Fig 1.
T-DNA insertion sites for five multiple-stress responsive genes.
The diagram on the left shows the SALK T-DNA insertion sites for the At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010 genes [20]. T-DNA insertion sites were obtained from the SIGnAL website (http://signal.salk.edu) whilst gene models were based on genomic DNA sequence information from the NCBI database. The gene models represent the mRNA (light grey) and protein coding (darker grey) regions of the five intronless genes. On the right, RT-PCR analyses using gene-specific primers (depicted as LP and RP relative to the T-DNA insertion in each gene model) show the loss of gene-specific mRNA transcripts in each homozygous, backcrossed T-DNA mutant, indicating that the selected five SALK lines are knockout lines. Alpha-(α)-Tubulin was used as a constitutive positive control with primers according to previous work [21]. Gene-specific RT-PCR primers are listed in S1 Table.
Fig 2.
Altered phenotypes for the transgenic lines overexpressing the At1g74450 open reading frame.
(A) Image showing a wildtype Col-0 plant on the left, a transgenic plant overexpressing the At1g74450 open reading frame (35S:ORF) in the middle and a SALK_145820 homozygous knockout plant for gene At1g74450 on the right. Arrows point to individual siliques. (B) Image of siliques corresponding to the plants shown in panel A.
Fig 3.
Primary inflorescence height of four independent transgenic lines overexpressing the At1g74450 open reading frame.
(A) Graph showing the average primary inflorescence height in cm for four independent 35S:ORF lines compared to wildtype Col-0. Bars indicate standard error for each line (2: n = 31, 3: n = 27, 4: n = 30, 5: n = 20, Col-0: n = 117). The asterisks indicate statistically significant differences (p<0.01) between each 35S:ORF line and wildtype Col-0 based on one-way ANOVA followed by post hoc t-tests (two-tailed distribution, two-sample unequal variance) and Bonferroni correction. (B) Composite gel image showing semi-quantitative RT-PCR results for the four independent 35S:ORF lines and wildtype Col-0. The top half of the gel image shows RT-PCR products after 28 cycles generated by constitutive control Alpha-(α)-Tubulin primers [21]. The lower half of the gel image shows RT-PCR products after 28 cycles generated by gene-specific primers.
Fig 4.
Localisation of GUS activity in pollen under the control of the At1g74450 promoter.
(A) Flower of prom:GUS line at stage 13–14 showing GUS expression in pollen after treatment with staining solution. (B) Flower of wildtype Col-0 at stage 13–14 showing no GUS activity after treatment with staining solution. Scale bar represents 1 mm.
Fig 5.
Phenotypical analysis of flowers and anthers from transgenic lines overexpressing the At1g74450 open reading frame.
(A-D) Flowers at developmental stages 13–14. (A) Flower from wildtype Col-0 showing stamens with pollen. (B) Flower from SALK_145820 homozygous knockout line for gene At1g74450 showing stamens with pollen. (C) Flower from 35S:ORF line 3 showing stamens with no pollen. (D) Flower from 35S:ORF line 2 showing stamens with some pollen. (E) Plant from 35S:ORF line 2 showing one elongated silique flanked by undeveloped short siliques. (F-K) Anthers dissected from flowers stage 12–13 and treated with Alexander stain. (F) Anther from wildtype Col-0 showing viable pollen. (G) Anther from 35S:ORF line 3 showing no viable pollen. (H) Anther from 35S:ORF line 3 showing mostly non-viable and some viable pollen. (I) Anther from 35S:ORF line 5 showing mostly non-viable and some viable pollen. (J) Anther from 35S:ORF line 4 showing non-viable and viable pollen. (K) Anther from SALK_145820 homozygous knockout line for gene At1g74450 showing viable pollen. Scale bars represent 1 mm (A-D), 1 cm (E) and 100 μm (F-K).
Fig 6.
Localisation of GFP in epidermal cells at the root apical meristem and transition zone.
(A-F) Confocal images of the transgenic line expressing the At1g74450 open reading frame fused with C-terminal GFP and driven by the ubiquitin-10 promoter (pUB10:ORF:C-GFP). (A, C and E) Images showing the same cells in one location of the root. (B, D and F) Images showing cells in a second location of the root. (A and B) GFP fluorescence is localised to the cytosol of root cells in two locations of the root. (C and D) Staining of mitochondria using MitoTracker Orange in two locations of the root. (E and F) Composite images showing no co-localisation of GFP signal with mitochondria when merging either panels A and C (E) or panels B and D (F). Scale bars represent 10 μm.
Fig 7.
Analysis of At1g74450 protein co-localisation with FM4-64 or FM4-64 + BFA.
(A-F) Confocal images of the transgenic line expressing the At1g74450 open reading frame fused with C-terminal GFP and driven by the ubiquitin-10 promoter (pUB10:ORF:C-GFP). (A and D) GFP fluorescence localisation. (B) 2 μM FM4-64 staining after 30 min. (E) 2 μM FM4-64 + 50 μM BFA staining after 1 hour. (C) Composite image showing no co-localisation of GFP signal with FM4-64 when merging panels A and B. (F) Composite image showing no co-localisation of GFP signal with FM4-64 + BFA when merging panels D and E. Scale bars represent 10 μm.
Fig 8.
Ruthenium red-stained inner seed coat mucilage layer after imbibition of wildtype and At1g74450 35S:ORF seeds.
(A) Wildtype Arabidopsis ecotype Columbia-0 (top row) and At1g74450 35S:ORF line 2 (bottom row). (B) Col-0 (top row) and 35S:ORF line 3 (bottom row). (C) Col-0 (top row) and 35S:ORF line 5 (bottom row). Scale bars represent 500 μm.
Fig 9.
Quantification of the inner seed coat mucilage layer depicted in Fig 8.
Graph showing the average surface areas in mm2 of the red/pink zone, the purple zone and the combined total layer for three independent At1g74450 35S:ORF lines compared to wildtype Col-0. Bars indicate standard error for each line (2: n = 3, 3: n = 3, 5: n = 3, Col-0: n = 9). The asterisks indicate statistically significant differences (p<0.01) between each 35S:ORF line and wildtype Col-0 based on one-way ANOVA followed by post hoc t-tests (two-tailed distribution, two-sample unequal variance) and Bonferroni correction.