Skip to main content

A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves

Abstract

Background

Bamboo is a perennial and renewable biomass forest resource and its leaf flavonoid is an antioxidant for biological and pharmacological research. The established genetic transformation and gene editing systems in bamboo are significantly limited by the dependence on bamboo regeneration capability. The way to improve the flavonoid content in bamboo leaves through biotechnology is still not feasible.

Results

Here, we developed an in-planta, Agrobacterium-mediated gene expression method for exogenous genes via wounding and vacuum in bamboo. We demonstrated that the RUBY served as a reporter efficiently expressed in bamboo leaves and shoots, albeit unable to integrate into the chromosome. We have also developed a gene editing system by creating an in situ mutant of the bamboo violaxanthin de-epoxidase (PeVDE) gene in bamboo leaves, with lower NPQ values under the fluorometer, which can serve as a native reporter for gene editing. Furthermore, the bamboo leaves with increased flavonoid content were achieved by knocking out the cinnamoyl-CoA reductase genes.

Conclusions

Our method can be applied for the functional characterization of novel genes in a short time and is helpful for bamboo leaf flavonoid biotechnology breeding in the future.

Background

Bamboo is an evergreen and renewable forest biomass resource, which is not only widely used in manufacturing timber, artwork, and papermaking, but also in biological and pharmacological research. Bamboo leaf flavonoids have a variety of activities such as antioxidant, anti-inflammatory, anti-ulcer, and antitumor, which can help reduce the risk of cardiovascular disease, Alzheimer’s disease, Parkinson’s disease, and diabetes in previous studies [1, 2]. Genetic transformation and gene editing systems in bamboo have been established via callus induction [3,4,5]. However, the application of the existing gene editing systems in bamboo is primarily restricted by the dependency on the regeneration capability of bamboo, which is highly time-consuming (lasting usually over one year), labor-intensive, and complicated. Bamboo has an unpredictable and very long flowering cycle, usually lasting in vegetative growth for a few decades before flowering, which makes it difficult to obtain suitable explant materials, including flowers and immature embryos for callus induction. Therefore, an easy and feasible method for rapid gene expression and gene editing is urgently necessary for bamboo research.

The Agrobacterium-mediated transient expression can be easily conducted without using expensive equipment or complicated procedures compared with other transient expression methods, such as biolistic bombardment, PEG-mediated gene transfer, and protoplast electroporation. After being infected by Agro-infiltration or Agro-injection, T-DNA will be transferred from the bacteria to plant cells. All pieces of foreign DNA are transcriptionally competent, only a small percentage of the T-DNA can be integrated into the host chromosome, leading to stably transformed cells that can subsequently be regenerated into transgenic plants [6]. Moreover, a previous study found that the non-integrated transient CRISPR/Cas9 expression can obtain mutation in tobacco [7]. Although Agrobacterium-mediated transient gene expression can be easily and widely performed in dicotyledons, the strategies for the transformation of monocots are relatively inefficient [8]. Therefore, transient gene expression and gene editing are challenging in monocots, especially bamboo.

Here, we describe an in-planta, Agrobacterium-mediated transient gene expression strategy for exogenous genes in bamboo. We have also developed a gene editing system by creating an in situ mutant of the bamboo violaxanthin de-epoxidase (PeVDE) gene in bamboo leaves, with lower values of non-photochemical quenching (NPQ). We further investigate that the edited PeVDE mutant can serve as a stable reporter for gene expression and gene editing without selection pressure and that the mutant of other genes can be easily detected by chlorophyll fluorescence imaging. To apply this technology in bamboo, we knock out cinnamoyl-CoA reductase (CCR) genes by CRISPR/Cas9 and generated the mutants with the increase of flavonoid content in bamboo leaves. Our method will be applied for the functional characterization of novel genes and in-planta gene editing in bamboo and other plants.

Methods

Plant materials and growth conditions

The calli induced from immature embryos of moso bamboo (P. edulis) were kindly provided by Ms. Guirong Qiao from the Chinese Academy of Forestry, Hangzhou, China. Moso bamboo seedlings were generated from seeds. After being soaked in water for two days, the seeds were sowed into a substrate with a mixture of soil and vermiculite (3:1) for germination and seedling growth under laboratory conditions at 18–25 °C. A 16 h light/8 h dark photoperiod, and the intensity of light in the light phase was 250–350 μmol m−2 s−1. The half-month-old seedlings with a height of 2–10 cm were used for Agro-infection.

