Strigolactone Signaling Is Required for Auxin-Dependent Stimulation of Secondary Growth in Plants

Corrections

PLANT BIOLOGY Correction for “Strigolactone signaling is required for auxin-de- 50, December 13, 2011, of Proc Natl Acad Sci USA (108:20242–20247; pendent stimulation of secondary growth in plants,” by Javier Agusti, first published November 28, 2011; 10.1073/pnas.1111902108). Silvia Herold, Martina Schwarz, Pablo Sanchez, Karin Ljung, The authors note that Fig. 3 appeared incorrectly. The cor- Elizabeth A. Dun, Philip B. Brewer, Christine A. Beveridge, Tobias rected figure and its legend appear below. This error does not Sieberer, Eva M. Sehr, and Thomas Greb, which appeared in issue affect the conclusions of the article. CORRECTIONS Fig. 3. Auxin levels and signaling are enhanced in max1 mutants. (A)Inpin1-613 and pin3-5 mutants, the acropetal progression of IC initiation is diminished. Plants were analyzed when shoots were 2, 5, 15, and 30 cm tall. (B) Comparison of levels of free IAA in wild type and max1-1 at different positions along the inflorescence stem. The first elongated internode above the rosette was counted as the first internode. IN, internode. (C and D) Analysis of DR5:GUS activity in wild-type (C) and max1-1 inflorescence stems (D). Rosette leaves have been removed for clarity. (E–H) Analysis of DR5rev:GFP activity at different positions of the inflorescence stem. (E and F) DR5rev:GFP detection 1 cm above the rosette in wild type (E)andmax1-1 (F). (G and H) DR5rev:GFP detection immediately above the uppermost rosette leaf of wild type (G) and max1-1 (H). [Scale bar in D (5 mm) also applies to C; scale bar in E (100 μm) also applies to F–H.]

www.pnas.org/cgi/doi/10.1073/pnas.1211779109

www.pnas.org PNAS | August 28, 2012 | vol. 109 | no. 35 | 14277–14279 Downloaded by guest on September 26, 2021 ECOLOGY, STATISTICS The authors note that the data for three of the eight datasets Correction for “A unifying approach for food webs, phylog- analyzed in the article (Skipwith, St. Martin, and Ythan) were eny, social networks, and statistics,” by Grace S. Chiu and incorrectly processed. As a result, Fig. 2, Table 1, and Table 2 Anton H. Westveld, which appeared in issue 38, September 20, appeared incorrectly. The corrected ﬁgure, its corrected corre- 2011, of Proc Natl Acad Sci USA (108:15881–15886; ﬁrst pub- sponding legend, and the corrected tables appear below. These lished September 6, 2011; 10.1073/pnas.1015359108). errors do not affect the conclusions of the article.

Fig. 2. Food web graphs displaying trophic structure from ﬁtting model 1 with phylogeny measure xij as the predictor. (SR) Feeding activity in Bay. The s axis refers to activity as prey, and the r axis, as predator. Label of node i is located at the mean of the bivariate posterior distribution (an “estimate”)of[si,ri]; this distribution describes the probability of the position of [si,ri]onthesr plane, given the knowledge of the observed food web. Distribution density appears as heat map for benthos-eating birds (“37”) and detritus (“48”). Legend for node labels appears in SI Text. (U, V) Preference of being consumed/consuming in

Skipwith. They are similarly interpreted as (SR), but referring to ui vectors for small oligochaetes (“2”), C. praeusta (“14”), L. marmoratus (“30”), and detritus (“37”), and vi vectors for A. juncea (“11”), A. germari (“19”), great diving beetle (“27”), and P. sagittalis (“31”). Nodes far apart in the latent u or v space differ substantially with respect to feeding preference.

