Switching the Direction of Stem Gravitropism by AlteringTwo Amino Acids in AtLAZY11[OPEN]

Takeshi Yoshihara and Edgar P. Spalding2,3

Department of Botany, University of Wisconsin, 430 Lincoln Drive, Madison, Wisconsin 53706

ORCID IDs: 0000-0002-4897-159X (T.Y.); 0000-0002-6890-4765 (E.P.S.).

From germination to flowering, gravity influences plant growth and development. A rice (Oryza sativa) mutant with a distinctlyprostrate growth habit led to the discovery of a gene category that participates in the shaping of plant form by gravity. Each so-called LAZY gene includes five short regions of conserved sequence. The importance of each of these regions in the LAZY1gene of Arabidopsis (Arabidopsis thaliana; AtLAZY1) was tested by mutating each region and measuring how well transgenicexpression of the resulting protein variant rescued the large inflorescence branch angle of an atlazy1 mutant. The effect of eachalteration on subcellular localization was also determined. Region I was required for AtLAZY1 to reside at the plasmamembrane, which is necessary for its function. Mutating region V severely disrupted function without affecting subcellularlocalization. Regions III and IV could be mutated without large impact on function or localization. Altering region II with twoconservative amino acid substitutions (L92A/I94A) had the profound effect of switching shoot gravity responses from negative(upward bending) to positive (downward bending), resulting in a “weeping” inflorescence phenotype. Mechanical weakness ofthe stem was ruled out as an explanation for the downward bending. Instead, experiments demonstrated that the L92A/I94Achange to AtLAZY1 reversed the auxin gradient normally established across stems by the gravity-sensing mechanism. Thisdiscovery opens up new avenues for studying how auxin gradients form across organs and new approaches for engineeringplant architecture for agronomic and other practical purposes.

The gravity vector is a source of information that theshoot system uses to orient growth of its components.For example, primary and secondary stems (Fukaki et al.,1996a, 1996b; Yamauchi et al., 1997; Roychoudhryet al., 2013; Yoshihara et al., 2013), leaf petioles (Manoet al., 2006),floral pedicels (Wei et al., 2010), andmonocottillers (Abe et al., 1996; Wu et al., 2013; Sang et al., 2014)each use gravity to guide growth at an angle that evo-lution has selected and the environment modified. Un-derstanding how plants decode the gravity vector toorient growth is thus an important topic in plant biology.A gravity-directed change in growth direction, or

gravitropism, begins with a perception process thatcauses a difference in the cell elongation rate across theorgan (Morita, 2010; Singh et al., 2017; Su et al., 2017).The growth differential across the organ, which pro-duces the appropriate degree of curvature, results from

the redistribution of the growth hormone auxin to thelower side of the organ. The perception event relies onstarch-filled plastids called statoliths settling to thelowest point in the sensory cells, which in Arabidopsis(Arabidopsis thaliana) inflorescence stems are the endo-dermal cells surrounding the vascular bundles in thegrowing region (Tasaka et al., 1999; Saito et al., 2005).The subsequent auxin redistribution phase relies on thePIN-FORMED (PIN) auxin transport proteins relocat-ing within the endodermal cells (Rakusová et al., 2015).As a result, growth-promoting concentrations of auxinaccumulate on the lower side of the organ and upwardbending ensues.A plant-specific LAZY gene family, discovered through

characterization of a rice (Oryza sativa) mutant with un-usually wide tiller angles (Li et al., 2007; Yoshihara andIino, 2007), encodes proteins that function in gravi-tropism. Studies of lazy mutants in Arabidopsis, maize(Zea mays), Medicago (Medicago truncatula), and ricehave begun to indicate where in a gravitropism path-way LAZY proteins function (Li et al., 2007; Yoshiharaand Iino, 2007; Dong et al., 2013; Yoshihara et al., 2013;Ge and Chen, 2016). Statoliths appear to sedimentnormally in Arabidopsis and rice lazy mutants that donot form a normal gravity-induced auxin gradient orgravitropic bending response. Etiolated hypocotyls ofan Arabidopsis lazy quadruple mutant (atlazy1;2;3;4)are essentially agravitropic but display a robust photo-tropic response (Yoshihara and Spalding, 2017), whichalso depends on PIN-mediated auxin redistribution(Haga and Sakai, 2013; Fankhauser and Christie, 2015).These and other results indicate that LAZY proteins

1This work was supported by the National Science FoundationPlant Genome Research Program (IOS-1031416 to E.P.S.).

2Author for contact: spalding@wisc.edu.3Senior author.The 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:Edgar P. Spalding (spalding@wisc.edu).

T.Y. and E.P.S. conceived the investigation of conserved residues;T.Y. performed all of the genetics, molecular biology, and phenotypemeasurements; E.P.S. developed the bending force assay; T.Y. andE.P.S. performed data analysis; T.Y. and E.P.S. wrote the manuscript.

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function in a gravity-specific process downstream ofstatolith sedimentation but upstream of the auxin re-distribution process.

LAZYgenes are foundonly in embryophytes (Yoshiharaet al., 2013). They encode moderate-sized proteins withno known or predictable biochemical function. Com-paring LAZY gene sequences from a variety of landplants identified five short regions of conserved se-quence ranging from region I at theN terminus to regionV at the C terminus (Yoshihara et al., 2013). The onlyrecognizable domain is an ethylene-responsive elementbinding factor-associated amphiphilic repression (EAR)motif (Yoshihara et al., 2013) that is generally associ-ated with transcriptional repression mechanisms (Kagaleet al., 2010). A well-established example is the binding ofTOPLESS proteins to the EAR motif of AUX/IAA pro-teins,which repress auxin-responsive genes (Salehin et al.,2015; Dinesh et al., 2016). The EARmotif of awheat LAZYprotein, TaDRO1, was found to interact with a wheatTOPLESS protein (Ashraf et al., 2019).

Dardick et al. (2013) showed that region II of LAZYgenes contains a GwL(A/T)GT motif, which placesLAZY genes within a larger family, IGT, to which theTiller Angle Control 1 (TAC1) gene belongs. Interest-ingly, TAC1 functionally opposes LAZY by increasingthe tiller angle in rice (Yu et al., 2007) and branch anglesin peach (Prunus persica) trees (Dardick et al., 2013) andArabidopsis inflorescences (Waite and Dardick, 2018).

Functional fusions of LAZY proteins with GFP showthey reside at the cell periphery (Li et al., 2007; Donget al., 2013; Yoshihara et al., 2013), colocalizing with aplasma membrane resident dye (Dong et al., 2013;Yoshihara et al., 2013). In Arabidopsis, AtLAZY1 alsoresides in the nucleus, but mutations that prevent nu-clear localization do not interfere with its branch angle-setting function (Yoshihara et al., 2013). Therefore, it isnot clear whether the potential interaction betweennuclear-localized auxin response factors and the EAR-like motif are relevant to the major role AtLAZY1plays in shoot gravity responses. The C-terminal do-main of AtLAZY1 also binds to microtubules (Sasakiand Yamamoto, 2015). Dong et al. (2013) showed thatZmLAZY1 binds to IAA17 in the nucleus and to areceptor-like kinase at the plasmamembrane. In studiesof rice, Li et al. (2019) found that OsLAZY1 interactswith a protein called OsBRXL4 at the plasma mem-brane. Narrower and wider tiller angles are associatedwith reducedOsBRXL4 expression and overexpression,respectively. More study of LAZY-interacting proteinspromises to clarify how and where in cells gravity sig-nals are converted into growth differentials.

Yoshihara et al. (2013) demonstrated that four LAZYgenes in Arabidopsis possess all five conserved regions(I–V). They are AT5G14090/LAZY1, AT1G17400/LAZY2, AT1G72490/LAZY4, and AT3G27025/LAZY6.One gene, At1G19115/LAZY3, lacks region III, whereasa sixth, AT3G24750/LAZY5, possesses only region V.Studies of these genes have found some unique andsome shared regions of expression in roots and shoots(Yoshihara and Spalding, 2017). Studies of knockout

mutants indicate that functions of the homologous genesalso overlap. For example, mutating AtLAZY1 caused alarge change in branch angle, but the primary inflores-cence stem remained vertical. Adding atlazy2 and atlazy4mutations to this background caused the entire inflo-rescence to become prostrate (Taniguchi et al., 2017;Yoshihara and Spalding, 2017), and possibly even toreverse its preferred growth direction, as has beenobserved in roots of the atlazy2;3;4/atngr1;2;3 triplemutant (Ge and Chen, 2016, 2019; Yoshihara andSpalding, 2017).

