Genome-wide Analysis of the WUSCHEL-related Homeobox Gene Family and Functional Characterization of VcWOX4b Regarding the Inhibition of Adventitious Root Formation in Blueberry (Vaccinium Spp.)

DOI: https://doi.org/10.21203/rs.3.rs-849933/v1

Abstract

Background: Blueberry (Vaccinium corymbosum L.) is one of the most important commercial fruit tree species. The development of high-quality seedlings is a prerequisite for fruit production. Stem cutting and tissue culture methods are widely applied for propagating blueberry seedlings. Both methods require adventitious roots (ARs), indicating ARs are critical for vegetative propagation. However, the underlying factors and molecular mechanisms regulating blueberry AR formation remain relatively unknown.

Results: In this study, the rooting abilities of differentially lignified cuttings from various cultivars or the same cultivars cultured differently were evaluated following an indole-3-butyric acid (IBA) treatment. Field-grown semi-lignified and tissue culture-grown cuttings formed ARs, but the latter had more pericycle and secondary xylem cells and formed ARs more easily and faster. WUSCHEL-related homeobox genes are commonly involved in vascular tissue development and early root meristem maintenance. On the basis of the available Vaccinium corymbosum genome data, 29 putative WOX genes with conserved homeodomains were identified and divided into three major clades (modern/WUS, intermediate, and ancient). These 29 WOX genes were differentially expressed in the root, shoot, leaf, flower bud, and fruit. Additionally, a qRT-PCR analysis revealed that five selected VcWOX genes were responsive to an IBA treatment during AR formation. Accordingly, VcWOX4b was functionally characterized. The overexpression of VcWOX4b in transgenic tobacco inhibited AR formation by altering vascular cell division and differentiation and the indole-3-acetic acid (IAA):cytokinin (CTK) ratio. These observations suggest that VcWOX4b regulates the IAA:CTK ratio to promote primary xylem cell differentiation, thereby inhibiting AR formation. However, an IBA treatment can induce AR formation by inhibiting VcWOX4b expression.

Conclusions: Current study elucidates the rooting abilities of various cultivars and the cytological characters of influence on AR formation of blueberry cuttings, which may provide novel insights into the selection of high-quality blueberry cuttings. VcWOX4b, VcWOX8/9a, VcWOX11/12c, and VcWOX13b might regulate blueberry AR formation in an IBA-dependent manner. Ectopic expression of VcWOX4b modulated the IAA:CTK ratio to promotes primary xylem cell differentiation, but inhibit secondary xylem cell differentiation, ultimately leading to decreased AR formation. 

1. Background

Blueberry (Vaccinium corymbosum L.) is an important commercial fruit tree species cultivated worldwide. Currently, methods involving stem cuttings and tissue culture are the primary means of propagating blueberry seedlings. Because unique characteristics are retained by stem cuttings, they are widely used in breeding programs to propagate elite genotypes. However, the rooting capacity of blueberry stem cuttings is affected by various factors, such as phytohormones, soils, varieties, and the extent of the lignification of the cuttings[1–3]. Plant tissue culture technology has been extensively employed for the large-scale production of genetically identical blueberry seedlings. Both methods require the development of adventitious roots (ARs), which is a critical step during vegetative propagation. However, the factors and molecular mechanisms underlying the regulation of AR formation remain poorly understood. To improve the efficiency of the rooting of stem cuttings, the cellular and genetic mechanisms that control blueberry AR formation must be revealed.

The formation of ARs has been divided into the following three steps: induction, initiation, and extension. To date, two types of AR founder cells have been identified in most woody plants. Dormant AR founder cells, which initiate in the stem, require appropriate environmental conditions to differentiate, resume growth, and form AR[4]. The other AR founder cells initiate from phloem or xylem parenchyma cells within or adjacent to vascular tissues, including the interfascicular cambium or the phloem/cambium junction[5]. Blueberry ARs initiate from non-root pericycle cells[6]. There are four main activation processes. The parenchyma cell differentiates into AR primordial cells, which then divide asymmetrically to form AR primordia. The cells in the AR primordia divide continuously to generate active meristems and break through the cortex to become new ARs. Therefore, enabling differentiated somatic cells to switch their fates and develop into root meristematic cells is the most complex and essential step.

Adventitious root formation is regulated by various hormones, and a high auxin concentration is necessary for root primordium formation. Exogenous indole-3-butyric acid (IBA) significantly stimulates the blueberry stem to form ARs[1]. The auxin transporters PIN1 and PIN2 regulate polar auxin transport, and mutations to these transporters adversely affect AR growth and elongation[7]. Additionally, the interaction between cytokinin (CTK) and auxin regulates AR growth and development. Overexpressing the gene encoding cytokinin oxidase/dehydrogenase (CKX) in tobacco and Arabidopsis decreases the CTK concentration and increases the number of ARs[7]. Abscisic acid (ABA) negatively regulates AR formation through its adverse effects on indole-3-acetic acid (IAA). Thus, a high IAA:ABA ratio promotes AR formation[8].

