Integration of biological and information technologies to enhance plant autoluminescence

Abstract

Autoluminescent plants have been genetically modified to express the fungal bioluminescence pathway (FBP). However, a bottleneck in precursor production has limited the brightness of these luminescent plants. Here, we demonstrate the effectiveness of utilizing a computational model to guide a multiplex five-gene-silencing strategy by an artificial microRNA array to enhance caffeic acid (CA) and hispidin levels in plants. By combining loss-of-function-directed metabolic flux with a tyrosine-derived CA pathway, we achieved substantially enhanced bioluminescence levels. We successfully generated eFBP2 plants that emit considerably brighter bioluminescence for naked-eye reading by integrating all validated DNA modules. Our analysis revealed that the luminous energy conversion efficiency of the eFBP2 plants is currently very low, suggesting that luminescence intensity can be improved in future iterations. These findings highlight the potential to enhance plant luminescence through the integration of biological and information technologies.

Figure 1.

Identification of multiplex genes strategy to improve the content of caffeic acid in plants. A) The processing and analysis of poplar RNA-Seq raw data. 366 RNA-Seq is downloaded from NCBI with the GEO accession number GSE78953. The processing of each RNA-Seq library includes the following steps: first, using trim-galore to remove adapters and low-quality RNA-Seq reads, followed by validation using FastQC; second, processing the data to obtain gene expression abundance using Hisat2 and FeatureCounts; and finally, conducting co-expression analysis using WGCNA on the data. B) Summary of the metabolic pathways of caffeic acid in plants. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-hydroxycinnamate:CoA ligase; C3H, coumarate 3-hydroxylase; C3′H, p-coumaroyl shikimate 3′ hydroxylase; C4H, cinnamic acid 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl CoA 3-O-methyltransferase; CCR, cinnamoyl-CoA reductase; CHI, chalcone isomerase; CHS, chalcone synthase; COMT, caffeate/5-hydroxyferulate 3-O-methyltransferase; DFR, fihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F5H, ferulate 5-hydroxylase; FLS, flavonol synthase; HCT, 4-hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyltransferase; C3′H, 4-coumaroyl shikimate/quinate 3′-hydroxylase; CSE, caffeoyl shikimate esterase. The four genes CCR, F3H, CHI and F5H serve as the reference gene. C) The Venn diagram of F3H and CHI genes from co-expression analysis in poplar. D) The Venn diagram of F5H and CCR genes from co-expression analysis in poplar. E) The correlation between relative expression range and standard deviation of 49 genes in poplar. The x-axis is the standard deviation of the normalized expression level by the mean value of each gene across all RNA-Seq data, and the y-axis is the expression range of each gene across all RNA-Seq data. F) The correlation between the percentage of caffeic acid content in tobacco change and the reciprocal of the reaction steps. The x-axis is the reciprocal of reaction steps between caffeic acid and substrate catalyzed by each gene, and the y-axis is the percentage change of caffeic acid in tobacco leaves after inhibition of corresponding gene expression. The size of the area of each point is equal to the product of the horizontal and vertical coordinates, indicating the size of the effect of inhibiting this gene on caffeic acid yield. The function of “reaction steps” is to calibrate the caffeic acid content in the instantaneous conversion test results. The higher influence weight indicates more caffeic acid increased after the inhibition of target genes in tobacco. CA (caffeic acid). G) The influence weight of different four-gene engineering combination strategies. The influence weight of the strategies is the summary of each gene's influence weight in tobacco.

Figure 2.

Optimization the strategies of accumulation caffeic acid and hispidin in plants. A) Schematic diagram of pre-amiR (precursor artificial microRNA) arrays and in vivo processing by the endogenous Csy4-processing system. HPLC analysis of amiR array strategies for accumulation of B) CA (caffeic acid) and C) hispidin contents in eFBP tobacco. The data from three individual experiments involving different plants were compared using t-tests on the same plants. Grey circles represent EV, and pink circles represent amiRs. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). D) The transcript level of F3H, FLS, CHS, and CCR by RT-qPCR in leaves from eFBP tobacco agroinfiltrated with EV (empty vector) and amiR-MYB11, respectively. The data from three individual experiments using different plants, grey circles representing EV, black circles representing amiR-MYB11. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). E) Suggested reaction catalyzed by TAL, HpaB and HpaC to convert tyrosine to caffeic acid. TAL, tyrosine ammonia lyase; HpaB, 4-hydroxyphenylacetate 3-monooxygenase; HpaC, 4-hydroxyphenylacetate 3-monooxygenase, reductase component. F) Introduction of the THH (TAL, HpaB and HpaC) pathway increased CA production in N. benthamiana. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01).

