Antagonistic activity of auxin and cytokinin in shoot and root organs

Abstract

The hormones auxin and cytokinin are essential for plant growth and development. Because of the central importance of root and shoot apical meristems in plant growth, auxin/cytokinin interactions have been predominantly analyzed in relation to apical meristem formation and function. In contrast, the auxin/cytokinin interactions during organ growth have remained largely unexplored. Here, we show that a specific interaction between auxin and cytokinin operates in both the root and the shoot where it serves as an additional determinant of plant development. We found that auxin at low concentrations limits the action of cytokinin. An increase in cytokinin level counteracts this inhibitory effect and leads to an inhibition of auxin signaling. At higher concentrations of both hormones, these antagonistic interactions between cytokinin and auxin are absent. Thus, our results reveal a bidirectional and asymmetrical interaction of auxin and cytokinin beyond the bounds of apical meristems. The relation is bidirectional in that both hormones exert inhibitory effects on each other's signaling mechanisms. However, this relation is also asymmetrical because under controlled growth conditions, auxin present in nontreated plants suppresses cytokinin signaling, whereas the reverse is not the case.

Keywords: apical meristems; auxin; cytokinin; hormonal antagonism; roots; shoots.

Figure 1

Auxin inhibits cytokinin signaling. (a) Expression pattern of ARR5p:GUS in 5‐day‐old Col‐0 (WT), axr3‐1, and axr3‐3 seedlings. (b) RNA gel blot analysis of the effect of treatment with the cytokinin BA on ARR5 transcript accumulation in Col‐0 and axr3‐3 seedlings. Plants were grown for 7 days and then treated with 5 μM BA for the denoted time interval. Methylene blue‐stained membrane region with ribosomal RNAs (rRNA) is shown as a loading control. Relative intensity (RI) of the ARR5 signal is shown below the loading control and is presented as mean ± SD of two biological replicates. Two different y axes are shown to allow the visualization of the difference in the untreated controls

Figure 2

Cytokinin‐related phenotypes of the axr3‐3 mutant. (a) Expression patterns of ARR5p:GUS in 4‐day‐old Col‐0 (WT), axr3‐3, axr3‐3 arr1‐1, and 35Sp:ARR5 axr3‐3 (ARR5 OE) seedlings. (b) Number of lateral roots emerging from the primary root of plants grown on vertically positioned plates for 18 days. The results are presented as mean ± SD (n ≥ 10). **p ≤ 0.01 compared to axr3‐3 (one‐way ANOVA with Bonferroni's multiple comparisons test). (c) Rosette diameter of 18‐day‐old plants grown on horizontal plates. The results are presented as mean ± SD (n ≥ 10). *p ≤ 0.05 and **p ≤ 0.01 compared to axr3‐3 (one‐way ANOVA with Bonferroni's multiple comparisons test). (d) Eighteen‐day‐old axr3‐3 and ARR5 OE axr3‐3 plants are shown to illustrate the differences in rosette sizes and root branching. (e) Relative anthocyanin content in 12‐day‐old plants. Pools of 10 plants (three biological replicates per line) were used for extraction and the results are presented as mean ± SD. **p ≤ 0.01 compared to Col‐0 (one‐way ANOVA with Bonferroni's multiple comparisons test)

Figure 3

ARF7 inhibits cytokinin signaling. (a) Expression analyses of ARR5p:GUS in 5‐day‐old Col‐0 (WT), arf7‐1, and the 35Sp:ARR5 arf7‐1 (ARR5 OE) seedlings. (b) Lateral root number on the main root of 18‐day‐old plants grown on vertical plates. The results are presented as mean ± SD (n ≥ 10). ns, not significant; ***p ≤ 0.001 compared to WT, ** (red), p ≤ 0.01 compared to arf7‐1 (one‐way ANOVA with Bonferroni's multiple comparisons test). (c) Relative anthocyanin content in 12‐day‐old seedlings. Pools of 10 plants were used for extraction and the results are presented as mean absorption at 530 nm per 10 seedlings (A530) ± SD (n = 3). ns, not significant; **p ≤ 0.01 compared to WT, *** (red), p ≤ 0.01 compared to arf7‐1 (one‐way ANOVA with Bonferroni's multiple comparisons test). (d) Representative 3‐week‐old plants illustrating the rosette size difference between the arf7‐1 arf19‐1 and arf7‐1 arf19‐1 arr1‐1 lines. (e) Root induction frequencies from root explants of 6‐day‐old seedlings incubated for 11 days on the denoted concentrations of NAA. The results are presented as mean ± SD (n ≥ 10)

Figure 4

Cytokinin inhibits auxin signaling. (a) Expression patterns of DR5p:GUS in 5‐day‐old Col‐0 seedlings cotreated for 4 hr with the denoted combinations of NAA and BA. (b) Expression patterns of DR5p:GUS in 4‐day‐old Col‐0 (WT) and phosphomimic 35Sp:ARR1 ^D94E (PM‐ARR1) seedlings treated with NAA. The length of the GUS reaction was timed to reveal the DR5p:GUS expression differences between the WT and 35Sp:ARR1 ^D94E backgrounds. (c) Expression patterns of DR5p:GUS in 5‐day‐old WT and arr1‐3 arr10‐5 arr12‐1 seedlings treated for 4 hr with 0.5 μM of NAA