mechanical induction of lateral root initiation in arabidopsis thaliana
TRANSCRIPT
Mechanical induction of lateral root initiationin Arabidopsis thalianaFranck Anicet Ditengoua,1, William D. Tealea, Philip Kocherspergera, Karl Andreas Flittnera, Irina Kneupera,Eric van der Graaffb, Hugues Nzienguia, Francesco Pinosaa, Xugang Lia, Roland Nitschkec, Thomas Lauxb,d,and Klaus Palmea,d,e,1
Institutes of aBiology II and bBiology III, Faculty of Biology, Albert-Ludwigs-Universitat of Freiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany; cLifeImaging Centre, Centre for Systems Biology, Habsburgerstrasse 49, D-79104 Freiburg, Germany; dFreiburg Institute of Advanced Sciences (FRIAS),Albert-Ludwigs-Universitat Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany; and eCentre for Biological Signalling Studies (bioss), Albert-Ludwigs-Universitat Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany
Edited by Mark Estelle, Indiana University, Bloomington, IN, and approved October 1, 2008 (received for review August 8, 2008)
Lateral roots are initiated postembryonically in response to envi-ronmental cues, enabling plants to explore efficiently their under-ground environment. However, the mechanisms by which theenvironment determines the position of lateral root formation areunknown. In this study, we demonstrate that in Arabidopsisthaliana lateral root initiation can be induced mechanically byeither gravitropic curvature or by the transient bending of a rootby hand. The plant hormone auxin accumulates at the site of lateralroot induction before a primordium starts to form. Here wedescribe a subcellular relocalization of PIN1, an auxin transportprotein, in a single protoxylem cell in response to gravitropiccurvature. This relocalization precedes auxin-dependent gene tran-scription at the site of a new primordium. Auxin-dependent nu-clear signaling is necessary for lateral root formation; arf7/19double knock-out mutants normally form no lateral roots but do soupon bending when the root tip is removed. Signaling througharf7/19 can therefore be bypassed by root bending. These datasupport a model in which a root-tip-derived signal acts on down-stream signaling molecules that specify lateral root identity.
Auxin � auxin transport � mechanosensory � organogenesis �plant gravitropism
P lants are immobile, with the vast majority relying on themechanical support an extensive root system provides. Lateral
roots, formed by postembryonic initiation and development, growin response to specific environmental cues. In this way, a plant canoptimize nutrient and water uptake, whilst avoiding physical ob-stacles. Thus, root architecture, an important agronomic trait, is toa large extent determined by the environment.
Lateral root initiation (LRI) is dependent on the coordinatedaction of many genes. Although more than seventy genes ofArabidopsis thaliana have been shown to affect lateral root devel-opment, auxin and its polar transport have emerged as centralregulators (1, 2). For example, specific Aux/IAA transcriptionalregulators appear necessary for lateral root growth. The gain-of-function mutation solitary root (slr-1) encodes a stabilized form ofone such Aux/IAA protein, IAA14, which binds and constitutivelyrepresses two auxin response factors (ARF7 and ARF19), tran-scriptional activators also required for lateral root development (3).Consequently, slr-1 plants form no lateral roots. A third gene family,named LATERAL ORGAN BOUNDARIES-DOMAIN (LBD), hasalso been implicated in auxin-dependent LRI, with LBDs 16 and 29acting as downstream effectors of ARF7 and ARF19 (4). Althoughsome very early events in lateral root development such as celldivision, redirection of auxin flux and cell differentiation are welldocumented (1, 2), the factors that determine where a new root willbe initiated are unknown. Recent work has revealed a genetic basisfor the initiation of lateral roots. Root waving, LRI, and a temporalpattern of auxin maxima (as measured by the auxin-sensitive DR5promoter) are correlated in the root tip, resulting in lateral rootsbeing spaced along the main axis in a regular left-right alternating
pattern. Under standardized experimental conditions which negateany extrinsic environmental effect on LRI, it has been proposedthat fluctuations in auxin distribution might mediate the lateralroots’ regular longitudinal spacing (5).
How are these cues integrated to shape the root system inresponse to the environment? The following experiments demon-strate that LRI is a consequence of root bending, either in responseto gravity or by direct manipulation. Here, we report on the seriesof molecular events that follow lateral root induction, with respectto auxin transport, auxin signaling, and the acquisition of stem cellidentity within the new lateral root primordium.