It is now clear that LAZY and related genes are im-portant mediators of gravity effects on plant architec-ture. In spite of the importance of the topic, themolecularmechanisms in which LAZY proteins participate re-main poorly understood. The work presented here ex-amines the functional consequences of rationally selectedand engineered mutations in conserved regions of theAtLAZY1 gene. The results include the discovery of asubtle change in the AtLAZY1 protein that reverses thedirection of shoot gravitropism.

RESULTS

Quantitative Transgenic Rescue of the atlazy1 BranchAngle Phenotype

The angle between the base of a branch and theprimary inflorescence was measured in 134 wild-typeand 141 homozygous atlazy1 plants, as shown inFigure 1A (inset). The average angle for the wild typewas 39.0° 6 0.5° and 81.4° 6 0.5° for atlazy1 (Fig. 1B),similar to previous reports (Yoshihara et al., 2013). Thewild type and atlazy1 data were normally distributed asdetermined by a Shapiro-Wilks normality test. Branchangle was also measured in atlazy1 plants transformedwith the wild-type AtLAZY1 gene under the control ofits native promoter (pAtLAZY1:AtLAZY1). Each T1plant is probably the product of a unique transforma-tion event (Desfeux et al., 2000). Thus, each of the 153 T1plants measured may be considered an independenttest of genetic rescue. The average branch angle of therescue-test population was 54.4° 6 1.4°, but fitting twoGaussian distributions to the data produced a proba-bility density function (PDF) that showed the popula-tion to be clearly bimodal (Fig. 1C). The Shapiro-Wilkstest indicated that these rescue-test data were not nor-mally distributed.Many of the transformants displayedawild-type branch angle, but many displayed amutantbranch angle (Fig. 1, B and C). The substantial portionof the pAtLAZY1:AtLAZY1/atlazy1 curve covering an-gles .60° indicates that the AtLAZY1 genomic frag-ment was often not fully effective. If this was solely dueto the random position of insertion in the genome,a similar PDF should be observed when a differentbut similarly functioning gene is used with the samepromoter. When AtLAZY2 was expressed under thecontrol of the AtLAZY1 promoter, the population oftransformants was unimodal, normally distributed,

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and similar to the wild type (Fig. 2, A and B). Thissuggests that the bimodality in the control rescue-testpopulation is not caused simply by a transgene posi-tion effect but by something specific to the AtLAZY1coding sequence, introns, untranslated regions of thetranscript, and/or the AtLAZY1 protein. These dataindicate that AtLAZY2, which is 19.8% identical toAtLAZY1 at the amino acid level, as determined by theT-COFFEE sequence alignment tool (Notredame et al.,2000), can perform the AtLAZY1 branch angle-relatedfunctions.AtLAZY1 is present at the plasmamembrane and the

nucleus of cells (Yoshihara et al., 2013). Mutation of theinternal nuclear localization signal from AtLAZY1eliminated nuclear accumulation of the protein but didnot affect its ability to rescue the atlazy1 branch anglephenotype (Yoshihara et al., 2013). AtLAZY2 is foundonly at the plasma membrane (Fig. 2C). Thus, rescue ofthe atlazy1 branch angle phenotype appears not to re-quire accumulation of anAtLAZYprotein in the nucleus.The results in Figures 1 and 2 establish a method for

structure-function analysis of the AtLAZY1 protein thatis based on (1) the AtLAZY1 promoter, (2) an atlazy1knockout mutant, (3) large numbers of independentlytransformed plants, and (4) branch angle PDFs toquantify the effectiveness of the transgene modification.

Structure-Function Analysis of the Five LAZYConserved Domains

Five short regions of sequence are conserved amongLAZY genes from diverse land plants (Yoshihara et al.,2013). Figure 3A shows the locations of conserved re-gions I–V in the 358-amino acid protein predicted fromthe AtLAZY1 gene sequence. The functional signifi-cance of these conserved regions in AtLAZY1 was in-vestigated by altering one or more of the most invariantamino acids in each and transforming atlazy1 plantswith the mutated versions controlled by the sameAtLAZY1 promoter used to produce the transgenicplant data in Figures 1 and 2. To determine whether thesite-directed mutagenesis prevented expression of theprotein in the plant, a GFP-tagged version of eachvariant controlled by a strong heat shock promoter wasvisualized with confocal fluorescence microscopy inetiolated Arabidopsis hypocotyls. This method wasnecessary because the natural AtLAZY1 expressionlevel is too low to be reliably detected with a fluores-cence protein tag. To test the effect of the mutationson subcellular localization, a GFP-tagged version was

Figure 1. Evaluation of rescue of the atlazy1 branch angle phenotype inthe T1 generation. A, Shoot architecture in the wild type, atlazy1, andatlazy1 rescued with pAtLAZY1:AtLAZY1 in the T1 generation. Shownfrom left are the wild type, atlazy1, and atlazy1 rescued with pAtLA-ZY1:AtLAZY1 in the T1 generation. The inset shows how the angle at thebase of the branch was measured with two 10-mm radii centered at theintersection of a main stem and a branch. B, Distributions of branchangles measured in populations of wild type, atlazy1, and atlazy1

rescued with pAtLAZY1:AtLAZY1 in the T1 generation. The horizontalline marks the mean, which is also given (6 SE). The numbers of samplesmeasuredwere as follows: 134 wild type, 141 atlazy1, and 153 rescuedlines. C, PDFs of branch angles measured in the populations of wild-type, atlazy1, and atlazy1 plants rescued with pAtLAZY1:AtLAZY1 inthe T1 generation. PDFs were obtained by fitting Gaussian distributionsto the data used in B.

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transiently overexpressed in Nicotiana benthamianaleaves and then examined by confocal fluorescencemicroscopy. Representative examples of subcellular

localization are shown in Figure 3, and more examplesare shown in Supplemental Figure S1. Figure 3B showsthat AtLAZY1 produced by atlazy1 plants transformedwith pAtLAZY1:AtLAZY1 was apparently producedand localized to the plasma membrane and nucleusconsistent with previous reports (Yoshihara et al.,2013; Taniguchi et al., 2017). Figure 3B also shows thatthe atlazy1 branch-angle phenotype was rescued bypAtLAZY1:AtLAZY1 to varying degrees (data replottedfrom Fig. 1, B and C). These results serve as the controlfor the following structure-function studies.

Invariant and highly conserved residues within re-gions I–V that were identified by comparing LAZYgenes from several species (Yoshihara et al., 2013) weresubjected to site-directed mutagenesis. The targetedamino acids were selected to test the function of each ofthe conserved regions. The N-terminal region I wasperturbed by changing amino acids 6–8 from Trp-Met-His to Ala-Ala-Ala, or W6A/M7A/H8A (Fig. 3C). Thischange to region I greatly diminished the gene’s abilityto rescue the atlazy1 branch angle defect, as indicated bycomparing the branch angle PDFs in Figure 3, B and C(see Supplemental Fig. S2 for a scatter plot of eachbranch angle value fromwhich the PDFswere derived).The W6A/M7A/H8A change to region I also reducedthe protein’s presence at the plasma membrane with-out affecting the nuclear pool (Fig. 3C; SupplementalFig. S1). This result indicates that the N terminus isrequired for plasma membrane localization and branch-angle function, consistent with previous findings (Fig. 2;Yoshihara et al., 2013).

To perturb region II, a three-amino acid sequence waschanged from Leu-Ala-Ile to Ala-Ala-Ala, or L92A/I94A(Fig. 3D). This change did not alter the subcellular lo-calization pattern of the protein but it completely pre-vented the protein from rescuing the branch angledefect (Fig. 3D). In fact, a substantial number of thebranch angles were.90°, i.e. were oriented downward.The notably unimodal function was shifted to greaterangles compared to the atlazy1 background in which ithad been placed. This two-amino acid variation of theprotein it replaced apparently caused a phenotypemore severe than complete loss of function. Two plantsthat showed greater branch angles than atlazy1 wereselected from the population of T1 plants expressingpAtLAZY1:AtLAZY1L92A/I94A. Two lines homozygous forthe transgene and homozygous for the atlazy1 back-ground mutation were obtained and named L1 and L2.The average branch angles of these homozygous linesbearing a two-amino acid variation of AtLAZY1 in anatlazy1 knockout background were shifted even furtherdownward, ;30° below the horizontal (SupplementalFig. S3). The branch angles in these lines were shifteddownward even more than that of an essentially agra-vitropic atlazy1;2;3;4 quadruple mutant (SupplementalFig. S3), which produces a prostrate inflorescence similarto that of the atlazy1;2;4 mutant described previously(Yoshihara and Spalding, 2017).

