Fig 1.
Arabidopsis ASNAP is constitutively expressed.
(A-H) Representative histochemical GUS staining of a seedling (A), root hairs (B), a cotyledon (C), trichomes on initiating leaves (D), an inflorescence (E), pollen grains and a pollen tube (F), a mature anther (G), and mature ovules (H) from ASNAPg:GUS transgenic plants. Bars = 1 mm for (A, E), 20 μm for (B, D, F, H), 200 μm for (C), 100 μm for (G).
Fig 2.
Functional loss of ASNAP causes reduced fertility.
(A) The ASNAP genomic structure. Inverted triangle indicates Cas9-target site. (B) Generation of the asnap mutants and the Cas9-resistant ASNAP (crASNAP). Dark blue indicates the protospacer sequence; red indicates the PAM sequence and pre-stop codon generated during genomic editing; pink indicates nucleotide insertions generated by CRISPR/Cas9 in the corresponding asnap mutants. Pre-stop codons were generated at the 730–732 (asnap-1 and asnap-3) or 709–711 bp (asnap-2) of the ASNAP CDS. (C-G) Representative seed set of wild type, asnap-1/+, asnap-2/+, and asnap-3/+. (G) Quantification of seed sets. Genotypes are indicated in (C-F). Results are means ± SD (n>10). Different letters indicate significantly different groups (One-Way ANOVA, Tukey’s multiple comparisons test, P<0.05). (H-I) Representative CLSM of an ovule from wild type (H) or asnap-1/+ (I) pollinated with wild-type pollen at 24 hours after pollination (HAP). Dotted line illustrate the embryo sac; arrowhead points to a single nucleus in the embryo sac; zg indicates the elongating zygote; pe indicates peripheral endosperm. Numbers at the bottom indicate displayed/total examined ovules. (J-K) Representative differential interference contrast (DIC) image of an ovule from wild type (J) or asnap-1/+ (K) pollinated with wild-type pollen at 24 HAP. Pink highlights the developing embryo in (J) or the embryo sac in (K). Numbers at the bottom indicate displayed/total examined ovules. Bars = 1 mm for (C-F); 20 μm for (H-K).
Table 1.
ASNAP is essential for both male and female transmission.
Fig 3.
ASNAP is essential for male gametophytic development.
(A-D) Alexander staining of a representative anther (A, B) or mature pollen grains (C, D) from wild type (A, C) or asnap-1/+ (B, D). (E-F) DAPI staining of mature pollen grains from wild type (E) or asnap-1/+ (F). Bright-field images are shown at the bottom of corresponding fluorescent images. Arrowheads point at aborted pollen grains. (G-J) Scanning electron micrographs (SEMs) of mature pollen from wild type (G, H) or asnap-1/+ (I, J). Aborted pollen grains are pseudo-colored in pink. (K-L) Representative semi-thin transverse sections of developing wild-type (K) or asnap-1/+ (L) anthers. From top to bottom: at stage 9, stage 10, stage 11, or stage 12. Defective microspores are pseudo-colored in pink. (M-Q) Transmission electron micrographs (TEMs) of microspores in asnap-1/+ at stage 10 (M, N), stage 11 (O, P), or stage 12 (Q). Wild-type-like microspores are shown in (M, O) while defective microspores are shown in (N, P). Two normally developed tricellular microspores are shown together with two aborted ones (pseudo-colored) in (Q). Bars = 100 μm for (A, B); 50 μm for (C, D, G, I); 20 μm for (E, F, K, L); 5 μm for (H, J); 2 μm for (M-P); 10 μm for (Q).
Fig 4.
Functional loss of ASNAP results in the arrest of female gametogenesis.
