Research. Roisin C. McGarry 1, Sarah F. Prewitt 1, Samantha Culpepper 1, Yuval Eshed 2, Eliezer Lifschitz 3 and Brian G. Ayre 1. Summary.

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1 Research Monopodial and sympodial branching architecture in cotton is differentially regulated by the Gossypium hirsutum SINGLE FLOWER TRUSS and SELF-PRUNING orthologs Roisin C. McGarry 1, Sarah F. Prewitt 1, Samantha Culpepper 1, Yuval Eshed 2, Eliezer Lifschitz 3 and Brian G. Ayre 1 1 Department of Biological Sciences, University of North Texas, 1155 Union Circle , Denton, TX , USA; 2 Plant Sciences, Weizmann Institute of Science, Rehovot , Israel; 3 Department of Biology, Technion Israel Institute of Technology, Haifa , Israel Author for correspondence: Brian G. Ayre Tel: bgayre@unt.edu Received: 16 March 2016 Accepted: 26 April 2016 doi: /nph Key words: architecture, branching pattern, development, domestication, flowering, phosphatidylethanolamine binding protein (PEBP), photoperiodism, signaling. Summary Domestication of upland cotton (Gossypium hirsutum) converted it from a lanky photoperiodic perennial to a day-neutral annual row-crop. Residual perennial traits, however, complicate irrigation and crop management, and more determinate architectures are desired. Cotton simultaneously maintains robust monopodial indeterminate shoots and sympodial determinate shoots. We questioned if and how the FLOWERING LOCUS T/SINGLE FLOWER TRUSS (SFT)-like and TERMINAL FLOWER1/SELF-PRUNING (SP)-like genes control the balance of monopodial and sympodial growth in a woody perennial with complex growth habit. Virus-based manipulation of GhSP and GhSFT expression enabled unprecedented functional analysis of cotton development. GhSP maintains growth in all apices; in its absence, both monopodial and sympodial branch systems terminate precociously. GhSFT encodes a florigenic signal stimulating rapid onset of sympodial branching and flowering in side shoots of wild photoperiodic and modern dayneutral accessions. High florigen concentrations did not alter monopodial apices, implying that once a cotton apex is SP-determined, it cannot be reset by florigen. GhSP is also essential to establish and maintain cambial activity. Dynamic changes in GhSFT and GhSP levels navigate meristems between monopodial and sympodial programs in a single plant. SFT and SP influenced cotton domestication and are ideal targets for further agricultural optimization. Introduction The success of modern agriculture is intimately tied to plant architecture: the shape of a plant, position of leaves and branches, and timing and distribution of reproductive structures are traits that affect crop productivity and management. The domestication of many crops resulted in more determinate architectures with compact growth habits, favorable and synchronous flowering times, and higher yields. Consequently, understanding the transition to flowering and determinate growth can benefit crop production. The positioning of vegetative and reproductive growth is well elucidated in Arabidopsis: TERMINAL FLOWER1 (AtTFL1) acts in the shoot apical meristem to maintain indeterminacy (Shannon & Meeks-Wagner, 1991; Alvarez et al., 1992; Conti & Bradley, 2007) whereas the closely related florigen, encoded by FLOWERING LOCUS T (AtFT), migrates to the meristem to activate the determinacy gene APETALA1 (Wigge et al., 2005; Corbesier et al., 2007). Arabidopsis, however, is monopodial, meaning that the axis of growth continues from a single point. Understanding the transition to reproductive growth in a sympodial system, where growth comprises a series of independent shoot initiations, requires further consideration. Research in tomato (Solanum lycopersicum) demonstrates that local balances of the FT ortholog SINGLE FLOWER TRUSS (SlSFT) to its antagonist, the TFL1 homolog SELF-PRUNING (SlSP), coordinates primary growth with regular sympodial cycles (Pnueli et al., 1998; Lifschitz et al., 2006, 2014; Shalit et al., 2009). In its simplest form, the SFT : SP balance model proposes that a high SFT : SP ratio in meristems promotes determinate growth and eventual termination of the apices whereas a low SFT : SP ratio promotes indeterminate growth. SP-like activities reside primarily in meristems whereas SFT-like activities may reside in the meristem but, importantly, are imported to the meristem from distal tissues in the form of systemic florigen. Florigen may be produced and imported in response to an environmental stimulus such as photoperiod (Chailakhyan & Krikorian, 1975) or in an age-related fashion, to control the switch from vegetative to reproductive growth, but also more broadly to alter the balance of indeterminate to determinate growth in all meristems. Speciesspecific variations in SFT and SP activity and transport are proposed to account for the diversity of observed growth patterns. It 244

2 New Phytologist Research 245 is clear that artificial selection of SFT and SP family members contributed to the domestication of many crops (Kojima et al., 2002; Bonnin et al., 2008; Kwak et al., 2008; Blackman et al., 2010; Pin et al., 2010; Tian et al., 2010; Danilevskaya et al., 2011; Comadran et al., 2012; Iwata et al., 2012; McGarry & Ayre, 2012b; Kloosterman et al., 2013; Mouhu et al., 2013; Ogiso-Tanaka et al., 2013; Rantanen et al., 2015). Upland cotton (Gossypium hirsutum) is the most important fiber crop with a worldwide annual impact estimated at $500 billion (Chen et al., 2007). Here we investigate if and how the levels of SFT and SP account for the balance of vegetative and reproductive growth in a woody perennial with complex growth habits. The main stem of cotton is monopodial and remains indeterminate throughout development, producing vegetative growth indefinitely (Fig. 1). In addition, cotton has two branching patterns: basal vegetative branches are monopodial and reiterate the main stem; distal fruiting branches are sympodial