Current Biology 23, 10571064, June 17, 2013 2013 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2013.04.061

Article A Cytokinin-Activating Enzyme Promotes Tuber Formation in Tomato

Tamar Eviatar-Ribak,1,3 Akiva Shalit-Kaneh,1,3 Louise Chappell-Maor,2 Ziva Amsellem,2 Yuval Eshed,2,* and Eliezer Lifschitz1,* 1Department of Biology, Technion, Israel Institute of Technology, Haifa 3200003, Israel 2Department of Plant Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel within the same family, tubers can be of different origin, e.g., the roots of radish or the stems of kohlrabi, both Brassicaceae family members. Tubers function as strong sinks for carbohydrates and accumulate species-specic proteins [1]. Some species exploit tubers to tide over for a dry season, whereas in others such as potato, they serve also as an alternative to sexual reproduction. Potato tubers are rounded, condensed shoots derived from the swollen tips of stolons: short plagiotropic shoots with suppressed leaves and dormant axillary buds [2]. The formation of potato tubers involves induction, initiation, and proliferation phases [3]. Induction of tuberization is species specic, and in potato it requires short days and is mediated by a leaf-derived systemic protein of the origen family [4]. No specic factor has thus far been associated with the control of tuber initiation or proliferation, and regulatory signals common to different storage organs have not been identied. In potato, stolons and tubers are restricted to basal juvenile nodes. However, under strong inductive conditions, when plants are subjected to stress conditions, or when supplied by a combination of sucrose, cytokinin (CK), and gibberellic acid inhibitors in culture, sessile (stalkless), minitubers are formed along the shoot [2]. Notably, such sessile aerial tubers store starch and express carbohydrate metabolism and defense proteins even though they lack some characteristics of underground tubers [5, 6]. Therefore, all axillary potato meristems are endowed with the potential to form tubers, which is suppressed in all but basal meristems and in all meristems of the closely related tomato [7, 8]. The body plan of owering plants is propagated through the hierarchical formation of apical, lateral, and intercalary pluripotent growth centers, called meristems. At rst, leaves, hosting transiently dormant axillary meristems, are formed on the anks of the shoot apical meristem (SAM). The axillary meristems, initially suppressed by systemic apical signals (apical dominance) [9], are gradually released to form higher-order appendages of differing fates: lateral shoots with variant vegetative-reproductive cycles or, depending on the context, owers, thorns, tendrils, or tubers. The formation of the functional organs of a plant can therefore be ascribed to a series of successive homeotic transformations of uncommitted meristems that might trace back to dynamic shifts in local balances of hormones from combined endogenous and systemic origins. Because the formation of tubers in potato, be it sessile or stolon borne, is conditioned on prior activation of axillary buds, it is possible that the evolutionary forces suppressing tuber differentiation are mediated by the same hormonal signals involved in bud activation [1012]. Here, we report that a unique type of CK signaling, activated by the LONELY GUY1 (LOG1) gene, is sufcient to endow tomato meristems with the potential to produce sessile minitubers, equivalent in many ways to potato aerial tubers. LOG1 catalyzes the conversion of CK ribosides to the biologically active CK [13]. The generation of homeotic tubers from naive meristems in the diploid, day-neutral, and genetically tractable tomato offers a new tool to explore the hidden potentials of axillary meristems, as well as the opportunity to reconstruct and dissect tuber initiation and proliferation.