Results and DiscussionLateral Root Positioning is Consistent with Root Shape. To investigatethe basis for the interaction between root architecture and theenvironment, we first examined the relationship between mechan-ical strain and LRI. It is known that in A. thaliana, when grown onhard agar plates tilted at 45°, lateral root establishment coincideswith a waving pattern in the growing root, with lateral roots alwaysemerging on the convex side of a wave (5). Both waving and thegravitropic response in roots are mediated by differential growth.This causes a reorientation of the root tip toward the gravity vectorand results in root bending. We therefore tested whether changingthe direction of root growth by rotating plants through an angle of135° affects lateral root formation. We observed the initiation of alateral root at the convex side of the gravity-induced curve [sup-porting information (SI) Fig. S1]. Auxin is a major regulator oflateral root development; the transcription of primary auxin re-sponse genes is among the first events of LRI (5). We thereforeanalyzed the formation of auxin-response maxima during grav-itropic curvature. Here, the auxin-sensitive DR5::GUS reporterfusion marked sites of LRI (Fig. S1B). Before rotation, DR5-drivennuclear-localized YFP (pDR5rev::3XVENUS-N7) fluorescence inthe distal elongation zone, was seen mainly in the protoxylem cellfiles, distributed equally between both poles (Fig. S2D). Five hoursafter rotation, the signal was detected in the epidermis and cortexof the concave side in the elongation zone as previously described(6). After the same time, an unequally distributed DR5 signal wasobserved also in the elongation zone, in protoxylem cells andpredominantly in the xylem pole of the convex side of the root(Fig. 1B).
Author contributions: F.A.D., W.D.T., and K.P. designed research; F.A.D., P.K., K.A.F., I.K.,E.v.d.G., H.N., F.P., and X.L. performed research; F.A.D., W.D.T., R.N., T.L., and K.P. analyzeddata; and F.A.D., W.D.T., and K.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/0807814105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
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We then tested whether a gravitropic response in the rootmeristem is necessary for LRI. We analyzed LRI in two agrav-itropic genotypes: aux1–7 and eir1–1, each lacking an auxin trans-port protein (7, 8). These proteins AUX1 and PIN2, are alsorequired for proper lateral-root spacing. We observed that in bothgenotypes, when visible, lateral roots developed on the convex sideof curves in 100% of roots analyzed (n � 75) (Fig. S3). This strictcorrelation was also seen when aux1–7 roots or eir1–1 roots formedloops (n � 75) (Fig. S3). We therefore conclude that lateral rootpositioning is consistent in relation to root shape, even in theabsence of a gravitropic response.
LRI Is Induced by Transient Manual Bending. To test the hypothesisthat LRI is caused by mechanical changes to the root, a root-bending assay was developed. When 10-day-old roots were bentwith fine forceps through 135° and immediately returned to theiroriginal position, we detected DR5-driven GUS activity at the siteof the bend after five hours, on the side that was convex when bent(Fig. 2B). Lateral root emergence then proceeded normally at the
site of bending, with a lateral root emerging after 72 hours (Fig. 2C).Seventy percent of roots (n � 45) responded in this way when bentwithin 1 mm of the root tip and only forty percent (n � 40) whenbent 3 mm away from the root tip. Fifteen percent (n � 33) ofunbent control roots developed a lateral root 1 mm behind the tipand only 5% did so further away (3 mm). These data demonstratedthat the application of a mechanical force induces LRI. The factthat, after bending, lateral roots were never positioned on theconcave side of the main root at the time of bending (in contrast tothe regular left-right pattern observed in unbent roots) rules out thepossibility that lateral initiation coincides with, but is independentfrom, root bending. Moreover, these data showed that the effi-ciency of LRI was dependent on distance from the root tip.
Coordination of the Early Events of LRI. To correlate DR5 expressionwith the expression of WOX5, a marker of the root quiescent center(9), pDR5::GFP plants (6) were subjected to a modified bendingassay (Fig. 3A). Here, roots were bent 1 mm behind the tip until thetip touched the rest of the root and immediately released. Rootsthen settled unaided to an angle of between 30° and 90° with respectto their original position. As with the first bending method de-scribed above, LRI and development proceeded normally. In allroot bending assays, the growth rate of the main was unaffectedindicating that no significant damage was caused by the bending(Table S1). In 80% (n � 25) of the roots analyzed, we detectedpDR5::GFP expression at between five and six hours after rootbending on the convex side of the curved root, at the site of thenewly initiated lateral root. A GFP signal was simultaneouslyobserved in the protoxylem, in two cells of the adjacent pericycle(the cell layer from which lateral roots are formed) and in either twoor three cells of the endodermis (Fig. 3A).
The homeobox gene WOX5, is expressed in the quiescent centerof the root tip and is required for stem cell maintenance in the rootmeristem (9). We constructed a pWOX5::GFP reporter and ana-lyzed its expression in response to manual root bending. Wedetected WOX5 promoter activity in two pericycle cells at the siteof LRI from five hours after a bending stimulus (Fig. 3 B-E). Auxin
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Fig. 1. PIN protein localization in gravistimulated roots of wild type andpPIN1::PIN1- GFP, pDR5rev::3XVenus-N7 transgenic lines. (A) root elongationzone 3 h after gravistimulation. Immunolocalization with anti-PIN1(green)and anti-PIN2 (red). Epidermis (e), cortex (c), endodermis (en), pericycle (p), px(protoxylem), arrows indicate inferred auxin fluxes, asterisk (*) and hash (#)indicate diffuse cytosolic PIN1 localization. (B) PIN1-GFP (green) and DR5-Venus (pink), 5 h after gravistimulation, DAPI labeled nuclei (blue), asterisk (*)indicates protoxylem cell with diffuse PIN1-GFP signal.