(A-F) CLSMs of representative wild-type (A-C) or asnap-1/+ (D-F) ovules at stage 3-I (A, D), stage 3-III (B, E), or stage 3-VI (C, F). ccn, central cell nucleus; cn, chalazal nucleus; dm, degenerating megaspore; en, egg cell nucleus; fm, functional megaspore; mn, micropyle nucleus; sn, synergid nucleus. Dotted lines in (B, E) illustrate the embryo sacs. Arrowheads point to the single nucleus in embryo sacs. Numbers at the bottom indicate displayed/total examined ovules. (G) Representative histochemical GUS staining of a DD45p:GUS (WT) or DD45p:GUS;asnap-1/+ (asnap-1/+) pistil at maturation. The percentage of GUS-positive ovules are means ± SD (n>10). (H) Overlaid CLSM images of a lysotracker red (magenta)-stained ES1p:NLS-YFP (WT) or ES1p:NLS-YFP;asnap-1/+ transgenic ovule (asnap-1/+) at stage 3-V. Numbers at the bottom indicate displayed/total examined ovules. (I-J) Histochemical GUS staining of a wild-type (I) or a asnap-1/+ pistil (J) pollinated with LAT52p:GUS pollen at 12 HAP. Percentage of ovules targeted by pollen tubes is shown at the bottom. Results are means ± SD (n>10). (K-L) Aniline blue staining of a wild-type (K) or a asnap-1/+ pistil (L) pollinated with wild-type pollen at 48 HAP. Arrowheads in (L) point at unfertilized ovules. Several overlapping high-magnification images were taken and overlaid to show the whole pistil in (I-L). Percentage of fertilized ovules is shown at the bottom. Results are means ± SD (n>10). Wild type and asnap-1/+ are significantly different at both pollen tube attraction and fertilization (t-test, P<0.05). Bars = 10 μm for (A-F); 100 μm for (G, I-L); 20 μm for (H).
Fig 5.
Downregulating ASNAP with a gametophyte-specific promoter mimics ASNAP loss-of-function.
(A-E) Representative silique from the wild type (A), self-fertilized GPR1p:amiR-ASNAP#1 (amiR#1) (B), amiR#1 pollinated with wild-type pollen (C), self-fertilized amiR#2 (D), or amiR#2 pollinated with wild-type pollen (E). (F-K) Alexander staining of a dehiscing anther (F-H) or pollen grains (I-K) from wild-type (F, I), amiR#1 (G, J) or amiR#2 (H, K) plants. (L-N) DAPI staining of mature pollen from wild-type (L), amiR#1 (M) or amiR#2 (N) plants. (O) Transcript abundance of ASNAP in wild type and two lines (#1 and #2) of the amiR plants. RNAs were extracted from inflorescences. Results are means ± SE (n = 3). Different letters indicate significant different groups (One-Way ANOVA, Tukey’s multiple comparisons test, P<0.05). (P-R) Percentage of seed set (P), of pollen viability by alexander staining (Q), or of tricellular pollen by DAPI staining (R). Results are means ± SD (n>10) for (P). Results are means ± SE (n = 4 involving over 300 pollen grains) for (Q, R). Different letters indicate significantly different groups (One-Way ANOVA, Tukey’s multiple comparisons test, P<0.05). Bars = 1 mm for (A-E); 100 μm for (F-H); 50 μm for (I-K); 20 μm for (L-N).
Fig 6.
Complementation of ASNAP loss-of-function fully by the ASNAP genomic fragment whereas partially by each of the two splicing variants.
(A-D) Representative silique from the wild type (A), ASNAPg;asnap-1 (B), UBQ10p:GFP-crASNAP.1;asnap-1/+ (C), or UBQ10p:GFP-crASNAP.2;asnap-1/+ (D). (E-L) Alexander staining of a dehiscing anther (E-H) or pollen grains (I-L) from wild-type (E, I), ASNAPg;asnap-1 (F, J), UBQ10p:GFP-crASNAP.1;asnap-1/+ (G, K), or UBQ10p:GFP-crASNAP.2;asnap-1/+ (H, L) plants. (M-P) DAPI staining of mature pollen from wild-type (M), ASNAPg;asnap-1 (N), UBQ10p:GFP-crASNAP.1;asnap-1/+ (O), or UBQ10p:GFP-crASNAP.2;asnap-1/+ (P) plants. DAPI channel and transmission channel images are shown at the top and the bottom, respectively. (O-P) insets are corresponding GFP channel images. (Q-S) Percentage of seed set (Q), of pollen viability by alexander staining (R), or of tricellular pollen by DAPI staining (S). Results are means ± SD (n>10) for (Q). Results are means ± SE (n = 4 involving over 300 pollen grains) for (R-S). Different letters indicate significantly different groups (One-Way ANOVA, Tukey’s multiple comparisons test, P<0.05). Bars = 1 mm for (A-D); 100 μm for (E-H); 50 μm for (I-L); 20 μm for (M-P).
