves as a positional signal during guard cell differentiation, and a candidate for the receptor for this putative signal has been identified in the form of the leucine-rich receptor-like protein encoded by the TOOMANYMOUTHS (TMM) gene (Yang and Sack, 1995), which appears to act downstream of SDD1 (von Groll et al. 2002). TMM is related to CLAVATA2 (CLV2), which acts as a receptor for a peptide ligand encoded by the CLAVATA3 (CLV3) gene and regulates the maintenance of the shoot apical meristem (Kayes and Clark, 1998). Thus other members of the CLV3-like (CLE) gene family, of which at least 10 are expressed in developing leaves (Sharma et al., 2003), are potential ligands for TMM and potential targets for SDD1 processing activity (Bergman, 2004). Most members of the CLE family are predicted to be targeted to the secretory pathway (Sharma et al., 2003), so the extracellular location of the SDD1 protein in Arabidopsis leaves (von Groll et al., 2002) is consistent with this model.
developed for SDD1, it is possible that one or more of the five CLE genes that are expressed preferentially in roots (Sharma et al., 2003) could encode targets for processing by AIR3. Since AIR3 does not appear to be expressed within the developing lateral root, but rather in a group of cortical and epidermal cells overlying the developing lateral root primordium (Figure 4A and Neuteboom et al., 1999b), a role in generating an extracellular signal would explain its ability to influence the activity of the lateral root meristem from a distance. This would be analogous to the way in which the CLV3 peptide acts as a short-range intercellular signal between the outermost layers of the shoot meristem and the underlying cell layers (Lenhard and Laux, 2003).
If AIR3 and SDD1 are processing proteases, then it is clear from the phenotypes of the 35S::AIR3 lines (this paper) and the 35S::SDD1 lines (von Groll et al., 2002) that their specificity of action (in modulating lateral root growth and stomatal patterning, respectively) does not depend on the specificity of their expression patterns. This surprising observation could be explained if it is the putative targets of SDD1 and AIR3, and/or the receptors for the peptide ligands generated by their activity, that are expressed in the required cell-specific manner. Future studies will focus on identifying the target(s) of AIR3 and the downstream components of the signaling pathway of which it is part.
involved in modulating processes within the adjacent lateral root primordium which determine its subsequent meristematic activity (for example by generating a larger meristem). This would imply a kind of pre-programming of the pre-emergent lateral root which could potentially be acted on by a variety of extrinsic factors (such as NO3- or
phosphate) and intrinsic (hormonal) factors that are known to affect lateral root growth rates (Casimiro et al., 2003). Our speculative model for the mode of action of AIR3 is illustrated in Figure 5.
Figure 5. Model for the mode of action of AIR3.
Expression of the AIR3 gene is restricted to a cluster of cortical and epidermal cells overlying the developing lateral root primordium (Neuteboom et al., 1999b). The encoded subtilisin-like protease is proposed to be responsible for processing a precursor protein, generating a diffusible peptide signal. This signal moves through the extracellular matrix and is perceived by receptors on the surface of the cells in the adjacent lateral root primordium. Perception of the signal initiates a signal cascade that promotes meristem development and the future meristematic activity of the mature lateral root. The alternative possibility that AIR3 is responsible for inactivating a negative signal, rather than generating a positive one, should also be considered.
Role of AIR3 in the nitrate regulation of lateral root growth
When the 35S::AIR3 lines were cultured on a range of NO3- concentrations, it
was revealed that the responsiveness of lateral root growth to the NO3- was altered
compared to control lines, an effect which was most noticeable at high [NO3-]. In the
control lines, growth of lateral roots was inhibited at 25 mM NO3- compared to 1 mM
stimulated by increasing the [NO3-] to 25 mM (Figures 2 and 3). This result suggests
that in wild-type plants AIR3 has a role in mediating the inhibitory effect of high [NO3-]
on lateral root development. To investigate this possibility further we examined the ability of a high [NO3-] to regulate the expression of AIR3. The results of a
histochemical analysis of an Arabidopsis line carrying an AIR3::GUS construct showed that AIR3 was strongly down-regulated in the vicinity of the developing lateral root (Figure 4). Previous studies have led to a model in which the accumulation of NO3- in
the shoot, accentuated under high rates of NO3- supply, generates a long-distance
signal that has a systemic inhibitory effect on lateral root development (reviewed in Forde, 2002). If AIR3 has a positive regulatory effect on lateral root growth, then the ability of high [NO3-] to down-regulate its expression would provide the basis for a
pathway through which the systemic inhibitory effect of NO3- could operate. It would
also explain why constitutive expression of AIR3 protected the lateral roots from the inhibitory effect of a high rate of NO3- supply (Figures 2 and 3).
Shoot-derived auxin has been shown to promote lateral root outgrowth in Arabidopsis (Bhalerao et al, 2002) and a model has been proposed in which the accumulation of high levels of tissue NO3- in the shoot inhibit lateral root development
by reducing the flux of auxin from shoot to root (Forde, 2002). Given the known auxin-inducibility of AIR3 (Neuteboom et al., 1999b), it was possible that its down-regulation in high [NO3-] was due to an auxin-mediated effect of this kind. However, this
hypothesis seems less likely in view of the finding that the expression of another auxin-regulated gene, AIR1, which is expressed in the same spatial pattern as AIR3 (Neuteboom, 2000), was not affected by the high [NO3-] treatment (Figure 4E).