P. aureosulcata f. spectabilis (Jinxiangyuzhu) and P. aureosulcata f. aureocarlis (Huangganjingzhu) were planted in the garden of the International Center for Bamboo and Rattan, in Beijing, China. The emerging bamboo shoots with a height of 5–20 cm were used for Agro-infection.

Plasmids and transformation.

The pHDE-35S::RUBY construct carrying the RUBY reporter gene under the control of the CaMV 35S promoter (35S::RUBY) was purchased from Addgene (Catalog number: 160908). The pC1300-Ubi::Cas9 [9] plasmid was kindly provided by Ms. Guirong Qiao.

The sgRNA guiding sequences of PeVDE and PeCCRs were inserted into the pC1300-Ubi::Cas9 vector, between the two AarI sites using annealed oligonucleotides (Additional file 3: Table S2).

The plasmids were individually transformed into the AGL1, GV3101, LBA4044, and EHA105 strains of A. tumefaciens. Agrobacterium in YEP medium with the antibiotics was grown at 28 °C to an OD600 of 0.8. Suspensions of Agrobacterium were resuspended in the infiltration medium [10 mM MgCl2 and 10 mM MES-KOH (pH 5.6)] to an OD600 of 0.6.

Agrobacterium-mediated in-planta transformation system

Transformation of bamboo seedlings: The seedlings were transferred from the normal culture conditions to that of higher humidity (RH > 90%) and lower illumination intensity (less than 50 μmol m−2 s−1) for 2 h. Prior to infiltration, the top part of bamboo seedlings were wound by a sharp needle (the positions were marked by red triangles in Fig. 1a). Then, the upper part of the seedlings were immediately immersed into suspensions of Agrobacterium and immediately transferred to vacuum conditions of 25–27 inches of Hg for 2 min. After infiltration, the treated seedlings were immediately placed in dim light or dark conditions (less than 50 μmol m−2 s−1) at room temperature (18–25 °C) with high humidity (RH > 90%) for 1–2 days.

Fig. 1
figure 1

Expression of RUBY and accumulation of betalain in bamboo leaves and young shoots. a Bamboo (Phyllostachys edulis) seedlings ready for infection with Agrobacterium. The red triangles indicate the positions of the wounds. b, c Phenotype of P. edulis leaves showing betalain accumulation after 3 days (b) and 5 days (c) of infection. d Bamboo (P. aureosulcata f. spectabilis) shoots in the fields, ready for infection with Agrobacterium by injection. e, f Young shoots showing betalain accumulation. The blue triangles represent the areas with betalain accumulation

Transformation of bamboo shoots under field conditions: A syringe was used for injecting the Agrobacterium suspension into the shoot tips. The syringe needle was used to pierce into the shoot tips directly without stripping the sheaths. The depth of the injection was adjusted to ensure that the suspension reached the hollow pith and the space inside the sheaths.

Primer design, PCR, and sequencing

The specific primers for amplification of the PeVDE, PeCCRs, and RUBY fragments were designed manually. All the primer sequences used for gene cloning and PCR analyses were listed in the Additional file 3: Table S2. High-sensitive Polymerase Chain Reaction (PCR) was performed using the PrimeSTAR Max DNA polymerase (Takara, Japan), which has high fidelity and amplification efficiency in gene cloning. The direct PCR products were sent to TsingkeBiotechnology (Beijing, China) for deep sequencing.

Measurement of NPQ values

The in vivo PSII chlorophyll fluorescence of bamboo leaves was measured with the actinic light of an IMAGING-PAM fluorometer (Walz, Effeltrich). Prior to measurements, the bamboo seedlings were placed under conditions of high light intensity (1200 μmol m−2 s−1) for 2 h. The intensity of the actinic light was set to 800 μmol m–2 s–1. The NPQ was calculated using the formula: NPQ = (FmFm’)/Fm’, where Fm represented the maximum fluorescence in the dark-adapted state, and Fm’ represented the maximum fluorescence in any light-adapted state [10]. The values of NPQ were monitored from the visual interface of the ImagingWin software (Walz, Effeltrich, Germany).

