Functional characterization of genes mediating cell wall metabolism and responses to plant cell wall integrity impairment
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Plant cell walls participate in all plant-environment interactions. Maintaining cell wall integrity (CWI) during these interactions is essential. This realization led to increased interest in CWI and resulted in knowledge regarding early perception and signalling mechanisms active during CWI maintenance. By contrast, knowledge regarding processes mediating changes in cell wall metabolism upon CWI impairment is very limited.
To identify genes involved and to investigate their contributions to the processes we selected 23 genes with altered expression in response to CWI impairment and characterized the impact of T-DNA insertions in these genes on cell wall composition using Fourier-Transform Infrared Spectroscopy (FTIR) in Arabidopsis thaliana seedlings. Insertions in 14 genes led to cell wall phenotypes detectable by FTIR. A detailed analysis of four genes found that their altered expression upon CWI impairment is dependent on THE1 activity, a key component of CWI maintenance. Phenotypic characterizations of insertion lines suggest that the four genes are required for particular aspects of CWI maintenance, cell wall composition or resistance to Plectosphaerella cucumerina infection in adult plants.
Taken together, the results implicate the genes in responses to CWI impairment, cell wall metabolism and/or pathogen defence, thus identifying new molecular components and processes relevant for CWI maintenance.
KeywordsCell wall Cell wall integrity Cell wall metabolism Cell wall signalling Plant pathogen-interaction Bioenergy production
ACTIVATED DISEASE RESISTANCE-LIKE 1
arabidopsis Gβ 1
ARABINOGALACTAN PROTEIN 7
Alcohol insoluble residue
BOTRYTIS INDUCED KINASE 1
CALCINEURIN INTERACTING KINASE 26
Catharanthus roseus RLK1-like kinase
Cell wall damage
Cell wall integrity
DIRIGENT PROTEIN 7
days post infection
Fourier-Transform Infrared Spectroscopy
gramm / Liter
Guanosine nucleotide exchange factor
High-performance anion-exchange chromatography with pulsed amperometric detection
irregular xylem 1
MID1-COMPLEMENTING ACTIVITY 1
MATING-INDUCED DEATH1 / CALCIUM CHANNEL1
MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2
MECHANOSENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 2
MECHANOSENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 3
Plectosphaerella cucumerina BMM
PHYTOCHROME KINASE SUBSTRATE1
Plant Natriuretic Peptides
- PSAT 1
PHOSPHOSERINE AMINO-TRANSFERASE 1
PHLOEM INTERCALATED WITH XYLEM / TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR RECEPTOR-CORRELATED 3
- qRT – PCR
quantitative reverse transcription polymerase chain reaction
- RBOH D/F
NADPH-oxidases like RESPIRATORY BURST OXIDASE HOMOLOGUE
- S. cerevisiae
SOMATIC EMBRYOGENESIS RECEPTOR KINASE 4
SALT OVERLY SENSITIVE5
Ultra-fast liquid chromatography
UDP GLYCOSYLTRANSFERASE 76B1
WALL STRESS RESPONSE
Plant cell walls are involved in all interactions between plants and their environment. Examples include pathogen infection or exposure to drought, where wall composition and structure change to prevent water loss, pathogen susceptibility or at least limit further pathogen spread [1, 2]. These changes of the walls are exemplified by reinforcement with Callose during infection or modifications of pectic polysaccharides to prevent water loss during exposure to drought stress [3, 4]. Cell walls are extremely plastic, undergoing dynamic changes to enable plant cells to expand and differentiate during growth and development . Controlled deposition of cellulose microfibrils through interactions between cellulose synthases and microtubules during cell expansion exemplify the changes in cell wall organization, permitting tightly controlled cell expansion [6, 7]. Deposition of suberin and lignin during formation of the Casparian strip in pericycle cells of the primary root exemplifies modifications of cell walls during cell differentiation [8, 9, 10]. These examples illustrate processes active during plant-environment interactions and development, enabling cell walls to fulfill their respective biological functions.
How do cell walls perform these various functions, which sometimes involve opposite performance requirements, while simultaneously maintaining their functional integrity? The available evidence supports the existence of a dedicated mechanism, which is monitoring the functional integrity of the plant cell wall and initiates adaptive changes in cellular and cell wall metabolism to maintain cell wall integrity (CWI) [11, 12, 13, 14, 15]. Studies of the mode of action of the CWI maintenance mechanism often investigate the responses to cell wall damage (CWD), which can be generated by cell wall degrading enzymes (cellulase, pectinase etc.) or compounds like isoxaben (ISX) . ISX inhibits specifically cellulose production during primary cell wall formation in elongating plant cells [17, 18, 19]. Established responses to CWD include growth inhibition involving cell cycle arrest, changes in the levels of phytohormones like jasmonic acid (JA), salicylic acid (SA) and cytokinins (CKs) as well as changes in cell wall composition involving pectic polysaccharides, lignin and Callose deposition [20, 21, 22, 23, 24].
