Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1–3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.


Introduction
Plants have evolved complex physiological and biochemical mechanisms to adapt to environmental stresses, including injury by abiotic or biotic factors.Callose is synthesized intracellularly, hydroxyproline-rich glycoproteins are insolubilized, and the cell wall and membrane are modified in response to wounding in plants.In addition, auxin accumulates on the upper side of the wound, promoting cell proliferation to reconnect the damaged tissues.In agriculture, this reconnection is used for grafting, in which cells divide and reconnect in the injured region, and vascular cells differentiate and regenerate.During tissue reunion in incised stems of Arabidopsis thaliana (L.) Heynh.(Arabidopsis), the cell division of pith tissue begins 3 days after incision (DAI) and reunion is completed at 7 DAI (Asahina et al. 2011).During grafting, the cells proliferate at the interface between the scion and rootstock, and new vascular tissue is formed (Melnyk et al. 2015).In Arabidopsis, auxin is indispensable in tissue reunion and grafting (Asahina et al. 2011;Nanda and Melnyk 2018).During grafting and repair of wounded stems, auxin promotes cell division, cell expansion, tissue regeneration, and vascular tissue connection (Nanda and Melnyk 2018).During this process in Arabidopsis, auxin accumulates on the upper side of the incision and induces tissue reunion (Asahina et al. 2011).It induces the expression of Arabidopsis NAC domain-containing proteins (ANACs), such as ANAC071, ANAC096, and ANAC011, which are crucial for tissue reunion (Matsuoka et al. 2021).In grafted Arabidopsis hypocotyls, auxin induces cell proliferation in vascular tissue (Matsuoka et al. 2016).High cambial activity 2 (HCA2) is induced at the grafting site by auxin and promotes phloem reconnection (Melnyk et al. 2018).Auxin influx and efflux proteins are related to tissue regeneration and vascular tissue connections.The auxin efflux proteins PIN-FORMED 1 (PIN1) and ATP-binding cassette subfamily B 1 (ABCB1) regulate grafting (Armengot et al. 2016).In wounded pea epicotyls, PsPIN1 is relocated along the wound (Sauer et al. 2006), triggering the differentiation of xylem cells.In addition, DR5::GUS staining is observed around wounded Arabidopsis stems and the auxin flows longitudinally along wounds (Mazur et al. 2016).
Polar auxin transport by PIN and ABCB proteins is crucial for plant development (Geisler et al. 2017;Sauer and Kleine-Vehn 2019).Auxin diffusion through plasmodesmata (PD) is also important in plant development, including for the appearance of lateral roots and phototropism of the hypocotyl (Band et al. 2021).During lateral root development and emergence, auxin accumulation in the lateral root primordium (LRP) zone and root tips is regulated by plasmodesmata-located protein 5 (PDLP5) (Sager et al. 2020).During phototropism of the hypocotyl, disrupting the function of glycan synthase like 8 (GSL8) affects auxin diffusion and inhibits bending (Han et al. 2014).Thus, PD closure by callose accumulation is associated with the control of symplastic auxin translocation.In addition to plant hormones, such as auxin, transcription factors, mRNA, and ions are transported through PD (Storme and Geelen 2014).PD also play a role in symplastic connection.During the grafting process, newly formed PD connect cells of the scion and stock (Chambaud et al. 2022;Kollmann et al. 1985;Kurotani and Notaguchi 2021;Van der Schoot et al. 1995).In one study, at the heterograft interface, fluorescence by carboxyfluorescein diacetate, a symplasmic tracer, was detected 3 days after grafting or later (Notaguchi et al. 2020).Therefore, several materials are symplastically transported after the new formation of PD.The control of PD opening and closing is involved in callose accumulation at the PD neck region.GSLs, plasmodesmata callose binding protein (PDCB), PDLP, and AtBG_PP contribute to PD callose accumulation (Wu et al. 2018).PDCBs have callose-binding domains and have been implicated in PD closure (Storme and Geelen 2014).PDCB1 regulates plasma membrane movement by inducing PD closure.Its overexpression increases callose deposition and limits GFP spread (Simpson et al. 2009).PDCB1 has > 50% amino acid sequence similarity to PDCB2 and PDCB3 (Simpson et al. 2009).Simpson et al (2009) showed that plants overexpressing PDCB1 or PDCB3 grow normally, while PDCB2 overexpression is lethal.Therefore, PDCB2 may play an important role in plant development.However, its function in the regeneration process is not yet clear.
To understand the function of PDCB2 during tissue reunion in the Arabidopsis incised stem model, we analyze the pdcb2 mutant phenotype and DR5::GUS distribution patterns.we showed that PDCB2 contributes to the regulation of auxin response and size of cambium/phloem region in the incision site.

