MYB93 regulates responses to environmental sulphur in Arabidopsis and tomato

MYB93 regulates responses to environmental sulphur in Arabidopsis and tomato | 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 MYB93 regulates responses to environmental sulphur in Arabidopsis and tomato Xulyu Cao , Helen B. Wilkinson , Bethany Hutton , Nancy McMulkin , View ORCID Profile Neil S. Graham , Ross Etherington , Alice Oliver , Clare M. Clayton , Harjeet Kaur , View ORCID Profile Juliet C. Coates doi: https://doi.org/10.1101/2025.03.04.641362 Xulyu Cao 1 College of Resources and Environment, Academy of Agricultural Sciences, Key Laboratory of Efficient Utilization of Soil and Fertilizer resources, Southwest University , Chongqing, 400716, China 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Helen B. Wilkinson 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bethany Hutton 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nancy McMulkin 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Neil S. Graham 3 Plant and Crop Sciences Division, School of Biosciences , Sutton Bonington Campus, University of Nottingham , LE12 5RD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Neil S. Graham Ross Etherington 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alice Oliver 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Clare M. Clayton 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Harjeet Kaur 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Juliet C. Coates 2 School of Biosciences, University of Birmingham , Birmingham B15 2TT, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Juliet C. Coates For correspondence: j.c.coates{at}bham.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Sulphur (S) is an important nutrient that has wide-ranging effects on plant health and metabolism. Several classes of transcription factor respond to S deprivation, including R2R3-MYBs. In Arabidopsis , the AtMYB93 transcription factor-encoding gene is upregulated by S deprivation. At MYB93 has a non-redundant function in lateral root development and redundant functions in suberin biosynthesis alongside related MYB transcription factors, but At MYB93’s role in S signalling, and how it relates to lateral root development, is unknown. We show that the transcriptome of Atmyb93 mutant roots implicates At MYB93 in responses to S, including changes in S transport and metabolism, and flavonoid- and carbohydrate metabolism. Elemental analysis demonstrates that the Atmyb93 mutant has elevated shoot S levels while tomato Sl MYB93-overexpressing plants have reduced shoot S. We uncover a stimulatory effect of S deprivation on adventitious root development. However, Atmyb93 mutants do not show significant changes in sensitivity to S with respect to lateral-or adventitious root development, most likely due to some functional redundancy. Moreover, AtMYB93 promoter activity is not spatially regulated by S deprivation. Taken together, our data suggest that AtMYB93 has a role in mediating root responses to S in alongside other root transcription factors. Introduction Plants require a range of nutrients to grow and develop optimally. The soil provides macronutrients and micronutrients, in relatively large and small amounts respectively. The key macronutrients that plants obtain from soil are nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S). S is often considered a ‘neglected’ macronutrient: S fertilisation can improve plant yield, abiotic- and biotic stress tolerance ( Ali et al. 2021 ; Narayan et al. 2023 ; Sarda et al. 2013 ; Zenda et al. 2021 ) although S deprivation may improve resistance to some pathogens ( Criollo-Arteaga et al. 2021 ). In terms of human nutrition, plants are the major source of methionine, an essential S-containing amino acid ( Kopriva et al. 2019 ). Problems with S deficiency in crops are increasing globally due to the decreasing amount of rainfall-derived S in combination with the continued development of high-yielding crop varieties ( Sharma et al. 2024 ) ( Zenda et al. 2021 ) ( Sarda et al. 2013 ) ( Grant et al. 2012 ). Crop requirements for S vary, with Brassicas (especially oilseed rape) requiring high S, cotton and sugarcane requiring medium S and cereals having relatively low S requirements ( Zenda et al. 2021 ). However, naturally high S Brassica crops used for animal feed can promote risk of goitre and kale anemia ( Smith 1980 ; Smith et al. 1974 ; Paxman and Hill 1974 ). Moreover, S levels can affect crop quality, for example high S can cause increased acrylamide formation in cooked potatoes ( Elmore et al. 2007 ; Elmore et al. 2010 ). In marine ecosystems, sulfide stress in seagrass beds, caused by microbial anaerobic digestion of sulfate in sediments, increases with elevated temperature ( Zhang et al. 2024 ). Thus, S levels in plants may require careful management on a case-by-case basis. S deficiency in plants has wide-ranging effects including reduced chlorophyll, changes in sugar- and amino acid metabolism, changes in phenylpropanoid metabolism (particularly flavonoids) and changes in antioxidant levels (e.g. ( Lunde et al. 2008 ; Wawrzynska et al. 2022 ; Bielecka et al. 2014 ; Chandra and Pandey 2014 ; Forieri et al. 2017 ; Henriquez-Valencia et al. 2018 ; Canales et al. 2020 ; Robe et al. 2020 ; Nikiforova et al. 2003 ; Wang et al. 2022 ). Moreover, various S-containing compounds affect plant hormone biosynthesis directly or indirectly ( Wawrzynska and Sirko 2024 ). Several classes of transcription factor (TF) have been identified that may co-ordinate responses to S stress. The ETHYLENE INSENSITIVE LIKE (EIL) TF family member SULFUR LIMITATION 1 (SLIM1/EIL3) is a key regulator of metabolism under S deficiency in Arabidopsis and tomato roots ( Maruyama-Nakashita et al. 2006 ; Canales et al. 2020 ) partnering with EIL1 ( Dietzen et al. 2020 ). Bioinformatic analysis suggested NUCLEAR FACTOR Y-A2 (NFY-A2) and the circadian regulator REVEILLE2 (RVE2) as ‘hub’ genes co-ordinating expression of S-response genes ( Henriquez-Valencia et al. 2018 ). S availability affects expression of Arabidopsis R2R3-MYB family TFs that regulate biosynthesis of glucosinolates, S-containing secondary metabolites with defence functions, likely via SLIM1 ( Frerigmann and Gigolashvili 2014a , b ; Li et al. 2013 ). Moreover, S stress increases expression of a suite of Arabidopsis root TFs including