inflorescences with each sympodial unit consisting of a terminal boll-generating flower, a subtending leaf and an axillary bud that generates the next sympodial unit (Gore, 1935). Wild cotton species are mostly short-day photoperiodic, with monopodial vegetative branches forming as they are released from apical dominance and sympodial fruiting branches forming in the young axils first activated by short-day stimulation. During domestication, cotton underwent artificial selection for day-neutral flowering with fruiting branches initiating as early as the fifth node. Irrespective of flowering time, both ancestral and domesticated cotton maintain robust monopodial and asynchronous sympodial growth simultaneously. AtFT expression in cotton altered plant architecture and reduced indeterminate, perennial traits (McGarry & Ayre, 2012a). In photoperiodic cotton, AtFT expression uncoupled flowering from photoperiod, and in domesticated day-neutral lines, high florigen synchronized flowering and compressed plant growth habits (McGarry & Ayre, 2012a; McGarry et al., 2013). These data suggested that manipulating florigen could enhance characteristics desired for breeding and annual cultivation, such as a higher boll-to-canopy ratio, compact stature, and more synchronized flowering and fruit set (McGarry & Ayre, 2012a). However, not all aerial meristems were responsive: the main stem and basal branches of photoperiodic and day-neutral lines remained monopodial, suggesting further regulation beyond simple activation of the florigenic signal. In order to further understand plant architecture, the transition to flowering, and the roles of FT (SFT) and TFL1 (SP) homologs in governing these developmental events, we identified all members of the CETS (CENTRORADIALIS/TFL1/SP, which includes FT-SFT-like genes) gene family in upland cotton. We functionally characterize the GhSFT and GhSP orthologs in G. hirsutum and demonstrate their contribution to regulated monopodial and sympodial growth, and, importantly, show how changes to GhSP and GhSFT levels navigate meristems between monopodial and sympodial programs in a single plant. Consistent with a florigenic activity, GhSFT, on the one hand, promotes determinate sympodial growth and flowering in side shoots of both photoperiodic and domesticated accessions, but does not (a) (e) 5 5 (d) (b) (f) (c) ~20 Fig. 1 Growth habits in modern and ancestral cotton (Gossypium hirsutum). (a c) Ball and stick diagrams illustrating growth habits in domesticated and wild cotton. Triangles indicate the axis of growth from meristems; monopodial indeterminate meristems are colored blue and sympodial meristems are red; balls represent determinate floral buds. The node of the main stem producing the first reproductive branch is indicated numerically. (a, d) Domesticated variety Delta Pine 61 (DP61) has a short, bushy stature with broad leaves, and produces many flowers and fruits. Reproductive branches initiate at approximately node 5 of the main stem. (b, e) Ancestral short-day photoperiodic accession Texas 701 (TX701). When grown in noninductive long days, TX701 produces vegetative growth exclusively. (c, f) TX701 grown under inductive short days transitions to reproductive growth at approximately node 20 while maintaining robust vegetative growth. Fruiting branches arise from older vegetative branches, as illustrated in (c). Plants in (d, e) are c. 11 wk post germination, and (f) is c. 20 wk post germination. In (d, f), arrows point to a small number of the many reproductive structures; bars, 10 cm.

3 246 Research New Phytologist influence established monopodial apices. GhSP, on the other hand, profoundly impacts all shoot meristems, including cambia, and in its absence, all apical and axillary meristems convert precociously to determinate flowers. Our data demonstrate that these gene products control cotton branching patterns and suggest that they may have been targets for artificial selection during domestication from wild short-day perennials to compact day-neutral cotton with annual characteristics. Furthermore, manipulating these two gene products impacts cotton architecture in ways that may benefit commercial field production. Materials and Methods Phylogenetic analysis Details are provided in Supporting Information Methods S1. Plasmid constructions All plasmid constructions were by standard molecular biology procedures (Sambrook et al., 1989). Oligonucleotides (Table S1) and cloning details are included in Methods S2. Virus inoculations Binary vectors were introduced to Agrobacterium tumefasciens strain GV3101 pmp90 by electroporation. Single colonies were used to inoculate cultures as described (Burch-Smith et al., 2006). TRV and dclcrv are bipartite viruses, and equal volumes of inoculum harboring each component of each virus were mixed, and infiltrated into cotyledons of Gossypium hirsutum (L) accessions TX701 and DP61 at 4 d post germination (dpg) using a 1- ml syringe. In a typical experiment, six seedlings of each accession were inoculated with each virus. Inoculated seedlings were covered overnight at room temperature, and maintained in a growth chamber at 25 C under short (10 h : 14 h) or long days (16 h : 8 h) for 3 wk to favor virus accumulation. Plants were then transplanted into 15-l pots with Fafard 52 or Metro-Mix 852 growth mix in the glasshouse (30 C) for long-day conditions (16 h : 8 h, light : dark) with supplemental light provided by a combination of metal halide and mercury lamps (up to 1300 lmol photons m 2 s 1 light intensity at leaf level), or in a growth room (30 C) for short-day conditions (10 h : 14 h, light : dark) with metal halide, mercury and T5 fluorescent lighting (200 lmol photons m 2 s 1 light intensity at leaf level). Each virus was tested in a minimum of two independent glasshouse trials. Gene expression In order to compare TRV and dclcrv, gene expression was analyzed by reverse transcription (RT) followed