A role for LAX2 in regulating xylem development and lateral ...genotypes were grown together and...

Annals of Botany 120: 577–590, 2017doi:10.1093/aob/mcx091, available online at www.academic.oup.com/aob

© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected]

A role for LAX2 in regulating xylem development and lateral-vein symmetryin the leaf

Guillermo S. Moreno-Piovano1,†, Javier E. Moreno1,†, Julieta V. Cabello1, Agust�ın L. Arce1, Mar�ıa E. Otegui2

and Raquel L. Chan1,*

1Instituto de Agrobiotecnolog�ıa del Litoral, Universidad Nacional del Litoral - CONICET, Facultad de Bioqu�ımica y CienciasBiol�ogicas, Colectora Ruta Nacional 168 km 0, Santa Fe, Argentina and 2Facultad de Agronom�ıa, Universidad de Buenos

Aires, CONICET, Argentina*For correspondence. E-mail [email protected]†G.S.M.P. and J.E.M. contributed equally to this work.

Received: 5 October 2016 Returned for revision: 23 February 2017 Editorial decision: 14 May 2017 Accepted: 9 June 2017

� Background and Aims The symmetry of venation patterning in leaves is highly conserved within a plant species.Auxins are involved in this process and also in xylem vasculature development. Studying transgenic Arabidopsisplants ectopically expressing the sunflower transcription factor HaHB4, it was observed that there was a significantlateral-vein asymmetry in leaves and in xylem formation compared to wild type plants. To unravel the molecularmechanisms behind this phenotype, genes differentially expressed in these plants and related to auxin influx wereinvestigated.� Methods Candidate genes responsible for the observed phenotypes were selected using a co-expression analysis.Single and multiple mutants in auxin influx carriers were characterized by morphological, physiological and molec-ular techniques. The analysis was further complemented by restoring the wild type (WT) phenotype by mutant com-plementation studies and using transgenic soybean plants ectopically expressing HaHB4.� Key Results LAX2, down-regulated in HaHB4 transgenic plants, was bioinformatically chosen as a candidategene. The quadruple mutant aux1 lax1 lax2 lax3 and the single mutants, except lax1, presented an enhanced asym-metry in venation patterning. Additionally, the xylem vasculature of the lax2 mutant and the HaHB4-expressingplants differed from the WT vasculature, including increased xylem length and number of xylem cell rows.Complementation of the lax2 mutant with the LAX2 gene restored both lateral-vein symmetry and xylem/stem arearatio in the stem, showing that auxin homeostasis is required to achieve normal vascular development. Interestingly,soybean plants ectopically expressing HaHB4 also showed an increased asymmetry in the venation patterning, ac-companied by the repression of several GmLAX genes.� Conclusions Auxin influx carriers have a significant role in leaf venation pattering in leaves and, in particular,LAX2 is required for normal xylem development, probablt controlling auxin homeostasis.

Key words: Auxin influx carriers; HD-Zip I; HaHB4; LAX2; venation symmetry; vascular patterning; xylemorganization.

INTRODUCTION

Organisms have evolved complex traits to cope with the sur-rounding environment. Biological systems show high resilienceto external perturbations that are somehow buffered by the reg-ulatory interaction of developmental networks (Masel andSiegal, 2009; Lempe et al., 2013; Payne and Wagner, 2015). Incertain scenarios, restoration of the individual’s homeostasiscannot be fully reached, leaving a permanent mark on develop-mental patterning (Leamy and Klingenberg, 2005). Ecologistshave largely recorded this environmental footprint in the devel-opment of organ symmetry, such as symmetry of wings andembryos in Drosophila (Klingenberg et al., 1998;Houchmandzadeh et al., 2002), mandibles in mice (Leamyet al., 2002), and leaf-lamina (Mu~noz-Nortes et al., 2014;Graham et al., 2015) and leaf-venation patterning (Aloni et al.,2003) in plants. Developmental gene regulatory networks playa central role in the resilience to environmental stress.

For example, the heat-shock protein 90 (Hsp90) chaperone ap-pears to be a conserved molecular capacitor in phylogeneticallydistant groups (Rutherford and Lindquist, 1998; Queitsch et al.,2002). These results underlined the role of Hsp90 in bufferingthe genetic variation within a population, and also highlightedthe fact that genetic variation can be exposed when bufferingsystems are strained by the environment. Plant ecologists havescored the impact of environmental stress on organs with bilat-eral symmetry, traditionally on leaves with a central vein.Recently, it was reported that slight differences in leaf bilateralsymmetry of Arabidopsis and tomato plants was correlated to adifferential auxin distribution on phyllotactic patterning(Chitwood et al., 2012). Plant symmetry has also been studiedon venation patterning (Aloni, 2010). In this regard, it has beenproposed that auxin distribution in the leaf lamina is involvedin the control of venation symmetry (Aloni, 2001; Aloni et al.,2003). These studies combined experiments with exogenous ap-plication of auxin and the use of the synthetic auxin-responsive

VC The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.All rights reserved. For Permissions, please email: [email protected]

Annals of Botany Page 1 of 14doi:10.1093/aob/mcx091, available online at https://academic.oup.com/aob

Moreno-Piovano et al. — LAX2 modulates leaf lateral-vein symmetry578

promoter DR5. This promoter fused to the GUS-reporter generevealed strong b-glucuronidase (GUS) activity on hydathodesof developing leaves. The high auxin accumulation at the stilldifferentiating hydathodes may regulate the differentiation ofupper secondary veins, thus controlling venation symmetry(Aloni et al., 2003). This model was supported by the followingobservations: (1) mutants with altered patterns of auxin accu-mulation translated into changes of the normal venation pattern-ing (Koizumi et al., 2000; Aloni et al., 2003), and (2)exogenous application of auxin-transport inhibitors or syntheticauxin analogues to Arabidopsis leaves caused an altered num-ber of secondary veins (Mattsson et al., 1999; Sieburth, 1999;Aloni, 2010).Auxins are exported and imported from cell to cell using ef-

flux and influx carriers: PIN and AUX/LAX, respectively.Auxin homeostasis in the leaf lamina is critical to achieve theappropriate growth response, and auxin transport appears to becritical for auxin homeostasis along the whole plant body.Auxin homeostasis includes auxin distribution, hormone syn-thesis and inactivation rates, and stability of receptors and re-pressors (Petrasek and Friml, 2009; Vanneste and Friml, 2009;Salehin et al., 2015). It is interesting to note that members ofboth the PIN and the AUX/LAX gene families have evolvedsub-functionalization. For instance, the aux1 mutant producedinhibition of root hair elongation and altered gravitropic re-sponse (R. Swarup et al., 2005, 2007), lax1 and lax3 mutantsproduced a decrease in lateral root formation (K. Swarup et al.,2008), and the lax2 mutant caused vascular breaks in cotyle-dons (Peret et al., 2012). In addition, leaf phyllotaxy was signif-icantly altered by mutations in AUX1 and LAX1 genes(Bainbridge et al., 2008). Recently, a direct connection betweenauxin influx carriers and vasculature development has beenshown in Arabidopsis. The quadruple aux1 lax1 lax2 lax3(hereafter quad) mutant, but not the single aux/lax mutants, de-veloped fewer and less-dense vascular bundles than the wildtype (WT) only when grown under short-day conditions(Fabregas et al., 2015). Yet, the vascular bundle organization ofthe quad mutant was uneven, with an increased number of pro-cambial and xylem cells (the latter of enhanced size) both inshoots and in roots (Fabregas et al., 2015).Transcription factors (TFs) are modular proteins able to artic-

