Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants
© Mann et al.; licensee BioMed Central Ltd. 2012
Received: 11 January 2012
Accepted: 25 April 2012
Published: 3 May 2012
The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues.
The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed.
From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.
KeywordsEndoplasmic reticulum targeting Fluorescent proteins GFP Marker genes OFP Orange fluorescent protein Reporter genes RFP Subcellular localization Transgenic plants Visual markers
Since the discovery and isolation of the green fluorescent protein (GFP) from the Pacific jellyfish Aequorea victoria, fluorescent proteins (FPs) have become an increasingly powerful tool for use in molecular biology [1–3]. The lack of a required substrate or co-factor along with the visible fluorescence that is emitted upon excitation of the fluorophore make FPs desirable tools and reporters for a wide variety of biological applications. Recent advances in imaging methods have also enhanced the applications of FPs in plant biology . In plant gene expression studies, the genes for FPs are often overexpressed alone or fused directly to other genes of interest to monitor spatial expression patterns, and entire vector sets have been constructed for the ease of this application [5, 6]. Additionally, FPs have been used as tracking agents to detect and improve the efficiency of transient expression and stable plant transformation systems. In this case, FPs are typically under the transcriptional regulation of highly constitutive promoters such as maize ubiquitin 1 promoter (ZmUbi1) or cauliflower mosaic virus (CaMV) 35S promoter [7, 8]. While whole cell or whole organism expression of FPs are common, fluorescent reporter genes have also been cloned under the control of tissue-specific promoters for discrete expression in transgenic plants, including in pollen [9, 10], endosperm and aleurone cells [11–13], roots [14, 15] and vascular tissues [16, 17]. FPs are useful to characterize inducible promoters  and can also be fused directly to sequence peptide tags at the N- or C-terminus of a gene sequence and targeted intracellularly to specific studies .
Although other FPs have been added to the color palette during the past 12 years, GFP has remained the most commonly used FP in these studies; GFPs generally form monomers in physiologically-relevant concentrations overexpressed in the cytosol, whereas native coral-derived FPs (yellow through far red) tend to autotetramerize. Nevertheless coral-derived FPs have gained wider use in recent years in transgenic plant studies simply because many of them are brighter, owing partially to their longer wavelengths. The longer wavelengths needed to excite RFPs also have much lower levels of autofluorescence in mature green tissue as compared to UV or blue light, which is used to visualize GFP . The greatest source of autofluorescence interference is chlorophyll autofluorescence (flue light excitation) in green tissues, which can obscure GFP fluorescence.
DsRed, derived from coral Discosoma sp. was the first coral-derived FP to be used in plants as a reporter gene [20, 21], and remains the most widely-used FP in biology after GFP. Systematic mutations have since been introduced into DsRed to improve its folding dynamics, solubilization, photostability and to render monomerization, and more recent mutations and improvements in DsRed have yielded derivative FPs with increased fluorescence intensity or brightness (e.g. increased extinction coefficient, quantum yield) and altered spectral properties (e.g. shifted excitation and emission wavelengths) for reporter gene applications . These include mRFP1  along with tdTomato, mStrawberry, and, mOrange [2, 23]. Furthermore, additional sources of coral- and other organism-derived FPs beyond Discosoma sp. are constantly being discovered and have recently been exploited to produce novel FPs, potentially resulting in improved FP reporter genes for plant biotechnology . Another gene in the toolbox is mEosFP, which has recently been used in plants in various organelle-targeted versions . EosFP is a green-to-red (actually orange) photoconvertable FP that fluoresces green when by blue light. When excited by 390 nm-405 nm light for a few seconds will convert to orange emission (581 nm maximum).