In addition to plant hormones, several groups of transcription factors, including those from the WRKY[9], NAM/ATAF/CUC (NAC)[10], and WUSCHEL-related homeobox (WOX) families[11], regulate critical processes mediating AR formation. The WOX gene family members, which encode plant-specific homeobox (HB) domain superfamily transcription factors, are involved in the specific maintenance of stem cells in primary and secondary plant meristems as well as in the early root meristem[12, 13]. The WOX genes have been identified in most plant species, including Arabidopsis[14], cotton [15], poplar[14], Brassica napus, [16] and cucumber[17]. Although information regarding the blueberry genome has been available for many years, there has yet to be a systematic analysis of the blueberry WOX gene family.

The WOX transcription factors are crucial regulators of root development and growth. The expression of PtWOX11 in poplar is limited to the quiescent central cells of the root tip and is strongly induced at injured sites during AR formation[18]. The PagWOX11/12a gene in the hybrid poplar is mainly expressed in the root tip and lateral root[19]. Both AtWOX11 and AtWOX12 contribute to the organogenesis of new roots via an auxin-dependent induction of gene expression in the leaf procambium or adjacent parenchyma cells at injured sites[20]. This represents the first step in the cell fate transformation necessary for AR formation.

In this study, we evaluated the rooting ability of semi-lignified stems harvested from 11 blueberry cultivars and explored the rooting process using adventitious shoots regenerated by tissue culture. We also performed a comparative anatomical analysis of the cuttings to clarify their rooting abilities. To analyze the regulators of blueberry AR formation, we identified the WOX genes in the blueberry genome. The spatiotemporal expression profiles of selected WOX genes during IBA-induced AR formation were determined by a quantitative real-time polymerase chain reaction (qRT-PCR) analysis. A blueberry WOX gene (VcWOX4) encoding an inhibitor of AR formation was identified. The results of this study may be useful for future investigations of blueberry AR formation.

2. Methods

2.1 Plant materials and treatment

Experiments were conducted using mature 5-year-old blueberry trees of the following cultivars grown at the Fruit Tree Experimental Station of Zhejiang Normal University (Jinhua, China): ‘Tiffblue (TFB)’, ‘Gardenblue (GDB)’, ‘Woodard (WDD)’, ‘Saphire (SPH)’, ‘Climax (CLM)’, ‘Premier (PRM)’, ‘Brightwell (BRW)’, ‘Powderblue (PDB)’, ‘O’Neal (ONL)’, ‘Star (STR)’, and ‘Emerald (EMD)’. Trees were not pruned or chemically treated during the experimental period. Wild-type (WT) ‘Xanthi’ tobacco (Nicotiana benthamiana) was used for genetic transformations.

Non-lignified or semi-lignified branches with approximately six axillary buds harvested from trees were used to evaluate the rooting ability of cuttings. The branches were cut into three parts, each with at least two buds. Half of each leaf was removed from the branches in the laboratory to prepare cuttings. Because these branches were collected from field-grown trees, they were defined as field-grown cuttings (FGCs). The ‘STR’ adventitious shoots (4 cm long) regenerated from calli induced on MWPM medium containing 2.0 mg·L− 1 zeatin were used to explore the rooting process. These shoots were defined as tissue culture-grown cuttings (TGCs). Each cutting was cultured vertically in medium comprising coco coir and half-strength MWPM solution with or without 2.0 mg·L− 1 IBA. All plant materials were cultivated in a climate chamber with a 16-h light (28°C)/8-h dark (25°C) photoperiod and 70 ± 5% relative humidity.

2.2 Evaluation of the rooting ability of the cuttings of various blueberry cultivars

General root architecture traits, including the total AR length (TARL) and total AR surface area (TARSA), were analyzed using the WinRHIZO scanner (EPSON Expression 12000XL). The total number of cuttings, rooting cuttings, ARs, and calli per treatment were recorded, after which the rooting rate (RR), average AR length (ARL), average number of ARs (ARN), callus rate (CR), and rooting index (RI) were calculated. Rooting ability was assessed according to the RI. To determine the effects of lignification on AR formation and the differences among blueberry cultivars, the analyses were performed at 60 days post-cutting. For the TGCs, the analyses were performed every 3 days. The data were analyzed using independent sample t-tests and the variance was analyzed using the IBM SPSS Statistics 26 software. Diagrams were plotted using the Origin 2020 software. Each biological replicate consisted of 20 randomly selected cuttings.

RR (%) = (number of rooted cuttings/total number of cuttings) × 100

ARL (%) = (total AR length/total number of cuttings) × 100

ARN (%) = (total number of ARs /total number of cuttings) × 100

CR (%) = (number of cuttings with a callus/total number of cuttings) × 100

RI (%) = RR × 50 + ARL × 25 + ARN × 25

2.3 Microstructural examination of cuttings

To analyze the microstructure of the vascular tissue, 1-cm sections from the bottom of the FGCs and TGCs as well as the tobacco AR base and tip were prepared. First, the samples were immediately fixed in FAA solution (formaldehyde/acetic acid/70% ethanol = 5:5:90, v/v/v), softened for about 10 days in 4% ethylenediamine, and then dehydrated using an ethanol gradient (75%, 85%, 95%, and 100%). After a vitrification step involving dimethylbenzene, the samples were embedded in paraffin. Transverse sections (10-µm thick) were cut using the RM2265 rotary microtome (Leica). The samples were examined using the ECLIPSE E200 light microscope (Nikon) and photographed[6].