EnvyGFP plasmid construction
To generate a gene for EnvyGFP, sfGFP (Fujii and Kodama 2015) provided by prof.Kodama (Utsunomiya University) was mutagenized by using PCR with Envy primers (Slubowski et al. 2015;Tseng et al. 2008; Table S1).Envy-GFP is more than twice as bright as sfGFP at 30 ℃, therefore EnvyGFP is superior to sfGFP in fluorescence observation (Slubowski et al. 2015).

Plasmid construction
To generate proPDCB2::GUS lines, PCR fragments of proPDCB2 (~2 kb upstream region) were isolated using the primers listed in Table S1.proPDCB2 was inserted into the pBI121 vector, under the control of the GUS reporter promoter.Arabidopsis (Col-0) was transformed using the floral dip method (Clough and Bent 1998) and selected with 50 µg/mL kanamycin.T3 or T4 homozygous lines were used.To generate SP-Envy-PDCB2 construction, we performed fusion PCR (Kuwayama et al. 2002).First, the signal peptide sequence of PDCB2 was generated by using DNA synthesis with PDCB2-sp primers (Table S1).Second, Envy sequence was amplified with primers Envy-F3 and GFP-R-linker from EnvyGFP plasmid.Finally, PDCB2 sequence without the signal peptide sequence was amplified with primers PDCB2-Fj and PDCB2-RP4 primers from cDNA.These DNA fragments were joined together by using PCR with BPr primers.The fragment joined was inserted into the Gateway TM pDONR TM /Zeo vector (Invitrogen TM ) by using Gateway TM BP Clonase TM II (Invitrogen TM ).SP-Envy-PDCB2 was inserted into the pH2GW7 vector (Invitrogen; Karimi et al. 2002) by using Gateway TM LR Clonase TM II (Invitrogen TM ).Arabidopsis (pdcb2-1 mutant) was transformed using the floral dip method (Clough and Bent 1998) and selected with 25 µg/mL hygromycin.T3 homozygous lines were used.

Plant inflorescence stem incision
The plant inflorescence stems were incised at 7-10 days after bolting.The first or second internode of the inflorescence stem was incised to half of its diameter under a stereomicroscope using a microsurgery knife (F•S•T, 15°, 5.0 mm blade) and grown for 0-7 days under the conditions described previously (Asahina et al. 2011).

Quantitative reverse transcription PCR
Incised inflorescence stems from seven plants were preserved in liquid nitrogen.Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen).Total RNA (400 ng or 1000 ng) was reverse transcribed using ReverTrace® (Toyobo).Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed in a total reaction volume of 15 µL consisting of 7.5 µL TB Green ProMix II (TaKaRa), 0.3 µL Rox, 1.0 µL cDNA, and 10 µM of each primer on a 7300 Real Time PCR System (Applied Biosystems).Gene expression was normalized relative to that of ACT2 (AT3G18780) in three independent replicates.

Callose staining in PD
Incised inflorescence stem samples were fixed in ice-cold Farmer's solution (3:1 [v/v] ethanol: acetic acid) for 2 h.The samples were rehydrated with 70% and 50% ethanol for 15 min each and washed with distilled water for 15 min.Next, the samples were stained with 0.1% aniline blue in 0.1 M K 3 PO 4 .Stained samples were observed using a confocal microscope (LSM700; Zeiss) with an excitation wavelength of 405 nm and a 455-510 nm band-pass filter.We followed the methods of Zavaliev and Epel (2015) and Zhao et al. (2017), and measured an area of 0.53 x 10 -2 mm 2 , and enumerated callose deposits per mm 2 using ImageJ software (NIH).First, background subtraction was performed on the sample images.In the background subtraction menu enter a value for the Rolling ball radius (30 for the sample images).Second, noise reduction by mean filter was performed on the images.In the mean menu enter the radius value (3 for the sample images).Third, Image thresholding was performed on the images.Finally, callose deposit on the images was counted by particle analysis.We counted callose deposits outside of the xylem and phloem.Amorphous spots > 20 µm 2 or those with a circularity index < 0.5 were not counted.

