The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+homeostasis

The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+ homeostasis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca 2+ homeostasis View ORCID Profile Xiaoyu Hou , View ORCID Profile Giorgia Tortora , View ORCID Profile Aline Herger , View ORCID Profile Stefano Buratti , View ORCID Profile Petre Dobrev , Roberta Vaculiková , View ORCID Profile Jozef Lacek , View ORCID Profile Alexandros Georgios Sotiropoulos , View ORCID Profile Gabor Kadler , View ORCID Profile Myriam Schaufelberger , View ORCID Profile Alessia Candeo , View ORCID Profile Andrea Bassi , View ORCID Profile Thomas Wicker , View ORCID Profile Alex Costa , View ORCID Profile Christoph Ringli doi: https://doi.org/10.1101/2025.06.25.660713 Xiaoyu Hou 1 Department of Physics, Politecnico di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xiaoyu Hou Giorgia Tortora 1 Department of Physics, Politecnico di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giorgia Tortora Aline Herger 5 Docworld AG , Steinhausen, Zug, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aline Herger Stefano Buratti 2 Department of Biosciences, Università degli Studi di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefano Buratti Petre Dobrev 4 Institute of Experimental Botany, Academy of Sciences of the Czech Republic , Prague, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Petre Dobrev Roberta Vaculiková 4 Institute of Experimental Botany, Academy of Sciences of the Czech Republic , Prague, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jozef Lacek 4 Institute of Experimental Botany, Academy of Sciences of the Czech Republic , Prague, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jozef Lacek Alexandros Georgios Sotiropoulos 6 University of Southern Queensland, Centre for Crop Health , Queensland, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexandros Georgios Sotiropoulos Gabor Kadler 7 University Hospital Zurich, Department of Intensive Care , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabor Kadler Myriam Schaufelberger 8 University Hospital Zurich, Department Pathology and Molecular Pathology , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Myriam Schaufelberger Alessia Candeo 1 Department of Physics, Politecnico di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alessia Candeo Andrea Bassi 1 Department of Physics, Politecnico di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrea Bassi Thomas Wicker Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Wicker Alex Costa 2 Department of Biosciences, Università degli Studi di Milano , Milan, Italy 3 Institute of Biophysics, National Research Council of Italy (CNR) , 20133, Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alex Costa Christoph Ringli 9 Department of Plant and Microbial Biology, University of Zurich, and Zurich-Basel Plant Science Center , Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christoph Ringli For correspondence: chringli{at}botinst.uzh.ch Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Calcium, as a cellular second messenger, is essential for plant growth. A tip-focused Ca 2+ gradient in polarized cells is considered to drive cell expansion. The cell wall polysaccharide pectin is a major Ca 2+ binding structure and Ca 2+ homeostasis is influenced by the cell wall architecture. LRR-extensin (LRX) proteins are extracellular regulators of cell wall development that are anchored in the cell wall by their extensin domain. The extensin-less LRX1ΔE14 variant of the root hair-expressed LRX1 of Arabidopsis induces a dominant-negative effect resulting in aberrant root hairs. In an effort to identify the underlying mechanism of the root hair defect caused by LRX1ΔE14 , we isolated a su ppressor of dominant- ne gative effect mutant, sune42 . It codes for the CATION CALCIUM EXCHANGER 4 (CCX4) that localizes to the Golgi apparatus and was shown to have Ca 2+ transport activity. A detailed investigation of the Ca 2+ dynamics revealed that LRX1ΔE14 coincides with a defect in tip-focused cytoplasmic Ca 2+ oscillation, and this effect is alleviated by the sune42 mutation. Additionally, reducing Ca 2+ availability influences the LRX1ΔE14 -induced root hair defect. We conclude that sune42 suppresses the root hair defect in LRX1ΔE14 through modulating cytoplasmic Ca 2+ dynamics, pointing at the importance of the Golgi apparatus for cellular Ca 2+ homeostasis. Introduction Plant cell growth is constrained by the cell wall, which undergoes periodic structural modifications to allow the cell to expand. The cell wall is a complex network of cellulose microfibrils embedded within a matrix of polysaccharides, interwoven with proteinaceous molecules ( Cosgrove, 2024 ). The cell wall-localized Leucine-rich repeat extensins (LRXs) are chimeric proteins that contain a C-terminal extensin domain and an N-terminal LRR domain, separated by a cysteine-rich domain ( Rubinstein et al., 1995 ; Baumberger et al., 2001 ; Ringli, 2010 ; Herger et al., 2019 ). LRXs influence cell wall formation by binding RALF (rapid alkalinization factor) peptides and compacting pectin ( Mecchia et al., 2017 ; Moussu et al., 2020 ; Moussu et al., 2023 ; Schoenaers et al., 2024 ). Genome analysis revealed the presence of eleven LRXs in Arabidopsis , among which LRX1 and LRX2 are predominantly expressed in root hairs ( Baumberger et al., 2003a ). Loss of LRX1 function results in shorter root hairs that often branch or burst ( Baumberger et al., 2001 ; Baumberger et al., 2003 ). The extensin domain of LRXs, which contains Ser-Hyp n repetitive sequences characteristic for structural hydroxyproline-rich glycoproteins (HRGPs) ( Borassi et al., 2016 ; Liu et al., 2016 ), anchors the protein in the extracellular matrix. It is essential for the function of LRX1, as LRX1 lacking the extensin domain (LRX1ΔE14) is unable to complement the lrx1 root hair phenotype ( Ringli, 2010 ). Furthermore, the expression of extensin-less LRXs in wild-type Col plants induces a dominant-negative effect ( Ringli, 2010 ; Dünser et al., 2019 ). Refined integration of extracellular and cytoplasmic events is needed for coordinating between cell wall enlargement and cell expansion. LRXs have been suggested to function in conjunction with the Catharanthus roseus Receptor-Like Kinase1-Like protein ( Cr RLK1L) FERONIA (FER) to translate the extracellular dynamics into cytoplasmic responses. lrx mutants phenocopy the fer-4 mutant, which shows root hair impairment similar to the lrx1 lrx2 mutant ( Herger et al., 2020 ) and shoot growth defects similar to the lrx3 lrx4 lrx5 mutant ( Draeger et al., 2015 ; Zhao et al., 2018 ; Dünser et al., 2019 ). The multitude of processes influenced by the LRX-RALF-FER module is only beginning to be understood ( Leiber et al., 2010 ; Schaufelberger et al., 2019 ; Song et al., 2022 ; Wang et al., 2022 ; Pacheco et al., 2023 ; Gupta et al., 2024 ). Tip-growing cells, like pollen tubes and root hairs, are featured for their rapid and unidirectional growth. Polarized cell growth relies on the apical fusion of secretory vesicles, which is under the control of the configuration of the actin cytoskeleton ( Campanoni and Blatt, 2007 ; Ketelaar, 2013 ). This process is intimately linked with Ca 2+ gradients and Ca 2+ oscillations in the cytoplasm ( Wymer et al., 1997 ; Zhang et al., 2017 ). Mutations in FER and several other CrRLK1L family members such as ERULUS and ANX1/2 dampen the tip-focused cytoplasmic Ca 2+ gradients in tip-growing cells ( Ngo et al., 2014 ; Kwon et al., 2018 ; Schoenaers et al., 2018 ; Gao et al., 2023 ; Schoenaers et al., 2024 ). A few Ca 2+ channels involved in CrRLK1L-mediated Ca 2+ signaling, like powdery mildew resistance locus (MLOs), have also been identified ( Ogawa et al., 2025 ). Other studies have revealed the essential roles of several cyclic nucleotide-gated channels (CNGCs) in polarized growth by modulating tip-focused Ca 2+ oscillations, among which CNGC6, CNGC9, and CNGC14 act together in controlling root hair polarity and integrity ( Zhang et al., 2017 ; Brost et al., 2019 ; Zhu et al., 2025 ). Importantly, organelles have been found to largely contribute to the cytoplasmic Ca 2+ signatures ( Trewavas et al., 1996 ; Kudla et al., 2010 ; Costa et al., 2018 ; Resentini et al., 2021 ). Organellar channels, transporters, and pumps involved in Ca 2+ transport (e.g. cation/Ca 2+ exchangers (CCXs), Ca 2+ -ATPases) have been reported to regulate a range of physiological processes ( Bose et al., 2011 ; Frei dit Frey et al., 2012 ; Pittman and Hirschi, 2016 ; Corso et al., 2018 ; Costa et al., 2023 ; Kanamori et al., 2023 ). In plants, the Golgi apparatus is known to be essential for protein posttranslational modification and trafficking ( Vitale and Galili, 2001 ) and cell wall polysaccharide