Plant-based production of recombinant proteins has emerged as an efficient and cost-effective alternative to microbial fermentation and mammalian cell culture systems. Chloroplasts harbor high plasmid copy numbers and can be stably transformed, making them efficient platforms for protein production. In the present study, we used green fluorescent protein (GFP) as a reporter to compare the three major chloroplast promoters (rrn, psbA, and rbcL) involved in protein production in Nicotiana tabacum cv. “Petit Havana.” Three chloroplast transformation vectors were constructed, each regulated by a different promoter, and the transformation was performed via biolistic particle bombardment. Transformants were selected based on spectinomycin resistance and were confirmed by PCR. Among the three promoters, psbA showed the highest transformation efficiency and protein expression levels. Reverse transcription quantitative PCR showed that the mRNA levels (relative to Actin) for psbA (218.21±19.64) were nearly twice that of rbcL (126.60±8.78), and five times that of rrn (43.27±1.57). This transcriptional hierarchy was also observed at protein level. Immunoblotting showed the GFP levels (relative to psbA) were: psbA (1.00), rbcL (0.87), and rrn (0.77), whereas quantification through ELISA revealed relative GFP concentrations of: 616.2±28.7 ng/g LFW for psbA, 510.3±32.4 ng/g LFW for rbcL, and 338.9±100.2 ng/g ng/g LFW for rrn. These quantitative results demonstrate the importance of promoter selection for efficient expression of recombinant proteins in chloroplasts and show that the psbA promoter is suitable for high-efficiency chloroplast expression systems, providing a foundation for advancing plant-based molecular farming.

Chrysanthemum is the most popular ornamental plant, after roses and lilies. The cauliflower mosaic virus (CaMV) promoter, which remains the most widely used promoter in dicotyledons, is a very strong promoter with sufficient effects in most crops. However, weak expression has often been reported in Chrysanthemum. Therefore, we searched for constitutive promoters available in Chrysanthemum. Based on the transcriptome analysis data of Chrysanthemum, nine constitutively expressed genes were selected, and each promoter region (1.0–3.0 kb) was isolated by genome walking. Only two of the nine promoters expressed GUS in tobacco and chrysanthemums. The major motif of the CmERF promoter (U41, 2060 bp) was related to the regulation of ethylene (ERELEE4) or gibberellin (PYRIMIDINEBOXOSRAMY1 and WRKY71OS). Similarly, the motif of the CmGA2 ox promoter (U47, 1060 bp) also contained gibberellin signaling factors, such as PRIMIDINEBOXHVEPB1 and WRKY71OS. Both promoters showed strong systemic expression in tobacco using GUS staining. Although weaker than in tobacco, significant expression was confirmed in the flowers and stems in chrysanthemum. The results of the GUS activity assay using chrysanthemum transformants showed that the transgenic line (#12) containing the U47 promoter had higher expression in all tissues than that containing the 35S-CaMV promoter. The U41 promoter was found to have a higher expression than the 35S-CaMV promoter in the stem.

Plants grown under stress conditions generate excessive reactive oxygen species resulting in cell death. Therefore, plants activate the protection mechanism via antioxidant accumulation. Anthocyanins are flavonoid-derived secondary metabolites with high antioxidant properties. In this study, we analyzed and characterized the promoter region of RsMYB1, a positive regulator of anthocyanin biosynthesis. The RsMYB1 promoter was designed with four different fragment lengths (MP1, -1034; MP2, -830; MP3, -633; and MP4, -430 bp), and then each RsMYB1 promoter region was fused into a GUS gene for Arabidopsis transformation. The expression patterns of the RsMYB1 promoter constructs were analyzed at different developmental stages and under various abiotic stresses. The GUS expression pattern steadily increased with plant growth, and coincided with enzyme activity and a histochemical GUS assay. In response to drought, salt, sucrose, and low temperature, the GUS transcript level was highly expressed in MP4 in parallel with GUS enzyme activity. These assays indicated that the proximal region (-430 to -1 bp) of RsMYB1 was the core sequence that was induced by salt and low temperature. The expression level of RsMYB1 in the leaves of radish was highly activated and was consistent with the anthocyanin content under salt and low temperature conditions. These results suggest that induction of the RsMYB1 gene can activate the biosynthesis of anthocyanins, which are expected to help plants adapt to stress conditions due to their antioxidant activity.