Fig 7.
Alternative splicing of α-SNAP is evolutionarily recurring.
Phylogenetic analysis of ASNAP orthologues using MEGA7.0. Arabidopsis protein sequences were obtained from TAIR whereas proteins from other species were obtained from the National Center for Biotechnology Information and Ensembl databases. Symbols of proteins from alternative splicing (gene symbol): NP_003818.2 and XP_011525739.1 for Homo sapiens α-SNAP.1 and α-SNAP.2 (8775); Pp3c18_14770V3.1 and Pp3c18_14770V3.2 for Physcomitrella patens α-SNAP.1 and α-SNAP.2 (Pp3c18_14770V3); XP_021307480.1 and XP_002466875.1 for Sorghum bicolor α-SNAP.1 and α-SNAP.2 (LOC8064551); Zm00001d033092_T001 and Zm00001d033092_T002 for Zea mays α-SNAP.1 and α-SNAP.2 (Zm00001d033092); XP_013742310.1 and XP_013742311.1 for Brassica napus α-SNAP.1 and α-SNAP.2 (LOC106445330); XP_033146543.1 and XP_009139115.1 for Brassica rapa α-SNAP.1 and α-SNAP.2 (LOC103863115); At3g56190.1 and At3g56190.2 for Arabidopsis thaliana α-SNAP.1 and α-SNAP.2 (At3g56190). Symbols of other α-SNAPs: AAA35029.1 for Saccharomyces cerevisiae; XP_005713623.1 for Chondrus crispus; OSTLU18863 for Ostreococcus lucimarinus; XP_001700026.1 for Chlamydomonas reinhardtii; AMTRs00007p00199530 for Amborella trichopoda; Os08g0282400 for Oryza sativa Japonica; BRADI_3g19810v3 for Brachypodium distachyon; Solyc06g050770.3 for Solanum lycopersicum; XP_004138403.1 for Cucumis sativus.
Fig 8.
ASNAP isoforms are targeted to distinct locations.
CLSM of root epidermal cells from the UBQ10p:GFP-ASNAP.1 or UBQ10p:GFP-ASNAP.2 transgenic plants. Seedlings at 4 days after germination (DAG) were pulse-labeled with 4 μM FM4-64 (FM4-64/1 min), 30 min after FM4-64 uptake (FM4-64/30 min), or FM4-64 for 5 min then treated with 50 μM BFA for 50 min (FM4-64/BFA), or FM4-64 for 5 min then treated with 33 μM WM for 30 min (FM4-64/WM). Arrowheads point at BFA compartments; white arrows point at enlarged PVC/MVB after WM treatment; yellow arrows point at PM-associated GFP signals. Bars = 10 μm.
Fig 9.
Both ASNAP isoforms interact with NSF.
(A) In vitro pull-down assay. NSF-His was used to pull-down ASNAP.1-GST, ASNAP.2-GST. Results are representative of three biological replicates. (B-C) FRET efficiency (B) or CLSM images of FRET assays (C). FRET signals are represented in pseudo-color, covering the full range of measured values within each dataset (max to min). Results are means ± SD (n > 30). Every combination was examined with three replicate experiments. Different letters indicate significantly different groups (One-Way ANOVA, Tukey’s multiple comparisons test, P<0.05). a.u. for arbitrary unit. Bars = 5 μm.