Summary Background: Dedicated storage organs in the form of tubers are evolutionary novelties that share a common function but originate in diverse species from different organs. Tubers in potato, Solanum tuberosum, are derived from the swollen tips of specialized basal lateral juvenile shoots, called stolons. Lateral buds of tomato, Solanum lycopersicum, a potato sibling species, only form regular shoots. The evo-devo mechanisms restricting tuber formation to basal juvenile axillary meristems of potato while completely inhibiting it in tomato meristems are not currently understood. Results: Ectopic expression of tomato LONELY GUY (LOG1), a cytokinin (CK) biosynthesis gene, imparts potential to the outgrowing juvenile tomato buds to generate, de novo, aerial minitubers (TMTs). TMTs are morphologically, developmentally, and metabolically homologous to aerial potato tubers and display a unique transcriptome with altered hormonal signaling networks. The new hormonal balance stimulates ectopic branching of dormant axillary meristems and loss of apical dominance without disruption of polar auxin transport and obviates the need for specic branching genes. miR156, a master regulator of juvenility, extends tuber-forming potential to distal axillary buds in both wild-type potato and tomato primed by LOG1 signaling. Conclusions: Ubiquitous activation of TLOG1 uncovered a developmentally suppressed tuber-forming potential within tomato axillary meristems. Other meristems in other plants may also carry hidden, suppressed organogenesis potentials. The unlocking of this potential by the activity of a single gene represents a prime example of an evolutionary novelty in the making and suggests that CKs may function as universal regulators of storage-organ formation in plants. Introduction Homologous plant organs, such as leaves, owers, roots, and plant vasculature, are generated by the same mechanisms in different plants. Notable exceptions are evolutionary novelties in the form of tubers, a generic name for dedicated storage organs and a major component of staple foods worldwide. These vary in their tissue and organ of origin, as manifested by the swelling roots of carrots and sweet potato versus the expanded hypocotyls of beets and the corms of yams. Even

3These authors contributed equally to this work *Correspondence: yuval.eshed@weizmann.ac.il (Y.E.), lifs@tx.technion.ac.il (E.L.)

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Figure 1. TLOG1 Induces De Novo Formation of Tubers in Tomato (A) Expression of the TLOG1 transgene (upper line) and the endogenous TLOG genes (lower lines) in transgenic and WT tomato meristems. Note the dynamic expression of WT TLOG1. XM, axillary meristem of the fourth leaf. VM, vegetative meristem; FM, oral meristem; ST, stem. Solid lines mark the TLOG1 plants, and the broken lines mark the NY progenitor background. See Table S6 for gene IDs. (B) Dormant CXMs (arrow) in a NY progenitor (WT) plant. (C) Ectopic branching and early TMTs in TLOG1 seedlings. (D) Basal TMTs (arrows) along the primary shoot of a young TLOG1 plant. (E) A TMT with two expanding leaves and a suppressed apex. (F) Chain tubers in TLOG1 plants. (G) TMTs in the CXMs of TLOG1 plants grown in articial culture medium. (H) An aerial tuber in a stressed potato plant. (I) An outgrowing lateral branch with initial TMTs in the CXMs of a sp pARR5:TLOG1 plant. (J and K) Expression patterns of the pARR5:GUS reporter in tomato stems. (J) pARR5:GUS marks vascular strands and stem-leaf junctions. (K) Top, unstained live image of anthocyanin-accumulating TMT primordia of TLOG1 plants expressing the pARR5:GUS reporter; bottom, same specimen after b-glucurodinase (GUS) staining. Arrowheads mark the cotyledons.

Results LOG1-Dependent CK Signaling Induces Axillary Tubers in Tomato In rice, where it was rst described, LOG1 is expressed in the apical meristems, where it promotes meristem maintenance [13]. Similarly, tomato TLOG1 is expressed in a dynamic pattern in vegetative, reproductive, and axillary meristems (Figure 1A). Expression of TLOG1 by the ubiquitous 35S cauliower mosaic virus promoter in tomato plants (New Yorker [NY], Figure 1A, top) triggered the complete loss of apical dominance and the associated formation of small, rounded, sessile, pigmented aerial minitubers (TMTs) in 9/12 independent T1 plants (Figures 1B1F). TMTs were rst formed from cotyledonary axillary meristems (CXMs) of juvenile seedlings (Figures 1B and 1C), which are typically never activated. Subsequently, in a diminishing acropetal pattern, TMTs are also formed from the rst 25 nodes (Figure 1D).