14278 | www.pnas.org Downloaded by guest on September 26, 2021 Table 1. Bayesian inference numerical summaries for selected food webs from ﬁtting statistical social network model 1, with phylogenetic similarity xij as the predictor Credible interval

Food web* Parameter Posterior median† Lower limit Upper limit Credibility‡

Bay β1 5.10 1.20 10.46 0.95

ρsr −0.46 −0.75 −0.07 0.95 ρ −0.82 −0.95 −0.11 0.75

Reef β1 2.16 0.06 4.21 0.90

ρsr −0.08 (interval includes 0) 0.50 ρ −0.29 −0.52 −0.02 0.60

Skipwith β1 13.08 (interval includes 0) 0.50

ρsr −0.94 −0.99 −0.11 0.85 ρ −0.97 −0.99 −0.67 0.99

St. Martin β1 −2.05 −4.23 −0.10 0.60

ρsr −0.34 −0.61 −0.02 0.85 ρ −0.30 (interval includes 0) 0.50

*Other food webs and zij appear in Table S2. †The posterior median can be considered a parameter “estimate.” ‡Credible intervals presented have approximately the highest credibility without including 0. High credibility for

an interval excluding 0 indicates statistical importance of the corresponding parameter to feeding potential (pij).

Table 2. Goodness-of-ﬁt summaries for selected food webs (others in Table S2) Model Predictor GoF* Model Predictor GoF

Bay Reef

1 xij 367 1 xij 524

1 zij 386 1 zij 526 1 — 394 1 — 533 † naive xij 719 naive xij 1273

naive zij 719 naive zij 1287

Skipwith St. Martin

1 xij 42 1 xij 286

1 zij 36 1 zij 275 1 — 33 1 — 286

naive xij 734 naive xij 679

naive zij 737 naive zij 678

*Derived from the Bayes factor on the model’s ability to predict the act of feeding (yij). When comparing between models, noticeably smaller GoF values suggest better ﬁt. † ′ Simple logistic regression ignoring network dependence—i.e., naively setting si þ rj þ uivj þ «ij =0foralli,j in model 1.

www.pnas.org/cgi/doi/10.1073/pnas.1212275109 CORRECTIONS

PNAS | August 28, 2012 | vol. 109 | no. 35 | 14279 Downloaded by guest on September 26, 2021 Strigolactone signaling is required for auxin-dependent stimulation of secondary growth in plants

Javier Agustia, Silvia Herolda,1, Martina Schwarza, Pablo Sancheza, Karin Ljungb, Elizabeth A. Dunc, Philip B. Brewerc, Christine A. Beveridgec, Tobias Siebererd,2, Eva M. Sehra,3, and Thomas Greba,4 aGregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Vienna, Austria; bUmeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden; cSchool of Biological Sciences, University of Queensland, St. Lucia QLD 4072, Australia; and dMax F. Perutz Laboratories, Department of Microbiology, Immunobiology, and Genetics, University of Vienna, 1030 Vienna, Austria

Edited by Athanasios Theologis, Plant Gene Expression Center, Albany, CA, and accepted by the Editorial Board November 8, 2011 (received for review July 21, 2011) Long distance cell-to-cell communication is critical for the de- the strigolactone (SL) biosynthesis and signaling pathway. SLs, a velopment of multicellular organisms. In this respect, plants are carotenoid-derived group of molecules, show all attributes of especially demanding as they constantly integrate environmental plant hormones, meaning that they travel through the plant (3) inputs to adjust growth processes to different conditions. One and are effective at low concentrations (4, 5). In addition to example is thickening of shoots and roots, also designated as shoot branching, SLs have been identified as germination secondary growth. Secondary growth is mediated by the vascular stimulants of parasitic plants and as triggers for the establish- cambium, a stem cell-like tissue whose cell-proliferating activity is ment of interactions with mycorrhizal fungi, in which context regulated over a long distance by the plant hormone auxin. How they were originally identified (reviewed in ref. 6). The SL sig- auxin signaling is integrated at the level of cambium cells and how naling pathway has, so far, been defined by a series of four genes cambium activity is coordinated with other growth processes are for which homologs have been characterized in Arabidopsis (7– largely unknown. Here, we provide physiological, genetic, and 10), pea (11), petunia (12), tomato (13), and rice (14). Also in pharmacological evidence that strigolactones (SLs), a group of plant rice, two additional genes associated with the SL pathway have hormones recently described to be involved in the repression of

