doi:10.1093/plphys/kiab102

PLANT PHYSIOLOGY 2021: 186: 832–835

LetterShohei Nosaki ,1,2 Mika K. KanekoKenji Miura 1,2,*,†
1234,3 Fuminori Tsuruta,1 Hideki Yoshida,1,2 Yukinari Kato

3,4andFaculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
Tsukuba-Plant Innovation Research Center, University of Tsukuba, Tsukuba 305-8572, Japan
Graduate School of Medicine, Tohoku University, Sendai 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, Sendai 980-8575, Japan*Author for communication: miura.kenji.ga@u.tsukuba.ac.jp†Senior author.
K.M. designed the original concept and the project; S.N., M.K.K., F.T., H.Y., and K.M. performed the experiments; S.N., Y.K., and K.M. analyzed the data;
S.N. and K.M. wrote the article. The authors have declared that no competing interests exist.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in theInstruction of Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Kenji Miura (miura.kenji.ga@u.tsukuba.ac.jp).

Dear Editor,
The overexpression of recombinant proteins can sometimes
result in the accumulation of unfolded or misfolded proteins,
triggering leaf necrosis, and/or dehydration and subsequentdegradation of recombinant proteins. To enhance the accumulation of active proteins, a fundamental and a simple
treatment to overcome leaf necrosis is required. In this study,
treatment of Nicotiana benthamiana plants with high concentrations (100 mM or more) of sodium ascorbate (AsA) via
foliar spraying after agroinfiltration suppressed the necrosis ofleaves expressing human Cul1 (hCul1) and F-box protein,
Fbxw7. The suppression of necrosis accordingly enhanced theaccumulation of recombinant proteins by three-fold or more.
Treatment with 200 mM AsA enhanced the formation andaccumulation of PMab-2, an anti-RAP (rat Aggrus/podoplanin[PDPN]) tag antibody with antigen-binding activity. Thus,
spray application of high concentrations of AsA is simple andeasily applicable in several kinds of facilities to effectively suppress leaf necrosis, leading to an increase in the accumulationof functional recombinant proteins.

Compared with traditional cell culture-based systems,
whole plant-based systems for the production of recombinant proteins have advantages, including cost-effectivenessand production scalability (Buyel et al., 2017). Transient
gene expression using a deconstructed viral vector is apromising technique for rapidly producing high amounts ofrecombinant proteins (Desai et al., 2010; Ma et al., 2013).
However, some recombinant proteins, such as hepatitis B
surface antigen and human growth hormone, causes
necrosis and/or dehydration of N. benthamiana leaves (Gilset al., 2005; Huang et al., 2008) and there has been no way
to prevent necrosis. To increase the yield of these recombinant proteins, a fundamental solution is required.
We constructed pBYR2HS-hCul1 and pBYR2HS-human
F-box protein (hFbxw7; Supplemental Figure S1) and transfected those to N. benthamiana leaves by agroinfiltration.
Leaves expressing hCul1 and hFbxw7 exhibited necrosis(Figure 1A; 0 mM). As necrosis appeared, expression of binding immunoglobulin protein (BiP) and H2O2 wasinduced (Supplemental Figure S2), suggesting thatendoplasmic reticulum (ER) stress-triggered accumulation

Received January 15, 2021. Accepted February 18, 2021. Advance access publication February 26, 2021
C American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Prevention of necrosis caused by transient expressionin Nicotiana benthamiana by application ofascorbic acidPlant Physiology, 2021, Vol. 186, No. 2

PLANT PHYSIOLOGY 2021: 186; 832–835| 833

of H2O2 (Ozgur et al., 2014). Treatment with ER stressinhibitors had no appreciable improvement in plants(Supplemental Figure S3).
In plants, high levels of reactive oxygen species (ROS) canserve as a trigger for programmed cell death (vanBreusegem and Dat, 2006). To remove ROS, a high concentration of AsA was applied to N. benthamiana plants
expressing green fluorescent protein (GFP), hCul1, andhFbxw7. Leaves expressing hCul1 and hFbxw7 treated with50 mM or less concentration of AsA via foliar spraying
exhibited necrotic symptoms. In contrast, leaves treatedwith AsA at a concentration 100 mM or higher appeared

healthy (Figure 1A). Consistently, accumulation of H2O2 wassignificantly reduced in leaves treated with 200 mM AsA(Supplemental Figure S2B).
To establish whether the alleviation of necrosis by AsA
also resulted in an enhancement in the protein expression,
soluble proteins were extracted from leaves and separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) . Peak GFP expression (4 mg/g fresh mass[FM]) occurs 3 d postagroinfiltration (dpa), and thereafter
declines (Yamamoto et al., 2018). At 7 dpa, only 2 mg/g
FM of GFP protein accumulated in the leaves, whereas
when AsA was applied at concentrations of at least

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Figure 1 Suppression of necrotic effects by foliar spray application of high concentration of AsA. A, Syringe agroinfiltration was performed to express GFP, hCul1, and hFbxw7 in the leaves of N. benthamiana, followed by foliar spray application of AsA at the indicated concentration at 2-dintervals. The plants were incubated at 20 C for 7 d. Soluble proteins from the indicated fresh mass (FM) of these leaves were separated by SDSPAGE. GFP (B) or hCul1 (D) were detected by Coomassie Brilliant Blue (CBB) staining or anti-DYKDDDDK antibody. NT indicates nontransfected
plants. The amount of GFP (C) or hCul1 (E) was determined from band intensities. N. benthamiana plants agroinfiltrated with different vectors
were incubated at 25 C following the foliar spray application of 200 mM AsA, and soluble proteins were extracted from leaves at 3, 5, or 7 d after
agroinfiltration. hCul1 (F) or hFbxw7 (H) were detected with anti-DYKDDDDK antibody. The amount of hCul1 (G) were determined from bandintensities. Data represent the means ± SD (n = 3–4) and significance was determined using unpaired Student’s t tests (*P 5 0.05) (C, E, G).

