Arabidopsis MAP3K16 and Other Salt-Inducible MAP3Ks Regulate ABA Response Redundantly

Abstract

In the Arabidopsis genome, approximately 80 MAP3Ks (mitogen-activated protein kinase kinase kinases) have been identified. However, only a few of them have been characterized, and the functions of most MAP3Ks are largely unknown. In this paper, we report the function of MAP3K16 and several other MAP3Ks, MAP3K14/15/17/18, whose expression is salt-inducible. We prepared MAP3K16 overexpression (OX) lines and analyzed their phenotypes. The result showed that the transgenic plants were ABA-insensitive during seed germination and cotyledon greening stage but their root growth was ABA-hypersensitive. The OX lines were more susceptible to water-deficit condition at later growth stage in soil. A MAP3K16 knockout (KO) line, on the other hand, exhibited opposite phenotypes. In similar transgenic analyses, we found that MAP3K14/15/17/18 OX and KO lines displayed similar phenotypes to those of MA3K16, suggesting the functional redundancy among them. MAP3K16 possesses in vitro kinase activity, and we carried out two-hybrid analyses to identify MAP3K16 substrates. Our results indicate that MAP3K16 interacts with MKK3 and the negative regulator of ABA response, ABR1, in yeast. Furthermore, MAP3K16 recombinant protein could phosphorylate MKK3 and ABR1, suggesting that they might be MAP3K16 substrates. Collectively, our results demonstrate that MAP3K16 and MAP3K14/15/17/18 are involved in ABA response, playing negative or positive roles depending on developmental stage and that MAP3K16 may function via MKK3 and ABR1.

Keywords: Arabidopsis thaliana; MAP kinase; MAP3K16; abiotic stress; abscisic acid (ABA).

Fig. 1. Expression pattern of MAP3K16

(A) Tissue-specific expression pattern of MAP3K16. Transcript levels were determined by semi-quantitative RT-PCR using RNA isolated from leaves (L), roots (R), flowers (F), and siliques (Si). (B) Tissue-specific expression pattern of MAP3K was determine by histochemical GUS staining of the transgenic plants harboring a 2.2 kb promoter-GUS construct. a, 8-day-old seedling. b, Roots of 8-day-old (left) and 39-day-old (right) plants. c, flowers. d, mature embryos.

Fig. 2. ABA sensitivity of MAP3K16 OX lines

(A) Expression levels of MAP3K16 in the transgenic lines determined by real-time RT-PCR. The error bars denote standard errors (B), (C) ABA sensitivity of seedling growth. Seeds were plated on MS media containing various concentrations of ABA and, after stratification, allowed to germinate and grow for 11 days in horizontal position (B) or 7 days in vertical position (C). (D) ABA sensitivity of seed germination. Mature, dry seeds were plated after stratification on MS medium containing various concentrations of ABA, and germination (radicle protrusion) rates were scored 2 days after plating. Experiments were performed in triplicates (n = 45 each), and the error bars indicate standard errors. (E) ABA sensitivity of cotyledon greening. Seeds were plated, and seedlings with green cotyledons were counted 11 days after plating. Each data point represents the percentage of seedlings with green cotyledons, and the error bars indicate standard errors (triplicates, n = 45 each). (F) ABA sensitivity of root growth. Seeds were germinated and grown for 3 days on ABA-free MS medium, transferred to ABA-containing media, and root elongation was measured 5 days after the transfer. Each data point represents the relative elongation rate compared with the control rate on ABA-free medium. Experiments were done in quintuplicates (n = 5 each), and the error bars denote standard errors.

Fig. 3. Stress responses of MAP3K16 OX lines

(A) Salt sensitivity of seed germination. Seeds were plated after stratification on media containing various concentrations of NaCl, and germination (radicle protrusion) was scores 3 days after the plating. Experiments were done in triplicates (n = 45 each), and the error bars denote standard errors. (B) Salt sensitivity of cotyledon greening. Seeds were plated as in (A), and seedlings with green cotyledons were counted 5 days after the plating. Each data point represents the percentage of seedlings with green cotyledons relative to the control rate on salt-free MS medium. Experiments were done in triplicates (n = 45 each), and the error bars denote standard errors. (C) Salt sensitivity of root growth. Seeds were germinated and grown for 3 days on salt-free MS medium, transferred to media containing NaCl, and root elongation was measured 5 days after the transfer. Each data point represents the relative elongation rate compared with the control rate on ABA-free medium. Experiments were done in quintuplicates (n = 5 each), and the error bars indicate standard errors. (D) Drought tolerance of MA3K16 OX lines. Plants were grown in soil for 7 days, withheld from water for 12 days, and then re-watered. The bottom panels show plants 2 days after the recovery. To minimize experimental variations, wild type and transgenic plants were grown in the same tray. (E) Survival rates of plants in (D) are presented. Experiments were done in triplicate (#49) (n = 20 each) or quadruplicate (#19) (n = 20 each). (F) Transpiration rates of wild type and MAP3K16 OX lines. Leaves of the same developmental stage (i.e., 5^th and 6^th true leaves from 20 day-old plants) were detached and weighed at 20 min interval after the detachment. The data represent relative (percentage) weight compared with the initial weight after the detachment. Experiments were done in triplicates (n = 15 each), and the error bars indicate standard errors.

