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Front Plant Sci
2019 Jan 01;10:1092. doi: 10.3389/fpls.2019.01092.
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Identification of Lysine Histidine Transporter 2 as an 1-Aminocyclopropane Carboxylic Acid Transporter in Arabidopsis thaliana by Transgenic Complementation Approach.
Choi J
,
Eom S
,
Shin K
,
Lee RA
,
Choi S
,
Lee JH
,
Lee S
,
Soh MS
.
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1-Aminocyclopropane-1-carboxylic acid (ACC), a biosynthetic precursor of ethylene, has long been proposed to act as a mobile messenger in higher plants. However, little is known about the transport system of ACC. Recently, our genetic characterization of an ACC-resistant mutant with normal ethylene sensitivity revealed that lysine histidine transporter 1 (LHT1) functions as a transporter of ACC. As amino acid transporters might have broad substrate specificity, we hypothesized that other amino acid transporters including LHT1 paralogs might have the ACC-transporter activity. Here, we took a gain-of-function approach by transgenic complementation of lht1 mutant with a selected set of amino acid transporters. When we introduced transgene into the lht1 mutant, the transgenic expression of LHT2, but not of LHT3 or amino acid permease 5 (AAP5), restored the ACC resistance phenotype of the lht1 mutant. The result provides genetic evidence that some, if not all, amino acid transporters in Arabidopsis can function as ACC transporters. In support, when expressed in Xenopus laevis oocytes, both LHT1 and LHT2 exhibited ACC-transporting activity, inducing inward current upon addition of ACC. Interestingly, the transgenic expression of LHT2, but not of LHT3 or AAP5, could also suppress the early senescence phenotypes of the lht1 mutant. Taking together, we propose that plants have evolved a multitude of ACC transporters based on amino acid transporters, which would contribute to the differential distribution of ACC under various spatiotemporal contexts.
Figure 1. Effect of supplemented amino acids on ACC-induced inhibition of hypocotyl and root growth. (A) Hypocotyl (left) and root length (right). The wild-type seedlings were grown under dark for 4 days on MS-sucrose media containing 1 µM of ACC supplemented with each of 20 types of 100 µM amino acid. Values are mean ± SD (n = 15). The significant difference of the length between seedlings grown on the media containing ACC only and ACC with amino acid is indicated with asterisks (t test, *p < 0.01). (B) Representative wild-type seedlings grown on MS-sucrose media containing either 1 or 10 µM of ACC supplemented without (upper) or with 100 µM of methionine (Met) (lower). Scale bar, 5 mm.
Figure 2. Transgenic complementation of lht1-101 by overexpression of LHT2, LHT3, or AAP5. (A) Restored ACC response of lht1-101 mutant by overexpression of LHT2. Left, Morphology of representative 4-day-old seedlings grown on MS-sucrose under dark media in the presence of 1 µM ACC. Note that the triple response of lht1-101 mutant was restored in the two independent LHT2-overexpressing transgenic lines (LHT2ox1 and LHT2ox3). Scale bar, 5 mm. Right, Root length of the transgenic lht1-101 seedlings. The seedlings were grown on MS-sucrose media supplemented with either Mock or 1 µM of ACC for 4 days under dark. Values are mean ± SD (n = 15). (B) Root length of LHT3 overexpressing transgenic lht1-101 seedlings. The seedlings were grown as described in (A). Values are mean ± SD (n = 15). (C) Root length of AAP5 overexpressing transgenic lht1-101 seedlings. Values are mean ± SD (n = 15). The seedlings were grown as described in (A).
Figure 3. ACC dose-response analysis of transgenic lht1-101 seedlings overexpressing LHT2. Wild-type, lht1-101, the transgenic lht1-101 seeds overexpressing LHT1 or LHT2 were grown on MS-sucrose media containing indicated concentrations (µM) of ACC. The seedlings were grown for 4 days in the dark. Values are mean ± SD (n = 15). A significant difference between ACC treated and mock-treated (0 ACC) seedlings was indicated with asterisks (t test, p < 0.01).
Figure 4. d-Amino acid response of transgenic lht1-101 seedlings overexpressing amino acid transporters. (A) Morphology of representative seedlings that have grown on MS-sucrose media containing mock, 1 mM of d-alanine (d-Ala), 3 mM of d-methionine (d-Met), or 3 mM of d-phenylalanine (d-Phe). Wild-type, lht1-101, and the transgenic lht1-101 plants overexpressing the amino acid transporter, e.g., LHT2, LHT3, or AAP5, were grown for 5 days under continuous light. Scale bar, 5 mm. (B) The root length of the seedlings grown as described in (A). The data are presented with box-and-whisker plot (n = 6-11). Different letters indicate significant differences at p < 0.01 according to one-way analysis of variance with Tukey honestly significant difference test. Evidence for normality of distribution and homoscedasticity is presented in Supplementary Table S2.
Figure 5. The early senescence syndrome of the transgenic lht1-101 mutant overexpressing LHT2. (A) Representative morphology of wild-type, lht1-101, and the transgenic plants overexpressing LHT2, LHT3, or AAP5 at 38 DAG (days after growth). For clarity, the inflorescence of each plant was removed. (B) Representative leaf morphology of the transgenic lht1-101 plants overexpressing LHT2 at 27 DAG. (C) Trypan blue staining. Cell death in the third leaf of each plant pictured in (B) was assayed with trypan blue. (D) Expression analysis of senescence-associated genes. The fourth leaves of the plants at 27 DAG were sampled for extraction of total RNA, which was subjected to qRT-PCR analysis. For each gene indicated, the relative expression was presented after normalization with the level of PP2A. Values are mean ± SD (n = 3, technical triplicate).
Figure 6. Amino acid selectivity of LHT1 and LHT2 in Xenopus oocytes. After perfusion with Ringer solution at pH 5.6, oocytes expressing LHT1 or LHT2 were treated with each amino acid (100 mM) or ACC (100 mM). For tyrosine, the currents were measured at 2.5 mM (limit of solubility). The resulting inward currents were recorded at −80 mV. Data represent the means ± SEM (n = 6–8 oocytes/four different frogs).
Figure 7. Concentration dependency and kinetic analysis of ACC-induced inward currents in LHT-expressing Xenopus oocytes. (A and B) The representative traces of LHT1- or LHT2-expressing oocytes after addition of various concentration of ACC. Exogenously applied ACC produced a reversible inward current. The holding potential was −80 mV. ACC induced the inward currents with the concentration-dependent manner in each LHT-expressing oocyte, respectively. (C and D) Kinetic analysis of ACC-induced inward currents. K0.5 value was calculated with Michaelis-Menten’s equation. Data represent the means ± SE.M (n = 6–8 oocytes/four different frogs).
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