Interaction between Glucose and Brassinosteroid during the … · Interaction between Glucose and Brassinosteroid during the Regulation of Lateral Root Development in Arabidopsis1

Interaction between Glucose and Brassinosteroidduring the Regulation of Lateral RootDevelopment in Arabidopsis1

Aditi Gupta2, Manjul Singh2, and Ashverya Laxmi*

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India

ORCID IDs: 0000-0002-3299-1625 (A.G.); 0000-0001-9547-269X (M.S.).

Glucose (Glc) plays a fundamental role in regulating lateral root (LR) development as well as LR emergence. In this study, weshow that brassinosteroid (BR) signaling works downstream of Glc in controlling LR production/emergence in Arabidopsis(Arabidopsis thaliana) seedlings. Glc and BR can promote LR emergence at lower concentrations, while at higher concentrations, bothhave an inhibitory effect. The BR biosynthesis and perception mutants showed highly reduced numbers of emerged LRs at all the Glcconcentrations tested. BR signaling works downstream of Glc signaling in regulating LR production, as in the glucose insensitive2-1brassinosteroid insensitive1 double mutant, Glc-induced LR production/emergence was severely reduced. Differential auxindistribution via the influx carriers AUXIN RESISTANT1/LIKE AUXIN RESISTANT1-3 and the efflux carrier PIN-FORMED2 playsa central role in controlling LR production in response to Glc and BR. Auxin signaling components AUXIN RESISTANT2,3 andSOLITARY ROOT act downstream of Glc and BR. AUXIN RESPONSE FACTOR7/19 work farther downstream and control LRproduction by regulating the expression of LATERAL ORGAN BOUNDARIES-DOMAIN29 and EXPANSIN17 genes. Increasing lightflux could also mimic the Glc effect on LR production/emergence. However, increased light flux could not affect LR production inthose BR and auxin signaling mutants that were defective for Glc-induced LR production. Altogether, our study suggests that, undernatural environmental conditions, modulation of endogenous sugar levels can manipulate root architecture for optimized developmentby altering its nutrient/water uptake as well as its anchorage capacity.

Plants constantly sense the changes in their environmentand transmit these signals as part of normal development.In plants, roots function as a vital system essential foroptimal growth and overall fitness. Root elongation, rootbranching via lateral root (LR) formation, and root direc-tion determine root system architecture, which allows theplant to adapt to continuously changing environmentalconditions as well as intrinsic cues (Malamy, 2005; Cuestaet al., 2013; Singh et al., 2014a). LR formation is an im-portant adaptive feature that provides flexibility to theentire root system toward changes in environmental con-ditions. LRs are initiated from pericycle cells adjacent toprotoxylem poles. In plants, many factors, such as light,nutrients, and phytohormones, play vital roles in modu-lating LR growth and development (Malamy, 2005; DenHerder et al., 2010; Garay-Arroyo et al., 2012). Auxin is oneof the major components regulating both primary root and

LR development (Lavenus et al., 2013). PIN-FORMED(PIN)-dependent auxin efflux and AUXIN RESISTANT1(AUX1)/LIKE AUXIN RESISTANT1 (LAX1)-mediatedauxin uptake are essential for LR initiation, primordiumdevelopment, and elongation (Benková et al., 2003; DeSmet, 2012; Marhavý et al., 2013). In Arabidopsis (Arabi-dopsis thaliana), LR initiation, patterning, and emergenceare regulated via multiple auxin response modules, whichinclude auxin-dependent degradation of auxin/indole-3-acetic acids (IAAs) such as SOLITARY ROOT (SLR)/IAA14, leading to the activation of AUXIN RESPONSEFACTOR7 (ARF7) and ARF19, which subsequently regu-late the activation of their target genes, such as members ofthe LATERAL ORGAN BOUNDARIES-DOMAIN (LBD)/ASYMMETRIC LEAVES2-LIKE (ASL) family and expan-sins, etc. (Okushima et al., 2007; De Smet, 2012; Lavenuset al., 2013). Several other phytohormones are also in-volved in regulating LR formation (Fukaki and Tasaka,2009). Brassinosteroids (BRs) regulate LR initiation posi-tively by increasing acropetal auxin transport (Bao et al.,2004). Similarly, the BR perception-defective mutantbrassinosteroid insensitive1 (bri1) also showed dramaticallydecreased numbers of LRs (Bao et al., 2004; Mouchelet al., 2006). Recently, the BR signaling negative regulatorBRASSINOSTEROID-INSENSITIVE2 was reported toregulate LR organogenesis by phosphorylating ARF7and ARF19 (Cho et al., 2014).

The optimal development of the root system is majorlyattuned to the availability of nutrients. Besides beingan important nutrient, Glc has also been assigned an

1 This work was supported by the Department of Biotechnology,Ministry of Science and Technology, Government of India (grant no.BT/PR3302/AGR/02/814/2011), by the National Institute of PlantGenome Research, and by the Council of Scientific and IndustrialResearch, India (research fellowships to A.G. and M.S.).

2 These authors contributed equally to the article.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ashverya Laxmi ([email protected]).

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important role as a signal molecule in plants (Ramonet al., 2008; Eveland and Jackson, 2012). In plants, threedistinct Glc signal transduction pathways operate: (1) theHEXOKINASE1 (HXK1)-dependent pathway governedby HXK1-mediated signaling function; (2) an HXK1-independent pathway where changes in gene expres-sion are modulated by Glc but are independent of HXK1;and (3) a glycolysis-dependent pathway that utilizes theSUCROSE NONFERMENTING RELATED KINASE1(SnRK1)/TARGET OF RAPAMYCIN (TOR) pathway(Fu et al., 2014). HXK1 was the first sugar sensor iden-tified in plants possessing dual properties as a Glc-metabolizing enzyme and a Glc sensor (Moore et al.,2003). Extracellular Glc is also sensed and transducedthrough heterotrimeric G-proteins, wherein REGULA-TOR OF G-PROTEIN SIGNALING1 (RGS1) acts as a Glcreceptor (Chen and Jones, 2004). In the presence of Glc,RGS1 and G-PROTEIN ALPHA-SUBUNIT1 (GPA1)form a complex that releases Gbg dimer, resulting inthe recruitment of WITH NO LYSINE kinase, whichphosphorylates RGS1. The phosphorylated AtRGS1

undergoes endocytosis, thus physically uncouplingGPA1 to self-activate and initiate downstream signaling(Urano et al., 2012). GPA1 also interacts with THYLA-KOID FORMATION1 (THF1), which undergoes rapiddegradation by Glc, thus playing a role in the sugar re-sponse (Huang et al., 2006). In Arabidopsis, SUCROSENONFERMENTING1 KINASE HOMOLOG10 (KIN10)and KIN11 are collectively designated as SnRKs, whichare central regulators of transcriptional responses towarddarkness, sugar, and stress conditions (Baena-Gonzálezet al., 2007). The TOR kinase system is another sensorrelated to sugar signaling that controls protein synthesis,cell growth, and proliferation in response to nutrients,growth factors, ATP, oxygen levels, and stress (Baena-González, 2010). TOR can sense photosynthetically gen-erated Glc and transduce the signal through glycolysisand mitochondrial energy relay (Xiong et al., 2013).