2.4 Identification of putative VcWOX genes in blueberry

Arabidopsis WOX protein sequences downloaded from The Arabidopsis Information Resource database were used as queries for a local BLAST screening of the blueberry genome database (https://www.vaccinium.org). The SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.sanger.ac.uk/search) online programs were used to verify all VcWOX protein sequences. Additionally, the ExPASy ProtParam online tool (http://www.expasy.org/tools/protparam.html/) was used to predict the molecular weight, isoelectric point, and grand average of hydrophilicity value of the VcWOX proteins.

2.5 Sequence analysis and phylogenetic tree construction

Conserved motifs were detected using MEME Suite 5.0.2 (http://meme-suite.org/tools/meme), with the following parameters: any number of repetitions per sequence; motif width range of 60–70 amino acids; and 11 as the maximum number of motifs. Next, the detected motifs were aligned using the MAST and Patmatch programs[21]. A multiple sequence alignment was performed using ClustalW in conjunction with MEGA X[22], and phylogenetic trees were constructed according to the neighbor-joining method, with P-distance and pairwise deletion.

2.6 DNA extraction, cDNA synthesis, and qRT-PCR

Total RNA was extracted from the cuttings using the Quick-RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA). The RNA quality was evaluated using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). First-strand cDNA was synthesized using the SuperScript® II Reverse Transcriptase kit (Invitrogen, USA). The qRT-PCR analysis was performed using the 7500 Real-time PCR system (Applied Biosystems). The 20-µL reaction solutions contained SYBR Premix Taq II (TaKaRa, Dalian, China). Relative gene expression levels were calculated according to the 2−ΔΔCt algorithm, with VcGAPDH used as the reference gene. The qRT-PCR analysis was completed using three biological replicates and three technical replicates per biological replicate. Details regarding the qRT-PCR primers are listed in Supplementary Table 1.

2.7 VcWOX4b gene cloning, vector construction, plant transformation, and screening of transgenic lines

The VcWOX4b coding sequence was amplified by PCR using gene-specific primers (Supplementary Table 2). The PCR product was cloned into GEC vector and then subcloned into pMDC32 to produce the 35Spro:VcWOX4b recombinant plasmid using the Gateway™ LR Clonase™ II Enzyme Mix (Invitrogen Shanghai Trading Co. Ltd., Shanghai, China). The 35Spro:VcWOX4b recombinant plasmid was inserted into tobacco via Agrobacterium tumefaciens-mediated transformation[23]. Thirty independent T0 transgenic lines were screened by RT-PCR and transplanted into soil.

2.8 Phenotypic analysis of transgenic tobacco roots

The roots of the T0 and T1 tobacco plants were examined. Tobacco cuttings with three leaves were prepared from 30-day-old seedlings by severing the stem above the base (3.0 cm from the apex). The cuttings were cultured vertically in half-strength MS medium. The number of cuttings, rooting cuttings, and ARs were recorded at 8 days post-cutting. The RR and ARN were then calculated. Each biological replicate comprised 20 randomly selected cuttings. Seeds from WT and T1 transgenic plants were germinated on half-strength MS medium. The primary root length and the number of lateral roots of the resulting seedlings were measured using Image J.

2.9 Determination of phytohormone contents during the AR formation of transgenic VcWOX4b plants

The phytohormone contents of the WT and transgenic tobacco seedlings were determined. A 1-cm section from the bottom of the seedling cuttings was sampled every 3 days and immediately frozen in liquid nitrogen. The hormones were separated using the Exion LC system (AB SCIEX) equipped with the Acquity UPLC BEH C18 column (2.1 mm × 100 mm, particle size: 1.7 mm) and then quantified on the basis of internal standards using the QTRAP 5500 LC-MS/MS system (AB SCIEX). The following parameters were used: optimized ion spray voltage: 5,500 V (positive ion mode) for CK, ABA and JA; 5,500 V (negative ion mode) for IAA and JA-Ile; turbo heater temperature: 600°C; nebulizing gas (Gas 1): 60 psi; heated gas (Gas 2): 60 psi. Data were processed using the MultiQuant software (version 3.0.2; AB SCIEX). The column was maintained at 40°C and the mobile phases for the analyses of CK and ACC consisted of water (A) and MeOH (B). The following multistep linear gradient elution was used: 5% B, 0–2.5 min; 5–20% B, 2.5–3 min; 20–50% B, 3–12.5 min; 50–100% B, 12.5–13 min; 100% B, 13–15 min; 100–5% B, 15–15.2 min; and 5% B, 15.2–18 min. The mobile phases for analyzing ABA, SA, JA, GA, IAA, and JA-Ile consisted of water and 0.1% formic acid (A) and MeOH and 0.1% formic acid (B). The following multistep linear gradient elution (flow rate of 0.3 mL min− 1) was used: 20% B, 0–1 min; 20–100% B, 1–7 min; 100% B, 7–9 min; 100–20% B, 9–9.3 min; and 20% B, 9.3–12 min[24].

3. Results

3.1 IBA significantly promoted AR formation in various blueberry cuttings

To clarify the effect of the lignification level on blueberry AR formation, the cuttings of non-lignified and semi-lignified branches of ‘EMD’ trees were analyzed. The RR was calculated at 60 days post-cutting. Rooting was detected for both cutting types in response to the treatment with 2.0 mg·L−1 IBA, but the RR of the semi-lignified cuttings was significantly greater than that of the non-lignified cuttings (Figure 1A). For both cutting types, the IBA treatment induced AR formation, although this induction was greater for the semi-lignified cuttings. The IBA treatment also significantly increased the ARN for the non-lignified and semi-lignified cuttings (Figure 1B).

