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Root system architecture is an important trait that determines the efficiency for water and nutrients acquisition in plants. Plants have evolved an array of mechanisms to modify their root system architecture in response to environmental stimuli (Malamy, 2005; Osmont et al., 2007; Nibau et al., 2008). This developmental plasticity is regulated by nutrient availability in soils in general (Forde, 2002; Desnos, 2008) and soil nitrogen status in particular (Zhang et al., 1999; Zhang & Forde, 2000; Linkohr et al., 2002). A dual effect of external nitrate on lateral root (LR) development in Arabidopsis thaliana has been recognized; a localized stimulatory effect of external nitrate on LR elongation (Zhang & Forde, 1998; Zhang et al., 1999; Linkohr et al., 2002), and a systemic inhibitory effect of globally high external nitrate concentrations (> 5 mm) on LR growth (Zhang & Forde, 1998; Zhang et al., 1999; Linkohr et al., 2002). The localized stimulatory effect of nitrate on LR development is mediated by a putative MADS-box transcriptional factor, a nitrate-inducible Arabidopsis gene (ANR1) (Zhang & Forde, 1998). In addition to ANR1, nitrate transporter, AtNRT1.1 (also called CHL1, Tsay et al., 1993), a dual-affinity nitrate transporter that mediates both low- and high-affinity nitrate transport across the plasma membrane (Tsay et al., 1993) may function as a nitrate sensor to participate in the stimulatory effect of low nitrate concentration on LR growth (Remans et al., 2006a). However, how plants sense the nitrate signal and transmit it to regulate the AtNRT1.1-ANR1 signaling pathway remains to be deciphered.
The systemic inhibitory effect of high nitrate concentration on LR occurs immediately after emergence of the LR primordium from the parent root, leading to accumulation of short LRs (Zhang et al., 1999). The internal nitrate concentration rather than accumulation of the products of nitrate assimilation is likely to be a critical factor modulating the nitrate-dependent response as the inhibitory effect is enhanced in a nitrate reductase-deficient mutant (Zhang et al., 1999). By contrast, the same nitrate reductase-deficient mutant is more sensitive to high external nitrate concentration than wild-type plants in terms of LR development (Zhang et al., 1999). These observations suggest that the systemic inhibitory effect of high nitrate concentration on LR development may result from the accumulation of nitrate inside plant tissues.
Long-distance signaling molecules that originate from shoots and are transported to the roots have been suggested to be associated with the suppression of LR development by high nitrate concentration (Zhang & Forde, 1998; Zhang et al., 1999). The involvement of auxin in high nitrate concentration-induced inhibition of root development has been suggested (Caba et al., 2000; Walch-Liu et al., 2006; Tian et al., 2008). These studies revealed that plants exposed to high nitrate concentration exhibited reduced indoleacetic acid (IAA) contents in roots because of either inhibition of auxin synthesis and/or inhibition of auxin transport from shoots to roots. In addition, there is also evidence suggesting that abscisic acid (ABA) may play a role in high nitrate-induced inhibition of root development. For example, the high nitrate-induced systemic inhibition of LR development is less in ABA synthesis and ABA-insensitive Arabidopsis mutants than in wild-type plants (Signora et al., 2001), and exogenous application of ABA can mimic high external nitrate concentration in suppression of LR meristem (De Smet et al., 2003). In addition to auxin and ABA, cytokinin (Sakakibara, 2003; Tian et al., 2005) and nitric oxide (Zhao et al., 2007) were also reported to be involved in nitrate-dependent root growth and development. Recent studies also indicated that the two nitrate transporters of NRT1.1 and NRT2.1, which underpin low and high affinity, and high-affinity nitrate transport across plasma membranes, respectively (Forde, 2000), play a signaling role in LR development in response to the external nitrate supply (Little et al., 2005; Remans et al., 2006a,b). Given that expression of NRT1.1 and NRT2.1 is sensitive to auxin (Guo et al., 2002) and ethylene (Leblanc et al., 2008), it is conceivable that the interactions between these phytohormones and nitrate transporters are of importance in modulation of nitrate-dependent root development.