The available evidence implicates receptor-like kinases (RLK) like MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2), FEI1, FEI2, THESEUS 1 (THE1) and FERONIA (FER) in CWI maintenance [16, 25, 26, 27, 28]. THE1 and FER belong to the Catharanthus roseus RLK1-like kinase (CrRLK1L) family, which has 17 members. These RLKs consist of an intracellular Serine / Threonine-kinase domain, a transmembrane domain and an extracellular domain exhibiting similarity to the malectin domain originally identified in Xenopus laevis . Currently it is not clear if malectin domains in CrRLK1Ls are either required for binding to cell wall epitopes, mediate protein-protein interaction or actually do both [28, 30, 31, 32, 33, 34, 35]. FER is required during gametophytic and root hair development, salt stress, JA signaling and coordination between abscisic acid- (ABA) and JA-based signaling processes [28, 36, 37, 38, 39, 40, 41, 42]. MIK2 and THE1 are required for root development, CWD-induced lignin and phytohormone production as well as resistance to the root pathogen Fusarium oxysporum [16, 25, 27, 33]. FEI1 and FEI2 have been originally identified through their impact on seedling root growth on medium containing 4.5% sucrose and subsequently implicated in a cell wall signaling pathway involving the SALT OVERLY SENSITIVE5 (SOS5) and FEI2 [43, 44, 45]. In parallel, ion-channels, like MID1-COMPLEMENTING ACTIVITY 1 (MCA1) and MECHANOSENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 2 (MSL2) and 3 (MSL3) were shown to contribute to activation of CWD-induced responses in plants [16, 23]. MCA1 was originally identified through its´ ability to partially complement a MID1/CCH1- deficient Saccharomyces cerevisiae strain . In yeast MID1/CCH1 form a plasma membrane-localized stretch-activated calcium channel required both for mechano-perception and CWI maintenance (Levin, 2011). CWD-induced responses in plants (like in yeast cells) seem also to be sensitive to turgor manipulation [11, 47]. The reason being that in Arabidopsis thaliana seedlings, exposed simultaneously to ISX and mild hyperosmotic conditions, most of the CWD-induced responses are suppressed in a concentration dependent manner [16, 48]. The early signals generated seem to be conveyed to downstream response mediators through changes in production of reactive oxygen species (ROS) and phytohormones (JA/SA/CKs) [23, 24]. Enzymes implicated in ROS production upon CWI impairment are NADPH-oxidases like RESPIRATORY BURST OXIDASE HOMOLOGUE (RBOH) D/F (after ISX-treatment) or RBOH H/J during pollen tube development . NADPH-oxidase activity in turn can be regulated via calcium binding, differential phosphorylation involving kinases controlled by changes in calcium levels (CALCINEURIN INTERACTING KINASE 26, CIPK26), activated in response to pathogen infection through phosphorylation involving BOTRYTIS INDUCED KINASE 1 (BIK1) or controlled via RHO GTPases, a ROPGEF and FER [37, 50, 51].
This abbreviated overview of molecular components active during plant CWI maintenance illustrates the increase in knowledge regarding putative CWI sensors and early signal transduction elements in recent years. Whilst it is fascinating to know about early CWD perception and signaling processes we also need to understand how signals generated lead to changes in cell wall composition and structure to dissect the mode of action of the CWI maintenance mechanism thoroughly. This is of particular interest in the context of targeted modification of biomass quality and improvement of food crop performance since the CWI maintenance mechanism seems to be an important component of cell wall plasticity [52, 53]. Cell wall plasticity in turn has been discussed as the root cause for the apparently limited success of efforts aimed at optimizing biomass quality that have been achieved so far .
We wanted to identify additional components and molecular processes, which are mediating responses to CWD and adaptive changes in cell wall metabolism. To achieve these aims we selected candidate genes using microarray-based expression profiling data deriving from ISX-treated Arabidopsis seedlings. FTIR spectroscopy was then used to identify candidate genes where insertions lead to cell wall changes on the seedling level. We performed in depth studies for four genes to validate the approach. These studies involved confirming that gene expression is responsive to ISX, determining if expression is controlled by THE1 and investigating how loss-of-function alleles for these genes affect cell wall composition in adult plants, resistance to the necrotrophic pathogen Plectosphaerella cucumerina and responses to ISX-induced CWD impairing CWI.
