PtHAK5, a candidate for mediating high-affinity K<sup>+</sup> uptake in the halophytic grass, <i>Puccinellia tenuiflora</i>

doi:10.15302/J-FASE-2018200

Front. Agr. Sci. Eng.

2018, Vol. 5

Issue (1) : 108-117 https://doi.org/10.15302/J-FASE-2018200

RESEARCH ARTICLE

PtHAK5, a candidate for mediating high-affinity K⁺ uptake in the halophytic grass, Puccinellia tenuiflora

Haili YANG, Weidan ZHANG, Weiwei CHAI, Wenying WANG, Li GAO, Jing ZHANG, Yongping WANG, Suo-Min WANG(

)

State Key Laboratory of Grassland Agro-ecosystems/College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China

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Abstract

Puccinellia tenuiflora is a typical salt-exclu-ding halophytic grass with strong salt-tolerance, which enhances tolerance by restricting Na⁺ influx as well as having a strong selectivity for K⁺ over Na⁺. The HAK5 K⁺ transporters generally modulate effective K⁺ acquisition in plants, especially under low K⁺ condition. In this study, PtHAK5 from P. tenuiflora was isolated by RT-PCR and characterized using yeast complementation. The results showed PtHAK5 consisted of 784 amino acids and shared over 80% homology with the identified high-affinity K⁺ transporter HAK5 from other higher plants. The expression of PtHAK5 rescued the K⁺-uptake-defective phenotype of yeast strain CY162. In conclusion, PtHAK5 is a candidate for mediating high-affinity K⁺ uptake under low K⁺ conditions.

Keywords K⁺ uptake PtHAK5 Puccinellia tenuiflora yeast complementation

Corresponding Author(s): Suo-Min WANG

Just Accepted Date: 27 December 2017 Online First Date: 08 February 2018 Issue Date: 21 March 2018

Cite this article:

Haili YANG,Weidan ZHANG,Weiwei CHAI, et al. PtHAK5, a candidate for mediating high-affinity K⁺ uptake in the halophytic grass, Puccinellia tenuiflora[J]. Front. Agr. Sci. Eng. , 2018, 5(1): 108-117.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2018200
https://academic.hep.com.cn/fase/EN/Y2018/V5/I1/108

Tab.1 Primers used in this work

Fig.1 The nucleic acid sequence and encoded amino acid sequence of PtHAK5 in Puccinellia tenuiflora. The full-length PtHAK5 sequence was used for BLAST search on the NCBI platform with sequences acquired from GenBank. The 2880 bp contained a 5′-untranslated region of 111 bp nucleotides, a predicted ORF of 2355 bp nucleotides encoding 784 amino acids and a 3′-untranslated region with 414 bp nucleotides.

Fig.2 Transmembrane domain prediction for PtHAK5 from Puccinellia tenuiflora. There are 11 highly conservative transmembrane domains predicted by TMHMM online biology platform. The abscissa indicates the amino acid site and contains a total of 784 amino acids.

Fig.3 Sequence alignment of PtHAK5 and HAK5-like proteins from higher plants. Sources of HAK5 and their GenBank accession numbers were as follows: OsHAK5 (Oryza sativa, BAD87321.1), ZmHAK5 (Zea mays, AHH35045.1), SlHAK5 (Solanum lycopersicum, ABF22603.1), AtHAK5 (Arabidopsis thaliana, AT4G13420). The sequences were aligned with DNAMAN 6.0 software. The 11 putative transmembrane domains (TM1–TM11) are underlined.

Fig.4 Phylogenetic tree analysis of PtHAK5 amino acid sequences. AtHAK5 from Arabidopsis thaliana, SlHAK5 from Solanum lycopersicum, OsHAK5 from Oryza sativa, ZmHAK5 from Zea mays.

Fig.5 Phylogenetic tree analysis of PtHAK5 and HAK/KUP/KT family members from Arabidopsis thaliana and Oryza sativa (rice). The phylogenetic tree was generated by MEGA 6.0 software using the maximum-likelihood method. Sources of K⁺ transporter amino acid sequence and their GenBank accession numbers are as follows: in A. thaliana, AtKUP1 (AT2G30070), AtKUP2 (AT2G40540), AtKUP3 (AT3G02050), AtKUP4 (AT4G23640), AtKUP5 (AT4G33530), AtKUP6 (AT1G70300), AtKUP7 (AT5G09400), AtKUP8 (AT5G14880), AtKUP9 (AT4G19960), AtKUP10 (AT1G31120), AtKUP11 (AT2G35060), AtKUP12 (AT1G60160), AtHAK5 (AT4G13420); and in O. sativa, OsHAK1 (NC_008397), OsHAK2 (BAF07234), OsHAK3 (AJ427974), OsHAK4 (AK071698), OsHAK5 (AP003272), OsHAK6 (NP_001045298), OsHAK7 (AJ427976), OsHAK8 (Q8VXB5), OsHAK9 (AJ427978), OsHAK10 (AJ427979), OsHAK11 (AJ427980), OsHAK12 (AJ427981), OsHAK13 (BAF20244), OsHAK14 (AJ427983), OsHAK15 (AJ427984), OsHAK16 (BAF12444), OsHAK17 (AJ427975), OsHAK18 (NP_001063938), OsHAK19 (BAF08880), OsHAK20 (BAF08882), OsHAK21 (BAH92235), OsHAK22 (EEC81370), OsHAK23 (BAF24960), OsHAK24 (BAF19271), OsHAK25 (EEC73938), OsHAK26 (BAF24119), OsHAK27 (BAF12443).

Fig.6 Complementation of the K⁺ uptake-deficient Saccharomyces cerevisiae mutant strain CY162 by expressing PtHAK5, AtAKT1, AtHKT1;1 and empty vector p416 GPD. Each yeast cell was plated on minimal AP medium containing four concentrations of K⁺ (0.1, 2, 10 and 100 mmol·L⁻¹) at 10-fold serial dilutions from OD₆₀₀ = 0.6 to 0.6 × 10⁻³. AP medium with 100 mmol·L⁻¹ K⁺ was used as a control, and CY162 expressing AtAKT1, AtHKT1;1 and p416 GPD were used as positive and negative controls, respectively. The triangles indicate that the cell concentration was diluted 10-fold from left to right.

Fig.7 Na⁺-induced growth-inhibition of Saccharomyces cerevisiae mutant strain G19 expressing PtHAK5, AtAKT1, At HKT1;1 and empty vector p416 G19. Yeast cells were plated on minimal AP medium containing 1 mmol·L⁻¹ K⁺ and various concentrations of Na⁺ (0, 25, 50 and 75 mmol·L⁻¹) at 10-fold serial dilutions from OD₆₀₀ = 0.6 to 0.6 × 10⁻³. AP medium without Na⁺ was used as a control, and G19 expressing AtHKT1;1, AtAKT1 and p416 GPD were used as positive and negative controls, respectively. The triangles indicate that the cell concentration was diluted 10-fold from left to right.

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