The plant hormone abscisic acid (ABA) has been found to play an important role in mediating the inhibitory effect of high [NO3-] on lateral root formation. The ABA
insensitive mutant abi4-1/2, abi5-1 and the ABA synthesis mutants aba1-1, aba2-3/4, and aba3-2, show reductions in the inhibitory effect of NO3- on lateral root elongation
(Signora et al., 2001) and exogenous ABA mimics the inhibitory effect of high [NO3-]
Materials and Methods
Growth of Arabidopsis seedlings
Seeds of Arabidopsis thaliana (L.) Heynh were surface-sterilized by incubation for 1 min in 70% (v/v) ethanol and 15 min in 1% (v/v) hypochlorite, 0.01% (v/v) Tween-20 followed by four rinses with sterilized water. For studies of root growth, the seed was sown on agar plates containing ½ Murashige and Skoog (MS) medium (containing approximately 20 mM NO3-+ 10 mM NH4+) supplemented with 2% (w/v) sucrose and
2.3 mM 2-morpholinoethane sulfonic acid (MES). In some experiments, particularly where the NO3- concentration was to be varied, we used a nutrient solution based on a
50-fold dilution of B5 medium (with 0.5% sucrose), as described by Zhang and Forde (1998). The plates were then placed at 4°C in the dark for up to 4 d to promote synchronous germination. Seedlings were subsequently cultured for up to 2 weeks on the surface of the vertically orientated plates at 21°C under a 16:8 h photoperiod at 100 µM photons.m-2.sec-1. At the end of the experiment the lengths of individual primary and lateral roots were measured using millimeter paper and a binocular microscope (100X magnification) or the root systems were imaged using a gel documentation system (BioRad EagleEye II) and root lengths and numbers were determined from the enlarged images.
Generation of AIR3 overexpressing lines
phenol and chloroform, and digested with NdeI and EcoRI. The resulting PCR fragment also included an 89 bp intron that is located within the first 42 bp of the coding region of the AIR3 gene. The fragment was ligated into the cDNA clone, which had been digested with the same restriction enzymes. The resulting 2592 bp hybrid cDNA with the first intron and a 19 bp A-tail was cloned into the pART7 plasmid to place it under control of the CaMV 35S promoter and the ocs terminator (Gleave, 1992). The EcoRI-KpnI restriction sites were finally used to excise the construct and introduce it into the pART27 binary vector as a NotI fragment. The binary 35S::AIR3 plasmid was mobilized to Agrobacterium tumefaciens strain MOG101 (Hood et al., 1993) using a triparental mating procedure (Ditta et al., 1980). Transformation of Arabidopsis thaliana ecotype C24 was performed as described by Vergunst et al., (1998). Transgenic plants were selected by their kanamycin resistant phenotype. A transgenic control (C1) was obtained by transforming C24 with an empty binary pART27 vector.
Histological analysis
For epidermal cell length measurements, the whole root was fixed in ethanol:acetic acid (3:1 v/v) for a minimum of 1 h and then small samples were mounted in clearing solution (chloral hydrate:water:glycerol, 8:3:1 v/v/v). A Zeiss Axioplan II microscope equipped with differential interference contrast (DIC) optics was used. For cytological analysis, root sections were prepared following the procedures described by Weijers et al. (2001).
Northern blots
Total RNA was isolated from 14 d-old seedlings which had been cultured on liquid B5 medium at 21°C with a 16:8 h photoperiod. Before extraction, one of the two samples of each line was induced for 24 h with 1 µM 1-NAA (1-naphthylacetic acid); the other was used as control. The material was frozen under liquid N2 and ground
electrophoresed and then transferred to a nylon membrane (GeneScreen Plus, NEN DuPont Boston, M.A, USA) by capillary blotting.
The northern blots were hybridized with a 32P-labeled AIR3 RNA probe made from a 1000-bp EcoRI/XhoI fragment containing the C-terminal part of the AIR3 coding sequence cloned in the pBluescript SK+ plasmid (Stratagene). The antisense probe was transcribed from from the EcoRI-linearized plasmid using T7 RNA polymerase. Labeling reactions were performed using the TransProbe T kit (Pharmacia). Pre-hybridization was performed for 2 h at 42°C in 50% deionized formamide, 5 X SSPE (1X SSPE = 180 mM NaCl, 1mM EDTA, and 10 mM sodium phosphate, pH 6.5), 2.5% (w/v) sodium dodecyl sulfate (SDS). The hybridization was performed in the same solution after addition of the probe. Blots were washed two times 30 min in 0.1 X SSPE, 0.1% (w/v) SDS at 65°C and then exposed to an X-ray film.
Identification and molecular analysis of an AIR3 knockout mutant
5’-AAACTCACTACGACTTTCTTGGTTCCTTC-3’ and the AIR3-3’ used in the screen for air3-T knockout and described above. RT-PCR was performed according to Weijers et al. (2001).