Fig. 2. Lateral root primordia induced after manual curvature. (A-C), LRI inmanually bent 10-day-old pDR5::GUS transgenic plants. (A) before bending,inset indicates root tip bent with fine forceps to 135° from its original position.(B) lateral root primordium as indicated by GUS signal (arrow) five hours afterroots were bent with forceps to 135° and immediately returned. (C) Lateralroot emergence after 72 h at the site of root bending (arrow).
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induces WOX5 expression (10). The simultaneous transcriptionfrom DR5 and WOX5 promoters in the pericycle suggests auxin actsvery early to specify the quiescent center of the new lateral root.
Signals from the Shoot or Root Tip are Not Required for LRI. In atwo-phase model which has been used to explain lateral rootformation during early seedling development, (i) lateral rootprimordium (LRP) initiation is dependent on an auxin source inthe root tip, and (ii) LRP emergence is dependent on leaf-derived auxin (11).
Roots of control pDR5::GFP plants, with shoots and root tipsintact, were subjected to the second bending assay (Fig. 4). After arecovery time of 24 h, a stage II primordium (after the firstpericlinal cell division, classification after ref. 12) was observed atthe site of the bend (Fig. 4B); GFP fluorescence was detectedexclusively at the convex side of the bent root in differentiatingxylem cells, the lateral root primordium cells, and in the adjacentendodermis. After two days, 8% of bent regions had emergedlateral roots (Table S2).
To test whether a signal from the shoot is necessary for LRI, wesubjected to bending pDR5::GFP plants 24 h after the removal oftheir aerial tissues (Fig. 4 D-F). After a further 24 h, GFP fluores-cence was detected as described for the uncut root (Fig. 4E).However, lateral root primordium development was arrested atstage I, the stage at which the first asymmetric anticlinal cell divisionis observed (Fig. 4E, Table S2). Subsequently, the root primordiadid not develop, even after a further five days (Fig. 4F). However,
when transferred onto growth medium supplemented with 1 �MIAA, plants first developed a single lateral root at the convex sideof the bend in every case (Fig. S4H).These results prove that auxinfrom the shoot is not necessary for LRI by mechanical stress, butis required for subsequent primordium outgrowth, consistent withresults recently reported elsewhere (13).
To test whether the basipetal transport of solutes from the roottip could regulate LRI after root bending, seedlings were bent afterthe removal of their root apical meristems (Fig. 4 G-I). After 24 h,this resulted in a larger number of lateral root primordia beinginitiated between the cut and the site of mechanical bending (Fig.4 G and I). Furthermore, these primordia developed more rapidlythan lateral roots of plants with intact root tips, reaching stage III(a primordium of three cell layers) after 24 h (Fig. 4H). After twodays, 100% of bent regions had emerged lateral roots (Table S2).Therefore, the root tip had an inhibitory effect on the initiation andsubsequent development of lateral root primordia after bending.
Lateral root development is disrupted by increased cytokininconcentration (14). However, it is unlikely that cytokinins form thistip-derived inhibitory signal. Bending a root with its tip removed inpresence of 100 nM N-benzyladenine (6-BA), a cytokinin concen-tration known to inhibit lateral root development (14), did notinfluence the number, the position or the frequency of initiationevents (Fig. S5).
To test whether this increase in lateral root number could havebeen caused by the removal of the major auxin sink followingexcision of the root apex and subsequent accumulation of auxin inthe distal elongation zone, we removed both the shoot and the roottip of DR5::GUS plants, using the previously described treatments
Fig. 3. pDR5::GFP and pWOX5-GFP expression after root bending. (A) Fivehours after root bending, DR5-GFP signal is present throughout the protoxy-lem (white arrows), except on the concave side of bends. DR5-GFP signal isdetected on the convex side of the bend in the developing lateral root (yellowarrows). (B-E) six-day-old pWOX5::GFP transgenic plants bent and left to growfor between 5 h and 24 h. (Scale bar, 20 �m.)
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Fig. 4. The origin of auxin during LRI and development. Six-day-oldpDR5::GFP plants with (A) root tips bent and left to grow for 24 h. (B)enlargement of boxed areas in A showing lateral root primordium (arrow-heads). (C) lateral roots 120 h after root bending. (D) pDR5::GFP seedlings werecut at the root-shoot junction and the roots allowed to grow vertically for 24 h.(E) enlargement of boxed areas in D showing lateral root primordium (arrow-heads). (F) No lateral root can be observed in plants described in D 120 h afterroot bending. (G) shoots intact and 24 h after the root tip was removed. (H)enlargement of boxed area in G showing lateral root primordium (arrow-head). (I) lateral roots 120 h after root bending. (Scale bar, 50 �m.)
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as controls. In this case, lateral roots were initiated and arrested ateither stage II or stage III ( Fig. S4D, Table S2); LRI thereforeproceeded in a similar manner in the absence of an aerial auxinsource, or a root tip-driven auxin sink. When repeated in thepresence of 1 �M IAA, lateral roots proliferated, but neveremerged on the concave side of bends (Fig. S4K). These resultsindicate that a balance between auxin from the shoot and an as yetunidentified signal from the root tip controls LRI and emergence.
