Abstract
Spatial organization of signaling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signaling but its exact role and spatial organization is obscure. Here, we show that, while auxin signaling levels increase in differentiating cambium descendants, a moderate level of signaling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by attenuating the activity of the stem cell promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signaling on distinct cambium features by specific signaling components and allow the conceptual integration of plant stem cell systems with distinct anatomies.
Introduction
In multicellular organisms, communication between cells is essential for coordinated growth and determination of cell fate. In plants in particular, the flexible regulation of cellular properties by hormone signaling is important throughout the whole life cycle. This is because plants are sessile and continuously adapt their growth and development to their local environment. The basis of this plastic growth mode are local stem cell niches at the tips and along plant growth axes, called meristems1. The tip-localized shoot and root apical meristems (SAM & RAM) are essential for primary, or longitudinal, growth of shoots and roots, respectively. In turn, the vascular cambium is the predominant lateral meristem forming a cylinder of indeterminate stem cells at the periphery of growth axes and mediating radial growth by producing the vascular tissues phloem and xylem in a bidirectional manner2, 3. This production is the basis of wood formation and is, thus, essential for the accumulation of a large proportion of terrestrial biomass.
The plant hormone auxin plays pivotal roles in local patterning and maintenance of stem cell niches in the SAM and RAM. In the SAM, auxin signaling is low in stem cells and increases during recruitment of cells for organ formation4, 5, 6. Cell wall modulation and the formation of vascular strands are two aspects promoted by auxin in this context7, 8. In contrast, a maximum of auxin signaling is present in the quiescent center (QC) and the surrounding stem cells in the RAM and cell differentiation is, at least partly, driven by a decrease in signaling levels9, 10. Therefore, the functions of auxin in both meristems are different and adapted to distinct niche requirements.
For the cambium, the role of differential auxin signaling along the radial sequence of tissues is still obscure. In Arabidopsis stems apex-derived auxin is transported basipetally and distributed laterally across the cambial zone by the auxin exporters PIN-FORMED1 (PIN1), PIN3, PIN4 and PIN711, 12. Indeed, direct auxin measurements in Populus and Pinus trees showed that the concentration of the major endogenous auxin indole-3-acetic acid (IAA) peaks in the center of the cambial zone and gradually declines towards differentiating xylem and phloem cells13, 14, 15. This observation prompted the idea that, in analogy to the situation in the RAM, radial auxin concentration gradients contribute to the transition of cambium stem cells to secondary vascular tissues16, 17. Consistently, ubiquitous repression of auxin responses by expressing a stabilized version of the auxin response inhibitor PttIAA3 reduces the number of cell divisions in the cambium region of hybrid aspen trees18. In addition, however, the zone of anticlinal cell divisions characteristic for cambial stem cells is enlarged in PttIAA3 overexpressing trees. This suggests that auxin signaling not only promotes cambium proliferation but also spatially restricts stem-cell characteristics within the cambium area18, 19. Indeed, especially xylem formation is associated with a local increase of auxin signaling in other contexts10, 20, 21, 22 which supports a role of auxin in the recruitment of cells for differentiation similarly as in the SAM. Therefore, it is currently unclear whether auxin signaling is predominantly associated with stem cell-like features or cell differentiation in the context of radial plant growth or how a positive effect on cambium proliferation and on the differentiation of vascular tissues is coordinated.
As a central cambium regulator, the WUSCHEL-RELATED HOMEOBOX4 (WOX4) transcription factor imparts auxin responsiveness to the cambium23. Equivalent to the role of WUSCHEL (WUS) and WOX5 in the SAM and RAM24, 25, WOX4 activity maintains stem cell fate23, 26. In turn, WOX4 transcription is stimulated by the leucine-rich repeat receptor-like kinase (LRR-RLK) PHLOEM INTERCALATED WITH XYLEM (PXY). Importantly, the expression domains of the WOX4 and PXY genes presumably overlap and are considered to mark cambium stem cells23, 26, 27, 28. However, a bipartite organization of the cambium zone was shown recently with PXY being expressed only in the xylem-facing part29. Whether this organization reflects the existence of two distinct stem cell pools feeding xylem and phloem production, respectively, has still to be determined.
Here, we identify functional sites of auxin signaling in the Arabidopsis cambium by local short-term modulation of auxin biosynthesis and signaling. We reveal that, while cambial stem cells do not appear to be a site of elevated auxin signaling, auxin signaling in these cells is required for cambium activity. By analyzing transcriptional reporters and mutants of vasculature-associated AUXIN RESPONSE FACTORs (ARFs), we identify ARF3, ARF4 and ARF5 as cambium regulators with different tissue-specificities as well as distinct roles in cambium regulation. Remarkably, whereas ARF3 and ARF4 act redundantly as more general cambium promoters, ARF5 acts specifically in cambium stem cells. In depth analysis of the auxin- and ARF5-dependent transcriptome in those cells, together with genetic analyses, indicates that the ARF5-dependent repression of WOX4 is an essential aspect of auxin signaling during cambium regulation.
