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. 2018 Nov 15;14(11):e1007795. doi: 10.1371/journal.pgen.1007795

Fig 2. ARF1 but not ARFA or ARFB is crucial for primary root growth and seed germination.

Fig 2

(A-P) Activation-impaired (T31N; TN) or hydrolysis-impaired (Q71L; QL) variants of ARF1, ARFA and ARFB were expressed from the estradiol-inducible system. (A) Primary root growth was severely affected by ARF1-TN-YFP, ARF1-QL-YFP and ARFA-QL-YFP but not by ARFA-TN-YFP, ARFB-TN-YFP or ARFB-QL-YFP. Number of seedlings analyzed is indicated above each column. (+), 20μM estradiol; (-), without estradiol. See also S1 Data. (B-N) Seed germination on estradiol-containing medium is inhibited by ARF1-TN-YFP (C, D), ARF1-QL-YFP (I, J) and ARFA-QL-YFP (K, L) but not by ARFA-TN-YFP (E, F), ARFB-TN-YFP (G, H) or ARFB-QL-YFP (M, N). Scale bar, 5 mm (B-N). (O, P) Western blot of estradiol-induced expression of activation-impaired (TN) or hydrolysis-impaired (QL) variants of ARF1, ARFA and ARFB in seedlings (O). Asterisk, expected size of approx. 47kDa. (P) γCOP as loading control; two asterisks, expected size of approx. 100kDa. COL, wild-type control. (Q-S) Quantitative secretion assay. (Q) Co-expression of the secretory reporter α-amylase with wild type (WT), activation-impaired (TN) or hydrolysis-impaired (QL) ARFA variants in tobacco protoplasts. Secretion index, ratio of extracellular to intracellular reporter activity; control, expression of α-amylase alone. (R) Co-expression of α-amylase and ARFA-QL with rising concentrations of ARFA-WT, ARFA-TN or ARF1-WT. Numbers below indicate the amount of respective plasmid used for protoplast transformation relative to ARFA-QL; constant amounts of α-amylase and ARFA-QL were transformed. (S) Co-expression of α-amylase and ARFA-QL with rising concentrations of ARFA-TN,QL. Numbers below indicate the amount of respective plasmid used for protoplast transformation relative to ARFA-QL; constant amounts of α-amylase and ARFA-QL were transformed. Panels below (Q-S): ARF protein expression levels evidenced indirectly by detection of GFP. Both ARF and GFP coding sequences are under control of the bidirectional mas promoter (consisting of a mas1’ and a mas2’ part) on the same plasmid, with mas1’ directing GFP expression and mas2’ directing ARF expression in a ratio of 1 to 10 [65]. Note full recovery of secretion by ARFA-TN (R) and ARFA-TN,QL (S) overexpression. See also S2, S3 and S4 Data.