To further evaluate the AR formation capacity of different blueberry cultivars (e.g., ‘TFB’, ‘GDB’, ‘WDD’, ‘SPH’, ‘CLM’, ‘PRM’, ‘BRW’, ‘PDB’, ‘ONL’, and ‘STR’), the semi-lignified cuttings were cultivated in substrates containing 2.0 mg·L−1 IBA. The AR formation capacity at 60 days post-cutting varied among the cultivars. Considering the RR, ARN, and ARL (Figure A-D), the rank order of the RIs (high to low) was ‘BRW’, ‘CLM’, ‘STR’, ‘ONL’, ‘SPH’, ‘TFB’, ‘PRM’, ‘WDD’, ‘GDB’, and ‘PDB’ (Figure 2E, F).

Because tissue culture methods can produce high seedling yields, they are commonly used to propagate blueberry seedlings. In this study, 4 cm long TGCs were cultivated in substrate containing 2.0 mg·L−1 IBA. At approximately 9 days post-cutting, a callus was detectable at the bottom of the stems. At 12 days post-cutting, ARs were detected. The CR, RR, ARN, ARL, and TARSA gradually increased as the incubation time increased (Figure3A-F). Moreover, the RI of the TGCs was 54.5%, which was significantly higher than the RI (20.8%) of the FGCs. These results indicate that ARs formed easily and quickly in the TGCs(Figure3G).

3.2 Increases in the number of pericyclic cells in tissue culture-grown semi-lignified cuttings likely enhance AR formation

Microstructural examinations of cuttings were performed to clarify why the rooting abilities varied among cuttings. The non-lignified and semi-lignified cuttings contained a similar number of pericycle cells. However, there were significant differences in the phloem cells between the two cutting types. Compared with the non-lignified cuttings, the semi-lignified cuttings had more layers of xylem cells, especially secondary xylem cells (Figure 4A, B). These results suggest that xylem cells significantly influence AR formation when there are no differences in the pericyclic cells.

Because the ARs of TGCs formed easily and quickly, the microstructures of the semi-lignified cuttings and TGCs were analyzed further. There was no noticeable difference in the proportion of secondary xylem cells in these two cutting types, implying the degree of lignification was similar (Figure 4B, C). However, significant differences were observed in the pericyclic cells. There were more pericyclic cell layers in the TGCs than in the semi-lignified cuttings, implying that an increase in the number of pericyclic cells promotes AR formation when the degree of lignification is appropriate.

3.3 Identification and classification of VcWOX transcription factors in blueberry

Arabidopsis WOX protein sequences were downloaded and used to identify WOX genes in the blueberry genome database. Twenty-nine putative WOX genes (516–1,335 bp long) with conserved homeodomains were identified. The deduced WOX protein sequences comprised 171–444 amino acids. To clarify the phylogenetic relationships among WOX genes, an unrooted phylogenetic tree consisting of WOX proteins from V. corymbosum, Arabidopsis, Oryza sativa, Actinidia chinensis, and Populus trichocarpa was constructed. The WOX transcription factors were classified into the following three subfamilies (Figure 5A): ancient clade, modern/WUS clade, and intermediate clade. The modern/WUS clade was the largest group, with 21 members from V. corymbosum, 8 members from Arabidopsis, 7 members from O. sativa, 11 members from A. chinensis, and 11 members from P. trichocarpa (Figure 5B). The intermediate and ancient clades included only six and two members from V. corymbosum, respectively. Moreover, paralogous and orthologous relationships between the WOX gene families of Arabidopsis and blueberry were revealed. Therefore, on the basis of the phylogenetic relationships, some of the putative blueberry WOX genes were named as follows: VcWUSa/VcWUSb, VcWox1a/VcWox1b/VcWox1c, VcWox2a/VcWox2b/VcWox2c/VcWox2d, VcWox3a/VcWox3b/VcWox3c/VcWox3d, VcWox4a/VcWox4b/VcWox4c/VcWox4d, VcWox5/7a/VcWox5/7b/VcWox5/7c/VcWox5/7d, VcWox8/9a/VcWox8/9b, VcWox11/12a/VcWox11b/12/VcWox11/12c/VcWox11/12d, and VcWox13a/VcWox13b. The increase in the number of genes was the result of gene family evolution. Additionally, analyses of physicochemical properties indicated that the VcWOX protein molecular weight ranged from 13.96 to 71.82 kDa, with isoelectric points between 5.08 and 9.58. The subcellular localization analysis suggested that all 29 VcWOX proteins are nuclear proteins (Supplementary Table S3). 

3.4 Detection of conserved residues and motifs in blueberry WOX proteins

To characterize putative conserved motifs in the VcWOX family, the 29 deduced VcWOX amino acid sequences were examined using MEME. Fifteen conserved motifs (1–8) were detected, ranging in size from 15 to 50 amino acids (Table 1). Motifs 1 and 2 were distributed in all 29 VcWOX proteins. In contrast, motif 3 was present only in the WUS clade proteins. Thus, the motifs in VcWOX proteins appear to be family-specific (Figure6A, B).