There are multilevel interactions between ethylene and auxin in the regulation of root development in Arabidopsis (Stepanova et al., 2005, 2007; Růžička et al., 2007; Swarup et al., 2007; Ivanchenko et al., 2008; Negi et al., 2008). Ethylene is closely associated with diverse physiological processes in plants, including root growth and development (Pierik et al., 2006). Ethylene is synthesized from methionine through S-adenosyl-l-methionine and 1-aminocyclopropane-1-carboxylic acid (ACC), which are catalysed by ACC synthase (ACS) and ACC oxidase (ACO) (Kende, 1993). Both ACS and ACO are encoded by multigene families and regulated by developmental and environmental factors (Wang et al., 2002). A number of key proteins involved in the ethylene signaling pathways have been identified (Alonso & Stepanova, 2004; Chen et al., 2005). The etr1 mutant has a defect in a membrane receptor for ethylene (Chen et al., 2005). The EIN2 protein acts downstream of ETR1 and resembles the NRAMP family of metal transporters (Alonso & Stepanova, 2004). The etr1 and ein2 mutants have been widely used in deciphering physiological functions of ethylene in the literature (Alonso et al., 1999; Binder et al., 2006; Buer et al., 2006; De Grauwe et al., 2007). There have been a number of reports demonstrating that ethylene is closely associated with responses of root development to nutritional deficiency, including phosphorus starvation, and deficiency in iron (Schmidt, 2001) and potassium (Shin & Schachtman, 2004). The possible involvement of ethylene in nitrate-dependent physiological processes has also been implicated, as shown by induction of ethylene evolution from alfalfa (Medicago sativa) (Caba et al., 1998) and chickpea (Cicer arietinum) (Nandwal et al., 2000) by high external nitrate concentration and increased sensitivity of maize to ethylene under nitrogen deficiency (Schmelz et al., 2003). However, there has been no detailed study to evaluate the role of ethylene in high nitrate concentration-induced suppression of root development.
To elucidate the mechanism underlying the systemic inhibition of lateral root development by high nitrate concentration, we examined the role of ethylene in responses of root development to high nitrate concentration in A. thaliana. More specifically, both Arabidopsis wild-types and mutants that are insensitive to ethylene (etr1-3 and ein2-1) and defective in nitrate transporters (chl1-5 and nrt2.1-1) were used to study the effects of high nitrate concentration on ethylene production and root development.
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Previous studies have shown that nitrate in the external medium have dual effects on LR development; localized nitrate supply stimulates LR growth and globally high concentration (> 5 mm) of nitrate suppresses LR growth (Forde, 2002; Zhang et al., 2007). Phytohormones such as ABA (Signora et al., 2001), auxin (Walch-Liu et al., 2006) and cytokinin (Sakakibara, 2003) may play regulatory roles in the nitrate-dependent LR growth and development. Given that root growth and lateral root formation is sensitive to ethylene (Lopez-Bucio et al., 2002) and that auxin and ethylene synergistically regulate root development, particularly lateral root development (Ivanchenko et al., 2008), it may be informative to examine whether ethylene is involved in the nitrate-dependent root development. In the present study, we demonstrated that exposure of Arabidopsis seedlings grown in low (0.1 mm) to high nitrate concentration (10 mm) evoked a rapid burst of ethylene production owing to upregulation of expression of ACS and ACO genes and increases in activities of ACS and ACO. Similar results, that high nitrate concentration induced ethylene production, have been reported in alfalfa (Caba et al., 1998) and chickpea (Nandwal et al., 2000). However, unlike these previous studies, we further identified that expression of genes encoding ACS and ACO, the two key enzymes responsible for ethylene synthesis, was transcriptionally upregulated by high nitrate concentrations (Fig. 4c,d). Moreover, we demonstrated that the high nitrate concentration-induced ethylene production occurred predominantly in both unemerged and emerged lateral roots, while ethylene levels in mature LR and PR were little changed, as shown by changes in activities of ethylene reporter EBS-GUS (Fig. 3). The differences in EBS-GUS activity between lateral and primary roots may underlie the observed the difference in sensitivity of root growth to the external nitrate concentrations between lateral and primary roots. Furthermore, it is interesting that the observed ethylene burst in response to high nitrate concentration did not turn on the ethylene signal in the primary root, leading to inhibition of root growth (cf. Fig. 1). This result suggests that different mechanisms may be involved in the perception and signaling of ethylene in primary and lateral roots.