Identification of candidate genes
Candidate genes selected from the transcriptomics / FTIR- based screen. Gene annotations are based on Araport11 and references listed. WSR: Wall Stress Response
DIR7, Disease resistance-responsive (dirigent-like protein) family protein
Paniagua et al., 2017 
Heavy metal transport / detoxification superfamily protein
De Abreu-Neto et al., 2013 
SFH19, Sec14p-like phosphatidylinositol transfer family protein
Vincent et al., 2005 
PXC3, Leucine-rich repeat protein kinase family protein
Wang et al., 2013 
FTIR-based analysis detects cell wall phenotypes in mutant seedlings
Performing detailed cell wall analysis for insertion lines in 23 candidate genes would be time consuming and possibly not very efficient. Previously, FTIR has been successfully used as an efficient approach to classify Arabidopsis mutants with altered cell wall architecture . We used this approach as foundation to facilitate identification of insertions in candidate genes leading to changes in cell wall composition or structure in Arabidopsis seedlings. FTIR spectra were collected for analysis from total cell wall material derived from 6 days-old, liquid culture grown Col-0 seedlings or seedlings with T-DNA insertions in the candidate genes. Initially only Col-0 samples were characterized to establish the variability observed in controls. Subsequently, twice the standard deviation of the Col-0 variability was used as a cut-off to identify insertions in candidate genes causing significant changes in the FTIR spectra. Based on this criterium FTIR spectra for 17 of the 23 insertion lines analyzed seemed to exhibit significant differences (Additional file 1: Figure S2). Pronounced differences were observed for seedlings with insertions in At5g24140 (SQUALENE MONOOXYGENASE2, SQP2, ) and At5g49360 (BETA-XYLOSIDASE1, ATBXL1, ), At1g07260 (UGT71C3, ), At1g74440 , At2g35730 [55, 63], At3g13650 (DIRIGENT PROTEIN 7, DIR7, ) and At4g33300 (ACTIVATED DISEASE RESISTANCE-LIKE 1, ADR1-L1, ) in the 1740–1600 cm− 1 and 1200–950 cm− 1 areas (Additional file 1: Figure S2a). These are supposedly characteristic for cellulose, elements of pectic polysaccharides (including alkyl esters in pectin as well as other esters, [59, 60, 65, 66] 2015). Insertion lines for At2g41820 (PHLOEM INTERCALATED WITH XYLEM / TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR RECEPTOR-CORRELATED 3, PXC3, ), At3g11340 (UDP GLYCOSYLTRANSFERASE 76B1, UGT76B1, ), At4g33420 (PEROXIDASE 47, PRX47, ), At4g35630 (PHOSPHOSERINE AMINO-TRANSFERASE 1, PSAT 1, ), At5g48460 (FIMBRIN 2, ATFIM2, ), At5g47730 (SEC14-HOMOLOGUE 19, SFH19, [56, 71]), At5g65390 (ARABINOGALACTAN PROTEIN 7, AGP7, ), At3g16560, At2g02950 (PHYTOCHROME KINASE SUBSTRATE1, PKS1) , At2g13790 (SOMATIC EMBRYOGENESIS RECEPTOR KINASE 4, SERK4) , exhibited apparent differences in the 1367–1200 cm− 1 area supposedly characteristic for C-H vibrations and CH2-bending in cellulose and hemicelluloses as well as bending of O-H groups in pyranose rings of pectins (Additional file 1: Figure S2b, ). The FTIR spectra for the other lines examined did not exhibit differences to Col-0 controls (Additional file 1: Figure S2c). The results from the FTIR-based analysis of the insertion lines suggested that cell wall composition or structure could be affected in seedlings with insertions for 17 of the 23 candidate genes examined. The insertions seemed to have distinct effects on cell wall composition / structure based on their apparent separation into three groups.