been recently described, indicating that the discovery of SL-re- PLANT BIOLOGY shoot branching, positively regulate cambial activity and that this lated genes is not yet completed (15, 16). The identification of function is conserved among species. We show that SL signaling in SL signaling in distantly related species, among them mono- and fi the vascular cambium itself is suf cient for cambium stimulation dicotyledonous species, suggests that this communication tool and that it interacts strongly with the auxin signaling pathway. Our represents an ancient invention and that it is widely distributed results provide a model of how auxin-based long-distance signaling within the plant kingdom. is translated into cambium activity and suggest that SLs act as The function of SL-related genes is characterized best in general modulators of plant growth forms linking the control of Arabidopsis where three of four MORE AXILLARY BRANCHES shoot branching with the thickening of stems and roots. (MAX) proteins (MAX1, MAX3, and MAX4) have been sug- gested to be involved in the biosynthesis of SLs starting from meristem | wood production | MORE AXILLARY BRANCHES carotenoids. This suggestion is based on their resemblance to carotenoid dioxygenases (MAX3 and -4) and to cytochrome P450 ormone-based long-distance signaling is essential for co- family members (MAX1), on corresponding biochemical activities Hordinating the growth and activity of different organs and (7–9)andonSLdeficiency of knockout mutants (4, 5, 17). In tissues during the life cycle of multicellular organisms. In par- contrast to max1,-3,and-4 mutants, max2 mutants are SL in- ticular, plants are demanding in this respect because, due to their sensitivewithregardtoaneffectonapicaldominance(4,5,9), sessile and indeterminate lifestyle, their reproductive success consistent with the idea that MAX2 functions as a receptor for SLs depends on the competence to adjust their developmental pro- or an unknown downstream product. Moreover, its similarity to F- grams to changing environmental conditions and requirements. box leucin-rich repeat proteins and its physical interaction with Auxin is one of the best-characterized hormones involved in compounds of Skp/Cullin/F-box (SCF) E3 ligase complexes sug- long-distance signaling, regulating a tremendous number of de- gests that MAX2 functions as an SL-dependent recruiting factor for velopmental processes. One of these is apical dominance, which, proteins destined for proteasomal degradation (10). indeed, represents a classical example for long-distance signaling max2 mutants (which were also isolated as ore9) (18) display in plants (1). In this case, apex-derived auxin is transported ba- various growth alterations in addition to a decrease of apical sipetally along the main shoot and this is required for sup- dominance, like delayed leaf senescence (18, 19), altered leaf pressing the outgrowth of axillary buds. Another process that depends on the basipetal transport of auxin is secondary growth (2). Secondary or lateral growth of growth axes is based on the Author contributions: J.A. and T.G. designed research; J.A., S.H., M.S., P.S., K.L., E.A.D., P.B.B., activity of the vascular cambium, a meristematic tissue organized C.A.B., T.S., and E.M.S. performed research; J.A. and T.G. analyzed data; and J.A. and T.G. as a cylinder encompassing the center of growth axes. Cambium wrote the paper. activity leads to the production of secondary vascular tissue, The authors declare no conflict of interest. which results in an increase of shoot or root diameter. The This article is a PNAS Direct Submission. A.T. is a guest editor invited by the Editorial interruptibility of the process by decapitation of the shoot tip and Board. fi the subsequent reversibility by arti cial auxin application is 1Present address: Institute of Chemical Engineering, Vienna University of Technology, shared between apical dominance and secondary growth (1, 2). Gumpendorferstraße 1a, 1060 Vienna, Austria. Even though both processes thus share upstream processes and, 2Present address: Department of Plant Sciences, Technische Universität München–Wei- presumably, regulators, how basipetal transport of auxin is henstephan, Liesel Beckmann Str. 1, 85354 Freising, Germany. translated into two different outputs and to what degree they are 3Present address: Roche Diagnostics, Engelhorngasse 3, 1211 Vienna, Austria. interconnected is unknown. 4To whom correspondence should be addressed. E-mail: [email protected]. fi Signi cant progress has been made in the understanding of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. molecular control of apical dominance by the characterization of 1073/pnas.1111902108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1111902108 PNAS Early Edition | 1of6 morphology (19), and hypocotyl elongation (20). In addition, the Results MAX2 gene is required for karrikin-induced seed germination in Strigolactone-Deficient Mutants Display a Reduction in Secondary postfire environments (21) and a role of the SL signaling path- Growth. To characterize long-distance regulation of the cam- way in the regulation of root system architecture was also de- bium, we studied the effect of SLs on cambium activity in wild- scribed recently (17, 22, 23). Intriguingly, in contrast to pea and type and max mutant Arabidopsis plants. Cambium activity was rice (4, 24), SLs are only reduced by 50% in Arabidopsis max1 and defined by the lateral extension of the tissue produced by the max4 mutants (17). Together, these observations suggest that the interfascicular cambium (IC) immediately above the uppermost broadness of SL-dependent processes is currently underestimated. rosette leaf (IC-derived tissue; ICD) and by the acropetal pro- One explanation for a broader role of the SL signaling path- gression of IC initiation along the inflorescence stem (34, 35). way in various developmental processes is an interactive mode of Quantifying these parameters, we observed that the IC-based action with the auxin signaling pathway. The interaction between tissue production was decreased by 30% in all max mutants (Fig. both pathways seems to exist on several levels. On the one hand, 1 A–E) and that the progression of IC initiation was reduced on the influence of SL signaling on auxin transport has been average by 40% (Fig. 1F). Consistent with a reduced cambium reported. max mutants in Arabidopsis display an enhanced ex- activity in SL-defective plants, qRT-PCR analyses demonstrated pression of PIN1 and PIN3, two members of the auxin efflux lower transcript levels of cambium-specific (35) and cell cycle-re- transporter family and show an increase in auxin transport ca- lated (36, 37) genes in max1-1 stems in comparison with wild type pacity (25–29). These observations indicate that SLs act in- (Fig. S1). Together, these observations demonstrate that plants directly on apical dominance by repressing auxin transport, with reduced SL signaling or biosynthesis show reduced cambium which impedes auxin synthesized in lateral buds to be trans- activity. ported into the main stem (25, 29, 30). On the other hand, SL- dependent inhibition of the outgrowth of side shoots functions Local GR24 Treatments Stimulate Cambium Activity. To test whether also in auxin-depleted plants and the application of SLs and the reduction in cambium activity in max mutants is based on auxin transport inhibitors, like N-1-naphthylphthalamic acid (NPA), a direct regulation of the vascular cambium by SLs, we treated results in different physiological responses (26). This suggests SL’s wild-type, max1-1, and max2-1 stems locally with the synthetic SL function to also be downstream of auxin signaling. In fact, MAX4 analog GR24. Stems were treated and analyzed in the first in- and RMS1, its homolog in pea, are inducible by auxin application ternode of the main inflorescence stem in a region where, with- (7, 31), and the same holds true for RMS5, the pea homolog of out treatment, no IC is established in any of the genotypes used MAX3 (32). Even though the significance of MAX4 inducibility in this study (Fig. 1G) (34). In contrast to mock treatments, lo- has been questioned (33), these observations indicate that SL cally applied GR24 induced cell divisions in interfascicular biosynthesis is stimulated by auxin. regions of plants of all three genetic backgrounds at the treat- In this study, we reveal a role of SL signaling in the regulation ment site (Fig. 2 A–C). The pattern of cell divisions was similar of secondary growth and show that the regulation of cambium to that observed during secondary growth initiation at the base of activity is an SL response, which is independent from the regu- the inflorescence stem of untreated plants (Fig. 1 A and B) (34), lation of apical dominance and conserved among species. On the and the effect was dose dependent (Fig. S2A). max1-1 mutants basis of genetic and pharmacological data, we propose that SLs were more sensitive than wild type and, consistent with the fulfill their roles directly in the cambium mainly downstream of employment of the canonical SL signaling pathway, the effect auxin signaling. was significantly weaker in max2-1 (Fig. 2C). Remarkably, max2-1