Measurement of flavonoid content

The total bamboo leaf flavonoid content was determined by aluminum chloride assay [11, 12].

Measurement of lignin content

The lignin content was determined by a lignin assay kit (Solarbio, China) according to the protocol provided by the manufacturer.

Results

Rapid and transient Agrobacterium-mediated in-planta gene expression in bamboo leaves and shoots

RUBY is a reporter with the function of converting tyrosine to vividly red betalain [13], which accumulated products can be visualized with the naked eye and will be a suitable reporter in transient gene expression system. We conducted moso bamboo (Phyllostachys edulis) callus transformation with RUBY (35S::RUBY) and GUS (35S::GUS) reporter genes by Huang et al. (2022) method, respectively. In our trial, the transformation completely didn’t work in the subcultured calli (> 12-month-old calli). Meanwhile, the method for dicot Agro-infiltration was also completely inefficient on bamboo seedlings.

Next, we developed an in-planta Agrobacterium-mediated method for the transient expression of the exogenous RUBY gene in the leaves and shoots of bamboo. The red color of the immature leaves was visible to the naked eye after three days of infection and became more vivid after the rolled leaves unfolded (blue triangle, Fig. 1b, c). The findings revealed that the exogenous RUBY gene was expressed and that betalain synthesis occurred in the immature leaves of the bamboo seedlings.

The percentage of bamboo seedlings that accumulated betalain after being infected by Agrobacterium GV3101 strain was the highest, with 85.20% in younger and 91.70% in elder seedlings respectively (Additional file 2: Table S1). The results also demonstrated that the Agrobacterium strain of GV3101 induced less physical injury than AGL1, EHA105, and LBA4404 (Additional file 1: Fig. S1). Furthermore, suspensions of Agrobacterium were injected with positive pressure into bamboo shoots harboring the RUBY gene under field conditions (Fig. 1d). As the shoots grew, betalain accumulation was evident in both the shoot sheaths and young lateral shoots after 15 days of infection (blue triangle, Fig. 1e, f; Additional file 1: Fig. S2 and S3).

High-sensitive PCR was performed using the template DNA extracted from the leaves showing betalain accumulation for determining the integration of the T-DNA into the bamboo genome. After 40 cycles of PCR, fragments of the RUBY gene were still not detected in the genomic DNA of bamboo leaves showing betalain accumulation after 30 days of infection (Additional file 1: Fig. S4), indicating that the host cell had either rejected the inserted T-DNA, or the number of integrated fragments were too low to be detected. We also observed that almost all the red betalain color will disappear in bamboo leaves after three months of infection. Therefore, the reporter gene, such as RUBY, can not stably exist in the non-integrated transient expression system.

In-planta gene editing of bamboo violaxanthin de-epoxidase gene (PeVDE)

Then, in order to investigate whether the transient expression of CRISPR/Cas9 could serve as an efficient gene editing system in bamboo, we selected the bamboo violaxanthin de-epoxidase gene (PeVDE), which is responsible for the synthesis of zeaxanthin to dissipate the excess absorbed light energy as thermal energy [14, 15], for trial gene editing studies. Two guide RNA (gRNA) target sites were designed in the first exon of PeVDE at the restriction sites of XbaI (gRNA-1) and AgeI (gRNA-2), upstream of the protospacer adjacent motif (PAM) (Fig. 2a). These restriction sites at the target areas might be lost due to mutations. Firstly, the Agrobacterium harboring PeVDE CRISPR/Cas9 was used in the transient expression by co-infiltrated with the Agrobacterium containing RUBY reporter with the ratio of 1:3. Compared to the infiltration only with RUBY reporter (Fig. 2b), we observed that certain areas in the leaf blades had lower non-photochemical quenching (NPQ) values after co-infiltration for five days (Fig. 2c). Due to the betalain accumulation hardly affecting the NPQ values of leaves (Fig. 2b), the areas with lower and higher NPQ values were separated and used for DNA extraction, respectively. The mutant of PeVDE sequences in those areas with lower NPQ values (Fig. 2d) was confirmed by digestion with the aforementioned restriction enzymes (Fig. 2e; Additional file 1: Fig. S5a), which indicated that the PeVDE was edited by the transient gene editing system. Meanwhile, the areas with lower NPQ values were used for DNA extraction and deep sequencing (Fig. 2f, g; Additional file 1: Fig. S5b, S5c). The results of sequencing further confirmed that the PeVDE was edited by the transient gene editing system and the mutation rate of gRNA-2 was 17.33% (2,296 mutant sequences in 13,246) (Fig. 2g). Similar results were obtained for gRNA-1 with an edited efficiency of 10.6% (1286 mutant sequences in 12,142), which was relatively lower than that of gRNA-2 (Additional file 1: Fig. S5). The results indicated that the PeVDE mutant decreased the leaf dissipation capacity of excess absorbed light energy, which could be clearly monitored by a chlorophyll fluorometer.