several R2R3-MYBs; in particular, AtMYB93 shows a sustained increase upon S deprivation and S resupply ( Bielecka et al. 2014 ). AtMYB93 is part of a flowering plant-specific clade (the S24 clade) of plant R2R3-MYB transcription factors ( Du et al. 2015 ; Gibbs et al. 2014 ). In Arabidopsis , the S24 clade consists of AtMYB93 , AtMYB92 and AtMYB53 ( Du et al. 2015 ; Gibbs et al. 2014 ). All three Arabidopsis S24 R2R3-MYB s show root-enriched expression with AtMYB93 being expressed exclusively in the root ( Gibbs et al. 2014 ). The function of the three Arabidopsis S24 MYB genes is not fully redundant. AtMYB93 , but not AtMYB92 , is a negative regulator of LR development and only AtMYB93 expression is induced by auxin ( Gibbs et al. 2014 ). As mentioned above, AtMYB93 is significantly upregulated upon S deprivation with persistent elevated expression upon S resupply in 7-day old seedlings ( Bielecka et al. 2014 ). AtMYB53 is upregulated by S deprivation to a lesser extent than AtMYB93 and is not persistently upregulated upon S resupply ( Bielecka et al. 2014 ). AtMYB92 shows a limited S deprivation response: upregulation in seedlings when seeds are germinated directly on S-deficient medium but not when S-replete seedlings are transferred to S-deficient medium after 8 days of growth ( Nikiforova et al. 2003 ). Interestingly, S deprivation inhibits LR development in young Arabidopsis seedlings ( Dan et al. 2007 ; Dong et al. 2019 ; Joshi et al. 2019 ) although the converse occurs in older plants ( Kutz et al. 2002 ). AtMYB93 is expressed only in the root endodermis ( Gibbs et al. 2014 ), a single-celled layer separating the outer epidermis and cortex from the inner pericycle and vasculature. The endodermis forms a selective barrier to passage of solutes, nutrients and water between the soil and the root vasculature (and hence the shoot) ( Andersen et al. 2015 ; Geldner 2013 ). AtMYB93 promoter activity is restricted to a very small number of endodermal cells overlying lateral root primordia (LRP) during early lateral root (LR) development ( Gibbs et al. 2014 ; Shukla et al. 2021 ). S24 MYB genes and the most closely related Arabidopsis genes in clades S10, S11 and S42 ( Du et al. 2015 ) are involved in synthesis of the lipophilic biopolymer suberin that surrounds mature root endodermal cells and is present in fruit and in seeds ( Andersen et al. 2015 ; Vishwanath et al. 2015 ; Barberon et al. 2016 ). MYB genes act, potentially in a hierarchical fashion ( Xu et al. 2022 ; Xu et al. 2023 ), to enhance suberin production. Analysis in tomato, apple and Arabidopsis identified a conserved cross-species gene expression ‘signature’ for suberin biosynthesis ( Gou et al. 2017 ; Lashbrooke et al. 2016 ). Apple MdMYB93 promotes biosynthesis and cell export of suberin in the fruit ( Legay et al. 2016 ). AtMYB39 ( SUBERMAN ), AtMYB41 , AtMYB53 , AtMYB92 and AtMYB93 are all expressed in the root endodermis and can promote suberin production ( Cohen et al. 2020 ; Hu 2018 ; Kosma et al. 2014 ; Shukla et al. 2021 ; To et al. 2020 ; Wang et al. 2020 ). Lipid-, wax-, fatty acid and suberin biosynthesis genes are differentially expressed in an Atmyb93/Atmyb92/Atmyb53 triple mutant and an At MYB53-overexpressing line ( Klein 2019 ). At MYB41, At MYB93, At MYB53, At MYB92 function redundantly to promote suberin biosynthesis in the root endodermis with At MYB41 having the dominant role ( Shukla et al. 2021 ). SlMYB92 promotes suberin biosynthesis in the tomato exodermis ( Canto-Pastor et al. 2024 ), while three MYB s, OsMYB93a , OsMYB41 and OsMYB76 also regulate root suberin biosynthesis in a monocot, rice ( Huang et al. 2024 ). Expression of AtMYB93 , AtMYB53 , AtMYB9 and two other MYB transcription factors is upregulated in the kcs1-5 mutant that has impaired synthesis of very long chain fatty acids (VLCFAs) ( Uemura et al. 2023 ), which are components of suberin ( Serra and Geldner 2022 ). Interestingly, an Arabidopsis mutant lacking all four of At MYB41, At MYB93, At MYB53 and At MYB92 has reduced suberin but has no change in lateral root development ( Shukla et al. 2021 ), suggesting the functions of At MYB93 in suberin formation and root development are separable. The function of At MYB93 and its relatives in plant responses to S has not been investigated. Given that AtMYB93 has a non-redundant root function, highly specific root expression and a unique pattern of regulation by hormones and environmental signals compared to other closely related MYB genes, we hypothesise that AtMYB93 has functions in addition to suberin biosynthesis during Arabidopsis root development. Results Transcriptome analysis of plants lacking AtMYB93 reveals a potential function in responding to S To ascertain how AtMYB93 carries out its function, we conducted transcriptome analysis comparing gene expression in roots of wild type (Col-0) and Atmyb93 seedlings using RNAseq ( Fig. 1 ; Online Resource 2). A total of 255 genes were differentially expressed (p<0.001) between Col-0 and Atmyb93 , of which 44 were downregulated (including AtMYB93 itself) and 211 were upregulated (Online Resource 2). Download figure Open in new tab Fig. 1 Differentially expressed genes in the Atmyb93 mutant are enriched for functions relating to S-, phenylpropanoid/flavonoid- and carbohydrate metabolism, and responses to a range of environmental stimuli. S-related differentially expressed genes in Atmyb93 largely also show differential expression in response to S deprivation. a) and b) Gene Ontology (GO) Biological Process (BP) term enrichment for genes (a) significantly downregulated (q value < 0.05) in the Atmyb93 mutant defined by biological process and (b) significantly upregulated (q value < 0.05) in the Atmyb93 mutant. In each panel, GO term enrichment was calculated using PlantRegMap (p<0.01) and represented with ReviGO, based on the semantic distances between GO terms. LogSize represents the log10 (number of annotations for GO Term ID in selected species in the EBI GOA database). c) Genes with S-related functions significantly differentially expressed in the Atmyb93 mutant, showing the gene, gene name, function and log2 fold-change (down, blue and up, yellow) in Atmyb93 or under S stress in the root. Data in the right-hand column highlights genes that also have a fold-change change >1 (down, blue or up, yellow) under S stress in either endodermis (endo, 3h