# Phenotypic Characterization of TransgenicMiscanthus sinensis Plants Overexpressing Arabidopsis Phytochrome B

Authors: Ok-Jin Hwang; Soo-Hyun Lim; Yun-Jeong Han; Ah-Young Shin; Do-Soon Kim; Jeong-Il Kim
Journal: International Journal of Photoenergy
 (2014)

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## Abstract

Phytochromes are dimeric pigment proteins with reversible photochromism between red and far-red light-absorbing forms. They are photoreceptors that regulate various aspects of plant growth and development and have been used for biotechnological applications to improve agricultural performance of crops.Miscanthus species have been suggested as one of the most promising energy crops. In this paper, Arabidopsis phytochrome B (PHYB) gene was introduced into Miscanthus sinensis using Agrobacterium-mediated transformation method that we developed recently, with the herbicide resistance gene (BAR) as a selection marker. After putative transgenic plants were selected using the herbicide resistance assay, genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by Northern blot analysis. Compared to nontransformed control plants, transgenic plants overexpressing PHYB showed phenotypes with increased phytochrome B function, which includes increased chlorophyll content, decreased plant height, and delayed flowering. Therefore, these results suggest that Arabidopsis phytochrome B is functional in M. sinensis and provide a method to develop Miscanthus varieties with enhanced agricultural performance using phytochromes.


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## Body

## 1. Introduction

Phytochromes are photoreceptors that regulate various aspects of plant growth and development in response to red and far-red light signals from the environment [1, 2]. They are dimeric chromopeptides with each monomer (120–130 kDa) possessing a covalently linked open-tetrapyrrole chromophore called phytochromobilin via a thioether linkage to a cysteine residue [3]. The most important characteristic of phytochromes is its reversible photochromism: the property of changing color on photon absorption and of reverting to the original form on the absorption of another photon. Phytochromes are synthesized in red light-absorbing Pr form (λ
max
⁡
=
660 nm) which can be phototransformed into far-red light-absorbing Pfr form (λ
max
⁡
=
730 nm) upon exposure to red light. The functional activities of the phytochromes are modulated by the photochromic transformation between these two forms [4, 5]. Generally, the Pr-to-Pfr phototransformation of phytochrome is known to induce the highly regulated signaling network for photomorphogenesis in plants [6–8].Phytochrome-mediated photomorphogenic responses include germination, stem and leaf growth, chloroplast development, biosynthesis of chlorophylls and other pigments, shade avoidance, circadian rhythm, and flowering [9]. In the aspect of biotechnological applications of phytochromes, physiological analysis of plants overexpressing phytochrome transgenes has previously revealed the potential of transgenic approaches to modifying the architecture of crop plants [10]. As an example, tobacco plants expressing oat phytochrome A transgene (PHYA) exhibit proximity-conditional dwarfing which leads to up to 20% improvement in harvest index [11]. In principle, phytochromes mediate plant shade avoidance responses in which plants react to far-red radiation reflected from neighbors and induce elongation growth to prevent overtopping by competitors growing alongside [12]. If the shade avoidance responses are induced, the reallocation of resources into elongation growth might result in reduced leaf and storage organ production with a decrease in crop yield [13]. Thus, the shade avoidance responses can be detrimental in agriculture because it limits the planting density. Therefore, it has suggested that plant productivity can be enhanced by reducing perception of the neighbors (i.e., suppression of shade avoidance responses) through the manipulation of phytochrome red/far-red light perception [14].Miscanthusspecies are tall perennial rhizomatous grasses with C4 photosynthesis which tend to give high biomass yields annually over a wide range of climates [15, 16]. Thus, they are suggested as one of the most promising energy crops for biomass production [17]. So far, the triploid hybridMiscanthus×giganteusbetween the diploidM. sinensisand the tetraploidM. sacchariflorusis currently the commercially grown species in the genus [18, 19]. However, this hybrid is sterile and lacks genetic variation, so the necessity for seed-propagated varieties in genetically stable and fertile species, such asM. sinensis, has been raised [20].The genetic improvement by conventional breeding has been used to improve traits of many species, but its success has been limited by barriers such as sexual reproduction and the relatively long time periods required for breeding programs. More recently, genetic engineering methods with the help of plant transformation technologies have been used to improve many species more efficiently, whereby useful traits have been introduced from a broader range of sources within an economically viable time frame [21]. In the case ofMiscanthusspecies, the genetic transformation systems have been established very recently using particle bombardment-mediated andAgrobacterium-mediated methods [22, 23]. However, there is no report thus far of transgenic Miscanthus plants with a useful gene other than selective marker genes. Therefore, genetically engineered Miscanthus plants need to be further developed by introducing a useful gene using the established transformation method.The objective of this study was to develop transgenicM. sinensis plants overexpressingArabidopsis phytochrome B gene (PHYB) using theAgrobacterium-mediated genetic transformation method that we developed recently. In addition, the phenotypes of the transgenic plants were investigated in terms of phytochrome function, which includes chlorophyll content, plant height, and flowering time. In the present study, we successfully obtained transgenicM. sinensis plants with a vector harboringPHYB as well as the herbicide resistance gene (BAR) as a selection marker and found that the overexpression ofPHYB increased chlorophyll content, reduced plant height, and delayed flowering inM. sinensis plants. Therefore, the present work provides a method for the development of genetically engineeredMiscanthus varieties with enhanced agricultural performance using phytochromes.