synthesis ( Cosgrove, 2024 ; Gupta et al., 2024 ). These processes are tightly linked with Ca 2+ , but little is known about Ca 2+ homeostasis and signaling in the Golgi apparatus ( Costa et al., 2018 ; Pirayesh et al., 2021 ). The estimated resting free Ca 2+ in the Golgi is slightly higher than that in the cytoplasm and transient Ca 2+ changes in the Golgi during stress responses have been observed ( Ordenes et al., 2012 ). A recent study has shown that a Golgi-localized CCX member in Arabidopsis , CCX4, mediates Ca 2+ transport and is involved in Ca 2+ response and osmotic tolerance ( Kanamori et al., 2023 ). To seek for novel modulators of the LRX1-related signaling pathway, a suppressor screen was performed on the LRX1ΔE14 -expressing line that displays a defect in root hair development. This led to the identification of sune42 ( suppressor of dominant-negative effect of LRX1ΔE14_42 ), which harbors a mutation in the gene Cation Calcium Exchanger 4 ( CCX4 ). We found that the previously reported role of CCX4 in influencing NPR1-dependent salicylic acid (SA) signaling ( Fujikura et al., 2020 ) is not involved in suppressing the root hair defect in LRX1ΔE14. Our study shows that LRX1ΔE14 is impaired in Ca 2+ oscillation and mutation in CCX4 likely alleviates this derangement induced by LRX1ΔE14 . Our data imply that sune42 mutation might restore the LRX1ΔE14 root hair phenotype through regulating Ca 2+ dynamics by altering the flux of Ca 2+ between the Golgi and the cytoplasm. Materials and Methods Plant material and growth conditions Arabidopsis thaliana Col was used for all experiments. The p LRX1 :: LRX1ΔE14 line (referred to LRX1ΔE14 ) is described in Ringli, 2010 . sune42 (in Col), lrx1 sune42 , fer-5 sune42 were obtained by crossing sune42 with wild-type Col, lrx1 ( Baumberger et al., 2001 ), and fer-5 (Duan et al., 2010), respectively. ccx4 and npr1 were generated by Crispr CAS9 -guided mutagenesis. LRX1ΔE14 sune42 was later crossed with npr1 to produce LRX1ΔE14 sune42 npr1. For the cytoplasmic calcium measurement, the transgenic line containing pUBQ10::R-GECO1 transgene ( Keinath et al., 2015 ) was crossed with sune42 , LRX1ΔE14, LRX1ΔE14 sune42 , and the resulting F2 populations were screened by phenotype and molecular markers for the individual mutations and transgenes. Unless otherwise detailed, Arabidopsis seeds were surface sterilized with 1% (v/v) sodium chlorite, 0.03% (v/v) Triton X-100 and washed three times with sterile water. After plating, they were stratified for 2 days at 4°C in darkness and subsequently grown vertically on ½ MS/Vitamins with 2% (w/v) sucrose, 10 mg/L myo-inositol, 0.5 g/L MES pH 5.7, and 0.6% (w/v) Gelrite (Duchefa). All seedlings were grown in a long-day regime (16-h light/8-h dark) at 22°C. Root hairs were visualized 5 days after germination. For propagation, transformation, and crossing, seedlings were transferred to soil and grown in the growth chamber at 22°C with a 16-h light/8-h dark cycle. Plasmid construction For CRISPR Cas9 mutagenesis, the pKI1.1 (Tsutsui and Higashiyama, 2016) plasmid digested with AarI was ligated with the double-stranded oligos CCX4_gRNA_F/R targeting the CCX4 gene and NPR1_gRNA_F/R targeting NPR1 ( Table 1 ). For the complementation assay, the CCX4 promoter and coding sequence of CCX4 were separately amplified using the primers SpeI_CCX4_F and CCX4_BamHI_R for fragment 1, primers CCX4_BamHI_F and CCX4_AscI_R for fragment 2 ( Table 1 ), and cloned into pJET vector with SpeI/BamHI and BamHI/AscI , respectively. After sequencing, correct clones of the two fragments were fused by a BamHI restriction site in both clones to generate pJET-pCCX4::CCX4 . pCCX4::CCX4 was then cut from pJET with SpeI and AscI and cloned into pGPTV-Bar-ocsTerminator ( Becker et al., 1992 ). View this table: View inline View popup Table 1 Primers used in this study. EMS mutagenesis The ethyl methanesulfonate (EMS) mutagenesis and whole genomic sequencing was performed as described elsewhere ( Guérin et al., 2025 ). Seeds from a line expressing LRX1::LRX1ΔE14 were incubated in 100 mM phosphate buffer overnight. The next day, seeds were incubated in 100 mM phosphate buffer containing 0.2% EMS for 8 h on a shaker. The M1 seeds were rinsed 15 times with 300 mL of water and then grown directly on soil in 240 pots, each containing 10 plants, to propagate to the M2 generation. Each pot represents a batch of M2 seeds. On average, around 20 seeds per M2 plant (200 seeds per batch) were screened for seedlings with a suppressed LRX1ΔE14 root hair phenotype. Selected putative mutant M2 seedlings were propagated and confirmed in the M3 generation. Positive lines were crossed with the non-mutagenized parental LRX1ΔE14 line and propagated to the F2 generation that was analysed for segregation of the sune mutant phenotype. Whole genome sequencing, CAPS marker design Whole-genome sequencing was performed as described in ( Guérin et al., 2025 ). Ten F2 seedlings from the first backcross of LRX1ΔE14 sune42 with the parental LRX1ΔE14 line exhibiting a LRX1ΔE14 sune42 phenotype were selected and pooled for DNA extraction. Whole-genome sequencing of the DNA of LRX1ΔE14 sune42 , along with the non-mutagenized LRX1ΔE14 line, was performed at Novogene using Illumina short-read technology. Raw sequence reads from the pooled LRX1ΔE14 sune42 mutants were trimmed with the Trimmomatic (version 0.38) with the parameters LEADING:10, TRAILING:10 SLIDINGWINDOW:5:10, MINLEN:50. Trimmed sequence reads were mapped with the bwa software (version 0.7.17-r118) to the Arabidopsis Columbia reference genome (TAIR version 10) using default parameters. Resulting BAM files were sorted and duplicates removed with samtools (version 1.9). New read groups were assigned to the reads with the Picard software (version:2.27.5). Sequence variants were called with the GATK software (version 4.2). The vcftools software (version 0.1.16) was used to filter the vcf files using the parameters (--max- meanDP 7 –remove-indels). The analysis revealed three SNPs (single nucleotide polymorphisms) on chromosome 1 linked with the sune42 mutation. Using this information, CAPS (cleaved amplified polymorphic sequences) markers were established, and co-segregation of the SNPs with the LRX1ΔE14 sune42 phenotype was analysed. For sequencing of the LRX1ΔE14 construct in the identified sune mutants, the construct was PCR-amplified using LRX1_F1 and LRX1_TermR primers ( Table 1 ), targeting the promoter and terminator of LRX1, respectively. Due to the repetitive nature and length of the extensin coding sequence ( Herger et al., 2019 ), the endogenous LRX1 was not amplified. For selection of sune42 plants lacking LRX1ΔE14 , a segregating F2 population of a backcross of LRX1ΔE14 sune42 x Col was selected by PCR with the primers LRX1_F1 and LRX1_TermR that detect LRX1ΔE14 ( Table 1 ). Plant transformation and selection Arabidopsis transgenic lines were obtained by standard floral dipping method mediated by Agrobacterium tumefaciens (strain GV3101). For the complementation lines, T1 seeds of plants transformed with pCCX4::CCX4 in pGPTV-Bar were selected on Basta selection media. The CRISPR/Cas9 vector pKI1.1R ( Tsutsui and Higashiyama, 2017 ) carries a FASTRED seed selection marker allowing selection based on RFP fluorescence. DNA from the cauline leaves of the inflorescences of T1 plants were extracted for genotyping using the primers CCX4_F2/R2 for ccx4 ( Table 1 ) and primers NPR1_F/R for npr1 . To detect the sune42 mutation, the PCR product of sune42cat_F and sune42cat_R was digested with SacI, which only cuts the wild type. The ccx4 crispr allele was detected using the primers CCX4_F2 and CCX4_R2 , and the product of only the wild type is cut by NcoI . The lrx1 mutation was detected by PCR using the primers LRX1_F2 and LRX1_R2 , followed by a digestion with EagI , which only cuts the mutant. The npr1 crispr allele was detected by sequencing the PCR product obtained with the primers NPR1_CC2_seq_F and NPR1_CC2_seq_R . Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) Phytohormone analysis was performed as described by Schmidt et al. (2024). Briefly, approximately 20 mg Fresh Weight of plant tissue was homogenized with 1.5 mm zirconium oxide beads using a FastPrep-24 instrument (MP Biomedicals) with 100 µL of 1 M HCOOH in water and a mixture of internal standards. After centrifugation at 17500 rpm for 20 minutes and re-extraction with additional 100 µL of 1 M HCOOH, the combined supernatant was applied to an SPE Oasis HLB 10 mg 96-well plate (Waters) pre-equilibrated with 100 µL of acetonitrile followed by the same volume of water and 1 M HCOOH. The supernatant was pushed through the SPE plate using a Pressure+96 manifold (Biotage). The 96-well SPE plate was washed three times with 100 µl of water. Samples were eluted with 100 µL 50% of acetonitrile/water (v/v). An aliquot of the eluate was injected into the LCMS system consisting of a UHPLC 1290 Infinity II (Agilent) coupled to a 6495 Triple Quadrupole mass spectrometer (Agilent). MS analysis was performed in MRM mode, two transitions per compound, using an isotope dilution method. Data acquisition and processing were performed using Mass Hunter software B.08 (Agilent). RNA extraction and qRT-PCR mRNA extraction was done with the dynabeads mRNA DIRECT Kit (Invitrogen) according to the manufacturer’s protocol, with 25 µL of oligo(dT)25 per extraction. Roots of 10-day-old seedlings were frozen and ground in liquid nitrogen immediately upon collection. Around 20 mg of root tissue was used for each sample and 4 biological replicates of each treatment were included. SA-responsive gene expression levels were assessed by qRT-PCR, with a CFX96 Real-Time System C1000TM Thermal cycler (Bio-Rad). Around 40 ng of mRNA was reverse transcribed using the iScript advanced kit (BioRad). qRT-PCR primers target WRKY53 (WRKY53_F/R) and WRKY70 (WRKY70_F/R) and the reference gene ACTIN2 (ACTIN_F/R) are listed in Table 1 . Four μL of 20-fold diluted cDNA was used for a total volume of 10 μL qRT-PCR reaction using KAPA SYBR FAST qPCR Master Mix (KK4601, Sigma–Aldrich) and 250 µM of each primer. Three technical replicates per sample were included. Root growth assay Surface-sterilized seeds were grown on ½ MS agar plates supplemented with 2% sucrose, containing the chemical or corresponding solvent as control. For ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA) treatment, 1 mM EGTA was used, whereas for the salicylic acid (SA) treatment, 10 μM SA in EtOH was used and EtOH was added to the media of the control group. Root hair phenotypes were visualized after 5 days of growth. The plates were scanned and root (hair) length was measured by Fiji imageJ ( Schindelin et al., 2012 ). Microscopy and quantitative imaging analysis Experiments were conducted over a period of two weeks using two different sets of samples prepared according to the protocol described in (Romano Armada et al., 2019 ). The fluorescence light sheet setup implemented is a modified version of the single-sided illumination setup described in ( Candeo et al., 2017 ). In the current setup, a laser at 561 nm is used as excitation source for the creation of the light sheet illumination. The detection unit, held orthogonally to the excitation axis, has been optimised to perform imaging of samples encoding the intensiometric calcium indicator R-GECO1 and includes a 10X water-dipping microscope objective (NA=0.3, UMPLFLN 10X W, Olympus) directly followed by a long pass filter at 600 nm (FELH0600 - Ø25.0 mm Longpass Filter, Cut-On Wavelength: 600 nm, Thorlabs), a tube lens (U-TLU-1-2, Olympus) and the camera (Neo 5.5 sCMOS, 2560×2160 pixels, ANDOR). Experiments were performed according to the protocol described in ( Candeo et al., 2017 ). For each sample, 270 time points were recorded with a sampling time of 3 seconds acquiring 15 planes spaced 3 µm apart for each time point with an exposure time of 100 ms. Maximum Intensity Projection (MIP) of the 15 slices was then computed for each time point stack obtaining a time-lapse dataset of 270 frames. In order to follow the calcium dynamics over time in the elongating root hairs tips, registration of images was needed. The registration process was performed using the “napari-roi-registration plugin” ( https://github.com/GiorgiaTortora/napari-roi-registration ), developed for the multidimensional image viewer napari and based on the openCV python library, which allows to identify and follow an unlimited number of Regions of Interest (ROIs) simultaneously. In one frame of the 270-images stack, ROIs have been manually selected in correspondence of the root hair tips of interest and have been then registered automatically by the plugin. After registration, information related to the intensity and displacement of ROIs were extracted and saved using another feature of the napari-roi-registration plugin. Calcium variations over time were calculated from the raw intensities as the normalized R-GECO1 fluorescence intensity ΔF/F 0 at each time point with the baseline F 0 determined as the mean fluorescence value over time. Starting from the extracted normalized intensities, Fourier analysis was performed to determine the main oscillation frequencies in the detected calcium signals ( Candeo et al., 2017 ). For each intensity signal, the fast Fourier transform was computed using a Python built-in function, in order to move from the time domain to the frequency domain and identify the characteristic oscillation frequency for each root hair. Fourier transform is a complex function composed of a real and an imaginary part, therefore, to identify the main oscillation frequencies, the power spectrum was extracted from the Fourier transform as: Where N is the number of time points and G ( f k ) is the Fourier transform defined in a discrete way as: Finally, for each line group, the Power Spectral Density was computed as the mean of all the power spectra in that group and the error was calculated as the standard deviation. Kymograph images with median filter radius=1.0 were generated from MIP time-lapses (x, y, t) using the Kymograph Builder plugin by Fiji, to provide a space–time representation of selected pixels. Pixels were selected by tracing a line along the root hair, and a new image was generated, displaying the temporal variation in the intensities of the pixels beneath the selected line. Each column of pixels in the kymograph represents a single time point of the time-lapse dataset, while the vertical axis shows the intensities of the pixels along the line. Results sune42 suppresses the dominant-negative effect mutant LRX1ΔE14 Expressing the truncated LRX1 lacking the extensin domain ( LRX1ΔE14 ) in wild-type Col induces aberrant root hairs that are misshaped, bulge, or burst ( Figure 1A ). LRX1ΔE14 seedlings have very few root hairs that are elongated and their average length is shorter than Col ( Figure 1B,C ). This suggests that LRX1ΔE14 exerts a dominant-negative effect, likely through interfering with the process involving the endogenous LRX1. Hence, the LRX1ΔE14 line is a genetic tool to investigate the physiological processes that are involved in the LRX1-related network. An ethyl methanesulfonate (EMS) mutagenesis was performed on the LRX1ΔE14 line to screen for suppressors of the dominant-negative effect of LRX1ΔE14 ( Guérin et al., 2025 ). EMS-mutagenized LRX1ΔE14 seeds were propagated to the M2 generation and M2 plants with reconstituted root hairs were selected for further analyses. Among those, LRX1ΔE14 sune42 ( su ppressor of dominant- ne gative effect 42 ) displays a wild type-like root hair phenotype ( Figure 1A ). A more detailed quantification revealed that the root hair phenotype of LRX1ΔE14 is largely but not completely suppressed by sune42 , with root hairs that are slightly shorter and more misshaped compared to wild-type Col ( Figure 1B,C ). To exclude intragenic suppression of LRX1ΔE14 , the construct LRX1:LRX1ΔE14 was sequenced in LRX1ΔE14 sune42 and revealed to be unaltered. Download figure Open in new tab Figure 1. sune42 suppresses the root hair defect of LRX1ΔE14. (A) Representative images of roots of 5-day-old Arabidopsis seedlings of the different lines. Seeds were germinated and vertically grown on ½ MS plates. Root hairs of LRX1ΔE14 are severely defective compared to wild-type Col and restored by the sune42 mutation, as well as the ccx4 crispr mutation representing the CRISPR/Cas9 -induced second allele of ccx4. The sune42 single mutant shows a wild type-like root hair phenotype. LRX1ΔE14 sune42 x LRX1ΔE14 is the F1 of a backcross confirming sune42 being recessive and LRX1ΔE14 sune42; pCCX4::CCX4 demonstrates complementation of sune42 by the wild type copy of CCX4 , inducing an LRX1ΔE14 -like phenotype. Scale bar = 0.5 mm. (B) Violin plot shows the average length of elongated root hairs. Significant differences are indicated by letters (n>150 per genotype, one-way ANOVA with Tukey’s unequal N-HSD post hoc test, P 150 per genotype). A backcross of LRX1ΔE14 sune42 with the LRX1ΔE14 parental line revealed that sune42 is a recessive mutation, as all the F1 seedlings exhibited a defective root hair phenotype resembling LRX1ΔE14 ( Figure 1A ). Of a segregating F2 population, seedlings displaying a wild type-like LRX1ΔE14 sune42 mutant phenotype were selected, pooled, and used for whole-genome sequencing (details see Materials and Methods). In comparison with LRX1ΔE14 , which was previously sequenced ( Guérin et al., 2025 ) , LRX1ΔE14 sune42 revealed two non-silent single nucleotide polymorphisms (SNPs) in two nearby genes: in the gene coding for the putative cation/Ca Ca 2+ exchanger CCX4 ( At1g54115 ) resulting in Gly187Glu ( Suppl. Figure S1 ) and in the so far uncharacterized gene At1g55750 (Ser502Leu). CAPS markers were established for the two mutations and tested on sune42 -like seedlings of the segregating F2 population mentioned above. Among 82 F2 plants tested, only the SNP in ccx4 showed complete linkage, suggesting that the ccx4 mutation represents sune42 . The LRX1ΔE14 sune42 line was transformed with a wild-type pCCX4::CCX4 construct, and several independent transgenic plants developed seedlings displaying LRX1ΔE14 -like root hair development ( Figure 1 ). Additionally, CRISPR/Cas9 -mediated mutagenesis produced an additional ccx4 crispr allele ( Suppl. Figure S1 ) that also alleviates the LRX1ΔE14 root hair defect ( Figure 1 ), further confirming that CCX4 represents the sune42 locus. A sune42 single mutant was obtained by crossing LRX1ΔE14 sune42 with wild-type Col and selecting for sune42 and the absence of LRX1ΔE14 in the segregating F2 generation. As shown in Figure 1 , the sune42 single mutant develops wild type-like root hairs, suggesting that sune42 does not affect the root hair phenotype in the absence of LRX1ΔE14. The visual phenotype was confirmed to be equal to the wild type by quantifying root hair defects ( Figure 1B,C ). sune42 partially suppresses the root hair defect in lrx1 and fer-5 The dominant-negative effect induced by LRX1ΔE14 is likely due to its interference with the endogenous LRX1-RALF-FER network ( Herger et al., 2020 ). Therefore, we examined the impact of the sune42 mutation on LRX1 and FER functions in lrx1 sune42 and fer-5 sune42 double mutants. We observed partial alleviation of the typical lrx1 and fer- 5 root hair defects, with more elongated root hairs in lrx1 sune42 and fer-5 sune42 compared to lrx1 and fer-5 , respectively. Deformed root hairs, however, were still present in lrx1 sune42 and fer-5 sune42 ( Figure 2 ). This suggests that sune42 partially suppresses the lrx1 and fer-5 phenotypes. Download figure Open in new tab Figure 2 sune42 partially suppresses fer-5 and lrx1 root hair phenotypes. (A) Representative images of 5-day-old Arabidopsis seedlings roots of Col, fer-5 , fer-5 sune42 , lrx1 , and lrx1 sune42. All seedlings were grown vertically on ½ MS plates. fer-5 and lrx1 show aberrant root hair phenotypes. For both mutants, sune42 results in slightly more/longer root hairs. Scale bar = 0.5 mm. (B) Quantification of the length of elongated root hairs is shown with the violin plot. Letters represent significant differences (n>150 per genotype, one-way ANOVA with Tukey’s unequal N-HSD post hoc test, P 150 per genotype). sune42 alters salicylic acid signaling It was previously reported that the loss of CCX4 function leads to increased salicylic acid (SA) accumulation and expression of SA-responsive genes ( Fujikura et al., 2020 ). Also, the LRX-FER module has been shown to modulate SA levels in the shoot ( Zhao et al., 2021 ). With SA being important for root development and growth ( Pasternak et al., 2019 ; Tan et al., 2020 ; Zhou et al., 2022 ), the involvement of SA signaling in the effect of sune42 on root hair formation was investigated. First, SA levels were determined in Col, LRX1ΔE14, and LRX1ΔE14 sune42 seedlings. Compared to LRX1ΔE14 , the sune42 mutation does not have a significant effect on SA levels in the root tissue but increases the SA levels in the shoot compared to LRX1ΔE14 , in line with other findings ( Fujikura et al., 2020 ) ( Figure 3 ). Download figure Open in new tab Figure 3 sune42 causes increased shoot salicylic acid (SA) levels in LRX1ΔE14 . Quantification of SA content in root (A) and shoot (B) tissues of 10-day-old Arabidopsis seedlings. The box plots represent the median (central line), 25th and 75th percentiles (limits), minimum/maximum values (whiskers), letters represent significance levels of statistical analyses (one-way ANOVA with Tukey’s unequal N-HSD post hoc test, P < 0.01, n=4). Plots showing primary data from one individual measurement. Long-distance transport through the vascular network has been extensively studied for SA ( Anfang and Shani, 2021 ), and it is possible that sune42 causes accumulation of SA in the shoot that is transported to the root, where it promotes SA signaling and thereby influences root hair development. To explore if the root SA signaling is affected by the shoot SA levels, we measured SA-responsive gene expression in root tissue. qRT-PCR on mRNA extracted from seedling roots shows that the SA-responsive genes WRKY70 and WRKY53 ( Yu et al., 2001 ; Li et al., 2004 ) are both downregulated in LRX1ΔE14 in comparison with the wild type Col, and sune42 rescues the gene expression level ( Figure 4A ), suggesting a possible correlation between the root hair phenotype and SA signaling. Download figure Open in new tab Figure 4 sune42 suppresses LRX1ΔE14 root hair phenotype independent of SA-induced NPR1 signaling. (A) qRT-PCR analyses showing transcript levels of SA responsive genes WRKY53 and WRKY71 in Col, LRX1ΔE14, LRX1ΔE14 sune42, LRX1ΔE14 sune42 npr1, and npr1 lines. mRNA was extracted from 10-day-old Arabidopsis seedlings and reverse transcribed to cDNA for analyses. ACTIN2 was used as an internal control. All values are normalized to ACTIN2 transcript levels. Values are means of four biological replicates ± SD, letters indicate significance levels of statistical analyses (one-way ANOVA with Tukey’s unequal N-HSD post hoc test, P < 0.01) (B) Sequencing chromatogram showing the mutation of CRISPR/Cas9 -mediated mutagenesis 603 bp downstream of the start codon. The insertion of one base pair at the codon Thr202 induces a frame shift and a stop codon after 8 amino acids. (C) Roots of 5-day-old vertically grown Arabidopsis seedlings. The root hair formation phenotype of LRX1ΔE14 is suppressed by LRX1ΔE14 sune42 and not further modified by LRX1ΔE14 sune42 npr1 crispr . Scale bar=0.5 mm. sune42 does not modulate LRX1ΔE14 root hair defect through SA-dependent NPR1 signaling To investigate the biological relevance of SA signaling in the LRX1ΔE14- induced root hair defect, we first tested the effect of exogenous SA on the root hair development in Col and LRX1ΔE14 . As shown in Suppl. Figure S2 , the presence of 10 μM SA did not cause any observable changes in Col and LRX1ΔE14 in terms of root hair development compared to mock treatment. A mutation in ccx4 has been shown to cause an SA-induced growth alteration that depends on the SA signaling component NPR1 ( Wu et al., 2012 ; Fujikura et al., 2020 ). To test whether the sune42 -induced effect on root hair formation is dependent on NPR1, a CRISPR/Cas9 -mediated frame shift mutation in NPR1 was induced in wild-type Col and LRX1ΔE14 sune42 backgrounds, causing a single-nucleotide insertion 603 bp downstream of the start codon ( Figure 4B ). qRT-PCR shows that the expression of SA-responsive genes, WRKY53 and WRKY70, is decreased in the NPR1 loss-of function mutant background npr1 crispr , in the context of the single mutant compared to Col as well as in the LRX1ΔE14 sune42 npr1 crispr compared to LRX1ΔE14 sune42 ( Figure 4A ). However, the root hair phenotype of LRX1ΔE14 sune42 remains unaffected in the LRX1ΔE14 sune42 npr1 crispr line ( Figure 4C ), implying that the sune42 -mediated suppression of LRX1ΔE14 is independent of the SA-dependent NPR1 signaling. Ca 2+ deficiency promotes the root hair emergence in LRX1ΔE14 CCX proteins have been implicated in Ca 2+ transport activities ( Corso et al., 2018 ; Kanamori et al., 2023 ). On the other hand, mutations in LRX genes have been shown to be affected in Ca 2+ homeostasis ( Fabrice et al., 2018 ). This led us to hypothesize that the dominant-negative effect in LRX1ΔE14 might be linked to impaired intracellular Ca 2+ homeostasis, which would then be rescued by mutations in CCX4 . To test that, we first investigated the effect of modified Ca 2+ availability on the LRX1ΔE14 root hair phenotype. Col and LRX1ΔE14 seedlings were grown in the presence of the calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA). Root hair elongation was similarly inhibited in both Col and LRX1ΔE14 ; yet, LRX1ΔE14 seedlings showed evidently more root hair emergence in the presence of 1 mM EGTA ( Figure 5 ), which causes a reduction of free Ca 2+ from 1.5 to 0.6 mM ( https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-NIST.htm ), indicating that limited Ca 2+ availability rescues the root hair formation defect in LRX1ΔE14 to a level that is close to the wild type. Download figure Open in new tab Figure 5 EGTA treatment leads to increased LRX1ΔE14 root hair emergence. (A) Representative images of 5-day-old Col and LRX1ΔE14 roots vertically grown on ½ MS plates with or without 1 mM EGTA. Scale bar = 500 μm. (B) Quantification of root hair density where only emerging root hair structures were counted (t-test, *:p<0.5, ***:p80 root hairs of four different seedlings). (C) Quantification of root hair length (t-test, ***:p80 root hairs of four different seedlings). sune42 stabilizes tip-focused Ca 2+ oscillation in LRX1ΔE14 The EGTA assay implies that the dominant-negative phenotype of LRX1ΔE14 is affected by the Ca 2+ availability. To further understand how root hair development is affected by Ca 2+ , we examined the root hair tip-focused cytosolic Ca 2+ dynamics by comparing Ca 2+ oscillations in lines expressing the calcium indicator R-GECO1 driven by the ubiquitin10 promoter ( Keinath et al., 2015 ) by time-lapse single-cell Light Sheet Fluorescence Microscopy (LSFM) imaging. In this experimental set-up, the root hair phenotypes remained with the LRX1ΔE14 showing impaired root hair formation, and alleviation of the root hair formation defect by sune42 ( Suppl. Figure S3 ). Kymographs were used to visualize temporal variations in the fluorescence signal. They provide a tool for a space-time representation from a time-lapse image sequence. In agreement with published data using the Cameleon YC3.6 Ca 2+ indicator ( Monshausen et al., 2008 ; Candeo et al., 2017 ), the tip-focused cytosolic Ca 2+ of Col root hairs oscillates with a period which ranges from 18 up to 33 s ( Figure 6A ). Intriguingly, time-lapse experiments revealed altered Ca 2+ oscillations in the root hairs of LRX1ΔE14 ( Figure 6A , Suppl. Video S1). While in root hairs of wild-type Col plants, a predominantly fast periodic oscillation is present, in LRX1ΔE14 specimens a slow oscillation seems to be superimposed to the fast one. This effect is not evidently observable in sune42 and LRX1ΔE14 sune42. To further compare the behaviour of the different populations, Fourier analysis was performed. The distribution of oscillation frequencies revealed a dominant frequency band extending from 0.057 to 0.03 Hz (from 18 to 33 s) in Col, while in LRX1ΔE14 two distinct frequency bands were identified. In particular, a high frequency band similar to the main one of Col but slightly shifted towards lower values (from 0.050 to 0.025 Hz) is found together with a secondary drastically slower frequency band extending from 0.016 to 0.006 Hz (from 1 to 2.8 min) ( Figure 6B ), suggesting that LRX1ΔE14 displays a combination of low and high Ca 2+ oscillation frequencies ( Figure 6B ). By contrast, the line LRX1ΔE14 sune42 shows a wild type-like Ca 2+ oscillation pattern with a dominant frequency band extending from 0.055 to 0.025 Hz that overlaps with the frequency distribution of Col and a significant attenuation of the lower frequency band which also appears to be shifted to higher values ( Figure 6B ). Interestingly, the sune42 single mutant shows only minimal alteration in Ca 2+ oscillations compared to Col ( Figure 6A,B ). Taken together, our findings provide a correlation between root hair development and tip-focused Ca 2+ oscillation that are both altered in LRX1ΔE14 but reconstituted in LRX1ΔE14 sune42 . Download figure Open in new tab Figure 6 The sune42 mutation alleviates the impaired tip-focused calcium oscillation in the root hairs of LRX1ΔE14 . (A) Kymographs (left) and plotted data (right) of Ca 2+ dynamics over time in growing root hairs of R-GECO1 -expressing Arabidopsis seedlings of the different lines. Each line in the kymograph corresponds to a single time point in the time-lapse, while the vertical axis represents the intensity values. The root transition zone was used as normalized R-GECO1 fluorescence intensity (ΔF/F 0 ). The sampling time was 3 seconds, and the measure was repeated 270 times. (B) Mean power spectral density obtained with Fourier transform spectral analysis. Dots of mean magnitude are connected to curves. Error bars are mean ± SD. (n=105 WT, 79 LRX1ΔE14, 67 LRX1ΔE14 sune42, and 75 sune42 root hairs from more than 10 independent 5-day-old seedlings per genotype). Discussion Mutations in ccx4 cause a suppression of the dominant-negative effect of LRX1ΔE14 on root hair development. CCX4 was demonstrated to be involved in Ca 2+ transport ( Kanamori et al., 2023 ), and CCX2 revealed to be important for Ca 2+ homeostasis ( Corso et al., 2018 ). CCX4 contains two highly conserved Ca 2+ - and Na + -binding motifs, GNG(A/S)PD in α-1 and (G/S)(N/D)SxGD in α-2 ( Suppl. Figure S1B ), that were identified in the Na + /Ca 2+ exchanger HsNCKX6 ( Cai and Lytton, 2004 ), suggesting the potential role of CCX4 in Ca 2+ transport. sune42 possesses a missense mutation in the α-1 repeat of CCX4 , resulting in Gly187Arg. Together, this points at Ca 2+ fluxes being part of the LRX1ΔE14-mediated defect in root hair development, which is supported by the distorted cytosolic Ca 2+ oscillations in root hairs of the LRX1ΔE14 line compared to the wild type. Ca 2+ is a key modulator in controlling the polarity and the expansion rate of tip-growing cells ( Monshausen et al., 2008 ; Candeo et al., 2017 ). Indeed, several cyclic nucleotide-gated channels (CNGCs) have been demonstrated to coordinate Ca 2+ oscillations and root hair growth ( Zhang et al., 2017 ; Brost et al., 2019 ; Zhu et al., 2025 ). Since ccx4 alleviates both the modified cytosolic Ca 2+ oscillations ( Figure 6 ) and root hair growth defect in the LRX1ΔE14 line ( Figure 1 ), the correlation is established, but it does not prove that LRX1ΔE14 induces the altered Ca 2+ dynamics. It is worth noting that mutations in pollen-expressed LRX8,9,10,11 induce aberrant Ca 2+ accumulation and limiting Ca 2+ availability alleviates the growth defects in these mutants ( Fabrice et al., 2018 ), suggesting a link between LRX-related processes and Ca 2+ dynamics. The positive effect of the Ca 2+ chelator EGTA on root hair formation ( Figure 5 ) suggests that, also here, limiting Ca 2+ availability helps overcoming the root hair defect by altered LRX activity. The activity of the Golgi-localized CCX4 tempers changes in Ca 2+ dynamics ( Kanamori et al., 2023 ) and appears to be involved in changing Ca 2+ fluxes, comparable to the function of other members of the CCX family ( Corso et al., 2018 ). This underlines the importance of organellar Ca 2+ pools for regulating cytoplasmic Ca 2+ homeostasis. In plants, a large amount of Ca 2+ is bound in the cell wall via ionic interactions with galacturonic acid that is found in de-methylated pectin and Arabinogalactan proteins ( Willats et al., 2001 ; Lopez-Hernandez et al., 2020 ). De-methylated pectin is bound by the LRX/RALF complex, which results in pectin compaction ( Moussu et al., 2023 ; Schoenaers et al., 2024 ). It is conceivable that altering LRX/RALF/pectin interacting by modifying LRX availability has an impact on pectin dynamics and thus Ca 2+ homeostasis, similar to the ability of RALF peptides to alter cytoplasmic Ca2+ concentrations ( Haruta et al., 2008 ). A more detailed analysis of the impact of LRXs on pectin dynamics will possibly provide clues on the observation that lrx mutants ( Fabrice et al., 2018 ) and LRX1ΔE14 impact Ca 2+ dynamics. The xs2 mutant is an allele of ccx4 and develops a defect in cell expansion due to the overaccumulation of SA signaling ( Fujikura et al., 2020 ). This point needed to be further investigated, since ( Zhao et al., 2021 ) showed that the salicylic acid (SA) and jasmonic acid (JA) pathways are constitutively upregulated in the lrx3 lrx4 lrx5 mutant, providing a potential link between LRX activity and SA signaling. Our data show that SA-responsive genes are downregulated in LRX1ΔE14 but upregulated again by the sune42 mutation, as is the SA level in the shoot ( Figure 3 and Figure 4 ), confirming the influence of SA signaling by ccx4 . While a mutation in NPR1 ( Wu et al., 2012 ), the key responsive regulator of SA signaling, rescues the cell expansion defect in xs2 shoots ( Fujikura et al., 2020 ), our results show that a mutation in NPR1 does not influence the root hair phenotype of LRX1ΔE14 sune42 ( Figure 4 ). This suggests that the effect of sune42 on the LRX1ΔE14 -mediated root hair defect is independent of the altered SA signaling. The effect of the ccx4 mutations is likely to be multifaceted. The Golgi apparatus is not only an organelle with high Ca 2+ concentration that helps regulating cytoplasmic [Ca 2+ ] ( Ordenes et al., 2012 ), but it is also central to posttranslational protein modifications and the synthesis of numerous polysaccharides destined for the cell wall ( Cosgrove, 2024 ). Glycosylation is the major intracellular posttranslational modification of cell wall proteins ( Nguema-Ona et al., 2014 ). In mammalian cells, these processes are influenced by Ca 2+ , which modifies vesicle transport dynamics ( Gerdes et al., 1989 ) and many glycosyltransferases and glycosidases involved in glycosylation ( Bai et al., 2006 ). Altering Golgi-localized processes can thus lead to changes in the cell wall. rol16 ( repressor of lrx1_16 ) is affected in the Golgi-localized Apyrase 7 ( Gupta et al., 2024 ), confirming that the effect of modified LRX1 function can be influenced by processes emanating from the Golgi. LRX proteins, in conjunction with the plasma membrane-localized receptor kinase FER, influence vacuole development and cell growth, resulting in comparable mutant phenotypes of fer and higher-order lrx mutants ( Dünser et al., 2019 ; Herger et al., 2020 ). While sune42 has a strongly alleviating effect on LRX1ΔE14 ( Figure 1 ), its impact on the lrx1 and fer-5 mutant phenotypes is limited ( Figure 2 ). This suggests that the LRX1ΔE14 dominant-negative and the lrx1 loss-of-function mutant have different causes. Interestingly, the truncated, extensin-less, variant of the root/shoot-expressed LRX4 ( Draeger et al., 2015 ), LRX4ΔE , also induces a dominant-negative phenotype, but at the same time renders the plant hypersensitive to RALF peptides ( Dünser et al., 2019 ). These plant peptide hormones are bound by LRX proteins ( Mecchia et al., 2017 ; Moussu et al., 2020 ), impact pectin compaction, and inhibit root growth ( Abarca et al., 2021 ; Moussu et al., 2023 ; Schoenaers et al., 2024 ). This suggests that extensin-less LRXs, LRX4ΔE and LRX1ΔE14 , are present but not anchored in the cell wall, changing their spatial distribution and making them available for interaction partners at incorrect locations. Thus, while the LRX1ΔE14 -induced root hair phenotype is comparable to a mutant lacking root hair-expressed LRXs ( Baumberger et al., 2003 ), LRX1ΔE14 actively interferes with the LRX1-related process in root hairs. The first identified sune mutant, sune82 , is affected in His biosynthesis and impacts the TOR (Target of Rapamycin) network ( Shi et al., 2018 ; Guérin et al., 2025 ). In contrast to sune42 characterized here, sune82 also suppresses lrx1 ( Guérin et al., 2025 ). Thus, it is important to use several approaches to identify suppressor mutants with different targets in the processes modified by LRX1. Depending on where in the process they take influence, these suppress only one or both types of distorted LRX1 activity. This will help us better understand the plethora of cellular events involving LRXs. Competing interests The authors declare no competing interest. Author contributions XH: CRISPR/Cas9 mutagenesis, CCX4 complementation tests, salicylic acid assays, Ca 2+ deficiency assays GT: Ca 2+ oscillation measurements AH: EMS mutagenesis, sune mutant screen SB: Ca 2+ oscillation measurements PD: Hormonal content analysis RV: Hormonal content analysis JL: Hormonal content analysis AS: whole-genome sequencing GK: genetic analysis MS: sune mutant analysis AC: Ca 2+ measurements TW: whole-genome sequencing, supervising AC: Ca 2+ measurements, supervising AB: Ca 2+ measurements, supervising CR: conceptualizing research, supervising, funding All authors contributed to the writing process Data availability All data are shown. Raw data will be made available upon request. Supporting information Supplemental Figures 1 - 3 are part of this manuscript and shown below. Supplemental Video S1 Video of Ca 2+ oscillations, monitored by R-GECO1 fluorescence in the four genotypes wild type (Col), LRX1ΔE14 , LRX1ΔE14 sune42 , and sune42 . These are accessible as a separate file accompanying this manuscript. Download figure Open in new tab Supplemental Figure S1. Scheme of CCX4 genomic sequences and amino acid sequence alignment. (A) Schematic diagram of the intronless CCX4 ( At1g54115 ) genomic sequence of 1935 bp, showing the missense mutation site of the EMS allele sune42 (G to A mutation at position 560 from the start codon resulting in a Gly187Glu substitution) and the CRISPR/Cas9 -guided insertion of one A at position 1115 relative to the start codon that leads to a shift in the ORF after Pro372 and an early stop codon after 48 base pairs. (B) Sequencing chromatograms of the wild-type, sune42 , and the CRISPR/Cas9 -induced ccx4 allele. Arrows indicate altered positions, brownish colorings show altered sequences. (C) Amino acid sequence alignment of CCX4 with its homologs from Arabidopsis AtCCX1, AtCCX2, AtCCX3 and its ortholog from Human HsNCKX6. Blue boxes indicate the conserved α repeats. The alignment was done with Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) ( Sievers et al., 2011 ). Identical, conserved, and similar positions in the alignment are indicated by asterisks, colons, or single dots, respectively. Download figure Open in new tab Supplemental Figure S2. Exogenous SA does not affect the root hair development. Col and LRX1ΔE14 Arabidopsis seedlings were grown on ½ MS plates with or without 10 μM SA for 5 days in a vertical orientation. Representative images of mock (left) and 10 μM SA (right) of each genotype are shown in pairs. Both Col and LRX1ΔE14 do not show altered root hair phenotype upon 10 μM SA treatment. Similar results were observed among three independent experiments. Download figure Open in new tab Supplemental Figure S3. Difference in root hair formation between genotypes are maintained under LSF microscopy. For Light Sheet Fluorescence (LSF) microscopy analysis, seedlings needed to be growth into the agar. Under these conditions, the LRX1ΔE14 line still showed strongly impaired root hair formation compared to the wild type, and this effect is suppressed by sune42 . The pie-charts depict root hair length distributions in the different lines (n>66). Root hairs longer than 21 μm (purple and pale green sectors) in the wild type or in the presence of sune42 were not detected in LRX1ΔE14 . Acknowledgements We thank the infrastructure team at IPMB was continuous support with growth facilities. This work was supported by the University of Zurich and the Swiss National Science Foundation grants Nr 31003A_166577/1 and 310030_192495 to CR; the Ministero dell’Istruzione, dell’Università e della Ricerca — Fondo per Progetti di Ricerca di Rilevante Interesse Nazionale 2022 to A.C. and S.B; the Agritech National Research Center, funded by the European Union NextGenerationEU (Piano Nazionale di Ripresa e Resilienza (PNRR) — Missione 4, Componente 2, Investimento 1.4 – D.D. 1032 17/06/2022, CN00000022) to A.C.; and the Fondazione Fratelli Confalonieri to S.B.. Funder Information Declared Swiss National Science Foundation, https://ror.org/00yjd3n13 , 31003A_166577/1 , 310030_192495 Ministero dell’Istruzione, dell’Università e della Ricerca – Fondo per Progetti di Ricerca di Rilevante Interesse Nazionale 2022 European Union NextGenerationEU (Piano Nazionale di Ripresa e Resilienza (PNRR) , 1032 17/06/2022, CN00000022 Fondazione Fratelli Confalonieri References 1. ↵ Abarca , A. , Franck , C.M. , and Zipfel , C . ( 2021 ). Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides . Plant Physiol . 187 , 996 – 1010 . OpenUrl CrossRef PubMed 2. ↵ Anfang , M. , and Shani , E . ( 2021 ). Transport mechanisms of plant hormones . Curr. Opin. Plant Biol . 63 , 102055 . OpenUrl CrossRef PubMed 3. ↵ Bai , C. , Xu , X.L. , Chan , F.Y. , Lee , R.T. , and Wang , Y . ( 2006 ). MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans . Eukaryotic cell 5 , 238 – 247 . OpenUrl Abstract / FREE Full Text 4. ↵ Baumberger , N. , Ringli , C. , and Keller , B . ( 2001 ). The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana . Genes Dev . 15 , 1128 – 1139 . OpenUrl Abstract / FREE Full Text 5. ↵ Baumberger , N. , Steiner , M. , Ryser , U. , Keller , B. , and Ringli , C . ( 2003 ). Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development . Plant J . 35 , 71 – 81 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Baumberger , N. , Doesseger , B. , Guyot , R. , Diet , A. , Parsons , R.L. , Clark , M.A. , Simmons , M.P. , Bedinger , P. , Goff , S.A. , Ringli , C. , and Keller , B . ( 2003a ). Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice: a conserved family of cell wall proteins form a vegetative and a reproductive clade . Plant Physiol . 131 , 1313 – 1326 . OpenUrl Abstract / FREE Full Text 7. ↵ Becker , D. , Kemper , E. , Schell , J. , and Masterson , R . ( 1992 ). New plant binary vectors with selectable markers located proximal to the left T-DNA border . Plant Mol. Biol . 20 , 1195 – 1197 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Borassi , C. , Sede , A.R. , Mecchia , M.A. , Salgado Salter , J.D. , Marzol , E. , Muschietti , J.P. , and Estevez , J.M . ( 2016 ). An update on cell surface proteins containing extensin-motifs . J. Exp. Bot . 67 , 477 – 487 . OpenUrl CrossRef PubMed 9. ↵ Bose , J. , Pottosin , II , Shabala , S.S. , Palmgren , M.G. , and Shabala , S . ( 2011 ). Calcium efflux systems in stress signaling and adaptation in plants . Front. Plant Sci . 