GSK3 activity is a cell fate switch that balances the ratio of vascular cell type

The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as “highways” for transport, and companion cells (CCs), which serve as “gates” to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. In this study, we develop a novel culture system for CC differentiation named VISUAL-CC, which reconstitutes the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess much more SEs and less CCs in planta. Conversely, gsk3 gain-of-function mutants induced by phloem-specific promoter partially increased the CC ratio. Taken together, GSK3 activity appears to function as a cell fate switch in the phloem, thereby balancing the SE/CC ratio.


Introduction
Multicellular organisms possess a variety of functional cells with a proper ratio for their life maintenance. In plants, the phloem tissue is composed of major two cell types; sieve elements (SEs) as conductive tubes and phloem companion cells (CCs) as helper cells for phloem transport. Phloem CCs function to support neighbouring SEs through connected plasmodesmata. Although they function together with each other to ensure phloem transport, it has long been a deep mystery how the ratio of SE/CC is strictly controlled in the phloem. Recent technical advances enabled to identify various regulators that control SE differentiation 1 . In contrast to SEs, understanding of the molecular mechanism underlying CC differentiation remains a long-standing challenge.
Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) is a culture system that can artificially mimic plant vascular development 2 . In the VISUAL system, mesophyll cells are reprogrammed into vascular stem cells, and then differentiated into xylem vessel elements or phloem SEs within a couple of days.
VISUAL enables the molecular genetic studies of vascular development, leading to the in-depth understanding of regulatory network especially for phloem SE differentiation.
Even in VISUAL, differentiation into phloem CCs rarely occurs 2 , which makes it difficult to study CC development in detail.
In this study, we develop a new culture system for CC differentiation named VISUAL-CC by modifying the conventional VISUAL method. Based on comprehensive gene expression analysis in VISUAL-CC, here we reveal that GLYCOGEN SYNTHASE KINASE 3 (GSK3) activity plays an important role in determining the SE/CC ratio. In vivo genetic analyses confirm the importance of GSK3 as a cell fate switch in phloem development.

VISUAL-CC is a novel culture system for CC differentiation
The conventional VISUAL system can induce ectopic xylem (XY) or phloem SEs via the stage of vascular stem cell (Fig. 1a). Toward the understating of CC differetniation, we modified the VISUAL based on a luciferase-based screen with SUCROSE-PROTON SYMPORTER 2 (SUC2) pro:ELUC, a specific CC marker 3 (Fig. 1b). In this screen process, vascular stem cells were induced by the conventional VISUAL method in advance, subsequently were exposed to a variety of culture media. After a series of screens with different media, we could induce pSUC2:ELUC activity (Fig. 1c) and ectopic expression of the pSUC2:YFPnls marker in cotyledons within 4 days using CC medium (Fig. 1d). Hereafter, we refer to this culture system as "VISUAL-CC". To further investigate the spatial pattern, a dual phloem marker line expressing pSUC2:YFPnls and SIEVE-ELEMENT-OCCLUSION-RELATED 1 (SEOR1) pro:SEOR1-RFP 5 , a specific SE marker was established. Detailed observation of the dual phloem marker line by confocal microscopy after tissue-clearing treatment (ClearSee) 4 revealed that CCs expressing pSUC2:YFPnls (green) are detected only in dividing cell-clusters and are always limited to the cells adjacent to SEs expressing pSEOR1:SEOR1-RFP 5 (red) (Fig. 1e). Thus, CC and SE markers appeared next to each other after several rounds of cell division (Fig. 1e). Observations using a field emission scanning electron microscope (FE-SEM) or transmission electron microscopy (TEM) consistently visualized CC-like cells with dense cytoplasm adjacent to SEs with brighter cytoplasm (Fig. 1f). These cells showed minor vacuolation and developed the branched plasmodesmata typically seen in SE-CC complexes in vivo (Fig. 1f-h). In VISUAL, SMXL4 and SMXL5 are known as important regulators for early phloem SE development ( Supplementary Fig. 1a) 6,7 . In VISUAL-CC, the double mutant smxl4 smxl5 significantly suppressed CC differentiation ( Supplementary Fig. 1b), suggesting that SE and CC differentiation shares a common developmental process from vascular stem cells. Taken together, these results suggest that VISUAL-CC can reconstitute the SE-CC complex formation.

