Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction

Abstract

Together, auxin and cytokinin regulate many of the processes that are critical to plant growth, development, and environmental responsiveness. We have previously shown that exogenous auxin regulates cytokinin biosynthesis in Arabidopsis thaliana. In this work, we show that, conversely, the application or induced ectopic biosynthesis of cytokinin leads to a rapid increase in auxin biosynthesis in young, developing root and shoot tissues. We also show that reducing endogenous cytokinin levels, either through the induction of CYTOKININ OXIDASE expression or the mutation of one or more of the cytokinin biosynthetic ISOPENTENYLTRANSFERASE genes leads to a reduction in auxin biosynthesis. Cytokinin modifies the abundance of transcripts for several putative auxin biosynthetic genes, suggesting a direct induction of auxin biosynthesis by cytokinin. Our data indicate that cytokinin is essential, not only to maintain basal levels of auxin biosynthesis in developing root and shoot tissues but also for the dynamic regulation of auxin biosynthesis in response to changing developmental or environmental conditions. In combination with our previous work, the data suggest that a homeostatic feedback regulatory loop involving both auxin and cytokinin signaling acts to maintain appropriate auxin and cytokinin concentrations in developing root and shoot tissues.

Figure 1.

Induction of IAA Biosynthesis by Cytokinin. (A) The de novo IAA biosynthesis rate was analyzed in specific tissues after incubation of 10 DAG Arabidopsis seedlings in a medium containing 30% ²H₂O. Seedlings were divided into (a) apex and young developing leaves (leaf 3 and smaller), (b) the first two true leaves, and (c) the root system after incubation. (B) IPT8/pga22 seedlings (10 DAG; either intact seedlings or seedlings dissected into root and shoot before incubation) were incubated first for 12 h in a medium containing 5 μM 17-β-estradiol and then for 24 h in a medium containing 30% ²H₂O and 5 μM 17-β-estradiol (+ est). As a control, seedlings were incubated without estradiol in the medium (− est). The IAA biosynthesis rate was analyzed in the different tissues described in (A). (C) Wild-type Ws seedlings 10 DAG were divided into root and shoot and incubated for 24 h in liquid medium containing 30% ²H₂O alone or with 5 μM 17-β-estradiol, 10 μM BAP, or 10 μM c/t-Z as indicated. The IAA biosynthesis rate was analyzed in the different tissues described in (A). Data are mean ± sd of three independent experiments (n = 3). P values in (B) (+ est versus − est) and (C) (+ est, + BAP, or + c/t-Z versus − est) were determined by two-tailed Student’s t test assuming equal variances (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Figure 2.

IAA Biosynthesis in Auxin and Cytokinin Signal Transduction Mutants. De novo IAA biosynthesis rate (A) and IAA concentration (B) were analyzed in root tips of auxin and cytokinin signal transduction mutants after treatment of 6 DAG seedling roots for 24 h with liquid medium containing 30% ²H₂O with or without 5 μM t-Z. Data are mean ± sd of three independent experiments (n = 3). P values (+ t-Z versus – t-Z) were determined by two-tailed Student’s t test assuming equal variances (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Figure 3.