Decapitation and phytohormone application
For qRT-PCR, the distal region of the first internode was removed 7-10 days after bolting, and 10 -3 M indole-3-acetic acid (IAA) or distilled water (DW) with lanolin (1:3, v/v) was immediately applied.For histochemical analysis of GUS activity, the distal region of the first internode was removed 7-10 days after bolting, and 10 -3 M IAA or distilled water was immediately applied to the wound.The remainder of the first internode was partially incised 24 h after application (Asahina et al. 2011).

Analysis of the number of cells and length of the cambium/phloem
We analyzed the cross-section of incised stems 200-400 µm above the incision.The tangential line (black dot line in Fig. S3) was defined as the position of the maximum tangential width of the vascular tissue.The lateral line was perpendicular to the tangential line and was defined as the position of the maximum lateral width of the vascular tissue.The length of the cambium/phloem was measured using ImageJ (analysis area: lateral area indicated by a red line in Fig. S3).

PDCB2 was expressed in the incised side during the tissue reunion process
From among those reported by Han et al. (2019), we determined proteins associated with PD expressed during the tissue reunion process via microarray analysis (Asahina et al. 2011).PDCB2 expression was markedly increased (Table 1).We also analyzed the expression levels of PDCBs at the top and bottom of intact grafted stems on Arabidopsis hypocotyls by RNA-Seq analysis (Melnyk et al. 2018: GSE107203).The expression of PDCB2 was again markedly increased (Fig. S1).Therefore, we focused on PDCB2 and investigated its function during tissue reunion.First, PDCB2 expression at 0-7 days after incision was determined by qRT-PCR in incised and intact (non-incised) stems (Fig. 1a).PDCB2 expression increased at 0-3 days after incision, but decreased 3-7 days after incision (Fig. 1b).Next, we examined its expression in the upper (incised upper) and lower (incised lower) sections of the incised stem and in the intact stem (Fig. 1c).At 3 days after incision, its expression was elevated in the incised upper stem compared to the incised lower stem (Fig. 1d).These results suggest that PDCB2 functions in the upper part of the incision.Next, to localize PDCB2 expression, we examined the activity of proPDCB2::GUS.In longitudinal and crosssections 3 days after incision, proPDCB2::GUS staining was observed in the cortex and endodermis on the incised side (Fig. 1e-g).Therefore, PDCB2 was expressed in cortex cells during tissue reunion.Auxin plays an important role in tissue reunion (Asahina et al. 2011).To evaluate the relation between PDCB2 expression and auxin, we analyzed PDCB2 expression in decapitated stems.Decapitated plants showed decreased PDCB2 expression in the incised stem, but the expression was not restored by IAA (Fig. 1h).Thus, auxin did not have any effect on the expression of PDCB2.In addition, we examined proPDCB2::GUS activity in decapitated stems.Decapitated proPDCB2::GUS stems showed decreased GUS activity similar to qRT-PCR (Fig. 1i-k).

Decreased callose deposition in the endodermis/ cortex on the incised side 1 day after incision in the pdcb2 mutant
PDCB2 has callose-binding activity (Simpson et al. 2009), and we investigated callose accumulation 1 day after incision.Aniline blue fluorescence was observed in the phloem on the opposite of incised side (Fig. 2a, e), but in the endodermis/cortex and phloem on the incised side in the WT (Fig. 2b, f).We analyzed whether callose deposited in the endodermis/cortex localizes to PD using by 35S::MP17-GFP.MP17 is the protein that accumulates in secondary branched PD and is known as a PD marker protein (Hofius et al. 2001;Kronberg et al. 2007;Vogel et al. 2007).Aniline blue and 35S::MP17-GFP fluorescence were coincident (Fig. S2).Stained spots were enumerated using ImageJ based on a method described previously (Zavaliev and Epel 2015).In WT, the accumulation of callose in the endodermis/cortex on the incised side was increased compared to the opposite of incised side (Fig. 2i).Next, we examined callose accumulation in the pdcb2 mutant.Aniline blue fluorescence was observed on the incised side only in the phloem of the pdcb2 mutant (Fig. 2d, h).In addition, callose accumulation in the endodermis/cortex on the incised side was reduced in the pdcb2 mutant compared to the WT (Fig. 2i).