by PCR. Sink leaves of mature plants inoculated with TRV:MgChl, dclcrv: amgchl and controls were harvested into liquid nitrogen and homogenized with a 2010 Geno-Grinder (Spex SamplePrep, Metuchen, NJ, USA). Total RNA was isolated (Methods S2) and treated with DNaseI (Zymo Research, Irvine, CA, USA, and Mo Bio Products, Carlsbad, CA, USA). RNA was quantified by nanodrop and 1 lg of total RNA from each sample was reverse transcribed using Superscript III (Invitrogen) and random primers (New England Biolabs, Ipswich, MA, USA). Each cdna was tested in separate 10 ll PCR reactions using Phire DNA Polymerase (New England Biolabs) with GAPDH, Rep and TRV CP primer sets (Table S1). The GhSFT and GhSP manipulation experiments were analyzed quantitatively by RT and quantitative (q)pcr. The apex of the monopodial main stem (c. 0.5 cm) of inoculated and uninoculated seedlings was harvested into acetone at 15 d post inoculation (dpi) and homogenized with a Mixer Mill MM400 (Retsch, Newtown, PA, USA). Plants lacking apices were transferred to the glasshouse for phenotype confirmation. RNA isolation, DNAse treatment and quantification were as described. Two micrograms of total RNA was used in RT with Superscript IV (Invitrogen) and random primers (New England Biolabs). Detailed information of primers (Table S1) are included in the Methods S3. Ten-microliter qpcr reactions used SYBR-Green (Sigma) on an Viia TM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a fast cycle (initial denaturation at 95 C for 30 s, followed by 40 cycles at 95 C for 5 s, 63 C for 15 s and 72 C for 10 s) and melt curve analysis (95 C for 15 s, 60 C for 1 min and 95 C for 15 s). Three biological samples and two technical replicates were included for each virus in each cotton accession. Data were analyzed by the DDCt method using GhpolyUBQ as the reference and variation is expressed as the SE of the mean. Two micrograms of RNA, isolated from the apex of the monopodial main stem (c. 0.5 cm) of inoculated and uninoculated seedlings at 15 dpi, from three biological replicates per treatment per accession were used to prepare Illumina TruSeq stranded mrna libraries (Illumina, San Diego, CA, USA) according to the manufacturer s protocols. Additional details are provided in the Methods S4. Spatial expression of GhSFT and GhSP in TX701 and DP61 grown in long and short days was determined quantitatively by RT-qPCR. Further details are provided in the Methods S3. Photosynthesis Photosynthesis measurements were recorded on domesticated G. hirsutum accession Coker 312. Plants were inoculated with TRV and TRV:GhSP; each virus was inoculated on three plants and there were four uninoculated controls. Each week, the terminal leaf (node 5) of TRV:GhSP plants and the corresponding leaf of TRV-infected and uninoculated plants were analyzed by infrared gas exchange using a LiCor LI-6400XT. Gas exchange measurements were determined with 400 lmol s 1 of CO 2, a gas flow rate of 500 mmol s 1 and photosynthetically active radiation at 500 lmol m 1 s 1 to reasonably mimic environmental conditions used during growth. Chamber block temperature and relative humidity were established by ambient air conditions, and were 30 C and c. 60%, respectively. Plants were allowed to acclimate for 120 s in the chamber before taking measurements. The acclimatization time was determined empirically. Two measurements were taken per plant at 30 s intervals.

4 New Phytologist Research 247 Microscopy Free-hand sections (c. 1 mm thick) of the main stem at nodes 3 5 were obtained from mature plants ( dpi) and visualized with an SMZ1500 stereomicroscope (Nikon, Melville, NY, USA). Images were captured using the natural autofluorescence of lignin polymers in the plant cell walls with UV epifluorescence ( nm excitation, nm emission) on an Eclipse E600 compound microscope (Nikon) equipped with a SPOT Insight 2 CCD camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA) and an X-cite 120 Fluor System (Exfo Life Sciences Division, Quebec, Canada). Photography Pictures were taken with a Canon SureShot A360. Results Gossypium hirsutum accessions demonstrate diverse architectures We selected two architecturally diverse G. hirsutum accessions: domesticated, day-neutral DeltaPine 61 (DP61) and wild, photoperiodic Texas701 (TX701). DP61 has a short, bushy growth habit, produces sympodial fruiting branches by approximately node 5 of the main stem, and the leaves from the main stem, vegetative and sympodial branches have three shallow lobes (Fig. 1a, d; McGarry & Ayre, 2012a). By contrast, TX701 is tall with pronounced apical dominance and has short vegetative branches. Under noninductive long days, TX701 remains vegetative, and leaves from the main stem and vegetative branches have five deep lobes (Fig. 1b,e). By contrast, when grown under inductive short days, TX701 produces sympodial fruiting branches by approximately node 20 of the main stem (Fig. 1c,f), and the subtending leaves of sympodial units become progressively more lanceolate in shape (Fig. 1f). Both domesticated and wild accessions demonstrate continued monopodial growth from the main stem and vegetative branches following the transition to flowering, and sympodial units within fruiting branches may reiterate indefinitely. Identifying and cloning the Gossypium CETS gene family FT and TFL1 of Arabidopsis, and SFT and SP in tomato, belong to the CENTRORADIALIS/TERMINAL FLOWER 1/SELF- PRUNING (CETS) gene family, and as CETS proteins harbor a predicted phosphatidylethanolamine binding domain, they are also referred to as phosphatidylethanolamine binding proteins (PEBP). To identify the cotton CETS family members, the Arabidopsis FT polypeptide sequence was queried against the A 2 genome of diploid G. arboreum (Li et al., 2014), the D 5 genome of diploid G. raimondii (Wang et al., 2012) and the AD 1 tetraploid genome of G. hirsutum (Zhang et al., 2015) using tblastn. Eight loci were identified