S deprivation) or a whole root time series (time of largest increase shown) published by ( Iyer-Pascuzzi et al. 2011 ) and visualized at the eFP browser ( Winter et al. 2007 ). Two genes, At1g22150 SULTR1;3 and At1g61740 sulfite exporter, show different regulation in the endodermis compared to the whole root When analysed for Gene Ontology (GO) term enrichment, genes downregulated in the Atmyb93 mutant compared to wild type showed enrichment of Biological Process (BP) terms associated with (i) S transport and metabolism, (ii) carbohydrate and sugar metabolism, (iii) phenylpropanoid and flavonoid metabolism and (iv) responses to stimuli including light, auxin and karrikin ( Fig. 1a ). Genes upregulated in Atmyb93 compared to wild type were enriched for GO-BP terms associated with (i) carbohydrate (including trehalose) metabolism, transport and responses and (ii) responses to stimuli including carbohydrates and hypoxia/oxygen as well as other abiotic stimuli ( Fig. 1b ). These changes are reminiscent of both S deprivation responses in other studies, as outlined in the introduction to this paper, and of the transcriptome of slim1 and eil1 mutant plants [25]. We collated the differentially expressed genes with known functions in S transport and metabolism ( Fig. 1c ) and compared their differential expression in Atmyb93 with their responses to S deprivation in whole root tissue and root endodermal tissue ( Iyer-Pascuzzi et al. 2011 ; Winter et al. 2007 ). Four of the six genes with S-related functions downregulated in Atmyb93 showed upregulation under S deprivation, while three of the four S-related genes upregulated in Atmyb93 showed downregulation under S deprivation ( Fig. 1c ). Two genes, At1g22150 SULTR1;3 and At1g61740 sulfite exporter, show different regulation in the endodermis compared to the whole root ( Fig. 1c ). There is no overlap between Atmyb93 differentially expressed genes (Online Resource 2) and the largely suberin-related genes differentially expressed in the Atmyb53/92/93 triple mutant ( Klein 2019 ). Collectively, these data suggest that At MYB93, directly or indirectly, regulates genes related to S-deprivation responses in Arabidopsis . MYB93 homologues in dicots show both root-specific expression and upregulation by S deprivation Our previous data demonstrated that At MYB93 shows the most root-specific expression of Arabidopsis S24 clade of R2R3-MYB genes ( Gibbs et al. 2014 ). To determine whether expression of MYB93 -related genes outside Arabidopsis is root-specific, we analysed tissue-specific expression of three tomato MYB93 homologues. The closest tomato MYB93 homologue is Sl04g074170 while Sl04g056310 has previously been annotated as SlMYB53L ( Canales et al. 2020 ) and Sl05g051550 has previously been annotated as SlMYB92 ( Canales et al. 2020 ; Canto-Pastor et al. 2024 ). Note that Sl04g074170 is different to the ‘ SlMYB93 ’ identified in Kajala et al (2021) , Sl11g011050 , which is closest in protein sequence to At MYB48 ( Kajala et al. 2021 ). All three SlMYB93 homologues show root enrichment ( Fig. 2a ). We made transgenic Arabidopsis plants heterologously overexpressing SlMYB93 ( Fig. 2c ) and showed that SlMYB93 can reduce lateral root density and root length ( Fig. 2c,d ) similarly to AtMYB93 overexpression ( Gibbs et al. 2014 ). Download figure Open in new tab Fig. 2 Tomato S24-related MYB93 homologues are root-enriched and Sl MYB93 functions in Arabidopsis similarly to At MYB93. a) Root-enriched expression of tomato MYB93 homologues demonstrated by RT-PCR. R, root; S, Stem; L, leaf; F, flower; TF, 4 days post anthesis (DPA) tomato fruit; IMG, immature green; MG, mature green; Br, breaker; RR, red ripen; SD, seed. SlMYB93 , Solyc04g074170; SlMYB53L , Solyc04g056310; SlMYB92 Solyc05g051550. The ACTIN2 gene is shown as a control. b) RT-PCR showing heterologous expression of SlMYB93 in Arabidopsis using a p35S::SlMYB93 construct (3 independent transgenic lines) compared to a wild type control (WT, Col-0). c) Lateral root density of 3 independent transgenic Arabidopsis lines overexpressing SlMYB93 ( Sl MYB93-OX). d) Primary root length of 3 independent transgenic Arabidopsis lines overexpressing SlMYB93 . 8-day old seedlings from 3 combined biological repeats are shown with data points (small coloured points) for each repeat coloured differently. Larger coloured circles represent the means of the biological repeats and black bars represent the overall mean and standard deviation of the means. In c) and d) 8-day old seedlings from 3 combined biological repeats are shown with data points (small coloured points) for each repeat coloured differently. Larger coloured circles represent the means of the biological repeats and black bars represent the overall mean and standard deviation of the means. The number of seedlings per treatment ranges from 35-75; these are the same seedlings in both panels To extend our analysis beyond Arabidopsis and tomato, we analysed publicly available transcriptome data and demonstrated that root-specific or root-enriched expression of putative MYB93 homologues is present in a wide range of, but not all, dicot species ( Fig. 3 ). In poplar and cassava, which each have 2 closely related MYB93 homologues, only one homologue shows root enrichment or specificity, whereas both soybean homologues show root specificity ( Fig. 3 ). Dicot MYB92/53 -like genes also show root enrichment ( Fig. 3 ). Some dicot AtMYB93 -like genes from outside the MYB93 clade show root enrichment ( AtMYB48 and Sl11g011050 ) ( Fig. 3 ). Some monocot AtMYB93 -related genes show root enrichment to a lesser extent: all rice homologues ( OsMYB93a (Os08g37970), OsMYB93b (Os06g17780) and OsMYB41 (Os02g51799) ( Huang et al. 2024 )) are root-enriched as is one barley homologue and one maize homologue but not the genes in Brachypodium ( Fig. 3 ). Overall, root specificity/enrichment is most pronounced in dicot AtMYB93 homologues (compared to MYB92 and MYB53 ). Download figure Open in new tab Fig. 3 Expression of flowering plant MYB93 homologues: root-enrichment and dicot-specific response to S deprivation. Phylogeny of AtMYB93 -related genes: the clade in which the Arabidopsis S24 genes fall is coloured in pink, a moncot AtMYB93 -related clade is purple and a clade containing the additional suberin-producing Arabidopsis MYBs is in black. Arabidopsis genes are shown in green and tomato