Loss of PDCB2 causes the increase in size of the cambium/phloem region during tissue reunion
To understand the function of PDCB2 during the tissue reunion process, we performed histochemical analysis of pdcb2 mutant 7 days after incision, the day on which tissue reunion was completed.In the WT, there was no difference in area between the incised and the opposite of incised side, but in the pdcb2 mutant, the incised side was clearly swollen and the area was larger (Fig. 3a, b).The reason for the larger area on the incised side was that the cambium/phloem area on this side was larger than that of the WT.Although the endodermis/cortex area appears to be enlarged in the pdcb2 mutant, measurements of the length of the endodermis/cortex area showed no significant difference (Fig. S5).Thus, in other tissues, neither cell size nor number differed between WT and pdcb2 mutant.In longitudinal sections, cell division in the cambium/phloem was significantly more active in the pdcb2 mutant than in the WT, and the area of the pdcb2 mutant was larger than that of the WT.And the lateral length of the cambium/phloem on the incised side in pdcb2 mutant was significantly increased compared to WT (Fig. 4).Then, complementation assays were also performed using 35S::SP-Envy-PDCB2 (pdcb2 mutant background) (Fig. 4e, f).35S::SP-Envy-PDCB2 (pdcb2 mutant background) showed restoration of the level of lateral length of the cambium/phloem comparable to WT (Fig. 4j).There was no change in the number of cells (Fig. S4).In the upper region of the incision, the expression of TDR/PXY and WOX4, which contribute to the proliferation of cambium cells, was increased (Fig. 5).The levels of both TDR/PXY and WOX4 were also higher in non-incised pdcb2 than the WT, although there is no statistical significance (Fig. 5).
These observations indicate that loss of PDCB2 caused the cambium/phloem region to become laterally larger during tissue reunion compared to WT.

pdcb2 mutation affects the DR5 expression on the incised side of the cortex
Auxin is also the most important hormone during tissue reunion (Asahina et al. 2011;Matsuoka et al. 2021).In addition, it induces cell expansion (Teale et al. 2006) and may cause the expansion of vascular cells in the pdcb2 mutant (Fig. 4).Therefore, we analyzed the relationship All experiment was biologically repeated at least 6 times independently with similar results.Amorphous spots whose area is larger than 20 μm 2 or circularity is smaller than 0.5 were not counted.Statistical analysis was performed by Welch's t-test.*Highly significant at P < 0.05 compared with the opposite of incised side between auxin and PDCB2 by examining the GUS signal pattern of DR5::GUS in the pdcb2 mutant background 3 days after incision compared to WT.We visualized crosssections of the WT and pdcb2 mutant.In WT, the highest level of GUS signal was detected in the endodermis/cortex on the incised side, with signal also detected in the xylem and cambium/phloem (Fig. 6a, c).In contrast, in the pdcb2 mutant, the highest level of GUS signal was detected in the cambium/phloem on the incised side, and signal was also detected in the xylem, but at very low levels in the endodermis/cortex (Fig. 6b, d).

Loss of PDCB2 affects PIN3 expression
The loss of PDCB2 affected the DR5 expression pattern (Fig. 6).Next, we focused on PIN3, which is associated with lateral auxin transport (Ding et al. 2011;Willige et al. 2013).In incised stems, PIN3 expression was significantly different 3 days after incision compared to non-incised stems (Fig. 7a).We analyzed the distribution of PIN3 expression using proPIN3::GUS.GUS signals in proPIN3::GUS plants 3 days after incision were detected in the upper part of the incision (Fig. 7b).
In cross-sections of proPIN3::GUS, GUS signals were localized in the phloem, endodermis, and cortex on the incised side (Fig. 7c, d).Next, to evaluate the relation between PIN3 expression and auxin, we analyzed PIN3 expression in decapitated stems by analysis of qRT-PCR and GUS assay.Decapitated plants showed decreased PIN3 expression in the incised stem, which was partially restored by IAA (Fig. 7e).However, PIN3 expression levels in the decapitated stem applied with IAA were lower than the non decapitated stem (Fig. 5e).Analysis of proPIN3::GUS activity was similar to qRT-PCR (Fig. 7f-h).Next, we examined the expression of PIN3 in the pdcb2 mutant 3 days after incision.The expression of PIN3 in the incised upper region 3 days after incision was increased compared to the incised region but decreased compared to the WT in the pdcb2 mutant (Fig. 7i).Furthermore, in the pdcb2 mutant, proPIN3::GUS staining was observed in the phloem close to the incision, which was different from WT (Fig. 7j-k).We also observed that the PIN3 signal was weaker in pdcb2 mutants than in WT in proPIN3::GUS plants in pdcb2 mutants background, even at the tissue level (Fig. S6).