ulate the perception of environmental signals to cellular re-sponses. The homeodomain-leucine zipper (HD-Zip) TF familycoordinates different aspects of the plant biology including, butnot limited to, plant biotic and abiotic stress tolerance, leafshape and polarity, vascular development, and root and tri-chome formation (Capella et al., 2015b). The HD-Zip familycomprises four subfamilies, I–IV. HD-Zip subfamily I (hereaf-ter HD-Zip I) TFs were initially associated with plant responsesto the environment and more recently these regulatory proteinshave been found to participate in developmental processes(Perotti et al., 2017).HaHB4 is an HD-Zip I TF from Helianthus annuus, defined

by phylogenetic analysis as an uncommon member given its ex-tremely short and divergent carboxy-terminus (Arce et al.,2011). HaHB4 ectopic expression improved drought and salin-ity tolerance in transgenic Arabidopsis and soybean plants withno penalty on yield under control conditions (Dezar et al.,2005; Arcadia-Biosciences, 2015). In Arabidopsis, this in-creased tolerance is indeed part of a complex phenotype that

includes delayed senescence and enhanced resistance to herbiv-ory (Manavella et al., 2006, 2008). However, the complex phe-notype triggered by HaHB4 in transgenic plants is not wellunderstood.In the current study, Arabidopsis and soybean transgenic

plants expressing HaHB4 were used as a prospecting tool to un-cover novel traits related to signal transduction pathways regu-lated by this TF. A co-expression analysis performed on genesdifferentially expressed in Arabidopsis HaHB4-transgenicplants allowed us to identify LAX2 as a potential down-regulated target of HaHB4. Then, the phenotypes of single andmultiple mutant alleles of the AUX/LAX gene family were char-acterized. An increased asymmetry in the venation patterningof Arabidopsis lax2 mutant alleles was observed. We also re-vealed the functional role of the auxin influx carrier LAX2 as anegative regulator of xylem development in Arabidopsis plants.The asymmetric venation phenotype was also observed in35S:HaHB4 Arabidopsis leaves as well as in soybean plants ex-pressing HaHB4 under the control of its native promoter.Finally, we linked this venation phenotype to the LAX2 auxininflux carrier based on HaHB4-mediated down-regulation ofLAX2 expression in Arabidopsis and GmLAX genes in soybean.

MATERIALS AND METHODS

Plant material, growth conditions and treatments

Arabidopsis thaliana plants were grown on Klasmann SubstratNo. 1 compost (Klasmann-Deilmann GmbH, Geeste, Germany)in a growth chamber at 22–24 �C under long-day conditions(16/8-h light/dark cycles) with a light intensity of 120 mmolm�2 s�1 in 8 � 7-cm pots. In all Arabidopsis experiments theCol-0 ecotype was used as the WT control and four plants wereplanted per pot.A total of six Arabidopsis mutant genotypes were grown; the

mutants used were the lax2-1, lax2-2, lax1, lax3, aux1-21 andquad, all in the Col-0 background. Arabidopsis null mutantlines lax2-1 (dSpm line) and lax2-2 (GK_345D11) were de-scribed previously (Peret et al., 2012). Arabidopsis plants bear-ing the HaHB4 cDNA driven by the 35S cauliflower mosaicvirus promoter were also previously described (Dezar et al.,2005).In the case of soybean (Glycine max ‘Williams 82’), two ge-

notypes were evaluated. Seeds of the WT and those expressingthe sunflower HaHB4 cDNA under its native promoter (namedhere as b10H event) were obtained from INDEAR (Rosario,Argentina). Soybean plants of each genotype were grown in20-litre pots (five plants per pot) up to the V7 developmentalstage. Pots bearing transgenic or control plants were arrangedin a glasshouse under long-day conditions (approx. 15/9-h light/dark cycles), with daily temperatures fluctuating between 19 �C(during the night) and 40 �C (during the day). Although thetemperature amplitude reached almost 30 �C in a day, all plantgenotypes were grown together and exposed to the sameconditions.Two water regimes (irrigated and water deficit) were im-

posed between developmental stages V5 and V7, representing14 d of treatment in December–January at our latitude. Duringthis period, one group of plants (20 plants of each genotype) re-ceived adequate water supply whereas irrigation was arrested in

Page 2 of 14 Moreno-Piovano et al. — LAX2 modulates leaf lateral-vein symmetry

the second group (20 plants). Plant measurements were taken atthe V7 stage in both groups.

RNA isolation and expression analyses by real time RT-PCR

Total RNA for real-time RT-PCR was isolated fromArabidopsis leaves or stems using Trizol reagent (Invitrogen,Carlsbad, CA, USA) according to the manufacturer’s instruc-tions. Total RNA (1lg) was reverse-transcribed usingoligo(dT)18 and M-MLV reverse transcriptase II (Promega,Fitchburg, WI, USA).

Total RNA from soybean was isolated from the terminalfoliole of the last fully developed leaf of three individual soy-bean plants (V7 stage). The samples were processed using thesame reagents and protocol described for Arabidopsis tissue.

Quantitative real-time PCR (RT-qPCR) was performed usinga Mx3000P Multiplex qPCR system (Stratagene, La Jolla, CA,USA) as described by Capella et al. (2015a) and using the pri-mers listed in Supplementary Data Table S1. Transcript levelswere normalized by applying the DDCt method. Actin tran-scripts (ACTIN2 and ACTIN8 for Arabidopsis and GmActin forsoybean) were used as internal standards to normalize differ-ences in template amounts. Three biological replicates, ob-tained by pooling tissue from three to four individual plants andtested in duplicate, were used to calculate the standarddeviation.

Genetic constructs and Arabidopsis plant transformation

A Gateway-compatible plasmid containing the LAX2 cDNAwas obtained from ABRC (Ohio State University, Columbus;clone U15627) and recombined into the pJV126 expressionvector (gift of Dr D. Weigel, Max Planck Institute, Tübingen-Germany) using the Gateway LR Clonase II Enzyme Mix(Thermo Fisher, Waltham, MA, USA) according to the manu-facturer’s instructions. The pJV126 vector is derived from thepGreen vector series: genetic fragments were recombined byGateway (Life Technologies) cloning into a modified pGreenvector (pFK210) conferring resistance to BASTA (Hellenset al., 2000). A 35S CaMV promoter drives the expression of anN-terminal fusion protein with mCitrine, here named35S:mCitrine:LAX2. A sequence-verified clone was used fortransforming Agrobacterium tumefaciens strain LBA4404 andthen generating transgenic Arabidopsis plants using the floraldip method (Clough and Bent, 1998). To screen for high-expressing lines, we followed a two-step selection process.First, T1 plants were grown in soil for 7 d and watered with asolution containing 0�3 lM glufosinate (Liberty; BayerCropScience, Leverkusen, Germany), and second, we used aLeica TCS SP8 Compact confocal microscope to screen forhighly fluorescent seedlings that accumulated mCitrine-LAX2fusion protein. T2 plants containing a single transfer DNA in-sertion (as determined by 3 : 1 segregation of herbicide resis-tance) were further used in experiments. All experiments weredone with at least three independent transgenic lines probablycontaining a single T-DNA. Multiple tandem insertions cannotbe ruled out with this selection procedure and therefore severalindependent lines were evaluated simultaneously. Homozygous

T3 lines were further used to analyse transgene expression lev-els and plant phenotypes.