Material and Methods
Plant Material
All plant lines used in this study were Arabidopsis thaliana (L.) Heynh. plants of the accession Columbia (Col-0), except for the mp-B4149 mutant, which has the Utrecht background30, 31. The arf3-1 (SAIL_1211_F06, N878509), ett-13 (SALK_040513, N540513), mp-S319 (SALK_021319, N521319), wox4-1 (GK_462GO1, N376572) and pxy-4 (SALK_009542, N800038) mutants, as well as the pDR5rev:GFP reporter line (N936132), were ordered from the Nottingham Arabidopsis Stock Centre (NASC). The mp-B4149 and arf4-2 (SALK_070506) mutant were provided by Dolf Weijers (University of Wageningen, The Netherlands) and Alexis Maizel (COS Heidelberg, Germany), respectively. Genotyping was performed by PCR using primers listed in Table S1. Genotyping of mp-B4149 was done as described previously33 with the modification of using the MP_for8/MP_rev8 primer pair for amplification.
Plant Growth and Histological Analyses
Plants destined for histology were grown and analyzed as described previously23, 28, 34. In brief after 3 weeks of growth in short day (SD) conditions (8 h light and 16 h dark) plants were transferred to long day (LD) conditions (16 h light and 8 h dark) to induce flowering and used for histology at a height of 15-20 cm. Stem segments of at least 1 cm in length (incl. the stem base) were harvested, embedded in paraffin and sectioned using a microtome (10 μm sections). After deparaffinization, sections were stained with 0.05% toluidine blue (Applichem), fixed with Entellan (Merck) and imaged using a Pannoramic SCAN digital slide scanner (3DHistech). Pictures were analyzed as described previously34 using Pannoramic Viewer 1.15.4 software (3DHistech). For quantitative analyzes at least five plants were analyzed for each data point.
Statistical Analyses
Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp. Means were calculated from measurements with sample sizes as indicated in the respective figure legends. In general, all displayed data represents at least two independent, technical repetitions, unlike otherwise indicated. Error bars represent ± standard deviation. All analysed datasets were prior tested for homogeneity of variances by the Levene statistic. Significant differences between two datasets were calculated by applying a Welch’s t-tests or Student’s t-test depending on the homogeneity of variances. The significance thresholds were set to p-value < 0.05 (indicated by one asterisk). For multiple comparisons between three or more datasets, a One-way ANOVA was performed, using a confidence interval (CI) of 95% and a post-hoc Bonferroni for comparisons of data sets of homogenous variances or a post-hoc Tamhane-T2 in case variances were not homogenous.
Sterile culture
Adventitious root formation in the strong arf5 mutant allele mp-B4149 was induced with some minor modifications as described previously35, 36. Seeds were liquid sterilized by 70% ethanol and incubation in 5% sodiumhyperchloride followed by three washes with ddH2O. After stratification at 4°C in the dark for 3 days, seeds were sown on ½ Murashige-Skoog (MS) medium plates (incl. B5 vitamins) in rows and grown vertically. After 7 days of growth under short-day (SD; 8 h light, 16 h dark) conditions, rootless mutant as well as wild type looking seedlings from the segregating population were bisected with a scalpel as described previously36 and transferred to adventitious root inducing medium (½ MS (incl. B5 Vitamins) + 1.5 % sucrose + 3 μg/ml indole butyric acid + 0.7 % agar + 50μg/ml ampicillin35). After additional two weeks of growth under SD conditions, successfully rooted seedlings were transferred to soil and grown under SD conditions for one additional week before they were transferred to LD conditions. Plants that survived the transfer, were genotyped for mp-B4149 and only wild type and homozygous mutant plants were used for histological analysis at a plant height of 15-20 cm as described in the previous section.