## 2. Materials and Methods

### 2.1. Preparation of RecombinantArabidopsis Phytochrome B Proteins

Chromophore-assembled holoproteins ofArabidopsis phytochrome B (phyB) were expressed, reconstituted, and purified as previously described [24, 25]. Full-lengthArabidopsis PHYB gene was subcloned into aPichia expression vector pPIC3.5K (Invitrogen). Ten amino acid streptavidin affinity tag from pASK75 vector (Biometra) was attached to the 3′ end of thePHYB gene. The primers 5′-CTCCCCGGGTACGTAACCATGGTTTCCGGAGTCGGGG-3′ (forward,SmaI/SnaBI) and 5′-TCGCAGCGCTATATGGCATCATCAGCATCATG-3′ (reverse,Eco47III) were used for the subcloning. The pPIC3.5K construct bearingPHYBwas then transformed intoPichia cells by means of a Micropulser Electroporation apparatus (Bio-Rad). Recombinant phytochrome proteins were expressed in thePichia expression system (Invitrogen) and purified by streptavidin affinity chromatography (IBA), according to the procedure described by the manufacturer. Phycocyanobilin (PCB) was prepared fromSpirulina platensis extracts by methanolysis as described [25] and used as chromophores for the holo-phytochrome assembly. Crude extracts were prepared by breakingPichia cells in liquid nitrogen using a homogenizer (Nihonseiki Kaisha). The phytochrome samples were then precipitated by adding 0.23 g/L ammonium sulfate and resuspended in a buffer (100 mM Tris, pH 7.8, 1 mM EDTA), the PCB chromophores in DMSO were added to the samples at a final concentration of 10 μM, and the mixture was incubated forin vitro reconstitution on ice for 1 h. After dialysis to remove free chromophores, the samples were loaded onto affinity chromatography columns and holo-phyB proteins were purified.


### 2.2. Zn2+ Fluorescence and Spectroscopic Analysis

For Zn2+ fluorescence assay to assess the chromophore ligation, the protein samples were analyzed on a 10% SDS-PAGE gel and the gel was soaked in 20 mM zinc acetate/150 mM Tris-HCl (pH 7.0) for 5–30 min at room temperature with gentle shaking. Zinc fluorescence of holo-phytochromes was visualized under UV light (312 nm). For the spectroscopic analysis of recombinant phyB, absorption spectra were recorded by a diode array UV/VIS spectrophotometer (Cary) after red or far-red light irradiation. All the spectroscopic experiments were carried out under the green safety light condition, which consisted of a white fluorescent lamp equipped with a plastic filter (Rosco) with a maximal transmittance at 500 nm, and a fiber optic illuminator system (Cole-Parmer) equipped with 656 and 730 nm interference filters (Oriel) was used as a light source. The light intensity was 8 W/m2 for red light and 6 W/m2 for far-red light. The samples were illuminated with red for 15 min or far-red light for 10 min. A difference spectrum was obtained by subtracting the Pfr spectrum from the Pr spectrum and used to determine the interpeak distance between Pr and Pfr absorption wavelength maxima (Δ of maxima) and the ratio of Pr and Pfr absorbance peaks (APfr/APr).


### 2.3. Plasmid and Agrobacterium Strain Used for Genetic Transformation

Arabidopsis PHYB gene (AT2G18790) was subcloned into the binary vector pCAMBIA3301 withBamHI/SmaI under the control of maize ubiquitin promoter (Pubi) andAgrobacterium tumefaciens NOS gene terminator. pCAMBIA3301 carriesBAR gene for herbicide resistance as a selectable marker, and theBAR gene encodes a phosphinotricin acetyltransferase that confers resistance to the herbicide, phosphinotricin (PPT). The binary vector DNA was then used to introduce intoA. tumefaciens strain EHA105 by the freeze-thaw method [26].


### 2.4. Generation of Putative TransgenicM. sinensis Plants

SNU-M-045 germplasm seeds ofM. sinensis that have been maintained in Seoul National University were used for the transformation, and tissue culture and genetic transformation ofM. sinensis were performed as described [23]. Mature seeds-derived embryogenic calli andA. tumefaciens EHA105 harboring pCAMBIA3301 withPHYB were used for the genetic transformation. Embryogenic calli were induced and cultured on the callus induction medium (MS salts and vitamins, 3% sucrose, 3 mg/L 2,4-D, 750 mg/L MgCl2
·6H2O, 25 mM L-proline, 0.2 g/L Gelrite, pH5.7) for 8 to 10 weeks. The embryogenic calli were then immersed inAgrobacterium suspensions and incubated for 15 min with gentle shaking followed by removal of excess bacteria and air-drying on filter paper. Infected calli were then transferred onto cocultivation medium (MS salts and vitamins, 2% sucrose, 1% glucose, 3 mg/L 2,4-D, 400 μM acetosyringone, 3 g/L Gelrite, pH  5.7) and incubated in the dark at 25°C for 5 days. During the transformation process, GUS staining assays were performed to check whether or not the embryogenic calli were transformed. After transformation, transgenic calli were selected on the selection medium containing 5 mg/L PPT, and transgenic shoots were induced on the regeneration medium with 3 mg/L PPT. Plantlets with well-developed roots were established in soil and grown for 2 weeks, prior to herbicide treatment. Herbicide resistance assay was then performed by spraying 0.4% (v/v) BASTA (which contains 18% glufosinate ammonium), and the herbicide resistance of putative transgenic plants was determined after 14 days.