VISUAL-CC can induce known CC-related genes expression
To validate the promoter-based assay, we compared SUC2 mRNA accumulation with the promoter:LUC activity in the same sample ( Fig. 2a, b). qRT-PCR analyses of VISUAL-CC samples and samples cultured in the conventional VISUAL medium (VISUAL, V) as negative controls revealed a strong correlation between promoter activity and mRNA level of SUC2 ( Fig. 2b; r = 0.97, P < 0.005) (Fig. 2b). We used these data for classification of VISUAL-CC samples into strong LUC activity (CC-strong, S) and moderate LUC activity (CC-moderate, M), according to their SUC2 levels ( Fig. 2a, b; Supplementary Fig. 2). Expression of SISTER OF ALTERED PHLOEM DEVELOPMENT (SAPL), another CC marker gene 8 , also showed a strong correlation with SUC2 expression (Fig. 2c; r = 0.91, P < 0.005). A microarray analysis using the same samples was performed to obtain a comprehensive gene expression profile ( Supplementary Fig. 3a). As expected, genes previously characterized as CC-related, including SULFATE TRANSPORTER 2;1 (SULTR2;1) 9 , SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1) 10 , C-TERMINALLY ENCODED 11,12 and MYB-RELATED PROTEIN 1 (MYR1) 13 , showed similar expression patterns to SUC2 and SAPL (Fig. 2d). Consistently, quantitative RT-PCR assay for these genes validated the microarray result ( Supplementary Fig. 4). By utilizing the variation in expression observed between samples (S, M, V), we identified 186 VISUAL-CC inducible genes that satisfied the following patterns of expression levels: S > M > V and S/V > 4 ( Fig. 2E and Supplementary Fig. 2). According to the previous dataset of root cell type-specific transcriptome 14 , these genes were mainly expressed in root CCs or phloem pole pericycles (PPPs; Fig. 2f). PPPs are also known to participate in phloem unloading from SEs in roots via intervening plasmodesmata 15 (Fig. 2f). Here we grouped 67 genes as VISUAL-CC-related (VC) genes based on CC-preferential expression (Supplementary Table 1). Transporter genes were over-represented among these VC genes, reflecting the functional aspect of phloem transport ( Supplementary   Fig. 3b, c).

SE and CC differentiation tends to show negative correlation in VISUAL-CC
We previously identified 137 VISUAL-XY-related (VX) genes and 218 VISUAL-SE-related (VS) genes using VISUAL microarray data 2 . Then, expression of VX and VS genes was examined in the VISUAL-CC transcriptome dataset. Although there was no regular pattern of VX gene expression, expression of VS genes was very low in the S samples in contrast to that of VC genes (Fig. 2g). Correlation analysis among these gene sets revealed that expression levels of VC genes negatively correlate with those of VS genes (Supplementary Fig. 5b; r = -0.91, P < 0.05) but not with those of VX genes (Supplementary Fig. 5a; r = -0.28, P > 0.05). To further assess this tendency, we calculated the quantitative expression levels of vascular marker genes in individual samples. Although there was a strong correlation between expression of SAPL (CC) and SUC2 (CC) ( Fig. 2c; r = 0.91, P < 0.005), no correlation was found between IRREGULAR XYLEM 3 (IRX3) 16  Bikinin is a specific inhibitor of plant GSK3s 19 and thus these results suggest that GSK3 activity plays a role in determining the SE/CC ratio. GSK3 activity correlates with the expression of brassinosteroid biosynthetic genes, as a negative feedback regulation.
Indeed, DWARF4 (DWF4) 20 , one of brassinosteroid biosynthetic genes, was down-regulated following bikinin treatment in a dose-dependent manner (Fig. 3f). In the VISUAL-CC transcriptome, expression of brassinosteroid biosynthetic genes tended to be higher in the S samples and lower in the M samples ( Supplementary Fig. 8), also suggesting the relationship between the GSK3 activity and the SE/CC ratio. Previous studies have reported 113 bikinin-suppressed genes 19 , then we investigated the correlation between these genes and VC genes in VISUAL-CC transcriptome data. The bikinin-suppressed genes showed higher expression in the S samples (Fig. 2h)  ( Supplementary Fig. 6b). By contrast, expression of TOUCH 4 (TCH4), a typical GSK3-supprressed gene 23 , showed a significant negative correlation with SUC2 expression ( Fig. 3h; r = -0.72, P < 0.05). These results strongly suggest that the SE/CC ratio is largely dependent on GSK3 activity in vitro (Fig. 3i).