Plasmid construction
The pPXY:CFP and pWOX4:YFP reporters were described previously23, 28. To avoid diffusion, all fluorescent proteins were targeted to the endoplasmatic reticulum (ER) by fusing them to the corresponding sequence motif (ER + HDEL motif37). For generating pDR5revV2:YFP (pKB46), we initially inserted the ADAPTOR PROTEIN-4 MU-ADAPTIN (AP4M, At4g24550) terminator, amplified from genomic DNA using the At4g24550_for1/At4g24550_rev1 primer pair, into pLC075 containing the DR5revV2 promoter fragment38 using BamHI/XhoI restriction sites. A fragment carrying the ER-EYFP-HDEL coding sequence (CDS) was inserted in the resulting pLC075:AP4Mterm using the BamHI restriction site, to obtain pLC075:YFP:AP4Mterm. The complete reporter fragment was inserted in the binary vector pGreenII01739 using KpnI/XhoI restriction sites. For generating p35S:Myc-GR-bdl (pKB9), the Myc-GR-bdl fragment was amplified from genomic DNA of pRPS5a:Myc-GR-bdl40 using the Myc_for1/BDL_rev3 primer pair. The resulting fragment was inserted in the pGreen0229 vector39 containing the 35S promoter (pGreen0229-35S) using XbaI/BamHI restriction sites. To produce pAlcA:iaaM(pKB2) the iaaM CDS was amplified from piaaM (pIND:IND-iaaM)41 using the IAAMfor1/IAAMrev1 primer pair and introduced into pGreen0229-AlcA42 using AatII/EcoRI restriction sites. pWOX4:AlcR (pTOM55) was produced by amplifying the WOX4 promoter using the primers WOX4for11/WOX4ref9 and inserting the resulting fragment into pAlcR-GUS42 using SpeI/NotI sites. The pPXY:Myc-GR-bdl (pKB45) construct was generated by cloning the Myc-GR-bdl fragment, amplified from pKB9 using the Myc_for5/BDL_rev7 primer pair, in pGreen0229 containing the PXY promoter (pTOM5028) using NcoI/Cfr9I restriction sites. The promoter regions of BDL43 were amplified from genomic DNA using the BDL_for2/BDL_rev4 & BDL_for3/BDLrev5 primer pairs. Both fragments were cloned into pGreenII0179 using NotI/XbaI & Cfr9I/KpnI restriction sites, respectively. The resulting plasmid (pKB27) was used to produce the pBDL:YFP (pKB28 using ER-EYFP-HDEL) and pBDL:Myc-GR-bdl (pKB29) constructs by inserting fragments carrying the respective CDSs using NcoI/Cfr9I restriction sites. For generating ARF3, ARF4 and ARF5 reporter constructs, promoter regions of the three genes were amplified from genomic DNA using the ARF3for1/ARF3rev1 & ARF3for2/ARF3rev2, ARF4for1/ARF4rev1 & ARF4for2/ARF4rev2 and MP_for7/MP_rev5 & MP_for5/MP_rev6 primer pairs. Both fragments were cloned for each gene into pGreen0229 using NotI/BamHI & BamHI/KpnI (ARF3), NotI/SpeI & Cfr9I/KpnI (ARF4) and NotI/BamHI & BamHI/ApaI (ARF5) restriction sites. The resulting plasmids (pKG40 (ARF3), pKG41 (ARF4) & pKB3 (ARF5)) were used to produce the pARF3:YFP (pKB30), pARF4:YFP (pKB31), pARF5:YFP (pKB24) and pARF5:mCherry (pKB4) constructs by inserting fragments carrying the respective CDSs using BamHI, SpeI and NdeI/XhoI restriction sites, respectively. To produce p35S:GR-ARF3 (pKB42) and p35S:GR-ARF5 (pKB17) we amplified the GR open reading frame from pKB9 using the GR_for1/GR_rev3 primer pair and inserted the resulting fragment in pGreen0229-35S using XbaI/Cfr9I restriction sites. An additional unannotated SalI restriction site in the pGreen0229 backbone was removed by PCR-based silent mutagenesis using the NOS-mut_for1/NOS_mut_rev1 primer pair. In the resulting pGreen0229-35S:GR (pKB41) vector we inserted the ARF3 and ARF5 CDS, amplified from cDNA using the ARF3_for4/ARF3_rev4 and MP_for16/MP_rev14 primer pair, using SalI/Cfr9I and SalI/EcoRI restriction sites, respectively. pPXY:GR-ARF3 (pKB43) was generated by cloning the GR-ARF3 fragment, amplified from pKB42 using the GR_for5/ARF3_rev4 primer pair, in pTOM50 using NcoI/Cfr9I restriction sites. For generating pPXY:GR-ARF5ΔIII/IV (pKB25), the GR-ARF5ΔIII/IV fragment with a stop codon was amplified from pKB17 using the MP_for18/MP_rev16 primer pair and inserted in pTOM50 using NcoI/Cfr9I restriction sites. For generating the pWOX4:LUC (firefly);p35S:LUC (Renilla) (pKB55) reporter the pZm3918:LUC (firefly) fragment in pZm3918:LUC (firefly);p35S:LUC (Renilla) (pGreen-LUC-REN) was excised by digest with KpnI/XbaI and replaced by a pWOX4:LUC (firefly) fragment previously excised from pWOX4:LUC (pMS80) using KpnI/NheI restriction sites. To produce p35S:ARF3 (pKB44) the ARF3 CDS was amplified from pKB42 using the ARF3_for6/ARF3_rev4 primer pair and introduced it in pGreen0229-35S using XbaI/Cfr9I restriction sites. For generating p35S:ARF5ΔIII/IV (pKB40) the ARF5ΔIII/IV CDS was amplified from pKB25 using MP_for17/MPrev15 and introduced in pGreen0229-35S using XbaI/EcoRI restriction sites. All constructs were sequenced and after plant transformation by floral dip44, single copy transgenic lines were identified by Southern blot analyses and representative lines used for crosses and further analyses. All primers mentioned in this section are listed in Table S1.