### 2.5. Molecular Analyses of TransgenicM. sinensis Plants

Genomic PCR, Southern blot, and Northern blot analyses of transgenic plants were performed as previously described [27, 28]. For genomic PCR analysis, total genomic DNA was isolated from the leaves of greenhouse-grown plants, and the coding region for thePHYB orBARtransgene was amplified by PCR from either genomic DNA or a positive-control vector, using the following sets of oligonucleotide primers: 5′-TCGGCGTTCCGGTCCATGGTTAG-3′ (forward) and 5′-GCAGCGCTCAGTGTCTGCGTTCTCAAAACG-3′ (reverse) forPHYB, 5′-CTACCATGAGCCCAGAACGACG-3′ (forward) and 5′-CTGCCAGAAACCCACGTCATGCCAGTTC-3′ (reverse) forBAR. The actin gene (ACT) ofM. sinensis was also amplified using the same template and the primers 5′-AACTGGGATGATATGGAGAA-3′ (forward) and 5′-CCTCCAATCCAGACACTGTA-3′ (reverse) and then run as a loading control of genomic DNA. The PCR products ofPHYB,BAR, andACT were expected to be 1106, 421, and 1,046 bp, respectively.For the Southern blot analysis, the genomic DNA was digested with eitherHindIII orEcoRI, and hybridizations were carried out with theBARgene probe that was labeled with [α
32P] dCTP using the Radiprime II Random Prime Labeling System (Amersham Biosciences). For Northern blot analysis, total RNA was extracted from leaves using Trizol reagent (Invitrogen), and hybridizations were carried out with [α
32P] dCTP-labeledPHYBprobe.


### 2.6. Measurement of Chlorophyll Content

Chlorophyll (chl) content was measured with the third leaf of the tillers harvested on DOY (day of year) 273. The leaves were extracted with 80% acetone and the solution absorbance was measured at 645 nm (A
645) and 663 nm (A
663). Total chlorophyll content was calculated from the equation: total chlorophyll (μg/mL)=
20.2
×
A
645
+
8.02
×
A
663. The relative chlorophyll content was then calculated by setting the chlorophyll content of nontransgenic control plant to 100%.


### 2.7. Phenotypic Analyses of TransgenicM. sinensis Plants

M. sinensis plants in soil were grown and vegetatively propagated in a culture room (24–26°C with a 16 h photoperiod). For the investigation of phenotypes, the plants were then transferred and planted from the culture room onto an isolated LMO greenhouse of Seoul National University in Suwon, Korea (N 37° 16′ 15.08′′, E 126° 59′ 22.02′′). Plant heights were measured as the lengths from the ground to the top of each plant. Heading and flowering dates were defined as the emergence of the panicle tip from the flag leaf sheath and the opening of the first flower, respectively.


### 2.8. Statistical Analysis

The results of the physiological parameters were analyzed using ANOVA with IBM SPSS statistics 20 software. Significant difference from the control value(s) was determined atP
<
0.05 level. All of the data represented the mean ± SD or SE of at least three independent experiments.


## 2.1. Preparation of RecombinantArabidopsis Phytochrome B Proteins

Chromophore-assembled holoproteins ofArabidopsis phytochrome B (phyB) were expressed, reconstituted, and purified as previously described [24, 25]. Full-lengthArabidopsis PHYB gene was subcloned into aPichia expression vector pPIC3.5K (Invitrogen). Ten amino acid streptavidin affinity tag from pASK75 vector (Biometra) was attached to the 3′ end of thePHYB gene. The primers 5′-CTCCCCGGGTACGTAACCATGGTTTCCGGAGTCGGGG-3′ (forward,SmaI/SnaBI) and 5′-TCGCAGCGCTATATGGCATCATCAGCATCATG-3′ (reverse,Eco47III) were used for the subcloning. The pPIC3.5K construct bearingPHYBwas then transformed intoPichia cells by means of a Micropulser Electroporation apparatus (Bio-Rad). Recombinant phytochrome proteins were expressed in thePichia expression system (Invitrogen) and purified by streptavidin affinity chromatography (IBA), according to the procedure described by the manufacturer. Phycocyanobilin (PCB) was prepared fromSpirulina platensis extracts by methanolysis as described [25] and used as chromophores for the holo-phytochrome assembly. Crude extracts were prepared by breakingPichia cells in liquid nitrogen using a homogenizer (Nihonseiki Kaisha). The phytochrome samples were then precipitated by adding 0.23 g/L ammonium sulfate and resuspended in a buffer (100 mM Tris, pH 7.8, 1 mM EDTA), the PCB chromophores in DMSO were added to the samples at a final concentration of 10 μM, and the mixture was incubated forin vitro reconstitution on ice for 1 h. After dialysis to remove free chromophores, the samples were loaded onto affinity chromatography columns and holo-phyB proteins were purified.


## 2.2. Zn2+ Fluorescence and Spectroscopic Analysis

For Zn2+ fluorescence assay to assess the chromophore ligation, the protein samples were analyzed on a 10% SDS-PAGE gel and the gel was soaked in 20 mM zinc acetate/150 mM Tris-HCl (pH 7.0) for 5–30 min at room temperature with gentle shaking. Zinc fluorescence of holo-phytochromes was visualized under UV light (312 nm). For the spectroscopic analysis of recombinant phyB, absorption spectra were recorded by a diode array UV/VIS spectrophotometer (Cary) after red or far-red light irradiation. All the spectroscopic experiments were carried out under the green safety light condition, which consisted of a white fluorescent lamp equipped with a plastic filter (Rosco) with a maximal transmittance at 500 nm, and a fiber optic illuminator system (Cole-Parmer) equipped with 656 and 730 nm interference filters (Oriel) was used as a light source. The light intensity was 8 W/m2 for red light and 6 W/m2 for far-red light. The samples were illuminated with red for 15 min or far-red light for 10 min. A difference spectrum was obtained by subtracting the Pfr spectrum from the Pr spectrum and used to determine the interpeak distance between Pr and Pfr absorption wavelength maxima (Δ of maxima) and the ratio of Pr and Pfr absorbance peaks (APfr/APr).


## 2.3. Plasmid and Agrobacterium Strain Used for Genetic Transformation

Arabidopsis PHYB gene (AT2G18790) was subcloned into the binary vector pCAMBIA3301 withBamHI/SmaI under the control of maize ubiquitin promoter (Pubi) andAgrobacterium tumefaciens NOS gene terminator. pCAMBIA3301 carriesBAR gene for herbicide resistance as a selectable marker, and theBAR gene encodes a phosphinotricin acetyltransferase that confers resistance to the herbicide, phosphinotricin (PPT). The binary vector DNA was then used to introduce intoA. tumefaciens strain EHA105 by the freeze-thaw method [26].