To establish the links between the high nitrate concentration-induced ethylene production in LRs and inhibition of LR growth by high nitrate concentration, both pharmacological and genetic approaches were employed. Suppression of ethylene production by inhibiting ACS and ACO with AVG and Co2+ markedly alleviated the high nitrate-induced reductions in LR length and number (Fig. 6). More specifically, we found that the ameliorative effect of AVG and Co2+ on LR growth and development lies in their effects on lateral roots immediately after emergence of LR primordia from the parental roots (Fig. 5). Our results (cf. Fig. 1d) and previous studies (Zhang et al., 1999) have shown that high nitrate concentration mainly inhibited growth of immature LRs emerging from the primary root, leading to reductions in numbers of visible LRs (<0.5 mm), while the high nitrate concentration had no effect on LR initiation (Fig. 1d). In addition, inhibition of LR length by the high nitrate concentration was reversible upon transferring the seedlings grown in HN medium to the LN medium (Fig. S1). This result confirms results reported by Zhang & Forde (2000). The ethylene production evoked by high nitrate concentration can also be reversed when seedlings were shifted from high to low nitrate concentration (Fig. S2). Together, these findings suggest that the inhibitory effect of high nitrate concentration on immature LRs is likely to result from elevated ethylene production. Furthermore, our observations that reductions in LR length and number in ethylene-insensitive mutants etr1-3 and ein2-1 were less than those in wild-type plants (Fig. 6) in response to high nitrate concentration highlight the important roles of ETR1- and EIN2-dependent ethylene signaling pathways played in nitrate-dependent LR development.
There is ample evidence that nitrate transporters of NRT1.1 and NRT2.1 are involved in the signaling cascades of the nitrate-dependent root growth and development (Orsel et al., 2004; Little et al., 2005; Remans et al., 2006a,b; Walch-Liu & Forde, 2008). For example, NRT1.1 is associated with the localized stimulatory effect of nitrate supply through putative interactions with ANR1 (Remans et al., 2006a). NRT2.1 has been shown to repress lateral root initiation (Little et al., 2005). However, previous studies mainly focus on the role of nitrate transporters in localized nitrate effect and their response to low nitrate concentration. No detailed study has been conducted to investigate the involvement of nitrate transporters in sensing and responding to high nitrate concentration. In the present study, we revealed that the transcript levels of AtNRT1.1 and AtNRT2.1 genes displayed contrasting responses to the high nitrate concentration (i.e. expression of AtNRT1.1 and AtNRT2.1 was upregulated and downregulated, respectively, in response to a shift from low to high nitrate concentration (Fig. 8a). These observations are in good agreement with those reported results in the literature (Orsel et al., 2004; Little et al., 2005; Remans et al., 2006a,b). More importantly, we found that the expression AtNRT1.1 and AtNRT2.1 was sensitive to ACC and AVG such that ACC and AVG rapidly upregulated and downregulated expression of AtNRT1.1 and AtNRT2.1 at low and high nitrate concentrations, respectively (Fig. 8b). These findings are indicative that the high nitrate concentration-induced AtNRT1.1 and AtNRT2.1 expression may occur via ethylene. This argument is strengthened by the findings that expression of AtNRT1.1 and AtNRT2.1 in ethylene-insensitive mutants of ert1-3 and ein2-1 was no longer responsive to the high nitrate concentration (Fig. 8c). Leblanc et al. (2008) recently reported similar effects of ACC and AVG on expression of BnNRT1.1 and BnNRT2.1 in oilseed rape. However, unlike our study, they did not compare the effects of ACC and AVG on expression of BnNRT1.1 and BnNRT2.1 in low and high nitrate concentrations; rather, they studied responses of BnNRT1.1 and BnNRT2.1 expression to ACC and AVG in nitrate concentration at 1 mm exclusively, and a longer duration of treatments with ACC and AVG (> 24 h) was used in their study.