Confocal microscopy
For imaging fluorescent reporter lines in the stem rough hand sections were taken with a razor blade (Wilkinson Sword) and analyzed using an LSM 780 spectral confocal microscope (Carl Zeiss) equipped with the Zen 2012 software (Carl Zeiss). Stem sections (except for pARF5:mCherry) were counterstained for 5 min with 5 μg/ml propidium iodide (PI; Merck) dissolved in tap water. PI was excited at 561 nm (DPSS laser) and detected at 590-690 nm. YFP was analysed with excitation at 514 nm (Argon laser) and detection at 516-539 nm. CFP was excited at 458 nm (Argon laser) and detected at 462-490 nm, while GFP was excited at 488 nm (Argon laser) and detected at 499-544 nm. mCherry reporter activity was analyzed with excitation at 561 nm (DPSS laser) and detection at 597-620 nm. Transmitted light pictures were generated using the transmission photo multiplier detector (T-PMT) of the microscope. 5-day-old Arabidopsis seedlings were counterstained with the cell membrane dye FM® 4-64 (Thermo Fisher Scientific) to visualize cell borders as described previously45. FM® 4-64 was excited at 561 nm (DPSS laser) and detected at 653-740 nm.
Pharmacological treatments
Stock solutions of 25 mM Dex (VWR) dissolved in 100% Ethanol and 10 mM cycloheximide (Cyclo; Carl Roth) dissolved in ddH2O were freshly prepared prior to use. For long-term Dex-treatments, plants were initially grown for three weeks without treatment in SD conditions to circumvent growth defects during early plant development. Plants were then transferred to LD conditions and watered twice a week with either 15 μM Dex (25 mM Dex stock diluted in tap water) or mock solution (equal amount of 100 % Ethanol in tap water) until they reached a height of 15-20 cm and were harvested for histology. For short-term Dex-treatments 15-20 cm tall plants were dipped headfirst in 15 μM Dex (25 mM Dex stock diluted in tap water + 0.02% Silwet) or Mock solution (equal amount of 100 % Ethanol in tap water + 0.02% Silwet) for 30 sec. Subsequently, plants were transferred to LD growth conditions, watered with 15 μM Dex or Mock solution and incubated until harvest of second internodes for RNA isolation. For additional short-term Cyclo treatment, 10 mM Cyclo stock was added to the 15 μM Dex and Mock solution to a final concentration of 10 μM and the plants were treated in the same way as described before. For inducing the AlcA/AlcR system42, plants where grown for three weeks in SD, transferred to long day until bolting. When plants where 0.5 – 3 cm tall they were put under a plastic dome together with 2 x 15 ml 70% ethanol in round petri dishes and left overnight. Plants where harvested 10 days after induction and wild type plants were around 25 cm tall.
Transient reporter activity assays
Transient activity assays were performed as previously described46. In brief protoplasts derived from an Arabidopsis (Col-0) dark-grown root cell suspension culture (kindly provided by Claudia Jonak) were isolated and transfected as previously described47. For transfection, we used 10 μg of reporter construct (pKB55) containing p35S:LUC (Renilla) as an internal control and 10 μg of each effector construct. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. Luminescence was measured using a Synergy H4 Hybrid Multiplate Reader (BioTek). For each reporter/effector combination 3-5 technical replicates were done and the experiments repeated at least three times. For experimental analysis Firefly Luciferase activity was normalized to Renilla Luciferase activity.
RNA Preparation and qRT-PCR
Frozen plant material from second internodes or the stem base (incl. 5 mm above) of 15-20 cm tall plants (three biological replicates (three plants each) per genotype/treatment) were pulverized with pestle and mortar and RNA was isolated by phenol/chlorophorm extraction as described previously48 with the modification of two additional concluding 70% EtOH washes. RNA elution in RNase-free water was followed by treatment with RNase-free DNase (Thermo Fisher Scientific) and reverse transcription (RevertAid First Strand cDNA Synthesis Kit; Thermo Fisher Scientific). cDNA was diluted 1:25 prior to amplification. qRT-PCR was performed using SensiMix™ SYBR® Green (Bioline Reagents Ltd) mastermix and gene specific primers (listed in Table S1), in a Roche Lightcycler480 following the manufacturer’s instructions. Experiments were performed in triplicates with plant material of three plants being pooled for each replicate. Two reference genes (ACT2 and EIF4a) were used to normalize our signal. Error bars: ± standard deviation. Raw amplification data were exported and further analysis and statistical tests were done using Microsoft Excel 2010.