Microarray data analysis and clustering

The transcriptome analysis of 35S:HaHB4 Arabidopsisplants was previously reported (Manavella et al., 2006). Thesedata were used in an exploratory analysis in which genes re-sponding to HaHB4 overexpression but not to drought stress inWT or in 35S:HaHB4 plants were selected. More precisely, thechosen set of genes satisfied the following condition: an abso-lute log2 expression ratio greater than 0�5 in 35S:HaHB4 vs.WT plants, and lower than 0�5 in stressed vs. non-stressedplants. This resulted in a list of 497 genes, which was furtherused in a co-expression analysis with public expression datasetsusing the R environment for statistical computing (https://www.r-project.org/). This analysis consisted in first retrieving publicArabidopsis microarray experiments from the AtGenExpressproject (http://jsp.weigelworld.org/AtGenExpress/resources/).The gene expression datasets considered were: abiotic stress inroots, shoots and cell cultures (Kilian et al., 2007), pathogentreatment and the developmental series (Schmid et al., 2005).Expression profiles of the 446 genes with measurements in themicroarrays, i.e. presenting probesets in the Affymetrix ATH1microarray, were then pairwise correlated. Correlations wereconverted to distances by subtracting their absolute value from1, and the resulting matrix was used to perform a hierarchicalclustering with the average agglomeration method. Gene clus-ters were then defined by cutting the tree at 0�4, and their geneswere analysed using the GeneMANIA server (Warde-Farleyet al., 2010). Description of the cluster is given inSupplementary Data Table S2.

Histology and microscopy

Arabidopsis inflorescence stem sections were harvestedfrom the base of the first internode of 30-cm-high stems.Soybean stem sections were collected from the base of the epi-cotyl of V7 plants. In all cases, sections 0�5–1 cm in lengthwere fixed at 24 �C for 1 h in a solution containing 3�7 % form-aldehyde, 5 % acetic acid and 47�5% ethanol, and then dehy-drated through a graded series of ethanol solutions (70, 80, 90,96 and 100%; 30min each) followed by 1 h in 100% xylene.The samples were placed into plastic moulds and finally em-bedded with 100% Histoplast (Biopack, Argentina). Eachblock was incubated overnight at room temperature to ensuresolidification. Transverse stem sections (10 mm thick) were ob-tained using a Leica Microtome (RM2125; Leica, Wetzlar,Germany). Cross sections were mounted on slides coated with50mg mL–1 poly-D-lysine (Sigma Chemical Co., St. Louis,MO, USA) in 10 mM Tris-HCl, pH 8�0, and dried for 16 h at37 �C. After removing the paraffin with 100% xylene for15min at room temperature, sections were rehydrated using agraded series of ethanol (100, 96, 90, 80, 70 and 50%; 1mineach) to finish in distilled water. Samples were then stainedwith 0�1% Toluidine blue, rinsed, and mounted on Canadianbalsam (Biopack) for microscopic visualization in an EclipseE200 Microscope (Nikon, Tokyo, Japan) equipped with aNikon Coolpix L810 camera. Toluidine blue was used to

Moreno-Piovano et al.— LAX2 modulates leaf lateral-vein symmetry Page 3 of 14

Moreno-Piovano et al. — LAX2 modulates leaf lateral-vein symmetry 579

promoter DR5. This promoter fused to the GUS-reporter generevealed strong b-glucuronidase (GUS) activity on hydathodesof developing leaves. The high auxin accumulation at the stilldifferentiating hydathodes may regulate the differentiation ofupper secondary veins, thus controlling venation symmetry(Aloni et al., 2003). This model was supported by the followingobservations: (1) mutants with altered patterns of auxin accu-mulation translated into changes of the normal venation pattern-ing (Koizumi et al., 2000; Aloni et al., 2003), and (2)exogenous application of auxin-transport inhibitors or syntheticauxin analogues to Arabidopsis leaves caused an altered num-ber of secondary veins (Mattsson et al., 1999; Sieburth, 1999;Aloni, 2010).Auxins are exported and imported from cell to cell using ef-

flux and influx carriers: PIN and AUX/LAX, respectively.Auxin homeostasis in the leaf lamina is critical to achieve theappropriate growth response, and auxin transport appears to becritical for auxin homeostasis along the whole plant body.Auxin homeostasis includes auxin distribution, hormone syn-thesis and inactivation rates, and stability of receptors and re-pressors (Petrasek and Friml, 2009; Vanneste and Friml, 2009;Salehin et al., 2015). It is interesting to note that members ofboth the PIN and the AUX/LAX gene families have evolvedsub-functionalization. For instance, the aux1 mutant producedinhibition of root hair elongation and altered gravitropic re-sponse (R. Swarup et al., 2005, 2007), lax1 and lax3 mutantsproduced a decrease in lateral root formation (K. Swarup et al.,2008), and the lax2 mutant caused vascular breaks in cotyle-dons (Peret et al., 2012). In addition, leaf phyllotaxy was signif-icantly altered by mutations in AUX1 and LAX1 genes(Bainbridge et al., 2008). Recently, a direct connection betweenauxin influx carriers and vasculature development has beenshown in Arabidopsis. The quadruple aux1 lax1 lax2 lax3(hereafter quad) mutant, but not the single aux/lax mutants, de-veloped fewer and less-dense vascular bundles than the wildtype (WT) only when grown under short-day conditions(Fabregas et al., 2015). Yet, the vascular bundle organization ofthe quad mutant was uneven, with an increased number of pro-cambial and xylem cells (the latter of enhanced size) both inshoots and in roots (Fabregas et al., 2015).Transcription factors (TFs) are modular proteins able to artic-

ulate the perception of environmental signals to cellular re-sponses. The homeodomain-leucine zipper (HD-Zip) TF familycoordinates different aspects of the plant biology including, butnot limited to, plant biotic and abiotic stress tolerance, leafshape and polarity, vascular development, and root and tri-chome formation (Capella et al., 2015b). The HD-Zip familycomprises four subfamilies, I–IV. HD-Zip subfamily I (hereaf-ter HD-Zip I) TFs were initially associated with plant responsesto the environment and more recently these regulatory proteinshave been found to participate in developmental processes(Perotti et al., 2017).HaHB4 is an HD-Zip I TF from Helianthus annuus, defined

by phylogenetic analysis as an uncommon member given its ex-tremely short and divergent carboxy-terminus (Arce et al.,2011). HaHB4 ectopic expression improved drought and salin-ity tolerance in transgenic Arabidopsis and soybean plants withno penalty on yield under control conditions (Dezar et al.,2005; Arcadia-Biosciences, 2015). In Arabidopsis, this in-creased tolerance is indeed part of a complex phenotype that

includes delayed senescence and enhanced resistance to herbiv-ory (Manavella et al., 2006, 2008). However, the complex phe-notype triggered by HaHB4 in transgenic plants is not wellunderstood.In the current study, Arabidopsis and soybean transgenic

plants expressing HaHB4 were used as a prospecting tool to un-cover novel traits related to signal transduction pathways regu-lated by this TF. A co-expression analysis performed on genesdifferentially expressed in Arabidopsis HaHB4-transgenicplants allowed us to identify LAX2 as a potential down-regulated target of HaHB4. Then, the phenotypes of single andmultiple mutant alleles of the AUX/LAX gene family were char-acterized. An increased asymmetry in the venation patterningof Arabidopsis lax2 mutant alleles was observed. We also re-vealed the functional role of the auxin influx carrier LAX2 as anegative regulator of xylem development in Arabidopsis plants.The asymmetric venation phenotype was also observed in35S:HaHB4 Arabidopsis leaves as well as in soybean plants ex-pressing HaHB4 under the control of its native promoter.Finally, we linked this venation phenotype to the LAX2 auxininflux carrier based on HaHB4-mediated down-regulation ofLAX2 expression in Arabidopsis and GmLAX genes in soybean.