## 2.4. Generation of Putative TransgenicM. sinensis Plants

SNU-M-045 germplasm seeds ofM. sinensis that have been maintained in Seoul National University were used for the transformation, and tissue culture and genetic transformation ofM. sinensis were performed as described [23]. Mature seeds-derived embryogenic calli andA. tumefaciens EHA105 harboring pCAMBIA3301 withPHYB were used for the genetic transformation. Embryogenic calli were induced and cultured on the callus induction medium (MS salts and vitamins, 3% sucrose, 3 mg/L 2,4-D, 750 mg/L MgCl2
·6H2O, 25 mM L-proline, 0.2 g/L Gelrite, pH5.7) for 8 to 10 weeks. The embryogenic calli were then immersed inAgrobacterium suspensions and incubated for 15 min with gentle shaking followed by removal of excess bacteria and air-drying on filter paper. Infected calli were then transferred onto cocultivation medium (MS salts and vitamins, 2% sucrose, 1% glucose, 3 mg/L 2,4-D, 400 μM acetosyringone, 3 g/L Gelrite, pH  5.7) and incubated in the dark at 25°C for 5 days. During the transformation process, GUS staining assays were performed to check whether or not the embryogenic calli were transformed. After transformation, transgenic calli were selected on the selection medium containing 5 mg/L PPT, and transgenic shoots were induced on the regeneration medium with 3 mg/L PPT. Plantlets with well-developed roots were established in soil and grown for 2 weeks, prior to herbicide treatment. Herbicide resistance assay was then performed by spraying 0.4% (v/v) BASTA (which contains 18% glufosinate ammonium), and the herbicide resistance of putative transgenic plants was determined after 14 days.


## 2.5. Molecular Analyses of TransgenicM. sinensis Plants

Genomic PCR, Southern blot, and Northern blot analyses of transgenic plants were performed as previously described [27, 28]. For genomic PCR analysis, total genomic DNA was isolated from the leaves of greenhouse-grown plants, and the coding region for thePHYB orBARtransgene was amplified by PCR from either genomic DNA or a positive-control vector, using the following sets of oligonucleotide primers: 5′-TCGGCGTTCCGGTCCATGGTTAG-3′ (forward) and 5′-GCAGCGCTCAGTGTCTGCGTTCTCAAAACG-3′ (reverse) forPHYB, 5′-CTACCATGAGCCCAGAACGACG-3′ (forward) and 5′-CTGCCAGAAACCCACGTCATGCCAGTTC-3′ (reverse) forBAR. The actin gene (ACT) ofM. sinensis was also amplified using the same template and the primers 5′-AACTGGGATGATATGGAGAA-3′ (forward) and 5′-CCTCCAATCCAGACACTGTA-3′ (reverse) and then run as a loading control of genomic DNA. The PCR products ofPHYB,BAR, andACT were expected to be 1106, 421, and 1,046 bp, respectively.For the Southern blot analysis, the genomic DNA was digested with eitherHindIII orEcoRI, and hybridizations were carried out with theBARgene probe that was labeled with [α
32P] dCTP using the Radiprime II Random Prime Labeling System (Amersham Biosciences). For Northern blot analysis, total RNA was extracted from leaves using Trizol reagent (Invitrogen), and hybridizations were carried out with [α
32P] dCTP-labeledPHYBprobe.


## 2.6. Measurement of Chlorophyll Content

Chlorophyll (chl) content was measured with the third leaf of the tillers harvested on DOY (day of year) 273. The leaves were extracted with 80% acetone and the solution absorbance was measured at 645 nm (A
645) and 663 nm (A
663). Total chlorophyll content was calculated from the equation: total chlorophyll (μg/mL)=
20.2
×
A
645
+
8.02
×
A
663. The relative chlorophyll content was then calculated by setting the chlorophyll content of nontransgenic control plant to 100%.


## 2.7. Phenotypic Analyses of TransgenicM. sinensis Plants

M. sinensis plants in soil were grown and vegetatively propagated in a culture room (24–26°C with a 16 h photoperiod). For the investigation of phenotypes, the plants were then transferred and planted from the culture room onto an isolated LMO greenhouse of Seoul National University in Suwon, Korea (N 37° 16′ 15.08′′, E 126° 59′ 22.02′′). Plant heights were measured as the lengths from the ground to the top of each plant. Heading and flowering dates were defined as the emergence of the panicle tip from the flag leaf sheath and the opening of the first flower, respectively.


## 2.8. Statistical Analysis

The results of the physiological parameters were analyzed using ANOVA with IBM SPSS statistics 20 software. Significant difference from the control value(s) was determined atP
<
0.05 level. All of the data represented the mean ± SD or SE of at least three independent experiments.