Lowering of Cytokinin Levels Leads to a Downregulation of IAA Biosynthesis. (A) Analysis of CKX3 transcript levels by qPCR after incubation of 6 DAG Arabidopsis wild-type Columbia and pMDC7:AtCKX3 seedlings with 5 μM 17-β-estradiol (est) for 0, 3, 6, 12, and 24 h. Error bars indicate sd (n = 3). (B) Quantification of the nucleotide (tZMP and iPMP) and riboside (tZR and iPA) cytokinin precursors after incubation of 6 DAG pMDC7:AtCKX3 seedlings with 5 μM 17-β-estradiol for 0, 3, 6, 12, and 24 h. Error bars indicate sd (n = 3). (C) IAA biosynthesis rate in 6 DAG pMDC7:AtCKX3 seedlings incubated with medium containing 30% ²H₂O with or without 5 μM 17-β-estradiol for 12 and 24 h. Error bars indicate sd (n = 3). (D) pMDC7:AtCKX3 seedlings 10 DAG were divided into root and shoot and incubated for 12 h in liquid medium containing 30% ²H₂O with or without 5 μM 17-β-estradiol. The IAA biosynthesis rate was analyzed in the different tissues described in Figure 1A. Error bars indicate sd (n = 5). (E) Col-0 and 35S:AtCKX1 seedling roots 6 DAG were incubated for 24 h in liquid medium containing 30% ²H₂O. The IAA biosynthesis rate was analyzed in root tips. Error bars indicate sd (n = 5). Data are mean ± sd of independent experiments. P values in (C) to (E) (+ est versus – est) were determined by two-tailed Student’s t test assuming equal variances (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Figure 4.

IAA Biosynthesis Is Downregulated in the Root Apex of ipt Mutant Lines. The de novo IAA biosynthesis rate was analyzed in root tips of ipt mutant lines after incubation of 6 DAG seedling roots for 24 h with liquid medium containing 30% ²H₂O. Data are mean ± sd of three independent experiments (n = 3). P values for the mutant lines versus the wild type were determined by two-tailed Student’s t test assuming equal variances (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Figure 5.

IAA Biosynthesis in Specific Cell Types after Cytokinin Treatment. (A) Intact roots from 8 DAG Arabidopsis seedlings expressing GFP in specific cell types of the root apex were incubated for 16 h in liquid medium (30% ²H₂O, 30 mM sucrose, 4.4 g MS, and 2.6 mM MES) containing 10 μM NPA and 10 μM t-Z. IAA biosynthesis rates were analyzed in isolated cells from the roots after enzymatic protoplasting and fluorescence-activated cell sorting. A general increase of IAA biosynthesis was observed in all cell types examined, but there were no significant differences between the four GFP-expressing lines. The data were processed in a single factor analysis of variance, which gave a P value of 0.38. Data are mean ± sd, and the number of biological and technical replicates was between 2 and 4, and 1 and 3, respectively. (B) GFP expression pattern in the Arabidopsis lines used in this study.

Figure 6.

qPCR Analysis of Auxin-Related Genes. Wild-type Arabidopsis (Columbia) seedlings were grown in LD for 7 DAG. The seedlings were then incubated for 0.5, 1, 2, 4, or 6 h in liquid medium with or without 5 μM t-Z. The root tips were harvested and genes related to auxin biosynthesis (A) or auxin signaling (B) were analyzed by qPCR. Error bars indicate se (n = 3).

Figure 7.

IAA Biosynthesis Pathways in Arabidopsis Regulated by Cytokinin. Putative IAA biosynthesis pathways in Arabidopsis, with genes shown to be differentially regulated in the microarray and/or qPCR analysis indicated (up,red; down, blue). Genes and pathways that are more strongly upregulated are indicated with red arrows and a pink background, respectively. A dotted arrow indicates that there is more than one enzymatic step involved in the pathway.

Figure 8.

A Model of the Homeostatic Feedback Loop Regulating IAA and Cytokinin Concentrations in the Arabidopsis Root Apex. Cytokinin (CK) acts as a positive regulator of auxin (IAA) biosynthesis in the root apex, possibly by ATR1 coinduction of the CYP79B2/B3 and NIT3 genes. On the other hand, auxin can act as a repressor of cytokinin biosynthesis via downregulation of specific IPT genes. Both auxin and cytokinin signaling is important for these interactions, and our data suggest that specific auxin/IAA genes (IAA17/AXR3 and IAA3/SHY2) and cytokinin signaling components (AHK4 and ARRs) are involved. Changes in auxin transport (PIN efflux carriers) and in the degradation of auxin (GH3s) and cytokinin (CKXs) may also play important roles in the modulation of the response.