The featured domain sequences usually determine the primary function of a plant transcription factor. An analysis of the exon and intron structures revealed that VcWOX genes contain 2–4 exons and 1–3 introns (Figure6C). By comparing the amino acid sequences of VcWOX and AtWOX proteins, the conserved amino acid residues within the homeodomain were identified for each clade. Residues 4R, 5W, 7P, 11Q, 15L, 23G, 27P, 35I, 39L, 43G, 49N, 50V, 53W, 54F, 55Q, 56N, and 60R were conserved in the 29 VcWOX sequences (Figure7).

Table 1 

Sequence of the conserved motif of VcWOX proteins

Conserved Motif

Motif sequence

Motif 1

ILEELYRGGMRTPTADQIQQITAQLSKYGKIEGKNVFYWFQNHKARERQK

Motif 2

SGEGGNSPGSSRWNPTPEQJR

Motif 3

RRIETLELFPJHPTG

Motif 4

YQPPGFMTVFINGVPTEVGRGPIDIKGMFGEDLVLVHSSGIPVPFNDYGF

Motif 5

GALDPLAPELPGGSSSACTAATADAAFDLKSFIAPESGPQGYLSNEQKTP

Motif 6

LRRKLMNKQMMHQQHVLYNPQQQQQQNHHHHSHLLHQFPGHYTPVGRGFL

Motif 7

PRASAASTITTISLDTRGQMEKEVEESPYKRKCRTWTFEGLEEEKRHCKD

Motif 8

CEYVDKSEPKTYPPHYLKMLEQGPTKP

3.5 Expression profiles of VcWOX genes in different blueberry organs

Gene expression patterns in different organs may provide critical information regarding gene functions related to organ development. The VcWOX expression data for various ‘Draper’ blueberry organs (i.e., root, salt-treated root, leaf, methyl jasmonate-treated leaf, flower bud, and fruit) were downloaded from the Vaccinium genome database (https://www.vaccinium.org). The heatmap of VcWOX expression generated using TBtools revealed the diversity in the expression levels among untreated and treated organs (Figure 8). More specifically, VcWOX4b, VcWOX5/7c, and VcWOX11/12c were highly expressed in the root, but their expression levels decreased in response to the salt treatment. Hence, these three genes might affect root development and responses to salt stress. The VcWOX4a, VcWOX4b, VcWOX4c, VcWOX4d, VcWOX13a, and VcWOX13b genes were highly expressed in the shoot. During fruit development, the VcWOX11b, VcWOX11d, and VcWOX13b expression levels were upregulated in the pink stage, but downregulated in the ripe stage, suggesting that these genes regulate blueberry fruit development. Both VcWOX1 and VcWOX3 were mainly expressed in the leaves. The VcWOX1a and VcWOX3a expression levels decreased at 1 h after the methyl jasmonate treatment, but then subsequently increased at 8 h before decreasing again at 24 h.

3.6 Expression level analysis of selected VcWOX genes during AR formation in blueberry

The root VcWOX expression levels determined on the basis of transcriptome data may not fully reveal the potential functions of these genes related to root formation and development. Therefore, the VcWOX4b, VcWOX5/7c, VcWOX8/9a, VcWOX11/12c, and VcWOX13b expression levels were verified by a qRT-PCR analysis (Figure 9). Total RNA was extracted from the IBA-treated stems, which were then harvested at 0, 3, 6, 9, 12, 15, 18, 21, and 24 days post-treatment. The VcWOX4b and VcWOX13b expression levels were downregulated continuously following the 2.0 mg L−1 IBA treatment (Figure 9A, B), indicating IBA decreased the expression of these genes during AR formation. Additionally, VcWOX8/9a expression was significantly upregulated (7.0 times) at 6 h, but was then downregulated (Figure 9C). The VcWOX11/12c expression levels were upregulated continuously, peaking at 18 h (Figure 9D). These results suggest that IBA activated the expression of VcWOX8/9a and VcWOX11/12c, which might be involved in AR formation. The VcWOX5/7a expression levels fluctuated after the IBA treatment (Figure 9E).

3.7 Overexpression of VcWOX4b resulted in multiple developmental defects in transgenic plants

The VcWOX4b gene was primarily expressed in the root and shoot. Moreover, its expression level decreased in response to the IBA treatment during AR formation. To further elucidate the regulatory function of VcWOX4b in blueberry, VcWOX4b was cloned and inserted into pMDC32 to produce the 35Spro:VcWOX4b recombinant plasmid. A total of 30 transgenic tobacco plants were produced via A. tumefaciens-mediated transformation (Figure10).

Some regenerating T0 plants had abnormal roots (Figure10A). Specifically, the number and length of ARs decreased significantly and the tips were swollen, indicating that VcWOX4b inhibited AR formation and growth (Figure10C). This abnormal root development caused most of these plants to die after they were transferred to soil. Only one severely defective T0 plant survived in soil, but it exhibited extreme dwarfism and was unable to produce flowers or seeds. The VcWOX4b expression levels varied among the transgenic lines, but some of the surviving transgenic lines with low VcWOX transcript levels grew to maturity and produced some T1 seeds. To investigate the effects of VcWOX4b on the root system, T1 transgenic and WT seeds were germinated on half-strength MS medium(Figure 10B). The primary root of the VcWOX4b-overexpressing transgenic seedlings was significantly shorter than that of the WT seedlings (Figure 10D), implying that VcWOX4b repressed primary root growth.