Previous studies suggested a prominent effect of Glcon root growth and development in Arabidopsis (Mishraet al., 2009; Booker et al., 2010; Kircher and Schopfer,2012; Xiong et al., 2013; Yuan et al., 2014). Increasing

Figure 1. Exogenous Glc induces LR emergence in Arabidopsis. Five-day-old light-grown wild-type (Col-0) seedlings weretransferred to 1/2 MS medium supplemented with different concentrations of Glc (0%, 0.5%, 1%, 3%, 4%, and 5% [w/v]) for4 d, and the number of emerged LRs was measured. A, Number of emerged LRs per seedling in wild-type Col-0 at different Glcconcentrations as observed after 4 d. Five-day-old light-grown seedlings of DR5::GUS were transferred to either sugar-free 1/2MS medium (0% [w/v] Glc) or 1/2 MS medium containing 3% (w/v) Glc for 4 d, and GUS expression patterns were observed.B, Detailed analysis of LRP development stages in DR5::GUS seedling roots treated without or with Glc. I to VII are LRPdevelopment stages; E, emerged LRs. C, DR5::GUS expression at sites of LR initiation (arrowheads) in the absence or presenceof Glc. D, Quantification of emerged LR count per seedling in wild-type Col-0 at the indicated concentrations of various sugaranalogs after 4 d. Besides Suc and Glc, other sugar analogs could not increase LR emergence. 3 OMG, 3-O-Methyl-D-glucopyranose. Data shown are averages of two biological replicates, each having at least 15 seedlings; error bars represent SE

(Student’s t test, P , 0.05; *, control versus treatment).

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concentrations of Glc could significantly increase LRnumber in Arabidopsis seedling root. However, not muchis known about the mechanism of this sugar/Glc regula-tion of LR development (Mishra et al., 2009). Sugars werepreviously shown to exhibit cross talk with phytohor-mones and other metabolic pathways (Gibson, 2004;Ramon et al., 2008). These and various other factors form acomplex signal response network to bring about optimumgrowth changes to enable better fitness in plants. Glc andauxin can interact extensively to control many aspects ofroot architecture, such as root elongation, LR production,root hair development, and root directional growth(Mishra et al., 2009; Mudgil et al., 2009; Booker et al.,2010; Yuan et al., 2014). However, there are very fewreports in the literature that either directly or indirectlylink BR responses to sugar signaling (Laxmi et al., 2004;Rognoni et al., 2007; Gupta et al., 2012; Singh et al.,2014a). In this study, we aim to analyze the nature of theinteraction between Glc and BR signaling and find itsmolecular bases during the regulation of LR emergenceusing the model plant system Arabidopsis.

RESULTS

Glc Induces LR Production via the HXK1-DependentSignal Transduction Pathway

Wild-type Columbia-0 (Col-0) seeds were germinatedand grown vertically on one-half-strength Murashige andSkoog (1/2 MS) medium containing 1% (w/v) Suc andsolidified with 0.8% (w/v) agar for 5 d in the light. Later,5-d-old homogenously grown seedlings were transferred toeither sugar-free 1/2MSmedium (0% [w/v] Glc) or 1/2MSmedium containing increasing concentrations of Glc(0.5%, 1%, 3%, 4%, and 5% [w/v]) for 3 to 4 d. The numberof emerged LRs per seedling as well as the LR densityincreased upon treatment with increased Glc concentra-tions in a dose-dependent manner (Fig. 1A; SupplementalFig. S1, A and B). Along with complete root, LR emer-gence from the old and new sections of root were alsoanalyzed separately, and it was found that exogenous Glcapplication could induce LR emergence from both oldand new sections of roots (Supplemental Fig. S1C).To check whether Glc is affecting the LR number byincreasing the initiation of new lateral root primordia(LRP) or accelerating the growth of preexisting primordia,5-d-old light-grown Auxin-responsive promoter DR5::GUS seedlings were transferred to either sugar-free 1/2MS medium (0% [w/v] Glc) or 1/2 MS medium con-taining 3% (w/v) Glc for 4 d. After GUS histochemicalstaining, the number of LRP belonging to various stagesof LR development was counted according to a pre-viously reported classification (Malamy and Benfey, 1997).The presence of Glc increased the total number of LRP ascompared with 0% (w/v) Glc (Fig. 1B). When the distri-bution of various LRP developmental stages was ob-served, it was found that 4 d after treatment, earlier stagesof LRP development (stages I–III) were diminished,whereas the later stages (stages IV–VII) as well as thenumber of emerged LRs were increased in Glc-treated

seedlings as compared with the untreated control (Fig.1C). These results suggest that Glc accelerates the growthof preexisting LRP and also promotes de novo LR for-mation to regulate root system architecture development.

In order to find if this induction in LR number is Glcspecific or if other sugars also could cause a similarresponse, the effect of various sugar analogs was ob-served. The seedlings could only show pronounced LRproduction in Suc- and Glc-containing media, whereasother slowly metabolizable sugars displayed a signifi-cantly lower number of emerged LRs even at higherconcentrations as compared with Suc and Glc (Fig. 1D;Supplemental Fig. S2). We used mannitol as an osmotic

Figure 2. Involvement of Glc signaling components during LR emer-gence. Analysis of Glc-induced LR emergence is shown in the wildtype (Col-0, Landsberg erecta [Ler], andWassilewskija [Ws]) versus theHXK1-dependent Glc signaling mutant gin2-1 (A), HXK1ox (B), andthe HXK1-independent Glc signaling mutants rgs1-1, rgs1-2, thf1-1,gpa1-1, and gpa1-2 (C). The HXK1-dependent Glc sensor mutantgin2-1 displayed a significantly reduced response to Glc-induced LRemergence, whereas the HXK1 overexpression mutant HXK1ox pro-duced a significantly higher number of emerged LRs at increased Glcconcentration as compared with their respective wild types. TheHXK1-independent signaling mutants showed no major change interms of emerged LR count as compared with their respective wildtypes. These results suggest that Glc affects LR emergence mainly viaHXK1-dependent components of Glc signaling. Data shown are av-erages of two biological replicates, each having at least 15 seedlings;error bars represent SE (Student’s t test, P , 0.05; *, control versustreatment; and **, the wild type versus mutant).

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control, and it could not promote LR production, sug-gesting that osmotic changes in the medium are not solelyresponsible for the observed response. 3-O-Methyl-D-glucopyranose could not even support root growth andthus caused no induction in LR production.

In plants, Glc signal perception and transductionoccur either through direct sensing via known Glcsensors such as HXK1 (Moore et al., 2003) and RGS1(Chen et al., 2003) or indirectly via energy and metabolitesensors (Sheen, 2014). The HXK1 mutant glucose insensi-tive2 (gin2) exhibits compromised responses toward Glc-induced LR production and LR density, whereas theHXK1 overexpression mutant HXK1ox produced a sig-nificantly higher number of emerged LRs and showedhigher LR density at increased Glc concentrations (Mishraet al., 2009; Fig. 2, A and B; Supplemental Fig. S3). TheHXK1-independent pathway involves a G-protein sig-naling component wherein AtRGS1 acts as a Glc sensor(Urano et al., 2013). To elucidate the role of HXK1-independent Glc signaling components, Glc regulation

of LR production in the HXK1-independent signalingmutants rgs1-1, rgs1-2, gpa1-1, gpa1-2, and thf1-1 wasmeasured. All the HXK1-independent signaling mutantstested showed no major change in terms of emerged LRcount or LR density as compared with their respectivewild types (Fig. 2C; Supplemental Fig. S4). Altogether, ourresults suggest that Glc mainly involves HXK1-dependentcomponents of Glc signaling to affect LR emergence inArabidopsis seedlings. However, we cannot rule out thepossible involvement of sugar metabolism-derived sig-naling in LR development.