MATERIALS AND METHODS

Plant material, growth conditions and treatments

Arabidopsis thaliana plants were grown on Klasmann SubstratNo. 1 compost (Klasmann-Deilmann GmbH, Geeste, Germany)in a growth chamber at 22–24 �C under long-day conditions(16/8-h light/dark cycles) with a light intensity of 120 mmolm�2 s�1 in 8 � 7-cm pots. In all Arabidopsis experiments theCol-0 ecotype was used as the WT control and four plants wereplanted per pot.A total of six Arabidopsis mutant genotypes were grown; the

mutants used were the lax2-1, lax2-2, lax1, lax3, aux1-21 andquad, all in the Col-0 background. Arabidopsis null mutantlines lax2-1 (dSpm line) and lax2-2 (GK_345D11) were de-scribed previously (Peret et al., 2012). Arabidopsis plants bear-ing the HaHB4 cDNA driven by the 35S cauliflower mosaicvirus promoter were also previously described (Dezar et al.,2005).In the case of soybean (Glycine max ‘Williams 82’), two ge-

notypes were evaluated. Seeds of the WT and those expressingthe sunflower HaHB4 cDNA under its native promoter (namedhere as b10H event) were obtained from INDEAR (Rosario,Argentina). Soybean plants of each genotype were grown in20-litre pots (five plants per pot) up to the V7 developmentalstage. Pots bearing transgenic or control plants were arrangedin a glasshouse under long-day conditions (approx. 15/9-h light/dark cycles), with daily temperatures fluctuating between 19 �C(during the night) and 40 �C (during the day). Although thetemperature amplitude reached almost 30 �C in a day, all plantgenotypes were grown together and exposed to the sameconditions.Two water regimes (irrigated and water deficit) were im-

posed between developmental stages V5 and V7, representing14 d of treatment in December–January at our latitude. Duringthis period, one group of plants (20 plants of each genotype) re-ceived adequate water supply whereas irrigation was arrested in


the second group (20 plants). Plant measurements were taken atthe V7 stage in both groups.

RNA isolation and expression analyses by real time RT-PCR

Total RNA for real-time RT-PCR was isolated fromArabidopsis leaves or stems using Trizol reagent (Invitrogen,Carlsbad, CA, USA) according to the manufacturer’s instruc-tions. Total RNA (1lg) was reverse-transcribed usingoligo(dT)18 and M-MLV reverse transcriptase II (Promega,Fitchburg, WI, USA).

Total RNA from soybean was isolated from the terminalfoliole of the last fully developed leaf of three individual soy-bean plants (V7 stage). The samples were processed using thesame reagents and protocol described for Arabidopsis tissue.

Quantitative real-time PCR (RT-qPCR) was performed usinga Mx3000P Multiplex qPCR system (Stratagene, La Jolla, CA,USA) as described by Capella et al. (2015a) and using the pri-mers listed in Supplementary Data Table S1. Transcript levelswere normalized by applying the DDCt method. Actin tran-scripts (ACTIN2 and ACTIN8 for Arabidopsis and GmActin forsoybean) were used as internal standards to normalize differ-ences in template amounts. Three biological replicates, ob-tained by pooling tissue from three to four individual plants andtested in duplicate, were used to calculate the standarddeviation.

Genetic constructs and Arabidopsis plant transformation

A Gateway-compatible plasmid containing the LAX2 cDNAwas obtained from ABRC (Ohio State University, Columbus;clone U15627) and recombined into the pJV126 expressionvector (gift of Dr D. Weigel, Max Planck Institute, Tübingen-Germany) using the Gateway LR Clonase II Enzyme Mix(Thermo Fisher, Waltham, MA, USA) according to the manu-facturer’s instructions. The pJV126 vector is derived from thepGreen vector series: genetic fragments were recombined byGateway (Life Technologies) cloning into a modified pGreenvector (pFK210) conferring resistance to BASTA (Hellenset al., 2000). A 35S CaMV promoter drives the expression of anN-terminal fusion protein with mCitrine, here named35S:mCitrine:LAX2. A sequence-verified clone was used fortransforming Agrobacterium tumefaciens strain LBA4404 andthen generating transgenic Arabidopsis plants using the floraldip method (Clough and Bent, 1998). To screen for high-expressing lines, we followed a two-step selection process.First, T1 plants were grown in soil for 7 d and watered with asolution containing 0�3 lM glufosinate (Liberty; BayerCropScience, Leverkusen, Germany), and second, we used aLeica TCS SP8 Compact confocal microscope to screen forhighly fluorescent seedlings that accumulated mCitrine-LAX2fusion protein. T2 plants containing a single transfer DNA in-sertion (as determined by 3 : 1 segregation of herbicide resis-tance) were further used in experiments. All experiments weredone with at least three independent transgenic lines probablycontaining a single T-DNA. Multiple tandem insertions cannotbe ruled out with this selection procedure and therefore severalindependent lines were evaluated simultaneously. Homozygous

T3 lines were further used to analyse transgene expression lev-els and plant phenotypes.

Microarray data analysis and clustering

The transcriptome analysis of 35S:HaHB4 Arabidopsisplants was previously reported (Manavella et al., 2006). Thesedata were used in an exploratory analysis in which genes re-sponding to HaHB4 overexpression but not to drought stress inWT or in 35S:HaHB4 plants were selected. More precisely, thechosen set of genes satisfied the following condition: an abso-lute log2 expression ratio greater than 0�5 in 35S:HaHB4 vs.WT plants, and lower than 0�5 in stressed vs. non-stressedplants. This resulted in a list of 497 genes, which was furtherused in a co-expression analysis with public expression datasetsusing the R environment for statistical computing (https://www.r-project.org/). This analysis consisted in first retrieving publicArabidopsis microarray experiments from the AtGenExpressproject (http://jsp.weigelworld.org/AtGenExpress/resources/).The gene expression datasets considered were: abiotic stress inroots, shoots and cell cultures (Kilian et al., 2007), pathogentreatment and the developmental series (Schmid et al., 2005).Expression profiles of the 446 genes with measurements in themicroarrays, i.e. presenting probesets in the Affymetrix ATH1microarray, were then pairwise correlated. Correlations wereconverted to distances by subtracting their absolute value from1, and the resulting matrix was used to perform a hierarchicalclustering with the average agglomeration method. Gene clus-ters were then defined by cutting the tree at 0�4, and their geneswere analysed using the GeneMANIA server (Warde-Farleyet al., 2010). Description of the cluster is given inSupplementary Data Table S2.

Histology and microscopy

Arabidopsis inflorescence stem sections were harvestedfrom the base of the first internode of 30-cm-high stems.Soybean stem sections were collected from the base of the epi-cotyl of V7 plants. In all cases, sections 0�5–1 cm in lengthwere fixed at 24 �C for 1 h in a solution containing 3�7 % form-aldehyde, 5 % acetic acid and 47�5% ethanol, and then dehy-drated through a graded series of ethanol solutions (70, 80, 90,96 and 100%; 30min each) followed by 1 h in 100% xylene.The samples were placed into plastic moulds and finally em-bedded with 100% Histoplast (Biopack, Argentina). Eachblock was incubated overnight at room temperature to ensuresolidification. Transverse stem sections (10 mm thick) were ob-tained using a Leica Microtome (RM2125; Leica, Wetzlar,Germany). Cross sections were mounted on slides coated with50mg mL–1 poly-D-lysine (Sigma Chemical Co., St. Louis,MO, USA) in 10 mM Tris-HCl, pH 8�0, and dried for 16 h at37 �C. After removing the paraffin with 100% xylene for15min at room temperature, sections were rehydrated using agraded series of ethanol (100, 96, 90, 80, 70 and 50%; 1mineach) to finish in distilled water. Samples were then stainedwith 0�1% Toluidine blue, rinsed, and mounted on Canadianbalsam (Biopack) for microscopic visualization in an EclipseE200 Microscope (Nikon, Tokyo, Japan) equipped with aNikon Coolpix L810 camera. Toluidine blue was used to



differentially stain primary and secondary cell walls. Primarycell walls are pink and secondary cell walls stain blue or blue-green. In this way, it is possible to distinguish between phloemand xylem cells since cells found in phloem have primary cellwalls (cellulose) whereas xylem cells have both primary andsecondary cell walls rich in lignin. Forty vascular bundlesof eight cross-sections were evaluated for each genotype andtreatment (40 vascular bundles per genotype/treatment).Quantification of vascular bundle (VB) parameters (xylem ra-tio, number of cells and VB length) was performed usingImageJ software (Schneider et al., 2012) as follows: both xylemlength and number of xylem cell rows were measured on astraight line traced from the last procambium cell layer to theinner xylem cells facing the centre of the stem. The xylem/stemarea ratio is the proportion that the xylem represents on the totalstem area (xylem area/total stem area).