Fig. 2
figure 2

In-planta gene editing of PeVDE gene in bamboo using CRISPR/Cas9 technology. a Structure of the PeVDE gene and sequences of the target sites. The red triangles represent the forward and reverse primers. bd NPQ imaging of leaves following infection with Agrobacterium harboring different constructs: 35S::RUBY (b), the mixture of 35S::RUBY and PeVDE CRISPR/Cas9 (c), PeVDE CRISPR/Cas9 (d). The numbers represent the NPQ values of the infected and non-infected areas. e Results of PCR of genomic DNA obtained from wild-type (WT) non-infected and infected leaves using gRNA-2 after 5 or 10 days of infection. + and—represent PCR products with or without AgeI digestion, the different bands are indicated with red arrows. f, g Deep sequencing results of the wide-type (f) and mutated PeVDE clones edited using gRNA-2 (g). Portions of sequences in red, blue, and grey indicate the target sites, PAM, and insertions, respectively. The red dashes indicate deleted nucleotides

In-planta gene editing of bamboo cinnamoyl-CoA reductase genes with the edited PeVDE mutant as a reporter

Furthermore, we applied the edited PeVDE mutant as a reporter in the non-integrated transient gene expression system. Cinnamoyl-CoA reductase (CCR) catalyzes the reduction reaction to synthesize p-coumaraldehyde, caffealdehyde, coniferaldehyde, 5H coniferaldehyde, and sinapaldehyde in the monolignol pathway for lignin biosynthesis [16]. Inhibiting CCR could promote flavonoid biosynthesis and reduce lignin production; opposite results were observed under the background of overexpressed CCR in apple [17]. Here, all the CCR genes in moso bamboo were genome-wide identified and the gRNA target sites of 11 PeCCRs were designed according to their sequences encoding the conserved motif of KNWYCYGK, which was critical for the catalysis of CCRs [18] (Fig. 3a; Additional file 3: Table S2). Five gRNAs were designed to target the five groups respectively. Specifically, CCR1 gRNA targeted the genes of PH02Gene02696.t1 and PH02Gene11141.t1; CCR2 gRNA targeted the genes of PH02Gene06795.t1, PH02Gene42850.t1, PH02Gene20528.t1, and PH02Gene28903.t1; CCR3 gRNA targeted the genes of PH02Gene42960.t1 and PH02Gene04803.t1; CCR4 gRNA targeted the gene of PH02Gene32193.t1 and PH02Gene45555.t1; CCR5 gRNA targeted the gene of PH02Gene42984.t1 (Additional file 3: Table S2). Then, the CRISPR/Cas9 vector carrying the gRNAs of PeVDE and PeCCRs respectively (Fig. 3b) was transfected into Agrobacterium for the transformation of bamboo leaves.

Fig. 3
figure 3

Edited PeVDE mutant served as a reporter for in-planta gene editing of PeCCRs. a Gene structures of PeCCRs and the region in genome encoding the conserved motif of KNWYCYGK. b The CRISPR/Cas9 constructs carrying the gRNAs of PeVDE and PeCCRs. c Images of leaves after infection with Agrobacterium harboring the constructs. White triangles represent areas with lower NPQ values. d, e Total flavonoid (d) and lignin (e) content in the infected leaves with lower NPQ values. * and ** indicate significant differences at p ≤ 0.05 and p ≤ 0.01 by ANOVA, respectively. Data are means ± AVEDEV (n = 3). Exact p values from CCR3 and CCR4 are as follows: d 0.0242 and 0.0069, e 0.0264 and 0.026