Glc Signals Interact with BR Signaling duringLR Production

Plant hormones such as auxin, BR, cytokinin, and eth-ylene have been shown to play important roles affectingroot growth and development (Li et al., 2005; Garay-Arroyo et al., 2012; Jung and McCouch, 2013). The effect

Figure 3. BR signaling dependence of the Glc response for LR emergence. A, Comparison of the effects of different phyto-hormones on wild-type (Col-0) seedlings to determine their roles in LR emergence. Besides auxin, a very low concentration ofBR (10 nM) could induce emerged LR count, whereas ACC and BAP significantly reduced emerged LR count. B, Five-day-oldlight-grown seedlings of wild-type Col-0 were transferred to 1/2 MS medium supplemented with different concentrations of Glc(0%, 1%, 3%, and 5% [w/v]) and BR (0 M, 10 nM, 100 nM, and 1 mM) for 4 d, and the number of emerged LRs was measured. Thelower concentrations of both BR (10 nM) and Glc (1% and 3% [w/v]) are promotive, whereas higher concentrations (100 nM and1 mM BR, 5% [w/v] Glc) are inhibitory. C, The BR biosynthesis inhibitor brassinazole (BRZ; 1 mM) could inhibit LR production/emergence in wild-type (Col-0) seedlings even at higher Glc concentrations. D, Five-day-old light-grown wild-type seedlings(Enkheim [En2], Ws, and Col-0) and BR biosynthesis as well as signaling mutant seedlings were transferred to 1/2 MS mediumsupplemented with different concentrations of Glc (0%, 1%, 3%, and 5% [w/v]) and studied for LR emergence. The BR bio-synthesis mutant cpd and the BR perception mutant bri1-6 were found to be resistant, bak1-1 was found to have less sensitivity,and bzr1-1D with constitutively higher BR signaling showed hypersensitivity for Glc regulation of LR emergence as comparedwith the wild type. Values represent averages from two biological replicates, each having 15 seedlings; error bars represent SE

(Student’s t test, P , 0.05; *, control versus treatment; and **, the wild type versus mutant).


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of various phytohormones, such as auxin, ethylene,cytokinin, abscisic acid, and BR, on LR developmenthas been studied (Bao et al., 2004; Laplaze et al.,2007; Lewis et al., 2011; Duan et al., 2013; Lavenuset al., 2013). For consistency, we checked the in-volvement of various phytohormones during LRproduction and observed the effect of increasingconcentrations of auxin (IAA), BR (24-epibrassinolide),cytokinin (6-benzylaminopurine [BAP]), or ethylene(1-aminocyclopropane-carboxylic acid hydrochloride[ACC]) on LR emergence. Besides auxin, a very lowconcentration of BR (10 nM) could induce LR production,while ACC and BAP showed mainly an inhibitory effecton the number of emerged LRs and LR density (Fig. 3A;Supplemental Fig. S5).To find the interaction between Glc and BR on LR

production, 5-d-old light-grown wild-type seedlings weretransferred to independent and combined treatments ofdifferent concentrations of Glc and BR for 4 d. Lowerconcentrations of both BR (10 nM) and Glc (1% and 3%[w/v]) were stimulatory for emerged LR count as well asLR density, whereas higher concentrations of BR (100 nMand 1 mM) were inhibitory at all the Glc concentrationstested (Fig. 3B; Supplemental Fig. S6A). Inhibition of BRbiosynthesis using brassinazole could strongly reduce Glc-mediated LR production both in terms of number ofemerged LRs and LR density (Fig. 3C; Supplemental Fig.S6B), suggesting an additive interaction between Glc andBR in regulating LR production. The Glc sensitivity of BRbiosynthesis- and signaling-defective mutants waschecked to investigate the possible involvement of BRsignaling components during the Glc regulation of LRemergence. The BR biosynthesis mutant constitutive pho-tomorphogenic dwarf (cpd) produced more LRs at 0% (w/v)Glc as compared with the wild type but was found to beless sensitive for higher concentrations of Glc for LRproduction both in terms of emerged LR count and LRdensity (Fig. 3D; Supplemental Fig. S7). Similarly, the BRsignaling mutant bri1-6 was resistant and bri1-associatedkinase1 (bak1-1) was found to be less sensitive to the Glcregulation of LR production both in terms of emerged LRcount and LR density as compared with their respectivewild types (Fig. 3D; Supplemental Fig. S7). The brassinazoleresistant1-1D (bzr1-1D) mutant, which has constitutivelyhigher BR signaling, was found to be more responsive forthe Glc regulation of LR production both in terms ofemerged LR count and LR density (Fig. 3D; SupplementalFig. S7).Similarly, 5-d-old light-grown seedlings of the wild

type, the HXK1-dependent Glc sensor mutant gin2-1,and the HXK1-independent Glc sensor mutant rgs1-1were transferred to different concentrations of Glc- andBR-containing 1/2 MS medium and kept in controlledgrowth conditions for 3 to 4 d. The gin2-1 mutantshowed an attenuated response to exogenous BR ascompared with that of the wild type, whereas rgs1-1showed a wild-type-like response for Glc/BR regulationof emerged LR count as well as LR density (Fig. 4;Supplemental Fig. S8, A and B). These results suggestthat an optimal level of HXK1-mediated Glc signaling is

essential for exhibiting BR responses properly, and anyperturbation in the level leads to an altered response toBR. A whole-genome transcription profiling study alsorevealed that Glc and BR together could regulate a largenumber of genes involved in various aspects of rootgrowth and development (Supplemental Table S1). Real-time expression analysis suggested that exogenous Glcapplication could significantly affect transcript levels ofmany key genes involved in BR biosynthesis, perception,signaling, and early response (Fig. 5). The Glc regulationof most of these BR genes was found to be either reducedor completely abolished in the HXK1-dependent Glcsignaling pathway mutant gin2-1 (Fig. 5; SupplementalFig. S9). These results further strengthened our hypoth-esis that BR interacts with Glc signaling in an HXK1-dependent manner.

Figure 4. BR regulation of LR emergence is dependent on HXK1-mediated Glc signaling. Five-day-old light-grown seedlings of the wildtype (Col-0 and Ler), the HXK1-dependent Glc sensor mutant gin2-1,and the HXK1-independent Glc sensor mutant rgs1-1 were transferredto 1/2 MS medium supplemented with different concentrations of Glc(0%, 1%, 3%, and 5% [w/v]) and BR (0 M, 10 nM, 100 nM, and 1 mM).A, Quantification of the LR count of wild-type Ler and HXK1-dependentpathway mutant gin2-1 upon combined BR and Glc treatments. TheHXK1-dependent pathway mutant gin2-1 was found to be less sensitive toBR as comparedwith the wild type. B, For LR emergence, the rgs1-1mutantshowed a similar response to BR application at all the Glc concentrationstested. Values represent averages from two biological replicates, eachhaving 15 seedlings; error bars represent SE (Student’s t test, P , 0.05;*, control versus treatment; and **, the wild type versus mutant).

