Venation patterning in leaves of adult plants

The study of venation patterning was performed on a fullydeveloped leaf of a 30-d-old plant. Based on previous morpho-logical studies on Arabidopsis plants (Boyes et al., 2001;Farmer et al., 2013), we chose the rosette true leaf No. 5. LeafNo. 5, considering only true leaves (Farmer et al., 2013), ofArabidopsis plants was harvested and cleared overnight in70 % ethanol. Leaves were imaged using a Leica MZ10F ste-reomicroscope coupled to a DFC7000 T camera (Leica). Inmost cases, pictures of the general leaf aspect were obtained bymerging two photographs (Leica merge tool), the upper andlower half of the leaf. In some cases, when the software failedto merge them, the two leaf pictures were manually overlaid.Venation patterning was recorded in leaf No. 5 using two com-plementary traits: (1) secondary vein-attachment-site distanceand (2) lateral-vein asymmetry index. Vein-attachment-site dis-tance was scored as the distance between the attachment pointsof the two secondary veins of the third pair on the midvein us-ing ImageJ software. In addition, Arabidopsis lateral-veinasymmetry index was scored as the fraction between the num-ber of vein pairs asymmetrically distributed on the midvein di-vided by the total number of vein-pairs in the leaf. In the caseof soybean leaves, venation patterning was recorded countingthe fraction of vein pairs asymmetrically developed (asymmet-ric vein pairs/total vein pairs) on the midvein of the terminalfoliole of the last fully developed leaf. In all cases, a total of sixplants of each genotype were scored.

Plant phenotyping

Different plant architecture parameters were scored on 35-d-old Arabidopsis plants. Age of plants was counted as days aftersowing. Measurements were performed at least on 12 plants pertreatment. Evaluated traits were inflorescence stem height (witha ruler), main stem width (with a Vernier caliper), number ofsecondary branches, number of secondary stems, total siliquenumber and yield.Leaf conductance was measured at the V7 stage in the termi-

nal foliole of the last fully developed leaf of soybean plants, ex-cluding the mid-rib area. Measurements were performed nearmidday by means of an SC-1 Leaf Porometer (Decagon

Devices Inc., Pullman, WA, USA), on both leaf sides (adaxialand abaxial) and using an integration time of 30 s. Three potsfor each combination of genotype (W82 and b10H plants) andsoil water condition (irrigated or water-stressed) were analysed.In each pot, representing an individual replica, the average ofthree plants was considered as a value. Standard deviation wascalculated taking the values of three pots.

Statistical analysis

Venation, xylem patterning and xylem/stem ratio were ana-lysed using a one way ANOVA with genotype as the main fac-tor. Significant (P< 0�05) differences between means wereanalysed using post-hoc Tukey comparisons. In soybean plantsgrowing under irrigated or water deficit conditions, datawere analysed using a two-way ANOVA with water regimeand genotype as factors. When interaction terms were signifi-cant (P< 0�05), differences between means were analysed us-ing Tukey comparisons. When needed, appropriatetransformations of the primary data were used to meet the as-sumptions of the analysis. In all cases, differences betweenmeans are indicated by different letters.

RESULTS

Ectopic expression of HaHB4 in Arabidopsis leaves inducessimilar changes in venation patterning to those observed in auxininflux carrier mutants

The expression of the sunflower HD-Zip I-encoding geneHaHB4 under the 35S CaMV promoter generated a wide tran-scriptional rearrangement affecting different plant traits such asdrought tolerance, ethylene sensitivity and jasmonate-induceddefences (Dezar et al., 2005; Manavella et al., 2006, 2008). Tofurther understand the molecular physiology behind this com-plex phenotype, we performed a new analysis on the previouslyreported microarray results of Arabidopsis 35S:HaHB4 plants(Manavella et al., 2006), which involved two-colour microar-rays with the following transcriptome comparisons: WT plantsin control vs. drought conditions, WT vs. 35:HaHB4 plants incontrol conditions, and 35:HaHB4 plants in drought vs. controlconditions. Briefly, the procedure consisted in selecting a set ofgenes responding to HaHB4 overexpression but not to droughtstress in WT or in 35S:HaHB4 plants, and then using them in aco-expression study with publicly available expression datasets.This allowed us to identify gene co-expression clusters that, af-ter functional enrichment analysis, could potentially help us indissecting the physiological role of HaHB4 in transgenic plants.This approach identified a gene cluster including the LAX2(like-AUXIN RESISTANT 2) gene and other auxin-relatedgenes, such as TAR2 and AIR9 (Fig. 1A). AUX/LAX transcriptlevels were evaluated by RT-qPCR in lax2-1, lax2-2 mutantsand 35S:HaHB4 plants resulting in significantly different pat-terns (Fig. 1B). Whereas AUX1 and LAX1 expression was simi-lar between genotypes, LAX3 expression was significantly up-regulated in 35S:HaHB4 (OE) plants. On the other side, LAX2was the only AUX/LAX gene family member showing a strongrepression by HaHB4 (Fig. 1B), validating expression valuesobserved in the microarray (log2 fold change ¼ 0�58 6 0�17).This result led us to explore the relationship among AUX/LAX


genes, in particular between LAX2 and the HaHB4-inducedphenotypes.

It is well known that plant water transport depends on bothxylem organization and vein architecture (for a review, seePrado and Maurel, 2013). Given that 35S:HaHB4 plants haveenhanced drought tolerance and that auxin modulates venationpatterning in leaves and vascular development, these auxin-related characteristics were further investigated in AUX/LAXgene mutants and in 35S:HaHB4 plants. To evaluate the leafvenation patterning, we defined two robust parameters. First,we scored the distance between the attachment points of the lat-eral veins of the third pair on the midvein (Fig. 2A). Second,we recorded the lateral-vein asymmetry index as a ratio be-tween the number of asymmetric lateral vein pairs divided bythe total number of lateral vein pairs in the leaf lamina (Fig.2A). All these parameters were scored on leaf numbers 5 and 6of 30-d-old Arabidopsis plants because at this stage the leaflaminae were fully expanded (Boyes et al., 2001). For simplic-ity, we show here only data for leaf number 5. Lateral-veinasymmetry index was also recorded on leaf number 5 at youn-ger stages. At these earlier stages, we were not able to detect

significant differences on leaf asymmetry, probably becauseleaf expansion was not complete, and therefore lateral-vein at-tachment sites were still too close to detect the differences. It isnoteworthy to mention that although vein patterning seems tobe a robust developmental response, all the analysed plant ge-notypes had a certain level of variation, even leaves of WTplants (Fig. 2C, D). We found that 35S:HaHB4 leaves hadgreater asymmetry in the venation pattern compared to thevein-attachment sites of WT leaves (Fig. 2B). In this regard, thedistance between the attachment sites of the third vein pair andthe lateral-vein asymmetry index of 35S:HaHB4 leaves weresignificantly higher than in the WT (Fig. 2C, D).Since LAX2 is repressed in 35S:HaHB4 transgenic plants, we

wondered whether auxin influx carriers belonging to the AUX/LAX gene family may contribute to the modulation of the vena-tion patterning in leaves. As observed for 35S:HaHB4 leaves,the venation patterning of all single mutants (aux1-21, lax2-1and lax2-2 and lax3) but lax1 and the quad mutant showed anincreased asymmetry compared to WT leaves (Fig. 2B–D). Theincreased asymmetry in venation patterning of lax mutants wasthe result of a higher distance between the attachment sites ofthe third vein-pair (Fig. 2C) and a higher lateral-vein asymme-try index (Fig. 2D). Based on these results, we showed that theectopic expression of HaHB4 as well as aux/lax mutations pro-moted changes in venation asymmetry in Arabidopsis leaves.