The Role of Auxin Transporters in LRI. It has been shown that lateralroots of the aux1 genotype are formed in an unequal left-rightdistribution along the main root and do not develop on every curve(5), with nine-day-old aux1-7 seedlings forming approximately halfthe number of lateral root primordia than wild type (WT); aux1-7LRP also display a developmental delay in the transition from stageI to stage II (15). These data are consistent with our observationthat a pAUX1::AUX1-YFP reporter is only induced in pericycle cellsat the site of LRI seven hours after a bending stimulus (at the timea stage I LRP is formed), supporting the hypothesis that AUX1 isnot directly involved in the first events of LRI but in the develop-ment of stage I primordia (Fig. S6). That aux1 seedlings form lessLRP in total could be related to a permissive function of abackground expression of AUX1 observed in the vasculature of theregion in which lateral roots are formed or to other associatedfunctions, for example in post-phloem unloading in the root tip (16)or auxin loading in shoot tissues (15) (Fig. S6).
We observed that lateral root induction by mechanical stress wasalso less efficient in both eir1- 1 and aux1–7 when compared to WT;approximately half of the aux1–7 and eir1–1 seedlings developedlateral roots (Table S3). To test whether abnormal auxin transportfrom the root apical meristem of aux1–7 or eir1–1 precludes normallateral root distribution, we removed the root tip in these genotypes.In this case, both mutants developed lateral roots in response tobending with an increased frequency (Table S3). We thereforeconclude that LRI by mechanical stress is independent of AUX1and PIN2, but the root tip is necessary for subsequent lateral rootoutgrowth.
We analyzed DR5-dependent gene expression to visualize lateralroot primordia in the eir1–1 genotype (Fig. S4 I and J). Afterbending intact eir1–1DR5::GUS roots, all displayed lateral rootprimordia at the expected position on the convex side of curves,proving that LRI per se is not dependent on the action of PIN2 (Fig.S4 I and J). Taken together, these results suggest that LRI is adirect consequence of root bending. These data lead us to hypoth-esize that a physical strain caused by either manual root bending orgravitropic curvature initiates a lateral root.
PIN Proteins Direct Auxin Flux to the Site of LRI. Both the gravitropicresponse and manual bending result in an asymmetric distributionof DR5-dependent transcription in differentiating xylem cells, butin different regions of the root (Fig. S1 and Fig. 2). However, unlikeyounger vasculature in the elongation zone, the xylem cells affectedby the manual bending assay do not express an important auxin-transport related protein, PIN1. We therefore used the morephysiologically relevant gravitropic induction of LRI to examinelocal auxin transport dynamics shortly before the initiation of lateralroot primordia.
First, we inspected root tips (n � 30) of straight-grown four-day-old seedlings (Fig. S2). PIN1 and PIN2 localization were used toindicate the direction of auxin flux (17). Throughout the stele, andin the endodermis and cortex of the meristematic zone, PIN1 waslocalized exclusively to the basal (lower) membrane of cells (17)(Fig. S2A). In the elongation zone (as defined by the slightly biggersize of the cells), PIN1 was absent from the cortex. Here, PIN1 waslocalized in the basal membrane of endodermal cells (Fig. S2A). Inthe pericycle cell file of the elongation zone, PIN1 was localizedboth basally and laterally toward provascular cell file. In themeristematic zone, PIN2 was localized apically (at the top of cells)
in epidermis and lateral root cap cells, and co-localized with PIN1in the basal (lower) membrane of cortex cells forming a previouslyreported loop of auxin flow (17) (Fig. S2B). PIN2 was absent fromthe endodermis of the meristematic zone. Further from the root tipbut still in the meristematic zone, PIN2 was localized apically in theepidermis, directing auxin toward the distal elongation zone. Be-tween the meristematic and elongation zones, PIN2 was localizedto both the apical and basal side of one, and sometimes two corticalcells. In the epidermis and cortex of the elongation zone, PIN2 waslocalized increasingly apically as the distance from the root tipincreased. In the endodermis of the elongation zone, PIN2 waslocalized laterally toward the stele in four or five cells (Fig. S2B).In the stele, PIN2 was entirely absent.
These data revealed the presence of an as yet unreported loop ofauxin flux in 100% of the plants, with PIN2 and PIN1 directingauxin toward the stele in the lower part of the elongation zonethrough the endodermis and pericycle respectively (Fig. S2C). Inaccordance with these observations, auxin (as visualized by a DR5reporter fusion) is localized to both protoxylem cell files throughoutthe root tip (Fig. S2D).
The presence of this PIN-dependent route by which auxin can becirculated to the elongation zone is potentially significant for therecently proposed genetically preprogrammed ‘‘priming’’ of peri-cycle cells necessary for LRI (5), and should be taken into accountwhen designing future mathematical models to predict PIN-dependent auxin flux in the root tip.