BRI1 Is Epistatic to HXK1 for the Regulation ofLR Production

To investigate the relationship between Glc andBR signaling at the genetic level, the double mutantgin2-1bri1-6 was generated by making a genetic crossbetween homozygous gin2-1 and bri1-6 mutant plants(Supplemental Fig. S10A). Phenotypically, the homozy-gous gin2-1bri1-6 double mutant was found to havesimilar defects to its bri1-6 parent (Supplemental Fig.S10B).The homozygous gin2-1bri1-6 double mutant dis-played similar defects to bri1-6 in terms of emerged LRcount per seedling as well as LR density (Fig. 6A;

Supplemental Fig. S10C). As the gin2-1 and bri1-6 mu-tants were in different ecotype backgrounds (i.e. Ler andEn2, respectively), we also used wild-type segregants asa control for the gin2-1bri1-6 double mutant. Analysis ofLR development in wild-type segregant, ecotypes Lerand En2, gin2-1, bri1-6, and gin2-1bri1-6 seedlings uponcombined Glc and BR treatments was performed. In thegin2-1 mutant, exogenous application of Glc and BRcould still enhance LR emergence, albeit to a significantlylesser extent than both wild-type segregant and Ler;however, the complete resistance of bri1-6 and the gin2-1bri1-6 double mutant toward Glc-BR regulation of LR

Figure 5. Exogenous Glc applicationcan transcriptionally regulate most ofthe BR-related genes in an HXK1-dependent manner. Relative expressionis shown for genes involved in BR bio-synthesis (A), BR perception (B), BRsignaling (C), and early BR responses(D) in 5-d-old seedlings of wild-typeLer and the HXK1-dependent pathwaymutant gin2-1 after treatment with3% (w/v) Glc as compared with the 0%(w/v) Glc treatment condition. DET2,DEETIOLATED2; DWF4, DWARF4;BKI1, BRASSINOSTEROID INSEN-SITIVE1 KINASE INHIBITOR1; BSU1,BRASSINOSTEROID INSENSITIVE1SUPPRESSOR1;BEE2,BRASSINOSTEROIDENHANCED EXPRESSION2; BIM1,BRASSINOSTEROID INSENSITIVE1-ETHYLMETHANESULFONATE-SUPPRESSOR1-INTERACTING MYC-LIKE1. Data shownare averages of two biological replicates;error bars represent SE (Student’s t test,P , 0.05; *, control versus treatment;and **, the wild type versus mutant).

Figure 6. Phenotypic analysis of the homozygous gin2-1bri1-6 double mutant. A, Difference between emerged LR count in 10-d-oldlight-grown ecotypes Ler and En2, Glc sensor mutant gin2-1, BR perception mutant bri1-6, and homozygous double mutant gin2-1bri1-6 seedlings growing on 1/2 MS medium. B, Analysis of the Glc and BR sensitivity of wild-type (WT) segregant, ecotypes Ler andEn2, Glc sensor mutant gin2-1, BR perception mutant bri1-6, and homozygous double mutant gin2-1bri1-6 seedlings in termsof emerged LR count. Values represent averages from two biological replicates, each having 15 seedlings; error bars represent SE(Student’s t test, P , 0.05; *, control versus treatment; and **, the wild type versus mutant).


Gupta et al.







production, both in terms of emerged LR count as wellas LR density, further proved that BRI1 works down-stream of HXK1 during the Glc regulation of LR devel-opment (Fig. 6B; Supplemental Fig. S10D).

Glc-BR Interaction in Controlling LR Production MayInvolve Changes in Differential Auxin Distribution

Auxin gradient maintenance is well studied in termsof controlling several molecular responses that coordi-nate root development in plants. During LR develop-ment, auxin acts as a key signal that triggers the initialmitotic division of LR founder cells in the pericycle tissueand also the cell patterning in LRP (Benková et al., 2003;Casimiro et al., 2003). The PIN auxin efflux carriers aswell as the auxin uptake carriers AUX1 and LAX3 havepreviously been reported to play important roles in LRemergence and development (Benková et al., 2003;Swarup et al., 2008; De Smet, 2012; Marhavý et al., 2013).Both BR and Glc were previously reported to affect ba-sipetal auxin transport in Arabidopsis root (Li et al., 2005;Mishra et al., 2009). In our whole-genome transcriptionprofiling study, we found that Glc and BR together couldregulate the transcript levels of many auxin-related genes(Supplemental Table S2). To check whether Glc and BRinteract by means of auxin gradient maintenance, inde-pendent as well as combined effects of Glc and BR werestudied in mutants with defects in auxin transport. Theauxin transport-defective mutants ethylene insensitiveroot1 (eir1) and multiple drug resistance1 (mdr1-1) showedsignificantly fewer emerged LRs as well as reduced LRdensity upon Glc treatment as compared with their re-spective wild types (Fig. 7A; Supplemental Fig. S11A).Similarly, in the auxin influx-defective mutants aux1-7and lax3, Glc- and BR-regulated LR emergence was sig-nificantly reduced as compared with the wild type (Fig.7B; Supplemental Fig. S11B). Furthermore, exogenousBR and Glc application could also regulate the tran-script levels of the auxin efflux carriers PIN1, PIN2,P-GLYCOPROTEIN1 (PGP1), and MDR1 and the auxininflux carriers AUX1, LAX2, and LAX3 in Arabidopsisseedling roots (Supplemental Fig. S12, A and B). BR alonewas able to induce the gene expression of efflux carriers,while Glc alone or Glc and BR in combination could in-duce the gene expression of all the efflux carriers testedand the members of AUX1/LAX family except LAX1.AGRIS promoter sequence analysis revealed the presenceof sugar/Glc-inducible cis-elements in all these genes butnot in the LAX1 gene promoter (Supplemental Fig. S12C).These results indicate that Glc and BR could alter differ-ential auxin distribution to control LR development.

The AUX/IAA-ARF7-ARF19 Module Works Downstreamof Glc and BR during LR Production

The molecular mechanism for auxin-regulated LRorganogenesis is well studied, and the presence andabsence of auxin is known to regulate many genes

involved in LR development. The generated auxin signalstrigger the degradation of AUX/IAA repressors (AXR2,AXR3, SLR/IAA14, etc.), which subsequently activatesthe auxin response factors ARF7 and ARF19, leading tothe activation of downstream target genes and tran-scription factors involved in LR development (Lavenuset al., 2013). To study the involvement of this moduleduring Glc-BR regulation of LR production, we studiedindependent as well as combined effects of Glc and BRupon LR emergence as well as LR density in the auxinsignaling mutant axr6-1, the AUX/IAA gain-of-functionmutants axr2-1, axr3-1, and slr-1, and also in the non-phototrophic hypocotyl4arf19 (nph4arf19) double mutant.

Figure 7. BR and Glc control of LR development is defective in auxintransport mutants. Five-day-old light-grown seedlings of the wild type(Col-0 and Ws) and auxin transport-defective mutants eir1, mdr1,aux1-7, and lax3 were transferred to 1/2 MS medium supplementedwith different concentrations of Glc (0%, 1%, and 3% [w/v]) and BR(0 M and 10 nM) for 4 d, and LR emergence was measured. A, The auxintransport-defective mutant eir1 showed significantly less sensitivity forboth Glc and BR as compared with the wild type, whereas the sensi-tivity of mdr1 was found to be comparable to that of the wild type interms of LR emergence. B, Auxin influx carrier mutants aux1-7 andlax3 were found to be less responsive for both Glc and BR in terms ofLR emergence as compared with the wild type, suggesting an impor-tant role for auxin influx during Glc-BR control of LR development.Values represent averages of two biological replicates, each having 15seedlings; error bars represent SE (Student’s t test, P , 0.05; *, controlversus treatment; and **, the wild type versus mutant).