The variants with mutations in region III (P185A/L186A) or region IV (H283A/R284A/K285A) rescued

Figure 2. AtLAZY2 at the plasma membrane replaces AtLAZY1 func-tion. A, Branch angles measured in populations of the wild type,atlazy1, and atlazy1 transformed with pAtLAZY1:AtLAZY2 in theT1 generation. The horizontal line marks the mean, which is alsogiven (6 SE). The numbers of samples measured were as follows: 49 wildtype, 60 atlazy1, and 82 atlazy1plants rescuedwith pAtLAZY1:AtLAZY2.B, PDFs of branch angles shown in A. The distribution of pAtLA-ZY1:AtLAZY2/atlazy1 branch angles was unimodal. C, Subcellular lo-calization of 35S:AtLAZY2-mRFP examined in Nicotiana benthamianaleaf epidermal cells. Subcellular localization of AtLAZY2-mRFP wasexamined by Agrobacterium tumefaciens-mediated transient expres-sion of 35S:AtLAZY2-mRFP and 35S:TGA5-eGFP in N. benthamianaleaf epidermal cells. The red and green channels show AtLAZY2-mRFPand the TGA5-eGFP nuclear markers, respectively. Bars 5 25 mm.AtLAZY2-mRFP localized to the plasmamembrane and not the nucleus.

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the atlazy1 branch angle defect slightly lesswell than thecontrol construct and showed typical wild-type sub-cellular localization (Fig. 3, E and F). These data setsappeared bimodal and did not pass a Shapiro-Wilkstest of normality. Regions III and IV are not as highlyconserved as the others (Yoshihara et al., 2013). Perhapsin certain environmental conditions regions III and IVwould be found to have a more significant impact onaspects of shoot architecture that gravity influences.Region V, a signature motif of LAZY proteins

(Yoshihara et al., 2013), is the longest conserved region,and therefore, three distinct variants (W346A, T349A/D350A, and V355A/L356A) were created to test its rolein setting branch angles. None of these changes af-fected production of the protein in Arabidopsis or its

subcellular localization in N. benthamiana leaf cells(Fig. 3, G–I–I). The first two (variants V-1 and V-2) weremarkedly less able to rescue the atlazy1 branch angledefect. The third (V-3) showed no rescue ability.Thus, region V is critical to the AtLAZY1 branch an-gle function.Only the W6A/M7A/H8A alteration in region I had

a qualitative effect on subcellular localization (Fig. 3C).It appeared to eliminate location at the plasma mem-brane, and it lacked branch angle-setting function. Re-gion I probably cannot anchor the AtLAZY1 protein tothe membrane by itself because region I fused to en-hanced GFP (eGFP) appeared to remain in the cyto-plasm, similar to the free-eGFP control (SupplementalFig. S4).

Figure 3. Mutagenesis of AtLAZY1conserved regions: effects on branchangle and subcellular localization. A,Schematic showing amino acid posi-tions of the fiveAtLAZY1protein regions(AT5G14090 gene model) conservedamong homologs, as defined byYoshihara et al. (2013). B to I, Effects ofmutagenesis on the function ofAtLAZY1 and its subcellular localiza-tion. The functions of the engineeredvariants were evaluated as shown inFigure 1. Selected conserved aminoacid sequences were replaced with al-anines by site-directed mutagenesis ofpAtLAZY1:AtLAZY1, and the engi-neered variants were expressed in anatlazy1 knockout background. Shownfor each variant are the amino acidsequence of the conserved region, thealtered sequence in magenta and inletter/number code, the PDFs derivedfrom the branch angle measurements,the subcellular localization in Nicoti-ana benthamiana epidermal cells (topright), and expression in Arabidopsishypocotyls (bottom right). PDFs of thewild type and atlazy1 are representedas dashed lines. Orange lines show thePDFs of the T1 population expressingthe variants. The data in B are takenfrom Figure 1C. The data in B, D, and Fdid not pass a Shapiro-Wilks test ofnormality and are considered bimodal.The numbers of samples ranged from134 for the wild type to 170 for the V-1 population. The effects of conservedregions on subcellular localizationwere examined by transiently express-ing the variants in N. benthamiana leafepidermal cells and in stably trans-formed Arabidopsis seedlings. Bars 510mm (forN. benthamiana) and 40mm(for Arabidopsis).

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The structure-function results indicate that regions I,II, and V play a critical role in determining lateralbranch angle. Region II mutations created a special ef-fect that was further investigated.

A Two-Amino Acid Change to Region IIReverses Gravitropism

The downward orientation of branches inpAtLAZY1:AtLAZY1L92A/I94A/atlazy1 T1 plants (here-after L92A/I94A) may be the result of a reversal in thenormal negative gravitropism mechanism. To test this,plants were grown in a normal orientation illuminatedfrom above (Fig. 4A), or upside down and illuminatedfrom below (Fig. 4B). The wild-type primary inflores-cence stem and its branches exhibited strong nega-tive gravitropism (upward bending). Regardless ofthe imposed orientation, atlazy1 inflorescence branchesemerged from the main axis at a near-horizontal angle.By contrast, the stem, branches, and even pedicelssubtending the flowers of the L92A/I94A plants alignedpositively (downward) with the gravity vector whethergrown right-side up or upside down. The interpretationthat wild-type negative gravitropismwas weakened bythe loss of AtLAZY1 and switched to a positive orien-tation by the L92A/I94A alteration was tested by ro-tating normally grown plants by 90°. Shoots of L92A/I94A responded with a phase of normal (negative)gravitropism that was followed by bending in the op-posite direction (positive or reverse gravitropism) tobring the apex near to the original, horizontal position(Fig. 4C). A possible complication to address was thatsagging due to a mechanical weakness of L92A/I94Ashoots contributed to the appearance of reverse, posi-tive gravitropism. Results of double reorientationassays argued against this possibility. When wild-typeplants were inverted, the primary inflorescence and

lateral branches curved to become upright and whenthey were returned to their original orientation, theshoots reversed the direction of new growth to be-come upright again (Supplemental Fig. S5). L92A/I94A branches grown upside down did not sag whenreturned to the normal orientation. Their new growthwas distinctly downward, displaying reverse (posi-tive) gravitropism (Supplemental Fig. S5).

The possibility that mechanical weakness contrib-uted wholly or in part to the apparent reverse gravi-tropism of L92A/I94A inflorescences was directly testedwith an electronic force transducer. The experimentwas designed to measure the stiffness of primary in-florescence stems that were at least 22 cm long, a pointin development when the apex of L92A/I94A inflores-cences, grown with support as shown in Figure 4A,clearly displayed downward growth. The custom de-vicemeasured the force required to flex the free end of astem segment by 2 mm, as shown in Figure 5, A and B.The method accurately quantified the difference inwild-type stem stiffness between the apical 3 cm (upperstem) and 7 cmbelow the apex (lower stem; Fig. 5C). Theresults obtained with the homozygous region II variants(L1 andL2)were not significantly different from thewildtype, or from the essentially prostrate atlazy1;2;3;4 qua-druple mutant. The results in Supplemental Figure S5and Figure 5C indicate that differences in mechanicalstrength of the stems do not explain the downward or-ientation of shoots homozygous for a region-II variationof AtLAZY1. Instead, the evidence indicates that thesubtle L92A/I94A variation in the AtLAZY1 proteinreversed the gravitropic response.

The reverse gravitropism explanation was testedby measuring the reorientation time course of primaryinflorescences following a 90° rotation. The apical 7 cmof the primary inflorescence was excised from plantsat developmental stages before (7–10 cm) or after(22–25 cm) the point when L92A/I94A shoots display

Figure 4. Downward orientation of inflores-cence stem caused by mutating region II (L92A/I94A). A, Shoot system architecture of wild-type(WT), atlazy1, andpAtLAZY1:AtLAZY1L92A/I94A/atlazy1 (L92A/I94A) plants grown in a normalorientation for 4 weeks with light supplied fromabove, as shown by the white arrow. Bar 5 2cm. B, Lateral organ orientations in wild-type,atlazy1, and L92A/I94A plants grown upside-down after their branches reached ;1–3 cmwith light supplied frombelow (see arrow). Notethat branches and pedicels oriented horizontallyin atlazy1 and downward in pAtLAZY1:AtLA-ZY1L92A/I94A/atlazy1, though tips of shoots areoriented toward the light source in both cases.Bar 5 3 cm. C, Gravitropism in excised wild-type, atlazy1, and L92A/I94A inflorescencestems. Images taken immediately after 90°rotation (0 h; blue) and 18 h after rotation(orange) were superimposed. Bar 5 5 mm.