TMTs initially accumulated high levels of anthocyanins and developed two enlarged leaves (Figure 1E) as reported a century ago in describing a potato eld under ooding: the abnormal tubers were purple in color; each one had several suppressed buds (eyes), and one or two minute green leaves in each eye [14]. Subsequent to their formation, TMTs sprouted, and the emerging leaves hosted secondary TMTs (Figure 1F) analogous to chain tubers in potato [2]. Sessile aerial tubers similar to stress-induced tubers [2] were formed when cuttings of short-day-grown potato plants were placed in water for just a few days (Figure 1H). Morphologically similar tubers were formed by TLOG1 seedlings, but not by progenitor seedlings, when grown on hormone-free medium (Figure 1G; Figures S1AS1C available online). Importantly, expression of TLOG1 by the ARR5 promoter was also sufcient to promote TMTs and promiscuous branching (Figures 1I1K). The pleiotropic effects of TLOG1 overexpression, collectively referred hereafter as the TLOG1 syndrome, also

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Figure 2. Metabolic Hallmarks of Tomato Tubers (AC) Cross-sections treated with Lugols staining. (A) Starch-particle accumulation in tomato TMTs. (B) Absence of starch accumulation in TLOG1 stems. (C) Starch in stolon-borne aerial potato tubers (see Figure 5). (D) Expression proles of genes encoding storage proteins and carbohydrate metabolism enzymes in axillary buds of progenitor and TLOG1 plants. Gradually maturing TMTs (TITIV) are described schematically in Figure 2F and Figure S2. For expression values, see Table S7. (E) Hierarchical clustering of progressively mature TMTs with samples from progenitor (black) and equivalent TLOG1 organs (red). TMTs and upper axillary bud (LXM) fated to form TMTs are clustered together. Numbers indicate bootstrap values. Tissue origin is marked on the shoot scheme at the right. XM/LXM, axillary meristems of the fourth leaf in WT and TLOG1 plants, respectively; CXM/LCXM (TI), cotyledonary axillary meristems; ST/LST, stems; VM/LVM, vegetative meristems; FM/LFM, oral meristems. Note the pigmentation in the epidermal cells of the TI apex. (F) A schematic presentation of the TMTs used for NGS analysis and the respective control. (G) Expression proles of genes encoding thylakoid-related proteins. (H) Expression proles of ANL2, the tomato KNOTTED2 (TKN2), and a selected histone gene. For other avonoid and CK-regulated genes, see Table S7.

include attenuated apical meristems, late owering, and simplied leaves (Figures S1DS1L), which will be further discussed only in relation to the formation of TMTs. In retrospect, in a blind screen for a tuber-promoting agent, the most likely candidate should be the one that performs best in a nontuberized sibling species. TMTs Have Novel Transcriptomes and Carry Metabolic Hallmarks of Potato Tubers Lugols staining showed that TMTs accumulate starch granules, demonstrating their role as strong sinks for carbohydrates, a prime aspect of tubers (Figures 2A2C) [15]. By contrast, stems and apices of TLOG1 plants do not accumulate starch, indicating that TMTs are metabolically distinguishable from their organ of origin. We thus probed the transcriptomes of TMTs for expression of marker genes that characterize storage organs in general. Deep RNA sequencing was performed from sequential developmental stages of cotyledonary TMTs (TITIV in Figure 2F; Figures S2AS2G), from axillary bud #4 of the same TLOG1 plants that are fated to form TMTs, and from equivalent buds of progenitor plants. Mining of these transcriptomes revealed that TMTs express progressively higher levels of SUCROSE SYNTHASE and other carbohydrate metabolic genes typical of potato tubers (Figure 2D; Figure S2H) [15, 16]. In general, storage organs express different proportions of carbohydrate metabolism and defense storage genes but can compensate for a genetic deciency in one protein by elevated synthesis of other proteins [17]. Notwithstanding this, we were still surprised by the high levels of transcripts encoding carbohydrate metabolism and defense genes expressed in TMTs (Figure 2D; Figures S2I and S2J; Table S1; Table S7). Comparison of 13,000 orthologous genes from the transcriptomes of TMTs and potato tubers revealed tuber markers common to both