2 , 85 . OpenUrl PubMed 10. ↵ Brost , C. , Studtrucker , T. , Reimann , R. , Denninger , P. , Czekalla , J. , Krebs , M. , Fabry , B. , Schumacher , K. , Grossmann , G. , and Dietrich , P . ( 2019 ). Multiple cyclic nucleotide-gated channels coordinate calcium oscillations and polar growth of root hairs . Plant J . 99 , 910 – 923 . OpenUrl CrossRef PubMed 11. ↵ Cai , X. , and Lytton , J . ( 2004 ). The cation/Ca(2+) exchanger superfamily: phylogenetic analysis and structural implications . Mol Biol Evol 21 , 1692 – 1703 . OpenUrl CrossRef PubMed Web of Science 12. ↵ Campanoni , P. , and Blatt , M.R . ( 2007 ). Membrane trafficking and polar growth in root hairs and pollen tubes . J Exp Bot 58 , 65 – 74 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Candeo , A. , Doccula , F.G. , Valentini , G. , Bassi , A. , and Costa , A . ( 2017 ). Light sheet fluorescence microscopy quantifies calcium oscillations in root hairs of Arabidopsis thaliana . Plant Cell Physiol . 58 , 1161 – 1172 . OpenUrl CrossRef PubMed 14. ↵ Corso , M. , Doccula , F.G. , de Melo , J.R.F. , Costa , A. , and Verbruggen , N. ( 2018 ). Endoplasmic reticulum-localized CCX2 is required for osmotolerance by regulating ER and cytosolic Ca2+ dynamics in Arabidopsis . Proc. Natl. Acad. Sci. U.S.A . 115 , 3966 – 3971 . OpenUrl Abstract / FREE Full Text 15. ↵ Cosgrove , D.J . ( 2024 ). Structure and growth of plant cell walls . Nature reviews. Molecular cell biology 25 , 340 – 358 . OpenUrl CrossRef PubMed 16. ↵ Costa , A. , Navazio , L. , and Szabo , I . ( 2018 ). The contribution of organelles to plant intracellular Calcium signalling . J. Exp. Bot . 17. ↵ Costa , A. , Resentini , F. , Buratti , S. , and Bonza , M.C . ( 2023 ). Plant Ca(2+)-ATPases: From biochemistry to signalling . Biochim. Biophys. Acta-Mol. Cell Res . 1870 , 119508 . OpenUrl CrossRef 18. ↵ Draeger , C. , Fabrice , T.N. , Gineau , E. , Mouille , G. , Kuhn , B.M. , Moller , I. , Abdou , M.-T. , Frey , B. , Pauly , M. , Bacic , A. , and Ringli , C . ( 2015 ). Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth . BMC Plant Biol . 15 , doi: 10.1186/s12870-12015-10548-12878 . OpenUrl CrossRef 19. ↵ Dünser , K. , Gupta , S. , Herger , A. , Feraru , M.I. , Ringli , C. , and Kleine-Vehn , J . ( 2019 ). Extracellular matrix sensing by FERONIA and Leucine-Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsis thaliana . EMBO J . 38 , e100353 . OpenUrl Abstract / FREE Full Text 20. ↵ Fabrice , T. , Vogler , H. , Draeger , C. , Munglani , G. , Gupta , S. , Herger , A.G. , Knox , P. , Grossniklaus , U. , and Ringli , C . ( 2018 ). LRX proteins play a crucial role in pollen grain and pollen tube cell wall development . Plant Physiol . 176 , 1981 – 1992 . OpenUrl Abstract / FREE Full Text 21. ↵ Frei dit Frey , N. , Mbengue , M. , Kwaaitaal , M. , Nitsch , L. , Altenbach , D. , Häweker , H. , Lozano-Duran , R. , Njo , M.F. , Beeckman , T. , Huettel , B. , Borst , J.W. , Panstruga , R. , and Robatzek , S. ( 2012 ). Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development . Plant Physiol . 159 , 798 – 809 . OpenUrl Abstract / FREE Full Text 22. ↵ Fujikura , U. , Ezaki , K. , Horiguchi , G. , Seo , M. , Kanno , Y. , Kamiya , Y. , Lenhard , M. , and Tsukaya , H . ( 2020 ). Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling . PLoS Genet . 16 , e1008873 . OpenUrl CrossRef PubMed 23. ↵ Gao , Q. , Wang , C. , Xi , Y. , Shao , Q. , Hou , C. , Li , L. , and Luan , S . ( 2023 ). RALF signaling pathway activates MLO calcium channels to maintain pollen tube integrity . Cell Res . 33 , 71 – 79 . OpenUrl CrossRef PubMed 24. ↵ Gerdes , H.H. , Rosa , P. , Phillips , E. , Baeuerle , P.A. , Frank , R. , Argos , P. , and Huttner , W.B . ( 1989 ). The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation . J. Biol. Chem . 264 , 12009 – 12015 . OpenUrl Abstract / FREE Full Text 25. ↵ Guérin , A. , Levasseur , C. , Herger , A. , Renggli , D. , Sotiropoulos , A.G. , Kadler , G. , Hou , X. , Schaufelberger , M. , Meyer , C. , Wicker , T. , Bigler , L. , and Ringli , C . ( 2025 ). Histidine limitation alters plant development and influences the TOR network . J. Exp. Bot . 76 , 1085 – 1098 . OpenUrl CrossRef PubMed 26. ↵ Gupta , S. , Guérin , A. , Herger , A. , Hou , X. , Schaufelberger , M. , Roulard , R. , Diet , A. , Roffler , S. , Lefebvre , V. , Wicker , T. , Pelloux , J. , and Ringli , C . ( 2024 ). Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX/RALF/FER growth regulatory module . PLoS Genet . 20 , e1011087 . OpenUrl CrossRef PubMed 27. ↵ Haruta , M. , Monshausen , G. , Gilroy , S. , and Sussman , M.R . ( 2008 ). A cytoplasmic Ca2+ functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings: Identification of AtRALF1 peptide . Biochemistry 47 , 6311 – 6321 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Herger , A. , Dünser , K. , Kleine-Vehn , J. , and Ringli , C . ( 2019 ). Leucine-rich repeat extensin proteins and their role in cell wall sensing . Curr. Biol . 29 , R851 – R858 . OpenUrl CrossRef PubMed 29. ↵ Herger , A. , Gupta , S. , Kadler , G. , Franck , C.M. , Boisson-Dernier , A. , and Ringli , C . ( 2020 ). Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis . PLoS Genet . 16 , e1008847 – e1008847 . OpenUrl CrossRef PubMed 30. ↵ Kanamori , K. , Nishimura , K. , Horie , T. , Sato , M.H. , Kajino , T. , Koyama , T. , Ariga , H. , Tanaka , K. , Yotsui , I. , Sakata , Y. , and Taji , T . ( 2023 ). Golgi apparatus-localized CATION CALCIUM EXCHANGER4 promotes osmotolerance of Arabidopsis . Plant Physiol . 194 , 1166 – 1180 . OpenUrl 31. ↵ Keinath , N.F. , Waadt , R. , Brugman , R. , Schroeder , J.I. , Grossmann , G. , Schumacher , K. , and Krebs , M . ( 2015 ). Live cell imaging with R-GECO1 sheds light on flg22-and chitin-induced transient Ca2+ (cyt) patterns in Arabidopsis . Mol. Plant 8 , 1188 – 1200 . OpenUrl CrossRef PubMed 32. ↵ Ketelaar , T . ( 2013 ). The actin cytoskeleton in root hairs: all is fine at the tip . Curr. Opin. Plant Biol . 16 , 749 – 756 . OpenUrl CrossRef PubMed 33. ↵ Kudla , J. , Batistic , O. , and Hashimoto , K . ( 2010 ). Calcium signals: the lead currency of plant information processing . Plant Cell 22 , 541 – 563 . OpenUrl Abstract / FREE Full Text 34. ↵ Kwon , T. , Sparks , J.A. , Liao , F.Q. , and Blancaflor , E.B . ( 2018 ). ERULUS is a plasma membrane-localized receptor-like kinase that specifies root hair growth by maintaining tip-focused cytoplasmic calcium oscillations . Plant Cell 30 , 1173 – 1177 . OpenUrl FREE Full Text 35. ↵ Leiber , R.M. , John , F. , Verhertbruggen , Y. , Diet , A. , Knox , J.P. , and Ringli , C . ( 2010 ). The TOR pathway modulates the structure of cell walls in Arabidopsis . Plant Cell 22 , 1898 – 1908 . OpenUrl Abstract / FREE Full Text 36. ↵ Li , J. , Brader , G. , and Palva , E.T . ( 2004 ). The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense . Plant Cell 16 , 319 – 331 . OpenUrl Abstract / FREE Full Text 37. ↵ Liu , X. , Wolfe , R. , Welch , L.R. , Domozych , D.S. , Popper , Z.A. , and Showalter , A.M . ( 2016 ). Bioinformatic identification and analysis of extensins in the plant kingdom . PLoS One 11 , e0150177 . OpenUrl CrossRef PubMed 38. ↵ Lopez-Hernandez , F. , Tryfona , T. , Rizza , A. , Yu , X.L. , Harris , M.O.B. , Webb , A.A.R. , Kotake , T. , and Dupree , P . ( 2020 ). Calcium Binding by Arabinogalactan Polysaccharides Is Important for Normal Plant Development . Plant Cell 32 , 3346 – 3369 . OpenUrl Abstract / FREE Full Text 39. ↵ Mecchia , M.A. , Santos-Fernandez , G. , Duss , N.N. , Somoza , S.C. , Boisson-Dernier , A. , Gagliardini , V. , Martinez-Bernardini , A. , Fabrice , T.N. , Ringli , C. , Muschietti , J.P. , and Grossniklaus , U . ( 2017 ). RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis . Science 358 , 1600 – 1603 . OpenUrl Abstract / FREE Full Text 40. ↵ Monshausen , G.B. , Messerli , M.A. , and Gilroy , S . ( 2008 ). Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis . Plant Physiol . 147 , 1690 – 1698 . OpenUrl Abstract / FREE Full Text 41. ↵ Moussu , S. , Broyart , C. , Santos-Fernandez , G. , Augustin , S. , Wehrle , S. , Grossniklaus , U. , and Santiago , J . ( 2020 ). Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth . Proc. Natl. Acad. Sci. U.S.A . 117 . 42. ↵ Moussu , S. , Lee , H.K. , Haas , K.T. , Broyart , C. , Rathgeb , U. , De Bellis , D. , Levasseur , T. , Schoenaers , S. , Fernandez , G.S. , Grossniklaus , U. , Bonnin , E. , Hosy , E. , Vissenberg , K. , Geldner , N. , Cathala , B. , Höfte , H. , and Santiago , J. ( 2023 ). Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction . Science 382 , 719 – 725 . OpenUrl CrossRef PubMed 43. ↵ Ngo , Q.A. , Vogler , H. , Lituiev , D.S. , Nestorova , A. , and Grossniklaus , U . ( 2014 ). A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery . Dev. Cell 29 , 491 – 500 . OpenUrl CrossRef PubMed Web of Science 44. ↵ Nguema-Ona , E. , Vicré-Gibouin , M. , Gotté , M. , Plancot , B. , Lerouge , P. , Bardor , M. , and Driouich , A . ( 2014 ). Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function . Front. Plant Sci . 5 , 499 . OpenUrl CrossRef PubMed 45. ↵ Ogawa , S.T. , Zhang , W. , Staiger , C.J. , and Kessler , S.A . ( 2025 ). MLO-mediated Ca2+ influx regulates root hair tip growth in Arabidopsis . bioRxiv , 2025.2004.2008.647801 . 46. ↵ Ordenes , V.R. , Moreno , I. , Maturana , D. , Norambuena , L. , Trewavas , A.J. , and Orellana , A . ( 2012 ). In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics . Cell calcium 52 , 397 - 404 . OpenUrl CrossRef PubMed Web of Science 47. ↵ Pacheco , J.M. , Song , L. , Kuběnová , L. , Ovečka , M. , Berdion Gabarain , V. , Peralta , J.M. , Lehuedé , T.U. , Ibeas , M.A. , Ricardi , M.M. , Zhu , S. , Shen , Y. , Schepetilnikov , M. , Ryabova , L.A. , Alvarez , J.M. , Gutierrez , R.A. , Grossmann , G. , Šamaj , J. , Yu , F. , and Estevez , J.M. ( 2023 ). Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana . New Phytol . 238 , 169 – 185 . OpenUrl CrossRef PubMed 48. ↵ Pasternak , T. , Groot , E.P. , Kazantsev , F.V. , Teale , W. , Omelyanchuk , N. , Kovrizhnykh , V. , Palme , K. , and Mironova , V.V . ( 2019 ). Salicylic acid affects root meristem patterning via auxin distribution in a concentration-dependent manner . Plant Physiol . 180 , 1725 – 1739 . OpenUrl Abstract / FREE Full Text 49. ↵ Pirayesh , N. , Giridhar , M. , Ben Khedher , A. , Vothknecht , U.C. , and Chigri , F . ( 2021 ). Organellar calcium signaling in plants: An update . Biochim. Biophys. Acta-Mol. Cell Res . 1868 , 118948 . OpenUrl CrossRef 50. ↵ Pittman , J.K. , and Hirschi , K.D . ( 2016 ). CAX-ing a wide net: Cation/H(+) transporters in metal remediation and abiotic stress signalling . Plant Biol . 18 , 741 – 749 . OpenUrl CrossRef PubMed 51. ↵ Resentini , F. , Ruberti , C. , Grenzi , M. , Bonza , M.C. , and Costa , A . ( 2021 ). The signatures of organellar calcium . Plant Physiol . 187 , 1985 – 2004 . OpenUrl CrossRef PubMed 52. ↵ Ringli , C . ( 2010 ). The hydroxyproline-rich glycoprotein domain of the Arabidopsis LRX1 requires Tyr for function but not for insolubilization in the cell wall . Plant J . 63 , 662 – 669 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Romano Armada , N. , Doccula , F.G. , Candeo , A. , Valentini , G. , Costa , A. , and Bassi , A. ( 2019 ). In Vivo Light Sheet Fluorescence Microscopy of Calcium Oscillations in Arabidopsis thaliana . Meth . Mol. Biol . 1925 , 87 – 101 . OpenUrl 54. ↵ Rubinstein , A.L. , Marquez , J. , Suarez-Cervera , M. , and Bedinger , P.A . ( 1995 ). Extensin-like glycoproteins in the maize pollen tube wall . Plant Cell 7 , 2211 – 2225 . OpenUrl Abstract / FREE Full Text 55. ↵ Schaufelberger , M. , Galbier , F. , Herger , A. , de Brito Francisco , R. , Roffler , S. , Clement , G. , Diet , A. , Hortensteiner , S. , Wicker , T. , and Ringli , C. ( 2019 ). Mutations in the Arabidopsis ROL17/isopropylmalate synthase 1 locus alter amino acid content, modify the TOR network, and suppress the root hair cell development mutant lrx1 . J. Exp. Bot . 70 , 2313 – 2323 . OpenUrl CrossRef PubMed 56. ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , Kaynig , V. , Longair , M. , Pietzsch , T. , Preibisch , S. , Rueden , C. , Saalfeld , S. , Schmid , B. , Tinevez , J.-Y. , White , D.J. , Hartenstein , V. , Eliceiri , K. , Tomancak , P. , and Cardona , A . ( 2012 ). Fiji: an open-source platform for biological-image analysis . Nat. Methods 9 , 676 – 682 . OpenUrl CrossRef PubMed Web of Science 57. ↵ Schoenaers , S. , Balcerowicz , D. , Breen , G. , Hill , K. , Zdanio , M. , Mouille , G. , Holman , T.J. , Oh , J. , Wilson , M.H. , Nikonorova , N. , Vu , L.D. , De Smet , I. , Swarup , R. , De Vos , W.H. , Pintelon , I. , Adriaensen , D. , Grierson , C. , Bennett , M.J. , and Vissenberg , K. ( 2018 ). The auxin-regulated Cr RLK1L kinase ERULUS controls cell wall composition during root hair tip growth . Curr. Biol . 28 , 722 – 732 . OpenUrl CrossRef PubMed 58. ↵ Schoenaers , S. , Lee , H.K. , Gonneau , M. , Faucher , E. , Levasseur , T. , Akary , E. , Claeijs , N. , Moussu , S. , Broyart , C. , Balcerowicz , D. , AbdElgawad , H. , Bassi , A. , Damineli , D.S.C. , Costa , A. , Feijó , J.A. , Moreau , C. , Bonnin , E. , Cathala , B. , Santiago , J. , Höfte , H. , and Vissenberg , K . ( 2024 ). Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly . Nat. Plants 10 , 494 – 511 . OpenUrl CrossRef PubMed 59. ↵ Shi , L. , Wu , Y. , and Sheen , J . ( 2018 ). TOR signaling in plants: conservation and innovation . Development 145 . 60. ↵ Sievers , F. , Wilm , A. , Dineen , D. , Gibson , T.J. , Karplus , K. , Li , W. , Lopez , R. , McWilliam , H. , Remmert , M. , Söding , J. , Thompson , J.D. , and Higgins , D.G . ( 2011 ). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Mol. Syst. Biol 7 , 539 . OpenUrl CrossRef PubMed 61. ↵ Song , L.M. , Xu , G.Y. , Li , T.T. , Zhou , H.N. , Lin , Q.L. , Chen , J. , Wang , L. , Wu , D.S. , Li , X.X. , Wang , L.F. , Zhu , S.R. , and Yu , F . ( 2022 ). The RALF1-FERONIA complex interacts with and activates TOR signaling in response to low nutrients . Mol. Plant 15 , 1120 – 1136 . OpenUrl CrossRef PubMed 62. ↵ Tan , S. , Abas , M. , Verstraeten , I. , Glanc , M. , Molnár , G. , Hajný , J. , Lasák , P. , Petřík , I. , Russinova , E. , Petrášek , J. , Novák , O. , Pospíšil , J. , and Friml , J. ( 2020 ). Salicylic acid targets Protein Phosphatase 2A to attenuate growth in plants . Curr. Biol . 30 , 381 – 395.e388 . OpenUrl CrossRef PubMed 63. ↵ Trewavas , A. , Read , N. , Campbell , A.K. , and Knight , M . ( 1996 ). Transduction of Ca2+ signals in plant cells and compartmentalization of the Ca2+ signal . Biochem. Soc. Trans . 24 , 971 – 974 . OpenUrl FREE Full Text 64. ↵ Tsutsui , H. , and Higashiyama , T . ( 2017 ). pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9 -Mediated Gene Knockout in Arabidopsis thaliana . Plant Cell Physiol . 58 , 46 – 56 . OpenUrl CrossRef PubMed 65. ↵ Vitale , A. , and Galili , G . ( 2001 ). The endomembrane system and the problem of protein sorting . Plant Physiol . 125 , 115 – 118 . OpenUrl FREE Full Text 66. ↵ Wang , P. , Clark , N.M. , Nolan , T.M. , Song , G. , Whitham , O.G. , Liao , C.Y. , Montes-Serey , C. , Bassham , D.C. , Walley , J.W. , Yin , Y. , and Guo , H . ( 2022 ). FERONIA functions through Target of Rapamycin (TOR) to negatively regulate autophagy . Front. Plant Sci . 13 , 961096 . OpenUrl CrossRef PubMed 67. ↵ Willats , W.G.T. , McCartney , L. , Mackie , W. , and Knox , J.P . ( 2001 ). Pectin: cell biology and prospects for functional analysis . Plant Mol. Biol . 47 , 9 – 27 . OpenUrl CrossRef PubMed Web of Science 68. ↵ Wu , Y. , Zhang , D. , Chu , J.Y. , Boyle , P. , Wang , Y. , Brindle , I.D. , De Luca , V. , and Després , C. ( 2012 ). The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid . Cell Rep . 1 , 639 – 647 . OpenUrl CrossRef PubMed 69. ↵ Wymer , C.L. , Bibikova , T.N. , and Gilroy , S. ( 1997 ). Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana . Plant J . 12 , 427 – 439 . OpenUrl CrossRef PubMed Web of Science 70. ↵ Yu , D. , Chen , C. , and Chen , Z . ( 2001 ). Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression . Plant Cell 13 , 1527 – 1540 . OpenUrl Abstract / FREE Full Text 71. ↵ Zhang , S. , Pan , Y. , Tian , W. , Dong , M. , Zhu , H. , Luan , S. , and Li , L . ( 2017 ). Arabidopsis CNGC14 mediates calcium influx required for tip growth in root hairs . Mol. Plant 10 , 1004 – 1006 . OpenUrl CrossRef PubMed 72. ↵ Zhao , C. , Jiang , W. , Zayed , O. , Liu , X. , Tang , K. , Nie , W. , Li , Y. , Xie , S. , Li , Y. , Long , T. , Liu , L. , Zhu , Y. , Zhao , Y. , and Zhu , J.K . ( 2021 ). The LRXs-RALFs-FER module controls plant growth and salt stress responses by modulating multiple plant hormones . Natl. Sci. Rev . 8 , nwaa149 . OpenUrl CrossRef PubMed 73. ↵ Zhao , C.Z. , Zayed , O. , Yu , Z.P. , Jiang , W. , Zhu , P.P. , Hsu , C.C. , Zhang , L.R. , Tao , W.A. , Lozano-Duran , R. , and Zhu , J.K . ( 2018 ). Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis . Proc. Natl. Acad. Sci. U.S.A . 115 , 13123 – 13128 . OpenUrl Abstract / FREE Full Text 74. ↵ Zhou , H. , Ge , H. , Chen , J. , Li , X. , Yang , L. , Zhang , H. , and Wang , Y. ( 2022 ). Salicylic acid regulates root gravitropic growth via clathrin-independent endocytic trafficking of PIN2 auxin transporter in Arabidopsis thaliana . Int. J. Mol. Sci . 23 . 75. ↵ Zhu , M. , Du , B.-Y. , Tan , Y.-Q. , Yang , Y. , Zhang , Y. , and Wang , Y.-F . ( 2025 ). CPK1 activates CNGCs through phosphorylation for Ca2+ signaling to promote root hair growth in Arabidopsis . Nat. Commun . 16 , 676 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted June 27, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+ homeostasis Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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