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downward growth. With the cut ends maintained in anutrient solution, excised wild-type inflorescences ofeither developmental age rapidly reoriented, overshot90°, and attained a steady vertical orientationwithin 6 h(Fig. 6A; Supplemental Video S1). The younger L1, L2,atlazy1, and wild-type stems responded similarly, ex-cept that the overshoot phase was dampened in themutants (Fig. 6A). However, the older L1 and L2 stemsegments displayed a normal (negative) gravitropismresponse until an angle of ;80° was achieved 3 h afterrotation. Thereafter, by approximately the time a wild-type stem would have reached 90°, the L1 and L2stems began to develop reverse (positive) gravitrop-ism (Fig. 6A), which continued over the next severalhours until the horizontal (0°) was approached. Theatlazy1;2;3;4 quadruple mutant response, representinga largely agravitropic control, was similar to thegravitropic response Taniguchi et al. (2017) reportedfor a triple mutant (lzy1;lzy2;lzy3) that is equivalent tothe atlazy1;2;4 mutant Yoshihara and Spalding (2017)characterized. These and other results indicate thatAtLAZY1/LZY1, AtLAZY2/LZY2, and AtLAZY4/LZY3are the major contributors to the inflorescence gravi-tropism response (Taniguchi et al., 2017; Yoshihara andSpalding, 2017).

The finding that a normal (upward bending)AtLAZY-dependent response fails to be maintained in L1 and L2(Fig. 6A) raises the possibility that the L92A/I94Amodification to AtLAZY1 impairs the function ofother contributing AtLAZY proteins. To explorethis possibility, crosses were performed to place thepAtLAZY1:AtLAZY1L92A/I94A in an atlazy1;2;3;4 back-ground. The L1/atlazy1;2;3;4 and L2/atlazy1;2;3;4 lineswere produced. They both showed substantially stron-ger reverse gravitropism than atlazy1;2;3;4 (Fig. 6B;Supplemental Video S2). Thus, impairment of otherAtLAZY homologs does not explain how the L92A/I94Amodification to AtLAZY1 switched the direction ofstem gravitropism. The genetic analysis was expandedto include AtLAZY6, for which limited knowledge iscurrently available. The atlazy1;2;3;4;6 quintuple mutant,which lacks all AtLAZY family function except the ab-errant AtLAZY5, did not display L92A/I94A-like reversegravitropism (Fig. 6C). Instead of 40° to 60° of down-ward bending in stems possessing the region II L92A/I94A variant, the quadruple and quintuple mutants,which probably lack all LAZY gene functions, onlytransiently exceeded 20° of reverse bending. These re-sults argue against the possibility that the L92A/I94Aeffects on branch angle and gravitropism are due to in-terference with other AtLAZY proteins.The robustness of the reverse gravitropism caused

by the region II variation was explored with a double-rotation experiment. Excised inflorescences from plantsat least 22 cm tall were rotated by 90° clockwise for 4 h,which is the point in time at which downward, reversegravitropism initiates (Fig. 6B). Then the stems wererotated a further 90° clockwise (a total of 180°). Theapices of L1/atlazy1;2;3;4 and L2/atlazy1;2;3;4 stems,now upside down, curved in the clockwise direction foranother 2 h. The tips deflected ;30° from the vertical,which would not have occurred if the downwardbending had been the consequence of sagging insteadof reverse gravitropism (Supplemental Fig. S6). Theatlazy1;2;3;4 quadruple mutant responded only weakly,consistent with its weak reverse gravitropism in re-sponse to a single rotation (Fig. 6B). These results(Fig. 6; Supplemental Fig. S6) demonstrate that replac-ing AtLAZY1 with AtLAZY1L92A/I94A reverses thegravity response of the inflorescence stem, which is aprofound alteration.

The L92A/I94A Change Reverses the Auxin Gradient, ButDoes Not Affect Auxin Responsiveness

A conservative alteration to the AtLAZY1 sequence(L92A/I94A) reversed the behavior of the shoot. Basedon how gravitropism is understood to occur, two dis-tinctly different explanations seemed possible. Onewould depend on the auxin response mechanism beinginverted. In this case, low auxin would promote growthand high auxin would inhibit growth. Alternatively,the auxin gradient that causes differential growthacross the organ could be reversed. In this scenario,

Figure 5. Mechanical strength of inflorescence stems. A, Diagram ofthe apparatus and bending stem segment. B, The force required to flexstem segments excised;3 cm (upper stem) and 7 cm (lower stem) fromthe shoot apical meristem was measured with an electronic forcetransducer. C, The mean force (6 SE) obtained from 6–14 separatesegments for each position and genotype are plotted. L1 and L2 refer toindependent transgenic lines that are homozygous for the L92A/I94Avariation in region II and homozygous for the atlazy1 knockout muta-tion. The genotype means in each position were not significantly dif-ferent as determined by two-way ANOVA.

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higher levels of auxin would accumulate on the upperflank rather than the lower flank of a branch, resultingin downward bending. To determine whether reversedauxin action or a reversed auxin gradient explains thebehavior of L92A/I94A plants, auxin was applied to oneside of growing inflorescence stems. The result wasunequivocal. Stems of wild-type, atlazy1, and L1 (a linehomozygous for both the pAtLAZY1:AtLAZY1L92A/I94Atransgene and the atlazy1 knockout mutation) plantsresponded similarly by bending away from the appliedauxin over a range of concentrations (Fig. 7). No switchin auxin responsiveness resulted from the L92A/I94Aalteration, which makes a reversed auxin gradient theexpected cause of the downward bending and reversegravitropism of L92A/I94A plants.

To test the reversed auxin gradient hypothesis,the expression level of an auxin-responsive gene wasmeasured in the upper and lower halves of inflores-cence stems by reverse-transcription quantitative PCR(RT-qPCR). IAA5 was previously shown to be a goodindicator of the gravity-induced auxin gradient ininflorescence stems (Taniguchi et al., 2014). L1/atlazy1:2;3;4 and L2/atlazy1;2;3;4, which showed pro-nounced reverse gravitropism (Fig. 6B), were used in

this experimentation. IAA5 expression was measuredat 1 and 3 h after rotation. Wild-type inflorescencestems showed an approximately 9-fold increase ofIAA5 expression in the lower half of stems 1 h afterrotation (Fig. 8A), as shown previously (Taniguchiet al., 2014, 2017). Unexpectedly, the basal expressionof IAA5 (expression at 0 h) was much lower inatlazy1;2;3;4, L1/atlazy1;2;3;4, and L2/atlazy1;2;3;4stems than in the wild type (Fig. 8A). SupplementalFigure S7 shows that the assaywas sensitive enough toreproducibly measure the 60%- to 80%-lower levels ofIAA5 expression in the mutants, so a normalizationapproach was taken to compare the gravity-inducedexpression gradients. The analysis showed that IAA5expression was higher in the upper side of the inflores-cence stems of L1/atlazy1;2;3;4, and L2/atlazy1;2;3;4plants at 3 h (Fig. 8B), the time at which reverse gravi-tropism started to become evident (Fig. 6B). The reverseauxin gradient did not develop in atlazy1;2;3;4 stems,and they did not display reverse gravitropism. There-fore, the auxin gradient through which gravity naturallydirects growth, diminished by loss of AtLAZY1, is re-versed by the L92A/I94A alteration of the AtLAZY1protein.

Figure 6. Gravitropism time courses. A, Gravitropic responses of segments excised from young inflorescences (7–10 cm long) orolder inflorescences (22–25 cm long). Two lines homozygous for the pAtLAZY1:AtLAZY1L92A/I94A transgene in the atlazy1knockout background (L1 and L2) and the atlazy1;2;3;4 quadruple mutant were measured. The effect of the L92A/I94A variantbecame clear in the older stems. The initial bending response reversed over hours. The means6 SE of.21 trials per genotype andage are shown. WT, Wild type. B, The pAtLAZY1:AtLAZY1L92A/I94A transgene caused downward bending in atlazy1;2;3;4. Theatlazy1 background of the L1 and L2 lines was replaced with atlazy1;2;3;4 for this experiment, which was conducted with stemsexcised from 22- to 25-cm inflorescences. The means 6 SE of .17 samples per genotype are shown. Asterisks mark time pointsthat are significantly different between atlazy1;2;3;4 and L1/atlazy1;2;3;4 or between atlazy1;2;3;4 and L2/atlazy1;2;3;4 (*P ,0.05). C, Gravitropism of atlazy1;2;3;4;6 inflorescence stems (22–25 cm long) compared to the response of atlazy1;2;3;4 stemsidentified no role for the AtLAZY6 gene. The mean 6 SE obtained for .17 samples per genotype is shown.