Fig. 1. Genetic analysis of the role of SL signaling and biosynthesis in cambium regulation. (A–D) Cross-sections from immediately above the uppermost rosette leaf of wild-type (A and B) and max1-1 (C and D) stems. Note that B and D are higher magnification images for areas labeled in A and C, respectively. The extension of the IC-derived tissue (ICD) is indicated by braces (}). [Scale bar in C (100 μm) also applies to A, and scale bar in D (50 μm) also applies to B.] Asterisks indicate the position of primary vascular bundles. (E) Quantification of lateral ICD extension immediately above the uppermost rosette leaf as indicated in A and C.(F) Quantification of the longitudinal progression of IC initiation, as illustrated in D.(G) Scheme of longitudinal IC extension in the Arabidopsis stem (red) and the relative position of the treatment zone described in Fig. 2 and Fig. 4.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1111902108 Agusti et al. Initially only active in the phloem of primary bundles, an APL: CFP reporter was also active in clusters of cells in interfascicular regions after stems were treated with GR24 (Fig. 2 F and G), indicating GR24-dependent formation of secondary vascular tissue. To characterize the spatial extension of the GR24-dependent effect, we treated two reporter lines expressing the β-glucuronidase (GUS) gene under the control of the PXY and APL promoters, respectively. GR24 treatments resulted in the induction of both reporters in the treatment zone and not extending outwards by more than 0.5 cm (Fig. 2 H and I). Im- portantly, our treatments did not repress the outgrowth of rosette branches (Fig. S2B). Together, these results demonstrate a local induction of the cambium-speciﬁc stem cell niche and of vascular tissue formation by GR24 applications and suggest that a local increase in SL levels is capable of stimulating secondary growth in Arabidopsis inﬂorescence stems independently from an effect on shoot branching.

