Decoding anthocyanin biosynthesis regulation in Asparagus officinalis peel coloration: Insights from integrated metabolomic and transcriptomic analyses.

Asparagus is a key global vegetable crop with significant economic importance. Purple asparagus, rich in anthocyanins, stands out for its nutritional value. Despite its prominence, the molecular mechanisms driving purple peel coloration in asparagus remain unclear. This study focuses on three asparagus varieties with distinct peel colors to analyze anthocyanins in both the metabolome and transcriptome, unraveling the regulatory mechanisms. Our findings identify 30 anthocyanins, categorized into five major anthocyanin aglycones across diverse asparagus peel colors. Notably, among the 30 differentially expressed metabolites (DEMs), 18 anthocyanins displayed significantly up-regulated expression in the 'Purple Passion' variety. Key contributors include Cyanidin-3-O-rutinoside-5-O-glucoside and Cyanidin-3-O-sophoroside. Cyanidin-3-O-glucoside is most abundant in 'Purple Passion', while Petunidin-glucoside-galactoside is the least. Analysis of differentially expressed genes (DEGs) displayed 21 structural genes in anthocyanin synthesis, with F3H, DFR, ANS, and one of three UFGTs showing significantly higher expression in the 'Purple Passion' compared to 'Grande' and 'Erasmus'. Additionally, transcription factors (TFs), including 38 MYB, 33 bHLH, and 13 bZIP, also display differential expression in this variety. Validation through real-time qPCR supports the idea that increased expression of anthocyanin structural genes contribute to anthocyanin accumulation. Transient overexpression of AoMYB17 in tobacco further showed that it had the vital function of increasing anthocyanin content. This study sheds light on the mechanisms behind anthocyanin coloration in three distinct asparagus peels. Therefore, it lays the foundation for potential genetic enhancements, aiming to develop new purple-fleshed asparagus germplasms with heightened anthocyanin content.

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (ß-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 µg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 µg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 µg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation.

In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/µg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells.