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Article

The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis

by
Qiqi Yang
1,†,
Qicheng Li
1,†,
Liyuan Gu
1,
Peng Chen
2,
Yu Zhang
1,
Yonghua Li
2,
Yun Chen
1,
Xia Ye
3,
Bin Tan
3,
Xianbo Zheng
3,
Jidong Li
1,* and
Jiancan Feng
3,*
1
College of Forestry, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, China
2
College of Landscape Architecture and Art, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, China
3
College of Horticulture, Henan Agricultural University, 95 Wenhua Road, Zhengzhou 450002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2022, 13(5), 723; https://doi.org/10.3390/f13050723
Submission received: 23 March 2022 / Revised: 1 May 2022 / Accepted: 3 May 2022 / Published: 5 May 2022
(This article belongs to the Special Issue Importance of Genetic Diversity for Forest and Landscape Restoration)
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Abstract

:
Jujube production is threatened by jujube witches’ broom (JWB) disease, which is caused by JWB phytoplasma. The jujube TCP transcription factor (TF) ZjTCP16 may be involved in the interaction of jujube plants with JWB phytoplasma. In this study, qRT-PCR proved that the expression pattern of ZjTCP16 was altered by JWB phytoplasma. The gene functions of ZjTCP16 were analyzed by its overexpression in Arabidopsis and jujube, as well as knock-down in. The overexpression of ZjTCP16 in Arabidopsis and jujube resulted in dwarfism and small leaves, while the zjtcp16 CRISPR mutants were higher than the WT. Microscopic observation of paraffin sections of jujube stems showed that ZjTCP16 affected the size of cells. The interactions of ZjTCP16 with ZjAS2 and ZjLOB in both the cytoplasm and nucleus were demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Yeast one-hybrid (Y1H) assays and qRT-PCR further confirmed that ZjTCP16 affected the expression of genes involved in leaf morphogenesis and cell proliferation (ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6) at the mRNA level through the ZjAS2 and ZjLOB pathways. In conclusion, ZjTCP16 regulates plant growth and cell size by altering the expression pattern of morphogenesis-related genes in jujube.

1. Introduction

Jujube (Ziziphus jujuba), also called Zao in Chinese, is the most ecologically and economically important member of the family Rhamnaceae [1,2]. Jujube has a long cultivation history of more than 7000 years, and grows in more than 40 countries including China, Korea, Japan, India, Australia and the United States [2]. The most destructive disease of jujube is jujube witches’ broom (JWB), which is related to the existence of JWB phytoplasma (‘Candidatus Phytoplasma ziziphi’) and brings heavy losses in yield and fruit production [3,4]. JWB-infected jujube trees show the modulation of fundamental plant developmental processes and typical disease symptoms that include witches’ broom (shoot proliferation), reduced leaf size, dwarf stature, stunted growth, and phyllody (leaf-like flowers) [5,6]. Studying the function of jujube genes involved in the response to JWB phytoplasma is an effective way to clarify the pathogenic mechanisms of JWB disease and will greatly enhance our understanding of how phytoplasma affects and reprograms plants [7,8].
Many reports have indicated that transcription factors (TFs) in the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family play important roles in phytoplasma or their secreted proteins responses. The SAP11 protein, secreted from Aster yellows witches’ broom (AY-WB) phytoplasma, was confirmed to induce smaller leaves, shoot proliferation, growth retardation and a low jasmonic acid (JA) level by binding, and it destabilizes AtTCP4 in Arabidopsis [9]. Wheat blue dwarf (WBD) phytoplasma secretes the SWP1 protein, which induces typical witches’ broom symptoms in Arabidopsis by degrading the key negative regulator of branching signals TCP18 (BRC1) [10]. SJP1 and SJP2, two effectors secreted by JWB phytoplasma in jujube, target the jujube TCP transcription factor ZjBRC1 and suppresses its expression, leading to witches’ broom symptoms [11].
In our previous study, 21 jujube TCP TFs were identified from the jujube genome. Of these, ZjTCP7 and ZjTCP16 were predicted to participate in the plant response to JWB phytoplasma [6]. An interaction network diagram of jujube TCP TFs and their down-stream genes was constructed, predicting that ZjTCP16 might interfere with leaf and stem development by interacting with the ZjAS2 and ZjLOB proteins, which would then alter the expression levels of genes involved in plant morphogenesis, such as ZjAS1 and KNOX family members [6].
A further study proved that the JWB phytoplasma effector Zaofeng6 interacts with ZjTCP7, a TCP transcription factor of BRC1 homologous gene in Arabidopsis. The interaction between Zaofeng6 and ZjTCP7 down-regulates genes in the strigolactone signaling pathway and induces shoot proliferation [12]. A weaker interaction signal was found between Zaofeng6 and ZjTCP16 by yeast two-hybrid (Y2H) [12]. This means that ZjTCP16 may be the target of phytoplasma effector Zaofeng6 and response for the infection of JWB phytoplasma.
In this study, we cloned and analyzed the protein structure of ZjTCP16, expressed ZjTCP16 in Arabidopsis and jujube, and verified the downstream genes and proteins that interact with ZjTCP16 by yeast one-hybrid (Y1H), Y2H and BiFC. This comprehensive study elucidates the possible function of ZjTCP16 in plant morphogenesis under phytoplasma infection.

2. Materials and Methods

2.1. Plant Material

Arabidopsis thaliana ecotype Col-0 plants were used for ZjTCP16 transformation and were cultured in a growth chamber [12]. In vitro-grown Z. jujuba ‘Huizao’ shoots used for ZjTCP16 cloning and transformation were maintained in tissue culture as previously reported [13]. Different tissues of JWB-infected Z. jujuba ‘Huizao’ trees from jujube orchards in Xinzheng county, Henan province, China, were collected for spatial expression analysis. The presence of phytoplasma in all the infected samples was verified by PCR with the primers R16mF2/R16mR1 [14]. N. benthamiana used for ZjTCP16 subcellular localization was grown in a temperature-controlled incubator as in a previous study [12].

2.2. Cloning, Sequence Analysis and Subcellular Localization of ZjTCP16

To clone the full-length cDNA sequence of ZjTCP16, total RNA extraction, reverse transcription in to cDNA and PCR were performed according to a previous study [8]. Sequences of PCR products were provided by Sangon Biotech Co., Ltd. (Shanghai, China).
The secondary structure of ZjTCP16 is predicted using online software PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/; accessed on 27 March 2020). The full CDS sequence of ZjTCP16 was cloned from the in vitro jujube shoots and then fused into the pSAK277-GFP vector with the recombination primers (Table S1). The Subcellular localization of ZjTCP16 was then carried out as reported [12]. The GFP empty vector was used as a negative control.

2.3. qRT-PCR Assay

Samples of different tissues such as flowers, young stems, young leaves (newly expanding leaves), old stems and old leaves (fully expanded leaves) from 5-year-old [6] JWB-infected jujube trees with typical witches’ broom (shoot proliferation) symptoms were observed, and healthy (PCR-negative) jujube trees were collected for ZjTCP16 spatial expression analysis. The qRT-PCR was performed according to Ye et al. [4]. The jujube actin gene was set as a reference for normalization of relative expression data [4].
qRT-PCR was used to analyze the transcript levels of genes involved in morphogenesis (ZjAS2, ZjAS2, ZjLOB, ZjKNAT1, ZjKNAT2 and ZjKNAT6). Total RNA was extracted from ZjTCP16-overexpressing (OE) and zjtcp16-mutant jujube shoots grown in sterile conditions in vitro cDNA synthesis and qRT-PCR were conducted according to Ye et al. [4]. The assay was replicated three times with three biological replicates for each sample type. The analysis of gene expression was conducted by the Duncan method using one-way ANOVA [4]. All PCR and qRT-PCR primers were designed by Primer Premier 5.0 (Premier, Quebec, QC, Canada) and are listed in Table S2.