PIN1 Relocalizes at the Site of LRI upon Gravitropic Curvature. Threehours after plants were rotated through 135°, significant changes inPIN1 localization in the root tip were observed. PIN1 was seen onboth the apical and basal side of a single protoxylem cell on theconvex side of the of the elongation zone after 3 h in 70% (n � 25)of the seedlings, (Fig. 1A [double-headed arrow], Fig. S7) and waspresent on only the apical side of between 1 and 3 basipetalprotoxylem cells on the convex side of the curving root after 5 h(Fig. 1B, Fig. S7). In these protoxylem cells, the gravity-inducedchange in PIN1 subcellular localization preceded by 2 h theasymmetrical distribution of DR5-driven nuclear-localized YFP(pDR5rev::3XVENUS-N7) fluorescence in the protoxylem poles ofthe bending region (Fig. 1B) compared to the unbent control (Fig.S2D). These data are consistent with a relocalization of PIN1 inresponse to gravitropic curvature serving to maintain locally auxinconcentration in a specific developing xylem cell. PIN1 has beenshown previously to mediate lateral root emergence (2). Therelocalization reported here therefore represents a second, earliermechanism by which PIN1 might influence lateral root develop-ment. Three hours after bending, in the protoxylem on the convexside of the curve, a diffuse cytosolic PIN1 localization was observedto be restricted to the cell immediately above that showing PIN1 onboth sides (Fig. 1A). After 5 h, PIN1 was lost entirely from theprotoxylem cell in which this diffuse cytosolic localization wasobserved (Fig. 1B [asterisk]).
Changes in PIN1 localization in the pericycle in response togravitropic curvature are restricted to the concave side of bends.Here, a similar diffuse cytosolic PIN1 signal was observed, but closeto the lateral wall in contact with the vasculature (Fig. 1A [hash]).This change in subcellular localization corresponded to a relativelyweak pDR5rev::3XVENUS-N7 signal (Fig. 1B) in adjacent vascularcells when compared to similar cells on the convex side of the root.
These data suggest that gravitropic root curvature initiatespositional cues in the protoxylem cell file which determine the siteof LRI. These cues act on an endogenous signaling cascade whichchanges the subcellular localization of PIN1 in the elongation zone.An investigation into decreased lateral root formation in weakalleles of the GNOM gene (which encodes an ARF-GEF necessaryfor PIN1 function) predicted that a relocalization of PIN1 in thepericycle would be necessary for LRI (18). Although in our studies,no relocalization was observed in the pericycle before the accumu-
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lation of a DR5-dependent signal, we suggest that a similar functioncan be attributed to PIN1 relocalization in the subtending protoxy-lem cell file. In contrast to the gnom phenotype, overexpression ofPIN1 in 35S::PIN1 plants initiates more primordia (2), suggestingthat PIN1 is important for LRI. However, plants of the pin1genotype initiate no fewer lateral roots than WT plants. Thissuggests a degree of functional redundancy among PIN proteins inlateral root development.
The relocalization of PIN1 upon bending in protoxylem cells ispredicted to focus auxin into the basipetal neighboring protoxylemcell. In the adjacent pericycle cells, PIN1 is localized to the lateralmembrane; also directing auxin into the same single protoxylem celland further increasing the auxin concentration therein. An increasein auxin concentration would be expected to inhibit the internal-ization of PIN1 (19). However, in this cell (into which PIN1 focusesauxin flux and is therefore inferred to contain an auxin concen-tration maximum), PIN1 becomes internalized. This unexpectedresponse could be a specific adaptation for LRI.
We then tested whether gravity-induced PIN1 relocalization inthe protoxylem is dependent on auxin signaling. ARFs are tran-scriptional regulators of early auxin response genes (20). Thearf7arf19 double mutant is severely impaired in lateral root forma-tion, a phenotype not observed in arf7 and arf19 single mutants,indicating that lateral root formation is redundantly regulated byARF7 and ARF19 (21). Arf7/19 double knockout plants(nph4–1arf19–1) were rotated through 135°. At the convex side ofthe induced bend, PIN1 was observed at the apical and basal sideof a single protoxylem cell after 5 h in 7 of 8 seedlings analyzed (Fig.S2E–G). No other PIN1 relocalization was observed. Nph4–1arf19plants are mildly agravitropic, explaining why PIN1 relocalizationoccurred after 5 h, as compared to after only 3 h in WT plants. Wetherefore conclude that the first stage of LRI, a gravity-inducedPIN1 relocalization in cells of the elongation zone, is independentof ARF7 and ARF19. These transcription factors are thereforenecessary for later stages of lateral root development. The LRIsignaling events before the relocalization of PIN1 are unknown.However, several candidate signaling components can be suggested.For example, cortical microtubule reorganization and mechanosen-sory ion channels could relay physical perturbations into a physi-ological response (22).