The axr6-1 mutant showed slightly less response, whileaxr2-1 and axr3-1 showed abrogated responses to Glc-BRcontrol of LR production both in terms of emerged LRcount as well as LR density as compared with that of thewild type. Both BR and Glc independently or in com-bination could not cause any LR emergence in slr-1 andnph4arf19 mutants (Fig. 8, A and B; Supplemental Fig.S13, A and B). We then checked the effect of Glc and BRon transcript levels of a few well-known downstreamtargets of ARF7 and ARF19, such as LAX3, LBD29, andEXPANSIN17 (EXP17), and found that Glc and BRcould differentially regulate the expression of thesegenes. However, in both slr-1 as well as the nph4arf19double mutant, the Glc- and BR-mediated regulation ofthese genes was significantly reduced (Fig. 8, C–E;Supplemental Fig. S13, C–E). These results suggestthat the Glc and BR regulation of LR development/emergence depends upon the SLR-, ARF7-, and ARF19-mediated auxin response module. Altogether, theseresults indicate that BR and Glc signaling pathwaysculminate to create a fine balance in auxin gradientacross the root to maintain optimum growth anddevelopment and that an intact auxin signaling path-way lies downstream of both Glc and BR signaling

pathways to execute Glc-BR-regulated LR production/emergence.

Relevance for Glc-Induced LR Production under NaturalEnvironmental Conditions

The availability of Glc under natural environmentalconditions depends on the presence of light, and Glc mightact as an intermediate during changing light conditions bywhich plants can modulate their root architecture. Lightlevels have been used in the past to modulate endogenoussugar levels in Arabidopsis (Freixes et al., 2002; Kircherand Schopfer, 2012). To test this hypothesis, we firstchecked if light could also modulate LR production similarto that of Glc. Increasing light intensity from 2,000 to 7,000lux could actually increase the number of emerged LRs in1/2 MS medium-grown wild-type (Col-0) seedlings (Fig.9A). Increased light flux application (7,000 lux) along withincreased Glc concentration displayed an additive effect onemerged LR count as well as LR density (Fig. 9B;Supplemental Fig. S14A). To determine if the light signal isactually transduced by Glc, the high light flux-induced LRproduction in the HXK1-dependent Glc signaling mutantgin2-1 was studied, and it was found that gin2-1 shows

Figure 8. BR and Glc control of LR development involves changes in auxin signaling and transport. A and B, Five-day-old light-grown seedlings of wild-type Col-0 and auxin signaling mutants axr2-1, axr3-1, axr6-1, and slr-1 (A) and nph4arf19 (B) weretransferred to 1/2 MS medium supplemented with different concentrations of Glc and BR for 4 d, and the number of emergedLRs was measured. The auxin signaling mutants axr3-1, slr-1, and nph4arf19 were found to be resistant for both Glc and BR ascompared with the wild type in terms of LR emergence. C to E, Five-day-old light-grown seedlings of wild-type Col-0, slr-1, andnph4arf19were transferred to 1/2 MS medium supplemented with different concentrations of Glc (0% and 3% [w/v]) and BR (0 M and10 nM) for 4 d, and transcript levels of a few well-known candidates regulating LR emergence, such as LAX3 (C), LBD29 (D), and EXP17(E), were checked in root tissue. BR as well as Glc regulation of these genes was found to be significantly reduced in both slr-1 andnph4arf19 mutants as compared with their respective untreated controls. Values represent averages of two biological replicates; errorbars represent SE (Student’s t test, P , 0.05; *, control versus treatment; and **, the wild type versus mutant).


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resistance toward increased light flux in terms of bothemerged LR count as well as LR density as comparedwiththe wild type (Fig. 9C; Supplemental Fig. S14B). Together,these results suggest that light may actually use Glc sig-naling components to modulate root architecture undernatural environmental conditions. Interestingly, higherlight flux application could not induce LR production/emergence in the mutants, which showed resistance to-ward Glc-induced LR emergence, such as bri1-6, axr3, slr-1,aux1-7, and lax3 (Fig. 9, D–F; Supplemental Fig. S14, C–E).These results further strengthened our hypothesis thatthe light signal is the upstream-most factor in theGlc-phytohormone scheme for the regulation of LRproduction/emergence in Arabidopsis early seedlings.Glc not only induced the LR emergence but also changedoverall root system architecture, as is evident by the in-creased surface area of the root system in seedlingstreated with 3% (w/v) Glc in contrast to the seedlingsgrowing in the absence of Glc (Supplemental Fig. S15). Thisincreased root system surface area enables better anchorageand support to the aboveground part and might alsofacilitate better nutrient and water uptake from the soil.

DISCUSSION

Roots act as a vital organ system for plants. LRs con-tribute as a major part of root biomass and perform keyfunctions, such as soil exploration, nutrient and wateruptake, and symbiosis with microorganisms (Den Herderet al., 2010; Cuesta et al., 2013). A variety of external aswell as internal factors, such as light, water, nutrients,phytohormones, etc., affect optimal root system architec-ture in plants (Malamy, 2005). Both sugars and BR arefundamental to plants and regulate a number of similarprocesses, such as root growth and development. Inter-actions of sugars with phytohormones such as ethyleneand abscisic acid have already been established (Gibson,2004), while Glc-auxin interaction has been decipheredusing a microarray approach (Mishra et al., 2009). Thereare only a few reports that highlight sugar and BR inter-action (Szekeres et al., 1996; Goetz et al., 2000; Laxmi et al.,2004; Bajguz, 2009; Vicentini et al., 2009; Vandenbusscheet al., 2011; Gupta et al., 2012). Recently, Glc-BR sig-naling interconnections during the directional growthof primary root have also been established (Singh et al.,

Figure 9. Photosynthetically generatedsugar signals can mimic Glc-inducedLR emergence. A, Comparison of LRemergence in wild-type (Col-0) seed-lings growing on 1/2 MS medium atincreasing light intensities (2,000–7,000lux). B, Effect of low light intensity(2,000 lux) and high light intensity(7,000 lux) on Glc-induced LR emer-gence in wild-type (Col-0) seedlings. C,Comparison of high-light flux-inducedLR emergence in the HXK1-dependentGlc signaling mutant gin2-1. The gin2-1mutant shows a perturbed response tohigh-light flux-induced LR emergence atall the Glc concentrations tested. D,Comparison of high-light flux-inducedLR emergence in the BR perceptionmutant bri1-6. The high-light flux ap-plication could not induce LR emer-gence in the bri1-6 mutant at any of theGlc concentrations tested. E, Compari-son of high-light flux-induced LR emer-gence in auxin signaling mutant axr3and slr-1 seedlings. The high-light fluxapplication could not induce LR emer-gence in these mutants. F, Comparisonof high-light flux-induced LR emergencein auxin influx carrier mutant aux1-7and lax3 seedlings. The high-light fluxapplication could not induce LR emer-gence in aux1-7 and lax3 to the extentseen in the wild type. Data shown areaverages of two biological replicates,each having at least 15 seedlings; errorbars represent SE (Student’s t test, P ,0.05; *, control versus treatment; and**, the wild type versus mutant).