Increased Auxin Content and Signaling in SL Mutant Inﬂorescence Stems. Previous reports showed that SL signaling can reduce auxin transport in the Arabidopsis stem (25–27, 29), presumably by suppressing members of the PIN family of auxin transporters (25, 28, 29). Furthermore, the analysis of auxin-sensitive reporters and measurements of auxin exported out of isolated stem fragments suggest that auxin levels are increased in max mutant backgrounds (25, 28). Because auxin is a positive key regulator of cambium activity (35, 41, 42), we thus analyzed

whether SLs stimulate cambium activity indirectly by repressing PLANT BIOLOGY auxin transport, which might result in enhanced auxin accumu- Fig. 2. GR24 treatments stimulate secondary growth in Arabidopsis in- lation in certain cell types. Initially, we analyzed pin1-613 and fl orescence stems. (A and B) Sections from GR24- (A) and mock-treated (B) pin3-5 mutants to see how changes in auxin transport capacities wild-type plants. Extension of tissue produced in interfascicular regions is influence cambium activity. Both mutants were chosen because indicated by the brace (}) in A.(C) Quantification of GR24-induced tissue production in interfascicular regions of wild-type, max1-1, and max2-1. Note PIN1 and PIN3 are strongly expressed in stems (Fig. S2C) and, at that in mock-treated plants no cell divisions were observed in any of the least for pin1 mutants, it has been shown that auxin transport genetic backgrounds (shown here for wild type). (D and E) PXY:CFP activity capacity along the stem is strongly impaired (43). Histological in mock- (D) and GR24-treated plants (E). Reporter gene-derived signal in analysis of the acropetal progression of IC initiation and ICD the fascicular cambium is indicated by arrows in D and in interfascicular extension revealed a decrease in both pin mutants (Fig. 3A and regions in E.(F and G) APL:CFP activity in mock- (F) and GR24-treated plants Fig. S3), implying that basipetal auxin transport along the stem is (G). Reporter gene-derived signal in the phloem of vascular bundles is in- positively correlated with cambium activity and that an enhanced dicated by arrows in F and in interfascicular regions in G.(H) APL:GUS activity auxin transport rate in max mutants is not causal for decreased in GR24- (Left) and mock-treated (Right) plants. (G) PXY:GUS detection in cambium activity. GR24- (Left) and mock-treated (Right) plants. Asterisks indicate the position of vascular bundles. [Scale bar in B (100 μm) also applies to A; scale bar in D Next, we measured the concentration of free indole-3-acetic (100 μm) also applies to E–G; scale bar in I (5 mm) also applies to H.] Note acid (IAA) in the stems of wild-type and max1-1 mutants at that sections in D–G were counterstained by propidium iodide (red), which different positions including the segment immediately above the highlights cell walls. uppermost rosette leaf displaying pronounced cambium activity (34). Our analyses revealed enhanced IAA concentration in max1-1 stems at all positions with an increasing difference to- plants were not fully GR24 insensitive with respect to the induction ward the shoot base, where max1-1 contained ∼5.5 times more of cambium-like cell divisions, which is in contrast to their in- IAA in comparison with wild type (Fig. 3B). To determine sensitivity with respect to other processes (5, 23) and suggests that whether enhanced IAA concentration also leads to enhanced there are factors acting in parallel to MAX2 in this case. Collec- auxin signaling, especially in segments with prominent secondary tively, these observations show that GR24 can locally stimulate growth, we analyzed DR5:GUS reporter activity visualizing auxin cambium-like cell divisions in the Arabidopsis inflorescence stem. signaling (44) in stems of both wild-type and max1-1 mutants. Next, we tested whether GR24-induced cell divisions represent Enhanced reporter gene activity was detected in max1-1 along secondary growth as observed under natural conditions by ana- the whole stem including the base, where secondary growth is lyzing the dynamics of cambium- and phloem-specific markers in most prominent (34) (Fig. 3 C and D). We also analyzed the response to GR24 treatments. PHLOEM INTERCALATED distribution of auxin signaling at tissue level resolution in wild- WITH XYLEM (PXY, also known as TDIF RECEPTOR, TDR) type and max1-1 backgrounds taking advantage of the DR5rev: is a receptor-like kinase expressed in cambium cells throughout GFP reporter (45). The analysis of stem cross-sections revealed a the Arabidopsis plant body (38, 39). A reporter line expressing the comparable pattern of reporter gene activity with more intense cyan fluorescent protein (CFP) under the control of the PXY activity in max1-1 than in wild type. Increased reporter gene promoter (PXY:CFP) visualized the fascicular cambium in pri- activity was observed in tissues including primary bundles along mary stems and, after GR24 treatments, was detected in inter- the whole stem and in interfascicular regions at the stem base fascicular regions indicating GR24-induced formation of the where IC formation takes place (Fig. 3 E–H). interfascicular cambium (Fig. 2 D and E). To visualize the for- Collectively, these results show that, even though secondary mation of secondary vascular tissue, we used the promoter of the growth is reduced, auxin concentration and signaling is enhanced APL gene encoding for a MYELOBLASTOSIS (MYB) tran- in the vasculature of plants with reduced SL biosynthesis. This scription factor specifically expressed in phloem tissues (40). suggests that the decrease in secondary growth observed in max