The aim of this study was to survey a sample of promising FPs that have seldom been used in plants to compare their performance as reporter genes. We also set out to improve them for use in applications where whole-plant fluorescence is of paramount importance (e.g., detecting inducible expression). We evaluated and modified the following FPs (maximal excitation and emission wavelengths in nm): DsRed2 (563, 582), tdTomato (554, 581) mOrange (548, 562) and pporRFP (578, 595). Whereas many of these proteins are often called red fluorescent proteins (RFPs), as Shaner et al.  rightly point out, their emissions are all orange. Therefore we will refer to these proteins as orange fluorescent proteins (OFPs). The extinction coefficients and quantum yields of these OFPs indicated that each should be useful as markers in plants, but all started with less brightness than tdTomato: DsRed2 (38% as bright), mOrange (52% as bright), and pporRFP (56% as bright). Since we are especially interested in their use as transgenic markers, and not as fusion protein candidates, tetramerization was not deemed to be a negative factor. However, we did use some monomeric protein-coding variants chosen because of their brightness. We compared non-targeted and endoplasmic reticulum- (ER-) targeted variants under the control of a constitutive promoter in identical DNA vector backbones. Transient expression in Nicotiana benthamiana using agroinfiltration and stable transgenic Arabidopsis thaliana and tobacco (Nicotiana tabacum) plants were assayed using epifluorescence and confocal microscopy, and spectrofluorescence measurements.
Results and discussion
Agroinfiltration-mediated transient expression of OFPs
Stable expression of unaltered OFP genes in tobacco
Plant codon optimization effects of pporRFP in stable transgenic plants
ER-targeting greatly enhances the fluorescence of tdTomato and mOrange in stable transgenic plants
OFPs have emerged as the real-time in vivo reporter genes of choice for plant transformation. Endoplasmic reticulum targeting allows the accumulation of greater OFP monomers than non-targeting in select OFPs. TdTomato-ER is the most brightly fluorescing FP marker gene ever characterized in transgenic plants, followed by mOrange-ER. These OFP variants will be especially valuable in quantifying inducible expression in plant organs.
Vector construction and Agrobacteriumtransformation
The coding region of pporRFP was amplified from the vector pGem-T-gbr15 using the forward primer 5′-ATGGCTCTTTCAAAGCAAAGTGG-3′ and reverse primer 5′- TTAGTGATGGTGATGGTGATGGG-3′. mOrange and tdTomato containing vectors were obtained from the laboratory of Roger Tsien (University of California San Diego). mOrange CDS was amplified from pRSET-mOrange using the forward primer 5′- ATGGTGAGCAAGGGCGAGGAGAATA-3′ and reverse primer 5′ TTACTTGTACAGCTCGTCCATGC-3′. The tdTomato CDS was amplified from pRSET-tdTomato using the forward primer 5′- ATGGTGAGCAAGGGCGAGGAGGT-3′ and reverse primer 5′-TTACTTGTACAGCTCGTCCATGC -3′. The resulting products were cloned into the entry vector pCR8/GW-TOPO (Invitrogen). DsRed2 coding sequence (Clontech) was recombined into the entry vector pDONR/Zeo from pET160/GW-DsRed2 using BP Clonase (Invitrogen). The N-terminal signal polypeptide sequence (MKTNLFLFLIFSLLLSLSSAEF) and C-terminal ER-retention polypeptide sequence (HDEL) were added to coding sequences through assembly and amplification PCR as described by Richardson et al . Common 5′assembly primer 5′ER01 (5′-CACCATGAAAACTAATCTTTTCTTGTTTCTTATCTTTTCACTTCTTTTGAGCTTAAGCTCTGCAG-3′) and 3′ assembly primer 3′ER20 (5′- TTACAACTCGTCATGCTTGTACAGCTCGTCCATGCCG-3′) were used in conjunction with sequence specific assembly primers in assembly PCR from a template of fluorescent protein coding sequence described above. Sequence specific assembly primers were as follows: mOrangeER (5′- GGCCATGTTATTCTCCTCGCCCTTGCTCACGAACTCTGCAGAGCTTAAGCTCAAAAGAA-3′), tdTomatoER (5′- CTCTTTGATGACCTCCTCGCCCTTGCTCACGAACTCTGCAGAGCTTAAGCTCAAAAG-3′), DsRed2ER (5′- GATGACGTTCTCGGAGGAGGCGAACTCTGCAGAGCTTAAGCTCAAAAGAA). A mutagenized ppor sequence was created using the GeMS program , utilizing Arabiopsis thaliana codon usage as a template, and a codon cutoff frequency of 0.2. Full length mutatgenized ppor product was assembled from partially overlapping 60-mer oligos designed via the program Gene Design . All products of assembly PCR were cloned into the directional entry vector pENTR/D-TOPO.