in each diploid genome and 16 were identified in tetraploid G. hirsutum, with eight in each A t and D t subgenomes corresponding to the eight in the extant diploids (Table S2). The predicted polypeptide sequences were analyzed phylogenetically with CETS proteins from Arabidopsis and tomato, and anchored with moss (Physcomitrella patens) (Methods S1; Figs S1, S2; from accessions included in Tables S2, S3). Consistent with relationships in other species, cotton CETS assort into three distinct clades: MOTHER OF FT AND TFL1- LIKE (MFT-L), FLOWERING LOCUS T-LIKE (FT-L) and TERMINAL FLOWER 1-LIKE (TFL1-L). The A 2 and D 5 diploid genomes, and both the A t and D t subgenomes of the AD 1 tetraploid, have two MFT-like genes, MFT-L1 and MFT- L2. Each has a single FT ortholog, referred to here as SFT as it is more closely related to tomato SFT than the Arabidopsis FT. Within the TFL1-like clade, two genes, TFL1-L1 and TFL1-L2 share homology with AtTFL1; two share homology with the Arabidopsis BROTHER OF FT AND TFL1 (AtBFT), BFT-L1 and BFT-L2; and one, SP, is orthologous to SlSP and ARABIDOPSIS THALIANA CENTRORADIALIS (ATC). An alternate assembly of G. hirsutum suggests duplication of an SP locus and a BFT-L1 locus, and more rearrangement of the genes among the A t and D t subgenomes (Li et al., 2015) (Table S2). GhSFT and GhSP are expressed in cottons with different shoot architectures In order to test if GhSP and GhSFT expression correlates with different cotton architectures, we examined the spatial profile of these two gene products in DP61 and TX701 plants grown under noninductive long days (16 h : 8 h, light : dark) and inductive short days (10 h : 14 h, light dark). Using RT-qPCR and RNA-seq to quantify transcript levels, we show that, in general, neither gene is strongly expressed throughout the shoot of mature plants (Fig. 2) nor in the apices of young plants (see uninoculated DP61 and TX701 in Table S4). GhSFT is expressed in all tissues tested in domesticated DP61 and photoperiodic TX701 grown under longday or short-day conditions (Fig. 2a,b), and this pattern is consistent with the expression of AtFT in Arabidopsis (Kardailsky et al., 1999; Kobayashi et al., 1999). In some aerial tissues, more GhSFT transcript is detected under short days than long days in both accessions, suggesting that it may experience photoperiodic regulation (Fig. 2a,b). GhSP is also expressed in all tissues tested in DP61 and TX701, but its expression is not affected by long vs short days (Fig. 2c,d). GhSFT expression spikes whereas GhSP expression is low in mature leaves of TX701 grown under inductive short days (Fig. 2b,d), consistent with mature leaves becoming more lanceolate in response to the short-day signal. This contrasts with GhSP expression exceeding that of GhSFT in mature TX701 leaves under noninductive long days (Fig. 2b,d). Mature TX701 leaves are deeply lobed, and these findings suggest that GhSP may be required to maintain this indeterminate leaf phenotype under long days. Importantly, we note that GhSP spikes in the monopodial main stem apex of TX701 grown under inductive short days, presumably to counteract increased GhSFT expression (Fig. 2b,d). Together, these findings support the notion that GhSP is present to counter-balance the GhSFT signal, especially in the monopodial main stem where wild-type plants strictly maintain indeterminacy.

5 248 Research New Phytologist (a) LD SD (b) GhSFT : UBQ GhSFT : UBQ (c) GhSP : UBQ Source leaf LD SD Sink leaf Floral bud Mono. main stem apex Veg. branch apex Fruit. branch apex (d) GhSP : UBQ Source leaf Sink leaf Mono. main stem apex Fig. 2 Relative expression of GhSFT and GhSP compared with GhpolyUBQ (UBQ) in the shoots of Gossypium hirsutum DP61 and TX701 plants grown under long (LD) and short (SD) days. Relative expression of GhSFT in (a) DP61 and (b) TX701 plants. Relative expression of GhSP in (c) DP61 and (d) TX701 plants. Expression was examined in mature source leaves, newly emerged sink leaves, floral buds, and the apices (< 1 cm) of monopodial main stems, vegetative branches and sympodial fruiting branches. TX701 plants grown under long days do not produce floral buds or sympodial fruiting branches and are not included in (b) or (d). Variation is expressed as the SE of the mean. Comparing expression tools for functional gene analysis in G. hirsutum Virus-based expression tools allow functional gene analyses in many cotton backgrounds whereas transgenic technologies are restricted to one (Coker 312) (Wallace et al., 2009). To date, two virus-based systems are reported: the disarmed geminivirus Cotton leaf crumple virus (dclcrv) is used for virus-induced flowering (McGarry & Ayre, 2012a), and dclcrv and tobravirus Tobacco rattle virus (TRV) are used for virus-induced gene silencing (VIGS) (Tuttle et al., 2008, 2012; Gao et al., 2011; Qu et al., 2012). No report directly compares the efficiency of each system for manipulating cotton gene expression. In order to determine the utility of each virus-based system, we compared the strength and duration of dclcrv and TRV in silencing identical gene sequences. The magnesium chelatase subunit (MgChl) was cloned into dclcrv in the antisense orientation and into TRV in the sense orientation, and delivered with appropriate controls to TX701 and DP61 seedlings. Plants inoculated with dclcrv:amgchl (a, antisense) produced chlorotic patches evident c. 12 dpi (Fig. 3a,b,e,f). This spotty silencing was observed in all inoculated plants (n = 6) and continued systemically in all new growth for the duration of the experiment (Fig. 3i). Thus, dclcrv provides partial but sustained silencing. All plants inoculated with TRV:MgChl (n = 6) demonstrated extensive chlorosis by 6 dpi which continued uniformly. Once systemic silencing was established, green sectors were not observed in the new growth (Fig. 3c,d,g,h). This silencing was sustained for one month and the lack of photoautotrophic tissue