species and some that are unique (Tables S2 and S3). When, for example, the expression of cell-cycle and histone genes in tomato and potato was compared, a similar pattern was found: upregulation during the initiation stage of tuber formation and downregulation toward maturation (Table S4). To further explore the novelty of TMTs, we performed hierarchical clustering (TM4 software) [18] and ordination analysis (multidimensional scaling [MDS]) [19] of pairs of transcriptomes from meristems of TLOG1 and NY plants (Figure 2E; Figure S2K and Supplemental Experimental Procedures). Hierarchical clustering grouped all samples according to their tissue origin, regardless of their genotype (Figure 2E): vegetative and oral apical meristems were grouped together, and a second clade was clustered with axillary buds. Within this clade, all TMT samples were highly related despite variations in their age and developmental status. Taken together, TMTs are regulated by novel transcriptomes, and their ontogeny and morphology are similar to those of potato aerial tubers (Tables S2S4). The TLOG1 Syndrome Faithfully Represents Increased CK Signaling Upon direct application to pea and Arabidopsis, CK activates axillary buds [12]; we showed that its application also activated the dormant CXM in tomato (Figure S2L). Activation of the ever-dormant CXMs and the precocious loss of apical dominance in TLOG1 plants is thus a prime signature of CK response [10, 12]. Other phenotypic and molecular aspects of the TLOG1 syndrome correlated with various CK targets, namely growth rate and cell-cycle genes [2022] (Figure S1, S2NS2S), accumulation of anthocyanin [23] (Figures 1C1F), and the corresponding upregulation of ANTHOCYANINLESS2 (ANL2) [24] and other avonoid biosynthesis genes (Figures 2G and S2Q). Leaves of TLOG1 plants were deep green

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Figure 3. An Autonomous Hormonal Balance Conditions TMT-Forming Meristems (A) TLOG1 is a suppressor of lateral inhibition by blind (see Figure S3). (B) ls inhibits formation and outgrowth of axillary buds by TLOG1 signaling. The arrow represents an escaper axillary shoot with a TMT initial. (C and D) Confocal images of PIN1 polarity in the primary apices of progenitor (C) and TLOG1 (D) plants. (E) PIN1 in the vascular strands of young seedlings (six leaves) of progenitor plants. (F) Three out of four sampling sites are indicated on the scheme. (G) PAT along the stem of a TLOG1 seedling. (H and I) NAA suppresses axillary-bud activation in decapitated 20-day-old progenitor and TLOG1 plants. Seedlings are shown 5 days after apical application of lanolin paste with (H) and without (I) 2.5 mM NAA. Bars represent 50 mm. The white circles mark SAMs.

(Figure S2M) and senesced more slowly, in line with the enrichment of thylakoid proteins in TLOG1 relative to its progenitor genotype (Figure 2H; Figure S2P; Table S7). Finally, TKNOTTED2 [25] was upregulated in all TLOG1 meristems with the exception of oral buds (Figure 2G), in line with activation of KNOTTED genes by CKs in Arabidopsis [26]. Tuber Formation Is Autonomously Determined in Each Axillary Bud The differentiation of tubers from the ever-dormant CXMs of TLOG1 plants and stolons from juvenile buds in potato require breakdown of apical dominance. To understand the relationship between the breakdown of apical dominance and the differentiation of tubers, we studied the phenotypic expression of branching suppressors in TLOG1 background and monitored possible perturbations in polar auxin transport (PAT). In blind (BL, MYB 2/3), distal and proximal leaves along the primary shoot host axillary meristems that remain mostly dormant, whereas centrally located leaves lack detectable meristems altogether [27]. We show that TLOG1 activated all bl axillary meristems in addition to inducing regular TMTs, thus indicating a rescue of a normal branching mode (Figure 3A

and Figure S3). However, TLOG1 signaling did not suppress the inorescence defects of bl. Branching in trifoliate (tf), a MYB factor and a moderate suppressor of branching [28], was also rescued by TLOG1. In contrast, TLOG1 failed to induce branching in LATERAL SUPPRESSOR (ls) [29], but when a bud escaped, it was associated with a TMT (Figure 3B). TLOG1 therefore assigns LS and BL to distinct phases of bud development and dormancy. The two genes are not required for TMT formation, but TLOG1 signaling is an effective suppressor of bl and tf, but not of ls. High CK signaling may directly or indirectly impact promiscuous branching and tuber differentiation in the bud by modifying or inhibiting the auxin signaling network [11, 12]. To examine the possibility that precocious branching and TMTs involve changes in PAT, we monitored the cellular localization of PIN1 [30, 31] in TLOG1 and progenitor seedlings (Figures 3C3G). We found that PIN1 was polarized in the SAMs of TLOG1 plants from their very inception. In organ primordia, PIN1 expression marked the developing provascular strands, which we refer to as Sachs canals (Figures 3C and 3D). No signicant deviations from the normal polarity or abundance of PIN1 were observed along the stems of TLOG1 seedlings, including in samples from the hypocotyl-epicotyl junctions