Transcriptional Profiling
10 μg of total RNA for each sample were treated with RNase-free DNase (Thermo Fisher Scientific) and purified using RNA-MiniElute columns (Qiagen) following the manufacturer’s protocol. Library preparation and next-generation-sequencing (NGS) was performed at the Campus Science Support Facilities (CSF) NGS Unit (www.csf.ac.at) using HiSeqV4 (Illumina) with single end 50-nucleotide reads. Reads were aligned to the Arabidopsis thaliana Columbia (TAIR10) genome using CLC Genomics Workbench v7.0.3 and analyzed using the DESeq package from the R/Bioconductor software49. Dex-treated samples were compared to mock-treated samples with a stringency of p-value < 0.05. Data processing was further analyzed using VirtualPlant 1.350 Gene Sect and BioMaps with a cut-off p-value < 0.05 and cut-off p-value <0. 01, respectively. Data was aligned to The Arabidopsis Information Resource (TAIR)-databases (77) and as background population for all analysis the Arabidopsis thaliana Columbia (TAIR10) genome was used. Further data processing was done in Microsoft Excel 2010.
Results
Local auxin responses in stem cells stimulate cambium activity
In Arabidopsis stems, the activity of the common auxin response marker pDR5rev:GFP32 was detected in vascular tissues (phloem and xylem) and cortical cells prior and during cambium initiation (Figures 1A, S1A)23. However, there was no overlap with pWOX4:YFP23 or pPXY:CFP28 reporter activities, the two canonical markers for cambium stem cells (Figure 1A-C, S1A-C)23. This suggested that auxin signaling in those cells occurs at low levels or is even absent. To decide between both possibilities, we generated a plant line expressing an endoplasmatic reticulum (ER)-targeted Yellow Fluorescent Protein (YFP) under the control of the high affinity DR5revV2 promoter which recapitulated the pattern of DR5revV2 activity previously reported in roots (Figures S1D-F)38. In the second internode of elongated shoots, pDR5rev:GFP and pDR5revV2:YFP activities were congruent but pDR5revV2:YFP activity also included the whole cortex as well as cambium cells marked by pPXY:CFP activity (Figure S1G-I). Immediately above the uppermost rosette leaf (denoted as stem base throughout the text), stem anatomy shows a secondary configuration, which is characterized by a continuous domain of cambium activity23. At this position, the expression domain of pDR5revV2:YFP was again broader than the domain of pDR5rev:GFP activity substantially overlapping with pPXY:CFP activity (Figure 1D-F). Based on these observations, we concluded that the auxin signaling machinery is active in PXY-positive cambial stem cells.
To see whether cambium activity was positively correlated with auxin levels in PXY-positive cells, we used the WOX4 promoter, whose activity fully recapitulated the PXY promoter activity (Figure S2A-I), for expressing a bacterial tryptophan monooxygenase (iaaM) in an inducible manner42. iaaM converts endogenous tryptophan to the IAA precursor indole-3-acetamide and was used before to boost endogenous IAA levels in Arabidopsis53. As a read out for cambium activity, we determined the amount of interfascicular cambium-derived (ICD) tissues34. Indeed, ethanol-based iaaM induction substantially stimulated the production of ICD tissues (Figure 1G, H, M, Figure S2J-N) demonstrating that an increase of auxin biosynthesis in PXY-positive stem cells stimulates cambium activity.
To determine to which extent downstream components of the auxin signaling cascade are required in those cells, we blocked ARF activity by expressing a dexamethasone (Dex)-inducible variant of the stabilized AUX/IAA protein BODENLOS (Myc-GR-bdl)40, 54 under the control of the PXY promoter. Consistent with a role of ARF activity in cambium regulation, Dex-treatments of pPXY:Myc-GR-bdl plants resulted in a strongly reduced amount of ICD tissues at the stem base (Figure 1I, J, M) but not in an altered overall growth habit (Figure S2O). Strikingly, Dex-treated pPXY:Myc-GR-bdl plants showed an even more pronounced repression of IC activity than the inhibition using the BDL promoter43 (Figure 1K, L, M, Figure S2P) whose activity was very broad including also PXY-positive cells (Figure S3A-F). These observations indicated that, local auxin signaling in PXY-positive stem cells stimulates cambium activity.
ARF3, ARF4 and ARF5 genes are expressed in cambium associated cells
To identify ARFs active in cambium stem cells, we mined public transcriptome datasets and found the ARF3/ETTIN, ARF4 and ARF5/MONOPTEROS genes to be co-induced with WOX4 and PXY during cambium initiation23. Indeed, pARF3:YFP, pARF4:YFP, pARF5:YFP promoter reporters were active in cambium-related cells at the stem base and the second internode (Figure 2A, D, G, Figure S4A, D, G). However, while pARF3:YFP and pARF4:YFP reporters were active in rather broad domains including the phloem, the xylem and, partly, pPXY:CFP-positive cells (Figure 2A-F, Figure S4A-F), pARF5:YFP was exclusively active in cells marked by pPXY:CFP activity (Figure 2G-I, Figure S4G-I). Moreover, in second internodes, pARF3:YFP and pARF4:YFP activities were both detected in the starch sheath, the innermost cortical cell layer which is considered to serve as the origin of the IC (Figure S4C & F arrows)55, 56 while pARF5:YFP activity was restricted to vascular bundles (Figure S4G-I). Indicating also a temporal difference between ARF3/4 and ARF5 activities, pARF3:YFP and pARF4:YFP reporters were active together with pPXY:CFP in interfascicular regions at positions approximately 5 mm above the stem base (Figure S5A-F) where cortical cells start dividing to form the IC34. In contrast, no pARF5:YFP activity was detected in the same cortical cells (Figure 2J-L). This observation suggested that ARF5 expression follows the expression of PXY during cambium initiation and is not active during early steps of cambium initiation. Consistently, in pxy-4 mutants where IC formation is largely absent in stems (Figure 5E; 57) a pARF5:mCherry promoter reporter was only active in vascular bundles but not in interfascicular regions (Figure S5G-I). Taken together, these observations were in line with a role of ARF3 and ARF4 as promoters of cambium activity and a role of ARF5 as a modulator of the established cambium.
