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. 2023 Nov 22;193(4):2292-2297.
doi: 10.1093/plphys/kiad449.

Reproductive defects in the abscission mutant ida-2 are caused by T-DNA-induced genomic rearrangements

Renate Alling  1   2 , Sergio Galindo-Trigo  1
Affiliations

Reproductive defects in the abscission mutant ida-2 are caused by T-DNA-induced genomic rearrangements

Renate Alling et al. Plant Physiol. .
No abstract available

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Conflict of interest statement

Conflict of interest statement. None declared.

Figures

Figure 1.
Figure 1.
T-DNA-induced genomic structural variation explains the ida-2 fertility defects and genetic linkage between the ida-2 insertion and chromosome 3. A) Representative images of fertilized seeds growing in dissected siliques of F1 plants of the indicated crosses. Many unfertilized ovules can be seen in the F1 generation of crosses between ida-2 and WT (Col-0) plants. Scale bars are 0.5 mm. B) Quantification of seed production per silique as a proxy for the ida-2 fertility defects. Data correspond to the average seed per silique in 5 siliques per plant. Five independent plants per cross were analyzed. Different letters indicate statistically significant differences in a 1-way ANOVA with post hoc pairwise comparisons using Bonferroni and Tukey tests (P < 0.05). See also Supplemental Fig. S2. C) Diagram of the detected T-DNA insertions in the ida-2 whole-genome sequencing analysis. Homozygous insertions were present in IDA, KNO1, and AT5G18550, although the read length (14 kb in average with PacBio HiFi) was insufficient to assemble the entire T-DNA. Arrows depict the pROK2 T-DNA sequence, whereas the dark grey boxes represent the pROK2 vector backbone. Dotted lines followed by a number indicate the nucleotide position based on the TAIR10 reference genome. D) The coincidence coefficient of the selected genomic features genotyped in 92 plants in the F2 population of the ida-2 × idl1cr1 cross shows the genetic linkage between the T-DNAs in chr1 and chr3 (IDA and KNO1, respectively). The T-DNA insertion in chr5 (AT5G18550) segregated independently from those in chr1 and chr3. E) Proposed karyotype in the ida-2 line based on the reproductive phenotype, segregation analyses, and whole-genome sequencing data.
Figure 2.
Figure 2.
The new idaCR allele is an effective alternative to ida-2. A) Diagram of the IDA WT gene and the idaCR allele. The light gray box highlights the IDA coding sequence, while the dark gray box indicates the stretch of nucleotides that encodes the mature and active IDA peptide. Underneath, the protospacer and protospacer-adjacent motif (PAM) sequences used to generate idaCR with CRISPR-Cas9 are shown. Below, the 1-nucleotide insertion and resulting frameshift in the idaCR line are represented. On the bottom, the resulting idaCR model is shown. After the 1-nucleotide insertion site, a frameshift includes a small tract of amino acids until a stop codon is reached. The rest of the IDA gene after the frameshift is therefore not translated, including the mature IDA peptide. B) Representative images of floral organ abscission phenotypes in the ida-2 and idaCR lines. Scale bars are 2 cm. C) Quantification of the floral organ abscission defect in ida-2 and idaCR plants. Five plants per line were analyzed. The graph shows the mean ± Se of floral organs attached to the flower at each floral position. D) Quantification of viable male and female gametophytes in stage 15 flowers of F1 plants from selfed Col-0, ida-2, idaCR, and their reciprocal crosses. Different letters indicate statistically significant differences in a 1-way ANOVA with post hoc pairwise comparisons using Bonferroni and Tukey tests (P < 0.05). Five plants per genotype were analyzed, averaging the number of pollen grains belonging to each category from 4 flowers per plant. Counts per flower ranged between 50 and 250 pollen grains. Dissected ovules from 3 flowers per plant were analyzed and assigned to viable or aborted categories based on their morphology, ranging between 36 and 180 ovules per plant. E) Representative images of viable and aborted gametophytes. Top left, pollen grains from F1 plants of the cross between ida-2 and Col-0 are shown. P corresponds to viable pollen grains; * marks aborted gametophytes. Top right, a viable and unfertilized ovule from an ida-2 plant is shown. SC, synergid cells; EC, egg cell; CC, central cell. The 2 images below show the most encountered aborted ovule phenotypes in the F1 generation of the cross between ida-2 and Col-0. We speculate they may correspond to early and late ovule abortion phenotypes (left and right, respectively). Scale bars are 20 µm.
See this image and copyright information in PMC

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