Culture for 30 days after infection, the seedlings were treated with light of high intensity (1200 μmol m−2 s−1) for 20 min for ensuring the absorption of excess light energy. The results of chlorophyll fluorescence measurement showed that no areas with decreased NPQ values were found in the leaves transfected by the CRISPR/Cas9 vector carrying the gRNAs of PeCCRs alone, which indicated that the edited PeCCRs had no effect on the NPQ value. Meanwhile, the areas of the leaves (indicated by white triangles) transfected by the CRISPR/Cas9 vector carrying the gRNAs of PeVDE and PeCCRs had lower NPQ values (right panel, Fig. 3c), which were detached for the validation of mutation and the determination of flavonoid and lignin.

The sequencing results revealed that the PeCCRs were edited by the transient gene editing system, and the mutation rates of the gRNAs for CCR1, CCR2, CCR3, CCR4, and CCR5 were 11.4% (138 mutant sequences in 1212) (Additional file 1: Fig. S6), 13.1% (228 mutant sequences in 1747) (Additional file 1: Fig. S7), 9.1% (513 mutant sequences in 5627) (Additional file 1: Fig. S8), 10.0% (331 mutant sequences in 3285) (Additional file 1: Fig. S9), and 9.3% (222 mutant sequences in 2687) (Additional file 1: Fig. S10) respectively. The results also indicated that the PeVDE mutant could be used as a reporter to screen the genome editing materials for other genes. Furthermore, we also checked the potential off-target editing by Cas9 in the transient gene editing system. In the CCR4 (PH02Gene32193.t1 and PH02Gene45555.t1) genes, the target site had only one base pair mismatched with the target site in CCR5 (PH02Gene42984.t1) gene, which was also recognized and cleaved by Cas9, generating the mutation rate of 1.4% (64 mutant sequences in 4684) (Additional file 1: Fig. S11). Also, the mutation rate of CCR5 by the gRNA of CCR4 was 1.3% (32 mutant sequences in 2432) (Additional file 1: Fig. S12).

The content measurement of flavonoid and lignin in the edited PeCCRs leaf areas showed that the mutants of CCR3 (PH02Gene42960.t1 and PH02Gene04803.t1) and CCR4 (PH02Gene32193.t1 and PH02Gene45555.t1) promoted the biosynthesis of flavonoid (Fig. 3d; Additional file 1: Fig. S13a) and reduced the biosynthesis of lignin (Fig. 3e; Additional file 1: Fig. S13b). In addition, VDE is a highly conserved protein with three conserved domains, namely, Cys-rich, lipocalin, and Glu-rich domains, in plants [14, 19], and the VDE mutant can also be used as a reporter in in-planta non-transgenic gene editing systems in other plants.

Discussion

The special biological characteristics of bamboo, such as the unpredictable flowering and long flowering cycle, have always been the main factor limiting the progress of bamboo breeding. In order to speed up the breeding process, people try to use modern biotechnology to carry out a lot of research work, such as space breeding, mutation breeding, and genetic engineering breeding. Recently, genetic transformation and gene editing systems have been reported in bamboo [3,4,5], and all of them are using callus for transformation through Agrobacterium. However, the callus induction and regeneration process is time-consuming, labor-intensive, and complicated. Also, these existing genetic transformation and gene editing systems are primarily restricted by the dependency on the regeneration capabilities of bamboo callus. Meanwhile, it is difficult to obtain suitable immature embryos for callus induction. It has been reported that the age of the callus is an important factor influencing the transformation efficiency [20]. The callus we used for transformation has been subcultured for more than one year, and our results show that the transformation is completely inefficient. Therefore, it is urgent to develop a rapid and efficient method for gene function identification in bamboo. Based on previous literature and experimental research, we successfully develop a time-saving in-planta gene expression strategy for exogenous genes in bamboo, without the complicated process of callus induction and regeneration. The RUBY served as a reporter that can efficiently express in bamboo leaves and shoots by Agrobacterium-mediated and vacuum transformation simultaneously, although the RUBY gene is unable to integrate into the chromosome of bamboo. This transient expression method will be widely applied for the functional characterization of novel genes in bamboo in the future.