Root Bending Induces Lateral Roots in Auxin-Insensitive Lateral Root-Deficient Genotypes. After its inferred accumulation at the site of anincipient lateral root primordium, auxin induces gene transcriptionto trigger lateral root growth. As the first stages of LRI areindependent of ARF7 and ARF19, we examined at which stage oflateral root development these proteins are involved. We bent rootsof two classes of auxin signaling mutants: aux/iaa and arf. Thegain-of-function mutant solitary root is impaired in its gravitropicresponse, synthesizing a stabilized form of IAA14, an Aux/IAA-family negative regulator of auxin signaling (23). This mutation alsoprevents the formation of lateral roots and root hairs (Fig. S8A).Manual bending induces a lateral root in the slr-1 mutant back-ground in 10% of cases (Fig. S8 B and C). These lateral roots areinitiated in an identical manner to the WT after manual bending.Root hairs are also initiated in slr-1 upon bending. However, theydo not develop normally and form densely packed balloon-likestructures (Fig. S8D).
To infer the expression pattern of IAA14 in mechanicallystimulated roots, we stained pIAA14::GUS plants. In unbent plants,we observed GUS activity in the lateral root cap and in epidermalcells of the distal elongation zone (Fig. S8E). Upon manual bending1.5 mm from the root tip, GUS activity was retained in epidermalcells on the concave side of the bend and detected in pericycle cellsin contact with the xylem pole on the convex side of the root at thesite of bending (Fig. S8H). Activity of the IAA14 promoter wassubsequently down-regulated upon the first cell divisions in thepericycle (Fig. S8I).
We tested whether lateral root development after manual bend-ing involves auxin-induced ARF7/ARF19-mediated transcription.Two independent lines of arf7arf19 double mutants, msg1–2arf19–1and nph4–1arf19–1, were used and gave similar results. Whensix-day-old seedlings were bent manually, 96.6% of WT plantsdeveloped lateral roots at the convex side after three days; almostno lateral root was observed in either arf7arf19 double mutant (Fig.S9 A–C, Table S4). However, when, after complete recovery, theroot tips of the same seedlings were removed, and roots were againbent and grown for a further 96 h, 50% of arf7arf19 mutant plantsdeveloped a lateral root at the convex side of the bend (Fig. S9 Dand E; Tables S4 and S5). Significantly, a lateral root also thenemerged from the initial bend, suggesting a primordium had beeninitiated but failed to emerge. This result supports the hypothesisthat specific pericycle cells can be marked as sites of LRI upstreamof auxin signaling.
The specific degradation of Aux/IAAs in pericycle cells is nec-essary for LRI (3). This degradation is a result of auxin accumu-lation in the same cells (5). Auxin accumulation therefore definesthe site of LRI, but the mechanisms involved in the selection of thissite remain unclear. Here we show that IAA14 is expressed at thesite of LRI in pericycle cells in response to an auxin maximumgenerated by manual bending, and subsequently disappears fromdividing lateral root founder cells. We therefore propose thatmechanical strain, caused either by gravitropic curvature or by theapplication of an external force, determines the site of LRI beforeauxin accumulation.
In the case of gravitropic curvature, auxin accumulation in thepericycle follows a reorientation of PIN1 in the subtending pro-toxylem cell file. No accompanying lateral reorientation of PIN1 inprotoxylem cells was observed. We therefore hypothesize that if thelateral movement of auxin from the protoxylem to the pericycle cellfile is significant, it happens either by diffusion or by the specificaction of another auxin transport protein. In the case of manualbending, no PIN1 relocalization was observed as it is not expressedin the bent portion of the root tip. However, the fact that a transientbending of the root is sufficient to induce LRI, and that the rate ofroot growth did not change, suggests that a signaling cascade isinitiated by root bending.
Auxin does not induce lateral roots in slr1 at physiologicallyrelevant concentrations (24), therefore the targeted degradation ofspecific Aux/IAA proteins mediate auxin’s ability to promotelateral root development. However, nuclear auxin signaling throughthe degradation of IAA14 is not strictly necessary for lateral rootformation, as removing the root tip of slr1 plants causes a limitednumber of lateral roots to appear (23). Similarly, we have shownthat if arf7/19 double knock-out plants are subjected to mechanicalstrain, either by manual bending or by gravitropic curvature, alateral root is initiated, but does not develop. Additional evidencethat the earliest stages of LRI are independent of Aux/IAAsdegradation comes from the observation that nuclei of adjacentpericycle cells move toward each other (an early event of LRI) whenIAA17 is stabilized in the pericycle (5). We therefore hypothesizethat a second, auxin-independent lateral root-promoting signaleither moves from the protoxylem or is generated directly in specificpericycle cells in response to a mechanical stimulus. The fact thatlateral roots form upon removal of the root tip in both arf 7/19 andslr1 genotypes suggests that this second signal acts downstream ofARF-dependent gene transcription and is inhibited by a third,mobile signal from the root tip. In this model, subsequent removalof the root tip would release a brake on the development ofquiescent, lateral root-marked pericycle cells, resulting in thedevelopment of a lateral root. This mobile inhibitory signal origi-nating in the root tip possibly acts to suppress downstream signalingin lateral root formation, for example via regulation of LBD genefunction (4). This auxin-independent signal could mediate theability of a main root to suppress the growth of lateral roots andinfluence apical dominance in the root.