2014a, 2014b). Here, we report that the metabolizablesugar Glc could modulate LR emergence in light-grownArabidopsis seedlings in an HXK1-dependent manner.We checked the combined effect of Glc and BR on LRemergence in Arabidopsis early seedlings to investigate apossible interconnection between these two signals in theregulation of LR development. Both Glc and BR act ag-onistically at lower concentrations for the regulation ofemerged LR count per seedling as well as LR density. BRand Glc could also regulate a large number of genes thatwere previously established to be involved in controllingvarious aspects of LR development. Moreover, the HXK1-dependent Glc signaling mutant gin2-1 showed an at-tenuated response to exogenous BR for LR emergence,whereas the Glc regulation of almost all the testedBR-related genes was also abolished in gin2-1 as com-pared with the wild type. Previously, we have shown thatthere is a direct involvement of both HXK1-dependentand independent pathways in the Glc-BR interaction inthe case of hypocotyl directional growth (Gupta et al.,2012), but in this study, we found that mainly the HXK1-dependent pathway is involved in the Glc-BR interactionduring LR development. In our previous study, we alsoreported that Glc increases BR signaling by enhancingBRI1 accumulation in the endocytic vesicle and at thesame time down-regulating BR biosynthetic genes, sug-gesting that BR signaling increases in Glc-treated seedlingroots (Singh et al., 2014a). On increasing concentrations ofGlc, the BR biosynthesis mutant cpd and perception mu-tant bri1-6were found to be resistant in terms of emergedLR count as well as LR density, suggesting that an intactBR biosynthetic and signaling pathway is needed for theproper Glc response, whereas the Glc-induced LR emer-gence and LR density in the bzr1-1D mutant, which hasendogenously high BR signaling, was found to be sig-nificantly greater as compared with the wild type. Thissuggests that enhancement of BR signaling might con-tribute positively to the regulation of LR production/emergence. The phenotype and Glc sensitivity analysisof the gin2-1bri1-6 double mutant further confirm that BRsignaling acts downstream of HXK1-mediated Glc sig-naling in regulating LR production.

The auxin carriers PINs and AUX1/LAX play keyroles during LR initiation bymaintaining cell type-specificauxin levels and distribution (Laskowski et al., 2008). Theauxin influx transporter AUX1/LAX family membersAUX1 and LAX3 have previously been implicated inregulating various stages of LR development (Swarupet al., 2008; Swarup and Péret, 2012). Both aux1 and lax3mutants have been shown to have reduced numbers ofLRs (Swarup et al., 2008). Auxin, at its site of action, ac-tivates a set of genes that regulate different steps of LRformation. Auxin triggers the degradation of AUX/IAAproteins, thereby derepressing the expression of thetranscription factors ARF7 and ARF19 (Lavenus et al.,2013). Several transcription factors of the LBD/ASLfamily are activated by ARF7 and ARF19, which posi-tively regulate LR production (Okushima et al., 2007;Lavenus et al., 2013). Auxin via ARF7 and ARF19 alsoinduces the expression of LAX3, creating a positive

feedback loop facilitating auxin influx in cells surround-ing LRP (Swarup et al., 2008). Auxin also induces cellwall-remodeling enzymes such as expansins in a LAX3-dependent manner to promote LR emergence (Laskowskiet al., 2006; Lavenus et al., 2013). Auxin also acts as acommon integrator to most endogenous and environ-mental signals that regulate LR development. The BRsignaling pathway has been reported to influence thefunctions of auxin and vice versa during root growth anddevelopment (Nemhauser et al., 2004; Mouchel et al.,2006; Hardtke et al., 2007; Vert et al., 2008). Similarly,sugar signals are also reported to interact with the auxinsignaling machinery (Moore et al., 2003; Gonzali et al.,2005; Ohto et al., 2006; Mishra et al., 2009; Mudgil et al.,2009; Booker et al., 2010; Sairanen et al., 2012; Yuanet al., 2014). Glc inhibits the G-protein complex-dependent

Figure 10. A testable model for BR and Glc control of LR emer-gence. Glc controls LR emergence through BR and auxin signalingmodules. Glc promotes BR signaling in an HXK1-dependent man-ner. BR promotes LR emergence by increasing auxin transport (Baoet al., 2004; Li et al., 2005). Auxin signal is relayed to the pericyclecells (mediated by AUX1, PIN2, LAX3, and MDRs), facilitating thedegradation of the AUX/IAAs (IAA14/SLR, etc.) involved in LR emer-gence. Degradation of AUX/IAAs activates ARF7/ARF19, which in turnregulates downstream target genes for LR emergence (LBD29/ASL16,EXP17, and other targets). Glc either directly or through BR can affectthe expression of downstream target genes such as LBD29, LAX3, andEXP17. Glc also enhances polar auxin transport (Mishra et al., 2009)and can induce auxin production directly (Sairanen et al., 2012), thusregulating LR emergence at multiple levels. Dotted arrows and ques-tion marks represent the possibility of additional routes and otherfactors.


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attenuation of auxin bimodality during LR development(Booker et al., 2010). Both Glc and auxin have previouslybeen shown to work synergistically at the whole-genomelevel (Mishra et al., 2009). Glc could effectively regulateauxin biosynthesis, degradation, signaling, as well astransport in Arabidopsis (Mishra et al., 2009; Sairanenet al., 2012). Both BR and Glc are also reported to affectbasipetal auxin transport in Arabidopsis root (Li et al.,2005; Mishra et al., 2009). There are also many reportsabout the interaction between auxin signaling and trans-port, with both Glc and BR independently and in com-bination, in controlling several developmental processessuch as primary root growth, LR production, hypocotyland root gravitropism, etc. (Mishra et al., 2009; Guptaet al., 2012; Singh et al., 2014a, 2014b).In our study, the attenuated Glc-BR sensitivity of

various auxin transport-defective mutants, such aseir1, mdr1-1, aux1-7, and lax3, proved the involvementof the intact auxin distribution machinery downstreamof Glc and BR during LR production. BR and Glc couldalso induce the transcript levels of many auxin effluxcarriers, such as PIN1, PIN2, PGP1, and MDR1, andauxin influx carriers, such as AUX1, LAX2, and LAX3.All of these observations suggest that the Glc-BR interactionmight involve polar transport-mediated differentialauxin distribution machinery to regulate LR emergence.The AUX/IAA gain-of-function mutants axr3 and slr-1as well as the nph4arf19 double mutant were found to behighly resistant to Glc- and BR-mediated LR production.The Glc-BR regulation of LAX3, LBD29, and EXP17transcripts was also found to be completely lost in slr-1and nph4arf19 roots, suggesting that this AUX/IAA-ARF7/ARF19 signaling module is involved in the Glc-BRregulation of LR production. Based on the previous reportsand our evidence here, we can say that the Glc-BR sig-naling interaction and subsequent modulation of auxintransport/responses play a major role in regulating LRemergence during early seedling development.