Agusti et al. PNAS Early Edition | 3of6 biosynthesis and signaling is important for auxin to stimulate vascular cambium activity. The fact that max mutants are not completely devoid of cambium activity might be explained by an SL-independent effect of auxin on the cambium or alternatively, by redundancy in the MAX pathway or a role of as yet uniden- tiﬁed factors. To determine whether SL and auxin signaling might act se- quentially or in parallel, we tested the genetic interaction between the axr1-3 and max mutants. axr1-3 mutants are impaired in auxin signaling (47) and we reasoned that mutant phenotypes should not be additive if both act through a common signaling pathway. Consistent with a role of AXR1 in cambium regulation, IC initiation and activity was reduced in axr1-3 mutants (Fig. S4). As expected, axr1-3 max double mutants displayed the same reduction in the NPA response as the corresponding single mutants (Fig. 4A). These ﬁndings support a sequential order of the two signaling pathways and, together with the observation of elevated auxin levels in max1-1 mutants and their reduced NPA responsiveness, suggest that SLs act downstream of auxin in a common signaling cascade. If this is the case, the axr1-3 mutation should not reduce GR24 responsiveness. Indeed, histological analyses showed that axr1-3, axr1-3 max1-1,andaxr1-3 max2-1 plants displayed the same GR24-induced response as wild type, max1-1,andmax2-1,respectively (Fig. 4B). Taken together, these results indicate that SLs function predominantly downstream of auxin in a signaling cascade that positively regulates secondary growth.

