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Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs) indispensable for chloroplast biogenesis. Salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce the expression of PhANGs, leading to photoinhibition of photosystem II and singlet oxygen (1O2) burst in chloroplasts. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1; 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. This earlier finding has initiated research on the potential role of GLK1/2 and EX1 in SA signaling. Consistent with this view, we reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities in Arabidopsis (Arabidopsis thaliana). Overexpression of LSD1 repressed GLK target genes, including PhANGs, whereas loss of LSD1 enhanced their expression. Remarkably, LSD1 overexpression inhibited chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. Subsequent chromatin immunoprecipitation coupled with expression analyses further revealed that LSD1 inhibits the DNA-binding activity of GLK1 toward its target promoters. SA-induced nuclear-targeted SIB1 proteins appeared to interrupt the LSD1-GLK interaction, and the subsequent SIB1-GLK interaction activated EX1-mediated 1O2 signaling, elucidating antagonistic modules SIB1 and LSD1 in the regulation of GLK activity. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)