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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (8): 2105-2121.doi: 10.3724/SP.J.1006.2023.24194

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Genome-wide identification of the HAK/KUP/KT potassium transporter family in foxtail millet and its response to K+ deficiency and high salt stress

DAI Shu-Tao1(), ZHU Can-Can1, MA Xiao-Qian2, QIN Na1, SONG Ying-Hui1, WEI Xin1, WANG Chun-Yi1, LI Jun-Xia1,*()   

  1. 1 Cereal Crops Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
    2 College of Agriculture, Henan University of Science and Technology, Luoyang 471023, Henan, China
  • Received:2022-08-24 Accepted:2023-02-10 Online:2023-08-12 Published:2023-02-21
  • Contact: LI Jun-Xia E-mail:daist82@163.com;lijunxia@126.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-06-14.5-B24);Funding of Joint Research on Agricultural Varieties Improvement of Henan Province(2022010401);Central Guidance on Science and Technology Development of Henan(Z20221341070);Innovation Team and Independent innovation Project of Henan Academy of Agricultural Sciences(TD2022033);Innovation Team and Independent innovation Project of Henan Academy of Agricultural Sciences(2022ZC07)

Abstract:

KT/HAK/KUP (HAK) family is the most abundant potassium (K+) transporter family in plants, which plays important roles in plant growth and environmental adaptation. Foxtail millet (Setaria italic L. Beauv) is a model plant for studies on stress resistance mechanisms. However, the HAK family has not yet been well characterized in foxtail millet. In this study, 29 Setaria italic HAK genes (SiHAKs) were identified based on genome-wide sequence information, and the basic characteristics, protein structure, chromosome location, gene replication, expression pattern, and responses to stress were comprehensively analyzed. The results showed as followes: (1) SiHAKs were divided into five phylogenetic clusters (Cluster I-Cluster V), containing 11, 9, 3, 3 and 3 members, respectively. Gene structure and conserved motif analyses indicated that SiHAKs was high conserved, and the conservatisms were as follows: Cluster III = Cluster V > Cluster II > Cluster I > Cluster IV. (2) Tandem replication was the main contribution to the amplification of SiHAKs. 15 SiHAKs were located in tandem replication. (3) 171 transcription factors, including a large number of members of ERF, NAC, MYB and WRKY families, may bind to the promoter sequences of SiHAKs, which may confer SiHAKs diverse response mechanisms to abiotic stress. (4) SiHAKs were divided into three groups (Group I, Group II, and Group III) by gene expression Cluster Ing. The relative expression patterns of most SiHAKs were consistent in the two varieties (Zhanggu and Yugu 1). The relative expression levels of SiHAKs from the five clusters were generally as follows: Cluster III > Cluster V > Cluster II > Cluster I> Cluster IV. (5) Eleven SiHAKs with high expression levels in root were selected to detect the responses to low K+ and high salt stress. Under K+ deficiency treatment, eight SiHAKs were markedly upregulated in the expression levels, one SiHAK was significantly decreased, and two SiHAKs had no obvious changes. Under high salt stress, Three SiHAKs were significantly increased in the expression levels, two SiHAKs were significantly decreased, and the remaining six SiHAKs had no obvious changes. Remarkably, SiHAK15 had the strongest response to K+ deficiency and high salt stress, its expression level was 151 times and 22 times of the control, respectively. (6) The differences of gene expression profiles reflected the function differences among SiHAKs from different clusters. SiHAKs from Cluster I were mainly expressed in root, implying their important roles in K+ absorption in root. SiHAKs from Cluster II generally had no tissue-specific expression characteristics, and might be involved in many biological processes, such as K+ absorption and transport, plant growth and development. SiHAKs from Cluster III were upregulated by K+ deficiency and high salt stress, inferring their potential roles in maintaining K+/Na+ balance and resisting salt stress. SiHAKs from Cluster IV were almost undetectable in gene expression in the tested tissues. SiHAKs from Cluster V had different responses to K+ deficiency and high salt stresses. Some of them were upregulated in gene expression, while others were inhibited, thus indicating that the members from Cluster V were functionally differentiated. This results not only provide the foundation for further functional studies of SiHAKs, but also provide the valuable guidance for the study of K+ efficient utilization and salt tolerance mechanism in plants.

