PubMed · 13858919
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J M ABELL. 1962. Perplexing plant.. https://pubmed.ncbi.nlm.nih.gov/13858919/
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A total of 118 NtMADS-box genes were identified in tobacco, revealing their potential roles in axillary bud development. Preliminary overexpression analysis indicated that NtMADS91 promotes axillary bud development. MADS-box transcription factors are core regulators of plant development, but their functions in axillary bud development in Nicotiana tabacum L. have not been systematically elucidated. In this study, 118 NtMADS-box genes were identified from the tobacco genome. Phylogenetic analysis classified them into type I (comprising the Mα and Mγ subfamilies) and type II (comprising the MIKC* and MIKCC clades). Promoter analysis revealed that cis-acting elements were predominantly associated with light and hormone responses. RNA-seq analysis of axillary buds after topping identified 60 differentially expressed NtMADS-box genes, from which 12 candidate genes with significant expression changes were selected. Tissue-specific qRT-PCR revealed that seven of these genes were preferentially expressed in axillary buds, with members of the SOC1 and SVP subfamilies accounting for the majority. Exogenous application of abscisic acid and the strigolactone analog GR24 significantly suppressed the expression of most candidate genes, including NtMADS91. The preliminary overexpression analysis suggested that NtMADS91 may promote axillary bud growth, increasing both the number and length of axillary buds. This study lays a foundation for future dissection of the regulatory mechanisms of the NtMADS-box gene family in axillary bud development and provides promising candidate genes for research related to tobacco axillary bud development.
Asparagine (Asn) is pivotal for plant nitrogen (N) metabolism and plays indispensable roles in plant growth, development, and stress tolerance. However, the systematic characteristics and core functions of asparagine synthetase genes (NtASNs) in tobacco remain unclear. Through a comprehensive genome-wide investigation, nine members of the NtASN gene family were identified. Subsequent CRISPR/Cas9-mediated knockout and overexpression assays of these NtASN genes revealed that NtASN1e, NtASN2a, and NtASN2b are the core genes responsible for Asn biosynthesis in tobacco. Their knockout reduced asparagine synthetase activity and Asn content, delayed seed germination by 2-3 days, and displayed elevated oxidative injury when exposed to salinity conditions. In contrast, overexpression of these genes elevated Asn accumulation. Subcellular localization analysis indicated that NtASN1e was localized to both the cytoplasm and chloroplasts, whereas NtASN2a exhibited dual localization in the cytoplasm and endoplasmic reticulum, and NtASN2b was mainly localized in the cytoplasm. This study systematically clarifies the evolutionary characteristics and core functions of the NtASN gene family and provides candidate genes for optimizing nitrogen metabolism and improving salt-stress adaptation in tobacco. These findings hold important practical significance for molecular breeding and product quality improvement in industrial crops.
Elucidating molecular interactions such as protein-DNA (PDIs) and protein-protein (PPIs) has traditionally relied on yeast-based 1-hybrid (1H) and 2-hybrid (2H) systems. To provide an alternative platform that better reflects the native cellular environment of plants, we optimized the tRUBY reporter system for 1H and 2H assays in Nicotiana benthamiana, enabling direct in planta analysis of PDIs and PPIs. Specifically, the 2A peptide sequence used for co-expressing the 3 betalain biosynthetic genes-responsible for the visible RUBY coloration-was replaced with T2A from the Thosea asigna virus in place of P2A or F2A from mammalian-pathogenic Picornaviridae viruses, improving biosafety for agricultural applications. The resulting tRUBY-1H and tRUBY-2H systems operate under near-physiological conditions with physiologically relevant expression levels, enabling quantitative, multiplexed, and directly compatible protein-level analyses, thereby offering high sensitivity and flexibility for advanced molecular studies. Ultimately, these systems demonstrate that the streamlined, cost-effective, and visually scorable in planta platform provided by RUBY is well-suited for intuitive, non-destructive monitoring of molecular interactions in plant tissues.