3.8 Overexpression of VcWOX4b altered vascular cell division and differentiation in transgenic tobacco

Because AR formation and growth was inhibited in the transgenic tobacco, the microstructures of the stem and root vascular tissues were characterized. There were significantly more vascular cells in the transgenic tobacco stem than in the WT tobacco stem, suggesting the overexpression of VcWOX4b promoted vascular cell division (Figure 11). Additionally, the vascular tissue of transgenic tobacco stems contained more cambial and primary xylem cells, but fewer secondary xylem cells, than the vascular tissue of WT tobacco stems (Figure 11). This indicated that the overexpression of VcWOX4b enhanced vascular cell differentiation. There were no noticeable differences in the pericycle cells between the WT and transgenic tobacco. Increases and decreases in the number of primary xylem cells and secondary xylem cells, respectively, may explain the decreased production of ARs by the transgenic tobacco.

The overexpression of VcWOX4b resulted in increased root thickness, decreased AR length, and swollen root tips. A microstructural analysis of the vascular tissue at the root base and tip revealed there were more cambial and xylem cells in the transgenic tobacco root base and tip than in the WT tobacco root base and tip. This observation suggests the overexpression of VcWOX4b accelerated vascular cell division and differentiation in the root, which altered the transgenic root phenotype (Figure 12).

3.9 VcWOX4b inhibited AR formation by modulating the IAA:CTK ratio

Hormone levels and proportions were analyzed in transgenic tobacco and WT cuttings at five time-points during AR formation. On the basis of the observations, tobacco AR formation was divided into the following three phases: induction (P1 to P2), initiation (P2 to P3), and extension (P3 to P5) . In transgenic tobacco, the ABA content decreased from P1 to P2, increased from P2 to P4, and decreased from P4 to P5. However, in WT tobacco, the ABA content increased from P1 to P2 and then decreased in the subsequent phases(Figure13A). The IAA content of transgenic tobacco was significantly lower than that of WT tobacco at P2, but it subsequently gradually increased and was significantly higher than that of WT tobacco at P3 and P5 (Figure 13B). The JA content of both WT and transgenic tobacco decreased to its lowest level at P3 and then gradually increased; however, the JA content was lower in transgenic tobacco than in WT tobacco. The JA-ILE content was significantly lower in transgenic tobacco than in WT tobacco at P2 and P5 (Figure 13D, E). The TZR, isopentenyl adenine (IP), and cis-anthocyanidin (CZ) contents were significantly higher in transgenic tobacco than in WT tobacco from P3 to P5 (Figure 13G–I). The TZ contents were substantially higher in transgenic tobacco than in WT tobacco at P3 and P4 (Figure 13J). The IPR content was significantly higher in transgenic tobacco than in WT tobacco from P2 to P5 (Figure 13K). In contrast, the CZR content in transgenic tobacco increased from P1 to P5. Moreover, the CZR content was higher in transgenic tobacco than in WT tobacco at P1 and P2, but the opposite pattern was observed at P3, P4, and P5 (Figure 13J). Plant growth and development are mediated by multiple hormones. The TZR, CZR, IPR, TZ, CZ, and IP contents increased as the CTK content increased(Figure13K). During AR formation, the IAA:CTK ratio exceeded 3.0 in WT tobacco, but it was significantly lower in transgenic tobacco (i.e., less than 1.5 from P2 to P5) (Figure 13L).

4. Discussion

4.1 A threshold number of xylem and pericycle cells is required for AR formation in blueberry

For both FGCs and TGCs, AR formation is a crucial step of the seedling propagation process. The essential difference between these two cutting types was the differentiation of the stem vascular tissue. In the present study, FGCs harvested from non-lignified or semi-lignified branches were examined; the semi-lignified cuttings had a higher RR. An increase in the number of xylem cells, especially secondary xylem cells, is critical for AR formation. Xylem efficiently transports water from the roots to the rest of the plant[25]. However, xylem is not sufficiently differentiated in the non-lignified cuttings, resulting in impeded water transport. Therefore, in addition to a lower RR, the non-lignified cuttings had a higher death rate than the semi-lignified cuttings during AR formation (data not shown). These results suggest a certain number of secondary xylem cells is required for AR formation in blueberry. However, adventitious rooting and xylogenesis are antagonistic events in the hypocotyls of pine[26].

With the exception of more layers of pericyclic cells in the TGCs, there were no significant differences between the field-grown semi-lignified cuttings and the TGCs. Previous research demonstrated that blueberry ARs are initiated from non-root pericycle cells[6, 27]. A certain proportion of pericycle cells is required for the AR formation of blueberry. Hence, the RI was significantly higher for the TGCs than for the FGCs. The TGCs were adventitious shoots regenerated from somatic embryos cultivated in MWPM medium supplemented with 2.0 mg L− 1 zeatin. The number of pericycle cells might be associated with highly active CTK. Moreover, the pericycle cells may be considered as founder cells in blueberry, in which auxin controls founder cell division and elongation[27]. Therefore, in the present study, 2.0 mg L− 1 IBA efficiently induced the AR formation of TGCs. Additionally, the ability to form ARs varied among the cuttings from diverse blueberry cultivars, which is consistent with the results of a previous study[6]. The optimal conditions for AR induction likely differ between blueberry cultivars, and will need to be determined in future studies.