A Testable Model for BR and Glc Regulation ofLR Emergence

A testable model based upon the above-mentionedfindings and previously published reports is presentedin Figure 10. Glc induces LR production/emergencemainly by using HXK1-dependent signaling pathways(Mishra et al., 2009; Figs. 1 and 2). Glc interacts with BRsignaling via the HXK1-mediated pathway to regulateLR production (Figs. 3, B and C, and 4), which also in-volves most of the known elements of BR signaling (Figs.3D and 5). Analysis of Glc-induced LR emergence in thegin2bri1-6 double mutant further confirms that BRI1 actsdownstream of HXK1 during LR production (Fig. 6). Ourresults also suggested that differential auxin distributionmay work farther downstream of Glc and BR, since theauxin transport mutants eir1-1, mdr1-1, aux1-7, and lax3display abrogated responses to Glc-BR regulation of LRproduction (Fig. 7). These Glc-BR-mediated changes inauxin level/distribution can finally culminate at the AUX/IAA-ARF7/ARF19 module of LR production (Fig. 8).

Adaptive Significance for Glc-Induced LR Production

Under natural environmental conditions, Glc as such isnot available to the root. We hypothesized that varyinglight conditions could modulate endogenous Glc levelsand signaling to regulate LR production. We could actu-ally show the similar effects of both increasing light in-tensity as well as Glc concentration upon LR production.Moreover, higher light intensity could not induce LRproduction in the Glc signaling mutant gin2-1 or in thoseBR and auxin signaling-defective mutants that were re-sistant to Glc-induced LR production as such. Together,these results suggest that light might use Glc signalingcomponents to modulate the root architecture under nat-ural environmental conditions. However, we cannot denythe possible involvement of sugar metabolism and energysignaling machinery along with HXK1-dependent signalsduring LR development, which in itself is an interestingaspect to work upon. In this work, we mainly focused onthe AtHXK1-dependent Glc signaling mode, and we areproposing the involvement of Glc signals upstream ofphytohormones such as BR and auxin in regulating LRproduction, eventually leading to optimal root architec-ture under changing light conditions. Furthermore, Glccould actually affect overall root system architecture byenhancing the average root system surface area, therebyproviding better adaptability in terms of nutrient/waterabsorption as well as better anchorage in the soil.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotypes Col-0, Ws, Ler, and En-2 wereused as wild-type controls. Seeds of gin2-1 (AT4G29130, CS6383), rgs1-1(AT3G26090, CS6537), rgs1-2 (AT3G26090, CS6538), gpa1-1 (AT2G26300,CS3910), gpa1-2 (AT2G26300, CS3911), thf1-1 (AT2G20890), bri1-6 (AT4G39400,CS399), bak1-1 (AT4G33430, CS6125), bzr1-1D (AT1G75080, CS65987), eir1-1(AT5G57090, CS8058), aux1-7 (AT2G38120, CS3074), axr2-1 (AT3G23050,CS3077), axr3-1 (AT1G04250, CS57504), axr6-1 (AT4G02570, CS3817), andnph4arf19 (AT5G20730, AT1G19220, CS24629) were obtained from theArabidopsis Biological Resource Center (http://www.arabidopsis.org/abrc/).Seeds of HXK1ox (AT4G29130, N70282) and DR5::GUS were obtained from theNottingham Arabidopsis Stock Centre (http://arabidopsis.info/). The followinglines were obtained from the original published sources: cpd (AT5G05690;Szekeres et al., 1996), mdr1-1 (AT3G28860; Noh et al., 2001), slr-1 (AT4G14550;Fukaki et al., 2002), and lax3 (AT1G77690; Swarup et al., 2008). All mutant lineswere in the Col-0 background except the following: the bri1-6 mutant was in theEn-2 background; bak1-1, gpa1-1, gpa1-2, and mdr1 were derived from the Wsbackground; and the gin2-1 mutant was in the Ler background.

Growth Conditions

Seeds were surface sterilized and imbibed at 4°C for 48 h. Imbibed seedswere germinated and grown vertically on square (120 3 120 mm) petri platescontaining 1/2 MS medium supplemented with 1% (w/v) Suc (pH 5.7) and0.8% (w/v) agar and grown vertically in a climate-controlled growth room(22°C 6 2°C, 16 h of light and 8 h of darkness, 60 mmol m22 s21 light intensity)for 5 d. Thereafter, seedlings were transferred to treatment medium and theirroot tips were marked. Seedlings were then grown vertically for the next 4 d ingrowth chambers. The two light intensities used in this study corresponded toapproximately 2,000 lux (approximately 30 mmol m–2 s–1) and approximately7,000 lux (approximately 100 mmol m–2 s–1). In all experiments, plates weresealed with gas-permeable tape to avoid ethylene accumulation. All chemicalswere purchased from Sigma except agar, which was purchased from Himedia.Epibrassinolide was prepared as a 1022

M stock solution in 50% (v/v) ethanol.




http://www.arabidopsis.org/abrc/

http://arabidopsis.info/


The following were prepared as 1022M stock solutions in dimethyl sulfoxide:

brassinazole, BAP, and IAA. ACC was prepared as a sterile 1022M aqueous

stock solution. 5-Bromo-4-chloro-3-indolyl-b-glucuronic acid was prepared asa 100 mg L21 stock solution in N,N-dimethylformamide. All treatment con-centrations for this study were chosen from previously published reports(Gupta et al., 2012; Singh et al., 2014a, 2014b).

Measurements

Five-day-old light-grown seedlings were transferred to treatment medium,and their root tips were marked at the back of the petri dish. Digital imageswere captured each day after transfer to treatment medium using a NikonCoolpix digital camera. The number of emerged LRs was quantified by countingdirectly using a Nikon Stereo-Zoom microscope 4 d after transfer to the treatmentmedium. Total root length of the seedling 4 d after transfer was measured usingthe ImageJ program from the National Institutes of Health. LR density wasquantified as the number of emerged LRs per cm of root length of the seedling. Thesurface area of the root system was measured using the ImageJ program.

Gene Expression Analysis

Imbibed seeds of the wild type (Col-0 and Ler), the HXK1-dependent sig-naling mutant gin2-1, and the auxin signaling-defective slr-1 and nph4arf19 mutantswere sown on square (120 3 120 mm) petri dishes containing 1/2 MS mediumsupplemented with 1% (w/v) Suc and 0.8% (w/v) agar and grown verticallyin culture room conditions (16 h of light and 8 h of darkness, 80 mmol m22 s21 lightintensity). Once the plant material was uniformly germinated, the experimental con-ditions were applied. Five-day-old uniformly grown seedlings of the wild type (Col-0and Ler) and the gin2-1mutant were washed seven times with sterile water, with thelast wash given by sugar-free liquid 1/2 MS medium to remove residual exogenoussugar. In order to deplete internal sugars, seedlings were grown in sugar-free liquid 1/2 MS medium for 24 h in the dark. All subsequent steps were performed in the dark,and the cultures were shaken at 140 rpm at 22°C. Briefly, seedlings were treated with0% (w/v)Glc-, 0% (w/v)Glc + 100 nM BR-, 3% (w/v)Glc-, and 3% (w/v)Glc + 100 nMBR-containing liquid 1/2 MSmedium for 3 h in dark. Similarly, for the analysis ofLR-specific genes, 5-d-old uniformly grown seedlings of Col-0, slr-1, and the nph4arf19double mutant were transferred to 0% (w/v) Glc-, 0% (w/v) Glc + 100 nM BR-, 3%(w/v) Glc-, and 3% (w/v) Glc + 100 nM BR-containing 1/2 MS medium solidifiedwith 0.8% agar (w/v) and grown vertically in culture room conditions for 72 h. Af-terward, either whole seedlings or root tissue from each sample were flash frozen inliquid nitrogen and stored at280°C. Total RNAwas isolated from frozen tissue usingthe RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s protocol. RNAwasquantified and tested for quality before it was used for subsequent analyses.