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DISCUSSION

The PDFs derived from branch angle measurementsmade onmany T1-generation plants expressingAtLAZY1variants were more useful than the mean value. Theydescribed the rescue ability of each AtLAZY1 variantmore comprehensively than the fairly commonpractice ofshowing a representative outcome which may be dif-ficult to objectively select. Our methodology revealedan intriguing bimodal distribution when AtLAZY1was expressed under the control of its own promoterin an atlazy1 knockout mutant. Perhaps expressionlevel varied between T1 plants and AtLAZY1 per-forms its function only when expressed above athreshold level. Perhaps some mRNA processing orposttranslational modification is triggered in someplants but not others. Whatever causes the bimodality,it clearly does not apply to the functionally relatedAtLAZY2 gene.Consistent with previous results (Yoshihara et al.,

2013), Figure 3C shows that loss of AtLAZY1 at theplasma membrane correlated with loss of branch-angle function. How AtLAZY1 associates with theplasma membrane, where it appears to function (Fig. 3;Yoshihara et al., 2013; Taniguchi et al., 2017), is notclear. It apparently lacks a membrane-spanning do-main. Lipid modification of the protein is a possibleexplanation, because the GPS-Lipid program devel-oped by Xie et al. (2016) identified the Gly at thefifth position in AtLAZY1 as a possible nonconsensusN-myristoylation site. Figure 3C shows that mutating theadjacent three amino acids greatly reduced plasma mem-brane association and function. Both AtLAZY2/LZY2

and AtLAZY4/LZY3 are plasma membrane localizedand both have a Gly at the fifth position like the pos-sible myristoylation site in AtLAZY1/LZY1. Arguingagainst this mechanism of membrane attachment is thefact that an N-terminal fragment of AtLAZY1 fused toeGFP was not associated with the plasma membrane(Supplemental Fig. S4). Future work should investigatethe possibility that at least some AtLAZY proteinslink to the plasma membrane via a posttranslationallipid modification, which probably involves short-ening the N terminus to make the Gly the secondposition (Hentschel et al., 2016).The role of nuclear-localized AtLAZY1 requires more

investigation. AtLAZY1 in the nucleus appeared to beineffective (Fig. 3C; Yoshihara et al., 2013); however, inrice, nuclear-localized OsLAZY1 appears to influencetiller angle (Li et al., 2019). The mechanism of LAZYaction may differ between species and organs. Evenits association with the plasma membrane may havea different molecular basis in different species. Somesequence-analysis tools find evidence for a singlemembrane-spanning domain in rice and maize LAZY1(Li et al., 2007; Dong et al., 2013). Membrane-spanningdomains are not predicted in AtLAZY1 or its Arabi-dopsis homologs.AtLAZY1 may associate with the plasma membrane

not by possessing a transmembrane span or by myr-istoylation but by binding to a membrane protein orcomplex. In support of this possibility, Li et al. (2019)reported that rice LAZY1 interacts with a protein calledOsBRXL4 to sequester OsLAZY1 at the plasma mem-brane and therefore away from its site of action in thenucleus. Another protein reported to interact with aLAZY1 protein at the plasma membrane is a maize ki-nase (Dong et al., 2013). The interacting maize kinase isrelated to the Arabidopsis PINOID kinase that partici-pates in directing auxin transport by affecting the lo-calization of PIN proteins (Michniewicz et al., 2007;Huang et al., 2010; Ganguly et al., 2012, 2014). An at-tractive hypothesis is that AtLAZY1 functions in aprotein complex at the plasma membrane that couplesthe presence of settled statoliths with the mechanismthat determines the localization of PIN auxin effluxregulators, especially PIN3. Somehow, in this scenario,the L92A/I94A variant of AtLAZY1 reverses the outputof the mechanism such that the PIN localization, andtherefore the auxin gradient, is reversed. Direct testsof this idea have been hampered by the difficulty ofvisualizing fluorescently tagged proteins in the en-dodermal cells near the center of thick, heavilypigmented stems.Altering two amino acids in the IGT domain in

region II of AtLAZY1 (L92A/I94A) reversed thegravitropic response of the inflorescence stem(Fig. 6B) and produced a weeping phenotype in theadult plant (Fig. 4A). The phenomenon of reversegravitropism due to mutations of LAZY genes hasbeen reported previously in roots (Ge and Chen, 2016;Yoshihara and Spalding, 2017). Taniguchi et al. (2017)showed that a C-terminal (region V) portion of

Figure 7. Normal growth response to auxin in L92A/I94A stems thatdisplay reverse gravitropism. Lanoline paste containing the indicatedconcentrations of auxin was applied to a flank of inflorescence stemsand images were captured 18 h later. A to C, Wild type (WT). D to F,atlazy1. G to I, pAtLAZY1:AtLAZY1L92A/L94A/atlazy1.

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AtLAZY2/LZY2 fused with mCherry causes upwardgrowth of roots, especially when expressed underthe control of a LZY2/AtLAZY2 promoter in anatlazy1;2;4/lzy1;2;3 background. It is not clear whetherhomologous mechanisms are responsible for the pre-viously reported examples of reverse root gravitropismand the reverse shoot gravitropism in L92A/I94A. Thetime-course analysis in Figure 6B shows that reverseshoot gravitropism began after a transient phase ofnegative (normal) gravitropism. A transient phase ofnormal, positive gravitropism did not precede the re-verse gravitropism in atlazy2;3;4/atngr1;2;3 roots (Geand Chen, 2016; Yoshihara and Spalding, 2017). Thetime course of the reverse gravitropism caused by theregion V LZY2-mCherry (CCL-mCherry) protein wasnot reported (Taniguchi et al., 2017), so it cannot becompared to the present shoot version of thephenomenon.

Gravitropism consists of a bending phase in whichan auxin gradient produces curvature and a subse-quent straightening phase during which the growthdifferential must reverse to produce an uprightstem. A mathematical model developed by Bastienet al. (2013) successfully simulates gravitropism inthe stems of many species by having the straighten-ing phase be a consequence of a proprioceptiveprocess sensitive to the bending produced by thefirst phase. Rakusová et al. (2016) present evidence

obtained with Arabidopsis hypocotyls indicating thatthe PIN3 auxin-efflux protein initially moves to thelower cell side after gravitropic stimulation to directauxin to the lower side of the hypocotyl to causethe upward bending phase. Then, in response tothe increased auxin concentration, PIN3 moves to theopposite cell side to dissipate the auxin gradient, ter-minate gravitropic bending, and produce a straighten-ing phase (Rakusová et al., 2016). Genetic andpharmacological results indicate that the cytoskeletonplays a role in the PIN repositioning and possibleproprioception required for the straightening response(Rakusová et al., 2011, 2019; Okamoto et al., 2015). Theregion II modification (L92A/I94A) may have im-paired the first phase and/or strengthened the secondphase, resulting in an overall reverse response (Fig. 6B)by directly or indirectly altering communication withthe cytoskeleton.

The results reported here may also pertain to rootgravitropism, which relies mostly on the AtLAZY2/LZY2/NGR1, AtLAZY3/LZY4/NGR3, and AtLAZY4/LZY3/NGR2 genes and PIN3 redistribution (Taniguchiet al., 2017; Ge and Chen, 2019). If mutations equivalentto L92A/I94A in these genes reverses root gravitrop-ism, then perturbations of region II may be a powerfultool for studying the fundamental basis of gravitysensing, which was critical to the success of embryo-phytes after the colonization of land.

Figure 8. Expression of an auxin-responsivegene, IAA5, in the upper and lower halves ofinflorescence stems measures the gravity-induced auxin gradient. A, A PCR-basedmethod quantified the levels of IAA5 in theupper and lower halves of inflorescence stemsafter the indicated period of gravistimulation.The values were divided by the average of thewild-type (WT) upper and lower pools at 0 h.Shown are the mean 6 SE obtained from threebiological replicates, each measured threetimes. An asterisk indicates a statistically sig-nificant difference (*P, 0.05). Note that basalexpression level of IAA5 was lower in themutants. B, To highlight the gradient ratherthan the absolute expression level, the meanexpression of IAA5 between the lower andupper halves was normalized to 1. Statisticallysignificant differences in IAA5 expression be-tween lower and upper halves were observedin the wild type and reversed gradients wereobserved in the two homozygous lines, whichalso displayed strong reverse gravitropism.Student’s t test. (***P , 0.001, *P , 0.05).