Genetic manipulation of GSK3 activity alters the in vivo SE/CC ratio
Then, we analysed the role of GSK3s in in vivo secondary phloem development in Arabidopsis hypocotyls. In hypocotyls, SEs are characterized by vacant cytoplasm whereas CCs are deeply stained with toluidine blue and they usually appear as pairs in a transverse section (Fig. 4a). Inhibition of GSK3 activity by bikinin treatment induced clusters of SEs and far fewer CCs (Fig. 4a). Bikinin treatment consistently reduced expression of pSUC2:YFPnls and resulted in clusters of pSEOR1:SEOR1-RFP signals in the dual phloem marker line (Fig. 4b, c), indicating that bikinin promotes SE formation and decreased CC number in vivo. Next, we confirmed the function of GSK3 proteins genetically using knock-out mutants of members of the SKII subfamily (BIN2, BIL1, and BIL2) and RNAi knock-down for SKI subfamily members (AtSK11, AtSK12, and AtSK13) 24 , because they are the main targets of bikinin 17 . The phloem tissue of the bin2 bil1 bil2 AtSK13RNAi quadruple mutant exhibited a slight but significant decrease in CC occupancy (40%) when compared with wild-type plants (44%) (Fig. 4d, e and Supplementary Fig. 9). The gsk hextuple mutant (quadruple + AtSK11, AtSK12RNAi) showed a reduction in CC occupancy (20%), resulting in more SEs and fewer CCs (Fig.   4d, e and Supplementary Fig. 9). Moreover, in the hextuple mutant, some of the PPP cells unexpectedly differentiated into ectopic SE-like cells (Fig. 4d, e). Previous studies have revealed that the vascular cells express SKII subgroup genes BIN2 and BIN2-LIKE2 (BIL2) 24 . In addition, expression of SKI subgroup genes pSK11:GUS and pSK12:GUS 25 was found in the vasculature including the phloem tissue (Fig. 4f).
Similarly to the GUS expression analysis, SKI/II genes expression was kept high in VISUAL time-course and in VISUAL CC transcriptome data ( Supplementary Fig. 10), indicating that 6 GSK3 members are present during phloem development. Next we investigated local GSK3 activity in the vasculature using pDWF4:GUS, which is an indicator of high GSK3 activity. Supporting with our idea, pDWF4:GUS expression was detected in the phloem CCs but not in SEs (Fig. 4f). Taken together, our results indicate that GSK3 activity is required for maintaining high CC occupancy in planta.
GSK3s function as signalling hubs to control xylem differentiation in the cambium 24 . Here to focus on phloem development, bin2-1, a stable form of GSK3 26 , was driven under promoters specific to each stage of phloem development. As we had previously demonstrated that the sequential genetic cascade in phloem SE differentiation is NAC020 (early), APL (middle), SEOR1 (late) 2 , we induced expression of bin2-1 under these different phloem promoters and investigated their phloem phenotype (Fig. 5a, b). Expression of bin2-1 driven by the APL and SEOR1 promoters did not affect phloem phenotypes, but pNAC020:bin2-1 slightly but significantly increased the ratio of CCs in the phloem (Fig. 5a-c and Supplementary Fig. 9). To confirm the results with the CC marker, number of pSUC2:YFPnls signal in WT and pNAC020:bin2-1 was quantified using confocal microscope (Fig. 5d). YFP-positive cell number estimated from 3D-reconstruction images was significantly higher in the pNAC020:bin2-1 than in the WT (Fig. 5e, f). All these results indicate that GSK3s function as cell fate switches for determining differentiation into phloem CCs or SEs, and that GSK3 activity, especially in the early phloem development, was important for ensuring the proper ratio between CCs and SEs (Fig. 5g).

BR-BES1 signalling does not participate in SE/CC fate determination
Finally, we examined the involvement of brassinosteroid (BR) in CC differentiation, because GSK3s function as signal mediators in BR signalling 26,27 . However, application of brassinolide (BL), an active BR, did not alter the SE/CC ratio in hypocotyls ( Supplementary Fig. 11). Moreover, bes1 bzr1 loss-of-function and bes1-D bzr1-D gain-of-function mutants for BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAXOLE RESISTANT 1 (BZR1), which are well-known transcription factors phosphorylated by GSK3s in BR signalling 28 , exhibited a normal phloem development in terms of the SE/CC ratio ( Supplementary Fig. 12). These results suggest the possibility that other signalling pathway(s) than BR participates in controlling the SE/CC ratio.

Discussion
Recent studies have revealed that CCs are not only of importance for phloem transport, but also act as a signal centre integrating environmental information into the developmental program 29 . We have established VISUAL-CC as a powerful tool for analysing the functional and developmental processes of CCs. During secondary vascular development, it has been widely believed that SEs and CCs are derived from the same phloem precursors via asymmetric cell division 30  Further studies on such interacting proteins and reverse genetic approaches combined with VISUAL-CC transcriptome data will be helpful for elucidating a novel signalling cascade controlling the SE/CC ratio.
GSK3s also function in animals as molecular switches determining differentiation into alternate cell types 32 , suggesting their common and important role as cell fate switches. On the other hand, GSK3 activity regulates asymmetric cell division in the stomata lineage by interacting with polarly localized proteins, which are required for specifying stomatal cell fate 33 . Further investigation with the context of asymmetric cell division will uncover the extent to which GSK3s serve as a common mechanism determining cell fate.