2.4. Generation of ZjTCP16-OE Lines of Arabidopsis and Z. Jujuba

To generate transgenic Arabidopsis lines, the full-length ZjTCP16 CDS sequence was amplified using the RNA samples from in vitro-grown jujube leaves. We connected the ZjTCP16-specific primer amplified product with pSAK277 overexpression (OE) vector (Table S3).
Agrobacterium tumefaciens carrying pSAK77-ZjTCP16 were used for floral dip transfection of Arabidopsis [15]. The ZjTCP16-overexpression (OE) jujube lines were obtained by leaf disc transformation of Z. jujuba ‘Huizao’ grown in vitro [13]. The insertion of the ZjTCP16 gene in selection-positive plants was confirmed by PCR using specific primers of the pSAK277 vector (Table S3).

2.5. Generation of zjtcp16 CRISPR Mutant Jujube Shoots

The zjtcp16 mutant jujube shoots were generated by CRISPR as described previously [12]. Cloning of coding sequence containing the target sequence of ZjTCP16 into the Cas9 vector with the primer pairs Cas9-ZjTCP16F/Cas9-ZjTCP16R was conducted (Table S3). The gene-edited mutant lines were verified by PCR and target locus sequencing. The sgRNA off target sites were predicted by CRISPOR (http://crispor.tefor.net/; accessed on 5 June 2021), detected by PCR amplification and sequencing. The primers used for amplifying the off-target areas are listed in Table S3.

2.6. Phenotypic Analysis and Microscopic Examination

The phenotypes of T2 transgenic Arabidopsis carrying ZjTCP16 were compared with pSAK277 empty vector (EV) and wild type (WT). Nine Arabidopsis seedlings of each line were observed for the growth indices such as leaf size, plant height and silique length at 25 and 50 days after root emergence. The phenotypes of ZjTCP16-OE transgenic, zjtcp16 CRISPR mutant and WT jujube shoots without roots were photographed 30 days after shoot induction. The stems of nine in vitro ZjTCP16-OE, zjtcp16 mutant and WT jujube shoots each were sampled, cut into 2 mm thin sections and sent to Servicebio Biotechnology Co., Ltd. (Wuhan, China) for paraffin section and microscopic observation.

2.7. Y1H and Y2H Assay

Y1H assays were based on a previous report [16]. We connected the full-length CDS of the genes (ZjAS1, ZjAS2, ZjLOB, ZjKNAT1, ZjKNAT2 and ZjKNAT6) with the pB42AD vector. The sequence of 300 to1500 bps upstream of the initiation codon of each gene (ZjAS1, ZjAS2, ZjLOB, ZjKNAT1, ZjKNAT2 and ZjKNAT6) was amplified from jujube genomic DNA by specific primers and fused with pLaczi vector. After three days of transformation, the transformants were coated on SD/-Trp-Ura (SD-TU) with 20 mg/L X-Gal for 3–4 days of screening.
Y2H assays were carried out as reported [10] to determine the interaction between ZjTCP16, ZjAS1, ZjAS2 and ZjLOB. The full-length cDNA of these genes was amplified and then ligated into pGBKT7 and pGADT7 vectors (ZOMANBIO, Beijing, China). All primers used for making Y1H and Y2H constructs are listed in Table S4.

2.8. BiFC Analysis

This study confirms the Y2H assays results by BiFC assays that were carried out according to Yan et al. [17]. ZjAS2 and ZjLOB gene cDNA sequences were cloned into pNC-BiFC-Ecn, while ZjTCP16 and ZjAS2 were cloned into pNC-BiFC-Enn. The primers used for BiFC assays are shown in Table S5. All of them were transformed into A. tumefaciens, which was then pressure-infiltrated by syringe into N. benthamiana leaves. Three leaves were infiltrated for the assays, with different combinations in different quadrants of each leaf. Leaves were viewed 48 h after agroinfiltration by confocal laser microscopy (Nikon Corporation, Tokyo, Japan).

3. Results

3.1. Sequence and Protein Structure Analysis and Subcellular Localization of ZjTCP16

The full-length CDS sequence of ZjTCP16 was cloned with the specific primer pair ZjTCP16F/ZjTCP16R (Table S1). A clear band at the predicted size (1368 bp) was observed (Figure 1a) and recovered for sequencing. The sequence of ZjTCP16 is predicted to encode a protein of 461 amino acids with several bHLH functional domains (Figure 1b). The TCP domain, which is a conserved domain in TCP transcription factors, was predicted from positions 35 to 287 (Figure 1b). In tobacco leaves transiently overexpressing a ZjTCP16-GFP fusion protein, fluorescence was observed in both the nucleus and the cytoplasm. These results indicated that ZjTCP16 could have functions in both the host cell membrane and nucleus (Figure 1c).

