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Summary Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.
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The Arabidopsis T‐DNA mutant SALK_008491 carries a 14‐kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress
Abstract In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO2fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known asnhd1‐1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independentnhd1null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream ofNHD1. BesidesNHD1, which encodes for a putative plastid Na+/H+antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.
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- Award ID(s):
- 1847193
- PAR ID:
- 10446065
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 6
- Issue:
- 7
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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