18822 � www.pnas.org�cgi�doi�10.1073�pnas.0807814105 Ditengou et al.
The evolutionary relationship between these distinct mecha-nisms of lateral root regulation (as well as the exact nature ofauxin-independent signals) and their relative developmental sig-nificance in different species are all areas for further study.
ConclusionsOur data demonstrate that bending causes the initiation of lateralroot primordia. Here, we report the earliest observable event ofLRI after gravitropic curvature to be a changing polar PIN positionin differentiating xylem cells. PIN1 relocalization is followed byauxin-dependent gene expression in the protoxylem. The signalingevents between the bending stimulus and PIN1 relocalization areunknown. We hypothesize that these signals are auxin-independentand act on cell polarity. Two further auxin-independent signalsaffect LRI, both acting downstream of ARF7/19-mediated tran-scription in the protoxylem pericycle cell file. Removing root tipsafter mechanically bending arf7/19 double mutants demonstratesthat the first is able to promote LRI, and acts antagonistically to thesecond, a root tip-derived inhibitory signal.
In nature, LRI is closely integrated with a range of environmen-tal stimuli such as water availability, nutrients, pathogens, andsymbionts. All of these factors, in combination with the plant’sgenetic background, influence the shape of the root system, makingthe control of root architecture an ideal model to study theenvironmental control of plant development. It is not yet clearwhether an ability to develop lateral roots upon bending of the mainroot confers a selective advantage. But it is tempting to speculatethat the efficient exploration of the root’s environment or increasedanchorage of the plant could be facilitated by such a response.
Materials and MethodsPlant Material and Growth Conditions. A. thaliana (L.) Heynh. ecotype Colum-bia (Col-0) and ecotype WS; pDR5::GFP (6), pDR5::GUS (24), pIAA14::GUS (a giftfrom H. Fukaki, Graduate School of Science, Kobe University, Kobe, Japan; 3);slr-1 (23); pin2/eir1–1 (8); pin2pDR5::GUS (25); pPIN1::GFP (2);pDR5rev::3XVENUS-N7 (a gift from M. Heisler, California Institute of Technol-ogy, Pasadena, CA; ref. 26); nph4–1arf19–1 (obtained from NASC, N24625),msg1–2arf19–1 (obtained from NASC, N24627); pWOX5::GFP was constructedas follows: a 4.46 kb 5� upstream promoter region of WOX5 was amplified
from WT (Col-0) genomic DNA and cloned into pGEM-T, producing AKS49. ForWOX-erGFP, the WOX5 promoter region was isolated from AKS49 and clonedin a pGreenII-derived vector, producing WOX5:Gal4VP16-UAS:erGFP (27, 28);Seeds were surface-sterilized and sown on solid Arabidopsis medium (AM) (2.3g/liter MS salts, 1% sucrose, 1.6% agar–agar (pH 6.0) adjusted with KOH). Aftervernalization for 2 days at 4 °C, seeds were germinated under long-day period(16 h light, 8 h darkness).
Hormone Treatment. Six-day-old pPIN::1GFP plants, grown on solid AM (de-scribed above), were transferred to solid AM supplemented or not with 1 �MIAA or 100 nM N-benzyladenine. After transfer, plants were grown for 48 h or72 h. Root tips were then removed at an average of 400 �m behind the tip.
Immunolocalization. Six-day-old seedlings were rotated through 135° for 5 h,then fixed with 4% paraformaldehyde in PBS (pH 7.3) and used for whole-mount in situ immunolocalization according to (29).
Microscopy. Histological detection of �-glucuronidase (GUS) activity was per-formed according to (30). Plants were mounted in chloral hydrate:glycerol:water (8:3:1, w/vol/vol). Plants containing fluorescent markers were fixed with4% formaldehyde and mounted in Prolong Gold antifade reagent containingDAPI (Molecular Probes). For light microscopy samples were observed with aZeiss Axiovert 200M MOT (Carl Zeiss MicroImaging) for high magnificationpictures. Low magnification views were taken with a Zeiss Stemi SV11 Apostereomicroscope (Carl Zeiss MicroImaging), viewed under differential inter-ference contrast (DIC) optics.
Fluorescent proteins were analyzed with a Zeiss LSM 5 DUO scanningmicroscope. To monitor GFP and DAPI fluorescence, we used multitracking inframe mode. GFP was excited using the 488 nm laser line in conjunction witha 505–530 band-pass filter. DAPI was excited with the 405 nm laser line andcollected using a 420–480 nm band-pass filter. For VENUS with GFP, bothfluorescent proteins were excited using the 458 nm laser line, and the emissionwas separated using the on-line unmixing feature of the Meta spectral ana-lyzer. Images were analyzed with the LSM image browser (Carl Zeiss Micro-Imaging) and representative of at least 20 individual plants.
ACKNOWLEDGMENTS. We thank Prof. Peter Schopfer and members of ourteam for helpful comments on the manuscript. We also gratefully acknowl-edge the excellent technical support from Katja Rapp and Bernd Gross. Thiswork was supported by the Collaborative Research Center 592, the ExcellenceInitiative of the German Federal and State Governments (EXC 294), Gradui-erten kolleg 1305, European Space Agency, Bundesministerium fur Forschungund Technik (BMBF), and Deutsches Zentrum fur Luft und Raumfahrt.