OFP-containing entry vectors were recombined into the plant binary destination vector pMDC32 . Features of this vector include constitutive expression of the gene of interest via a dual CaMV 35 S promoter and hygromycin selection of transgenic plant tissue. Binary vectors were transformed into Agrobacterium tumefaciens GV3850. See the Additional file 1 for vector construction diagrams. All nine expression vectors are available via MTA (See http://plantsciences.utk.edu/stewart.htm) as follows: mMDC32-DsRed2, mMDC32-tdTomato, mMDC32-mOrange, mMDC32-pporRFP, mMDC32-pporRFP-mut, mMDC32-DsRed2-ER, mMDC32-tdTomato-ER, mMDC32-mOrange-ER, and mMDC32-pporRFP-mut-ER.
Agroinfiltration of Nicotiana benthamiana was performed as described by Liu et al.  Stable transformation of tobacco cv Xanthi was performed using the Horsch et al. method. Stable transformation of Arabidopsis Col1 ecotype was performed using the floral dip method . Arabidopsis plants were grown in growth chambers and allowed to self-fertilize. Spectrofluorometry analysis was completed on Arabidopsis T2 generation seeds, screened on MSA media containing 50 mg/L hygromycin, resistant plants were transferred to potting media and grown in growth chambers (10 hr day length, 18°C/14°C day/night). Plants were 9-week-old rosettes when spectrofluometry was performed. Self-fertilized tobacco plants were grown in the greenhouse (16 hr. day, 27-30°C). Tobacco plants for spectrofluorometry analysis were started as T1 segregating seeds grown in potting media, screened for fluorescent protein expression using microscopy, transplanted to individual pots and grown to six-weed old stage under greenhouse conditions.
Epifluorescent and confocal microscopy and spectrofluorometry
Epifluorescent microscopy of plants was performed using the tdTomato filter set: 535/30 nm excitation and 600/50 nm band pass emission or the mOrange filter set: 535/30 excitation and 585/40 nm band pass emission filter (Olympus stereo microscope model SZX12, Olympus America, Center Valley, PA, USA). Confocal microscopy images were produced using a Leica TCS SP2 microscope (Buffalo Grove, IL. USA), which allows for adjustable bandwidths for the detected fluorescence. The samples were excited with a 543 nm HeNe laser and fluorescence emission was collected from 555–604 nm for mOrange, 570–620 nm for DsRed and tdTomato, and 590 – 610 nm for pporRFP. Chlorophyll autofluorescence was checked for each sample by exciting the sample with 488 nm light from an argon ion laser and collecting emission from 650–750 nm. When chlorophyll autofluorescence was imaged along with the fluorescent protein, images were collected using sequential scanning to prevent bleedthrough fluorescence. Fluorescence measurements (i.e., those results displayed in Figures 234 and 5) were made using spectrofluorometry according to methods described by Millwood et al.  but with an updated Fluorolog®-3 system (Jobin Yvon and Glen Spectra, Edison, NJ, USA). For each of the samples, the youngest fully expanded leaf was chosen to control for developmental stage.
Transgenic plants were statistically analyzed using a one-way analysis of variance in SAS where the response variable was fluorescence measurements from spectrofluorometry. If significant differences were found, mean separations were performed using Fisher’s LSD to determine which genotypes were significantly different at the P = 0.05- to P = 0.0001 levels.
We are most appreciative of Mikhail Matz for his collaboration and sharing pporRFP gene and Roger Tsien for sharing the tdTomato and mOrange FP genes and also for their helpful comments on the manuscript. We thank Matthew D. Halfhill for his contributions. We appreciate funding from the US Armed Forces Medical Intelligence Center, the USDA, the BioEnergy Science Center, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science, and the University of Tennessee. Binary vectors for heterologous expression are available for non-profit organizations (See http://plantsciences.utk.edu/stewart.htm).
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