genes are shown in blue. Semi-quantitative expression analysis from publicly available/published data is shown: R, root; S, shoot, F, fruit, silique or seed; End, endodermis; Exo, exodermis; Sul, S deprivation response. Pink, tissue expression; yellow, upregulation in response to S deprivation. Key to species: At, Arabidopsis thaliana ; Bn, Brassica napus; Br, Brassica rapa ; Gm, Glycine max (soybean); Potri, Populus trichocarpa (poplar); Manes, Manihot esculenta (cassava); Solyc Solanum lycopersicum (tomato); Md, Malus domestica (apple); Os, Oryza sativa (rice); Zm, Zea mays (maize); Bradi, Brachypodium distachyon; HORVU, Hordeum vulgare (barley). According to the notation of Huang et al (2024) , Os2g51799 corresponds to OsMYB41 , Os08g37970 corresponds to OsMYB93a and Os06g17780 corresponds to OsMYB93b We also analysed, where available, the cell type specificity of MYB93 homologues within the root and their response to S deprivation. Both AtMYB93 and Sl04g074170 show endodermal-specific and meristematic expression ( Fig. 3 ; ( Gibbs et al. 2014 ; Kajala et al. 2021 ; Waese et al. 2017 )). In contrast, Sl11g011050 is expressed in both exodermis and endodermis ( Fig. 3 ; ( Gibbs et al. 2014 ; Kajala et al. 2021 ; Waese et al. 2017 )). Sl04g056310 ( SlMYB53L ) is present in endodermis, exodermis and xylem ( Fig. 3 ; ( Gibbs et al. 2014 ; Kajala et al. 2021 ; Waese et al. 2017 )) and Sl05g051550 ( SlMYB92 ) is expressed in the exodermis ( Fig. 3 ; ( Canto-Pastor et al. 2024 ; Kajala et al. 2021 ; Waese et al. 2017 )). All three closest tomato MYB93 homologues are upregulated by S deprivation ( Fig. 3 ; ( Canales et al. 2020 )) with SlMYB93 and SlMYB53L showing the strongest response, similarly to Arabidopsis ( Fig. 3 ; ( Bielecka et al. 2014 )). Outside the MYB93 clade, AtMYB107 and Sl11g011050 also show some upregulation by S deprivation ( Fig. 3 ; ( Canales et al. 2020 )). However, no change in rice MYB93 -like genes is seen upon S deprivation ( Fig. 3 ; ( Wang et al. 2022 )). Taken together, these data suggest a conserved role for dicot MYB93 -like genes in regulating root responses to S deprivation, with the closest MYB93 homologues specifically functioning in the endodermis. Changes in MYB93 level alter plant shoot element composition To ask whether MYB93 ’s potential link to root S-deprivation responses is associated with changes in the shoot, we first measured the elemental composition of key macronutrients (Mg, P, S, K, Ca) and micronutrients (Mn, Fe, Zn, B, Cu, Mo) in Arabidopsis wild type and Atmyb93 mutant shoot tissue. Under normal growth conditions, the Atmyb93 mutant showed generally elevated macronutrient and micronutrient levels but significant differences in S, Mg and B ( Fig. 4a ; Online Resource 2). Next, we investigated elemental composition of transgenic tomato lines with increased SlMYB93 expression, SlMYB93-OX ( Fig. 4b,c ). The trends observed were in accordance with the Arabidopsis result, with two out of three independent SlMYB93-OX lines showing reduced shoot elements ( Fig. 4C ; Online Resource 2). Download figure Open in new tab Fig. 4 Arabidopsis Atmyb93 mutants and tomato overexpressing SlMYB93 show altered shoot element composition. a) Comparison of shoot element composition in Col-0 and Atmyb93 21-day old plants. Heatmap shows the percentage change of means from 3 combined biological repeats. Asterisks represent significant differences (p=0.04 for each) from Mann Whitney tests on the raw data. b) RT-PCR detection of SlMYB93 in the roots of wild type tomato plants and two independent SlMYB93 -overexpressing lines. c) Comparison of shoot element composition in wild type and SlMYB93- overexpressing tomato plants. Heatmap shows the percentage change of means from 3 combined biological repeats. Asterisks represent significant differences (all p=0.04) from Mann Whitney tests on the raw data One line showed a significant reduction in Mg and B and another showed a significant reduction in S, B and Cu ( Fig. 4C ; Online Resource 2). These data suggest that altering MYB93 function in the root can lead to elemental changes particularly to S, Mg and B in the shoot. Atmyb93 mutants respond to S deprivation To further investigate the link between At MYB93 and plant responses to S, we compared root development responses of wild type and Atmyb93 mutant plants under S deprivation. A reduction in lateral root number under S starvation has previously been reported ( Dong et al. 2019 ). A reduction in lateral root density and an increase in root length was seen in seedlings transferred to -S for 3 days after 5 days’ growth on S-replete medium that was slightly, but not significantly, less pronounced in the Atmyb93 mutant ( Fig. 5a,b ). We also observed that S deprivation increases the number of adventitious roots (AR) produced at the root-shoot junction (collet) in both wild type and Atmyb93 plants transferred to medium lacking S for 3 days after 5 days’ growth on S-replete medium, with no significant differences in AR number between wild type and Atmyb93 being seen ( Fig. 5c ). We also tested the sensitivity of Atmyb93 to O-acetyl serine (OAS), a compound that is part of the S assimilation pathway and increases in response to S deprivation ( Hirai et al. 2003 ; Hubberten et al. 2012a ; Hubberten et al. 2012b ). We did not observe significant effects of OAS on either the primary root length or lateral root density of wild type or Atmyb93 mutant plants ( Fig. 5d,e ). Download figure Open in new tab Fig. 5 S deprivation affects root development but Atmyb93 mutants do not show strong root or shoot phenotypes upon S deprivation or OAS treatment. a), b) Lateral root density (a) and primary root length (b) in wild type (Col-0) and Atmyb93 seedlings grown with S for 3 days then transferred to –S medium (or +S medium control) for 5 days. 2 combined biological repeats are shown with data points (small coloured points) for each repeat coloured differently. Larger coloured circles represent the means of the biological repeats and black bars represent the overall mean and standard deviation of the means. c) Formation of adventitious roots at the collet in wild type (Col-0) and Atmyb93 8-day old seedlings grown for 3 days with S before transfer to –S medium (or a +S medium control). 