## 3. Results and Discussion

### 3.1. Photochemical Properties ofArabidopsis phyB

There are five isoforms of plant phytochromes (phyA–phyE) in the dicot model plantArabidopsis thaliana, among which phyA and phyB have been known to play major roles in plants [29, 30]. For example, phyA regulates seed germination and seedling growth in response to far-red light (FR), whereas phyB regulates seed germination in response to red light (R), plant growth and shade avoidance in response to R/FR ratio, and flowering time [4]. Especially, it has been well known that phyB-deficient mutant (hy3) shows phenotypes of constitutive shade avoidance and early flowering [31], suggesting that phyB is necessary to suppress the shade avoidance responses and flowering in plants, which is necessary to improve plant productivity. Therefore, we planned to introduceArabidopsis phyB intoM. sinensis and to investigate the phenotypes of the monocot crop overexpressing dicot phyB.Before the genetic transformation, we first performed the photochemical analysis ofArabidopsis phyB using purified recombinant proteins, because phyB has not been characterized well due to its low abundance in native tissue [32]. In contrast, the spectral properties of phyA have been well characterized because of the relatively high abundance of this photoreceptor in dark-grown tissue (~100 times more than phyB). In the present study, we expressed and purified recombinant phyB proteins successfully using thePichia protein expression system and streptavidin affinity chromatography (Figure 1(a)). PurifiedArabidopsis phyB showed ligation with the chromophore phycocyanobilin (PCB) and normal patterns of absorbance and difference spectra (Figure 1). Spectroscopic analysis of the purified phyB proteins revealed that the absorbance wavelength maxima of Pr (λ
Pr) and Pfr (λ
Pfr) forms are 650 nm and 714 nm, respectively. Compared with the photochemical properties of oat phyA in a previous report [25],Arabidopsis phyB showed similar properties to phyA including Δ of maxima (64 m) and APfr/APr (1.09) obtained from the difference spectrum. The only observed difference was that the absorbance wavelengths of phyB were slightly blue-shifted (4–6 nm) compared with the absorbance peaks of oat phyA (654 nm for Pr and 720 nm for Pfr). Collectively, the results demonstrate that PCB-assembled recombinantArabidopsis phyB has a typical photochromism of plant phytochromes.Photochemical analysis of purifiedArabidopsis phyB proteins assembled with phycocyanobilin (PCB). (a) Purification of PCB-assembled phyB proteins by streptavidin affinity chromatography. SDS-PAGE gel was shown with Coomassie blue staining. Lane S: Fermentas prestained protein ladder; lane C: crude extract; lane F: flow-through from the affinity column; lanes 2–5: elution fractions of holo-phyB. Zinc-induced fluorescence assay (Zinc) was shown to confirm chromophore-assembled phyB proteins. (b) Absorption and difference spectra of the purified PCB-assembled phyB proteins. Absorption spectra of Pr (solid line) and Pfr (dotted line) were shown, and the difference spectrum (gray line) was obtained by subtracting the Pfr absorption spectrum from the Pr absorption spectrum.
(a)
(b)


### 3.2. Production of TransgenicM. sinensis Plants OverexpressingArabidopsis PHYB

Embryogenic calli derived from mature seeds and the binary vector pCAMBIA3301 harboring thePHYB gene (Figure 2(a)) was used for transformation by followingAgrobacterium-mediated transformation method that we recently developed [23]. Since the binary vector used for the transformation contained theBAR herbicide resistance gene, putative transgenic creeping bentgrass plants were identified by herbicide resistance assay. Thein vitro regenerated putative transgenic lines were grown in a greenhouse for two weeks and then sprayed with 0.4% (v/v) BASTA. Following herbicide treatment, nontransformed wild-type control plant (NT) died within 14 days, whereas all of the putative transgenic plants were resistant to the herbicide (Figure 2(b)). These results indicate that theBARgene was expressed in these transgenic plants. When theAgrobacterium-mediated transformation efficiency was calculated as a percentage of herbicide-resistant transgenic lines obtained from all infected calli, a transformation efficiency of 0.71% (4 independent lines out of 567 infected calli) was obtained, which is similar to the reported efficiencies (0.84~0.20%). These putative transgenic lines were used further for molecular and phenotypic analyses.Construct for the transformation and herbicide resistance assay of putative transgenic plants. (a) T-DNA region of the binary vector plasmid pCAMBIA3301 harboringArabidopsis PHYB gene. RB: right border; LB: left border; P
35
S: CaMV 35S promoter; Pubi: ubiquitin promoter; intron GUS: GUS coding region with catalase intron insertion;PHYB: full-length phyB gene ofArabidopsis;BAR: phosphinotricin acetyltransferase gene coding region; T1: CaMV 35S gene terminator; T2:A. tumefaciens NOS gene terminator. Arrows indicate directions of transcription. (b) Herbicide resistance assay. Numbers in lanes represent putative transgenic plants selected for this analysis. 0.4% BASTA was sprayed onto nontransformed wild-type control plant (NT) and the transgenic plants, and the herbicide resistance of the plants was determined 14 days later.
(a)
(b)


### 3.3. Molecular Analyses of TransgenicM. sinensis Plants withArabidopsis PHYB

To verify the insertion ofBARandPHYB transgenes in the putative transgenic plants, PCR assays on genomic DNA extracted from the leaves were performed. Integration of both transgenes was observed in all herbicide-resistant plants (Figure 3(a)), whereas no amplified band was observed in the PCR of the total DNA from nontransformed control plant (NT). The integration of foreign DNA into the plant genome was further confirmed by Southern blot hybridization using theBAR-specific probe (Figure 3(b)). The 1 and 3 transgenic lines showed the same band patterns in the blots, suggesting that they were the transgenic lines originated from the same callus (data not shown). Thus, two independent transgenic lines (1/3 and 4) contained single gene integration, whereas line 2 had a gene copy number of two and line 5 had a gene copy number of three. No hybridization signal was observed when probing DNA samples derived from nontransformed control plant (NT).Molecular analyses of representative transgenicMiscanthus plants withPHYB. (a) Genomic PCR analysis of transgenic lines. The coding regions of thePHYB andBAR transgenes were amplified by PCR from genomic DNA. The actin gene (ACT) ofM. sinensis plants was shown as a loading control of the genomic DNA. NT: nontransformed control plant; V: pCAMBIA3301 harboringPHYB gene that was used for transformation. (b) Southern blot analysis of transgenic lines. Genomic DNA (15 μg) from each transgenic plant was digested with eitherHindIII orEcoRI and then probed with theBAR gene. Numbers in lanes represent independent transgenic lines selected for analysis, and asterisks indicate theBAR bands. (c) Northern blot analysis of transgenic lines. Total RNA was isolated from the leaves of transgenic plants and thePHYB gene was used as a probe. Total RNA was shown as a loading control.
(a)
(b)
(c)The expression ofPHYB transgene was analyzed by Northern blot analysis (Figure 3(c)). While all of the transgenic plants expressed the integrated transgenes, no hybridization was detected in the nontransformed control plant (NT). However, the levels of transgene expression differed among transgenic lines. 1-1, 3-1, 3-2, and 4-3 lines with single gene integration expressed similar levels ofPHYB transcripts, while the 2-2 and 5-1 lines with two and three gene copy number showed reducedPHYB transcript levels. Since it has been known that transgenic plants with multiple integrated genes expressed lower transcript levels of the transgenes than those with single gene integration, the reduced expression ofPHYBtransgene in 2-2 and 5-1 lines was consistent with the previous findings that multiple gene copies frequently lead to cosuppression and gene silencing [33, 34]. Collectively, we obtained several independent transgenic lines with single transgene integration, which were used for further phenotypic analyses.


