Fig 1.
pgkc-1 mutant exhibits enhanced pollen germination and growth depolarization.
(A) WT pollen tube morphology. (B) pgkc-1 (SALK_066422C) pollen tube morphology. (C) Complemented pgkc-1 pollen tube morphology. Scale bar = 50 μm. (D) Pollen germination rate at 3 h and 9 h, respectively. pgkc-1 germinated at higher rates than WT and complemented pollen, especially at the early time point. (E) Pollen tube length of WT, pgkc-1 mutant, and genetically complemented pgkc-1 plants at 9 h after germination. (F) Pollen tube width of WT, pgkc-1 mutant, and genetically complemented pgkc-1 plants at 9 h after germination. Bars represent mean ± SEM. Asterisks indicate significant differences (** = p < 0.001) versus WT as determined by Student’s t-test.
Fig 2.
F-actin dynamics in the pgkc-1 mutant.
(A) Lifeact-mEGFP signal in WT and pgkc-1 pollen tubes with mock or 1.5 nM LatB treatment. Scale bar = 5 μm. (B) Average GFP signal intensity along WT and pgkc-1 pollen tubes with mock or 1.5 nM LatB treatment. Measurements were performed as described in Materials and Methods. Thirty-five pollen tubes were measured for each sample. The 0 μm indicates the position of the extreme tip. Orange line indicates WT pollen tube; red line indicates WT pollen tube treated with 1.5 nM LatB; gray line indicates pgkc-1 pollen tube; black line indicates pgkc-1 pollen tube treated with 1.5 nM LatB. Error bars on curves indicate standard error of the mean. (C and D) WT and pgkc-1 pollen tube growth when subjected to mock medium (C) or 1.5 nM LatB (D) treatment. Scale bar = 50 μm. (E to G) WT and pgkc-1 plant pollen germination (E), pollen tube length (F), and pollen tube width (G) when subjected to mock or 1.5 nM LatB treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s t-test (** = p < 0.001).
Fig 3.
Vesicle trafficking in the pgkc-1 mutant.
(A) EYFP-RABA4D signal in WT and pgkc-1 pollen tubes subjected to mock or 0.4 μM BFA treatment. Scale bar = 5 μm. (B) EYFP-RABA4D signal intensity. Measurements were performed as described in Materials and Methods. Fifteen to twenty pollen tubes from each sample were measured. 0 μm indicates the position of apical tip. Error bars on curves indicate standard error. (C and D) WT and pgkc-1 pollen tube morphology when subjected to (C) mock and (D) 0.4 μM BFA treatment. Scale bar = 50 μm. (E to G) WT and pgkc-1 plant pollen germination (E), pollen tube length (F), and pollen tube width (G) when subjected to mock or 0.4 μM BFA treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s t-test (** = p < 0.001).
Fig 4.
ROP1 signaling in the pgkc-1 mutant.
(A) Active ROP1 visualized by CRIB4-GFP signal in WT and pgkc-1 pollen tubes. Scale bar = 5μm. (B) Average CRIB4-GFP signal intensity along WT and pgkc-1 pollen tubes. (C) GFP-REN1 localization in WT and pgkc-1 pollen tubes. Scale bar = 5 μm. (D) Average GFP-REN1 signal intensity along WT and pgkc-1 pollen tubes. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip. Error bars on curves indicate standard error of the mean. (E to G) Pollen tube morphology of pgkc-1 (E), ren1-3 (F), and ren1-3/pgkc-1 double mutant plants (G). Scale bar = 50 μm. (H and I) Pollen tube length (H) and width I) of WT, pgkc-1, ren1-3, and ren1-3/pgkc-1 double mutant plants. Bars represent mean ± SEM. Asterisks indicate significant differences versus single mutant plant as determined using Student’s t-test (** = p < 0.001).
Fig 5.
Oligomycin treatment inhibited pollen tube growth in a manner distinct from the pgkc-1 mutant phenotype.
(A) WT pollen tubes subjected to mock treatment. (B) WT pollen tubes treated with 40 nM oligomycin. Scale bar = 50μm. (C) Average length of 50 pollen tubes. (D) Average width of 50 pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment group as determined using Student’s t-test (* = p < 0.05).
Fig 6.
Catalytically inactive mPGKc could not rescue the pgkc-1 mutant phenotype.
(A) Protein sequences of the conserved Glutamate179 regions of PGK proteins. AtPGKc, Arabidopsis thaliana cytosolic PGK; OsPGKc, Oryza sativa cytosolic PGK; DmPGK, Drosophila melanogaster PGK; ScPGK, Saccharomyces cerevisiae PGK; EcPGK, Escherichia coli PGK. Glutamate179 in AtPGKc is labeled with a red dot. (B) PGKc expression level. (C) Pollen germination rate. (D) Average length of pollen tubes. (E) Average width of pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus WT as determined using Student’s t-test (** = p < 0.001).
Fig 7.
Effects of disrupting GAPDH on pollen tube polarity.
(A) Glycolytic pathway of GAPDH and PGK. (B to G) Pollen tube morphology of WT (B), pgkc-1 (C), and ren1-3 (D) plants subjected to mock treatment; pollen tube morphology of WT (E), pgkc-1 (F), and ren1-3 (G) plants treated with 40 μM CGP 3466B. Both pgkc-1 and ren1-3 plants were dramatically depolarized by CGP medium. Scale bar = 50 μm. (H and I) Pollen tube length (H) and width (I) of WT, pgkc-1, and ren1-3 pollen tubes subjected to mock and 40 μM CGP treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus either single mutant as determined using Student’s t-test with either single mutant (** = p < 0.001). (J)-(M) Average signal intensity along WT pollen tubes subjected to mock or CGP treatment of (J) GFP- REN1, (K) CRIB4-GFP, (L) Lifeact-mEGFP, (M) EYFP-RABA4D. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip.
Fig 8.
gapcp1/gapcp2 double mutant is also defective in pollen tube polarity.
(A) WT pollen tubes. (B) gapcp1/gapcp2 double mutant pollen tubes. Scale bar = 50μm. (C) Average length of pollen tubes. (D) Average width of pollen tubes. Bars represent mean ± SEM. Asterisks indicate significant differences versus WT as determined using Student’s t-test (** = p < 0.001).
Fig 9.
Proposed model for potential role of glycolysis in pollen tube polarity.
Glycolysis is required for the association of the REN1 RopGAP with exocytic vesicles. REN1 RopGAP negatively regulates the Rho GTPase signaling, which coordinates pollen tube growth by coordinating actin dynamics and exocytosis. The mechanisms linking glycolysis with cell polarity remains elusive, which are possibly energization of unclear vesicle trafficking, or HXK signaling.