4.2 The blueberry WOX gene family expanded and contracted during evolution

The WOX gene family regulates various plant developmental processes, especially organ morphogenesis, in many species[14, 16, 17, 28–30]. Because the blueberry genome sequence has been published, systematic analyses of the WOX gene family in blueberry are possible[31]. On the basis of a comparison with the 15 WOX genes in Arabidopsis (diploid), 29 VcWOX genes were identified in blueberry (tetraploid), implying that the WOX gene family expanded in blueberry. Fifty-two WOX genes have been identified in B. napus (allotetraploid), suggesting gene expansion events are ubiquitous in polyploid plants[16]. Furthermore, polyploidization events appear to be the leading cause of the expansion of the WOX gene family. In Arabidopsis, WOX1, WOX4, WOX5, WOX9, and WOX11 are homologous or functionally redundant with WOX6, WOX14, WOX7, WOX8, and WOX12, respectively[32, 33]. The orthologous relationships of some WOX genes in blueberry and Arabidopsis cannot be distinguished by a phylogenetic analysis alone. Nevertheless, we named the blueberry WOX genes identified in this study (e.g., VcWox5/7a/VcWox5/7b/VcWox5/7c/ VcWox5/7d and VcWox8/9a/VcWox8/9b). Additionally, the rearrangement of the genome sequence after hybridization or chromosome doubling always leads to gene loss. Both VcWOX6 and VcWOX14 were not detected in blueberry; these genes were likely lost during blueberry hybridization or chromosome doubling.

The HB domain, which consists of 60–66 amino acids folded into a helix–turn–helix structure, is a common feature of WOX proteins[34]. The completely conserved residues 4R, 5W, 7P, 11Q, 15L, 23G, 27P, 35I, 39L, 43G, 49N, 50V, 53W, 54F, 55Q, 56N, and 60R were detected in the 29 VcWOX protein sequences. On the basis of the conserved domain architecture and high sequence similarity, the WOX proteins in blueberry, Arabidopsis, rice, poplar, and kiwifruit were classified into three subfamilies (i.e., ancient clade, modern/WUS clade, and intermediate clade). A MEME analysis revealed the differences in the motif compositions of the deduced polypeptides. Some motifs were present within the HB domain, which is associated with high-affinity binding to DNA[35].

4.3 VcWOX expression patterns differ among organs and are altered by hormones and abiotic stress

Gene expression patterns often provide clues regarding gene functions. Previous studies proved that the WOX family members play crucial roles in embryonic patterning, stem cell maintenance, and organ formation[12, 36]. Most of the VcWOX genes were expressed at low levels or were not expressed in specific organs. However, VcWUSa and VcWUSb were highly expressed in flower buds. The expression profiles also revealed the similarities and differences in the expression patterns among paralogous VcWOX genes. The VcWOX4a, VcWOX4b, VcWOX4c, and VcWOX4d genes were expressed in the shoot, but only VcWOX4b was highly expressed in both the root and shoot. Conserved expression patterns may reflect functional conservation, whereas divergent expression patterns may be indicative of the development of a new function[37]. The WOX gene expression features modified by chromosomal reduplication have been reported[16, 38]. Additionally, the transcription of WOX genes is regulated by stresses. For example, OsWOX3 and OsWOX5 expression in rice is induced by salt stress[39], whereas GhWOX10, GhWOX12, GhWOX13a, and GhWOX13b expression in cotton is responsive to drought or saline conditions[28]. The expression levels of VcWOX11/12a and VcWOX11/12b increase in salt-treated roots. Some WOX genes encode regulators of root formation, growth, and development[40, 41]. Because VcWOX4b, VcWOX5/7c, and VcWOX11/12c are mainly expressed in the root, they may modulate blueberry root growth and development. A qRT-PCR analysis verified that IBA activates the expression of VcWOX8/9a and VcWOX11/12c, but has the opposite effect on the expression of VcWOX4b and VcWOX13b during AR formation. Thus, these genes might regulate AR formation in an IBA-dependent manner.

4.4 Effects of VcWOX4b on vascular tissue differentiation and AR formation

Vascular stem cells in the procambium and cambium are involved in xylem and phloem differentiation[42]. In Arabidopsis, AtWOX4 is required for maintaining stem cells in the cambium[43]. In poplar, PtrWOX4a is highly expressed in the cambium and procambium[32]. The overexpression of SlWOX4 in tomato leads to an increase in the number of xylem and phloem cells[44]. Adventitious roots may be initiated from pericycle cells. Furthermore, AR formation and xylogenesis are antagonistic events in some forest tree species[26]. In the present study, the overexpression of VcWOX4b altered vascular cell division and differentiation in transgenic tobacco. Compared with the WT control, the transgenic tobacco had more primary xylem cells, but fewer secondary xylem cells in the vascular tissue.