ARF control of cambium proliferation
To find indications for these roles, we analyzed cambium activity in mutants for the respective ARF genes (Figure S5J-L). Consistent with a positive effect of ARF3, both weak and strong arf3 mutants58, 59 showed significantly reduced cambium activity (Figure 3A, B, E, F, M). This reduction was further increased upon depletion of ARF4 activity by introducing the arf4-2 mutation59 into the respective arf3 mutant backgrounds (Figure 3C, D, G, H, M). Consequently, we concluded that cambium activity is positively regulated by ARF3 and ARF4, which, as in other contexts59, 60, act in a concerted fashion.
In contrast, cambium activity was enhanced in the hypomorphic arf5 mutant mp-S31961 (Figure 3I, J, N) suggesting that ARF5 counteracts cambium proliferation. To confirm this role, we generated adult plants of the strong ARF5 loss-of-function mutant mp-B4149 30 and wild type plants through tissue culture35, 36. As before, mp-B4149 plants showed enhanced ICD formation comparable to mp-S319 mutants (Figure 3K, L, N). Further confirming a negative effect of ARF5 on cambium activity, ubiquitous expression of a Dex-dependent GR-ARF5 protein fusion using the 35S promoter62 led to significantly reduced tissue production under long-term induction (Figure 4A-C, J).
To test whether the identified ARFs function in PXY-positive stem cells, we first employed the PXY promoter to express GR-ARF5ΔIII/IV, a truncated variant of ARF5 lacking the domains III and IV releasing it from AUX/IAA-based repression63. Indeed, long-term Dex treatment of pPXY:GR-ARF5ΔIII/IV plants resulted in reduced cambium proliferation (Figure 4D-F, J) arguing for a stem cell-specific role of ARF5. In contrast, the same treatment of a pPXY:GR-ARF3 line, did not influence cambium activity (Figure 4G-I, J, see below) arguing against a rate-limiting role of the non-AUX/IAA-dependent64 ARF3 protein in those cells. Collectively, we concluded that ARF3 and ARF4 on one side and ARF5 on the other side represent two subgroups of ARF transcription factors with differences in both their spatio-temporal expression and roles in cambium regulation.
ARF5 restricts the number of undifferentiated cambium cells
To dissect the ARF5-dependent control of cambium stem cells, we took advantage of the DEX-inducibility of our pPXY:GR-ARF5ΔIII/IV and of a p35S:Myc-GR-bdl line. By determining transcript abundance of the direct ARF5 targets ATHB8 and PIN165, 66 at different time points after Dex treatment, 3 h of treatment was identified as being optimal for observing short-term effects on gene activity (Figure S6A, B). After establishing genome-wide transcript profiles at that time point, we identified a common group of 600 genes with altered transcript levels in both the pPXY:GR-ARF5ΔIII/IV and the p35S:Myc-GR-bdl line (p < 0.01; Figure 5A & Table S2). The 600 genes represented various functional categories including primary auxin response (IAAs, SAURs, GH3s), xylem and phloem formation (IAA20 & IAA3022, REV12, CVP2 & CVL167) and cell wall modifications (PMEs & EXPs) (Figure S6C & Table S3). Moreover, the 312 genes that were induced by pPXY:GR-ARF5ΔIII/IV and repressed by p35S:Myc-GR-bdl (Figure 5A; Table S2), overlapped significantly with a previously published set of ARF5-inducible genes from seedlings66 (Figure S6E) indicating that we indeed revealed ARF5-dependent genes in stems. Strikingly, while our expectation was that genes, which are induced by GR-ARF5ΔIII/IV induction would be repressed by the auxin signaling repressor bdl and vice versa, we observed 144 genes (24%) that were either induced (73 genes) or repressed (71 genes) by both constructs (Figure 5A). This indicated that in PXY-positive cells ARF5 antagonizes the effect of overall auxin signaling on a substantial subset of target genes. Since we observed opposing effects of ARF5 and total canonical auxin signaling on cambium activity, we suspected that genes integrating these effects are among the 144 genes behaving in an unexpected manner. Interestingly, 11 genes out of 144 were also found in a set of genes that are differentially expressed during IC formation57 one of them being WOX4 which was repressed by both GR-ARF5ΔIII/IV and Myc-GR-bdl induction (Figure 5B).
