Agrobacterium-mediated transformation of annatto (Bixa orellana L.): protocol optimization and overexpression of microRNA 156 in transgenic plants

Faria, Daniele Vidal; Freitas, Débora Márcia Silva; Duarte, Marcos Bruno Silva; Machado, Kleiton Lima Godoy; Matos, Elyabe Monteiro de; Batista, Diego Silva; Costa, Márcio Gilberto Cardoso; Viccini, Lyderson Facio; Nogueira, Fábio Tebaldi Silveira; Otoni, Wagner Campos

doi:10.1007/s11240-021-02211-2

Cited by 5 publications

(8 citation statements)

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“…We have previously generated B. orellana plants overexpressing the miR156 (Faria et al ., 2022). For phenotypic analyses, we selected two in vitro OE::156 lines with similar vegetative architecture and ramification patterns to avoid any bias that would affect subsequent growth ex vitro .…”

Section: Resultsmentioning

confidence: 99%

“…For phenotypic analyses, we selected two in vitro OE::156 lines with similar vegetative architecture and ramification patterns to avoid any bias that would affect subsequent growth ex vitro . Moreover, flow cytometric analyses of these two lines showed stability of nuclear DNA compared with non-transgenic lines (Faria et al ., 2022). To our knowledge, to date there are no studies characterizing the transition between juvenile and adult phases in B. orellana plants.…”

Section: Resultsmentioning

confidence: 99%

“…High levels of miR156, together with the lack of BoSPLs and BomiR172 (Fig. 4), suggested that even under greenhouse conditions, OE::156 annatto plants displayed similar overall gene regulation as in vitro -raised plants (Faria et al ., 2022). Importantly, BomiR172 was less abundant in OE::156 leaves, which might correlate with their loss of leaf cell maturation, as observed in rice and Arabidopsis (Zhu et al ., 2009; Jung et al ., 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Sequences of primers used in this study are listed in Table S1. Expression data were normalized using the 40S RIBOSOMAL PROTEIN S9 (BoRPS9) (Moreira et al ., 2018; Faria et al ., 2022) (Table S1), and expression levels of each gene and miRNA were calculated using the 2 −ΔΔCt method (Livak and Schmittgen, 2001). Differences were compared using Student’s t -test ( P ≤ 0.05).…”

Section: Methodsmentioning

confidence: 99%

“…We hypothesized that the miR156/SPL module regulates bixin production in annatto leaves in an age-dependent manner. To test this conjecture, we previously generated transgenic annatto plants overexpressing the miR156 (Faria et al ., 2022). Here, we showed that the loss of miR156-targeted SPL function prolonged the juvenile phase in annatto, leading to reduced bixin levels in leaves as late as 90 days post-germination.…”

Section: Introductionmentioning

confidence: 99%

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Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis inBixa orellanaL.

Machado

Faria

Duarte

et al. 2022

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Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. Annatto (Bixa orellana L.) is a source of bixin, an apocarotenoid used in the world's food industry worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for pharmaceutical, cosmetic, and textile industries. Here, we used genetic and molecular tools to unravel the role of the annatto age regulated miRNA156 (miR156) targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (BoSPL) genes in secondary metabolism. Low expression of several BoSPL genes in miR156 overexpressing annatto plants (OE::156) impacted leaf ontogeny, reducing bixin production and increasing abscisic acid (ABA) levels. Modulation of BoCCD4;4 and BoCCD1 expression, key genes in lycopene cleavage, was associated with diverting the carbon flux from bixin to ABA, whereas upregulation of lycopene β cyclase genes implies the xanthophyll biosynthetic pathway acted as a carbon sink in OE::156 plants. Proteomic analyses revealed low accumulation of most secondary metabolite-related enzymes in OE::156 plants, suggesting that miR156 targeted BoSPLs are required to activate several annatto secondary metabolic pathways. Our findings suggest that carbon flux in B. orellana OE::156 leaves was redirected from bixin to ABA production, indicating an age-dependent leaf dynamics of bixin biosynthesis. Importantly, our study opened a new venue to future annatto breeding programs aiming to improve bixin output.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%