Promoters are essential regulatory elements for efficiently expressing a gene of interest in a target tissue of a organism. Therefore, the identification of a suitable promoter is important in plant biotechnology. In this study, four promoters were selected and identified to be constitutively or tissue-specifically expressed in Brassica rapa bacterial artificial chromosome (BAC) clones using the results of transcriptomic analysis of Brassica rapa and open-source database information on Arabidopsis thaliana. The 2 kb region of the 5′ upstream was isolated from the Brassica rapa genomic DNA for each promoter. The four promoters were then fused to β-glucuronidase (gus) and green fluorescent protein (gfp) genes, and the recombinant transgenes were introduced into Arabidopsis. As a result of histochemical GUS staining, GFP fluorescence and RT-PCR, the gus gene was observed to be expressed constitutively in all tissues using all four promoters. A GUS activity assay using fluorescent 4-MUG revealed that the BR11 promoter showed similar activity to the CaMV35S promoter, while the BR4, BR15, and BR16 promoters showed 1.5, 4, and 18-fold higher activities than the CaMV35S promoter, respectively. These results indicate that these four promoters could be used to incorporate useful genes with enhanced function into crops of interest.

To identify and apply tissue-specific promoters is one of the major challenges in plants genetic engineering for optimizing efficient expression of interest genes in appropriate tissues. In this research, open-source database information of Arabidopsis thaliana was adapted to determine root-specific expressed promoter region. A total seven sequences that might function as a root-specific promoter element were initially isolated from Arabidopsis genomic DNA. Then seven promoters were cloned into pBGWFS7 in which β-glucuronidase (gus) and green fluorescent protein (gfp) genes were linked. The GUS activities were measured in different tissues of transgenic Arabidopsis by both histochemical GUS staining and fluorescent 4 methylumbelliferyl β-D-glucuronide (MUG) assay. To confirm root-specific expression, GFP-confocal microscope analysis was conducted in Arabidopsis transgenic plant. As a result, the five promoters showed strong GUS activity in the root tissue as compared with the CaMV35S promoter. To test crop application availability as a root-specific promoter, seven promoters were introduced into tomato plants and confirmed transient expression using Agrobacterium rhizogenesis ARqua1 root-nodule inducible strain. Two promoters showed that gus genes were specifically expressed in roots of transgenic tomato plant. Taken together, the novel seven promoters showed specific activity in root suggesting that it is applicable in crop improvement.

Anthocyanins are the major pigments contributing to flower coloration. A 1584 bp 5’ upstream sequence of ALCHS2 gene was isolated from Acapulco lily (Lilium Oriental hybrid cv. Acapulco). Computer-based analyses (GeneScan, AtPAN) predicted a CAATBOX1 and putative transcription factor-binding sites, including tissue-specific elements. When gALCHS7 promoter–gus fusion was introduced to petunia (‘Dream Red’), all ten putative transgenic plants showed localized GUS activity in the anther, but five of them also showed weak GUS activity in the ovule. No distinctive signal in the leaf and petal was detected in the same stage. To clearly determine the operation of the promotor region, anther and ovule tissues of transgenic line 6 were fixed in paraffin for dark-field analysis. At 1 cm length of floral bud, a GUS signal was not observed in the anther, but weak expression was observed in the ovule. Before anthesis, GUS protein was highly expressed in the pollen, endothecium, and epidermis. Fluorometric GUS assays of individual organs taken from four transgenic plants demonstrated that all lines showed high GUS activity in the anther compared to 35S CaMV promoter (pBI1 121), except line 34. Using the truncated promoters by cis-acting elements, we found that minimal region (gALCHS7-7, 270 bp) displayed GUS expression only in the anther, though at weaker activity than in the original promoter.