### 3.4. Phenotypic Analysis of TransgenicM. sinensis Plants OverexpressingArabidopsis PHYB

Phytochromes regulate photomorphogenic responses in plants, which include stem and leaf growth, biosynthesis of chlorophylls, and flowering. Especially, loss-of-function mutant of phyB shows dramatically enhanced elongation growth and induces flowering early [31]. In contrast, overexpression of phyB has been shown to induce a short hypocotyl phenotype inArabidopsis and tobacco plants [35, 36], and to influence photosynthetic performance with increased pigmentation in potato [37]. More recently, overexpression ofArabidopsis phyB in cotton has been reported to cause pleiotropic effects such as semi-dwarfism, decreased apical dominance, and increased yield [38]. Based on these previous reports, we investigated the chlorophyll content, plant height, and flowering time of the transgenicM. sinensis plants compared with nontransformed control plant.The results of chlorophyll content measurements showed that all the transgenic plants had increases in chlorophyll content relative to control plant, but increased levels were somewhat varied among transgenic lines (Figure4). The 1-1 and 4-3 lines with single gene integration showed approximately 28 and 26% increases in chlorophyll content, respectively, while the 2-2 and 5-1 lines with two and three gene copy number showed about 18 and 6% increases. These results were well corelated with the transcript levels ofPHYBexpression (Figure 3(c)), suggesting that chlorophyll biosynthesis is increased in the transgenic lines with higher expression of functional phyB. The results are also consistent with the previous reports of strong dependency of phytochrome function on the photoreceptor amounts [39, 40]. TransgenicM. sinensis plants overexpressingPHYB also showed shorter plant height than the control plant (Figures 5(a) and 5(b)). These results are consistent with the previous reports that overexpression ofArabidopsis phyB shows phenotypes of semidwarfism with decreased apical dominance in potato and cotton [37, 38]. In addition, the transgenic plants displayed significant delays in both heading and flowering (Figure 5(c)). On average, approximately 2 week delay in flowering was observed in the transgenic plants compared with the control plant. Again, transgenic lines with higherPHYB expression showed more delay in heading and flowering. In rice, phyB has been reported to suppress the expression ofHd3a, an ortholog of ArabidopsisFT (FLOWERING LOCUS T) that induces flowering in plants [41]. This result indicates that overexpression of phyB might suppress flowering in the monocot. Thus, delayed flowering in the transgenicM. sinensis plants overexpressingPHYB is consistent with this previous result. Collectively, the results suggest that overexpression ofArabidopsis phyB increases chlorophyll biosynthesis, delays flowering with reduced plant height inM. sinensis.Figure 4
Relative chlorophyll content of transgenicM. sinensis plants withPHYB. The third leaf of the tillers was used for the extraction of chlorophyll (chl). Relative chlorophyll content was calculated by setting the chlorophyll content of nontransformed control plant (NT) to 100%. Error bars indicate standard errors (n
=
5). Means with different letters are significantly different at P
<
0.05, using Duncan.Phenotypic analyses of transgenicM. sinensis plants overexpressingPHYB. (a) Representative picture of nontransformed control plant (NT) and transgenicM. sinensis plants (T). Blue and red arrows represent the control and transgenic plants, respectively. The pictures were taken on DOY (day of year) 273, and the black arrow indicates the difference of plant heights between the control and transgenic plants. Bar: 20 cm. (b) Measurements of plant heights. Error bars indicate standard deviations (n
=
3). Statistically significant changes compared with NT are indicated by*when P
<
0.05
,using LSD. (c) Measurements of heading and flowering time. Heading and flowering were observed and measured on date (DOY). Error bars indicate standard deviations (n
=
3). Means with different letters are significantly different at P
<
0.05, using Duncan.
(a)
(b)
(c)


