Wulff N et al. (2019), An Optimized Screen Reduces the Number of GA Tr...

XB-ART-56399

Front Plant Sci 2019 Jan 01;10:1106. doi: 10.3389/fpls.2019.01106.

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An Optimized Screen Reduces the Number of GA Transporters and Provides Insights Into Nitrate Transporter 1/Peptide Transporter Family Substrate Determinants.

Wulff N , Ernst HA , Jørgensen ME , Lambertz S , Maierhofer T , Belew ZM , Crocoll C , Motawia MS , Geiger D , Jørgensen FS , Mirza O , Nour-Eldin HH .

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Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes, wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities toward organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis suggests distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.

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Species referenced: Xenopus laevis
Genes referenced: gnas pkd2

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	Figure 1. GA membrane permeability is a function of pH and oxygen content. (A). Mock expressing oocytes (n = 7–9) were exposed to a mix of 50 µM GA1, 50 µM GA3, 50 µM GA4, 50 µM GA7, 50 µM GA8, 50 µM GA9, 50 µM GA19, and 50 µM GA24 in pH 4.7, 5.3, and 6.0 for 60 min and GA content was quantified by LC-MS/MS. (B). Chemical structures of the tested GAs. (C). GA content of mock injected oocytes at pH 4.7 as a function of GA oxygen content.
	Figure 2. Assessment of GA permeability and NPF transporter function. (A). Time course of NPF3.1 mediate GA3 transport over 180 min. NPF3.1 expressing oocytes were exposed to 50 µM membrane non-permeable GA3 in kulori pH 5 or 6. At each time point, oocytes (n = 6) were taken out for analysis. (B). Time course of GA9 membrane penetration over 180 min. Mock expressing oocytes were exposed to 50 µM GA9 in kulori pH 5 or 6. At each time point, oocytes (n = 6) were taken out for analysis. (C). NPF3.1 mediated GA9 transport at pH 5.5 and 6. Oocytes (n = 6) were exposed to 50 µM GA9 in kulori (Materials and Methods) pH 5.5 or 6 for 60 min. Oocyte GA content was analyzed using LC-MS/MS. Statistical assessment was performed with Holm Sidak two-way ANOVA p = 0.01.
	Figure 3. The proton potassium antiport function of NPF7.3 influences the ion-trap mechanism of membrane permeable weak acids. (A). Oocytes were exposed to 100 µM JA in kulori pH 5 for 60 min and analyzed in three technical replicates of five oocytes. Statistical assessment was performed with Holm Sidak one-way ANOVA p = 0.01. (B). Lower accumulation of membrane permeable phytohormones in NPF7.3 expressing oocytes compared to mock. Oocytes were exposed to 100 µM phytohormone in kulori pH 5 for 60 min (JA, JA-Ile, OPDA, ABA) or 90 min (GA4) and analyzed in three to four technical reps of four to five oocytes. Statistical assessment was performed with two-tailed t-tests p = 0.05 (C). Upper panel: Internal oocyte pH measured using three-electrode voltage-clamp of mock vs NPF7.3 expressing oocytes. Starting in ekulori pH 7.4, the external buffer was changed to ekulori pH 5.0 for 400 s followed by ekulori pH 7.4 for another 400 s. pH was measured continuously at a membrane potential of 0 mV. Representative measurements are shown. Lower panel: Mean values of cytosolic pH in mock or NPF7.3 expressing oocytes in standard ekulori buffer pH 7.4 or 400 s after incubation in ekulori pH 5.0 (n ≥ 3, mean ± SD). (D). JA content at defined cytosolic pH. Oocytes injected with 0.5 M TRIS 50 mM EGTA adjusted to pH 7.7 with 0.5 M MES (pH stabilized at ∼7.5), 0.5 M MES 50 mM EGTA adjusted to 5.7 with 0.5 M TRIS (pH stabilized at ∼6.25) or water (not stabilized: NS) was exposed to 100 µM JA in kulori pH 5 for 20 min and analyzed in 2 × 3 technical reps of four to five oocytes. Oocyte phytohormone content was analyzed using LC-MS/MS. Statistical assessment was performed with Holm Sidak two-way ANOVA p = 0.01. *significance is p = 0.01 in (A), p = 0.005 in (B), and p = 0.01 in (C).
