Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake

Abstract

Potassium (K(+)) is the most abundant cation in plants and is required for plant growth. To ensure an adequate supply of K(+), plants have multiple mechanisms for uptake and translocation. However, relatively little is known about the physiological role of proteins encoded by a family of 13 genes, named AtKT/KUP, that are involved in K(+) transport and translocation. To begin to understand where and under what conditions these transporters function, we used reverse transcription-PCR to determine the spatial and temporal expression patterns of each AtKT/KUP gene across a range of organs and tested whether selected AtKT/KUP cDNAs function as K(+) transporters in Escherichia coli. Many AtKT/KUPs were expressed in roots, leaves, siliques, and flowers of plants grown under K(+)-sufficient conditions (1.75 mm KCl) in hydroponic culture. AtHAK5 was the only gene in this family that was up-regulated upon K(+) deprivation and rapidly down-regulated with resupply of K(+). Ten AtKT/KUPs were expressed in root hairs, but only five were expressed in root tip cells. This suggests an important role for root hairs in K(+) uptake. The growth and rubidium (Rb(+)) uptake of two root hair mutants, trh1-1 (tiny root hairs) and rhd6 (root hair defective), were studied to determine the contribution of root hairs to whole-plant K(+) status. Whole-plant biomass decreased in the root hair mutants only when K(+) concentrations were low; Rb(+) (used as a tracer for K(+)) uptake rates were lower in the mutants at all Rb(+) concentrations. Seven genes encoding AtKUP transporters were expressed in E. coli (AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5), and their K(+) transport function was demonstrated.

Figure 1.

Gene structure of the Arabidopsis AtKT/KUPs. Prediction programs from TAIR (http://www.Arabidopsis.org) and TIGR (http://www.tigr.org) were used. Predictions were compared with full-length cDNAs except for the AtKT/KUP8, AtKT/KUP9, and AtKT/KUP12 genes for which full-length cDNA data were not available. Gray boxes, UTRs; black boxes, exons; solid lines, introns. Scale shown in base pairs.

Figure 2.

Relative expression levels of individual AtKT/KUP genes as determined by real-time RT-PCR. Plants were grown under control conditions (1.75 mm KCl) and harvested at flowering stage. Transcript abundance was quantified in total RNA from roots (R), older leaves (OL), younger leaves (YL), developing siliques (DS), and flower (F). Expression levels were calculated relative to ubiquitin. The results shown represent real-time RT-PCR analysis of the cDNA synthesized from one experiment. The mean ± se of three reactions are shown as error bars, and the numerical values for SE are shown above the columns where expression was detected. The same results were obtained on two other independent sets of cDNA synthesized for different sets of plants. If relative expression levels were below 0.010, transcripts were considered to be not detectable (ND).

Figure 3.

Temporal expression of AtHAK5 in roots (A) and older leaves (B) of flowering plants grown under hydroponic conditions for 6 weeks. The growth conditions included: K⁺ sufficient (1.75 mm) as a control (lane 1), K⁺ deprivation for 1 (lane 2) and 6 (lane 3) d, K⁺ deprived for 6 d then transferred to K⁺-sufficient conditions for 6 h (lane 4), and 30 h (lane 5). Plants were exposed to excess K⁺ (lanes 6 and 7) and 50 mm Na⁺ (lane 8) for 6 d. Real-time RT-PCR results are shown for roots (A) and older leaves (B). Shown above the real-time PCR data are the end point PCR products from each reaction after agarose gel electrophoresis. Values are mean ± se (n = 3) and representative of at least two independent experiments.

Figure 4.

Root and leaf expression patterns of AtKT/KUP genes in 1-week-old seedlings grown on vertically oriented agar plates and 6-week-old flowering plants grown in hydroponic solution, both containing 1.75 mm KCl. The diagram shows the regions of the root sampled (A). AtKC1 and SKOR were amplified to determine whether root hair cells were contaminated by other cell types (B). Results of RT-PCR on root hairs (RH), root tip (RT), whole roots (WR), and leaf (L) in 1-week-old and whole roots from 6-week-old plants. Forty cycles of PCR were used (C).

Figure 5.

Light microscope images showing the difference between root hairs of the Wassilewskija (Ws; wild type), trh1-1, and rhd6. Roots were grown in hydroponic culture for 6 weeks (large panels) and on vertically oriented agar plates for 1 week (small panels). A, Whole-plant biomass (B) and K⁺ content in roots (C) of Ws, trh1-1 (tiny root hairs), and rhd6 (root hair defective). Plants were grown with sufficient K⁺ (1.75 mm KCl) and under limiting conditions (100 μm KCl) for 21 d. Values are mean ± se. The numbers above the columns are the calculated percent of biomass or K⁺ content as compared with control plants grown with sufficient K⁺. Scale is same for all photos. Bars = 1 mm.

Figure 6.

⁸⁶Rb⁺ uptake rate at 20, 100, and 1,000 μm RbCl of Ws (wild type), trh1-1 (tiny root hairs), and rhd6 (root hair defective) exposed to K⁺ deprivation for 2 d before uptake was measured (n = 6 ± se).

Figure 7.

The complementation of the E. coli TK2420 strain by several AtKT/KUP transporters. The E. coli TK2420 is defective in three K⁺ transporters and was transformed with the empty pPAB404 plasmid or with the plasmid containing AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5. The strains were dropped on medium containing: A, 0.5 mm KCl with 0.5 mm isoprophyl-1-thio-β-d-galactropyranoside (IPTG); B) and 0.5 mm KCl without IPTG; and C, 30 mm KCl without IPTG.