After Agro-infection, the T-DNA fragments will be transferred to plant cells and only a small percentage of the fragments can be integrated into the host chromosome. In most cases, the maximum level of non-integrated transient gene expression occurs at 3–4 days after infiltration and fade rapidly after 5–6 days [8]. Thus, a stable reporter gene in the non-integrated transient gene expression system is an objective that plant biotechnologists pursue. The application of CRISPR/Cas9 always entails the stable integration of Cas9 endonuclease and gRNA genes into its chromosome, the transient expression fragments still can obtain mutation in plants [7]. Although the mutant of the phytoene desaturase (PDS) gene caused by the non-integrated gene editing can stably exist after the degradation of the non-integrated T-DNA fragments [7], the pds mutant has the disability of catalyzing the desaturation of colorless phytoene into z-carotene and the albinism retards the plant growth [21, 22]. Zeaxanthin is synthesized by violaxanthin de-epoxidase (VDE) and its content is highly correlated with the value of NPQ, which can dissipate the excess absorbed light energy as thermal energy to prevent photoinhibition [14, 15, 19]. We have developed an in-planta gene editing system via transient expression of CRISPR/Cas9 in bamboo leaves. The edited PeVDE mutant has the phenotype of lower NPQ value and which can be easily detected by chlorophyll fluorometer. Furthermore, gene editing of PeCCRs was performed with the edited PeVDE as a native reporter, the different mutation rates of CCR1 to CCR5 were confirmed by sequencing, and the increased flavonoid content and decreased lignin content were achieved in the edited bamboo leaves.

In our trials, the transient expression only occurs in the leaves, shoot sheaths, and young lateral shoots, whose tissues cannot generate germline-edited or transgenic progeny in bamboo. While the bamboo species has unique characteristics of a long and unpredictable flowering events, in-planta gene editing is not needed to obtain germline-edited or transgenic progeny in bamboo. In our future study, we will focus on the shoot meristem cells where gene editing can take place. Newly formed shoots derived from gene-edited shoot meristem cells will therefore be edited. Such a procedure can be easy and simple to perform because it requires no tissue culture, plant regeneration, or genetic transformation. Thus, our study develops a new reporter for screening the mutant of other genes in the transient gene expression system of bamboo, which can be applied for the functional characterization of novel genes and in-planta gene editing in bamboo and other plants.

Conclusions

In conclusion, GV3101 is an effective Agrobacterium strain, which can meditate the expression of RUBY used as a reporter for monitoring transient in-planta gene expression in bamboo with visibly vivid red coloration. Furthermore, gene editing is successfully conducted in bamboo leaves, the edited PeVDE mutant of bamboo can be used as a native reporter gene for screening mutant tissues by gene editing with non-integrated transient gene expression system. The increased flavonoid content is successfully achieved by gene editing of PeCCRs with the edited PeVDE as a native reporter. The method provides a non-transgenic strategy for in-planta gene editing, which can be used as a platform for the functional characterization of endogenous genes.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon request, and detailed information of the primer and gRNA sequences used in this study is provided in the Additional file documents.

References

  1. Nirmala C, Bisht MS, Bajwa HK, Santosh O. Bamboo: A rich source of natural antioxidants and its applications in the food and pharmaceutical industry. Trends Food Sci Technol. 2018;77:91–9. https://doi.org/10.1016/j.tifs.2018.05.003.

    Article  CAS  Google Scholar 

  2. Li H, Chen Y, Peng Q, Tan X, Chen G, Zhou H, et al. Flavonoids from bamboo leaves improve the stability of unsaturated fatty acids in the lipids of walnut emulsions. Ind Crop Prod. 2022;178:114609. https://doi.org/10.1016/j.indcrop.2022.114609.

    Article  CAS  Google Scholar 

  3. Ye S, Cai C, Ren H, Wang W, Xiang M, Tang X, et al. An efficient plant regeneration and transformation system of ma bamboo (Dendrocalamus latiflorus Munro) started from young shoot as explant. Front Plant Sci. 2017;8:1298. https://doi.org/10.3389/fpls.2017.01298.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ye S, Chen G, Kohnen MV, Wang W, Cai C, Ding W, et al. Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro). Plant Biotechnol J. 2020;18:1501. https://doi.org/10.1111/pbi.13320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Huang B, Zhuo R, Fan H, Wang Y, Xu J, Jin K, et al. An efficient genetic transformation and CRISPR/Cas9-based genome editing system for moso bamboo (Phyllostachys edulis). Front Plant Sci. 2022;13:822022. https://doi.org/10.3389/fpls.2022.822022.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005;1:1–14. https://doi.org/10.1186/1746-4811-1-13.