## 3.1. Photochemical Properties ofArabidopsis phyB

There are five isoforms of plant phytochromes (phyA–phyE) in the dicot model plantArabidopsis thaliana, among which phyA and phyB have been known to play major roles in plants [29, 30]. For example, phyA regulates seed germination and seedling growth in response to far-red light (FR), whereas phyB regulates seed germination in response to red light (R), plant growth and shade avoidance in response to R/FR ratio, and flowering time [4]. Especially, it has been well known that phyB-deficient mutant (hy3) shows phenotypes of constitutive shade avoidance and early flowering [31], suggesting that phyB is necessary to suppress the shade avoidance responses and flowering in plants, which is necessary to improve plant productivity. Therefore, we planned to introduceArabidopsis phyB intoM. sinensis and to investigate the phenotypes of the monocot crop overexpressing dicot phyB.Before the genetic transformation, we first performed the photochemical analysis ofArabidopsis phyB using purified recombinant proteins, because phyB has not been characterized well due to its low abundance in native tissue [32]. In contrast, the spectral properties of phyA have been well characterized because of the relatively high abundance of this photoreceptor in dark-grown tissue (~100 times more than phyB). In the present study, we expressed and purified recombinant phyB proteins successfully using thePichia protein expression system and streptavidin affinity chromatography (Figure 1(a)). PurifiedArabidopsis phyB showed ligation with the chromophore phycocyanobilin (PCB) and normal patterns of absorbance and difference spectra (Figure 1). Spectroscopic analysis of the purified phyB proteins revealed that the absorbance wavelength maxima of Pr (λ
Pr) and Pfr (λ
Pfr) forms are 650 nm and 714 nm, respectively. Compared with the photochemical properties of oat phyA in a previous report [25],Arabidopsis phyB showed similar properties to phyA including Δ of maxima (64 m) and APfr/APr (1.09) obtained from the difference spectrum. The only observed difference was that the absorbance wavelengths of phyB were slightly blue-shifted (4–6 nm) compared with the absorbance peaks of oat phyA (654 nm for Pr and 720 nm for Pfr). Collectively, the results demonstrate that PCB-assembled recombinantArabidopsis phyB has a typical photochromism of plant phytochromes.Photochemical analysis of purifiedArabidopsis phyB proteins assembled with phycocyanobilin (PCB). (a) Purification of PCB-assembled phyB proteins by streptavidin affinity chromatography. SDS-PAGE gel was shown with Coomassie blue staining. Lane S: Fermentas prestained protein ladder; lane C: crude extract; lane F: flow-through from the affinity column; lanes 2–5: elution fractions of holo-phyB. Zinc-induced fluorescence assay (Zinc) was shown to confirm chromophore-assembled phyB proteins. (b) Absorption and difference spectra of the purified PCB-assembled phyB proteins. Absorption spectra of Pr (solid line) and Pfr (dotted line) were shown, and the difference spectrum (gray line) was obtained by subtracting the Pfr absorption spectrum from the Pr absorption spectrum.
(a)
(b)


## 3.2. Production of TransgenicM. sinensis Plants OverexpressingArabidopsis PHYB

Embryogenic calli derived from mature seeds and the binary vector pCAMBIA3301 harboring thePHYB gene (Figure 2(a)) was used for transformation by followingAgrobacterium-mediated transformation method that we recently developed [23]. Since the binary vector used for the transformation contained theBAR herbicide resistance gene, putative transgenic creeping bentgrass plants were identified by herbicide resistance assay. Thein vitro regenerated putative transgenic lines were grown in a greenhouse for two weeks and then sprayed with 0.4% (v/v) BASTA. Following herbicide treatment, nontransformed wild-type control plant (NT) died within 14 days, whereas all of the putative transgenic plants were resistant to the herbicide (Figure 2(b)). These results indicate that theBARgene was expressed in these transgenic plants. When theAgrobacterium-mediated transformation efficiency was calculated as a percentage of herbicide-resistant transgenic lines obtained from all infected calli, a transformation efficiency of 0.71% (4 independent lines out of 567 infected calli) was obtained, which is similar to the reported efficiencies (0.84~0.20%). These putative transgenic lines were used further for molecular and phenotypic analyses.Construct for the transformation and herbicide resistance assay of putative transgenic plants. (a) T-DNA region of the binary vector plasmid pCAMBIA3301 harboringArabidopsis PHYB gene. RB: right border; LB: left border; P
35
S: CaMV 35S promoter; Pubi: ubiquitin promoter; intron GUS: GUS coding region with catalase intron insertion;PHYB: full-length phyB gene ofArabidopsis;BAR: phosphinotricin acetyltransferase gene coding region; T1: CaMV 35S gene terminator; T2:A. tumefaciens NOS gene terminator. Arrows indicate directions of transcription. (b) Herbicide resistance assay. Numbers in lanes represent putative transgenic plants selected for this analysis. 0.4% BASTA was sprayed onto nontransformed wild-type control plant (NT) and the transgenic plants, and the herbicide resistance of the plants was determined 14 days later.
(a)
(b)


## 3.3. Molecular Analyses of TransgenicM. sinensis Plants withArabidopsis PHYB

To verify the insertion ofBARandPHYB transgenes in the putative transgenic plants, PCR assays on genomic DNA extracted from the leaves were performed. Integration of both transgenes was observed in all herbicide-resistant plants (Figure 3(a)), whereas no amplified band was observed in the PCR of the total DNA from nontransformed control plant (NT). The integration of foreign DNA into the plant genome was further confirmed by Southern blot hybridization using theBAR-specific probe (Figure 3(b)). The 1 and 3 transgenic lines showed the same band patterns in the blots, suggesting that they were the transgenic lines originated from the same callus (data not shown). Thus, two independent transgenic lines (1/3 and 4) contained single gene integration, whereas line 2 had a gene copy number of two and line 5 had a gene copy number of three. No hybridization signal was observed when probing DNA samples derived from nontransformed control plant (NT).Molecular analyses of representative transgenicMiscanthus plants withPHYB. (a) Genomic PCR analysis of transgenic lines. The coding regions of thePHYB andBAR transgenes were amplified by PCR from genomic DNA. The actin gene (ACT) ofM. sinensis plants was shown as a loading control of the genomic DNA. NT: nontransformed control plant; V: pCAMBIA3301 harboringPHYB gene that was used for transformation. (b) Southern blot analysis of transgenic lines. Genomic DNA (15 μg) from each transgenic plant was digested with eitherHindIII orEcoRI and then probed with theBAR gene. Numbers in lanes represent independent transgenic lines selected for analysis, and asterisks indicate theBAR bands. (c) Northern blot analysis of transgenic lines. Total RNA was isolated from the leaves of transgenic plants and thePHYB gene was used as a probe. Total RNA was shown as a loading control.
(a)
(b)
(c)The expression ofPHYB transgene was analyzed by Northern blot analysis (Figure 3(c)). While all of the transgenic plants expressed the integrated transgenes, no hybridization was detected in the nontransformed control plant (NT). However, the levels of transgene expression differed among transgenic lines. 1-1, 3-1, 3-2, and 4-3 lines with single gene integration expressed similar levels ofPHYB transcripts, while the 2-2 and 5-1 lines with two and three gene copy number showed reducedPHYB transcript levels. Since it has been known that transgenic plants with multiple integrated genes expressed lower transcript levels of the transgenes than those with single gene integration, the reduced expression ofPHYBtransgene in 2-2 and 5-1 lines was consistent with the previous findings that multiple gene copies frequently lead to cosuppression and gene silencing [33, 34]. Collectively, we obtained several independent transgenic lines with single transgene integration, which were used for further phenotypic analyses.