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Figure 4. TLOG1 Directs Novel, Meristem-Specic, Hormonal Balances (AD) TLOG1 directs novel, meristem-specic, hormonal balances. An intragenotype MDS ordination of meristem samples based on the best 5,000 distinguishing genes (A and B) or 137 hormonal genes (C and D) in NY (A and C) and TLOG1 (B and D) plants. Red and black symbols indicate TLOG1 and NY plants, respectively. (EI) TLOG1 directs meristem-specic expression of CK signaling (E and F), auxin signaling (G), and GA signaling and metabolism genes (H and I). Continuous lines denote TLOG1 samples; broken lines denote progenitor samples. x axis, tissue of origin; y axis, expression levels at FPKM (see Experimental Procedures).

(Figures 3E3G). In support of a normal PAT system, 1-naphthaleneacetic acid (NAA) application arrested the outgrowth of CXMs in decapitated NY seedlings and similarly suppressed TMTs in TLOG1 plants (Figures 3H and 3I). Thus, although apical dominance in TLOG1 plants was disturbed, PAT was not altered along the shoot. Taken together, bud activation and tuberization in TLOG1 plants are likely to be due to a novel organ-autonomous, TLOG1-driven CK/auxin balance, but not a result of disruptive PAT. Unique Steady-State Hormonal Balance In pursuit of a correlation between the distinct TLOG1-induced responses of different meristems and their transcription proles, we examined, using MDS, the proximity between transcriptomes of the ve pairs of meristems, described in Figure 2E, from progenitor and TLOG1 plants. Primary vegetative (VM) and owering (FM) apices were found to be similar to each other, regardless of their genotypic status, whereas axillary meristems were more similar to each other in TLOG1, compared to their NY counterparts (Figures 4A and 4B). In NY plants, CXM was found to be more similar to stem (ST). Even when analyzing a subset of genes such as these involved in CK, auxin, and gibberellic acid (GA) responses, the same relative rations were maintained (Figures 4C and 4D). The heatmap and the corresponding hierarchical clustering (Figure S4) that are based on the same set of hormonal genes tested in Figures 4C and 4D further corroborate these modied meristem-specic interrelations. At the single-gene level, this assertion is best exemplied by the disproportionate induction of CK response inhibitors (Figure 4E), by upregulation of THK4/CRE1 and THP5 in the CXM of TLOG1 plants and in a subset of TLOG1 meristems (Figure 4F), and by the altered expression of the negative auxin network regulators class (Figure 4G) and of genes of the GA

network (Figures 4H and 4I). Interestingly, by analogy with TMTs, a spontaneous gain-of-function allele of the widely expressed HK4/CRE1 of Lotus japonicus correlated with specic and local differentiation of root nodules [22]. Hence, TLOG1 meristems maintain novel homeostatic proportions of primary hormonal signaling components, which probably explain their unique developmental fates, growth dynamics, and branching patterns. miR156 Links Juvenility with Tuber Differentiation In potato, only axillary meristems hosted by the basal simple and juvenile leaves develop tuber-bearing stolons [2] (Figure S5). The TLOG1 syndrome involves precocious branching from juvenile axillary meristems, increased leaf simplication, and, importantly, delayed owering (Figures S1JS1L). Juvenility and late owering in maize, Arabidopsis, and tomato are regulated by the miR156 system [3234]. In tomato and Arabidopsis, miR156 induces pigmentation of epidermal tissues, and similarly, peels of tomato TMTs and wild-type (WT) potato tubers accumulate high levels of anthocyanins (Figure S2G) [33, 35]. Finally, in tomato, overexpression of miR156 induces precocious branching and simpler juvenilelike leaves (Figures 5A5C and Figure S5), just like with TLOG1. We therefore explored the interaction between TLOG1 and miR156, a regulator of juvenility with broad conservation. Whereas 35S:miR156 tomato plants did not produce tubers, 35S:TLOG1/+ 35S:miR156/+ plants generated TMTs at every nodal bud along primary and secondary shoots, in addition to augmented pigmentation and promiscuous branching (Figures 5F5I). Thus, by extending the juvenile stage, miR156 enhanced the tuber-forming potential of tomato meristems. If this interpretation is correct, miR156 is expected to induce tuber-bearing stolons from distal potato meristems.