1. De Smet I, Vanneste S, Inze D, Beeckman T (2006) LRI or the birth of a new meristem.Plant Mol Biol 60:871–87.
2. Benkova E, et al. (2003) Local, efflux-dependent auxin gradients as a common modulefor plant organ formation. Cell 115:591–602.
3. Fukaki H, Nakao Y, Okushima Y, Theologis A, Tasaka M (2005) Tissue-specific expres-sion of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidop-sis. Plant J 44:382–395.
4. Okushima Y, Fukaki H, Onoda M, Theologis A, Tasaka M (2007) ARF7 and ARF19regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis.Plant Cell 19:118–130.
5. De Smet I, et al. (2007) Auxin-dependent regulation of lateral root positioning in thebasal meristem of Arabidopsis. Development 134:681–690.
6. Ottenschlager I, et al. (2003) Gravity-regulated differential auxin transport fromcolumella to lateral root cap cells. Proc Natl Acad Sci USA 100:2987–2991.
7. Pickett FB, Wilson AK, Estelle M (1990) The aux1 mutation of Arabidopsis confers bothauxin and ethylene resistance. Plant Physiol 94:1462–1466.
8. Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR (1995) Genetic analysis ofethylene signal transduction in Arabidopsis thaliana: Five novel mutant loci integratedinto a stress response pathway. Genetics 139:1393–1409.
9. Sarkar AK, et al. (2007) Conserved factors regulate signalling in Arabidopsis thalianashoot and root stem cell organizers. Nature 446:811–814.
10. Gonzali S, et al. (2005) A turanose-insensitive mutant suggests a role for WOX5 in auxinhomeostasis in Arabidopsis thaliana. Plant J 44:633–45.
11. Bhalerao RP, et al. (2002) Shoot-derived auxin is essential for early lateral root emer-gence in Arabidopsis seedlings. Plant J 29:325–332.
12. Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots ofArabidopsis thaliana. Development 124:33–44.
13. Swarup K, et al. The auxin influx carrier LAX3 promotes lateral root emergence. NatCell Biol. 10:946–954.
14. Li X, Mo X, Shou H, Wu P (2006) Cytokinin-mediated cell cycling arrest of pericyclefounder cells in LRI of Arabidopsis. Plant Cell Physiol 47:1112–1123.
15. Marchant A, et al. (2002) AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling.Plant Cell 14:589–597.
16. Swarup R, et al. (2001) Localisation of the auxin permease AUX1 suggests two func-tionally distinct hormone transport pathways operate in the Arabidopsis root apex.Genes Dev 15:2648–2653.
17. Blilou I, et al. (2005) The PIN auxin efflux facilitator network controls growth andpatterning in Arabidopsis roots. Nature 433:39–44.
18. Geldner N (2004) The plant endosomal system—its structure and role in signal trans-duction and plant development. Planta 219:547–560.
19. Paciorek T, et al. (2005) Auxin inhibits endocytosis and promotes its own efflux fromcells. Nature 435:1251–1256.
20. Guilfoyle TJ, Hagen G (2007) Auxin response factors. Curr Opin Plant Biol 10:453 460.21. Wilmoth JC, et al. (2005) NPH4/ARF7 and ARF19 promote leaf expansion and auxin
induced lateral root formation. Plant J 43:118–130.22. Fischer K, Schopfer P (1998) Physical strain-mediated microtubule reorientation in the
epidermis of gravitropically or phototropically stimulated maize coleoptiles. Plant J15:119–123.
23. Fukaki H, Tameda S, Masuda H, Tasaka M (2002) Lateral root formation is blocked bya gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of. Arabidopsis PlantJ 29:153–168.
24. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress expressionof reporter genes containing natural and highly active synthetic auxin responseelements. Plant Cell 9:1963–1971.
25. Sabatini S, et al. (1999) An auxin-dependent distal organizer of pattern and polarity inthe Arabidopsis root. Cell 99:463–472.
26. Heisler MG, et al. (2005) Patterns of auxin transport and gene expression duringprimordium development revealed by live imaging of the Arabidopsis inflorescencemeristem. Curr Biol 15:1899–1911.
27. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: A versatileand flexible binary Ti vector for Agrobacterium-mediated plant transformation. PlantMol Biol 42:819–832.
28. Sabatini S, Heidstra R, Wildwater M, Scheres B (2003) SCARECROW is involved inpositioning the stem cell niche in the Arabidopsis root meristem. Genes Dev 17:354–358.
29. Friml J, et al. (2004) A PINOID-dependent binary switch in apical-basal PIN polartargeting directs auxin efflux. Science 306:862–865.
30. Scarpella E, Francis P, Berleth T (2004) Stage-specific markers define early stepsof procambium development in Arabidopsis leaves and correlate terminationof vein formation with mesophyll differentiation. Development 131:3445–3455.
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