Tissue-Specific SL Signaling. MAX2 activity can be found in a broad spectrum of tissues (10) and the other MAX genes can influence shoot branching over long distances (3). To dissect whether SL signaling in the vascular cambium is sufficient for stimulating cambium activity, we expressed MAX2 under the control of the (pro)cambium-specific WOX4 promoter (48) in max2-1 mutants. As controls, we also established lines expressing MAX2 in the starch sheath, phloem, and xylem, with the help of the SCR (49), Fig. 3. Auxin levels and signaling are enhanced in max1 mutants. (A)In APL (40), and NST3 (50) promoters, respectively. RT-PCR with pin1-613 and pin3-5 mutants, the acropetal progression of IC initiation is primers specific for the respective constructs demonstrated that diminished. Plants were analyzed when shoots were 2, 5, 15, and 30 cm tall. all transgenes were actively transcribed (Fig. S2D). We analyzed (B) Comparison of levels of free IAA in wild type and max1-1 at different fl positions along the inflorescence stem. The first elongated internode above the lateral ICD extension at the base of the main in orescence the rosette was counted as the first internode. IN, internode. (C and D) stem from two independent lines for each construct. This anal- Analysis of DR5:GUS activity in wild-type (C) and max1-1 inflorescence stems ysis demonstrated that only the lines expressing MAX2 in the (D). Rosette leaves have been removed for clarity. (E–H) Analysis of DR5rev: vascular cambium displayed secondary growth at wild-type-like GFP activity at different positions of the inflorescence stem. (E and F) levels (Fig. 4C). Lines expressing MAX2 within the phloem or the DR5rev:GFP detection 1 cm above the rosette in wild type (E) and max1-1 (F). starch sheath displayed no difference compared with max2-1, (G and H) DR5rev:GFP detection immediately above the uppermost rosette leaf of wild type (G) and max1-1 (H). [Scale bar in D (5 mm) also applies to C; whereas only a minor increase could be observed in lines scale bar in E (100 μm) also applies to F–H.] expressing MAX2 in the xylem (Fig. 4C). All in all, our results show that SL signaling within the vascular cambium is sufficient to promote cambium activity arguing that MAX2-dependent SL mutants is not a consequence of decreased auxin levels, sup- signaling regulates the process in a cell-autonomous manner. porting a direct role of SL signaling in the regulation of cambium activity independently or downstream of auxin accumulation. SL-Dependent Cambium Regulation Is Conserved Between Species. To elucidate whether SLs fulfill a positive role in cambium Single Signaling Pathway for Auxin and SL in Secondary Growth regulation in woody species displaying prominent secondary Regulation. Local treatments of stems by NPA are able to block growth, we performed local GR24 treatments on stems of Eu- basipetal auxin transport leading to auxin accumulation above calyptus globulus. As a result, we observed a significant increase in the treatment zone and local initiation of secondary growth the production of secondary vascular tissues and in particular, the (36, 41, 46). We took advantage of this effect to decipher the lateral extension of the cambium zone was increased (Fig. S5). To interaction of auxin and SL signaling pathways. We speculated see whether the endogenous SL pathway itself is involved in that if SL signaling is important for a positive effect of auxin on cambium regulation in species other than Arabidopsis, we quan- cambium activity, then max mutants should exhibit either no tified cambium activity in the pea mutant rms1-1, impaired in the response or, at least, a weaker response than wild type. To test activity of the MAX4 ortholog (7, 32). Our analysis revealed a this hypothesis, we performed local NPA treatments of wild- significant reduction in cambium activity in comparison with wild type, max1-1,andmax2-1 stems. We observed that interfa- type (Fig. S6). Collectively, these results suggest a conserved role scicular tissue production in NPA-treated max1-1 and max2-1 for SLs in the regulation of cambium activity across species, in- mutants was reduced by 75% (Fig. 4A), indicating that SL cluding species with prominent wood production.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1111902108 Agusti et al. Fig. 4. Genetic interaction between axr1 and max mutants and the effect of tissue-specific SL signaling. (A) Quantification of NPA-induced tissue production in interfascicular regions of wild type, max1-1, max2-1, axr1-3, axr1-3 max1-1, and axr1-3 max2-1.(B) Quantification of GR24-induced tissue production in PLANT BIOLOGY interfascicular regions of wild type, max1-1, max2-1, axr1-3, axr1-3 max1-1,andaxr1-3 max2-1. Note that in mock-treated plants no interfascicular cell divisions were observed in any of the genetic backgrounds (shown here for wild type). (C) Quantification of lateral ICD extension immediately above the uppermost rosette leaf in wild-type, max2-1, and max2-1 plants carrying APL:MAX2, NST3:MAX2, SCR:MAX2,orWOX4:MAX2 transgenes. For each construct, two independent transgenic lines were analyzed. In all cases, n = 10.