    Article  CAS  Google Scholar 

  7. Chen L, Li W, Katin-Grazzini L, Ding J, Gu X, Li Y, et al. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants. Hortic Res. 2018;5:13. https://doi.org/10.1038/s41438-018-0023-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lee W-S, Hammond-Kosack KE, Kanyuka K. In planta transient expression systems for monocots. In: Azhakanandam K, Silverstone A, Daniell H, Davey MR, editors. Recent advancements in gene expression and enabling technologies in crop plants. New York: Springer; 2015.

    Google Scholar 

  9. Wang C, Shen L, Fu Y, Yan C, Wang K. A simple CRISPR/Cas9 system for multiplex genome editing in rice. J Genet Genomics. 2015;42:703–6. https://doi.org/10.1016/j.jgg.2015.09.011.

    Article  PubMed  Google Scholar 

  10. Klughammer C, Schreiber U. Complementary PS II quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry and the saturation pulse method. PAM Application Notes. 2008;1:201–47.

    Google Scholar 

  11. Wang Y, Gao S, He X, Li Y, Zhang Y, Chen W. Response of total phenols, flavonoids, minerals, and amino acids of four edible fern species to four shading treatments. PeerJ. 2020;8:e8354. https://doi.org/10.7717/peerj.8354.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Shraim AM, Ahmed TA, Rahman MM, Hijji YM. Determination of total flavonoid content by aluminum chloride assay: a critical evaluation. LWT. 2021;150:111932. https://doi.org/10.1016/j.lwt.2021.111932.

    Article  CAS  Google Scholar 

  13. He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020;7:152. https://doi.org/10.1038/s41438-020-00390-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gao Z, Liu Q, Zheng B, Chen Y. Molecular characterization and primary functional analysis of PeVDE, a violaxanthin de-epoxidase gene from bamboo (Phyllostachys edulis). Plant Cell Rep. 2013;32:1381–91. https://doi.org/10.1007/s00299-013-1450-1.

    Article  CAS  PubMed  Google Scholar 

  15. Lou Y, Sun H, Zhu C, Yang K, Li X, Gao Z. PeVDE, a violaxanthin de-epoxidase gene from moso bamboo, confers photoprotection ability in transgenic Arabidopsis under high light. Front Plant Sci. 2022;13:927949. https://doi.org/10.3389/fpls.2022.927949.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Lacombe E, Hawkins S, Van Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, et al. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J. 1997;11:429–41. https://doi.org/10.1046/j.1365-313X.1997.11030429.x.

    Article  CAS  PubMed  Google Scholar 

  17. Hu Y, Cheng H, Zhang Y, Zhang J, Niu S, Wang X, et al. The MdMYB16/MdMYB1-miR7125-MdCCR module regulates the homeostasis between anthocyanin and lignin biosynthesis during light induction in apple. New Phytol. 2021;231:1105–22. https://doi.org/10.1111/nph.17431.

    Article  CAS  PubMed  Google Scholar 

  18. Zhou R, Jackson L, Shadle G, Nakashima J, Temple S, Chen F, et al. Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula. Proc Natl Acad Sci USA. 2010;107:17803–8. https://doi.org/10.1073/pnas.1012900107.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Goss R, Latowski D. Lipid dependence of xanthophyll cycling in higher plants and algae. Front Plant Sci. 2020;11:455. https://doi.org/10.3389/fpls.2020.00455.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Patel M, Dewey RE, Qu R. Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell Tissue Organ Cult. 2013;114:19–29. https://doi.org/10.1007/s11240-013-0301-7.

