Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum.

BACKGROUND: DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. RESULTS: This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum 'CM334' and C. annuum 'Dempsey'. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. CONCLUSIONS: Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.

Functions of the Plant Qbc SNARE SNAP25 in Cytokinesis and Biotic and Abiotic Stress Responses.

Eukaryotes transport biomolecules between intracellular organelles and between cells and the environment via vesicle trafficking. Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNARE proteins) play pivotal roles in vesicle and membrane trafficking. These proteins are categorized as Qa, Qb, Qc, and R SNAREs and form a complex that induces vesicle fusion for targeting of vesicle cargos. As the core components of the SNARE complex, the SNAP25 Qbc SNAREs perform various functions related to cellular homeostasis. The Arabidopsis thaliana SNAP25 homolog AtSNAP33 interacts with Qa and R SNAREs and plays a key role in cytokinesis and in triggering innate immune responses. However, other Arabidopsis SNAP25 homologs, such as AtSNAP29 and AtSNAP30, are not well studied; this includes their localization, interactions, structures, and functions. Here, we discuss three biological functions of plant SNAP25 orthologs in the context of AtSNAP33 and highlight recent findings on SNAP25 orthologs in various plants. We propose future directions for determining the roles of the less well-characterized AtSNAP29 and AtSNAP30 proteins.