Discussion
Microarray data of Arabidopsis with incised stems indicated that PDCB2 expression was markedly increased during tissue reunion, especially in the cortex and endodermis on the incised side (Table 1, Fig. 1).Therefore, we investigated the function of PDCB2 contributing to the control of symplastic transport by PD closure during the tissue reunion process.PDCBs are localized to PD and induce PD closure by binding PD callose (Han et al. 2019).Overexpression of PDCB1 which is the most highly conserved homolog of PDCB2, increased callose accumulation and inhibited molecular diffusion through the PD of the epidermis (Simpson et al. 2009).The defect of PDCB2 results in reduced callose deposition in the endodermis and cortex of the incision plane during the early stages of tissue reunion (Figs. 1, 2).These results implicate PDCB2 in callose accumulation in the endodermis/cortex on the incised side during tissue reunion, and this callose reduction in PD in the pdcb2 mutant may affect PD-mediated material transport in this region.In the present study, we found that loss of PDCB2 increased the size of the cambium/phloem and increased cell division during tissue reunion (Figs. 3, 4).And, the increased expression of TDR/PXY and WOX4, factors important for cell proliferation in the procambium, in the pdcb2 mutant (Fig. 5) indicates that PDCB2 deficiency promotes vascular tissue development.The PD-mediated transport by PDCB2 is prevented, thereby promoting vascular TDR/PXY and WOX4 are induced by auxin during the process of tissue recombination (Matsuoka et al. 2021).The DR5 auxin response was altered in the pdcb2 mutant at 3 days after incision compared to the WT (Fig. 6c, d).After incision, DR5::GUS signals were strongly observed in the endodermis and cortex in the WT, whereas endodermis/cortex GUS signals were weak but were strong in the cambium/phloem region in the pdcb2 mutant (Fig. 6a-d).Our results suggest that the loss of PDCB2 may have affected auxin translocation to the endodermis and cortex, resulting in higher relative concentrations of auxin in cambium/phloem.In the pdcb2 mutant, the endodermis/cortex also appeared to be enlarged, although not significantly (Fig. 3).Auxins may have different functions in cell proliferation in these tissue types, but this would not interfere with consideration of the function of the cambium/phloem.In addition, PIN3 is expressed in the affected cambium/phloem region (Fig. 7c, d, Fig. S6).It is therefore possible that the reduced expression of PIN3 due to PDCB2 deficiency affects the expansion of cambium/phloem region via abnormal auxin translocation to the endodermis and cortex.In addition, PIN3 is expressed in the affected cambium/phloem region (Fig. 7c, d), and pin3 mutant shows similar enlarged cambium/phloem as pdcb2 (Figs.S5, S7).Thus, although there is no direct evidence, it is possible that the reduced expression of PIN3 due to PDCB2 deficiency affects the expansion of cambium/phloem region via abnormal auxin translocation to the endodermis and cortex.Also, callose deposition by PDCB2 may contribute to the level of PIN3 protein.Żabka et al. (2016) suggested that an enhanced level of PIN2-like proteins (PIN2-LPs), which are recognized by antibodies against PIN-FORMED2 (PIN2) in Arabidopsis, are correlated with enhanced deposition of callose in Chara vulgaris.The normal function of the PIN protein may be affected by this structural change in the cell wall to include callose.Therefore, reduced levels of PIN3 expression or PIN3 protein may impact auxin transport within the vascular bundle, resulting in an expansion of the cambium/phloem region.Decapitation experiments show that PDCB2 expression is not significantly induced by IAA treatment (Fig. 1h) but PIN3 expression is partially induced (Fig. 7e).The auxin inducibility of PDCB2 may not be as sensitive as that of PIN3.Further investigation is required to elucidate the correlation between PIN3 and vascular formation, as well as the relationship between PIN3 and PDCB2.
During the tissue reunion process of incised Arabidopsis stems, Matsuoka et al. (2021) reported that auxin induced ANAC071 and ANAC096, which promote the proliferation of cambium-like cell which redifferentiated into xylem and phloem vessels.In wounded Arabidopsis stems, auxin induces vascular cambium regeneration and vessel formation (Mazur et al. 2016).Nanda and Melnyk (2018) suggested that during grafting, auxin contributes to callus formation, tissue fusion, and vascular bundle reconnection.High temperature promotes vascular reconnection by increasing the auxin response (Serivichyaswat et al. 2022).DOF transcription factors are induced by auxin and promote wound healing (Zhang et al. 2022).Thus, auxin contributes to vascular reconnection in tissue reunion process of wounded stem and grafting.To change the expression of DR5 auxin response in pdcb2 mutant, we considered a defect of PDCB2 might also have some effect on tissue reunion process (Fig. 6).Also, auxin regulates the size and shape of cells in various organs (Brophy 2022;Luo et al. 2018;Teale et al. 2006).In addition, auxin is involved in vascular development and affects vascular cell size (Agustí and Blázquez 2020).Overexpression of IAA1 with domain II mutation, which impairs various auxin responses, decreases the size of phloem and xylem cells of inflorescences (Ku et al. 2009;Park et al. 2002).In leaf, this overexpression affects phloem in particular and causes the formation of curly leaves (Ku et al. 2009).Also, loss of LATERAL ORGAN BOUNDARIES DOMEIN4 (LBD4) and PXY causes the change in phloem distribution but no change in the number of phloem cells compared with lbd4 and pxy single mutant (Smit et al. 2020).In addition, LBD4 and PXY are induced by auxin (Smit et al. 2020;Suer et al. 2011).These previous reports show auxin contributes to the size of phloem and xylem.
In this study, pdcb2 mutant showed a significant increase of TDR/PXY and WOX4 expression levels in the upper region of incision (Fig. 5).In contrast, TDR/PXY and WOX4 expression levels tended to be higher in non-incised pdcb2 mutant than in WT, but the difference was not significant.The increase of TDR/PXY and WOX4 expression may cause the expansion of the cambium/phloem region.Also, these genes are induced by auxin during the tissue reunion process (Matsuoka et al. 2021).Our results show that the change in auxin response pattern by defect of PDCB2 may cause the increase of TDR/PXY and WOX4 and be due to the increase in size of the cambium/phloem.Therefore, during the tissue reunion process, PDCB2 may control the appropriate size of cambium/phloem and adjust the cost balance by controlling DR5 auxin response pattern.