	Figure 4. Transporter expression and oocyte maturity influence internal oocyte pH. Internal oocyte pH measured using three-electrode voltage-clamp of mock versus expressing oocytes. (A). after 30 min resting in ekulori pH 7.4 and additional 5 min clamped at 0 mV with pH 7.4 ekulori perfusion. (B). Change in internal pH after additional 5 min perfusion with pH 5 ekulori. Statistical assessment was performed with Holm Sidak two-way ANOVA p = 0.05. a and b indicates statistical indifference.
	Figure 5. Quantitative screen of the NPFs for GA transport of not membrane non-permeable GAs. Due to logistic considerations, the 53 NPF members were screened in two portions on the same day and normalized to the transport of NPF4.1 of membrane non-permeable GAs. NPF4.1 was chosen for normalization as it is a well characterized GA transporter (Kanno et al., 2012; Saito et al., 2015; Tal et al., 2016). For each transporter the proportion of each GA imported into oocytes is given in proportion to total imported amount of GAs. Statistical significant transport (Holm Sidak one-way ANOVA p = 0.05) is indicated with one asterisk for GA1, two asterisks for GA3, three asterisks for GA8, and four asterisks for GA19. Two assays of 28 and 29 genes, respectively, were performed on same day on the same oocyte batch. Both assays included NPF3.1, NPF4.1, and Mock to normalize. Oocytes (n = 5–6) were exposed to a mix of 50 µM GA1, 50 µM GA3, 100 µM GA4, 50 µM GA8, 50 µM GA19, and 50 µM GA24 in pH kulori 5.5 (Materials and Methods) for 60 min. Oocyte GA content was analyzed using LC-MS/MS.
	Figure 6. Quantitative screen of the NPF proteins for GA transport of membrane permeable GAs. Due to logistic considerations, the 53 NPF members were screened in two portions on the same day and normalized to the transport of NPF4.1 of membrane permeable GAs. NPF4.1 was chosen for normalization as it is a well characterized GA transporter (Kanno et al., 2012; Saito et al., 2015; Tal et al., 2016). For each transporter the proportion of each GA imported into oocytes is given in proportion to total imported amount of GAs. Statistical significant transport (Holm Sidak one-way ANOVA p = 0.05) is indicated with one asterisk for GA4 and two asterisks for GA24. Two assays of 28 and 29 genes, respectively, were performed on same day on the same oocyte batch. Both assays included NPF3.1, NPF4.1, and Mock to normalize. Oocytes (n = 5–6) were exposed to a mix of 50 µM GA1, 50 µM GA3, 100 µM GA4, 50 µM GA8, 50 µM GA19, and 50 µM GA24 in pH kulori 5.5 (Materials and Methods) for 60 min. Oocyte GA content was analyzed using LC-MS/MS.
	Figure 7. Sequence logos for the GA transporting (GA+) versus GA non-transporting (GA-) NPF proteins. Numbers correspond to the amino acid position of Arabidopsis thaliana NPF6.3 and the relative position of the binding site residues. The figure was made in the WebLogo program (Crooks et al., 2004).
	Figure 8. PCA of the 51 NPF sequences expressed by z-scales. GA transporting transporters are shown as green dots and squares and GA non-transporting transporters as red dots, triangles, and squares. Glucosinolate transporting genes and peptide transporting genes are shown as squares and triangles, respectively. The four clusters are marked by ellipses. PC1 and PC2 refer to the first and second principal components, respectively.
	Figure 9. Sequence logos for the transporters in the four clusters. The figure was made in the WebLogo program (Crooks et al., 2004). Red, blue, and black arrows mark positions unique for cluster I, cluster II, and for clusters I and II, respectively.
	Figure 10. 3D structure of the NPF6.3 [PDB: 4OH3 (Sun et al., 2014)]. Residues unique for cluster I are shown as green sticks, the residue unique for cluster II is shown as a blue stick and residues unique for clusters I and II are shown as yellow sticks.

References [+] :

Abramson, Structure and mechanism of the lactose permease of Escherichia coli. 2003, Pubmed