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Figure 5. miR156 Links Tuberization Juvenility and CK Signaling

with

(A) A compound leaf of WT tomato. (B) A leaf of a 35S:miR156 tomato plant. (C) A leaf of a TLOG1 tomato plant. (D) WT potato shoot. Note the age-dependent changes in leaf morphology along the shoot. (E) A shoot of a 35S:miR156 potato plant. (F and G) An upper part of a TLOG1 shoot. (H and I) miR156 promotes TMTs in upper nodes of TLOG1 plants. (J) Tuber-bearing aerial stolons (arrows) in a 35S:miR156 transgenic potato plant. For more responses of tomato and potato to altered miR156 activity, see Figure S5.

Consistent with this hypothesis, 35S:miR156 potato plants, under short-day and low-light conditions, produced, in addition to basal tubers, stolon-borne aerial minitubers from almost all of their distal buds (Figure 5J and Figure S5). The formation of tuber-bearing stolons in potato but only of sessile tubers in tomato demonstrates the developmental uncoupling of the two processes [2] and suggests that tomato stolon development is more robustly suppressed than tuberization. The observation that TLOG1 and miR156 inhibit owering but promote tuber differentiation suggests that at some developmental junctions, the regulation of the reproductive phase intersects with that of tuberization. Discussion Overexpression of TLOG1 was observed to cause de novo formation of organized homeotic tubers and reshape shoot architecture by modifying apical dominance; it also induced late owering and attenuated the growth of all aerial meristems. These changes were accompanied by meristem-specic transcription proles and hormone-signaling networks and by a sensitized response to miR156-regulated systems. CK regulates cell proliferation and tissue differentiation in many organs and plants [36], but the induction of TMTs, a completely new homeotic organ, is a novelty unparalleled by any other plant hormone. In other cases where CK stimulated the formation of organs, normal endogenous programs were ectopically duplicated [6, 22, 37, 38]. Other forms of elevated CK signaling, such as CK application or overexpression of IPT [39, 40] or KNOTTED genes [41], were insufcient to promote tubers in tomato, probably due to inappropriate local hormonal balances or to changing ratios among CK metabolites [42]. In TLOG1 plants, the transgene is expressed at high levels throughout (Figure 1A), but the response is tissue and organ specic: axillary meristems are activated, but oral, apical, and leaf meristems are attenuated. TLOG1 and miR156

additively suppress owering and promote tuber formation. TLOG1 is a suppressor of blind in axillary meristems, but not in sympodial meristems. In support of the meristem-specic responses to similar buildups of hormonal balances, TLOG1 disrupts apical dominance (Figures 3H and 3I), but PAT remains normal (Figures 3C3G). The systemic hormones auxin, cytokinin, and strigolactone are involved in regulating apical dominance [11, 12]. The systemic protein origen, a potent generic growth hormone, also regulates shoot architecture through relaxation of apical dominance upon induction of oral transition [43]. Hence, by analogy to origen, the long-range physiological impact of auxin and CK is primarily the consequence of systemic modications of their local ratios rather than their absolute levels [43]. Stolon-borne tubers in potato are restricted to basal juvenile meristems, but, conditions permitting, any potato meristem can generate aerial tubers [2]. Because such tubers are sessile, the differentiation of stolons is regulated in a meristem-specic manner by a developmental mechanism that is obviated by a level of high miR156 activity (Figure 5J). In tomato, tubers are not formed under any circumstances, a developmental blockade which is obviated, primarily in basal nodes, by ubiquitous TLOG1 signaling (Figure 1), whereas miR156 promotes only sessile tubers, and only in meristems primed by TLOG1 signaling. The formation of TMTs and of stolon-borne aerial tubers in miR156 potato and the differential response of meristems in the two species to miR156 unearthed hidden potentials shared by their meristems while also uncovering the genetic barriers underlying the developmental segregation of axillary meristems along the shoot. The ectopic activation of the normally dormant cotyledonary buds and the unlocking of the tuberforming potential of meristems in tomato, together with the ectopic formation of stolons in potato, indicate that each axillary meristem is endowed with a unique developmental potential. The patterning of lateral-meristem diversication suggests the developmental fate of each axillary meristem was subjected, as speciation itself, to the selective forces of evolution. The extant suppressed potential of tomato meristems depends on local hormonal balances and is extremely labile, as suggested by the ease with which it is disrupted by TLOG1 signaling. It is probable that the evo-devo adjustments

