Cytokinin-regulated targets of Cytokinin Response Factor 6 are involved in potassium transport

Abstract

Cytokinin (CK) is a plant hormone crucial to plant development and growth. Cytokinin Response Factor 6 (CRF6) is a CK-induced transcription factor that is part of the CK signaling cascade. While the role of CRF6 has been examined in oxidative stress response, there has been surprisingly little investigation of CRF6 in the context of CK signaling, including identifying CK-regulated targets of CRF6. Here, we conduct a transcriptomic study of Arabidopsis examining the CRF6 mutant (crf6) in the presence and absence of CK, revealing 163 downstream CRF6-dependent CK-regulated differentially expressed genes (DEGs). 15.3% of these DEGS were found as overlapping with larger number of standardly identified CK-regulated DEGs, suggesting that CRF6 is involved in regulating a subset of downstream CK responses through these gene targets. The general transcriptional regulation of CRF6-dependent CK-regulated DEGs indicates that CRF6 may function as a negative regulator of CK response. We investigated one subset of CRF6 CK-dependent targets (SKOR, HAK5, and NRT1. 5) involved in an underexamined functional role of CK response: the uptake and transportation of potassium. To determine how CK and CRF6 are involved in potassium acquisition and distribution, ionomic and physiological experiments were conducted on plants grown in media with sufficient and deficient potassium concentrations and in the presence and absence of CK. In order to investigate how CK alone affects potassium transport, similar experiments were performed on skor, hak5, and nrt1.5 mutant lines of these CRF6-dependent CK-regulated targets. These findings indicate novel connections between CK and potassium transport, which appear to be regulated in a CRF6-dependent manner.

Keywords: CRF6; Cytokinin; Cytokinin Response Factor; potassium; transport.

FIGURE 1

(a) Heat map depicting the fold change in expression of genes in WT and crf6 mutant plants when treated and untreated with CK and separated into induced and repressed categories. (b) Venn diagrams illustrating the crossover between the genes between genes that were induced (addition of CK caused 1.5 times greater transcription of the gene) and were repressed (addition of CK caused transcription of the gene to be reduced to half or lower) and the genes that were differentially altered in transcription in the crf6 mutant plants

FIGURE 2

FV/FM measurements were taken to gauge the overall health of the plants after 10 days. Asterisks on comparison bars indicate significance level; *p ≤ .05, **p ≤ .005, ***p ≤ .0005. Comparison bars between treatments within a line are indicated closer to the x axis with each end indicating the compared data. The asterisks above the bars indicate the significance level of the data as compared to the same treatment in WT

FIGURE 3

Root growth, in millimeters, 10 days after transfer to treatment and control plates. Asterisks indicate significance level; *p ≤ .05, **p ≤ .005, ***p ≤ .0005 when an ANOVA Ad Hoc test was used. Asterisks above the error bars indicate significance of the length of the roots of that line as compared to that of the wildtype grown in the same treatment. The asterisks on comparison bars closer to the x axis indicate significance of differences within lines between different treatments. All plant roots grown on standard potassium media were significantly different from those grown on treatment plates (p < .0005)

FIGURE 4

Rosette diameter, in millimeters, 10 days after transfer to treatment and control plates. Asterisks indicate significance level; *p ≤ .05, **p ≤ .005, ***p ≤ .0005, when an ANOVA Ad Hoc test was used. Asterisks above the error bars indicate significance of the length of the roots of that line as compared to that of the wildtype grown in the same treatment. The asterisks on comparison bars closer to the x axis indicate significance of differences within lines between different treatments. All plant rosettes grown on standard potassium media were significantly wider than those grown on treatment plates (p < .0005)

FIGURE 5

Potassium levels as measured by ICP‐OES collected from A. shoots and B. roots. Asterisks indicate significance level; *p ≤ .05, **p ≤ .005, ***p ≤ .0005 when an Ad Hoc test was used to compare that line, treatment, and tissue type to the WT of the same treatment and tissue type