Key words: foxtail millet, potassium transporter, KT/HAK/KUP family, salt stress, the relative expression analysis

Table S1

Sequence information for primers used for SiHAK genes expression analysis"

基因
Gene
引物名称
Primer name
序列
Sequence (5°-3°)
SiHAK2 SiHAK2-F CGGCGTCCTCTCCTTCGTCT
SiHAK2-R CCCGTGGCCTTCTCTTCCTC
SiHAK3 SiHAK3-F TCTTTATTCACTGCTTTGCCG
SiHAK3-R TTCTGAGTTGGTGCCCCTCTA
SiHAK4 SiHAK4-F AAAAGTTAGCCTGCTTCCGAA
SiHAK4-R GCCGTCACCAATCACCATAGA
SiHAK6 SiHAK6-F AGGTTTCTCTTTCGTCGGGTC
SiHAK6-R TTCAAATGCTTGGGGGTTCTC
SiHAK7 SiHAK7-F ACGTGACCCTGACATAAGCAAA
SiHAK7-R TCAAGAGCCAGAGCACAACAAT
SiHAK10 SiHAK10-F CACGGAGAGCAACGAGGAGA
SiHAK10-R GCAGAGGAGGGAGTAGAGCG
SiHAK15 SiHAK15-F GAATCCCCAAATGTAAGCCG
SiHAK15-R CATGATGTATGCCACCCCAG
SiHAK16 SiHAK16-F CTCTTGCACTGGGTTGTTTTCC
SiHAK16-R GCATTTCCTATCTGGCTTTGGT
SiHAK18 SiHAK18-F ACCTTCGCTCCTGTCATCTCG
SiHAK18-R CCATCCTTCCCATTCCTTTTG
SiHAK23 SiHAK23-F CCCCCACATCTTCTCCCACT
SiHAK23-R CTTGTACCCGTACCTCGCCA
SiHAK24 SiHAK24-F GCATCCCACCCATATTCCCT
SiHAK24-R TACCCATACCGTGCCACACA
Si-actin-2 Si-actin-2-F ATCCAGCCCCTTGTATGTGA
Si-actin-2-R ACGCCCAACAATACTTGGAA

Table 1

Basic information of SiHAK genes and proteins in foxtail millet"

基因名称
Gene name
基因序列号
Gene ID
染色体位置
Chromosome position
基因组大小
Genome size (bp)
编码序列长度
Coding sequence
length (bp)
氨基酸
Amino
acid (aa)
分子量
Molecular
weight (kD)
等电点 Isoelectric
point (pI)
亚细胞定位
Subcellular location
SiHAK1 Seita.1G175000 Chr.1:25211256..25214882 forward 3627 2193 731 80.02 8.26 质膜 Plasma membrane
SiHAK2 Seita.1G307200 Chr.1:37040523..37045883 forward 5361 2322 774 86.24 7.85 质膜 Plasma membrane
SiHAK3 Seita.2G129100 Chr.2:14902256..14908174 reverse 5919 2364 788 88.61 8.49 质膜 Plasma membrane
SiHAK4 Seita.2G192700 Chr.2:28886553..28891290 reverse 4738 2568 856 94.46 7.47 质膜 Plasma membrane
SiHAK5 Seita.2G224400 Chr.2:32661560..32666230 forward 4671 2133 711 78.65 9.21 质膜 Plasma membrane
SiHAK6 Seita.2G327100 Chr.2:41466190..41473814 reverse 7625 2559 853 94.42 5.94 质膜 Plasma membrane
SiHAK7 Seita.2G427100 Chr.2:47923110..47928818 forward 5709 2352 784 87.76 8.86 质膜 Plasma membrane
SiHAK8 Seita.2G432200 Chr.2:48265283..48269697 reverse 4415 2364 788 87.21 7.61 质膜 Plasma membrane
SiHAK9 Seita.4G119700 Chr.4:12129620..12135065 reverse 5446 2331 777 85.78 8.25 质膜 Plasma membrane
SiHAK10 Seita.4G209800 Chr.4:32743375..32748151 forward 4777 2448 816 90.27 8.47 质膜 Plasma membrane
SiHAK11 Seita.4G266200 Chr.4:38390011..38398958 reverse 8948 2322 774 85.41 8.22 质膜 Plasma membrane
SiHAK12 Seita.5G168000 Chr.5:16861878..16864997 reverse 3120 2442 814 89.65 8.60 质膜 Plasma membrane
SiHAK13 Seita.5G439500 Chr.5:45554118..45559684 forward 5567 2325 775 86.47 8.52 质膜 Plasma membrane
SiHAK14 Seita.5G441200 Chr.5:45651197..45655632 reverse 4436 2205 735 80.50 8.50 质膜 Plasma membrane
SiHAK15 Seita.5G444100 Chr.5:45784733..45789729 forward 4997 2361 787 88.43 9.07 质膜 Plasma membrane
SiHAK16 Seita.6G027600 Chr.6:1995986..2001010 reverse 5025 2367 789 88.43 8.34 质膜 Plasma membrane
SiHAK17 Seita.6G205800 Chr.6:32522339..32526598 forward 4260 2220 740 82.47 8.84 质膜 Plasma membrane
SiHAK18 Seita.7G082300 Chr.7:18653291..18658866 forward 5576 2322 774 86.58 8.64 质膜 Plasma membrane
SiHAK19 Seita.7G082600 Chr.7:18675471..18680567 forward 5097 2373 791 88.54 8.82 质膜 Plasma membrane
SiHAK20 Seita.7G082700 Chr.7:18682739..18686823 forward 4085 2331 777 87.11 8.92 质膜 Plasma membrane
SiHAK21 Seita.7G082800 Chr.7:18693088..18697351 forward 4264 2286 762 85.03 6.99 质膜 Plasma membrane
SiHAK22 Seita.7G232600 Chr.7:29611687..29616556 reverse 4870 2550 850 94.73 6.15 质膜 Plasma membrane
SiHAK23 Seita.7G235500 Chr.7:29787315..29792347 reverse 5033 2412 804 89.58 8.34 质膜 Plasma membrane
SiHAK24 Seita.9G182300 Chr.9:12585631..12590025 forward 4395 2415 805 91.00 8.64 质膜 Plasma membrane
SiHAK25 Seita.9G182400 Chr.9:12603097..12608101 forward 5005 2433 811 91.93 9.02 质膜 Plasma membrane
SiHAK26 Seita.9G182500 Chr.9:12637593..12642369 forward 4777 2403 801 90.86 8.84 质膜 Plasma membrane
SiHAK27 Seita.9G182600 Chr.9:12646706..12651914 forward 5209 2415 805 90.42 8.82 质膜 Plasma membrane
SiHAK28 Seita.9G412000 Chr.9:46973981..46979758 reverse 5778 2415 805 89.58 6.82 质膜 Plasma membrane
SiHAK29 Seita.J004400 scaffold_11:162273..168095 reverse 5823 2682 894 98.77 8.68 质膜 Plasma membrane