Because ARF5 induction resulted in both, WOX4-repression and the induction of xylem- and phloem-related genes (Figure S6), we reasoned that the repressive effect of ARF5 on cambium proliferation was due to an influence on the transition of cambial stem cells to vascular cells. To test this, we analyzed the stem cell marker pWOX4:YFP in mp-S319 mutants. Indeed, the radial extension of the pWOX4:YFP domain was increased in mp-S319 plants (Figure 5C-E) suggesting that the number of undifferentiated cambium cells was higher when ARF5 activity was reduced. Consistently, when analyzing the anatomy of the cambium zone predominantly the size of the domain of undifferentiated cells was increased (Figure 5F-H) resulting specifically in an increased ratio of the domains of undifferentiated cells to xylem cells (Figure 5I). This indicated that ARF5 predominantly fulfils its function by promoting the transition of undifferentiated stem cells to differentiated xylem cells.
WOX4 mediates ARF5 activity
Considering the role of WOX4 as a mediator of auxin responses23 and its response to GR-ARF5ΔIII/IV induction, we hypothesized that ARF5 acts on cambium activity by regulating WOX4. Indeed, the expression domain of the transcriptional pWOX4:YFP reporter23 almost perfectly overlapped with pARF5:mCherry at the stem base (Figure 6A-C). Moreover, WOX4 transcript levels were increased in mp-S319 mutant stems (Figure 6D) demonstrating that the endogenous ARF5 gene is required for the regulation of WOX4. Importantly, the negative effect of GR-ARF5ΔIII/IV induction on WOX4 activity was also observed in the presence of the protein biosynthesis inhibitor cycloheximide (Cyclo) (Figure 6E), which was in line with a direct regulation of WOX4 by ARF5. Consistently, transient expression of ARF5ΔIII/IV in cultured cells had, similar as on other genes directly repressed by ARF568, a strong effect on the activity of a pWOX4:LUC promoter reporter, while this effect was only minor when ARF3 was expressed (Figure 6F). This suggested that, in comparison to ARF3, ARF5 substantially influenced the activity of the WOX4 promoter. Indeed, neither cambium-specific nor global induction of GR-ARF3 activity led to a significant change in WOX4 expression in wild type or arf3;arf4 double mutants although IPT3, a putative downstream target of ARF369, was induced (Figure S7A-C). Furthermore, WOX4 expression was not significantly altered in the arf3;arf4 double mutant (Figure S7D) making it rather implausible that ARF3 and ARF4 act on cambium activity by regulating WOX4.
To analyze the relevance of the observed effect of ARF5 on WOX4 activity we determined ICD extension in mp-S319 and wox4-1 single and double mutants. While mp-S319 showed enhanced cambium activity (Figure 6G, H, M), cambium activity was similar in wox4-1 single and in mp-S319;wox4-1 double mutants (Figure 6I, J, M) suggesting that WOX4 is required for an ARF5-dependent repression of cambium activity. In comparison, depletion of ARF5 activity in mp-S319;pxy-4 double mutants lead to a mild suppression of cambium defects observed in pxy-4 single mutants (Figure 6K, L, M57), suggesting that the epistatic relationship between WOX4 and ARF5 is specific.
Discussion
Similar to apical meristems, the regulation of the vascular cambium has been tightly associated with the plant hormone auxin for several decades13, 17, 70. However, spatial organization of functional signaling domains and the role of auxin signaling in controlling different aspects of cambium activity remained unknown. Here, we show that auxin signaling takes place in cambium stem cells and that this signaling is crucial for cambium activity. We also show that not only stem cell activity in general but also the balance between undifferentiated and stem cells depends on the auxin signaling machinery with ARF5 fulfilling a rather specific and WOX4-dependent role in this respect. Thus, auxin-related signaling controls distinct aspects of cambium activity important for a dynamic tissue production and a complex growth process.
The concentration of IAA peaks in the center of the cambial zone in Populus and Pinus13, 14, 15 and transcriptional profilings indicated a spatial correlation of this peak with the expression of auxin signaling components17, 71. However, genes responding to auxin were rather expressed in developing xylem cells arguing that sites of intense auxin signaling and of downstream responses do not necessarily overlap18. Consistently, our analysis of the highly sensitive auxin response marker pDR5revV2:YFP revealed a moderate auxin response in PXY-positive stem cells and a higher response in differentiated vascular tissues. Importantly, the auxin response in the PXY-positive region is overall pivotal for cambium activity since its local repression resulted in reduced tissue production similar as found in wox4 or pxy mutants defective for canonical regulators of stem cell activity23, 28, 57. This demonstrates that, in the cambium, auxin signaling promotes stem cell activity in a cell-autonomous manner. Interestingly, ARF5 and auxin signaling acts upstream of WOX5 in the context of RAM organization24, 72 but differentiation of distal root stem cells is promoted by ARF10 and ARF1672, 73. In comparison, ARF5 restricts the stem cell domain in the SAM by repressing stem cell-related features5. In the RAM and the SAM, ARF5 expression is found next to the expression domains of their central regulators WOX5 and WUS, respectively5, 24, 40, whereas it overlaps completely with the domain of WOX4 expression in the cambium. Thus, a division of labor of different auxin signaling components is found in various plant meristems and recruitment of distinct factors and adaptation of expression domains seem to have happened during the evolution of those systems.
