- Top of page
- Experimental procedures
Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post-embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10-fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA-induced lateral root inhibition is mediated by an auxin-independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin-dependent regulatory pathway for the post-emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3-1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA-sensitive, auxin-independent checkpoint for lateral root development in Arabidopsis at the post-emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.
- Top of page
- Experimental procedures
The root system of most ‘higher’ plants consists of three types of roots, i.e. the main (also known as tap or primary), lateral and adventitious root (Fitter, 1991). The meristem of the main root is formed during embryogenesis. In contrast, the meristems of lateral and adventitious roots are formed post-embryogenically during the lifetime of a plant and their numbers vary according to the age and the growth conditions (Malamy and Benfey, 1997a; Van den Berg et al., 1998).
Lateral roots (LR) arise from a specific differentiated layer of cells encircling the vascular stele, the pericycle. The developmental process begins in most plants with asymmetric transverse divisions of a limited number of pericycle cells adjacent to the two xylem poles of the parent root (Casimiro et al., 2001). Some of the newly divided cells undergo further periclinal divisions leading to the formation of a dome-shaped LR primordium (LRP), which then grows through the outer layers of the parent root and eventually emerges. Soon after the emergence, the LRP is activated and forms a functional LR meristem (Malamy and Benfey, 1997b).
Auxin plays a key role in LR development. It has been known for more than 50 years that auxin stimulates LR formation (Torrey, 1950). Increasing auxin supply via either exogenous auxin application or an enhancement of endogenous auxin synthesis results in a significant increase in the number of LRs (Boerjan et al., 1995; Kares et al., 1990; Klee et al., 1987; Laskowski et al., 1995). A large body of evidence indicates that auxin regulates at least three developmental stages: initiation, LRP establishment, and the activation of LR meristems. Cell divisions in the pericycle represent the first step of LR development and are stimulated by auxin. In Arabidopsis, radish and many other plant species, exogenous auxin application can induce LRP formation from almost all the pericycle cells adjacent to the xylem poles (Blakely et al., 1982; Laskowski et al., 1995).
A requirement for auxin during LRP establishment is demonstrated in experiments with excised Arabidopsis or radish root segments (Laskowski et al., 1995). In the presence of exogenous auxin, LRPs of any developmental stage in the excised segments continued their development to form LRs. However, in the absence of auxin, only primordia that had at least 3–5 cell layers developed into LRs, and no further development occurred in primordia of less than three cell layers. These observations suggested that LR development prior to the 3–5 cell layer stage was auxin dependent and that development beyond this stage was either auxin independent or auxin self-sufficient (i.e. the primordium is capable of synthesising the required amount of auxin de novo).
The phenotype of the aberrant lateral root formation 3 (alf3-1) mutant indicates that auxin is also required for lateral root development beyond the 3–5 cell layer stage. In the absence of exogenous auxin, LRs in alf3-1 arrest soon after emergence from the parent roots. This developmental arrest can be rescued by exogenous IAA or indole, a precursor of IAA biosynthesis. This observation suggests that auxin is required at the post-emergence stage (Celenza et al., 1995).
Two recent studies suggest that auxin may also play an important role in LR emergence (Bhalerao et al., 2002; Marchant et al., 2002). During early seedling development (3–10 days after germination (DAG)) the emergence of the first LR coincides with the formation of the first true leaves (Marchant et al., 2002). The removal of apical tissues prior to the formation of the first true leaves has very little effect on LR initiation but inhibits LR emergence (Bhalerao et al., 2002), indicating that the emergence of LRs requires leaf-derived IAA.
In addition to auxin, many environmental factors also affect LR development. An excellent example is the effect of nutrients (Drew, 1975; Drew and Saker, 1975; Drew et al., 1973; Leyser and Fitter, 1998; Malamy and Ryan, 2001). It has been known for decades that, in soil or growth medium with patchy nutrient distribution, LRs preferentially proliferate in the nutrient-rich zone (Drew and Saker, 1975; Drew et al., 1973). The biological significance of this developmental plasticity is easy to understand as it enables the plant to explore the available nutrients in the surrounding soil environment more efficiently. However, the mechanisms by which plants incorporate such signals into the LR developmental process are poorly understood.
We reported previously that, in Arabidopsis thaliana, nitrate availability affects LR development in two different ways, i.e. a localised stimulatory effect and a systemic inhibitory effect. The localised stimulatory effect acts mainly on the elongation of lateral roots and is mediated by a putative MADS-box transcriptional factor, ANR1 (Zhang and Forde, 1998). The systemic inhibitory effect is only observed at high concentrations of nitrate and occurs at a specific developmental point, immediately after the emergence of the LRP from the parent root (Zhang et al., 1999, 2000). Interestingly, this inhibitory effect was significantly reduced in all known ABA synthesis mutants (aba1-1, aba2-3, aba2-4 and aba3-2) and two ABA-insensitive mutants, abi4 and abi5 (Signora et al., 2001). The results led us to investigate whether ABA plays a direct role in LR regulation. In this paper, we present evidence for the existence of a novel ABA-sensitive, auxin-independent checkpoint during LR development in Arabidopsis.