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of seemingly serendipitous, but context- and cytokinin-dependent, shifts in hormonal balances [12, 44] function as universal determinants of storage-organ formation in plants. TMTs provide an integrative framework for testing this hypothesis and for exploring the potential of axillary meristems to generate alien organs. Genetic analysis of the pathways that transform competent meristematic domains to tuber-initiating sites [3, 4] is hindered in potato and other tuber forming plants by intractable genetic systems [15]. The generation of TMTs in the diploid genetically tractable tomato offers a complementary experimental platform. At the same time, our results suggest new genetic opportunities to reconstruct full-size, stolon-borne, ground-bound, and dormant tubers from naive buds of tomato, essentially reconstructing the path for domestication and the evolution of one single unique morph. After all, domestication in potato started with tubers the size of TMTs [45].
Experimental Procedures The cultivars NY (sp background) and Money Maker (MM) and the blind, trifoliate, and lateral suppressor mutant lines were obtained from the C.M. Rick Tomato Genetics Resource Center at UC Davis. Other lines mentioned in the text were bred for this work. Plant transformations of tomato NY and e were performed as previously described [46, 47]. potato Desire The TLOG1 coding sequence was amplied as an XhoI-blunt complementary DNA fragment from RNA extracted from apices of NY seedlings and then subcloned into the XhoI-SmaI site of the pART7 intermediary vector, containing the 35S promoter and the OCS terminator. The nal construct was then transferred as a NotI fragment into the ART27 binary vector for transformation. The miR156a gene was amplied from the Arabidopsis genome as an XhoI-BamHI fragment and was similarly subcloned into ART7, and then into ART27. The T1 sp 35S:TLOG1 plants that formed TMTs exhibited slow growth rate, small apical meristems, reduced complexity of inorescences and leaves, and variable degrees of sterility. We chose the one with the best fertility and regularity of organs, hereafter designated as TLOG1, for all the experiments reported here. The pPIN1:PNI1-GFP reporter gene in the MM background, the generous gift of Cris Kuhlemeier, was used to generate 35S:TLOG1 plants expressing the PIN1 reporter. The pARR5:GUS entry clone was the gift of Joe Kieber. Confocal imaging was done with a Zeiss 510 LSM Meta using the water objective LCI Plan-Neouar 253/0.8 Imm. Korr. Excitation was set at 488 nm with 34% laser. For next-generation sequencing (NGS) analysis, axillary and apical meristems were manually dissected using a stereomicroscope and a ne surgical blade (K2-5000, Katena). All samples were harvested at 11:00 a.m., transferred immediately into liquid nitrogen, processed using the RNeasy Plant Mini Kit, and treated with the DNase Set (QIAGEN). RNA samples were processed at the Technion LS&E Infrastructure Unit for messenger RNA (mRNA) sequencing using both Illumina HiSeq 2000 and Illumina Genome Analyzer IIx sequencing platforms. The mRNA prep kit (Illumina), the standard cluster-generation kit (Illumina), and the sequencing kit (Illumina) were used for constructing libraries for single-read sequencing, cluster amplication, and nal sequencing by synthesis. Image analysis, base calling, and extraction of 50 bp reads were performed using the Illumina pipeline, and phi X 174 was used as a technical control. Reads from mRNA sequencing data were aligned to the International Tomato Annotation Group (ITAG) v.2.3 of the tomato genome (http:// solgenomics.net/organism/solanum_lycopersicum/genome) with the Burrows-Wheeler Alignment tool program, allowing an up to 1 mismatch per read. After mapping, raw gene counts (gene-expression levels) were counted by Cufinks v.1.3.0 and then normalized (also by Cufinks) to FPKM values (fragments per kilobase per million mapped fragments) using the following formula: FPKM = raw count/(transcript length 3 number of mapped reads in millions). The FPKM values were used directly for additional data analyses. Gene annotation and the corresponding Arabidopsis thaliana codes were obtained from the Sol Genomics ITAG 2.3 release. Hierarchical clustering was performed on all genes with TM4 software [18]. Bootstrapping values, presented at branch points in the dendograms, were generated by the support-tree option (1,000 iterations) and optimized