Fig. 4. ABA and salt sensitivity of MAP3K16 KO line

(A) Schematic presentation of T-DNA insertion site in the map3k16 mutant. (B) Left, Disruption of the MAP3K16 gene was confirmed by PCR, using forward (F) and reverse (R) primer set (Supplementary Table S2). Right, Semi-quantitative RT-PCR to confirm the null expression of MAP3K16. (C) Growth of the map3k16 plants on media containing ABA. Plants were grown for 6 days after plating. (D), (E) Seeds were plated in media containing various concentration of ABA, and germination (radicle protrusion) and cotyledon greening were scored one day and 5 days, respectively, after the plating. Experiments were done in triplicates (n = 45 each), and the error bars denote standard errors. (F) Growth of the map3k16 plants on media containing NaCl. Plants grown for 4 days after plating are shown. (G), (H) Seeds were plated in media containing various concentration of NaCl, and germination and cotyledon greening were scored two days and three days, respectively, after the plating. Experiments were done in triplicates (n = 45 each), and the error bars represent standard errors. The error bars for map3k16 in (G) and (H) are smaller than the data point symbols.

Fig. 5. ABA sensitivity of MAP3K14/15/17/18 OX lines

(A), (D), (G), (J) Expression levels of MAP3K14/15/17/18, respectively, in transgenic lines determined by real-time RT-PCR. Experiments were done in duplicates, and the error bars represent standard errors. (B), (E), (H), (K) Seedlings of MAP3K14/15/17/18 OX lines grown for 7–9 days after plating on media containing ABA as indicated. (C), (F), (I), (L) Cotyledon greening of the MAP3K14/15/17/18 OX line seedlings. Each data point represents the percentage of seedlings with green cotyledons counted 5 days (C, F, I) or 7 (L) days after plating. Experiments were done in triplicates (n = 45 each), and the error bars indicate standard errors.

Fig. 6. Interaction of MAP3K16 with MKKs

(A) Interactions between MAP3K16 and MKK2/3/4/5 were examined by two-hybrid assay. Full-length MA3K16 was employed as bait, and MKKs were employed as prey. Yeast transformants were grown on SC-LWU plates, and the LacZ reporter activity was determined by X-gal overlay assay. (B) Liquid β-galactosidase assay. The LacZ reporter activity was determined by liquid assay using O-nitrophenyl-β-D-galactopyra-no-side (ONPG) as a substrate. Four independent transformants were assayed for each pair of constructs, and the numbers indicate the LacZ activity in Miller unit. The error bars indicate standard errors. (C) Bimolecular fluorescence complementation assay (BiFC). The interaction between MAP3K16 and MKK3 was examined by BiFC as described in the Materials and Methods. Protoplasts, which were prepared from tobacco (N. benthamiana) leaves infiltrated with pSPYNE-35S and pSPYCE-35S constructs, were observed under microscope.

Fig. 7. Interaction of MAP3K16 with ABR1

(A) The interaction between MAP3K16 and ABR1 was investigated by two-hybrid assay, using ABR1 as bait and MAP3K16 as prey. FL, full-length. KD, kinase domain. (B) MBP (maltose binding protein) pulldown assay was performed using MBP-tagged ABR1 and in vitro-translated MAP3K16 labeled with ³⁵S. Lane 1, MBP only. Lane 2, MBP-ABR1. Lane 3, in vitro translation product of MAP3K16. Lane 4, negative control for in vitro translation. (C) Bimolecular fluorescence complementation assay (BiFC). The interaction between MAP3K16 and ABR1 was investigated by BiFC, as described in the “Materials and Methods”. Protoplasts prepared from tobacco (N. benthamiana) leaves infiltrated with the pSPYNE-35S and pSPYCE-35S constructs pair were observed under microscope. (D) Schematic diagram of MA3K16 domain structure. The numbers indicate amino acid position. (E) Schematic diagram of ABR1 domain structure and the various fragments used in the two-hybrid assay in (F). The numbers indicate the amino acid position. (F) Two-hybrid assay to determine the interaction domains of ABR1 shown in (E). Full-length MA3K16 was used as bait, and various portions of ABR1 as prey. Transformants were grown on SC-HLWU plates containing 0.5 mM 3-aminotriazole (3-AT), and the LacZ reporter activity was determine by X-gal overlay assay.

Fig. 8. Kinase activity of MAP3K16

(A) In vitro assay to investigate the kinase activity of MAP3K16. Recombinant MAP3K16 protein (~ 0.5 μg) tagged with maltose-binding protein was employed in an assay in which myelin basic protein (MyBP) was used as a substrate. The right panel shows an autoradiogram, and the left panel shows a gel stained with coomassie brilliant blue R (CBB). The same amount of MyBP (~ 1 μg) was used in lanes 1–4. In lanes 2 and 4, recombinant ABR1 (~1 μg) was also added as a substrate. (B) Phosphorylation of ABR1 by MAP3K16. In vitro kinase assay was carried out to study phosphorylation of ABR1 by MAP3K16 (kinase domain, KD). Approximately 1 μg of MAP3K16 was used, and the amount of ABR1 in lanes 3 and 4 was 0.8 μg and 1.6 μg, respectively. Left panel, CBB-stained gel. Right panel, autordiogram. The X-ray film exposure time was longer (~2. 5 times) than in (A). (C) Phosphorylation of MKK3 by MAP3K16 (kinase domain) was investigated by in vitro kinase assay. Assays were performed as in (A) and (B). Left panel, CBB-stained gel. Right panel, autoradiogram. The numbers in the autoradiograms indicate the position of size markers. The assay components in each reaction mixture are indicated above the autoradiograms in (A), (B), and (C).