ARF5 plays a major role in translating auxin accumulation into the establishment of procambium identity in embryos and leaf primordia (reviewed in 74). However, ARF5 is also tightly associated with xylem formation via its direct targets TMO5 and ATHB865, 75, 76. In fact, we found both genes and their targets ACAULIS5 (ACL5), SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3) and BUSHY AND DWARF2 (BUD2)21, 76 to be induced upon GR-ARF5ΔIII/IV induction. Together with the observation that the domain of WOX4-positive cells is enlarged in arf5 mutants, this suggests that ARF5 promotes the transition of stem cells to xylem cells. Because we also revealed a negative effect of ARF5ΔIII/IV induction on WOX4 activity, as well as a responsiveness of the WOX4 promoter in transient expression systems and an epistatic genetic relationship between WOX4 and ARF5, we propose that ARF5 fulfils its function partly by attenuating WOX4 activity. Therefore, ARF5 acts as one hub modulating the activity of a multitude of genes in PXY-positive cells to foster the transition from stem cells to differentiated vascular cells. Consistent with the possibility that ARF5 does not necessarily act as a transcriptional activator, it represses AUXIN RESPONSE REGULATOR15 (ARR15) and STOMAGEN in the SAM and in leaf mesophyll cells, respectively5, 68. As in our case observed for WOX4, both genes are yet induced by ARF5 in transient expression systems68. The role of ARF5 in transcriptional regulation does therefore depend on the target promoter and the respective cellular environment77. Remarkably, not only xylem-related but also phloem-related genes are activated in stems when inducing GR-ARF5ΔIII/IV plants. This would argue for a general role of ARF5 in promoting vascular differentiation and for the existence of one pool of stem cells marked by PXY promoter activity and feeding both xylem and phloem production. Alternatively, promotion of xylem differentiation is translated rapidly into the promotion of phloem differentiation by cell-to-cell signaling. The fact that the stem cell-to-phloem ratio is not altered in arf5 mutants would argue for the latter option.
Consistent with a crucial role of cell-autonomous auxin signaling in cambium stem cells, ARF3, ARF4 and ARF5 expression was found in PXY-positive cells with ARF5 being exclusively active in those. ARF3 and ARF4 have previously been shown to act in part redundantly in the establishment of abaxial identity in lateral organs59. In line with this function, we found both genes being mostly expressed distally of the cambium in phloem-related cell types. In fact, the lack of any effect on cambium or WOX4 activity when modulating ARF3 activity exclusively in PXY-positive cells, suggests that at least ARF3 functions outside of this domain when regulating cambium activity and that ARF transcription factors positively regulating WOX4 transcription still have to be discovered. Whether the phloem-related expression of ARF3 and ARF4 modulates the activity of cambium regulators expressed in areas distally to the PXY expression domain like MORE LATERAL GROWTH1 (MOL1)29 or CLAVATA/ESR41/44 (CLE41/44)26, 27, 78 remains to be determined. Transcriptionally, at least, our modulation of auxin signaling had no effect on the activity of MOL1 or CLE41/42 genes.
Collectively, we found a role of auxin signaling in the cambium sharing features with both the situation in the RAM where auxin regulates cell divisions79 and the SAM where auxin, and particular ARF5, is strongly correlated with cell differentiation4. Thereby, we enlighten a long-observed role of auxin signaling in plant development and reveal that its function is partly specific in different stem cell niches. The involvement of different auxin signaling components regulating individual aspects of meristem activity may provide a setup required for regulating a complex developmental process by one simple signaling molecule.
Acknowledgements
This work was supported by the SFB 873 of the German Research Foundation (DFG) and a Heisenberg professorship to T.G. (DFG, GR2104/5-1). arf4-2 mutants and the pRPS5a:Myc-GR-bdl line were kindly provided by Alexis Maizel (COS Heidelberg, Germany) and Gerd Jürgens (University of Tübingen, Germany), respectively. mp-B4149 mutant seeds and the pLC075 construct were obtained from Dolf Weijers (University of Wageningen, The Netherlands). Armin Djamei (GMI, Austria) donated the pGreen-LUC-REN construct. An established Arabidopsis (Col-0) dark-grown root cell suspension culture was a kind gift from Claudia Jonak (GMI, Austria). We thank members of the Greb lab for helpful discussions on the manuscript.
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