5 combined biological repeats are shown with data points (small coloured points) and distributions (shaded areas) for each repeat coloured differently. Larger coloured circles represent the means of the biological repeats and black bars represent the standard deviation of the means. d), e) Primary root length (d) and lateral root density (e) of wild type (Col-0) and Atmyb93 seedlings grown for 8 days on 0.1mM O-acetyl serine (OAS). 2 combined biological repeats are shown with data points (small coloured points) for each repeat coloured differently. Larger coloured circles represent the means of the biological repeats and black bars represent the overall mean and standard deviation of the means. In all panels, letters indicate differences p<0.05 in post-hoc Dunn’s test carried out after a Kruskal-Wallis test. For adventitious roots, seedling numbers range from 9 to 20 per treatment in a biological repeat. For primary/lateral root experiments ±S, seedling numbers range from 30 to 98 per treatment depending on the biological repeat. For OAS experiments, seedling numbers range from 18 to 20 per treatment in one biological repeat and 56 to 59 in the other Collectively, these results show that S deprivation can increase adventitious root formation at the root-hypocotyl junction and suggest that, while Atmyb93 may show slight insensitivity to S deprivation, factors in addition to AtMYB93 also contribute to root S deprivation responses. S stress does not affect the spatial localisation of AtMYB93 promoter activity As S deprivation increases AtMYB93 gene expression ( Bielecka et al. 2014 ) and as AtMYB93 transcriptional activity is restricted to a very few cells in the root, we investigated whether endodermal localization of AtMYB93 changes under S deprivation using transgenic Arabidopsis expressing pAtMYB93::GUS ( Gibbs et al. 2014 ). No change in the localisation and extent of AtMYB93 promoter activity was seen under S stress ( Fig. 6 ). Download figure Open in new tab Fig. 6 Spatial localization of AtMYB93 promoter activity is not changed by S stress. At MYB93 promoter activity visualised in wild type plants expressing pAtMYB93::GUS ( Gibbs et al 2014 ) under normal sulphur levels (+S) and S deprivation (-S) in 7-day old roots. 2 examples of each treatment are shown. Cells expressing pAtMYB93::GUS are highlighted with arrows. Developing Lateral Root Primordia are highlighted with arrowheads. Scale bars represent 50μm Discussion The Atmyb93 mutant transcriptome highlights root responses to S Despite the known (albeit redundant) role for At MYB93 in endodermal suberin production ( Klein 2019 ; Shukla et al. 2021 ) the Atmyb93 mutant root transcriptome does not show changes in suberin metabolism or transport genes. This is in agreement with an experiment that identified genes differentially expressed between wild type and Atmyb93 root segments containing gravity-induced 46h lateral root primordia, which identified cell wall metabolism as an enriched biological process but no suberin metabolism genes ( Uemura et al. 2023 ). However, there is almost no gene overlap between the dataset from root segments at this very specific, synchronised developmental time point ( Uemura et al. 2023 ) and our whole-root dataset (Online Resource 2). Instead, genes differentially expressed in Atmyb93 mutant whole roots compared to wild type are enriched in GO terms for S transport and metabolism, carbohydrate metabolism, flavonoid-and phenylpropanoid metabolism and responses to oxygen and other stimuli. This profile is similar to S deprivation response transcriptomes (e.g. ( Bielecka et al. 2014 ; Canales et al. 2020 ; Chandra and Pandey 2014 ; Forieri et al. 2017 ; Henriquez-Valencia et al. 2018 ; Lunde et al. 2008 ; Robe et al. 2020 ; Wawrzynska et al. 2022 )). The trend of down- and upregulated genes suggests that the Atmyb93 mutant is in a ‘S-replete’ state as the key S deprivation response genes APR2 and SDI2 are downregulated in Atmyb93 but upregulated upon S stress ( Fig. 1c ; ( Maruyama-Nakashita et al. 2003 ; Aarabi et al. 2016 )). The two sulphate transporters downregulated in the Atmyb93 mutant are the vascular transporters SULTR1;3 (phloem) and SULTR3;5 (xylem, pericycle and LRP), which mediate long-distance transport from source to sink and from root to shoot, respectively ( Kataoka et al. 2004 ; Yoshimoto et al. 2003 ). The genomic region of At1g61740 sulfite exporter, which is upregulated in Atmyb93 , was also identified as binding to At MYB93 in a DNA-affinity purification-sequencing (DAP-seq) experiment ( O’Malley et al. 2016 ). Interestingly, root to shoot movement of sulphate under drought stress affects leaf ABA biosynthesis and stomatal closure via S-incorporation into the amino acid cysteine ( Batool et al. 2018 ; Cao et al. 2014 ). As AtMYB93 is expressed only in a very few cells of the root ( Gibbs et al. 2014 ) it may be mediating localised changes in or responses to S levels in the root that affect responses to the environment. AtMYB93 , S stress and root development At MYB93 is a known negative regulator of lateral root development ( Gibbs et al. 2014 ) that is upregulated by S stress (( Bielecka et al. 2014 ); this work). Previous research has shown that S stress can reduce LR development ( Dan et al. 2007 ; Dong et al. 2019 ; Joshi et al. 2019 ) and enhance primary root elongation ( Zhao et al. 2014 ). Although we hypothesised that these changes in root development upon S stress would be mediated by Atmyb93 , we did not see clear differences in S deprivation root phenotypes in wild type or Atmyb93 plants in our experiments. Previous experiments showing LR changes in wild type plants either measured lateral root number (rather than density) and lateral root length ( Joshi et al. 2019 ), or changes were measured only in the context of S resupply ( Dong et al. 2019 ), or in much older (15 day) seedings ( Dan et al. 2007 ). This suggests that S regulation of LR development may have context-dependent nuances, only some of which involve At MYB93, or involve At MYB93 working partly in concert with other related R2R3 MYBs. Moreover, S deprivation does not extend the localisation of pMYB93::GUS expression beyond those endodermal cells overlying lateral root primordia, similarly to what is seen with ABA ( Gibbs et al. 2014 ), suggesting that the extent of AtMYB93 promoter activity is tightly restricted even under stress conditions. S stress promotes flavonoid biosynthesis and increases anthocyanin levels (( Dong et al. 