    Article  CAS  Google Scholar 

  21. Tian L. Recent advances in understanding carotenoid-derived signaling molecules in regulating plant growth and development. Front Plant Sci. 2015;6:790. https://doi.org/10.3389/fpls.2015.00790.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Bai C, Capell T, Berman J, Medina V, Sandmann G, Christou P, Zhu C. Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance. Plant Biotech J. 2016;14:195–205. https://doi.org/10.1111/pbi.12373.

    Article  CAS  Google Scholar 

Download references

Funding

We thank the financial support from the National Key Research and Development Program of China (Grant No. 2021YFD2200502), the National Natural Science Foundation of China (Grant No. 31971736).

Author information

Authors and Affiliations

Authors

Contributions

HS and ZG conceived and designed the experiments and wrote the manuscript. HS and SW performed the experiments. HS, SW, CZ, YL, and KY contributed to the data analysis. ZG supervised the project. All authors provided feedback on the manuscript.

Corresponding author

Correspondence to Zhimin Gao.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors affirm consent for publication.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Fig. S1. The leaf phenotype of Phyllostachys edulis seedlings with betalain accumulation. The Agrobacterium tumefaciens strains of AGL1, LBA4404, EHA105, and GV3101 harboring 35S::RUBY construct were used to infiltrate by vacuum respectively. The red triangles indicated the positions of the wounds. Fig. S2. The shoot phenotype of Phyllostachys aureosulcata Spectabilis’ with betalain accumulation after infection for 15 days. a, Shoots injected with Agrobacterium-free suspension. b, Shoots injected with the buffer solution with Agrobacterium suspension harboring 35S:RUBY construct. Black box, Magnified image. Fig. S3. The shoot phenotype of Phyllostachys aureosulcata 'Aureocarlis' with betalain accumulation after infection for 15 days. a, Shoots injected with Agrobacterium-free suspension. b, Shoots injected with the buffer solution with Agrobacterium suspension harboring 35S:RUBY construct. Black box, Magnified image. Fig. S4. RUBY reporter gene detected in the red leaf areas with betalain accumulation using PCR. Lane1, the non-infected leaves as the negative control; Lane2 and 3, the betalain accumulated leaves after Agrobacterium infection for three days and 30 days respectively; Lane4, the 35S::RUBY plasmid as positive control. PeActin (GenBank Accession No. GU434145) was used as endogenous control under the same PCR conditions. Fig. S5. The detection of gene-edited target1 of PeVDE in bamboo leaves. a, Results of PCR of genomic DNA obtained from wild-type (WT) non-infected and infected leaves using gRNA-1 after infection for 5 or 10 days. + and - represent PCR products with or without XbaI digestion. The different bands were indicated with red arrows. b–c, Deep sequencing results of wide-type (b) and mutated PeVDE clones edited using gRNA-1 (c). Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S6. Deep sequencing results of gene edited of CCR1 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S7. Deep sequencing results of gene edited of CCR2 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S8. Deep sequencing results of gene edited of CCR3 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S9. Deep sequencing results of gene edited of CCR4 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S10. Deep sequencing results of gene edited of CCR5 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S11. Deep sequencing results of CCR4 by the gRNA of CCR5 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S12. Deep sequencing results of CCR5 by the gRNA of CCR4 in bamboo leaves. Portions of sequences in red, blue, and grey indicated the target sites, PAM, and insertions, respectively. The red dashes indicated the deleted nucleotides. Fig. S13. The measurement of flavonoid and lignin content in the bamboo leaves. a, Total flavonoid content in the infected leaves with lower NPQ values. b, Lignin content in the infected leaves with lower NPQ values. The green and red filled columns represented the decrease and increase of flavonoid content compared with the control, respectively. Data are means ± AVEDEV (n=3). * and ** indicated significant differences at p<0.05 and p<0.01 by ANOVA, respectively. Exact p values from CCR1 to CCR5 were as follows: a 0.0945, 0.0358, 0.0242, 0.0069, 0.2445, b 0.853, 0.9798, 0.0264, 0.026, 0.9718.

Additional file 2:

Table S1. The percentage of seedlings accumulated betalain after being infected by Agrobacterium strains harboring 35S:RUBY construct.

Additional file 3:

Table S2. The sequence information of primers.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, H., Wang, S., Zhu, C. et al. A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves. Plant Methods 19, 20 (2023). https://doi.org/10.1186/s13007-023-00993-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13007-023-00993-4

Keywords