In plants, WOX proteins play a vital role in root growth and development. Earlier studies confirmed that PtoWOX4 is expressed in poplar roots[32]. Additionally, the ARN and ARL reportedly increase and decrease, respectively, in LkWOX4-overexpressing plants[41]. These findings indicate that LkWOX4 is involved in the AR developmental process in Larix kaempferi. The silencing of OsWOX4 by RNA interference leads to a significant increase in the primary root length. In contrast, its overexpression significantly inhibits primary root elongation. The expression of OsWOX4 modulates the extension of primary roots because of the associated activation of AUX1 expression[45]. The overexpression of AtWOX4 in Arabidopsis is detrimental to root elongation and root tip meristem enlargement[43]. In this study, we observed that the ARN and ARL of the transgenic samples decreased significantly, implying VcWOX4b inhibits AR formation.

Phytohormones interact with WOX4 to regulate vascular tissue growth and development. A WOX4 transcription factor is positively regulated by auxin, and MP regulates its downstream HD-ZIP III transcription factor to induce WOX4 expression, which is essential for the initiation and morphogenesis of the plant vascular cambium[46]. In rice, OsWOX4 can activate the expression of AUX1 and influence the elongation of the primary root by regulating auxin transport[45]. Moreover, LkWOX4 may regulate AR growth through IAA, JA, and ABA signaling pathways. Additionally, WOX4 is related to CTK[41, 47]. The IP and CZ contents are higher in calli overexpressing OsWOX4 than in control calli[45]. The overexpression of OsWOX4 also leads to upregulated expression of the CTK-related genes OSH1, OsLOGL3, and OsLOGL10 in transgenic rice plants [48]. Knocking out OsWOX4 results in decreased CTK levels[49]. In the present study, the CTK content was significantly higher in the transgenic tobacco than in the WT tobacco. CTK negatively regulate AR formation, possibly by minimizing the inductive effects of IAA. High IAA:CTK ratios can promote AR formation. The IAA:CTK ratio was clearly lower for the VcWOX4b-overexpressing transgenic tobacco than for the WT tobacco. We speculate that VcWOX4b alters the IAA:CTK ratio to inhibit AR formation and growth.

5. Conclusions

The cytological and molecular basis of AR formation during blueberry seedling propagation should be comprehensively characterized. In this study, an analysis of rooting abilities indicated that the TGCs, in which the number of pericycle and secondary xylem cells increased, developed roots relatively easily and rapidly. By screening the blueberry genome database, we identified 29 VcWOX genes with conserved homeodomains; the characteristics of these genes were similar to those of AtWOX genes. The expression of some VcWOX genes was markedly upregulated or downregulated in the root, shoot, leaf, flower bud, and fruit, indicating that they may be essential for the growth and development of these organs. Five selected VcWOX genes were responsive to IBA treatments during AR formation, of which VcWOX4b transcription was downregulated by IBA. The overexpression of VcWOX4b inhibited AR formation. It is possible that VcWOX4b regulates the IAA:CTK ratio to promote primary xylem cell differentiation, but inhibit secondary xylem cell differentiation, ultimately leading to decreased AR formation (Fig. 14).

Abbreviations

Full name

Abbreviation

Adventitious root

AR

Indole-3-butyric acid

IBA

Indole-3-acetic acid

IAA

Cytokinin

CTK

Cytokinin oxidase/dehydrogenase

CKX

Abscisic acid

ABA

NAM/ATAF/CUC

NAC

WUSCHEL-related homeobox

WOX

Homeobox

HB

Quantitative real-time polymerase chain reaction

qRT-PCR

Tiffblue

TFB

Gardenblue

GDB

Woodard

WDD

Saphire

SPH

Climax

CLM

Premier

PRM

Brightwell

BRW

Powderblue

PDB

O’Neal

ONL

Star

STR

Emerald

EMD

Wild-type

WT

Field-grown cuttings

FGCs

Tissue culture-grown cuttings

TGCs

Total AR length

TARL

Total AR surface area

TARSA

Rooting rate

RR

Average AR length

ARL

Average number of ARs

ARN

Callus rate

CR

Rooting index

RI

Pericycle

Pe

Cambial cells

Cc

Phloem

Ph

Xylem

Xy

Primary xylem

Pri-XY

Secondary xylem

Sec-Xy

Declarations

Availability of data and materials

The whole genome data of blueberry (Vaccinium spp.) is downloaded from the GDV database (https://www.vaccinium.org/crop/blueberry). The transcriptome data is obtained from GDV (https://www.vaccinium.org/transcriptData), and the accession number is PRJNA417702. The WOX protein sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa L.), poplar (Populus L.), and kiwifruit (Actinidia Chinensis) are downloaded from the PlantTFDB database (http://planttfdb.gao-lab.org/index.php). Public access to all databases is open. The datasets supporting the conclusions of this article are included within the article and are available from the corresponding author on reasonable request.

Acknowledgements

Not Applicable.

Funding

This study was supported by grants from the Key Research Project of Science Technology Department of Zhejiang Province (2016C02052-9), the Zhejiang Province Public Welfare Technology Application Research Project (LGN21C150011) and the Major Scientific and Technological Project of Zhejiang Province (2018C02007).

Author Contributions

The work presented here was a collaborative effort among all the authors. YZ and WG formulated the research topic, designed the methods and revised the manuscript. YG and KL conducted experiments, analyzed data and interpreted the results. YG wrote the manuscript. YY, XL and JL carried out the DNA and RNA extractions and qRT-PCR analyses. YL and YZ performed the bioinformatics analyses. All authors read and approved the final manuscript.

Ethics declarations

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

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