3.2. Expression of ZjTCP16 in Different Tissues of JWB-Positive and -Negative Jujube Trees

Orchard-grown jujube trees were phototyped as diseased based on physical symptoms and PCR for the phytoplasma gene. Transcript levels of ZjTCP16 in different tissues (flowers, young stems, young leaves, old stems and old leaves) of healthy and JWB-infected jujube trees seedlings were detected by qRT-PCR. In healthy jujube, ZjTCP16 was highly expressed in flowers and young leaves and was moderately expressed in stems (Figure 2a). In diseased jujube, the expression level of ZjTCP16 was still the lowest in the stem, while it showed the highest expression level in old leaves (Figure 2b). The results suggested that JWB phytoplasma altered the expression pattern of ZjTCP16.

3.3. Overexpression of ZjTCP16 Resulted in Dwarfism and Smaller Leaves in Arabidopsis

The coding sequence of ZjTCP16 was cloned into pSAK277 vector and expressed in Arabidopsis to explore the effect of ZjTCP16 on the growth of Arabidopsis. The growth index of ZjTCP16 -OE Arabidopsis lines, EV and WT, were recorded at each growth stage (Figure 3 and Figure 4). Over the entire growth cycle, most ZjTCP16-OE Arabidopsis lines were dwarfed and had smaller leaves. One week after transplant (WAT), ZjTCP16 overexpression significantly reduced the size of Arabidopsis rosette leaves (Figure 3a,b and Figure 4a–c). After bolting (3 WAT, 5 WAT), plant height and the size of leaves in bolts of ZjTCP16-OE Arabidopsis were distinctly reduced when compared with those in EV and WT (Figure 3b–d and Figure 4d–g). ZjTCP16 also had a great effect on the growth of Arabidopsis siliques. When compared with EV and WT, ZjTCP16-OE lines produced fewer and smaller siliques at five weeks after transplant (Figure 3e and Figure 4h,i). These results suggest that ZjTCP16 reduced leaf size and induced a dwarf phenotype in Arabidopsis.

3.4. Alteration of ZjTCP16 Expression Regulated Jujube Growth and Cell Size in Z. Jujube Shoots

To better study the effect of ZjTCP16 on jujube development, ZjTCP16-OE and zjtcp16 CRISPR mutant jujube lines were generated in vitro (Figure 5a,b,e). To detect off-target events, three putative off-target sites were predicted and checked by sequencing. The sequencing result did not show any mutation in these three sites (Figure S1). Morphologic and microscopic observation 50 days after shoot emergence suggest that the overexpression of ZjTCP16 in jujube shoots resulted in a dwarf phenotype, as shoot height was significantly lower than that of WT. On the other hand, the shoot height of zjtcp16 CRISPR mutants was higher than the WT (Figure 5c,e). The alteration of ZjTCP16 expression also resulted in changes in cell size in jujube shoots. The cell size in ZjTCP16-OE shoots was significantly smaller than in WT shoots, while the cell size of the zjtcp16 CRISPR mutant was significantly bigger (Figure 5d,f). These results suggest that changes in the expression level of ZjTCP16 altered jujube shoot growth and cell size.

3.5. ZjTCP16 Interacted with ZjAS2 and ZjLOB

The interactions of ZjAS2 and ZjLOB with ZjTCP16 were assessed by Y2H and BiFC assays (Figure 6). Y2H assays showed clear interactions of ZjTCP16 with ZjAS2 and ZjAS2 with ZjLOB, as well as a weaker interaction between ZjTCP16 and ZjLOB (Figure 6a). BiFC assay further confirmed the interaction between ZjTCP16, ZjAS2 and ZjLOB. When any two of these three genes are co-expressed in tobacco leaves, strong fluorescent signals can be observed in the nucleus and cytoplasm (Figure 6b).