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Chinese hamster ovary (CHO)-K1 cell-expressed rat PDPN as
did hybridoma-derived PMab-2 or plant-derived PMab-2. No
substantial changes were observed in the CHO-K1 cells
treated with PMab-2 (Supplemental Figure S4). Immunoblot
analysis revealed that rat PDPN was similarly detected by
PMab-2 derived from hybridoma cells and plant cells withor without AsA treatment (Figure 2D). These results indicatethat the application of AsA did not affect the function of arecombinant antibody. No apparent phenotypic difference
was observed when a high concentration of AsA wasapplied.
In this study, ER retention signal was fused with genes ofinterest. Some proteins require late posttranslational modification, such as the formation of complex glycan. These
modifications are typically performed downstream of the ER
along secretory pathways (Faye et al., 2005). Thus, the production of modified proteins is likely to be enhanced byspray application of AsA.
Overall, our research demonstrated the application ofhigh concentrations of AsA suppresses necrosis caused by
the expression of foreign recombinant proteins in N. benthamiana, increasing in protein accumulation and AsA did
not affect the antigen-binding activity of the recombinantantibody. The application of AsA by a foliar spray is simple,thus, it is easily applicable to several kinds of facilities.

Supplemental dataThe following supplemental materials are available.
Supplemental Figure S1. Schematic representation of theT-DNA regions of the plasmids pBYR2HS-hCul1 andpBYR2HS-hFbxw7.

Figure 2 Application of AsA increases the production of an antibody that retains binding activity. N. benthamiana leaves were infiltrated with amixed preparation of Agrobacterium harboring pBYR2HS-PMab2H and pBYR2HS-PMab2L, after which, the leaves were treated with water(Mock) or 200 mM AsA via foliar spray application. At 3 or 4 d postinfiltration, leaves were harvested. Total protein extracts were separated andwere transferred to PVDF membranes. The membranes were incubated with anti-mouse IgG(H) or anti-mouse IgG(L) to detect the HC (A) andLC (B) of PMab-2, respectively. C, Native PAGE was used to detect the tetrameric assembly of PMab-2 (two HCs and two LCs). D, Immunoblot
analysis with PMab-2 to detect rat PDPN. Soluble proteins from CHO-K1 cells (1) or CHO-K1 expressing rat PDPN (2) were separated on SDSPAGE. Rat PDPN was detected using PMab-2 derived from hybridoma cells (hybridoma) or N. benthamiana leaves treated without (Mock) orwith 200 mM AsA. Total proteins were visualized by CBB staining.

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100 mM, 3 mg/g FM of GFP expression in these leaves(Figure 1, B and C). Similarly, in leaves expressing hCul1, noclear bands (51 lg/g FM) were observed when 50 mM orless AsA was treated, but notable protein expression(11.4 lg/g FM when applied at 200 mM AsA) was observed
after treatment with AsA at concentrations of 100 mM or
higher (Figure 1, D and E), indicating high concentrations ofAsA have the effect of inhibiting protein degradation. Leaves
were incubated at 25 C for 3, 5, and 7 d and 200 mM AsA
was applied. A marked increase in the expression of hCul1
and hFbxw7 was observed in response to treatment withAsA, with peak hCul1 expression being attained at 3 dpa(Figure 1, F–H). These results indicate that the applicationof a high concentration of AsA can mitigate the necrosis
and markedly enhances protein expression.
Approximately 0.3 mg/g FM of PMab-2, an antibody
against the RAP epitope, which is in rat PDPN (Fujii et al.,2017), was accumulated in N. benthamiana without AsA
treatment (Miura et al., 2020). In this study, 200 mM AsA
was applied to N. benthamiana expressing the PMab-2
heavy chain (HC) and light chain (LC). The PMab-2 HC andLC were detected using anti-mouse IgG (H) and anti-mouse
IgG (L) antibodies, respectively (Figure 2, A and B). The fulltetrameric assembly of PMab-2 (2 HCs and 2 LCs) wasdetected by immunoblot analysis using native-PAGE(Figure 2C). Application of AsA increased the accumulationof PMab-2 HC and LC and enhanced the formation of tetrameric PMab-2. Without AsA application, the formationof antibodies was not stable and the peak of HC and LC
production may be shifted. PMab-2 was purified fromN. benthamiana. Fluorescence-activated cell sorting analysis
indicated that AsA-treated plant-derived PMab-2 recognizedNosaki et al.

Plant Physiology, 2021, Vol. 186, No. 2

Supplemental Figure S2. BiP expression and H2O2
accumulation were induced by hCul or hFbxw7.
Supplemental Figure S3. Necrosis was observed in leaves
treated with ER stress inhibitors.
Supplemental Figure S4. AsA-treated plant-derived
antibody retained its binding activity.
Supplemental Methods. The methodology used in thisstudy.

Funding

Conflict of interest statement. None declared.

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This work was supported by a JSPS Grant-in-Aid (19H04637 and20K21302) and Program on Open Innovation Platform with Enterprise,
Research Institute and Academia, Japan Science and Technology
Agency (JST-OPERA, JPMJOP1851 to K.M.), a Cooperative Research
Grant #2020 of the Plant Transgenic Design Initiative (PTraD) bythe Gene Research Center, Tsukuba-Plant Innovation Research Center(T-PIRC), University of Tsukuba, and AMED (Grant numbers
JP20am0101078 and JP20am0401013 to Y.K).

PLANT PHYSIOLOGY 2021: 186; 832–835