severely hampered growth. After this time, some plants developed branches that were fully green and resumed normal growth (Fig. 3j). This suggests that systemic silencing by TRV in cotton is highly potent because new growth was completely silenced, but is also somewhat unstable because escaping sectors could emerge. Consistent with the escaping/recovering sectors losing functional virus, we were unable to detect the viral gene encoding the TRV coat protein in new green tissues (Fig. 3k). Based upon these findings, we focused our experiments with TRV-mediated VIGS of GhSP and GhSFT. For further comparisons of the effectiveness of dclcrv and TRV for VIGS, dclcrv-mediated VIGS of GhSP and GhSFT are included in Figs S3 and S4. GhSP is required for monopodial and sympodial branching In order to test if GhSP promotes indeterminate growth in cotton, we cloned the GhSP coding sequence in TRV to silence its expression in TX701 and DP61 seedlings. All TX701 seedlings inoculated with TRV:GhSP flowered under noninductive long days (16 h : 8 h), with floral buds evident by 27 dpi, and, remarkably, the main stem terminating with a floral bud by node five (Fig. 4b). That is, the monopodial apex which normally remains indeterminate in even the earliest domesticated varieties was converted to a terminal flower. Furthermore, all axillary meristems, including those subtended by the cotyledons,

6 New Phytologist Research 249 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) uninoc dclcrv dclcrv: αmgchl TRV TRV: MgChl + _ GAPDH TRV CP dclcrv Rep Fig. 3 Comparing the efficiency of gene silencing by Cotton leaf crumple virus (dclcrv) and Tobacco rattle virus (TRV) in domesticated and wild Gossypium hirsutum.(a d) Infections in DP61, and (e j) infections in TX701. (a, e) dclcrv infected controls; (b, f) dclcrv:amgchl-infected plants show patchy chlorosis on the first and second true leaves, indicating the silencing of the MgChl endogene. (c, g) TRV-infected controls; (d, h) TRV:MgChl-infected plants show complete bleaching of the first and second true leaves, indicating systemic silencing. (i) TX701 infected with dclcrv:amgchl shows sustained silencing of the MgChl endogene, indicated by the spotty chlorosis on a slightly stunted plant. (j) Silenced sector in TX701 infected with TRV:MgChl recovered and resumed normal growth. Bars, 10 cm. (k) Viral gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) from systemic leaves of infected TX701 plants c. 11 wk post inoculation. Consistent with the loss of silencing, expression of the TRV coat protein (TRV CP) could not be detected c. 11 wk post inoculation, or in sectors of TRV:MgChl-infected plants that resumed normal growth, but this sequence was amplified from silenced leaves of a 10 d post inoculation seedling ( + ). By contrast, the persistent spotty silencing observed in dclcrv:amgchl-infected TX701 correlates with expression of the dclcrv Rep gene. Detection of GhGAPDH serves as an internal control, and the no template control is indicated ( ). produced determinate floral buds directly on the main stem. Because all meristems converted to a determinate floral fate, further vegetative growth did not occur in most GhSP-silenced plants once they reached node 5 (Fig. 4b, d f, i). (As with the breakthrough sectors reported in Fig. 3(j,k), some TRV:GhSPinfected plants eventually developed branches displaying normal growth, presumably due to loss of the silencing signal.) These highly-determinate phenotypes were also observed in TRV: GhSP-infected TX701 plants grown under inductive short days (10 h/14 h) and in TRV:GhSP-infected day-neutral DP61 grown under non-inductive long days (16 h/8 h; Fig. 5). As shown in Figs 4(f) and 5(g), the main stem terminal flower (i.e. the converted monopodial apex) reached anthesis first and floral maturation progressed basipetally, following the pattern of buds being released from apical dominance. These flowers produced mature bolls harboring fiber and seeds. Thus, silencing GhSP achieved more synchronous flowering, and these phenotypes were not observed among controls (compare Figs 4a,c, 5a,c with 4b,d, 5b,

7 250 Research New Phytologist (a) (b) (c) (d) (g) (e) (h) Relative expression (log 2 fold-change) SFT uninoc SP CLCrV: GhSFT (f) TRV: CLCrV:GhSFT + GhSP TRV:GhSP (i) Fig. 4 Silencing GhSP in Gossypium hirsutum accession TX701 demonstrates that GhSP is required for monopodial and sympodial growth. (a) Uninoculated TX701. (b) TRV: GhSP infection terminates growth by node 5 with a terminal flower, and all axillary meristems subsequently produce floral buds directly on the main stem. Arrows point to flowers developing in a basipetal pattern; the cluster at the top consists of a terminal flower and two axillary flowers. (c) TRV. (d) dclcrv:ghsft overexpression combined with TRV:GhSP silencing terminates growth with the formation of a terminal flower and vegetative growth is reduced compared with the TRV:GhSP infection alone. The cluster of a terminal flower and two axillary flowers is clearly visible. Plants in (a d) are the same age. (e) Floral buds arise in the axils of the cotyledons in the co-infected TX701 in noninductive long days; (f) the terminal flower at anthesis; (g) terminal flowers yielded mature bolls. (h) The observed changes to TX701 architecture correlate with changes in GhSFT and GhSP expression. Expression is measured relative to ubiquitin. Variation is expressed as the SE of the mean. (i) Ball and stick diagram illustrating determinate growth from TRV:GhSPinfected plants: balls represent determinate floral buds; see Fig. 1 for representation of uninfected plants. Bars, 10 cm. d,g) or when GhSFT was overexpressed (see later). RT-qPCR demonstrated that GhSP expression was reduced in the TRV: GhSP lines compared with other treatments (Figs 4h, 5e), and RNA-seq analysis indicated that TRV:GhSP treatment specifically targeted GhSP expression without off-target silencing of other