3.6. ZjTCP16 Altered the Expression Levels of Genes Involved in Plant Morphogenesis

The interaction between ZjAS2, ZjLOB and their downstream genes ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6 were confirmed by Y1H assay (Figure 7). The promoter regions of ZjAS2 and ZjKNAT1 interacted with ZjAS1, while ZjAS2 interacted with the promoter of ZjKNAT1, ZjKNAT2 and ZjKNAT6. These promoter-binding assays indicated that ZjAS1 may regulate the expression of ZjAS2 and ZjKNAT1 at the transcriptional level, while ZjAS2 may control the transcription of ZjKNAT1, ZjKNAT2 and ZjKNAT6. In addition, ZjKNAT6 and ZjLOB might also regulate the expression level of ZjKNAT1 (Figure 7a). These interactions were mapped with Cytoscape software (Figure 7b).
To further clarify the effect of ZjTCP16 on the expression level of genes involved in plant morphogenesis (ZjAS1, ZjAS2, ZjLOB, ZjKNAT1, ZjKNAT2 and ZjKNAT6) in jujube, the total RNA of ZjTCP16-OE, zjtcp16 mutant and WT jujube lines was extracted for qRT-PCR (Figure 7c). The expression of ZjAS1 and ZjAS2 was significantly increased in ZjTCP16-OE jujube lines, while obviously reduced in zjtcp16 mutant lines when compared with WT. On the contrary, ZjLOB showed higher transcript levels in zjtcp16 mutant lines compared to ZjTCP16-OE jujube lines and WT. The transcript level of ZjKNAT6 was markedly reduced in ZjTCP16-OE jujube lines (Figure 7c). ZjKNAT1 and ZjKNAT2 did not show significant changes in expression between the two sets of transgenic plants. These results further showed that ZjTCP16 affected the expression of genes involved in plant morphogenesis.

4. Discussion

4.1. ZjTCP16 Induced Dwarf and Smaller Leaf Phenotypes by Changing the Expression of Morphogenesis-Related Genes

As plant-specific transcription factors, members of the TCP family are widely involved in the whole life process of plants, such as leaf development, shoot apical meristem, biosynthesis of phytohormones, cell proliferation and circadian clock rhythms [18,19]. AtTCP4, the ortholog of ZjTCP16, functions in the control of leaf development, circadian cycle and flowering [20]. The hyper-activation of AtTCP4 reduces leaf size and promotes cell expansion in leaves [21]. AtTCP4 can interact with AS2, which then alters expression of KNOX genes and leaf development [22]. The activation of YUCCA5 transcription by AtTCP4 can promote cell elongation in Arabidopsis thaliana hypocotyls [23]. In this study, ZjTCP16 was shown to interact with KNOX gene family members ZjAS2 and ZjLOB by Y2H and BiFC and to alter the expression of the downstream genes ZjKNAT1, ZjKNAT2 and ZjKNAT6. Overexpression and mutation of ZjTCP16 altered plant height and leaf and cell size. Taken together, we deduced that ZjTCP16 might reduce leaf and plant size by changing the expression of morphogenesis genes.

4.2. Mutation of ZjTCP16 by CRISPR/Cas9-Mediated Gene Editing

CRISPR/Cas9 technology has been successfully applied to study gene function and to improve certain traits, including disease resistance in plants as diverse as Arabidopsis, rice, maize, tobacco, poplar, grape, apple and citrus [24,25]. CRISPR/Cas9 can efficiently induce targeted mutations by base-pairing engineered single-guide RNAs (sgRNAs) to the target DNA sites [26,27]. In poplar, knockout mutants of the TCP transcription factor BRANCHED1 and BRANCHED2 were obtained using CRISPR/Cas9. The mutants displayed a bud overgrowth phenotype, revealing that the BRANCHED genes function in bud outgrowth control [28]. A CRISPR/Cas9-mediated mutant of jujube TCP7 exhibited excessive shoot proliferation, similar to the disease symptom caused by the JWB effector Zaofeng6 [12]. In this study, a two-nucleotide deletion in the TCP domain was generated by CRISPR in the mutant line compared to the WT, which led to a frameshift mutation. The mutant jujube shoots exhibited abnormal phenotypes and altered expression of downstream genes compared to WT. The mutation further proved that the ZjTCP16 gene functions in plant growth and cell size regulation. The results provide a theoretical basis for clarifying the pathogenic mechanism of JWB and the cultivation of disease-resistant jujube varieties.