## 3.4. Phenotypic Analysis of TransgenicM. sinensis Plants OverexpressingArabidopsis PHYB

Phytochromes regulate photomorphogenic responses in plants, which include stem and leaf growth, biosynthesis of chlorophylls, and flowering. Especially, loss-of-function mutant of phyB shows dramatically enhanced elongation growth and induces flowering early [31]. In contrast, overexpression of phyB has been shown to induce a short hypocotyl phenotype inArabidopsis and tobacco plants [35, 36], and to influence photosynthetic performance with increased pigmentation in potato [37]. More recently, overexpression ofArabidopsis phyB in cotton has been reported to cause pleiotropic effects such as semi-dwarfism, decreased apical dominance, and increased yield [38]. Based on these previous reports, we investigated the chlorophyll content, plant height, and flowering time of the transgenicM. sinensis plants compared with nontransformed control plant.The results of chlorophyll content measurements showed that all the transgenic plants had increases in chlorophyll content relative to control plant, but increased levels were somewhat varied among transgenic lines (Figure4). The 1-1 and 4-3 lines with single gene integration showed approximately 28 and 26% increases in chlorophyll content, respectively, while the 2-2 and 5-1 lines with two and three gene copy number showed about 18 and 6% increases. These results were well corelated with the transcript levels ofPHYBexpression (Figure 3(c)), suggesting that chlorophyll biosynthesis is increased in the transgenic lines with higher expression of functional phyB. The results are also consistent with the previous reports of strong dependency of phytochrome function on the photoreceptor amounts [39, 40]. TransgenicM. sinensis plants overexpressingPHYB also showed shorter plant height than the control plant (Figures 5(a) and 5(b)). These results are consistent with the previous reports that overexpression ofArabidopsis phyB shows phenotypes of semidwarfism with decreased apical dominance in potato and cotton [37, 38]. In addition, the transgenic plants displayed significant delays in both heading and flowering (Figure 5(c)). On average, approximately 2 week delay in flowering was observed in the transgenic plants compared with the control plant. Again, transgenic lines with higherPHYB expression showed more delay in heading and flowering. In rice, phyB has been reported to suppress the expression ofHd3a, an ortholog of ArabidopsisFT (FLOWERING LOCUS T) that induces flowering in plants [41]. This result indicates that overexpression of phyB might suppress flowering in the monocot. Thus, delayed flowering in the transgenicM. sinensis plants overexpressingPHYB is consistent with this previous result. Collectively, the results suggest that overexpression ofArabidopsis phyB increases chlorophyll biosynthesis, delays flowering with reduced plant height inM. sinensis.Figure 4
Relative chlorophyll content of transgenicM. sinensis plants withPHYB. The third leaf of the tillers was used for the extraction of chlorophyll (chl). Relative chlorophyll content was calculated by setting the chlorophyll content of nontransformed control plant (NT) to 100%. Error bars indicate standard errors (n
=
5). Means with different letters are significantly different at P
<
0.05, using Duncan.Phenotypic analyses of transgenicM. sinensis plants overexpressingPHYB. (a) Representative picture of nontransformed control plant (NT) and transgenicM. sinensis plants (T). Blue and red arrows represent the control and transgenic plants, respectively. The pictures were taken on DOY (day of year) 273, and the black arrow indicates the difference of plant heights between the control and transgenic plants. Bar: 20 cm. (b) Measurements of plant heights. Error bars indicate standard deviations (n
=
3). Statistically significant changes compared with NT are indicated by*when P
<
0.05
,using LSD. (c) Measurements of heading and flowering time. Heading and flowering were observed and measured on date (DOY). Error bars indicate standard deviations (n
=
3). Means with different letters are significantly different at P
<
0.05, using Duncan.
(a)
(b)
(c)


## 4. Conclusions

In this work, we have successfully produced transgenicM. sinensisplants overexpressingArabidopsis PHYB gene. To our knowledge, this might be the first report to obtain genetically engineeredM. sinensisplants with a useful gene other than selectable marker genes using anAgrobacterium-mediated transformation method. Furthermore, phenotypic analysis of the transgenic plants demonstrated that overexpression of dicot phyB fromArabidopsis thaliana shows pleiotropic effects in the monocot cropM. sinensis, which includes increased chlorophyll content, reduced plant height, and delayed flowering. The observed phenotypes are consistent with previous results with the overexpression of phyB in other plants such as potato and cotton. Assuming that increased chlorophyll content induces efficient photosynthetic performance and delayed flowering increases biomass productivity, the transgenicM. sinensisplants developed in this work might be considered as a high-value added cultivar of energy crops. In addition, the genetically engineeredM. sinensisplants obtained in this work contained theBAR gene that confers herbicide-resistance to the plants. Therefore, theM. sinensisplants with increased phyB function and herbicide resistance reported herein offer economic and environmental advantages over wild-typeMiscanthus plants.



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*Source: 501016-2014-05-18.xml*