Arabidopsis 3β-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development
Abstract
:1. Introduction
2. Results
2.1. Generation of hsd1 and hsd2 Mutants via CRISPR/Cas9-Based Genome Editing
2.2. The hsd1 hsd2 Double Mutation Impairs Development of Male Gametophytes
2.3. The hsd1 hsd2 Double Mutation in Heterozygosity Does Not Affect Flower Formation
2.4. The hsd1 hsd2 Double Mutation Causes the Male Pollen Lethality
2.5. The Male Gametophytic Defect of the hsd1 hsd2/+ Mutant Was Complemented by the Pollen-Specific Expression of the HSD2 Gene
2.6. The hsd1 hsd2 Double Mutation Likely Caused the Embryo Lethality
3. Discussion
3.1. The CRISPR/Cas9-Genome Editing Failed to Create a True hsd1 hsd2 Double Mutant
3.2. HSD1 and HSD2 Function Redundantly in Pollen Development
3.3. HSD1 and HSD2 Are Essential for Embryonic Development
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
4.2. CRISPR/Cas9-Mediated Genome Editing to Create HSD1 and HSD2 Mutant
4.3. Total RNA Extraction and cDNA Synthesis
4.4. Plasmid Construction and Plant Transformation
4.5. Mutant Self-Inbred Offspring Separation Ratio and Forward and Reverse Cross Experiments
4.6. Flower Organ Phenotypic Observation and Pollen Alexander Staining
4.7. Phenotypic Observation of Pods and Statistical Analysis of Seed Abortion Rate
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Self-Cross | No. of Progeny | Genotypes of Progeny | ||
---|---|---|---|---|
HSD1 hsd2 or hsd1 HSD2 | hsd1/+ hsd2 or hsd1 hsd2/+ | hsd1 hsd2 | ||
hsd1/+ hsd2 | 178 | 52.2% (93) | 47.8% (85) | 0 |
hsd1 hsd2/+ | 200 | 51.5% (103) | 48.5% (97) | 0 |
Male | × | Female | Expected Heterozygosity | Observed Heterozygosity | TEM | TFF |
---|---|---|---|---|---|---|
hsd1 | × | WT | 56/56 | 56/56 | 100% | NA |
hsd2 | × | WT | 39/39 | 39/39 | 100% | NA |
hsd1/+ hsd2 | × | WT | 120/240 | 0/240 | 0 | NA |
WT | × | hsd1/+ hsd2 | 118/235 | 120/235 | NA | 104% |
hsd1 hsd2/+ | × | WT | 123/245 | 0/245 | 0 | NA |
WT | × | hsd1 hsd2/+ | 156/312 | 153/312 | NA | 96% |
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Pan, J.; Li, W.; Chen, B.; Liu, L.; Zhang, J.; Li, J. Arabidopsis 3β-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development. Int. J. Mol. Sci. 2023, 24, 15565. https://doi.org/10.3390/ijms242115565
Pan J, Li W, Chen B, Liu L, Zhang J, Li J. Arabidopsis 3β-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development. International Journal of Molecular Sciences. 2023; 24(21):15565. https://doi.org/10.3390/ijms242115565
Chicago/Turabian StylePan, Jiawen, Weifeng Li, Binzhao Chen, Linchuan Liu, Jianjun Zhang, and Jianming Li. 2023. "Arabidopsis 3β-Hydroxysteroid Dehydrogenases/C4-Decarboxylases Are Essential for the Pollen and Embryonic Development" International Journal of Molecular Sciences 24, no. 21: 15565. https://doi.org/10.3390/ijms242115565