4.3. The Expression of ZjTCP16 in Response to JWB Phytoplasma

The TCP TFs play major roles in regulating biosynthesis and signaling of different phytohormones including auxin, JA and brassinosteroid (BR). Thus, TCP TFs are hubs targeted by multiple effectors from different pathogens [29,30]. In recent research, jujube TCP TFs homologs, ZjTCP7 and ZjBRC1, have been identified as target of SAP11-like JWB phytoplasma effectors, Zaofeng6, SJP1 and SJP2 [11,12]. In an JWB phytoplasma infection experiment performed by disease bud grafting, the expression of ZjTCP16 in jujube leaves were upregulated at 13 and 20 weeks after grafting [6]. In this study, comparison of ZjTCP16 expression between different tissues of health and JWB diseased jujube trees revealed altered expression pattern induced by JWB phytoplasma. The response of ZjTCP16 to JWB phytoplasma infection and interaction with JWB phytoplasma effectors should be studied in further study.

5. Conclusions

ZjTCP16 regulates plant growth and cell size by altering the expression of genes in-volved in plant morphogenesis and cell proliferation such as ZjAS1, ZjKNAT1, ZjKNAT2 and ZjKNAT6 through the ZjAS2 and ZjLOB pathways.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f13050723/s1, Table S1: Primer design of Clone, sequence analysis and subcellular localization; Table S2: Primer design for qRT-PCR assay; Table S3: Primer design of transgenic plants generation and detection; Table S4: Primer design for Y1H and Y2H; Table S5: Primer design for BiFC assays. Figure S1: Map of sequence alignment for off-target detection.