HaHB4 and LAX2 control xylem development in theinflorescence stem of Arabidopsis

Given that HaHB4 repressed the expression of only oneAUX/LAX gene – LAX2 – and lax2 mutant alleles displayed asimilar phenotype to 35S:HaHB4 plants, we decided to furtherexplore the role of LAX2 in vascular development. For this, adetailed phenotypic comparison of lax2 mutant alleles (lax2-1and lax2-2) and 35S:HaHB4 plants was carried out. The mostrelevant resemblance between these genotypes was related tothe inflorescence stem width, which was significantly wider inthe three genotypes with respect to WT plants (Fig. 3A).However, other stem traits showed a significant change in35S:HaHB4 plants but remained constant in plants of the WTand lax2 genotypes (Fig. 3B and Fig. S1).To further examine stem-related traits, basal stem cross-

sections of the inflorescence stem were prepared, and differentVB attributes were evaluated, including area, length and num-ber of cell rows (Fig. 4B). Both the total xylem length and thenumber of xylem cell rows were significantly higher in lax2mutants and 35S:HaHB4 plants (Fig. 4A–D). In agreementwith a previous report, aux1, lax1, lax3 and quad mutant plantsgrowing under long day conditions did not differ in VB organi-zation (Fabregas et al., 2015). Furthermore, we recorded the xy-lem/stem area ratio calculated as the fraction of total xylemarea with respect to the total stem area ratio. This parameter re-flects the proportion of xylem in the total stem area. Again, nodifference was detected for this trait among the aux1, lax1 andlax3 single mutants (Fig. 4E), whereas a slight but significantdecrease was computed for the quad mutant as compared to theWT (Fig. 4). Notably, we observed a significant increase in theproportion of the mentioned ratio among the lax2 mutant and35S:HaHB4 plants as compared to stem cross-sections of the

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HB4

FIG. 1. HaHB4 represses the expression of LAX2 in Arabidopsis transgenicplants. (A) Co-expression of genes in the cluster generated using the GeneManiasoftware package. The cluster is statistically enriched in genes related to auxinhomeostasis, i.e. LAX2, TAR2 and AIR9 (P � 0�05). Black nodes represent thelist of query genes used in GeneMania. Grey nodes represent genes that were as-sociated by a co-expression pattern according to GeneMania. (B) Relative tran-script levels of AUX/LAX family members in rosette leaves of 20-d-old plants ofWT (Col-0), lax2-1, lax2-2 and HaHB4 (displayed here as HB4) plants. AUX/LAX transcript abundance was measured and expressed relative to the level de-tected in Col-0 plants. Different letters indicate significant differences between

means (P < 0�05, Tukey test).



Vein pair #

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asymmetric lateral-vein pairstotal lateral-vein pairs

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FIG. 2. Both lax2mutants and 35:HaHB4 leaves have enhanced asymmetry on lateral-vein attachment site of the midvein resulting in leaves with increased asymmetryin venation patterning. (A) Leaf diagram showing the parameters measured on the 5th leaf lamina. (B) Illustrative photographs of leaf number 5 and the correspondinginset showing a zoom on the third vein pair of Col-0, lax2-1, lax2-2, 35S:HaHB4, aux1-21, lax1, lax3, quad, arf6-2 and arf8-3 genotypes. Whole-leaf scale bar repre-sents 0�5 cm, whereas leaf-inset scale bar represents 1�0mm. (C) Average distance between the two attachment sites of the lateral veins of the third pair. (D) Lateral-

vein asymmetry index calculated as described in Methods. Thin bars represent s.e. Different small letters indicate significant differences (P< 0�05, Tukey test).


WT (Fig. 4E). This change was mostly explained by an in-creased number of xylem cell rows that caused an increased to-tal xylem length (Fig. 4C, D).

Enhanced xylem development in the lax2 mutant is not explainedby apparent changes in the expression patterns of HD-Zip III andKANADI TFs

Phloem and xylem development relies on a complex regula-tion between HD-Zip III and KANADI TFs, with a central rolefor the phytohormone auxin in activating cell differentiation(Huang et al., 2014; Ilegems et al., 2010). We wondered if thisincrease in xylem development might be the consequence of themissexpression of HD-Zip III and KANADI TFs in the inflores-cence stem. To answer this question, we quantified the expres-sion of several HD-Zip III and KANADI genes in the last 3 cmof the inflorescence stem, where vascular-bundle differentiationis still an on-going process (Ilegems et al., 2010; Lucas et al.,2013). RT-qPCR showed no differential expression for thesegenes, either in lax2-1 or in lax2-2mutants (Fig. 5). On the otherhand, HaHB4 up-regulated the expression of most HD-Zip IIIand KANADI genes in the same tissue samples (Fig. 5).

Complementation assays with LAX2 restores the xylem ratio inthe lax2-2 mutant

A transgenic complementation assay of the lax2-2 mutantwas conducted to test the functional role of LAX2 in changing

the xylem ratio in inflorescence stems, probably as a conse-quence of alterations in auxin transport (Peret et al., 2012). Inparallel with the lax2-2 mutant, we transformed plants of theCol-0 background as a control. In each case, we obtained 20 in-dependent transgenic lines. Eleven independent transgenic linesof each genetic background were screened using a confocal mi-croscope. From high-expressing lines, only those segregating asharbouring T-DNA insertion were kept for later studies. Withinthis group, we used RT-qPCR to screen T2 plants for lines pre-senting high levels of the mRNA encoding for the fusion proteinmCitrine-LAX2 (Supplementary Data Fig. S2A). We selectedtwo lines, A and B, for further characterization. Additionally,we checked the expression pattern of the other AUX/LAX familymembers. These lines showed a significant decrease in the ex-pression of the other AUX/LAX genes; this was particularly no-ticeable for the repression of AUX1 and LAX1 genes (Fig. S2B).Unexpectedly, AUX1 was also downregulated in lax2-2 mutantplants and this result cannot be easily explained; it is possiblethat a back negative feedback or cross regulation with anothergene may be occurring. The phenotypic characterization of thesetransgenic plants brought interesting but unexpected results.LAX2 expression under the CaMV 35S promoter significantlyincreased the inflorescence stem size (Fig. S3A). This incrementin stem area resulted in the expansion of xylem size, includingxylem length and number of xylem cell rows (Fig. 6A, B; Fig.S3B). However, the proportion of xylem to total stem area (xy-lem area/total stem area) was restored to WT levels in the lax2-2 plants overexpressing LAX2 (Fig. 6C). Moreover, LAX2 ec-topic expression in the lax2-2 genetic background also restoredthe leaf venation symmetry to WT levels (Fig. 7A), displaying asimilar distance between the attachment sites of the third veinpair (Fig. 7B), and a similar lateral-vein asymmetry index (Fig.7C) to WT leaves. These results suggested that LAX2 is able tomodulate xylem development.