Fig. 1

Chromosomal distribution of SiHAKs in foxtail millet The red font represents that the gene is located on the forward strand of the chromosome; the blue font represents that the gene is located on the reverse strand of the chromosome."

Fig. 2

Transmembrane structure of SiHAK proteins The red peaks represent predicted transmembrane helices."

Fig. 3

Phylogenetic tree of HAK proteins in foxtail millet, Oryza sativa, and Arabidopsis"

Fig. 4

Gene structure and conserved motifs of SiHAKs"

Fig. 5

Transcription factors (TFs) in the promoter regions of 29 SiHAKs A: the TF families that might bind to the promoter regions of 29 SiHAKs. YABBY: YABBY domain. WOX: WUSCHEL-related homeobox. TALE: three-amino-acid-loop-extension. GRAS: GAI, RGA, and SCR. CAMTA: calmodulin-binding transcription activator. C3H: p-coumarate 3-hydroxylase. ARR-B: type-B Arabidopsis response regulator. EIL: Ethylene-Insensitive-like. E2F/DP: E2 factor/dimerization partner. CPP: cystein-rich polycomb-like protein. BBR-BPC: barley B-recombinant/basic penta cysteine. B3: B3 domain. LBD: the lateral organ boundaries domain. HSF: heat shock factor. AP2: APETALA2. TCP: TEOSINTE BRANCHED1/CYCLOIDEA/proliferation cell factor. SBP: the squamosa promoter binding protein. MYB_related: myeloblastosis-related. MIKC_MADS: MIKC-type MADS (MCMI, AGAMOUS, DEFICIENS, and SRF4). bHLH: the basic helix-loop-helix. Trihelix: helix-loop-helix-loop-helix. GATA: GATA binding protein. HD-ZIP: homeodomain-leucine-zipper protein. G2-like: Golden 2-like. Dof: DNA binding with one finger. bZIP: basic leucine zipper. C2H2: Cys2/His2-type zinc finger protein. WRKY: WRKYGQK domain zinc finger motif. MYB: myeloblastosis. NAC: NAM, ATAF1/2 and CUC2. ERF: the ethylene response factor. B: the number of TFs in the promoter regions of different SiHAKs. The abscissa represents the number of TFs."

Fig. 6

Transposon components of the promoter sequences of SiHAKs"

Fig. 7

Relative expression pattern of 29 SiHAKs genes A: the relative expression pattern of the SiHAKs in four tissues (root, stem, leaf, and spike) in the cultivar Zhanggu. B: the relative expression profiles of the SiHAKs in 20 samples from different tissues or under different treatments in the cultivar Yugu 1. a: the etiolated seedling grown in the dark for 5 days; b: the germinated shoot in the dark for 6 days; c: the first leaf on a 2-week-old plant; d: the second leaf on a 2-week-old plant; e: the third leaf on a 2-week-old plant; f: the four leaf on a 2-week-old plant; g: the five leaf on a 2-week-old plant; h: the six leaf on a 2-week-old plant; i: the panicle at stage 1; j: the panicle at stage 2; k: the Young root; l: root under ammonium growth condition; m: root under drought condition; n: root under nitrate condition; o: root grown with urea as nitrogen source; p: young stem grown for a week; q: seedling grown under blue light; r: seedling grown under dark conditions; s: seedling grown under far-red light; t: seedling grown under red light."

Fig. 8

Relative expression levels of 11 SiHAKs in root under low potassium and high salt conditions Error bar represents the standard deviation of three independent experiments. * represents significant difference at 0.01 < P < 0.05; ** represents extremely significant difference at P < 0.01."

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