Author Contributions

Q.Y. and Q.L. performed experiments and co-wrote the manuscript, which made the same contribution to this work. J.L. and J.F. conceived this project and supervised the experiments. L.G., P.C., Y.Z., X.Y., B.T. and Y.C. analyzed the data. X.Z. and Y.L. contributed jujube materials and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (32071803) and the Henan Key Laboratory of Fruit and Cucurbit Biology (200903).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Yan Pu. The carrier used in BiFC experiment was provided by Yan Pu, Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences (ITBB, CATAS).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Characterization of ZjTCP16. (a): The PCR-amplified products using ZjTCP16-specific primers were separated by gel electrophoresis. The lane from left to right is DL2000 DNA Marker and four replicates of ZjTCP16 gene amplification. (b): Predicted secondary structure of ZjTCP16. The TCP domain is outlined with a box and is predicted to bind to DNA and interact with proteins. (c): Subcellular localization of ZjTCP16-GFP fusion protein.
Figure 1. Characterization of ZjTCP16. (a): The PCR-amplified products using ZjTCP16-specific primers were separated by gel electrophoresis. The lane from left to right is DL2000 DNA Marker and four replicates of ZjTCP16 gene amplification. (b): Predicted secondary structure of ZjTCP16. The TCP domain is outlined with a box and is predicted to bind to DNA and interact with proteins. (c): Subcellular localization of ZjTCP16-GFP fusion protein.
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Figure 2. Expression pattern analysis of ZjTCP16 in response to JWB phytoplasma. Different tissues of healthy (a) and JWB-phytoplasma-infected (b) jujube were sampled for RNA extraction and qRT-PCR. Expression levels of ZjTCP16 in different tissues of healthy and JWB-infected jujube trees, including flowers (HF/DF), young stems (HYS/DYS), young leaves (HYL/DYL), old stems (HOS/DOS) and old leaves (HOL/DOL). Different letters above the bars indicated significant difference (p < 0.05) as obtained by one-way ANOVA.
Figure 2. Expression pattern analysis of ZjTCP16 in response to JWB phytoplasma. Different tissues of healthy (a) and JWB-phytoplasma-infected (b) jujube were sampled for RNA extraction and qRT-PCR. Expression levels of ZjTCP16 in different tissues of healthy and JWB-infected jujube trees, including flowers (HF/DF), young stems (HYS/DYS), young leaves (HYL/DYL), old stems (HOS/DOS) and old leaves (HOL/DOL). Different letters above the bars indicated significant difference (p < 0.05) as obtained by one-way ANOVA.
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Figure 3. Overexpression of ZjTCP16 in Arabidopsis induced phenotype of dwarfism and reduced leaf size. (a): Phenotypes of wild-type Arabidopsis, empty vector (EV) and ZjTCP16-OE lines at 1 week after transfer (WAT) from germination medium. (b): Phenotypes of wild-type, EV and ZjTCP16-OE Arabidopsis at 3WAT. (c): Phenotypes of wild-type, EV and ZjTCP16-OE Arabidopsis at 5WAT. (d): Bolts of WT, EV and ZjTCP16-OE Arabidopsis at 5WAT; (e): Morphological observation of siliques of WT, EV and ZjTCP16-OE Arabidopsis at 5WAT.
Figure 3. Overexpression of ZjTCP16 in Arabidopsis induced phenotype of dwarfism and reduced leaf size. (a): Phenotypes of wild-type Arabidopsis, empty vector (EV) and ZjTCP16-OE lines at 1 week after transfer (WAT) from germination medium. (b): Phenotypes of wild-type, EV and ZjTCP16-OE Arabidopsis at 3WAT. (c): Phenotypes of wild-type, EV and ZjTCP16-OE Arabidopsis at 5WAT. (d): Bolts of WT, EV and ZjTCP16-OE Arabidopsis at 5WAT; (e): Morphological observation of siliques of WT, EV and ZjTCP16-OE Arabidopsis at 5WAT.
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Figure 4. Growth indices of the Arabidopsis overexpressing ZjTCP16. Bars represent the mean ± standard deviation of 9 plants. Asterisks indicated with WT (* = p < 0.05; one-way analysis of variance (ANOVA)) showed significant differences. (a): Average of rosette leaves number of WT, EV and ZjTCP16-OE Arabidopsis lines. (b): Average length of rosette leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (c): Average width of rosette leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (d): Average number of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (e): Average length of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (f): Average width of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (g): Average plant height of WT, EV and ZjTCP16-OE Arabidopsis lines. (h): Average number of siliques of WT, EV and ZjTCP16-OE Arabidopsis lines. (i): Average length of siliques of WT, EV and ZjTCP16-OE Arabidopsis lines.