sample leaf order; the average linkage method was used. MDS analysis was performed with the edgeR package [19]. The two-dimensional plot depicts the distance between each pair of samples, where the distance was dened as the root-mean-square of the common dispersion for the number of chosen genes for the given analysis (5,000 genes of 20,255 for Figures 4A and 4B; 5,000 of 20,209 for Figure S2K; and 137 of 137 for Figures 4C and 4D). These genes were chosen according to the tagwise dispersion of all the samples by the program. The subclassication of gene families or tomato homologs of Arabidopsis genes and the generation of the heatmaps are described in the Supplemental Experimental Procedures. Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, ve gures, and seven tables and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2013.04.061. Acknowledgments We thank Z. Lippman, C. Lichtenstein, M. Tsiantis, O. Leyser, and B. Hurvitz for valuable comments on the manuscript. We also thank C. Kuhlemeier for pPIN1:PIN1-GFP seeds, I. Nepomniashchi and A. Feldman for technical help, the crews in the Technion LSE and TGC centers for NGS analysis, and M. Shmoish and members of the Technion BKU for their outstanding support with the bioinformatic analyses. This work was supported by Israel Science Foundation (ISF), Binational Agricultural Research and Development (BARD), and the Technion Russell Berrie Nanotechnology Institute grants to E.L. and ISF and BARD grants to Y.E. Received: January 30, 2013 Revised: March 20, 2013 Accepted: April 23, 2013 Published: June 6, 2013 References 1. Shewry, P.R. (2003). Tuber storage proteins. Ann. Bot. (Lond.) 91, 755769. 2. Ewing, E.E., and Struik, P.C. (1992). Tuber formation in potato: induction, initiation and growth. In Horticultural Reviwes, Volume 14 (Oxford: John Wiley & Sons), pp. 89197. 3. Gregory, L.E. (1956). Some factors for tuberization in the potato plant. Am. J. Bot. 43, 281288. , E., Cue llar, C.A., Tamaki, S., 4. Navarro, C., Abelenda, J.A., Cruz-Oro Silva, J., Shimamoto, K., and Prat, S. (2011). Control of owering and storage organ formation in potato by FLOWERING LOCUS T. Nature 478, 119122. 5. Hendriks, T., Vreugdenhil, D., and Stiekema, W.J. (1991). Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol. Biol. 17, 385394. 6. Perl, A., Aviv, D., Willmitzer, L., and Galun, E. (1991). In vitro tuberization in transgenic potatoes harboring b-glucuronidase linked to a patatin promoter: effects of sucrose levels and photoperiods. Plant Sci. 73, 8795. 7. Xu, X., Pan, S., Cheng, S., Zhang, B., Mu, D., Ni, P., Zhang, G., Yang, S., Li, R., Wang, J., et al.; Potato Genome Sequencing Consortium. (2011). Genome sequence and analysis of the tuber crop potato. Nature 475, 189195. 8. Tomato Genome Consortium. (2012). The tomato genome sequence provides insights into eshy fruit evolution. Nature 485, 635641. 9. Cline, M. (1997). Concepts and terminology of apical dominance. Am. J. Bot. 84, 10641069. 10. Sachs, T., and Thimann, K.V. (1967). The role of auxins and cytokinins in the release of buds from dominance. Am. J. Bot. 54, 136144. 11. Domagalska, M.A., and Leyser, O. (2011). Signal integration in the control of shoot branching. Nat. Rev. Mol. Cell Biol. 12, 211221. 12. Dun, E.A.A., de Saint Germain, A., Rameau, C., and Beveridge, C.A. (2012). Antagonistic action of strigolactone and cytokinin in bud outgrowth control. Plant Physiol. 158, 487498. 13. Kurakawa, T., Ueda, N., Maekawa, M., Kobayashi, K., Kojima, M., Nagato, Y., Sakakibara, H., and Kyozuka, J. (2007). Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445, 652655. 14. Traylen, W. (1904). Aerial Tubers on the Potato. Nature 69, 465.

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