2019 ; Bielecka et al. 2014 ; Nikiforova et al. 2003 )). GO terms for phenylpropanoid- and flavonoid biosynthesis are enriched in the genes downregulated in the Atmyb93 mutant compared to wild type ( Fig. 1a ). The key flavonoid regulator AtMYB12 ( Mehrtens et al. 2005 ) is downregulated In the Atmyb93 mutant, along with genes in the flavonoid biosynthesis pathway ( CYP75B1 (a flavonoid 3’ hydroxylase), UGT78D2 , UGT84A1 , UGT84A2 , UGT90A1 ; Online Resource 2). Moreover, AtMYBL2 , an inhibitor of anthocyanin biosynthesis ( Dubos et al. 2008 ; Matsui et al. 2008 ), is upregulated in the Atmyb93 mutant (Online Resource 2). These data suggest that Atmyb93 mutant roots would contain reduced flavonoids including anthocyanins. These gene expression changes could at least in part explain the increased root phenotypes of the Atmyb93 mutants as flavonoids inhibit lateral root development ( Brown et al. 2001 ; Chapman and Muday 2021 ) while At MYB12 is known to regulates cell division orientation and cell differentiation in the root ( Wybouw et al. 2023 ). Phenylpropanoids (which include flavonoids and lignin precursors) are required for both lignin- and suberin production and maintenance in the endodermis ( Andersen et al. 2021 ). This could explain the role of AtMYB93 in suberin deposition. Moreover, the transcription factor DEWAX, a negative regulator of very long chain fatty acid (VLCFA) biosynthesis ( Go et al. 2014 ), is upregulated in the Atmyb93 mutant (Online Resource 2) suggesting that At MYB93 could affect VLCFA levels surrounding the root cells in which it is expressed. However, neither the quadruple AtMYB41/53/92/93 mutant with highly reduced suberin nor the Atmyb92 mutant (with reduced suberin ( Klein 2019 )) has a lateral root phenotype ( Gibbs et al. 2014 ; Shukla et al. 2021 ), arguing against a clear role for suberin itself in LR development. AtMYB12 is downregulated in the Atmyb39 / suberman mutant ( Cohen et al. 2020 ), but the lateral root phenotype of this mutant is not known. Interestingly, in our experiments S deprivation increases adventitious root formation at the root-hypocotyl junction. To our knowledge, this is the first time that S deprivation has been shown to affect adventitious rooting at the collet in Arabidopsis . S stress reduces crown root formation in rice ( Grewal et al. 2018 ) most likely via a non- MYB93 mechanism as OsMYB93 s are not upregulated by S stress ( Fig. 3 ). Flavonoids are known to inhibit AR formation in the hypocotyl in the presence of auxin (Correa Lda et al. 2012) although a mutant lacking flavonoids makes fewer ARs than wild type (Correa Lda et al. 2012). Whether the Atmyb93 adventitious root sensitivity is linked to flavonoids is unknown. AtMYB93 , shoot element composition, root architecture and root barriers The Atmyb93 mutant shows a general trend of elevated shoot elements, significantly S, Mg and B, while two tomato SlMYB93 -overexpressing lines show the opposite trend. The third SlMYB93 -overexpressing line broadly resembles wild type plants, perhaps due to position-specific insertion effects of the transgene ( Schnell et al. 2015 ). The role of suberin on S uptake and assimilation is not fully understood. S deprivation increases and extends suberisation in the root leading to fewer passage cells ( Barberon et al. 2016 ; Ogden et al. 2018 ) and loss of endodermal suberin enhances the S deficiency phenotype of an S transporter mutant ( Barberon et al. 2016 ) emphasising the requirement of an in-tact endodermal barrier for S uptake and retention. However, ionomic analysis shows shoot sulphate ion composition either not changing or being elevated in suberin-deficient transgenic plants made by expressing a suberin-degrading enzyme in the endodermis ( ELTP::CDEF1 and CASP1::CDEF1 respectively, ( Barberon et al. 2016 )). Furthermore, another ionomic analysis shows no change in S in ELTP::CDEF1 plants, a slight S reduction in the Atmyb41 / Atmyb53 / Atmyb92 / Atmyb93 quadruple mutant plants and no change/a slight reduction in S in plants overexpressing AtMYB41 in the endodermis ( ELTP::MYB41 and CDEF1::MYB41 respectively, ( Shukla et al. 2021 )). In both studies, the profiles of Mg and B are more in-line with our Atmyb93 mutant data, being generally elevated with reduced suberin and reduced with elevated suberin (( Barberon et al. 2016 ; Shukla et al. 2021 ); Fig. 4 ). Plants with elevated suberin due to AtMYB39 overexpression show reduced shoot elemental S (and several other elements), similarly to our SlMYB93 overexpression data, but the Atmyb39 mutant does not show elevated S ( Cohen et al. 2020 ). Conversely, the enhanced suberin mutant esb1 has elevated shoot sulphate levels ( Baxter et al. 2009 ). Together, these data imply that changes in suberin are not the primary driver of the changes in shoot S seen in the Atmyb93 mutant: these changes are more readily explained by alterations in root architecture and/or changes in S assimilation and transport caused by the perturbed Atmyb93 transcriptome. Although SULTR3;5 is downregulated in the Atmyb39 mutant as in Atmyb93 , AtSULTR3;5 and another S transporter AtSULTR1;2 are both downregulated in an At MYB39-overexpressing line and there is no other S-related overlap between the Atmyb93 and Atmyb39 mutant transcriptomes ( Cohen et al. 2020 ). Importantly, the fatty acid elongation required for suberin biosynthesis requires acetyl coenzyme A (acetyl coA), a S-containing compound ( Woolfson et al. 2022 ) suggesting a possible link between suberin biosynthesis and S metabolism. We suggest that AtMYB93 is indirectly involved, via phenylpropanoid metabolism, in increasing the amount and extent of suberisation specifically in its highly localised regions of expression during S deprivation, whilst also additionally responding to S levels to change S transport and metabolism. Conclusion and future directions We have shown that MYB93 regulates responses to S levels. The Atmyb93 transcriptome shows a S stress signature, and MYB93 homologues in two dicot plants ( Arabidopsis and tomato) are upregulated by S deprivation. Arabidopsis and tomato with perturbed MYB93 levels show changes in shoot S content. S deprivation changes root architecture in Arabidopsis but this is not mediated solely by AtMYB93 . At MYB93’s effect on lateral root development is likely via flavonoids rather than suberin as there is no evidence