Fig. 1
Fig. 1 PDCB2 expression in the cortex during tissue reunion.a Stem divided into incised and intact (non-incised) stems.b PDCB2 expression 0, 1, 3, 5, and 7 days after incision by using qRT-PCR.Values are means ± SE (n = 3) from biological replicates.Statistical analysis was performed by Welch's t-test.* Significant at P < 0.05 compared with non-incised; **Highly significant at P < 0.01 compared with non-incised.c Stem divided into the upper (incised upper) and lower (incised lower) parts of the incision, and intact stem (non-incised).d PDCB2 expression 3 days after incision by using qRT-PCR.Values are means ± SD (n = 3) from biological replicates.Statistical analysis was performed by Welch's t-test.*Significant at P < 0.05 compared with non-incised; **Highly significant at P < 0.01 compared

Fig. 2
Fig. 2 Callose deposition in the endodermis/cortex on the incised side in the upper region 1 day after incision.Callose accumulation by using aniline blue staining in endodermis/cortex region (red broken rectangle) of WT (a, b, e, f) and pdcb2 (c, d, g, h) on the opposite of incised side (a, c, e, g) and incised side (b, d, f, h).The images (e-h) indicate the typical magnified images of the area enclosed by orange rectangles (a-d).Black arrows indicate PD callose (g).Bars = 100 µm in (a-d).Bars = 10 µm in (e-h).(i) Number of callose deposits in

Fig. 3
Fig. 3 Histological analysis in pdcb2 mutant 7 days after incision.(a, b) Cross-sections in the upper region of the incised in WT (a) and pdcb2 mutant (b).c, d The longitudinal section near the area indicated by the red dot in the cross-sections (a, b) in WT (c) and pdcb2

Table 1
Expression of proteins associated with plasmodesmata during tissue reunion