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MATERIALS AND METHODS

Plant Materials and Growth Conditions

Ecotype Col-0 of Arabidopsis (Arabidopsis thaliana), obtained from theEuropean Arabidopsis Stock Centre, was the wild type used in all experi-ments. The transfer DNA insertion mutants inAtLAZY genes were obtainedas described in Yoshihara and Spalding (2017). To test the ability of pAt-LAZY1:AtLAZY1 and its variants to rescue atlazy1, T1 seeds were surfacesterilized and sown onMurashige and Skoog (MS) plates (0.05% [w/v] MESand 0.7% [w/v] agarose, pH 5.7 with KOH) supplemented with Suc (1%[w/v]) containing BASTA (7.5 mg/mL). Healthy seedlings were trans-planted to pots containing a commercial soil mixture (Fafard 3B mix,Sungro Horticulture) ;2 weeks after planting, then grown for 4–6 weeks ina growth chamber (22°C, ;90 mmol m22 s21 of white light, 16-h light/8-hdark cycles).

Branch Angle Measurements

Lateral branch angles were measured as described previously (Yoshiharaet al., 2013) with the following minor changes. Images of stems were obtainedwith a flatbed scanner. Branch angles were determined from the images bycentering a 10-mm circle at the center of the node and drawing a straight linethat bisected the region of the branch that intersected the circle. The angle be-tween this branch line and a radial line that best bisected the primary stem wasmeasured using ImageJ software (Schneider et al., 2012). The PDFs were de-termined by fitting two Gaussian distributions to a frequency histogram of theangle measurements for each genotype using the Origin 2018b software pack-age (OriginLab Corp.). Dividing the cumulative fitted Gaussian curve by thenumber of observations and the bin width produced the displayed plots. Thearea under each curve equals 1. The Shapiro-Wilks test of normality was per-formed on each of the data sets in Figure 3 using the Origin 2018b softwarepackage and a P-value of 0.05.

Stem Bending Force Measurement

Primary inflorescences 22–25 cm long were used for measurements ofbending force. Two 3-cm-long segments were excised from each primaryinflorescence. The apical end of one segment was a point 3 cm below the apexand that of the other was a point 7 cm below the apex, so the “upper stem”

segment represented 3–6 cm and the “lower stem” represented 7–10 cm fromthe tip. The basal end of the stem segment was inserted into a clamped,horizontal glass capillary tube (1.5-mm inner diameter) such that ;1 cm ofthe apical portion was exposed. Cotton thread was tied in a knot around thesegment ;3 mm from the apical end to prevent a wire hook that was con-nected to an overhead force transducer from slipping off the segment as thetransducer was slowly lifted with the aid of a manual micromanipulator. Acomputer-controlled charge-coupled device camera and macro zoom lensallowed the experimenter to observe when the free end of the segment hadbeen displaced upward by 2 mm as judged with the aid of a graph paperbackground in the scene. The voltage output of the transducer, which isproportional to force, was measured at 20 Hz during the lift with a customdigital acquisition platform similar to that described previously (Spalding,1995). The difference between the starting (free hanging) voltage and thesteady state voltage after lifting the free end by 2 mm was determined andconverted to Newtons using a linear calibration curved constructed withknown masses.

Inflorescence Gravitropism

Gravitropism of inflorescence stems was measured as described in Toyotaet al. (2013) with some modifications. The cut end of a 7-cm section excisedfrom the apex of an inflorescence stem was placed in a 0.2-mL PCR tubecontaining half-strength MS media supplemented with 0.5% (w/v) Suc. Thestem section was secured in the tube by wrapping the base with Parafilm M(Pechiney Plastic Packaging). The tubes with stems were maintained uprightin a custom rack to recover for 2 h in dim red light (4–6 mmol m22 s21) pro-duced by light-emitting diodes having a peak output at 660 nm. The tube rackwith stems was rotated by 90° while a charge-coupled device camera (Marlin;Allied Vision Technologies), with its internal infrared filter removed andoutfitted with an 8-mm lens (M0814-MP; Computar) fronted with an infraredlongpass filter made from infrared-transmitting, visibly opaque plexiglass

(ACRY11460; Ridout Plastics) acquired images. The sample was backlit withinfrared light-emitting diodes having a peak output at 940 nm. The cameraswere programmed to collect an image every 30 min for 18 h. Angle mea-surements were made manually from the time series of images using ImageJsoftware.

Plasmid Construction

All primers and DNA oligomers used in this study were synthesized byIntegrated DNA Technologies and listed in Supplemental Table S1. Promotersand genes were amplified from genomic DNA by PCR and the resulting frag-ments were subcloned into pGEM-T-Easy (Promega) for sequence confirmationbefore proceeding.

For the rescuing tests of the atlazy1 branch angle phenotype, pAtLA-ZY1:AtLAZY1was constructed. Awild-type genomic fragment ofAtLAZY1including 1.9 kb of the promoter region was amplified with a Pac I site at the59 end and a BamH I site at 39 end. These amplified fragments were subcl-oned and recloned between the Pac I/BamH I sites of the binary vectorpEGAD (Cutler et al., 2000). The GV3101 strain of Agrobacterium tumefacienswas transformed with the binary vector. Arabidopsis plants were trans-formed using the floral dip method (Clough and Bent, 1998). The Phusionsite-directed mutagenesis kit (Finnzymes) was used to introduce muta-tions in the conserved regions. The subcloned fragments of genomic DNAof AtLAZY1 in pGEM-T-Easy obtained as stated above were subjectedto site-directed mutagenesis and recloned into pEGAD after sequenceconfirmation.

For a promoter swapping construct, pAtLAZY1:AtLAZY2, the wild-typegenomic fragment (1.9 kb) of the promoter region of AtLAZY1, was amplifiedwith the Sma I site at the 59 end and the wild-type genomic fragment (1.8 kb) ofAtLAZY2 (At1g17400) from start codon to 39 untranslated region (UTR) wasamplifiedwith BamH I site at the 39 end. TheAtLAZY1 promoter was fusedwiththe AtLAZY2 coding region by the overlap-extension PCR method (Ho et al.,1989). The resulting fragment was subcloned and recloned between the Pac I/BamH I sites in the binary vector pEGAD.

To observe the effects of changes to AtLAZY1 on expression and localizationin Arabidopsis, the previously described pHSP18.2:AtLAZY1-eGFP in pGEM-T-Easy (Yoshihara et al., 2013) was used. The subcloned pHSP18.2:AtLAZY1-eGFPwas subjected to site-directed mutagenesis as described above and reclonedbetween Pac I/BamH I sites in the binary vector pEGAD.

Transient expression in Nicotiana benthamiana leaf epidermal cells wasused to investigate the subcellular localization of wild-type and mutatedAtLAZY1, wild-type AtLAZY2, the TGA5 nuclear marker, and an N-terminalportion of AtLAZY1. The 35S:AtLAZY2-mRFP construct was generated bythree-step PCR (Grandori et al., 1997). The entire AtLAZY2 genomic sequencefrom the start codon to the 39 UTR and an mRFP sequence were amplifiedusing the primers in Supplemental Table S1. The mRFP sequence wasobtained from the pSAT6-RFP-C1 vector (Tzfira et al., 2005). These amplifiedfragments were subcloned and combined into the Pac I/BamH I site ofpEGAD. To express TGA5 as a nuclear marker in N. benthamiana leaf epi-dermal cells, 35S:eGFP-TGA5 was constructed. TGA5 was amplified with theSma I and BamH I sites and subcloned. The Sma I–BamH I fragment of TGA5was recloned between the Sma I/BamH I sites of pEGAD. The N-terminal partof AtLAZY1 containing region I was fused with eGFP to see the effect of theN-terminal part on AtLAZY1 localization. Oligonucleotides codingN-terminal parts of AtLAZY1 were annealed to make double-stranded DNAand digestedwithAge I and recloned into theAge I site of pEGAD to obtain theconstructs N-terminal-AtLAZY1:eGFP.

An atlazy6 mutant was created using the CRISPR/Cas9 system as de-scribed in Fauser et al. (2014). The DNA oligomers containing the protospacerand AtLAZY6 complementary sequence (Supplemental Table S1) wereannealed to make double-stranded DNA and the resulting fragment wasinserted into the Bbs I site of the pChimera vector, producing the pEn-Chimera.The expression cassette of single-guide RNA created in pEn-Chimera wasexcised with Avr II and recloned into the Avr II/Avr II site of the pCAS9-TPCbinary vector (Fauser et al., 2014). A. tumefaciens cells were transformed withthe binary vector. T1 plants were selected on MS plates containing BASTAand T2 seeds were obtained. The genomic DNA of T2 plants corresponding tothe target region was sequenced andmutations were detected. Amutant witha thymine insertion that creates an early stop codon in AtLAZY6 was namedatlazy6 and used to create a quintuple lazy mutant by crossing. Cleaved am-plified polymorphic sequence (CAPS) markers were designed (Neff et al.,2002) to detect the mutation and used for genotyping (SupplementalTable S1).