CETS genes (Table S4). (We note MFT-L2 expression was reduced in infected compared with uninoculated DP61, but as this was not observed in TX701 and is inconsistent with the phenotypes reproduced in both accessions, it is likely an indirect effect.) These results show that GhSP is needed to maintain indeterminate growth in both sympodial and monopodial branch systems. The leaves subtending the terminal flowers in both TX701 and DP61 were unusually large and lanceolate rather than lobed (Figs 4f, 5b). All leaves on TRV:GhSP-silenced plants were larger, and developed and retained dark green color whereas leaves of the same age on control plants started to senesce. We hypothesized that because these were the only leaves to provide photoassimilate to maturing bolls, they were compensating for higher sink demands by enhancing photosynthesis. We measured photosynthesis by infrared gas exchange from the fifth leaf of TRV:GhSP-silenced, TRV-infected and uninoculated controls. Because productivity is fundamental to cotton cultivation, we conducted GhSP-silencing in G. hirsutum accession Coker 312, the domesticated and day-neutral variety which is the genetic standard from which cultivated transgenic cotton derives. TRV: GhSP-silenced Coker 312 plants showed identical phenotypes to GhSP-silenced DP61 and TX701, confirming the same effect across a diversity of lines. The dark green leaves of TRV:GhSPsilenced plants conducted more photosynthesis and were productive longer than the corresponding leaves from controls (Fig. 6; Table S5). This is likely a physiological response of the limited and fixed number of source leaves to the extreme sink demand of the maturing bolls following manipulation of SFT and SP levels. GhSFT plays a conserved role in regulating flowering in photoperiodic and day-neutral G. hirsutum In order to test whether GhSFT promotes flowering and determinate growth in day-neutral cotton, the GhSFT coding sequence (nts 1 388) was cloned in TRV for silencing in DP61 seedlings. All TRV:GhSFT-infected DP61 plants flowered significantly

8 New Phytologist Research 251 (a) (b) (c) Fig. 5 GhSP regulates monopodial and sympodial growth in domesticated Gossypium hirstum accession DP61. (a) Uninoculated DP61; (b) growth terminates with a floral bud by node 5 in TRV:GhSPsilenced DP61 and axillary meristems produce floral buds directly off the main stem. Leaves subtending the main stem flowers are lanceolate. Arrows point to flowers developing in a basipetal pattern; the cluster at the top consists of a terminal flower and two axillary flowers. (c) TRV-infected DP61; (d) co-infection of dclcrv:ghsft and TRV:GhSP terminates growth similar to TRV: GhSP infection. Plants in (a d) are the same age. (e) The changes in DP61 architecture correlate with changes in GhSFT and GhSP expression. Expression is measured relative to ubiquitin. Variation is expressed as the SE of the mean. (f) Floral buds arise in the axils of the cotyledons of the co-infected plant. (g) Flowering progresses basipetally in coinfected DP61. The terminus of the main stem reached anthesis first: the pink petals indicated the flower bloomed and closed 24 h before. The white flower at the lower node is opening and the next floral bud can be observed at the next lower node. (h) Flowers yield mature bolls. (i) Ball and stick diagram illustrating determinate growth from TRV:GhSP-infected plants; balls represent determinate floral buds; see Fig. 1 for representation of uninfected plants. Bars, 10 cm. (d) (f) (g) (h) (e) Relative expression (log 2 fold-change) uninoc CLCrV: GhSFT SFT TRV: GhSFT SP TRV: CLCrV:GhSFT+ GhSP TRV:GhSP (i) Net assimilation rate (μmol CO 2 m 2 s 1 ) Uninoculated TRV TRV:GhSP Days post inoculation Fig. 6 TRV:GhSP-silenced plants demonstrate greater photosynthetic productivity per unit area. TRV:GhSP-silenced plants displayed large, deep green leaves and stayed green longer than controls. Photosynthesis (net CO 2 assimilation) was measured weekly in mature leaves at the 5 th node of uninfected (n = 4), TRV- (empty vector; n = 3), and TRV:GhSP-infected (n = 3) Gossypium hirsutum plants until leaves of control plants senesced. Average values from biological replicates are shown here, and means with standard deviations are included in Supporting Information Table S5. later than controls (Fig. 7a,c,d,e; see also Fig. S4 for silencing with dclcrv:aghsft) and demonstrated more vegetative growth than controls. Further evidence of prolonged growth was seen in larger main stem leaves and elongated petioles (Fig. S5). Correlating with these phenotypes, GhSFT expression in the TRV:GhSFT lines was reduced compared with uninoculated plants (Fig. 5e). These indeterminate characteristics obtained by reducing GhSFT function emphasize that GhSFT retains a role in promoting determinate growth in domesticated, day-neutral cotton. In order to test if GhSFT has a florigenic function, the GhSFT coding sequence was cloned into dclcrv for gain-of-function analysis. This construct and the virus control were delivered to TX701 and DP61, and all plants were grown under 16 h long days. All photoperiodic TX701 plants inoculated with dclcrv: GhSFT flowered under noninductive long days, with short axillary floral branches developing as early as the fifth node (Fig. 8a, b). The dclcrv:ghsft-infected plants exhibited prolific flowering with internode length and subtending leaves reduced, and nearly all sympodial branches terminated with floral clusters rather than continued sympodial reiterations (Fig. 8c,d). Importantly, and in contrast to the GhSP-silenced plants, the main stem remained monopodial and indeterminate (Fig. 8a,d). Floral buds, including the earliest induced, were morphologically normal and yielded mature bolls (Fig. 8e). These phenotypes correlated with elevated GhSFT expression compared with uninoculated controls (Fig. 4h). None of the uninoculated or dclcrv-infected TX701 flowered under long days.

