Fig 1.
Characterization of the sterile phenotype in ice1-2.
(A) Structures of the ICE1 gene in ice1-2 mutant (SALK_003155). The scaled linear map depicts four exons as boxes and three introns as bold lines between boxes. The positions of qRT-PCR primers (indicated by arrows) and T-DNA insertion are shown. (B) Morphology of reproductive growth of Col-0, ice1-2 and c-ice1-2 plants. (C) Relative expression of ICE1 gene in inflorescences. The ACTIN2 gene (AT3G18780) was an internal control. SE, n = 3, *** p < 0.001. (D) Comparison of seed numbers per silique of each genotype. SE, n = 32, *** p < 0.001. (E) Morphology of siliques from Col-0, ice1-2 and c-ice1-2 fresh plants. (F) Comparison of silique length of each genotype. SE, n = 14.
Fig 2.
Stamen morphology and anther dehiscence in ice1-2.
(A) Developmental series of flowers at flower developmental stage 12–15 within a single inflorescence from Col-0, c-ice1-2 and ice1-2. A, anther; F, filament; Ov, ovary; Pa, stigmatic papilla; Sg, stigma; Sy, style; Pe, Petal; Pg, Pollen grain. (B) Flower cluster showing the developmental series used to quantitatively describe anther dehiscence. The number 0 indicates the beginning of flower stage 12; 2 indicates the end of stage 12; 1 (stage 12); 3 (stage 13); 4 (stage 14); 5 (stage 15); -1 (stage 11); -2 (stage 11); -3 (stage 10); -4 (stage 9). (C) The number of dehiscent anthers in plants (SE, n = 15–28 flowers, one inflorescence per plant was used, *** p < 0.001). (D) Scanning electron micrographs of the anther adaxial surface from flower stage 12–15. St, stomium; En, epidermis; L, locule, StR, stomium region; Pg, Pollen grain.
Fig 3.
Pollen phenotypes, viability and germination analysis in ice1-2.
(A) Alexander staining of the anther, DAPI staining of pollen at tricellular stage, scanning electron microscopy (SEM) of pollen grains from Col-0 and ice1-2. Vn, vegetative nuclei; Sc, sperm cells. (B) FDA (fluorescein diacetate) staining of pollen from Col-0, ice1-2 and c-ice1-2 at flower stage 13. (C) Comparison of viability of pollen from Col-0, ice1-2 and c-ice1-2. SE, n = 5, ** p < 0.01. (D) The in vitro germination of pollen from Col-0, c-ice1-2, ice1-2 and selected ice1-2 anthers with obviously open and enlarged stomium (ice1-2-o) at flower stages indicated. The germination rates are listed below photographs. SE, n = 3. (E) Aniline blue-stained pistils of Col-0 flowers at 2 h after pollination with pollen from Col-0, c-ice1-2, ice1-2 and ice1-2-o at flower stages indicated. (F) Scanning electron micrographs of the anther adaxial surface from Col-0, c-ice1-2, ice1-2 and ice1-2-o at flower stages indicated. Arrows indicate the stomium in the ice1-2 anther. St, stomium.
Fig 4.
The pollen inviability, low pollen germination rate and anther indehiscence in ice1-2 can be rescued when grown in low humidity.
(A) Flowers and anthers from Col-0 and ice1-2 plants grown under 40% or 80% relative humidity (RH), respectively. The insets (top left corner) exhibit magnification of anther phenotypes. (B) Comparison of dehisced anther numbers between Col-0 and ice1-2 per flower at flower stage 13 under 40% and 80% RH, respectively. (SE, n = 25–292 flowers, *** p < 0.001). (C) FDA (fluorescein diacetate) staining of pollen from Col-0 and ice1-2 at stage 13 under 40% RH. (D) The in vitro germination of pollen from Col-0 and ice1-2 at stage 13 under 40% RH (upper row). The aniline blue-stained pistils of Col-0 flowers at 2 h after pollination with pollen from Col-0 and ice1-2 at stage 13 under 40% RH are also shown (lower row). (E-G) Comparison of pollen viability (E) (SE, n = 5), pollen germination rates (F) (SE, n = 3), and seed numbers per silique (G) (SE, n = 20) between Col-0 and ice1-2 grown under 40% RH. (H) Manual pollination on the Col-0 plants grown in the normal condition with pollen from Col-0 and ice1-2 under 40% RH, respectively. Arrows indicate the normal siliques generated using ice1-2 pollen under 40% RH.
Fig 5.
The Fertility of ice1-2 can be rescued when grown in low humidity.
(A) Comparison of pollination in Col-0 and ice1-2 plants grown under 40% and 80% relative humidity (RH), respectively. SE, n = 62–292 flowers, *** p < 0.001. (B) Comparison of silique length in Col-0 and ice1-2 plants grown under 40% (SE, n = 73) and 80% RH (SE, n = 140), respectively. ** p < 0.01, *** p < 0.001. (C) The shoots of the Col-0 and ice1-2 plants grown under 40% RH. Arrows indicate the rescued siliques in ice1-2.
