Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Protein-protein interaction of NRT1.5 and AHA2. (A) THY.AP4 cells were transformed with NRT1.5 or NRT1.5 G209E fused to Cub and AHA2 fused to Nub. To detect protein-protein-interaction, 12 µl yeast cell culture with an OD 600 nm =1 and OD 600 nm =2, respectively, were dropped on YNB-Leu-Trp-His-Ade medium supplemented with 20 mM 3-amino-1,2,4-triazole (3-AT) and on YNB-Leu-Trp medium as growth control. The addition of 3-AT reduces non-specific yeast growth not based on protein-protein interactions. Plates were incubated at 30°C for three days and then photographed. (B) Confirmation of the interaction of NRT1.5 with AHA2 by bimolecular complementation assay. Shown are confocal microscopy images of N. benthamiana epidermis cells two days post-infiltration co-expressing non-fused nYFP, cYFP-NRT1.5 and RFP (top row), nYFP-AHA2, cYFP-NRT1.5 and RFP (center row), and nYFP-AHA2, cYFP-NRT1.5 G209E and RFP (bottom row). Column YFP: YFP fluorescence in yellow indicates interaction between proteins. Column RFP, RFP fluorescence in red visualizes the cytoplasm and the lumen of the nucleus as expression control. Column Chl, autofluorescence of chlorophyll (represented in violet). Column BF, bright field image. Column overlay: overlay of all 4 channels. Scale bar = 20 µm.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Transformation Assay, Cell Culture, Incubation, Confocal Microscopy, Expressing, Fluorescence

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Phenotyping of wild-type and mutant plants under low (LK) and high nutrient (HK) conditions. (A) RT-PCR analysis of NTR1.5 and AHA2 transcripts in Col-0 and the nrt1.5/aha2 double mutant. GAPC1 (At3g04120) was used as reference housekeeping gene (B) Rosette phenotypes, (C) Leaf fresh weight, and (D) Fraction of chlorotic leaves (%) of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants that were pre-germinated in fertilized soil for seven days and transferred to unfertilized soil type for another 30 days while watered with HK or LK medium, respectively. Shown are the means ± SD of the fraction of chlorotic leaves in 20 individual plants. Different letters above columns indicate statistically significant differences (Tukey’s test; P <0.05).

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Elemental analysis by ICP-OES of Arabidopsis Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants grown under low (LK) and high (HK) nutrient conditions. (A) Macronutrient and (B) micronutrient concentration in shoots of 30 days-old Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 plants grown in LK and HK medium. Different letters above columns indicate statistically significant differences (Tukey’s test; P <0.05) between mutants and Col-0 (means ± SD, n =8).

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Concentration Assay

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: Phenotypes of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 seedlings at 0K or HK supply. (A) Root phenotypes. Seedlings were pre-germinated in 0.5 × MS medium for five days and then transferred to 0K or HK media for fourteen days further. White arrows show lateral root formation. Dotted lines represent the maximum primary root growth of Col-0 in each condition. (B) Quantification of the density of lateral roots from (A) The number of lateral roots formed/main length of the primary root (cm) was plotted as lateral root density. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P < 0.05, (means ± SD, n ≥15). (C) Root hair phenotypes of seedlings in (A) . (D) Root hair density was determined as the number of hair roots in a 15 mm section from the starting point of the root tip under a light stereomicroscope (SZX12, Olympus) and quantified using Image J software. Results were expressed as the mean ± standard error. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P < 0.05, (means ± SD, n =15). (E) Shoot phenotypes of seedlings in A after a week of growth in the conditions indicated.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Software

Journal: Frontiers in Plant Science

Article Title: The K + transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 - deficiency

doi: 10.3389/fpls.2023.1287843

Figure Lengend Snippet: pH and plasma membrane potential are influenced by NRT1.5 and AHA2 expression. (A) Root growth phenotypes of Col-0, nrt1.5 , aha2 , and nrt1.5/aha2 in response to 5 µg/ml HygB. 5-day-old seedlings were transferred to 0.5 × MS medium pH 5.5 supplemented with or without 5 µg/ml HygB for fourteen more days. The white bar represents 1 cm. (B) Extracellular pH measurements in Col-0, nrt1.5 , aha2, nrt1.5/aha2 roots under HK and 0K nutrition supply. Ten days-old seedlings were incubated with 0.25 x MS liquid medium supplemented with 30 µg/ml of FITC-dextran for 16 h. Media pH was determined by a fluorescence standard curve with a pH range from 4.5 to 7.5. Different letters indicate statistically significant differences (Tukey’s test) between mutants and Col-0 with P <0.05, (means ± SD, n =12). (C) BY4741 wild-type and BYT12 mutant yeast strains were transformed with empty expression vectors (p425-TEF and p426-TEF) or expression constructs for the indicated proteins. Twelve 20 µl drops of untransformed (negative control) or transformed yeast cell suspension (OD 600 nm =1) were spotted along the HygB gradient (from 0 to 0.5 g/l) on SD-leu-ura pH 6.0, 0.1 M KCl plates and incubated at 30°C for two days. p425-TEF and p426-TEF are the empty expression vectors.

Article Snippet: pDONR222-NRT1.5 was generated by amplifying the full-length NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222. pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5-site-directed mutagenesis kit (New England Biolabs). pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.

Techniques: Membrane, Expressing, Incubation, Fluorescence, Mutagenesis, Transformation Assay, Construct, Negative Control, Suspension