Plant materials
Arabidopsis plants used in this study is Col-0 accession, except for gsk3 high-order mutants (Ws background). To construct the CC-reporter lines, approximately 2.0 kb of the SUC2 promoter region was cloned and then fused with ELUC (Toyobo) or YFP containing a nuclear localization signal. A genomic fragment of SEOR1, approximately 4.8 kb long and containing 1.6 kb of the promoter, was fused with RFP to make pSEOR1:SEOR1-RFP; this was subsequently transformed into pSUC2:YFPnls to generate the double phloem marker line. To construct pDWF4:GUS, approximately 1.9 kb of the DWF4 promoter region was cloned and introduced into pMDC163 vector to create a GUS-fusion gene.
Phloem-specific GSK3 activation lines were constructed by cloning bin2-1 (BIN2E263K) and fusing it with the NAC020 (2.4 kb), APL (2.9 kb), and SEOR1 (1.6 kb) promoters using the LR reaction (Thermo Fisher Scientific). The gsk quadruple and hextuple mutants (Ws accession) used in this study were as reported previously 23 . The smxl4 smxl5 mutants were as reported previously 6,7 . The bes1 bzr1 loss-of-function mutants and bes1-D bzr1-D gain-of-function mutants were as reported previously 33 .

LUCIFERASE measurement
In this study, ELUC with PEST domain (Toyobo) was used as a short half-life luminescent protein. pSUC2:ELUC seedlings were co-cultured with 200 µM D-luciferin (Wako) in white 24-well plates (PerkinElmer). The time-course of luciferase (LUC) activity was measured automatically using a TriStar2 LB942 (Berthold) within a growth chamber (Nihonika).

Microscopic observation
For deep imaging with confocal microscopes, isolated tissue samples were fixed for 3 hours under vacuum in a fixative solution (4% paraformaldehyde and 0.01% Triton X-100 in 1× PBS). Fixed samples were washed twice with 1× PBS and transferred to ClearSee solution (25% urea, 15% xylitol, 10% sodium deoxycholate). ClearSee solution was replaced with fresh solution every 2 days for 3 to 4 weeks. Calcofluor staining was performed 1 week before microscopic observations by adding 0.1% (w/v) calcofluor white to the ClearSee solution. The samples were stained overnight and then washed with ClearSee solution without calcofluor. Once the samples were stained, washing was continued as described above. Cleared samples were observed using LSM880 (Zeiss) or FV1200 (Olympus) confocal microscopes with Z-stack. For the quantification of YFP-positive cells, we counted the number of cells in a phloem pole of approximately 420 µm length of hypocotyls based on reconstructed 3D images.

Electron microscopy
Sample preparation for electron microscopy observation was modified slightly from a previous study 35 . Briefly, leaf disks induced by VISUAL-CC were fixed and embedded in resin. Thin sections (100 nm) were mounted on glass slides. Sections were stained with 0.4% uranyl acetate solution (UA) and a lead citrate solution (Pb), and then coated with osmium tetroxide. Observations of slides were made using a field emission scanning electron microscope (FE-SEM) (Hitachi SU 8220). Thinner (80 nm) sections were mounted on formvar-coated 1-slot copper grids, stained with 4% UA and Pb, and then observed using an 80 kV transmission electron microscope (JEOL JEM-1400 Flash).

qRT-PCR and Microarray experiments
Total RNA was extracted from four cotyledons using RNeasy plant mini kit (Qiagen) after LUC measurement. After reverse transcription reaction, qRT-PCR was performed using LightCycler 480II (Roche) by a universal probe method. Expression value was normalized with an internal control UBQ14. Microarray experiments were conducted with the Arabidopsis Gene 1.0 ST Array (Affymetrix) and analyzed with Subio platform and R gplots package. Primers used in this study were listed in Supplementary Table 2.

Cross section
Hypocotyls of 10-or 11-day-old seedlings were fixed with FAA (formalin:acetic acid:alcohol, 1:1:18) for 1 day. Fixed hypocotyls were subjected to an ethanol series (50%, 70%, 80%, 90%, 99.5%) each for 30 minutes and then transferred into Technovit 7100 solution without Hardener II for 1 day. After the pre-incubation, samples were embedded in a mixture of Technovit 7100 + Hardener II (12.5:1) and incubated at 37 °C for more than 1 hour to harden. Technovit samples were sliced into 2 µm sections using a LEICA RM2255 microtome and stained with 0.1% toluidine blue to enable CCs to be distinguished from SEs under microscopy. Cross sections for GUS-stained samples were made as reported previously 24 .     Asterisks indicate significant differences using the Student's t-test (*P < 0.05, n = 3).