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Transient and Stable Expression in N. benthamianaand Arabidopsis

Arabidopsis plants were transformed using the floral dip method (Cloughand Bent, 1998), and stable transformants harboring pHSP18.2:AtLAZY1-eGFPor its variants were obtained. Seedlings were grown on the surface of MS platessupplemented with Suc for 3 d in the dark. Heat shock was given by incubatingplates with seedlings in 37°C for 5–6 h. Fluorescence from eGFP was observedright after the heat shock. Leaves from N. benthamiana were injected with A.tumefaciens strain GV3101 harboring expression vectors as described previously(Yoshihara et al., 2013). The overnight culture of Agrobacteriumwas pelleted bycentrifugation and resuspended in deionized distilled water. Resuspendedculture was injected into the abaxial side of the leaf and the injected leaves wereleft attached until observation (for 24–30 h).

Microscopy

A Zeiss LSM 510 laser scanning confocal microscope was used to visualizefluorescent proteins inplant cells. Theoptics employedwere aPlan-Apochromat203 lens and a C-Apochromat 403 water immersion lens. The sample wasexcited with the 488-nm laser line for eGFP and the 543-nm laser line for mRFPfrom a 30-mW argon gas laser. Channel mode detection was used to record theemission of eGFP (excitation 488 nm, emission 500–550 nm) ormRFP (excitation543 nm, emission 565–615 nm) for subcellular localization.

Auxin Application

Auxin (Indole-3-acetic acid; Sigma-Aldrich) was dissolved in and dilutedwithethanol. The dilution series was mixed with lanolin to make up lanolin pastescontaining different concentrations of auxin. The final concentration of ethanol inthe lanolin pastes was the same for all treatments and ,0.1% (w/v). The lanolinpaste was applied to one side of the inflorescence stem about 3 cm from the tip asan;4-mm-long streak along the long axis. The plants used were 6–7 weeks old,because the inflorescence stems of pAtLAZY1:AtLAZY1L92A/I94A/atlazy1 plantsdepicted a clearly drooping phenotype at this age.

Transcript Analysis by RT-qPCR

Transcript analysis of IAA5 was performed mostly as described inTaniguchi et al. (2014). Inflorescence stems grown to 22–25 cm were grav-istimulated as in the time course imaging experiments described above. Aftergravistimulation, a segment of stem ;0.5–5.5 cm below the apical meristemwas cut into upper and lower halves along the longitudinal axis under adissection microscope. Tissue samples from 10–13 stems per genotype andtime point were pooled. Total RNA was extracted using an RNeasy PlantMini Kit (Qiagen), including the treatment with RNase-free DNase (Qiagen).RT-qPCR was carried out using an Mx3000P QPCR system (Stratagene) withthe KAPA SYBR FAST One-Step Universal Kit (KAPA Biosystems). TriplicateRT-qPCR assays were performed for each of three independently grown,treated, and harvested samples (biological replicates). ACTIN8 (ACT8) wasused as an internal control. Relative amounts of IAA5 transcript were cal-culated using Pfaffl’s model (Pfaffl, 2001). Primers used were as described inTaniguchi et al. (2014).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers AT5G14090 (LAZY1), AT1G17400 (LAZY2),AT1G19115 (LAZY3), AT1G72490 (LAZY4), AT3G24750 (LAZY5), andAT3G27025 (LAZY6).

SUPPLEMENTAL DATA

The following supplemental materials are available.

Supplemental Figure S1. Effects of mutation in AtLAZY1 conserved re-gions on subcellular localization: Additional examples to supplementthose shown in Figure 3.

Supplemental Figure S2. Measurements from which the PDFs in Figure 3were derived.

Supplemental Figure S3. Branch angle values in homozygous populationsof pAtLAZY1:AtLAZY1L92A/I94A/atlazy1.

Supplemental Figure S4. Subcellular localization of N-terminal AtLAZY1fused with eGFP.

Supplemental Figure S5. Shape of plants grown upside-down for 4 d andthen returned to right-side up for 2 d.

Supplemental Figure S6. Double rotation experiment designed to deter-mine whether reverse gravitropism of L1 and L2 (two lines homozygousfor pAtLAZY1:AtLAZY1L92A/I94A/atlazy1;2;3;4) induced by 90° rotationwould persist and therefore bend away from the plumb line after asecond 90° rotation.

Supplemental Figure S7. IAA5 expression in atlazy1;2;3;4, L1/atlazy1;2;3;4,and L2/atlazy1;2;3;4 measured by reverse-transcription quantitative PCR

Supplemental Video S1. Excised wild-type inflorescence stem performingnormal (negative) gravitropism.

Supplemental Video S2. Excised L1/atlazy1;2;3;4 inflorescence stem per-forming reverse (positive) gravitropism.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center (The Ohio StateUniversity, Columbus), the European Arabidopsis Stock Centre, and theInstitut Jean-Pierre Bourgin in the French National Institute for AgriculturalResearch for providing vectors and the seeds of Arabidopsis thaliana transferDNA insertion lines. Microscopy was performed at the Newcomb ImagingCenter, Department of Botany, University of Wisconsin–Madison. We alsothank Alexys Hoppman, Emma Keel, and Ashley Perry for technical assistance.

Received September 18, 2019; accepted November 25, 2019; published Decem-ber 9, 2019.

LITERATURE CITED

Abe K, Takahashi H, Suge H (1996) Lazy gene (la) responsible for both anagravitropism of seedlings and lazy habit of tiller growth in rice (Oryzasativa L.). J Plant Res 109: 381–386

Ashraf A, Rehman OU, Muzammil S, Léon J, Naz AA, Rasool F, Ali GM,Zafar Y, Khan MR (2019) Evolution of Deeper Rooting 1-like homoeologsin wheat entails the C-terminus mutations as well as gain and loss ofauxin response elements. PLoS One 14: e0214145

Bastien R, Bohr T, Moulia B, Douady S (2013) Unifying model of shootgravitropism reveals proprioception as a central feature of posturecontrol in plants. Proc Natl Acad Sci USA 110: 755–760

Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743

Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells ofArabidopsis at a high frequency. Proc Natl Acad Sci USA 97: 3718–3723

Dardick C, Callahan A, Horn R, Ruiz KB, Zhebentyayeva T, Hollender C,Whitaker M, Abbott A, Scorza R (2013) PpeTAC1 promotes the hori-zontal growth of branches in peach trees and is a member of a func-tionally conserved gene family found in diverse plant species. Plant J 75:618–630

Desfeux C, Clough SJ, Bent AF (2000) Female reproductive tissues are theprimary target of Agrobacterium-mediated transformation by the Ara-bidopsis floral-dip method. Plant Physiol 123: 895–904

Dinesh DC, Villalobos LIAC, Abel S (2016) Structural biology of nuclearauxin action. Trends Plant Sci 21: 302–316

Dong Z, Jiang C, Chen X, Zhang T, Ding L, Song W, Luo H, Lai J, Chen H,Liu R, et al (2013) Maize LAZY1 mediates shoot gravitropism and in-florescence development through regulating auxin transport, auxinsignaling, and light response. Plant Physiol 163: 1306–1322

Fankhauser C, Christie JM (2015) Plant phototropic growth. Curr Biol 25:R384–R389

Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleasesand nickases can be used efficiently for genome engineering in Arabi-dopsis thaliana. Plant J 79: 348–359

1050 Plant Physiol. Vol. 182, 2020

Yoshihara and Spalding

www.plantphysiol.orgon July 26, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Fukaki H, Fujisawa H, Tasaka M (1996a) Gravitropic response of inflo-rescence stems in Arabidopsis thaliana. Plant Physiol 110: 933–943

Fukaki H, Fujisawa H, Tasaka M (1996b) SGR1, SGR2, SGR3: Novel ge-netic loci involved in shoot gravitropism in Arabidopsis thaliana. PlantPhysiol 110: 945–955

Ganguly A, Lee S-H, Cho H-T (2012) Functional identification of thephosphorylation sites of Arabidopsis PIN-FORMED3 for its subcellularlocalization and biological role. Plant J 71: 810–823

Ganguly A, Park M, Kesawat MS, Cho H-T (2014) Functional analysis ofthe hydrophilic loop in intracellular trafficking of Arabidopsis PIN-FORMED proteins. Plant Cell 26: 1570–1585

Ge L, Chen R (2019) Negative gravitropic response of roots directs auxinflow to control root gravitropism. Plant Cell Environ 42: 2372–2383