Four candidate genes are selected for more detailed characterization
Quantitative gene expression analysis confirms transcriptomics results and suggests THE1 is controlling WSR gene expression
Identification of knockout and knockdown alleles for WSR genes
Using knockout (KO) or knockdown (KD) alleles generated through T-DNA insertions is a well-established and successful method to characterize genes of interest . We identified two independent T-DNA insertion lines for each of the four genes using the Arabidopsis Gene Mapping Tool (http://signal.salk.edu/cgi-bin/tdnaexpress). Plants homozygous for the insertions were isolated using PCR-based genotyping as well as insertion positions in the individual gene and their effects on transcript levels determined. For WSR1 the first insertion is located in the 5′ (wsr1–1, Salk_046217) and the second (wsr1–2, Salk_092919) in the 3′ untranslated region of the gene (Additional file 1: Figure S3a). The insertions in WSR2 were mapped to the first intron (wsr2–2, Salk_123509) and the third exon (wsr2–1, Salk_058271) (Additional file 1: Figure S3b). For WSR3 the insertions were located either in the promoter region (wsr3–2, SALK_079548) or in the 10th intron (wsr3–1, SALK_039575) (Additional file 1: Figure S3c). In the wsr4–2 (SALK_121365) allele the insertion is located in the 1st while the one giving rise to the wsr4–1 (SALK_082484) allele is located in the 2nd exon (Additional file 1: Figure S3d). To determine if the insertions affect transcript levels of the genes, we performed qRT-PCR using total RNA isolated from mock-treated 7 days-old seedlings. These experiments identified four bona fide KO- (wsr2–1, wsr2–2, wsr3–1, wsr4–2) and four KD-alleles (wsr1–1, wsr1–2, wsr3–2 and wsr4–1) for the candidate genes (Additional file 1: Figure S3a-d).
Responses to ISX-induced CWD are modified in seedlings with insertions in WSR1 or 4
WSR1, 2, 3 and 4 contribute to cell wall formation during stem growth
Plants with insertions in WSR1 exhibit pathogen response phenotypes
In plants a mechanism is existing, which monitors and maintains the functional integrity of the cell walls [12, 14]. This mechanism seems to exhibit similarities to the one described in S. cerevisiae and is capable of detecting CWD and initiating adaptive changes in cellular and cell wall metabolism to maintain the functional integrity of the wall . Understanding of the molecular mechanisms underlying CWD perception and the signaling cascades involved in regulating the CWD response in plants is increasing [16, 28]. However, our knowledge of the genes and molecular processes bringing about changes in cell wall metabolism in response to CWD and their function during growth and development is very limited. Here, we have determined if we can identify genes both mediating responses to CWD in seedlings and cell wall metabolism in adult plants by combining transcriptomics data from ISX-treated seedlings with FTIR-based cell wall analysis of seedlings carrying T-DNA insertions in candidate genes identified from the transcriptomics data. This approach identified 17 genes (out of 23 original candidates), where T-DNA insertions in the candidate genes seem to lead to cell wall defects based on FTIR results. The functions of these genes, belonging to different gene families, are often not well-understood (Additional file 1: Table S1). Very little is known about the biological function of At1g74440 beyond that it encodes an ER membrane protein. The gene has been implicated in biotic and abiotic stress responses mediated by Plant Natriuretic Peptides (PNPs) based on co-expression with AtPNP-A . ATFIM2 encodes a protein belonging to the Fimbrin family and seems to modulates the organization of actin filaments . SQE2 encodes a squalene epoxidase converting squalene into oxidosqualene, which forms the precursor of all known angiosperm cyclic triterpenoids . Triterpenoids are required for production of membrane sterols and brassinosteroids. PSAT1 encodes an amino transferase required for serine biosynthesis taking place in the chloroplast . Serine biosynthesis in turn is required during photorespiration, a prerequisite for carbohydrate metabolism and plant growth. While AGPs have been implicated in cell wall remodeling, very little information is available regarding the specific function of AGP7 in this context . AtBXL1 encodes an enzyme acting during vascular differentiation as a β-D-xylosidase while acting as an α-L-arabinofuranosidase during seed coat development . PRX47 encodes a putative peroxidase, is apparently expressed in differentiating vascular tissue in seedling roots and stems and involved in lignification . UGT71C3 and UGT76B1 encode UDP-glycosyltransferases (UGTs), which have been implicated in glycosylation of phytohormones and / or metabolites during the response to biotic and abiotic stress . UGT76B1 in particular seems to glycosylate isoleucic acid, which is required for coordination of SA- and JA-based defence responses active during infection by pathogens like Pseudomonas syringae and Alternaria brassicicola . ADR1-L1 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein and forms an important element of the effector-triggered immunity in plants [64, 79]. Reviewing the available knowledge provides further evidence that several of the genes are probably required for processes relevant for cell wall (PRX47, ATBXL1, AGP7), membrane (SQE2), photosynthetic (PSAT1) metabolism or light perception (PKS1). Intriguingly UGT76B1, UGT71C3, ADR1-L1, SERK4 and At3g16560 have been implicated before in the responses to abiotic or biotic stress, which also involves plant cell walls [61, 67, 79]. In our experimental conditions the seedlings are exposed to CWD but not biotic / abiotic stress, thus raising the possibility that these genes are actually responding to cell wall-related events, which may also occur during biotic and abiotic stress. More importantly the results suggest that the approach pursued here enables us to identify amongst the many genes in the Arabidopsis genome those that contribute to the responses to CWD and regulation of relevant aspects of cell wall and membrane metabolism.