9 252 Research New Phytologist (a) (b) (c) (d) 11 (e) 12 Node of first fruiting branch a a b CLCrV uninoc CLCrV GhSFT Q139D b TRV c TRV GhSFT Fig. 7 Silencing GhSFT influences flowering time in domesticated day-neutral DP61. (a) Uninoculated Gossypium hirsutum accession DP61 has many flowers (arrows). (b) DP61 infected with dominant negative dclcrv: GhSFTQ139D had fewer floral buds. (c) Flowering is limited in DP61 infected with TRV:GhSFT. (d) Ball and stick diagram illustrating the delayed onset of reproductive growth. Triangles indicate the axis of growth from meristems; monopodial indeterminate meristems are colored blue and sympodial meristems are red; balls represent determinate floral buds. The node of the main stem producing the first reproductive branch is indicated numerically. See Fig. 1 for representation of uninfected plants. (e) The time to flower is quantified by the node of first fruiting branch. Error bars indicate the standard deviation among replicates (n = 6). Significant differences among treatments, indicated with lowercase letters, were determined using the mean separation test by Tukey HSD at P < The day-neutral DP61 plants were expected to flower irrespective of day length. Overexpression of GhSFT with dclcrv:ghsft in DP61 resulted in more determinate, compact plants, with characteristics similar to those observed in TX701. The transition to flowering was earlier, at node 4 of the main stem, leaves were smaller, internodes were shorter and stems were thinner (Fig. S4), and RT-qPCR results correlated these phenotypes with greater GhSFT expression compared with uninoculated controls (Fig 5e). To more subtly perturb SFT function, we generated a dominant-negative allele expected to interfere with florigenic GhSFT. The dominantnegative construct dclcrv:ghsftq139d is akin to the AtFTQ140D mutation shown to convert AtFT to an AtTFL1- like function without adversely affecting ligand binding (Ho & Weigel, 2014). We anticipated that this adjustment should impact determinate growth patterns, but less severely than observed with the VIGS because expression and function of the endogenous GhSFT gene products were not directly manipulated. As expected, DP61 plants infected with dclcrv: GhSFTQ139D had fewer floral buds (Fig. 7a,b) and the node of first fruiting branch was significantly delayed compared with controls (Fig. 7e). Taken together, these findings demonstrate that GhSFT encodes a florigenic signal, and this is not limited to photoperiod induction in wild accessions: its function is retained and important to the early flowering and compact growth habit desired in day-neutral domesticated lines. We predicted that combined overexpression of GhSFT and silencing of GhSP would synergistically impact determinate growth, as reported in tomato and apple (Lifschitz et al., 2006; Yamagishi et al., 2014). Co-infections reflected anticipated changes in GhSFT and GhSP expression (Figs 4h, 5e). The main stem and all axillary meristems of co-infected plants terminated with floral buds directly from the main stem, and the terminal flower was evident by 26 dpi. Co-infected plants had less vegetative growth than TRV:GhSP plants (Fig. 4d,f). Notwithstanding, the determinate co-infected plants yielded mature bolls (Figs 4g, 5h). GhSFT and GhSP influence leaf marginal and cambial meristems Besides flowering and shoot termination, changes in GhSFT and GhSP expression affected leaf development and stem expansion. Overexpression of AtFT or inductive short days induced flowering in TX701, and the leaves of reproductive branches transitioned from five deep lobes to lanceolate (McGarry & Ayre, 2012a). Consistent with GhSFT functioning as the florigenic stimulus, viral delivery of GhSFT to TX701 in noninductive long days produced lanceolate leaves in reproductive branches whereas leaves from uninoculated or dclcrv-infected TX701 plants remained deeply lobed (Fig. 9). Lanceolate leaves were similarly observed when GhSP was silenced in DP61 (Fig. 5b) and on

10 New Phytologist Research 253 (a) (b) 5 (c) (d) (e) Fig. 8 GhSFT regulates flowering in photoperiodic Gossypium hirsutum accession TX701. (a) Overexpression of GhSFT from dclcrv in short-day photoperiodic TX701 uncouples flowering from photoperiod and results in precocious flowering. From left to right: an uninoculated TX701 plant, dclcrvand dclcrv:ghsft-infected TX701. Arrows point to floral buds. (b) Ball and stick diagram illustrating a sympodial branch terminating with a cluster of flowers and another sympodial branch dense with floral buds, but the main stem remains monopodial; triangles and balls as described in Fig 1. (c) Overexpression of GhSFT from dclcrv yielded clusters of floral buds on reproductive branches, terminating sympodial growth; the cluster consists of a terminal sympodial unit consisting of only a flower, and two sympodial units with a subtending leaf and extremely short internode. (d) The apex of dclcrv-ghsft infected TX701 grown under noninductive long days. Although the monopodial main stem is still indeterminate, fruiting branches were dense with sympodial units. (e) The GhSFT-induced floral buds were productive, and precocious floral buds yielded mature bolls. sympodial fruiting branches of TX701 infected with dclcrv: aghsp (Figs 9, S3) which, because of the weaker silencing, produced fruiting branches from the main stem axils rather than the main stem flowers that formed with TRV:GhSP. These changes in leaf morphology correlate with GhSFT and GhSP expression in mature TX701 leaves (Fig. 2b,d), and show that shifting SP and SFT levels influences the activity of leaf marginal meristems. SFT and SP also regulate stem growth. The main stems of mature TX701 plants overexpressing GhSFT from dclcrv: GhSFT were narrow compared with controls (Fig. 10a), but this was not observed in mature DP61 (Fig. 10b). Silencing GhSP in TRV:GhSP-infected TX701 and DP61 plants also restricted stem expansion. GhSP silencing in TRV:GhSP-infected plants showed significant disruption in the pattern and maturation of (a) (b) (c) (d) Uninoculated dclcrv dclcrv:ghsft dclcrv:αghsp Fig. 9 Overexpressing GhSFT and silencing GhSP with dclcrv promotes the transition to determinate, lanceolate leaves in photoperiodic Gossypium hirsutum accession TX701. Leaves from the main stem and vegetative branches of (a) uninoculated and (b) dclcrv-infected TX701 grown under noninductive long days remain deeply lobed. By contrast, the subtending leaves of sympodial fruiting branches are very small and lanceolate in dclcrv: GhSFT-infected TX701 (c), whereas this progression is more gradual along a sympodial branch from dclcrv:aghsp-infected TX701 plants (d).