Ge L, Chen R (2016) Negative gravitropism in plant roots. Nat Plants 2:16155

Grandori R, Struck K, Giovanielli K, Carey J (1997) A three-step PCRprotocol for construction of chimeric proteins. Protein Eng 10: 1099–1100

Haga K, Sakai T (2013) Differential roles of auxin efflux carrier PIN pro-teins in hypocotyl phototropism of etiolated Arabidopsis seedlings de-pend on the direction of light stimulus. Plant Signal Behav 8: e22556

Hentschel A, Zahedi RP, Ahrends R (2016) Protein lipid modifications—More than just a greasy ballast. Proteomics 16: 759–782

Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directedmutagenesis by overlap extension using the polymerase chain reaction.Gene 77: 51–59

Huang F, Zago MK, Abas L, van Marion A, Galván-Ampudia CS,Offringa R (2010) Phosphorylation of conserved PIN motifs directsArabidopsis PIN1 polarity and auxin transport. Plant Cell 22: 1129–1142

Kagale S, Links MG, Rozwadowski K (2010) Genome-wide analysis ofethylene-responsive element binding factor-associated amphiphilic re-pression motif-containing transcriptional regulators in Arabidopsis.Plant Physiol 152: 1109–1134

Li P, Wang Y, Qian Q, Fu Z, Wang M, Zeng D, Li B, Wang X, Li J (2007)LAZY1 controls rice shoot gravitropism through regulating polar auxintransport. Cell Res 17: 402–410

Li Z, Liang Y, Yuan Y, Wang L, Meng X, Xiong G, Zhou J, Cai Y, Han N,Hua L, et al (2019) OsBRXL4 regulates shoot gravitropism and rice tillerangle through affecting LAZY1 nuclear localization. Mol Plant 12:1143–1156

Mano E, Horiguchi G, Tsukaya H (2006) Gravitropism in leaves of Ara-bidopsis thaliana (L.) Heynh. Plant Cell Physiol 47: 217–223

Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A,Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F, et al (2007) An-tagonistic regulation of PIN phosphorylation by PP2A and PINOIDdirects auxin flux. Cell 130: 1044–1056

Morita MT (2010) Directional gravity sensing in gravitropism. Annu RevPlant Biol 61: 705–720

Neff MM, Turk E, Kalishman M (2002) Web-based primer design forsingle nucleotide polymorphism analysis. Trends Genet 18: 613–615

Notredame C, Higgins DG, Heringa J (2000) T-Coffee: A novel method forfast and accurate multiple sequence alignment. J Mol Biol 302: 205–217

Okamoto K, Ueda H, Shimada T, Tamura K, Kato T, Tasaka M, MoritaMT, Hara-Nishimura I (2015) Regulation of organ straightening andplant posture by an actin-myosin XI cytoskeleton. Nat Plants 1: 15031

Pfaffl MW (2001) A new mathematical model for relative quantification inreal-time RT-PCR. Nucleic Acids Res 29: e45

Rakusová H, Gallego-Bartolomé J, Vanstraelen M, Robert HS, Alabadí D,Blázquez MA, Benková E, Friml J (2011) Polarization of PIN3-dependent auxin transport for hypocotyl gravitropic response in Ara-bidopsis thaliana. Plant J 67: 817–826

Rakusová H, Abbas M, Han H, Song S, Robert HS, Friml J (2016) Ter-mination of shoot gravitropic responses by auxin feedback on PIN3polarity. Curr Biol 26: 3026–3032

Rakusová H, Fendrych M, Friml J (2015) Intracellular trafficking and PIN-mediated cell polarity during tropic responses in plants. Curr Opin PlantBiol 23: 116–123

Rakusová H, Han H, Valosek P, Friml J (2019) Genetic screen for factorsmediating PIN polarization in gravistimulated Arabidopsis thaliana hy-pocotyls. Plant J 98: 1048–1059

Roychoudhry S, Del Bianco M, Kieffer M, Kepinski S (2013) Auxincontrols gravitropic setpoint angle in higher plant lateral branches. CurrBiol 23: 1497–1504

Saito C, Morita MT, Kato T, Tasaka M (2005) Amyloplasts and vacuolarmembrane dynamics in the living graviperceptive cell of the Arabidopsisinflorescence stem. Plant Cell 17: 548–558

Salehin M, Bagchi R, Estelle M (2015) SCFTIR1/AFB-based auxin per-ception: Mechanism and role in plant growth and development. PlantCell 27: 9–19

Sang D, Chen D, Liu G, Liang Y, Huang L, Meng X, Chu J, Sun X, DongG, Yuan Y, et al (2014) Strigolactones regulate rice tiller angle by at-tenuating shoot gravitropism through inhibiting auxin biosynthesis.Proc Natl Acad Sci USA 111: 11199–11204

Sasaki S, Yamamoto KT (2015) Arabidopsis LAZY1 is a peripheral mem-brane protein of which the carboxy-terminal fragment potentially in-teracts with microtubules. Plant Biotechnol 32: 103–108

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ:25 years of image analysis. Nat Methods 9: 671–675

Singh M, Gupta A, Laxmi A (2017) Striking the right chord: Signalingenigma during root gravitropism. Front Plant Sci 8: 1304

Spalding EP (1995) An apparatus for studying rapid electrophysiological re-sponses to light demonstrated onArabidopsis leaves. Photochem Photobiol 62:934–939

Su SH, Gibbs NM, Jancewicz AL, Masson PH (2017) Molecular mecha-nisms of root gravitropism. Curr Biol 27: R964–R972

Taniguchi M, Furutani M, Nishimura T, Nakamura M, Fushita T, IijimaK, Baba K, Tanaka H, Toyota M, Tasaka M, et al (2017) The Arabi-dopsis LAZY1 family plays a key role in gravity signaling within sta-tocytes and in branch angle control of roots and shoots. Plant Cell 29:1984–1999

Taniguchi M, Nakamura M, Tasaka M, Morita MT (2014) Identification ofgravitropic response indicator genes in Arabidopsis inflorescence stems.Plant Signal Behav 9: e29570

Tasaka M, Kato T, Fukaki H (1999) The endodermis and shoot gravi-tropism. Trends Plant Sci 4: 103–107

Toyota M, Ikeda N, Sawai-Toyota S, Kato T, Gilroy S, Tasaka M, MoritaMT (2013) Amyloplast displacement is necessary for gravisensing inArabidopsis shoots as revealed by a centrifuge microscope. Plant J 76:648–660

Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, KrichevskyA, Taylor T, Vainstein A, Citovsky V (2005) pSAT vectors: A modularseries of plasmids for autofluorescent protein tagging and expression ofmultiple genes in plants. Plant Mol Biol 57: 503–516

Waite JM, Dardick C (2018) TILLER ANGLE CONTROL 1 modulates plantarchitecture in response to photosynthetic signals. J Exp Bot 69:4935–4944

Wei N, Tan C, Qi B, Zhang Y, Xu G, Zheng H (2010) Changes in gravi-tational forces induce the modification of Arabidopsis thaliana siliquepedicel positioning. J Exp Bot 61: 3875–3884

Wu X, Tang D, Li M, Wang K, Cheng Z (2013) Loose Plant Architecture1,an INDETERMINATE DOMAIN protein involved in shoot gravitrop-ism, regulates plant architecture in rice. Plant Physiol 161: 317–329

Xie Y, Zheng Y, Li H, Luo X, He Z, Cao S, Shi Y, Zhao Q, Xue Y, Zuo Z,et al (2016) GPS-Lipid: A robust tool for the prediction of multiple lipidmodification sites. Sci Rep 6: 28249

Yamauchi Y, Fukaki H, Fujisawa H, Tasaka M (1997) Mutations in theSGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravi-tropism. Plant Cell Physiol 38: 530–535

Yoshihara T, Iino M (2007) Identification of the gravitropism-related ricegene LAZY1 and elucidation of LAZY1-dependent and -independentgravity signaling pathways. Plant Cell Physiol 48: 678–688

Yoshihara T, Spalding EP (2017) LAZY genes mediate the effects of gravityon auxin gradients and plant architecture. Plant Physiol 175: 959–969

Yoshihara T, Spalding EP, Iino M (2013) AtLAZY1 is a signaling compo-nent required for gravitropism of the Arabidopsis thaliana inflorescence.Plant J 74: 267–279

Yu B, Lin Z, Li H, Li X, Li J, Wang Y, Zhang X, Zhu Z, Zhai W, Wang X,et al (2007) TAC1, a major quantitative trait locus controlling tiller anglein rice. Plant J 52: 891–898

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