We characterized four candidate genes in more detail. These had been selected based on the FTIR phenotypes apparently caused by insertions in the candidate genes and the limited detailed knowledge regarding their biological functions. qRT-PCR-based expression analysis of the four genes in ISX-treated seedlings yielded results similar to the data from the transcriptomics experiment. Experiments with loss- and gain-of-function alleles of THE1 showed that ISX-induced changes in the transcript levels of WSR1, 2, 3 and 4 are sensitive to an increase in the activity of THE1 (the1–4) while effects of reductions (the1–1) are less pronounced . These results are to be expected since complete loss of THE1 results in reduced responses to CWD but not complete losses, suggesting that the THE1-mediated CWI maintenance mechanism is either redundantly organized or other signaling mechanisms exist . However, the results support the notion that WSR gene expression is regulated by the THE1-mediated CWI maintenance mechanism and that WSR activity might be controlled on the transcriptional level.
Overview of the phenotypes observed for mutant lines of the different candidate genes examined. Statistically significant differences compared to the wild type are indicated with blue (increased) or red (decreased) arrows
To summarize, seedlings with T-DNA insertions in 17 of the 23 candidate genes that were selected in this study seemed to exhibit FTIR phenotypes. Gene expression analysis showed that WSR gene expression is modulated in response to ISX-induced CWD, with the modulation apparently sensitive to changes in THE1 activity. This connected the genes identified to the THE1-dependent CWI maintenance mechanism, suggesting that our approach has identified new components mediating CWI maintenance in Arabidopsis. Follow up studies with KO or KD lines for four candidate genes found cell wall phenotypes in adult plants for all four and effects on CWD responses for WSR1 and 4. These results also suggest strongly that a more detailed analysis of the remaining candidate genes identified, will probably yield interesting novel insights into the mode of action of the CWI maintenance mechanism and cell wall metabolism in general.
All chemicals and enzymes were purchased from Sigma-Aldrich unless stated otherwise.
Wild-type and mutant Arabidopsis thaliana lines used in this study were ordered from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/). Detailed information is listed in Additional file 1: Table S1. Seedlings were grown for 6 days in liquid culture (2.1 g/L Murashige and Skoog Basal Medium, 0.5 g/L MES salt and 1% sucrose at pH 5.7) before treatment with 600 nM isoxaben (in DMSO) as described . For cell wall analysis, plants were grown on soil (Pro-Mix HP) in long-day conditions (16 h light, 11000 Lux, 22 °C, 8 h dark, 20 °C, 70% relative humidity). For pathogen infection assays, plants were grown in phytochambers on sterile soil-vermiculite (3,1) under short-day conditions (10 h of light/14 h of dark) at 20–21 °C.
Pathogen infection assays
For Plectosphaerella cucumerina BMM pathogenicity assays, 18 days-old plants (n > 15) were sprayed with a spore suspension (4 × 106 spores/ml) of the fungus as previously described [82, 83]. Fungal biomass in planta was quantified by determining the level of the PcBMM β-tubulin gene by qPCR (forward primer: CAAGTATGTTCCCCGAGCCGT and reverse primer: GGTCCCTTCGGTCAGCTCTTC) and normalizing these values to those of UBIQUITIN-CONJUGATING ENZYME21 (UBC21, AT5G25760).