In addition to the dependence of AtNRT1.1 and AtNRT2.1 expression on nitrate concentration, we also demonstrated that mutants defective in NRT1.1 (chl1-5) and NRT2.1 (nrt2.1-1) exhibited altered sensitivity to the high nitrate concentration in terms of LR length and number (Fig. 7b,c). In comparison with wild-type plants (Col-0 and Ws), LR length in chl1-5 and nrt2.1-1 plants was less reduced in response to the high nitrate concentration (Fig. 7). These observations may be explained by the fact that mutation of low-affinity NRT1.1 transporter renders the chl1-5 unable to accumulate nitrate inside cells as the accumulation of nitrate inside root cells is implicated in the high nitrate-induced LR growth (Zhang et al., 1999). We have no explanation for the less inhibitory effect of high nitrate concentration on LR length in nrt2.1-1 than in wild-type plants. However, it is possible that mutation of NRT2.1 may also affect function of NRT1.1, leading to inhibition of nitrate accumulation as a recent study showed close interaction between the two nitrate transporters at transcription level (Muños et al., 2004). Further, the observation that LR number in nrt2.1 plants was no longer responsive to high nitrate concentration (Fig. 7c) is in agreement with the role of NRT2.1 in repression of LR initiation (Little et al., 2005). The rapid burst of ethylene production in response to high nitrate concentration and transcriptional dependence of AtNRT1.1 and AtNRT2.1 on ethylene seems to imply that ethylene acts e upstream of the nitrate transporters. Alternatively, the lower responsiveness of chl1-5 and nrt2.1 to high nitrate concentration than that of wild-type plants in terms of LR length and number may result from the differences in ethylene evolution between the mutants and wild-type plants in response to high nitrate concentrations. However, our observation that the mutants (chl1-5 and nrt2.1) did not differ from their corresponding wild-type counterparts in terms of high nitrate-induced ethylene production (data not shown) seems to discount this possibility.
In our study, Arabidopsis seedlings were first grown in the 1/2 MS medium that contained c. 20 mm nitrate for 4 d and were then exposed to the treatment regimes of LN and HN media. This may cause a recovery of root growth because of the reduction in nitrate concentration. However, we found that LR length, LR number and ethylene production were not affected by transferring seedlings from 20 mm nitrate to 10 mm nitrate media (data not shown). Conversely, seedlings grown in LN medium following 4-d growth in the 1/2 MS medium and in HN medium showed comparable responses to the high nitrate treatment in terms of LR length, LR number and ethylene production. Therefore, the experimental protocols used in the present study might not affect the overall conclusions that ethylene plays a regulatory role in mediation of high nitrate concentration-induced lateral root growth and development in Arabidopsis.
In conclusion, we demonstrated that exposure of Arabidopsis seedlings to high nitrate concentration led to a burst of ethylene production that occurred very rapidly and predominately in immature lateral roots. The elevated ethylene was closely associated with reductions in LR length and LR number, as demonstrated by mitigation of the inhibitory effect on LR with antagonists of ACS and ACO, and the lower sensitivity of ethylene insensitivity mutants than that of wild-type plants to nitrate concentration. We further revealed that nitrate transporters AtNRT1.1 and AtNTR2.1 were regulated transcriptionally by high nitrate concentration, and that the effects were mimicked by ACC at low nitrate concentration, and the dependence of AtNRT1.1 and AtNTR2.1 on high nitrate concentration was abolished by treatment with AVG. These findings indicate that inhibition of immature LR growth by high nitrate concentration is likely to result from elevated ethylene production, which in turn regulates NRT1 and NRT2, leading to the observed changes in root branching of Arabidopsis grown in high nitrate concentration.