The expression of HAK-type K(+) transporters is regulated in response to salinity stress in common ice plant

Abstract

Four transcripts homologous to K(+) transporters of the HAK/KT/KUP family have been characterized from the common ice plant (Mesembryanthemum crystallinum). We report tissue-specific expression of McHAK1 and McHAK4 transcripts abundant in roots, leaves, and stems. McHAK2 was predominantly present in stems and McHAK3 in root tissues. By in situ hybridizations, the McHAKs showed signals in the leaf vascular bundles, mesophyll, and epidermal cells as well as in epidermal bladder cells. In mature roots, transcripts were mainly localized to the vasculature, and in differentiated root tips, the strongest signals were obtained from the epidermis. Expression of McHAK1, McHAK2, and McHAK4 complemented a yeast mutant defective in low- and high-affinity K(+) uptake. Growth of the yeast mutant was restored at low-millimolar K(+) concentrations and was inhibited by Rb(+) and Cs(+) but was not affected by Na(+). Transcript levels of McHAK1 and McHAK4 increased by K(+) starvation and by salt stress of 400 mM NaCl in leaves and roots. Expression of McHAK2 and McHAK3 was stimulated in leaves and was transiently induced in roots in response to high salinity with prestress transcript levels restored in salt-adapted plants. We discuss possible roles for such transporters in ion homeostasis at high salinity.

Figure 1

Characterization of the McHAK transcripts from common ice plant. A, Alignment of the predicted amino acid sequences of McHAK1 (AF367864), McHAK2 (AF367865), McHAK3 (AF367866), McHAK4, and AtKUP3 (AAF19432). B, Hydropathy plot of McHAK1 and McHAK2 (Kyte and Doolittle, 1982). Putative transmembrane domains are indicated (I–XII). C, Phylogenetic tree of members of the HAK subfamilies (ClustalX, only full-length cDNAs included).(Figure continues on facing page.)

Figure 1

Characterization of the McHAK transcripts from common ice plant. A, Alignment of the predicted amino acid sequences of McHAK1 (AF367864), McHAK2 (AF367865), McHAK3 (AF367866), McHAK4, and AtKUP3 (AAF19432). B, Hydropathy plot of McHAK1 and McHAK2 (Kyte and Doolittle, 1982). Putative transmembrane domains are indicated (I–XII). C, Phylogenetic tree of members of the HAK subfamilies (ClustalX, only full-length cDNAs included).(Figure continues on facing page.)

Figure 2

Tissue-specific expression of McHAK genes. 3′-UTR regions of McHAK1, McHAK2, and McHAK3 and the EST sequence AI547371 of McHAK4 were hybridized to total RNA extracted from unstressed plants (except flower and seed pot). Tissues were specified as root (RT), stem (ST), leaf (LF), flower (FL), and seed pot (SP). Actin was used as a loading control. McHAK1, 2.8 kb (lower band, 2.7 kb); McHAK2, 2.9 kb; McHAK3, 2.8 kb (lower band, 2.7 kb); and McHAK4, 2.9 kb.

Figure 3

Northern-type hybridization of the expression of McHAK during NaCl stress. Six-week-old plants were stressed with 400 mm NaCl for the time indicated. Total RNA from different tissues were probed with either the 3′-UTR region of McHAK1, McHAK2, and McHAK3, respectively, or the EST fragment of McHAK4 (AI547371). Apparent M_rs are as in Figure 2.

Figure 4

Effects of NaCl treatment and K⁺ starvation on the expression of McHAK1 and McHAK4 in common ice plant. Transcript amounts were quantified by RT-PCR using template cDNA obtained from leaves (Lc) and roots (Rc) from nonstressed control plants and from leaves (Ls) and roots (Rs) from plants treated with 400 mm NaCl for 72 h. Plants were adapted to nutrition solution without K⁺ added (A) and to nutrition solution containing 3 mm K⁺ (B). C, Comparison of transcript amounts from plants adapted to 0 mm K⁺ and 3 mm K⁺. For PCR, gene-specific primers as outlined in “Materials and Methods” were used. Transcripts were amplified in the linear range of amplification with 26 cycles for both transcripts (A), 28 cycles for McHAK1 and 27 cycles for McHAK4 (B), and 27 cycles for both transcripts (C). Actin was amplified as a loading control.

Figure 5

Cell specificity of McHAK expression in common ice plant. A through G, In situ hybridization of McHAK4 to tissue cross sections from common ice plant adapted to nutrition solution without K⁺ added. A, Leaf, control; antisense. B, Leaf, 72 h 400 mm NaCl, antisense. C, Root tip, control; antisense. D, Root tip, 72 h, 400 mm NaCl; antisense. E, Root, 72 h, 400 mm NaCl; antisense. F, Leaf, control; sense. G, Root, 72 h, 400 mm NaCl; sense. H and J, In situ hybridization of McHAK1 and McHAK3 to tissue cross sections from common ice plant adapted to nutrition solution with 3 mm K⁺ added. H, Root tip, 12 h, 400 mm NaCl; McHAK1, antisense. I, Root tip, 12 h, 400 mm NaCl; McHAK3, antisense. J, Root tip, 12 h, 400 mm NaCl; McHAK3, sense. mp, Mesophyll; ph, phloem; xy, xylem; eb, epidermal bladder cell; ct, cortex; vb, vascular bundle.

Figure 6

Accumulation of K⁺ and Na⁺ in common ice plant plants grown hydroponically in nutrition solution without K⁺ added or in nutrition solution containing 3 mm K⁺. Plants were grown as nonstressed control plants or treated with 400 mm NaCl for 72 h (n = 6).

Figure 7

Complementation of a yeast TRK1/TRK2 mutant with McHAK1, McHAK2, and McHAK4. The yeast strain CY162 (Ko and Gaber, 1991) was transformed with either the empty vector pYES2 or with McHAK1, McHAK2, and McHAK4, respectively, cloned into the yeast expression vector pYES2. Positive transformants were grown on Gal-SC containing 3 mm K⁺ (A) or 7 mm K⁺ (B), or Gal-SC supplemented with 150 mm RbCl, 150 mm NaCl, or 15 mm CsCl.