Discussion exogenous SL applications can bypass auxin signaling in the in- In this work, we present the role of SLs and their interaction with duction of secondary growth. In the case of max1-1, GR24 the auxin signaling pathway in the control of secondary growth. treatments would stimulate cambium activity largely indepen- We report a significant reduction in cambium activity in SL-de- dently from an effect on the auxin signaling pathway. Together ficient mutants (Fig. 1) and show induction of secondary growth with the idea that there is SL production in the shoot (3) and the upon local treatments with the SL analog GR24 (Figs. 1 and 2). observation that the expression of MAX2 in the cambium is suf- Collectively, these results demonstrate a positive role of SL ficient to restore wild-type–like levels of cambium activity (Fig. 4), signaling in the regulation of cambium activity. these results argue for an important role of auxin-dependent Previous studies have shown that PIN protein levels and auxin stimulation of SL biosynthesis in the cambium for secondary transport capacity are enhanced in max1-1 mutant stems and that growth. As the evidence presented in this study is rather indirect, GR24 applications reduce PIN1 protein levels and dampen auxin we want to point out, however, that our interpretation does not transport (25, 29). It could, hence, be possible that an SL-in- exclude the possibility that SLs influence cambium activity also duced change in auxin transport is the reason for an effect of SL by regulating auxin transport. signaling on cambium activity. However, several observations Importantly, the reduction of cambium activity in the pea support an influence of SLs on cambium activity, which is mainly mutant rms1-1 (impaired in SL biosynthesis) as well as the pos- independent from regulating auxin transport. First, cambium itive effect of GR24 treatments on the production of secondary activity is reduced in pin1 and pin3 mutants (Fig. 3), indicating vascular tissue in Eucalyptus, demonstrate that the role of SL in that, in general, basipetal auxin transport along the stem is cambium activity is conserved among species and that it repre- positively correlated with cambium activity. Second, even though sents a common signaling mechanism for coordinating growth of cambium activity is decreased, auxin concentration and signaling different plant organs. is increased in the stem of max1-1 mutants. Third, genetic Conclusions analyses of the interaction between SL and auxin signaling in the context of cambium regulation are in line with a requirement of The converse effects of SLs on branching and secondary growth SL signaling for auxin becoming fully active on cambium cells suggest a central role for SL in regulating plant architecture. and show that SL responsiveness is not impaired in auxin sig- Shoot branching and secondary growth are both influenced by naling mutants (Fig. 4). Moreover, the axr1-3 max1-1 and axr1-3 environmental stimuli such as shading, temperature, or day max2-1 double mutants did not display additive phenotypes in length (52–55). On the basis of our finding that SLs are involved comparison with single mutants arguing for a linear relationship in the regulation of both these processes, we hypothesize that of both signaling pathways. SLs function as modulators used by plants to switch between two Recently, it was shown that auxin induces the expression of the SL growth forms: a bushier form displaying a strong outgrowth of biosynthesis genes MAX3 and MAX4 in the stem of Arabidopsis in many side shoots and a weak main stem, and a form in which the an AXR1-dependent manner (51). In agreement with that obser- main shoot dominates and displays enhanced secondary growth. vation, we propose that the reduced responsiveness of axr1-3 and The ability to oscillate to a certain extent between these growth max1-1 toward NPA is, at least partly, due to a reduced SL bio- forms could be regarded as being beneficial if, for example, synthesis. In the case of axr1-3, this model would imply that differences in the necessity to compete for light, or in the total

Agusti et al. PNAS Early Edition | 5of6 number of ﬂowers produced by the shoot system under changing plants of the accession Columbia were used with the exception of the PXY: environmental conditions, are considered. This feature could GUS reporter line, which is in the Ler background (38). represent a strategy in which the plant can choose the optimal ACKNOWLEDGMENTS. We are grateful to Ottoline Leyser for providing the way to grow to cope with the challenges imposed by a sessile max1-1, max2-1, max3-9, max4-1, axr1-3 max1-1, and axr1-3 max2-1 mutant lifestyle. lines; Christian Luschnig for providing the pin3-5 mutant; and Simon Turner for providing the PXY:GUS marker line. This work was supported by Austrian Materials and Methods Science Fund (FWF) Grants P21258-B03 and P23781-B16 (to M.S., P.S., and J.A.) the Wiener Wissenschafts-, Forschungs-, und Technologiefonds (T.S.), the Details about growth conditions, cloning and plant lines, histological anal- Australian Research Council (E.A.D., P.B.B., and C.A.B.), and the Swedish yses, pharmacological treatments, RT-PCR, auxin measurements, and other Governmental Agency for Innovation Systems and the Swedish Research techniques are in SI Materials and Methods. Arabidopsis thaliana (L.) Heynh. Council (K.L.).

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