For quantitative real-time PCR analysis, first strand complementary DNA wassynthesized by reverse transcription using 4 mg of total RNA in a 40-mL reactionvolume using the high-capacity cDNA Archive kit (Applied Biosystems) follow-ing the manufacturer’s protocol. Diluted complementary DNA samples (1:50 di-lution) were used for quantitative real-time PCR analysis. Each primer at 5 mM

was mixed with SYBR Green PCR master mix as per the manufacturer’s in-structions. The reaction was carried out on 96-/384-well optical reaction plates(Applied Biosystems) using the ABI 7900HT Fast Real-Time PCR System (AppliedBiosystems). To normalize the variance among samples, 18S ribosomal RNA wasused as the endogenous control. The mRNA levels for each candidate gene indifferent samples were determined using the delta delta cycle threshold method(Livak and Schmittgen, 2001). Relative expression values were calculated afternormalizing against the maximum expression value. Primers for real-time PCRwere designed for all the genes preferentially from the 39 end of the gene usingPrimer Express version 2.0 (PE Applied Biosystems) with default parameters. Theprimer sequences for all the genes tested are included in Supplemental Figure S16.

Forwhole-genome transcriptionprofiling experiments, 5-d-olduniformlygrownwild-type (Col-0) seedlings were starved in the dark and then treated with 0% (w/v) Glc-, 0%(w/v) Glc + 100 nM BR-, 3% (w/v) Glc-, and 3% (w/v) Glc + 100 nM BR-containingliquid 1/2 MS medium for 3 h. Microarray analysis was performed as described pre-viously (Mishra et al., 2009). Additional microarray data presentation andmanipulationwere assessed using Microsoft Excel. All data are Minimum Information about aMicroarray Experiment compliant. The raw data have been deposited in theArrayExpress database through MIAMExpress (accession no. E-MEXP-2714).

Crossing Arabidopsis Plants and Screening ofDouble Mutants

The double mutant gin2-1bri1-6 was generated by making a genetic crossbetween healthy plants of homozygous gin2-1 and bri1-6 mutants. Seeds from

successfully fertilized siliques were harvested, and the F1 generation wasallowed to self-pollinate. The segregating F2 populations were screened togenerate homozygous double mutant lines as well as wild-type segregants.The cleaved-amplified polymorphic sequence approach was used to genotypegin2-1 and bri1-6 mutations in the F2 generation (Supplemental Fig. S9, A and B).Genomic DNA from the crossed plants, mutant parents, and wild-type plants wasisolated, and HXK1 and BRI1 gene fragments were amplified with specific primersets. For homozygous gin2-1mutation confirmation, the amplifiedHXK1 fragment(400 bp) was purified and digested with HypCH4V enzyme, as the restriction sitefor this enzyme is perturbed in the homozygous gin2-1 mutant. Similarly, forhomozygous bri1-6 mutation confirmation, the amplified BRI1 fragment (559 bp)was purified and digested with enzyme Tsp45I, as the restriction site for this en-zyme is perturbed in the homozygous bri1-6 mutant. The primer sequences usedfor genotyping are included in Supplemental Figure S15.

GUS Histochemical Staining

GUS reporter activity was determined using a standard GUS histochemicalstaining procedure. Briefly, 5-d-old light-grown DR5::GUS seedlings weretransferred to 1/2 MS growth medium supplemented without or with Glc (0%and 3% [w/v]) and solidified with 0.8% (w/v) agar for 4 d. Seedlings aftertreatment were subsequently incubated at 37°C in a GUS staining solution[0.1 M sodium phosphate buffer, pH 7, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6,50 mM EDTA, and 1 mg mL21 5-bromo-4-chloro-3-indolyl-b-glucuronic acid]for 6 h. The seedlings were then kept in 70% (v/v) ethanol for the removal ofchlorophyll. Different stages of LR development were then observed andscored using a Nikon SMZ1500 Stereo-Zoom microscope, and photographswere taken with a Nikon Coolpix digital camera connected to a NikonSMZ1500 Stereo-Zoom microscope. The experiment was repeated twice, witheach replicate having at least 10 seedlings, yielding similar results.

Statistical Analyses

All experiments reported in this work were performed at least three times,yielding similar results. Data points are measures of at least 15 seedlings, unlessotherwise specified, from two to four experiments, and error bars represent SE.For quantitative real-time PCR results, the values represent averages of thetwo biological replicates (each with three technical replicates), and error barsrepresent SE. Statistical significance for all the experiments was evaluated us-ing Excel (Microsoft). For all experiments, statistical differences between bothcontrol/treatment and wild-type/mutant pairs were analyzed using Student’st test evaluation with paired two-tailed distribution. The P value cutoff wastaken at P , 0.05 except where stated otherwise. All end-point analyses weretaken 4 d after transfer to treatment medium unless otherwise specified,although plates were observed for longer periods up to 10 d.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Effect of Glc on LR emergence and LR density inArabidopsis seedlings.

Supplemental Figure S2. Effect of different sugar analogs on LR density.

Supplemental Figure S3. Glc regulation of LR density in HXK1-dependentsignaling mutants.

Supplemental Figure S4. Glc regulation of LR density in HXK1-independentsignaling mutants.

Supplemental Figure S5. Phytohormone regulation of LR density in thewild type.

Supplemental Figure S6. BR and Glc interaction in regulating LR density.

Supplemental Figure S7. Glc regulation of LR density in BR biosynthesisand signaling mutants.

Supplemental Figure S8. Quantification of Glc-BR regulation of LR den-sities in the wild type, HXK1-dependent, and HXK1-independent Glcsignaling mutants.

Supplemental Figure S9. Comparison of transcriptional regulation of theBR-related genes in wild-type Ler and HXK1-dependent pathway mu-tant gin2-1 seedlings upon treatment without or with Glc.


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Supplemental Figure S10. Homozygous gin2-1bri1-6 double mutant gen-otyping and phenotype analysis.

Supplemental Figure S11. Quantification of LR density in the wild typeand auxin transport-defective mutants.

Supplemental Figure S12. BR and Glc control of LR development involvespolar transport-mediated changes in auxin distribution.

Supplemental Figure S13. Quantification of LR density in the wild typeand auxin signaling mutants.

Supplemental Figure S14. Changes in light flux correlate with Glc-mediated effects on LR density.

Supplemental Figure S15. Glc affects overall root system architecture.

Supplemental Figure S16. List of primers used in this study.

Supplemental Table S1. Differential regulation of LR development-relatedgenes by Glc and BR independently and in combination.

Supplemental Table S2. Differential regulation of auxin response-relatedgenes by Glc and BR independently and in combination.

ACKNOWLEDGMENTS

We thank the National Institute of Plant Genome Research CentralInstrument Facility (Real-Time PCR Division) for assistance and the DNA andProtein Microarray Facility (University of California, Irvine) for conductingmicroarray experiments and initial data analysis.

Received December 23, 2014; accepted March 19, 2015; published March 25,2015.

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Interaction between Glucose and Brassinosteroid during the … · Interaction between Glucose and Brassinosteroid during the Regulation of Lateral Root Development in Arabidopsis1