Environmental regulation of lateral root initiation in Arabidopsis

Abstract

Plant morphology is dramatically influenced by environmental signals. The growth and development of the root system is an excellent example of this developmental plasticity. Both the number and placement of lateral roots are highly responsive to nutritional cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the "decision" to form a lateral root at a particular time and place. Lateral roots originate from differentiated cells in adult tissues. These cells must reenter the cell cycle, proliferate, and redifferentiate to produce all of the cell types that make up a new organ. Almost nothing is known about how lateral root initiation is regulated or coordinated with growth conditions. Here, we report a novel growth assay that allows this regulatory mechanism to be dissected in Arabidopsis. When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed. Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation. We have isolated a mutant, lateral root initiation 1 (lin1), that overcomes the repressive conditions. This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios. The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues. Therefore, these studies provide novel insights into the mechanisms that regulate the earliest steps in lateral root initiation and that coordinate plant development with the environment.

Figure 1

Phenotype of lin1. A, Wild-type Col seedlings (left) and lin1 seedlings (right) were grown for 10 d on repressive 4.5/0.02 media (see text). The roots were then cut with a razor blade approximately 0.5 cm from the root tip. Seven days after tip excision, the lin1 roots are highly branched, whereas few if any lateral roots can be observed in the wild-type seedlings. B, Close-up of the aerial parts of wild-type and lin1 seedlings at 17 d. The wild-type seedling leaves are red or brown, whereas the lin1 leaves are green. The aerial tissues of wild type and mutant are severely stunted by the nitrogen starvation conditions. A wild-type seedling grown for 17 d on standard media is shown for comparison. Bar = 2 mm.

Figure 2

Growth rate of wild-type (A) and lin1 (B) seedlings on 4.5/60 (squares) and 4.5/0.02 (white circles) media. The root tips of seedlings were marked each day beginning 4 d post-planting. After 13 d, the distances between each mark were measured for each plant. The average root growth for each time period is shown in the graph, with time point 5 indicating the average growth from d 4 to 5 and so on. Growth rates of wild-type seedlings were dramatically inhibited by the 4.5/0.02 media compared with the standard 4.5/60 media. In contrast, lin1 showed similar growth rates on both media. Rates are averages from 12 to 20 plants. Error bars = ±sd.

Figure 3

Development of wild-type and lin1 mutant seedlings on high Suc. Top, Wild-type seedlings; bottom, lin1 seedlings. Two plates of 50 seeds each were sown on standard Murashige and Skoog media (containing 4.5%, 10%, or 12% [w/v] Suc as indicated and 60 mm nitrogen [20 mm KNO₃ and 20 mm NH₄NO₃]) and grown for 12 d. All seeds germinated and seedlings produced healthy roots and green cotyledons and primary leaves when grown on 4.5% (w/v) Suc. On 10% (w/v) Suc, the plants were much smaller, with green cotyledons but only small primary leaves and reduced roots. On 12% (w/v) Suc, no primary leaves formed, the cotyledons were white, and there was very little growth of the primary root. These phenotypes were similar between wild-type and lin1 mutant seedlings. Shown are representative seedlings.

Figure 4

GUS expression in DR5 transgenic plants under different Suc to nitrogen ratios. A and B, Photos of representative 6-d-old wild-type seedlings grown on 0.5/0.02 media; C and D, Similar seedling grown on 4.5/0.02 media. The regions indicated by the red lines in A and C are shown in the micrographs in B and D, respectively. The arrows indicate the hypocoyl/root junction. A and B, The hypocotyls of seedlings grown on 0.5/0.02 show no staining. The blue spot at the junction in A and B is a lateral root primordium. C and D, The hypocotyls of seedlings grown on 4.5/0.02 media are intensely stained. The staining is in the lower half of the hypocotyl, starting at the hypocotyl root junction and extending up the hypocotyl. The extent of the stained region differs from seedling to seedling. Also, note the increased thickness of the hypocotyl stele in D. Bar in B and D = 1 mm.

Figure 5

Effects of auxin transport inhibitors on lateral root initiation in wild type and lin1. Five-day-old seedlings were transferred to media containing DMSO alone or the auxin transport inhibitors TIBA (20 μm) and NPA (10 μm) dissolved in DMSO. At d 12, the lateral roots and lateral root primordia produced in the new growth were counted on 10 seedlings. TIBA and NPA were highly effective in reducing lateral root initiation in wild-type seedlings grown on standard media or on 4.5/0.02 media when compared with DMSO controls. Initiation was also dramatically reduced in the new growth of lin1 roots. Error bars = ±sd.

Figure 6

Effect of NAA on primary root elongation in wild-type and lin1 seedlings. Seedlings were grown for 5 d on standard Murashige and Skoog media (4.5/60). They were then transferred to the same media containing various concentrations of NAA as indicated, and the position of the root tip was marked. Seven days later, the new growth was measured from the mark. Lengths are averages of 15 to 25 plants. Error bars = ±sd.