for direct suberin regulation by At MYB93: future work will investigate this link further. Materials and Methods Plant materials and growth conditions Arabidopsis ecotype Col-0 wild type, Atmyb93 mutant ( Gibbs et al. 2014 ), Atmpk3-1 mutant (SALK_151594, NASC ID N869692; ( Merkouropoulos et al. 2008 )) and pAtMYB93::GUS transgenic lines ( Gibbs et al. 2014 ) were grown in the glasshouse in Levington M3 compost/vermiculite mix at 22°C under 16h light. For growth on plates, Col-0, Atmyb93 , Atmpk3 seeds were sterilised for 10 minutes in 10% Parozone TM Bleach (Jeyes, Hemel Hempstead, UK) followed by 3 rinses in sterile distilled water and resuspension in 200µl distilled water. Seeds were vernalised at 4°C for 2 days in the dark and plated in rows to grow vertically at the top of half-strength Murashige and Skoog (MS) medium (M0404, Sigma-Aldrich, St Louis, Missouri, USA) pH5.6-5.8 with 1% agar. For RNAseq analysis, the medium used was Sigma-Aldrich MS M0404 and seedlings were grown for 7 days. For root assays of Col-0, Atmyb93 and Atmpk3 (comparing genotypes, or OAS treatment) the medium used was half-strength MS (Sigma-Aldrich M0404), with seedlings grown for 8 days. For S deprivation root assay experiments, the medium used was half-strength MS either containing (+S) or not containing (-S) S (MS basal salts MSP01 and MSP44; Caisson Labs, Smithfield, Utah USA) with 1% agar, with seedlings grown for 8 days. For some experiments, seedlings were initially germinated and grown on +S for 5 days and then transferred to -S for 3 days, using sterile forceps in a sterile laminar flow cabinet. All plates were sealed with micropore tape (3M, St Paul, Minnesota, USA). For Arabidopsis fresh weight and shoot element analysis, Col-0 and Atmyb93 were grown in Magenta pots (Sigma) containing 4 evenly spaced seedlings per pot. Pots were filled to half capacity (125ml) with 0.5 strength MS made using the Merck classic protocol (KGaA 2018) with 1% sucrose and 0.7% agar (Sigma-Aldrich CAS 9002-18-0). For medium without S, all sulphate ions were replaced with the chloride equivalent. Foil was wrapped round the part of the pot with agar to mimic soil darkness and facilitate root penetration of the agar. Pots were sealed with micropore tape (3M). Solanum lycopersicum cultivar Micro-Tom was grown in an incubator (24°C) or glasshouse (22°C) in Levington M3 compost/vermiculite mix under 16h light. Seeds were sown directly onto compost unless transformation was to be carried out (see tomato transformation section); soil-grown plants were used for elemental analysis, RT-PCR analysis and cloning (roots were washed before making RNA). RNA extraction and cDNA generation Plant tissue was ground in liquid nitrogen using RNAse-free ceramic pestles and mortars and RNA extraction was performed using an ISOLATE II Plant RNA kit (Bioline, Meridian Biosciences, Memphis, TN, USA). Transcriptome sequencing and analysis RNA from ∼30mg of tissue per sample of whole 7-day old roots of wild type and Atmyb93 (triplicate samples) was extracted and quality checked by both calculating the OD260/280 and OD 260/230 ratios of a 1µl sample using a Nanodrop analyser and by agarose gel electrophoresis to visualise ribosomal RNA integrity (Online Resource 1). Samples were sent to Novogene for further quality control (Agilent 2100 analysis), library preparation and sequencing. Briefly, sample mRNA was enriched using oligo(dT) coupled beads and RNA was fragmented using buffer containing 0.1mM ZnCl2. cDNA libraries were synthesised from mRNA fragments using random hexamers and reverse transcriptase followed by second strand cDNA synthesis using dNTPs, RNaseH and E. coli DNA polymerase I. The library was finished by carrying out end-repair, A-tailing, adaptor ligation, size selection and amplification by PCR. Library insert size and concentration was checked before Illumina sequencing. Raw data were transformed by base calling into sequenced reads (including sequence information and sequence quality information), which were exported in FASTQ format. For quality control, Phred scores were used to estimate the error rate of sequenced reads. Raw reads were filtered to remove adapters and low-quality reads. Cleaned FASTQ data were aligned to the Arabidopsis reference genome using HISAT2 ( Kim et al. 2019 ). Clean, mapped fragments representing transcript abundance were used to estimate the expression level of each gene in each sample via fragments per kilobase of transcript sequence per million fragments paired sequenced (FKPM) values using HTseq software in union mode ( Anders et al. 2015 ). Normalised FKPM values were subject to Pearson’s correlation analysis to estimate similarity between biological repeats. Differentially expressed genes (DEGs) were identified using DEseq2 software with the DEseq normalisation method with a false discovery rate (FDR) < 0.05. GO enrichment analysis was carried out separately on down- and up-regulated DEGs, using the GO enrichment tool in PlantRegMap ( Tian et al. 2020 ) with a cutoff of p<0.01. Enriched GO terms were visualised using ReviGo ( Supek et al. 2011 ). Phylogeny and in silico expression analysis of MYB93-related genes AtMYB93 -related protein sequences were selected as follows: (i) the Arabidopsis genes known to be involved in suberin formation (refs) that are (ii) also most closely related at the protein sequence level to At MYB93 (Clades S24, S10, S11, S42; Du et al 2015 ), (iii) sequences from representative dicots and monocots identified as being most similar to At MYB93 by BLASTP (Altschul et al), mostly from species for which published expression data was also available. Chosen species were Brassica napus , Brassica rapa , Glycine max (soybean), Populus trichocarpa (poplar), Manihot esculenta (cassava), Solanum lycopersicum (tomato), Malus domestica (apple), Oryza sativa (rice), Zea mays (maize), Brachypodium distachyon , Hordeum vulgare (barley). Sequences were obtained from NCBI, Phytozome and BrassicaDB ( http://brassicadb.cn/ #/ ). Sequences were aligned using MUSCLE ( https://www.ebi.ac.uk/jdispatcher/msa ) and a simple Neighbour-Joining tree was generated, which was visualized using FigTree ( http://tree.bio.ed.ac.uk/software/figtree/ ). Expression data was extracted from a combination of the ePlant ( Waese et al. 2017 ) and the Arabidopsis eFP browser RootII dataset ( Iyer-Pascuzzi et al. 2011 ; Winter