Figure 4. Growth indices of the Arabidopsis overexpressing ZjTCP16. Bars represent the mean ± standard deviation of 9 plants. Asterisks indicated with WT (* = p < 0.05; one-way analysis of variance (ANOVA)) showed significant differences. (a): Average of rosette leaves number of WT, EV and ZjTCP16-OE Arabidopsis lines. (b): Average length of rosette leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (c): Average width of rosette leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (d): Average number of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (e): Average length of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (f): Average width of leaves of WT, EV and ZjTCP16-OE Arabidopsis lines. (g): Average plant height of WT, EV and ZjTCP16-OE Arabidopsis lines. (h): Average number of siliques of WT, EV and ZjTCP16-OE Arabidopsis lines. (i): Average length of siliques of WT, EV and ZjTCP16-OE Arabidopsis lines.
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Figure 5. (a): DNA sequencing of the target locus in the zjtcp16 CRISPR mutant compared to WT. The alignment shows the single-guide RNA (sgRNA) binding sites including the corresponding protospacer-adjacent motif (PAM). Matching nucleotide residues at the same position are represented by the same color. (b): Protein sequence aligns between the target locus in the zjtcp16 CRISPR mutants and WT. (c): Average height of ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT. Bar = 1 cm. (d): Size of 50 cells in ZjTCP16-OE, zjtcp16 lines and WT. (e): Phenotype of ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT at 1 month; (f): Observation on the stem and cell section of the internode in ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT. Bar = 200 μm. (*: p < 0.05).
Figure 5. (a): DNA sequencing of the target locus in the zjtcp16 CRISPR mutant compared to WT. The alignment shows the single-guide RNA (sgRNA) binding sites including the corresponding protospacer-adjacent motif (PAM). Matching nucleotide residues at the same position are represented by the same color. (b): Protein sequence aligns between the target locus in the zjtcp16 CRISPR mutants and WT. (c): Average height of ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT. Bar = 1 cm. (d): Size of 50 cells in ZjTCP16-OE, zjtcp16 lines and WT. (e): Phenotype of ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT at 1 month; (f): Observation on the stem and cell section of the internode in ZjTCP16-OE jujube lines, zjtcp16 mutant lines and WT. Bar = 200 μm. (*: p < 0.05).
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Figure 6. ZjTCP16 interacted with ZjAS2 and ZjLOB in both cytoplasm and nucleus. (a): Y2H assays between ZjTCP16, ZjAS2 and ZjLOB. (b): BiFC assays between ZjTCP16, ZjAS2 and ZjLOB. In the top two panels, ZjTCP16 was fused to pNC-BiFC-Enn, and ZjAS2 or ZjLOB were fused to pNC-BiFC-Ecn, while in the lowest panel, ZjAS2 was fused to pNC-BiFC-Enn. Bars, 100 μm.
Figure 6. ZjTCP16 interacted with ZjAS2 and ZjLOB in both cytoplasm and nucleus. (a): Y2H assays between ZjTCP16, ZjAS2 and ZjLOB. (b): BiFC assays between ZjTCP16, ZjAS2 and ZjLOB. In the top two panels, ZjTCP16 was fused to pNC-BiFC-Enn, and ZjAS2 or ZjLOB were fused to pNC-BiFC-Ecn, while in the lowest panel, ZjAS2 was fused to pNC-BiFC-Enn. Bars, 100 μm.
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Figure 7. ZjTCP16 affected the expression of genes involved in morphogenesis and cell proliferation through the ZjAS2 and ZjLOB pathway. (a): Yeast one-hybrid (Y1H) assays of ZjAS2 and ZjLOB and their potential downstream genes. (b): Interaction network of genes involved in plant morphogenesis mediated by ZjTCP16. (c): Expression of genes involved in morphogenesis and cell proliferation in ZjTCP16-OE and zjtcp16 mutant jujube shoots. Bars represent the mean ± standard deviation of 9 plants. Asterisks indicate significant differences compared with WT (*: p < 0.05; one-way analysis of variance (ANOVA)).
Figure 7. ZjTCP16 affected the expression of genes involved in morphogenesis and cell proliferation through the ZjAS2 and ZjLOB pathway. (a): Yeast one-hybrid (Y1H) assays of ZjAS2 and ZjLOB and their potential downstream genes. (b): Interaction network of genes involved in plant morphogenesis mediated by ZjTCP16. (c): Expression of genes involved in morphogenesis and cell proliferation in ZjTCP16-OE and zjtcp16 mutant jujube shoots. Bars represent the mean ± standard deviation of 9 plants. Asterisks indicate significant differences compared with WT (*: p < 0.05; one-way analysis of variance (ANOVA)).
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Yang, Q.; Li, Q.; Gu, L.; Chen, P.; Zhang, Y.; Li, Y.; Chen, Y.; Ye, X.; Tan, B.; Zheng, X.; et al. The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis. Forests 2022, 13, 723. https://doi.org/10.3390/f13050723

AMA Style

Yang Q, Li Q, Gu L, Chen P, Zhang Y, Li Y, Chen Y, Ye X, Tan B, Zheng X, et al. The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis. Forests. 2022; 13(5):723. https://doi.org/10.3390/f13050723

Chicago/Turabian Style

Yang, Qiqi, Qicheng Li, Liyuan Gu, Peng Chen, Yu Zhang, Yonghua Li, Yun Chen, Xia Ye, Bin Tan, Xianbo Zheng, and et al. 2022. "The Jujube TCP Transcription Factor ZjTCP16 Regulates Plant Growth and Cell Size by Affecting the Expression of Genes Involved in Plant Morphogenesis" Forests 13, no. 5: 723. https://doi.org/10.3390/f13050723

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