ACT2-FOR, ACT2-REV, WSR1-FOR, WSR1-REV, WSR2-FOR, WSR2-REV,
WSR3-FOR2 (5′- TCTTATCCGGTTGCGGAAGG-3′),
FTIR spectroscopy and analysis
For FTIR analysis, 4 biological replicates per genotype and 5 technical replicates per biological replicate were collected (i.e. for each genotype 20 spectra were collected). Spectra for each technical replicate were measured from 800 to 5000 cm− 1 with 15 accumulations per measurement on a Bruker Vertex 70. All spectra were measured at 10 kHz, with a 10 kHz lowpass filter and the Fourier transform was carried out using Blackman-Harris 3-term. Atmospheric compensation was carried out on the data using OPUS version 5 (www.bruker.com). The spectra were cropped to the area between 802 cm− 1 to 1820 cm− 1 to cover informative wavenumbers as described in . Linear regression was carried out based on the first 10 points in either end of the spectra and used for baseline correction. The data was normalized to sum 1 with any negative values still present set to 0 for normalization purposes. Biological variation in the Col-0 controls was determined based on three independent experiments carried out with 4 biological replicates and 5 technical replicates per biological replicate (i.e. 20 spectra per experiment). The difference between the insertion lines and Col-0 was calculated by averaging all the technical repeats for a line and subtracting the corresponding average from Col-0. The difference between the insertion lines and Col-0 was plotted by wavelength. Two times the standard deviation of Col-0 was chosen as a cutoff as it would indicate significance if the natural variation is assumed to be symmetrical across Col-0 and the insertion line.
Cell wall analysis
Cell wall preparation and analysis were performed as described  with minor modifications. For analysis of stem cell wall composition, major stems of three 5 weeks-old plants per genotype were pooled to form one biological replicate. For analysis of leaf cell wall composition, whole leaf rosettes of three 5 weeks-old plants per genotype were pooled to form one biological replicate. Four biological replicates were analysed in all cases. Plant samples were immediately flash-frozen in liquid nitrogen after sampling and lyophilized. Dried material was ball-milled with zirconia beads in a Labman robot (www.labmanautomation.com), extracted three times with 70% ethanol at 70 °C and dried under vacuum. Starch was removed using a Megazyme Total Starch Kit according to the manufacturer’s instructions. After drying under vacuum, de-starched alcohol insoluble residue (AIR) was weighed out in 2 ml screw caps tubes for cell wall monosaccharide analysis (2 mg AIR) and GC vials for lignin analysis (1.2 mg AIR), respectively, with the Labman robot (0.2 mg tolerance). Cellulose, neutral sugars and uronic acids were determined following the published one-step two-step hydrolysis protocol . High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed on a Thermo Fisher Dionex ICS-5000 system with CarboPac PA-20 and PA-200 columns as described . Acetyl bromide soluble lignin was quantified as described .
JA and SA were extracted and analysed as described . Briefly, extraction was performed in 10% methanol / 1% acetic acid with Jasmonic-d5 Acid and Salicylic-d4 Acid (CDN Isotopes) as internal standards. Quantification was performed on a Shimadzu UFLC XR / AB SCIEX Triple Quad 5500 system using the following mass transitions: JA 209 > 59, D5-JA 214 > 62, SA 137 > 93, D4-SA 141 > 97.
Lignin detection in roots
Lignification in seedling roots (n > 15) was analysed 24 h after start of treatment. Lignified regions were detected with phloroglucinol-HCl, photographed with a Zeiss Axio Zoom.V16 stereomicroscope and quantified as described .
Statistical significance was assessed using Student’s t-test in Microsoft Excel (2-tailed distribution, two-sample unequal variance). Statistically significant differences are indicated by * p < 0.05, ** p < 0.01. Boxplots were generated using R package “boxplot” with default settings (range = 1.5*IQR).
Programming support from Ane-Kjersti Vie and help with lignin and phytohormone quantification from Trude Johansen and the PROMEC facility at NTNU are gratefully acknowledged.
TE, LK, NGB, LV, SB, EM, AM and TH contributed to experimental design. TE, LK, NGB, LV, SB, EM, AM, LJ and IC generated data. TE, LK, NGB, LV, SB, EM, AM and TH co-wrote the manuscript. All authors have read the final version of the manuscript and approved it.
This work was supported through Gatsby AdHoc funds and a grant from the Peder Sather Center for Advanced Study to T.H. and Chris Somerville. T.E. was supported through a EU Marie Curie Fellowship “SUGAROSMO-SIGNALLING” and a DFG postdoctoral fellowship (EN 1071/1–1). L.K. was supported by a Ph.D. Fellowship from the Porter Institute at Imperial College. N.G.-B. was supported through the EEA project grant CYTOWALL. L.D. and A.W were supported through postdoctoral fellowships provided by the Porter Institute at Imperial College and I.C. by a PhD fellowship provided by the Royal Thai government. Research by A.M. was supported by Spanish Ministry of Economy and Competitiveness (MINECO) grant BIO2015–64077-R. The different funders did not have any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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The authors declare that they have no competing interests.
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