et al. 2007 ) and papers where expression of MYB93-related genes or whole-plant responses to S deprivation are recorded ( Bielecka et al. 2014 ; Canales et al. 2020 ; Du et al. 2012 ; Jain et al. 2007 ; Kajala et al. 2021 ; Libault et al. 2009 ; Sekhon et al. 2011 ; Wang et al. 2022 ; Wilkins et al. 2009 ; Wilson et al. 2017 ). Data was visualised semi-quantitatively using colour intensity to provide an overall summary of trends. RT-PCR and semi quantitative RT-PCR For cloning and RT-PCR in tomato, cDNA synthesis was performed from RNA using the SuperScript TM III first-strand synthesis system (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). For expression analysis of SlMYB93 homologues, PCR was carried out on the cDNA using Taq polymerase using primers in Online Resource 2. Generation of an SlMYB93 overexpression construct and transformation into Arabidopsis and tomato Full-length SlMYB93 ( Solyc04g074170 ) cDNA was cloned from tomato roots cv. Ailsa Craig using a proofreading DNA polymerase (Phusion TM , New England Biolabs, Ipswich, MA, USA) and relevant primers (Online Resource 2) into a modified pBI121 vector (Clontech), mpBI12135S, with modifications to the left and right border (Yongsheng Liu personal communication; ( Cao et al. 2012 )). mpBI121 contains additional restriction sites compared to pBI121, including Kpn I, Sal I and Xho I, and the GUS reporter gene has been removed, so that only the NOS terminator remains at the end of the multiple cloning site. mpBI121 contains the nptII kanamycin resistance gene for selection in plants. SlMYB93 in mpBI121 was transformed into electrocompetent Agrobacterium strain GV3101 by electroporation at 2.5kV, 25μF capacitance and 400 Ο resistance.The p35S::SlMYB93 construct was transformed into Arabidopsis via floral dip ( Clough and Bent 1998 ). For transformation of p35S::SlMYB93 into tomato, Solanum lycopersicum cv. Micro Tom seeds were imbibed in water for 1h at 37°C and sterilised by soaking in 20% Parozone TM (Jeyes, Hemel Hempstead, UK) bleach for 20 min followed by 3 washes with 30ml autoclaved water and drying on autoclaved filter paper in a laminar flow hood. Seeds were then rinsed in sterile water at least 3 further times in fresh tubes to completely remove all bleach residues and air-dried. Seeds were placed on half-strength MS basal salts (M5524, Sigma-Aldrich, St Louis, Missouri, USA) containing 2% sucrose in Magenta pots (Sigma-Aldrich, St Louis, Missouri, USA) and cold-treated (4°C) in the dark for at 2-3 days before moving to a growth room at 22°C in the dark for 2 days. If most seeds germinated, pots were transferred to a 16h light /8h dark cycle for 4-5 days. From these plants, tomato transformation was carried out as in ( Cao 2022 ). Briefly, cotyledons were wounded and transformed with Agrobacterium containing p35S::SlMYB93 before rounds of kanamycin selection to generate transformed callus producing shoots, followed by transfer to rooting medium and further transfer of rooted, transformed plants to soil. Shoot element analysis of Arabidopsis ICP-MS Arabidopsis plants were grown in Magenta pots as detailed previously and harvested using tweezers at 21 days, before flowering had begun. Roots were removed with a razor blade and pooled fresh shoot tissue was heated at 60°C for 48h in an oven to remove all water. At least 100mg of dry shoot tissue for each condition was analysed by ICP-MS as in ( Thomas et al. 2016 ). Significant differences were identified by Mann Whitney U-tests comparing Atmyb93 mutants to wild type. Shoot element analysis of tomato by ICP-OES For tomato shoot element analysis, ∼20g fresh weight of young leaves (the first 10-12 leaves behind the growing tip) were harvested from ∼6-week old plants of each genotype, which were dried at 50°C in an oven for 48h. At least 0.2g dry weight of each type of leaf tissue was sent for analysis by inductively coupled plasma optical emission spectroscopy (ICP-OES) at Yara Analytical Services, York, UK. Briefly, samples were dried, milled, and sieved prior to analysis. Samples were digested in nitric acid using a microwave digester (CEM, Buckingham, UK). The digested samples were analysed on an Agilent ICP 5900 ICP-OES instrument (Agilent Technologies LDA UK Ltd, Stockport, UK) calibrated against standards of known concentrations. Significant differences between wild type and SlMYB93 -overexpressing lines were identified by Mann Whitney U-tests. Arabidopsis root assay quantification Arabidopsis seedlings growing vertically on +S or -S medium were photographed. Emerged lateral roots and adventitious roots (roots emerging from the collet) were counted by eye from plates and root length was measured from photographs using the freehand drawing tool in ImageJ ( https://imagej.net/ij/ ). Lateral root density was calculated for each seedling by dividing lateral root number by primary root length. All root data was visualised using SuperPlots (( Lord et al. 2020 ); https://huygens.science.uva.nl/SuperPlotsOfData/ ) Statistical significance was calculated via a Kruskal-Wallis test with a post-hoc Dunn’s test, or by a Mann-Whitney U-test for pairwise comparisons. AtMYB93 promoter activity Seedlings expressing pAtMYB93::GUS ( Gibbs et al. 2014 ) were grown vertically on 0.5MS (KGaA 2018) plates with or without S and with 1% agar (Sigma-Aldrich CAS 9002-18-0) for 5-8 days. GUS assays were carried out as in Gibbs et al (2014) . Images were acquired by light microscopy using a GXCAM-HiChrome-S camera (GT vision, Stansfield, UK). Funding and acknowledgements XC was funded by China Scholarship Council studentship 201606690032. CC and HW were funded by UK Biotechnology and Biological Sciences Research Council (BBSRC) doctoral training grant BB/M01116X/1. NSG was funded by BBSRC SARIC grant, BB/N004302/1. We thank Jasmine Carlson, Maxwell Ware, Ahmed Hussain, Lisa King, Elizabeth Chapman, Jessica Finch and Adam Elgey for related preliminary work on aspects of At MYB93 function and abiotic stress that informed the direction of this paper. Competing interests The authors declare no competing interests. Author Contributions XC and JCC designed research. All authors performed research and analysed data. XC, HW, AO, RE and JCC visualised data. JCC supervised the project. XC and JCC wrote the manuscript. All authors reviewed the manuscript. 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