Expression of HaHB4 in transgenic soybean increases vascularasymmetry in leaves

HaHB4 expression driven by its native promoter in transgenicsoybean plants improves yield and drought tolerance in the field(Chan and Gonzalez, 2015). This biotechnological event, origi-nally called b10H, was used to test the hypothesis of whetherHaHB4 is able to induce changes in soybean leaves similar tothose observed in transgenic Arabidopsis plants, i.e. changes inxylem ratio and vascular innervations in leaves. To test this hy-pothesis, we scored the vein asymmetry ratio in the terminalfoliole of the last fully developed leaf of V7 soybean plants(Glycine max ‘Williams 82’) grown in the glasshouse. We quan-tified the xylem ratio in basal epicotyl cross-sections and foundno difference between transformed and WT individuals(Supplementary Data Fig. S4A-C). On the other hand, we ob-served that vein attachment sites were more asymmetrically dis-tributed in the b10H soybean leaves than in the WT (Fig. 8A).Indeed, the vein asymmetry ratio was significantly higher in theformer than in the latter (Fig. 8B). Then, we wondered if waterdeficit might also influence this phenotypic trait. As expected,stomatal conductance was reduced among water-stressed plants(Fig. S5A). We did not find significant differences in stomatalconductance between WT and b10H genotypes grown under

B

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FIG. 3. Both lax2 mutants and 35:HaHB4 plants exhibit a wider inflorescencestem than WT plants. Genotype effects on both main stem height (A) and width(B) of WT, lax2-1 and lax2-2mutant alleles, and 35:HaHB4 plants. Thin bars rep-resent s.e. Different letters indicate significant differences (P< 0�05, Tukey test).



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FIG. 4. Both lax2 mutants and 35:HaHB4 plants show an enhanced proportion of xylem in the main inflorescence stem. (A) Illustrative photographs of basal shootcross-section of inflorescence stems together with a picture inset showing a zoom on a representative vascular bundle for analysed genotypes. Basal shoot scale bar rep-resents 0�5mm, whereas vascular-bundle inset scale bar represents 0�1mm. (B) Illustration of a vascular bundle showing measured parameters as follows: yellow lineis the total xylem area; red dots are xylem cell row along the main xylem axis; green dashed line illustrates a portion of the total stem area considered for xylem/stemarea ratio. Procambial cells are enclosed in orange. The blue area indicates phloem cells. Column bar graphs of: (C) xylem length, (D) number of xylem cell rows and(E) xylem/stem area ratio. Vascular bundle parameters were taken from pictures of basal shoot cross-section of inflorescence stems for different genotypes, including

Col-0, lax2-1, lax2-2, 35S:HaHB4, aux1-21, lax1, lax3 and quad. Thin bars represent s.e. Different letters indicate significant differences (P< 0�05, Tukey test).


water deficit (Fig. S5A). Further experiments under field condi-tions will be relevant to shed light on this subject. We observedthat the vein asymmetry ratio did not change significantly uponwater deprivation (Fig. S5B). In other words, the vein asymmetryratio was somehow independent of the plant water status. Thisresult confirmed that soybean plants bearing the HaHB4 genebehave similar to 35S:HaHB4 Arabidopsis plants, inducing anincreased asymmetry on leaf venation.

HaHB4 down-regulates the expression of GmLAX genes intransgenic soybean

We wondered if this increased asymmetry in the venationpatterning of b10H soybean leaves could be related, as well as

in Arabidopsis, to the repression of LAX genes. We developed aBLAT (BLAST-like Alignment Tool) search on the soybeangenome for exact or nearly exact coincidences representative ofrobust hits. This search allowed us to identify nine putativeGmLAX genes with high protein sequence similarity (> 95%)to the AtLAX2 protein sequence. We then selected at least onebest BLAT soybean hit for each member of the ArabidopsisAUX/LAX family and performed RT-qPCR measurements. Intotal, we tested the transcript levels of six out of nine putativeGmLAX genes in the same foliole used to estimate venation pat-terning. Based on the RT-qPCR quantifications we showed thatthe expression of four out of six GmLAX genes was signifi-cantly repressed in b10H leaves as compared to the control ge-notype (Fig. 8C).

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FIG. 5. Expression levels of Arabidopsis transcription factors involved in vascular development. Relative transcript levels of HD-ZIP III (A) and KANADI (B) geneswere quantified by RT-qPCR using RNAs isolated from the last 3 cm of inflorescence stem excluding flower buds. Different letters indicate significant differences

(P < 0�05, Tukey test).



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FIG. 4. Both lax2 mutants and 35:HaHB4 plants show an enhanced proportion of xylem in the main inflorescence stem. (A) Illustrative photographs of basal shootcross-section of inflorescence stems together with a picture inset showing a zoom on a representative vascular bundle for analysed genotypes. Basal shoot scale bar rep-resents 0�5mm, whereas vascular-bundle inset scale bar represents 0�1mm. (B) Illustration of a vascular bundle showing measured parameters as follows: yellow lineis the total xylem area; red dots are xylem cell row along the main xylem axis; green dashed line illustrates a portion of the total stem area considered for xylem/stemarea ratio. Procambial cells are enclosed in orange. The blue area indicates phloem cells. Column bar graphs of: (C) xylem length, (D) number of xylem cell rows and(E) xylem/stem area ratio. Vascular bundle parameters were taken from pictures of basal shoot cross-section of inflorescence stems for different genotypes, including

Col-0, lax2-1, lax2-2, 35S:HaHB4, aux1-21, lax1, lax3 and quad. Thin bars represent s.e. Different letters indicate significant differences (P< 0�05, Tukey test).


water deficit (Fig. S5A). Further experiments under field condi-tions will be relevant to shed light on this subject. We observedthat the vein asymmetry ratio did not change significantly uponwater deprivation (Fig. S5B). In other words, the vein asymmetryratio was somehow independent of the plant water status. Thisresult confirmed that soybean plants bearing the HaHB4 genebehave similar to 35S:HaHB4 Arabidopsis plants, inducing anincreased asymmetry on leaf venation.

HaHB4 down-regulates the expression of GmLAX genes intransgenic soybean

We wondered if this increased asymmetry in the venationpatterning of b10H soybean leaves could be related, as well as

in Arabidopsis, to the repression of LAX genes. We developed aBLAT (BLAST-like Alignment Tool) search on the soybeangenome for exact or nearly exact coincidences representative ofrobust hits. This search allowed us to identify nine putativeGmLAX genes with high protein sequence similarity (> 95%)to the AtLAX2 protein sequence. We then selected at least onebest BLAT soybean hit for each member of the ArabidopsisAUX/LAX family and performed RT-qPCR measurements. Intotal, we tested the transcript levels of six out of nine putativeGmLAX genes in the same foliole used to estimate venation pat-terning. Based on the RT-qPCR quantifications we showed thatthe expression of four out of six GmLAX genes was signifi-cantly repressed in b10H leaves as compared to the control ge-notype (Fig. 8C).