Figure 3.

eFBP2 effectively generates stronger autoluminescence across the genus of Nicotiana. A) Schematic illustrations of assembled DNA modules eFBP1 and eFBP2 for enhancing bioluminescence, T, terminator. B) Bioluminescent intensity analysis of infiltrated N. benthamiana leaves with FBP, eFBP1, and eFBP2 modules after 72 h, respectively. Scale bars, 1 cm. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). C) Analysis of the content of caffeic acid and hispidin from infiltrated leaves after 72 h. Error bars indicate means ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). D) Measurement of average photons from infiltrated Nicotiana plants (N. alata, N. tabacum) leaves with FBP and eFBP2 modules after 72 h respectively. Scale bars, 1 cm. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). E) Total amount of caffeic acid and hispidin in above leaves. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). RT-qPCR assay of amiR targets of multi-species in the genus Nicotiana as F)N. tabacum, G)N. alata, and H)N. benthamiana. Error bars indicating means ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01), and the statistical analyses described apply to panels F–H).

Figure 4.

Creation of enhanced bioluminescent plant. A) Representative bioluminescent image of eFBP and eFBP2 transgenic lines. WT as negative control. eFBP lines were described previously (Zheng et al. 2023). Scale bars, 1 cm. B) Statistical analysis of average photons emission from leaves and flowers of eFBP and eFBP2 transgenic lines. ns, No signal. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). C) Photos were taken for eFBP2, eFBP, and WT lines at 30 DAG in ambient light with 1/200 s exposure and in the dark with 10-sec exposure, respectively. Scale bars, 2 cm. D) LC–MS/MS analysis of caffeic acid and hispidin contents in leaves from eFBP and eFBP2 transgenic seedlings. ns, No signal. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). E) The performance of tobacco constitutively expressing eFBP2 at 80 DAG, captured with a Nikon D750 camera and 10-s shutter speed (see Materials and methods), Imaging was performed at room temperature. Scale bars, 3 cm. F) Total lignin content in leaves of eFBP and eFBP2 transgenic lines. Values are mean ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (**P ≤ 0.01). G) Phloroglucinol staining in cross-sections of WT and transgenic lines (eFBP and eFBP2). Scale bars, 0.1 cm. H) Average content of anthocyanin and flavonoids in eFBP and eFBP2 lines. Error bars indicate means ± SD (n = 3). Statistical significance was assessed using two-tailed t-tests (*P ≤ 0.05, **P ≤ 0.01). I) The ECE (energy conversion efficiency) of eFBP2 transgenic lines. The wavelength of green light emitted by FBP is λ₁ = 520 nm, the wavelength of red light emitted by gas exchange system is λ₂ = 620 nm. h, Planck constant, c, the speed of light. N₁, photons emission from eFBP2 transgenic plants. N₂, photons assimilated by eFBP2 transgenic plants.

Figure 5.

Integration of the FBP into engineered caffeic acid and hispidin metabolic network in plants. Biochemical reactions of fungal luciferin biosynthesis and recycling coupled with sustained caffeic acid in plants. Black solid arrows indicate a direct reaction, while grey dashed arrows represent multiple steps. Enzymes from microbes are highlighted in orange font, while compounds increased in the engineered network are in red font. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-hydroxycinnamate:CoA ligase; TAL, tyrosine ammonia lyase from Rhodotorula glutinis; HpaB, 4-hydroxyphenylacetate 3-monooxygenase; HpaC, 4-hydroxyphenylacetate 3-monooxygenase, reductase component; C3H, coumarate 3-hydroxylase; C4H, cinnamic acid 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl CoA 3-O-methyltransferase; CCR, cinnamoyl-CoA reductase; CHI, chalcone isomerase; CHS, chalcone synthase; COMT, caffeate/5-hydroxyferulate 3-O-methyltransferase; DFR, fihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F5H, ferulate 5-hydroxylase; FLS, flavonol synthase; HCT, 4-hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyltransferase; C3′H, 4-coumaroyl shikimate/quinate 3′-hydroxylase; CSE, caffeoyl shikimate esterase; HispS, hispidin synthase; NPGA, 4′-phosphopantetheinyl transferase; H3H, hispidin-3-hydroxylase; Luz, luciferase; CPH, caffeoylpyruvate hydrolase; ATP, adenosine triphosphate; Mal-CoA, malonyl CoA; CoA, co-enzyme A; NAD(P)H, nicotinamide adenine dinucleotide (phosphate). The green down arrows indicate silencing by amiRs.