Fig 6.
Stomata development of the anther is controlled by ICE1.
(A) GUS activity driven by ICE1 promoter is determined in the anther of flower stage 8 and 12. Strong GUS staining is shown in guard cells of stomata in the anther of flower stage 12 (indicated by red arrows). (B) Confocal images showing GFP-ICE1 accumulation (indicated by blue arrows) in the anther of flower stage 12. (C) Stomata numbers of anthers in flower stage 9–12. SE, n = 7–42 anthers. (D) Mode pattern of stomatal development in anthers. Diagram shows cell-state transitional steps within stomatal cell lineages. A subset of protodermal cells (white) assumes meristemoid mother cell (MMC) identity and executes an asymmetric entry division that creates meristemoids (M) (blue) and a sister cell, called stomatal-lineage ground cell (SLGC) (white). The meristemoids reiterate asymmetric amplifying division, but eventually differentiate into the guard mother cell (GMC) (green), which divides symmetrically once to form a stoma with differentiated guard cells (GCs) (red). (E) Scanning electron micrographs of the abaxial side of anthers at flower stage 8–14 in Col-0 (a-g). Anthers increase cell numbers from stage 8 to 9 (a-b). The stomatal lineage cells make their first appearance at about stage 9 (b). The number of stomatal lineage cells increases gradually during flower stage 9–12 (b-e). The majority of mature stomata are formed at about stage 12 (e). Inserts of anthers outline diagram show stomatal lineage cells with blue dots and mature stomata with red triangles. (F) Scanning electron micrographs of the abaxial side of anthers at flower stage 12 from Col-0, ice1-2 and c-ice1-2 plants. Cells colored in pink show distribution of mature stomata. Blue arrows indicate M and SLGC; green arrows indicate GMC; red arrows indicate GC. (G) Comparison of stomata numbers in Col-0, ice1-2 and c-ice1-2 plants at flower stage 12. SE, n = 30–42 anthers, *** p < 0.001).
Fig 7.
Guard cell expressed genes are overrepresented within ICE1-regulated genes in the anther.
(A) Number of down- and up-regulated genes (DG and UG) in anthers at flower stage 9–13 from ice1-2 compared with that in Col-0. The guard cell-expressed genes and the stamen-expressed genes are shown in circles of light green and dark green, respectively. (B) Heat map showing expression patterns of eight leaf stomatal development genes at the flower stage 10–13 in anthers from Col-0 and ice1-2 measured by qRT-PCR. The gene expression profiles were normalized with ACTIN2 gene (AT3G18780) and were plotted using Heatmapper (http://www2.heatmapper.ca/). (C) Regulatory network of stomatal development in the anther. Red shows up-regulated genes and blue shows down-regulated genes in the anther of ice1-2. When ICE1 is knocked-out, the differentiation from guard mother cells (GMCs) to guard cells (GCs) is blocked.
Fig 8.
ICE1 directly binds to the promoter of FAMA to activate its expression.
(A) Confocal images of FAMA protein accumulation (indicated by arrows) in the anther at flower stage 12 in FAMApro::FAMA-GFP plants. (B) The upstream region of 2.5 kb from transcription start site and ORF sequences of FAMA are shown with a black line and a blackish green box, respectively. The vertical lines indicate the E-box positions. Eight probes (P1 to P8) containing E-boxes are also exhibited. P6 contains two E-boxes. (C) Dual-LUC Assays in tobacco leaves. ICE1 driven by 35S promoter was served as the effector and LUC under control of FAMA promoter (2.5 kb upstream from transcription start site) was the reporter. (D) The relative activity (LUC/REN) is shown. The reporter co-transformed with pC1302 vector was used as the control. SE, n = 6, ** p < 0.01. (E-F) Electrophoretic mobility shift assay (EMSA) showing the binding activity of ICE1 to the probes with E-box elements at -582 to -613 bp (labeled as P3 in B) and -629 to -664 bp (labeled as P4 in B) upstream from transcription start site of FAMA, respectively. The sequences of P3 and P4 as well as mutated probes are listed. (G) EMSA showing the competition of ICE1-P3 interaction using P4. P4 has higher binding affinity with ICE1 than P3.
Fig 9.
Pathway enrichment and functional category of genes regulated by ICE1 in the anther.
(A-B) The enrichment analysis of down-regulated genes (A) and up-regulated genes (B) that are expressed in both of the guard cell and the stamen. The key clusters in the three categories identified by GO are shown in columns. The ratios of each cluster to the total gene number are shown with percentage. (C) Phenotype of impaired ion exchange in the ice1-2 mutant. Arrows indicate the wilted flower buds.
Fig 10.
ICE1 modulates water movement in the anther.
Hypothetical signal pathways deduced from GO enrichment analysis are highlighted in the yellow boxes. ICE1 modulates water transport and stomatal differentiation to control water transport in the stamen and evaporation through stomata in the anther, which can also be affected by ambient drought. On the other hand, ICE1 activates CBF signaling to protect floral tissues from drought and cold stresses.