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FIG. 5. Expression levels of Arabidopsis transcription factors involved in vascular development. Relative transcript levels of HD-ZIP III (A) and KANADI (B) geneswere quantified by RT-qPCR using RNAs isolated from the last 3 cm of inflorescence stem excluding flower buds. Different letters indicate significant differences




DISCUSSION

Here, we report that the ectopic expression of HaHB4 inArabidopsis and soybean plants repressed the expression ofLAX2, or AUX/LAX homologue genes from soybean, and in-duced changes in leaf venation patterning in both heterologousspecies. Such changes were also observed in lax2mutant plants,suggesting a key role of LAX2 in this developmental event. Itwas previously reported that auxin distribution in the leaf lam-ina is one of the major factors controlling venation symmetry(Aloni, 2001; Aloni et al., 2003). In the context of vascular de-velopment, it was also shown that the lax2 mutant exhibitedvascular breaks in cotyledons (Peret et al., 2012). More

recently, a computational approach suggested that AUX/LAXinflux carriers were required for vascular patterning (Fabregaset al., 2015). In this regard, it was experimentally shown thatthe quad mutant developed fewer and less-dense vascular bun-dles than the WT, not balanced with the formation of xylemcells (number and size) both in shoots and roots (Fabregaset al., 2015). This evidence supported a role for auxin influxcarriers on xylem development, and together with the fact thatmicroarray data and RT-qPCR matched HaHB4 in the ability todownregulate the expression of LAX2 in Arabidopsis plants, ledus to explore the venation patterning and xylem developmenton lax2 mutants. In agreement with our hypothesis, lax2-1 andlax2-2 mutant leaves had an altered symmetry in the distribu-tion of lateral-vein attachment sites and consequently in theirlateral-vein asymmetry index, similar to those observed in35S:HaHB4 plants (Fig. 2B–D). The differential phenotypes ofthe lax2 mutant were complemented with a WT copy of theLAX2 gene, indicating that auxin homeostasis in plant tissues iscritical to achieve a normal leaf venation symmetry and normalxylem development (Fig. 7). It is important to mention that aprevious report using an N-terminal translational fusionbetween AUX1 protein and the yellow fluorescent protein(N-YFP-AUX1) failed to restore the root gravitropic responseof the null aux1-22 mutant (Swarup et al., 2004). In this paper,we used a different vector to create an N-terminal fusion pro-tein between mCitrine and LAX2. This protein chimera wasable to restore venation patterning and xylem development ofthe lax2-2 mutant. The different behaviour of comparable con-structs might arise from intrinsic differences of the translationalfusion proteins or from expression levels derived from differentvector backbones. The overexpression of LAX2 complementedthe mutant phenotype but did not generate a differential oppo-site phenotype, in either the mutant or the WT backgroundplants. This observation can be explained by LAX2 expressionreaching a certain threshold level and other mechanisms con-trolling the activity and/or amount of auxin transporters. In thiscontext, it might be relevant to explore the ability of HaHB4 torestore the lax2 mutant phenotype. However, we can suggestthat a complementation with HaHB4, or with an ArabidopsisHD-Zip I gene, would not be able to restore the differentiallax2-2 phenotype because our evidence suggests that it wouldbe acting upstream of LAX2. However, this question is stillopen and further experiments are in course to obtain a better un-derstanding of this scenario.Our results suggest that LAX2 protein levels are required to

modulate AUX1 gene expression. As was previously suggestedby other research groups, there is a complex regulation of the dif-ferent auxin transporters and such regulation might be dependenton the cell niche of expression. Kasprzewska et al. (2015) de-scribed experiments performed using ProLAX1:GUS andProLAX2:GUS fusion constructs in which both LAX1 or LAX2promoter activities were similar in young true leaves of the quadmutant and wild-type plants. However, at the root cellular level,LAX2 and LAX3 were somehow absent in AUX1-expressingcells, either when LAX2 or when LAX3 were expressed under thecontrol of the AUX1 promoter or fused to YFP under the controlof a CAMV 35S promoter (Peret et al., 2012). Taken together,these results support the fact that AUX/LAX gene expression pat-terns and protein levels in the cell might be regulated by theabundance of other AUX/LAX members.

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a

b b b

a a a a

Num

ber

ofxy

lem

cel

l row

s

20

10

15

5

0

a

b b b

a

b

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b

Xyl

em le

ngth

(µm

)

250A

B

C

150

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100

50

0

a

b b c

b b b b

FIG. 6. Ectopic expression of LAX2 in Arabidopsis transgenic plants restoresthe xylem/stem area ratio of lax2 mutants. Column bar graphs represent (A) thexylem length, (B) the number of xylem cell rows and (C) the xylem/total stemarea ratio in basal shoot cross-sections of stems. Basal shoot cross-sections oftransgenic 35S:mCitrine:LAX2 stems were used to measure vascular bundle pa-rameters. Thin bars represent s.e. Different letters indicate significant differences



A Col-0

Leaf

#5

Leaf

#5

Inse

tIn

set

1·5

aa a a a

b b b

2ry

vein

-atta

chm

ent s

ites

dist

ance

(m

m)

Late

ral-v

ein

assy

met

ryin

dex

1·0

0·5

0

0·8

aa a a a

b b b

0·4

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0·2

0

lax2-1 lax2-2 HB4

OX-Col A OX-Col B

Col lax2-1

lax2-2

HB4

OX-Col

A B

OX-lax2

A B

OX-lax2 A OX-lax2 B

B

C

FIG. 7. Ectopic expression of LAX2 in Arabidopsis transgenic plants restores venation patterning in leaves of lax2 mutants. (A) Illustrative photographs of leaf num-ber 5 and the corresponding inset showing a zoom on the third vein pair. The final image resulted from merging two photographs. Whole-leaf scale bar represents2�0mm, whereas leaf-inset scale bar represents 1�0mm. (B) Average distance between the two attachment sites of the lateral veins of the third vascular pair.

(C) Lateral-vein asymmetry index. Thin bars represent s.e.. Different letters indicate significant differences (P < 0�05, Tukey test).



DISCUSSION

Here, we report that the ectopic expression of HaHB4 inArabidopsis and soybean plants repressed the expression ofLAX2, or AUX/LAX homologue genes from soybean, and in-duced changes in leaf venation patterning in both heterologousspecies. Such changes were also observed in lax2mutant plants,suggesting a key role of LAX2 in this developmental event. Itwas previously reported that auxin distribution in the leaf lam-ina is one of the major factors controlling venation symmetry(Aloni, 2001; Aloni et al., 2003). In the context of vascular de-velopment, it was also shown that the lax2 mutant exhibitedvascular breaks in cotyledons (Peret et al., 2012). More

recently, a computational approach suggested that AUX/LAXinflux carriers were required for vascular patterning (Fabregaset al., 2015). In this regard, it was experimentally shown thatthe quad mutant developed fewer and less-dense vascular bun-dles than the WT, not balanced with the formation of xylemcells (number and size) both in shoots and roots (Fabregaset al., 2015). This evidence supported a role for auxin influxcarriers on xylem development, and together with the fact thatmicroarray data and RT-qPCR matched HaHB4 in the ability todownregulate the expression of LAX2 in Arabidopsis plants, ledus to explore the venation patterning and xylem developmenton lax2 mutants. In agreement with our hypothesis, lax2-1 andlax2-2 mutant leaves had an altered symmetry in the distribu-tion of lateral-vein attachment sites and consequently in theirlateral-vein asymmetry index, similar to those observed in35S:HaHB4 plants (Fig. 2B–D). The differential phenotypes ofthe lax2 mutant were complemented with a WT copy of theLAX2 gene, indicating that auxin homeostasis in plant tissues iscritical to achieve a normal leaf venation symmetry and normalxylem development (Fig. 7). It is important to mention that aprevious report using an N-terminal translational fusionbetween AUX1 protein and the yellow fluorescent protein(N-YFP-AUX1) failed to restore the root gravitropic responseof the null aux1-22 mutant (Swarup et al., 2004). In this paper,we used a different vector to create an N-terminal fusion pro-tein between mCitrine and LAX

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A role for LAX2 in regulating xylem development and lateral ...genotypes were grown together and...

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