11 254 Research New Phytologist secondary growth in stems compared with controls of the same age (Fig. 10a d). Rather than the stem pattern of a typical woody perennial, the stems of GhSP-silenced plants had characteristics more typical of an herbaceous annual. Cortex was substantially expanded whereas layers of phloem fibers were reduced to a single layer; patterns of secondary xylem that make wood failed to develop and lignification of cell walls was observed only in dispersed short files consisting of no more than c. 10 cells. Collectively, these findings demonstrate that GhSFT restricts growth in all primary and secondary shoot meristems, and that GhSP is required to limit the role of GhSFT and maintain indeterminate vegetative growth at all shoot meristems. Although overexpressing SlSFT restricts normal radial expansion in tomato stems (Shalit et al., 2009), these findings from a herbaceous plant could not predict the impact of altered SP and SFT function observed in a woody perennial. Discussion Cotton is widely cultivated as an annual commodity crop, but this management strategy is at odds with its perennial and indeterminate growth habit. Because cotton flowering and fruit set are asynchronous, producers tend to extend the growing season to maximize yield. This compromises the cleanliness and quality of early-forming bolls, and harvesting late-forming bolls, which have inferior fiber characteristics, can reduce bale value. Furthermore, because vegetative growth continues after the transition to reproductive growth, resources are diverted from fiber and seed production (Oosterhuis, 1990). To control cotton s perennial growth habit, growth inhibitors are used during the growing season and defoliants are used in preparation for mechanical harvest (Jost et al., 2006; Cothren & Oosterhuis, 2010). These treatments further increase producer costs and have negative environmental consequences. Thus, cotton agriculture would benefit from a more annualized and determinate plant architecture. Based on research in tomato, it was proposed that the balance of determinate and indeterminate growth in all plants is controlled by the balance of the activities of the SINGLE FLOWER TRUSS (SFT ) and SELF-PRUNING (SP ) gene products (Pnueli et al., 1998; Lifschitz et al., 2006, 2014; Shalit et al., 2009). Support for this hypothesis can be found in the domestication of diverse crop plants, where, in many instances, the selection for determinate growth habits resulted from mutations in FT and TFL1 homologs (Danilevskaya et al., 2010; Tian et al., 2010; Blackman et al., 2011a; Comadran et al., 2012; Iwata et al., 2012; Koskela et al., 2012). The first recognition of the SFT : SP (a) i ii iii iv v (b) 5 Node 4 3 (c) (d) X P C X P C Fig. 10 GhSFT and GhSP affect stem radial expansion. (a) Cross-sections of nodes 3, 4 and 5 of mature Gossypium hirsutum TX701 plants (c. 94 d post inoculation, dpi) infected with different viruses. From left to right: (i) uninoculated; (ii) TRV; (iii) dclcrv:ghsft + TRV:GhSP; (iv) dclcrv:ghsft; and (v) dclcrv infected plants. Stem diameters from dclcrv:ghsft-infected plants are more narrow than uninoculated, dclcrv- and TRV-infected control plants. Stems from TX701 infected with dclcrv:ghsft + TRV:GhSP are narrow and appear herbaceous. (b) Cross-sections of mature (c. 110 dpi) DP61stems (node 5). Clockwise from top left: (1) uninoculated; (2) dclcrv:ghsft; (3) TRV:GhSP; and (4) TRV:GhSFT. The stem diameter of TRV:GhSPinfected DP61 is reduced and the stem tissues appear herbaceous in contrast to the other sections which show woody tissues. (c) The same stem crosssection from node 5 of uninoculated DP61 shown in (b). Multiple layers of phloem fiber bundles are organized between the xylem and periderm. (d) The same stem cross-section from node 5 of TRV:GhSP-infected DP61 shown in (b). Phloem fiber bundles are reduced to a single layer whereas the cortex between the fibers and periderm is expanded; files of xylem are small, disorganized, and not abundant; X, xylem; P, phloem fiber bundles; C, cortex. Bars: (a) 1 cm; (b) 1 mm; (c, d) 500 lm.

12 New Phytologist Research 255 ratio to impact plant architecture and benefit crop production is from identifying the gene responsible for the self-pruning (sp) phenotype of tomato (Yeager, 1927; Pnueli et al., 1998). The apparent linear shoot of wild tomato consists of a primary shoot with approximately nine vegetative nodes and a terminal compound inflorescence, and consecutive sympodial reiterations of three vegetative nodes capped by a compound inflorescence. In sp mutants, each progressive sympodium terminates earlier than the previous one, until the shoot axis terminates with two consecutive inflorescences. This results in a compact and synchronized growth habit well-adapted to mechanical harvesting, and the sp phenotype was rapidly bred into all industrial tomatoes (Rick, 1978). Other examples of artificial selection at SFT and SP loci are also now characterized. Glycine soja is the indeterminate wild progenitor of cultivated soybean, Glycine max. The transition from indeterminate to determinate soybean resulted from human selection of four single nucleotide substitutions in the GmTFL1 gene, each of which resulted in an amino acid change, and artificial selection for the determinate soybean growth habit occurred early in landrace radiation (Tian et al., 2010). The FT paralog HaFT1 from sunflower (Helianthus annuus) experienced a selective sweep during domestication and affects flowering by interfering with the action of florigenic HaFT4 (Blackman et al., 2010, 2011b). The growth cycle of sugar beet (Beta vulgaris) is regulated by FT paralogs: BvFT2 is essential for flowering in long days whereas BvFT1 prevents flowering in short days before vernalization by repressing BvFT2 expression (Pin et al., 2010). Pseudoresponse regulator BOLTING TIME CONTROL 1 (BvBTC1) is a clock gene acting upstream of BvFT1 and BvFT2 and regulates their expression. A partial loss-of-function allele of BvBTC1 experienced selection during domestication and is responsible for converting sugar beet from an annual to biennial growth habit (Pin et al., 2012). In many grains, pseudo-response regulators alter photoperiod response, thus moderating flowering time, by acting upstream of SFT homologs (Turner et al., 2005; Beales et al., 2007; Murphy et al., 2011; Gao et al., 2014). Cotton domestication converted a perennial, photoperiodic, tropical tree to a day-neutral shrub. How this conversion took place is not certain, but our findings suggest that regulation of GhSFT activities contributed to that process. GhSFT is indeed expressed in all tissues tested under noninductive long days, and with generally elevated levels in inductive short days (Fig. 2a,b). We show that GhSFT encodes the florigenic stimulus conserved in both photoperiodic ancestral plants and domesticated dayneutral varieties. Overexpressing GhSFT from dclcrv resulted in significantly earlier flowering uncoupled from photoperiod in wild TX701, and a more compact growth habit in both TX701 and in domesticated day-neutral DP61. Silencing GhSFT significantly delayed the onset of flowering in domesticated cotton, as quantified by the node of first fruiting branch (Fig. 7e). Together, these results suggest that the plants are constantly primed to flower with GhSFT, and when GhSFT is silenced, the plants are less primed. That a domesticated, day-neutral variety of G. hirsutum still responds to a component of a photoperiodic signal is significant, and suggests that plants exhibiting strong GhSFT expression may have been selected during domestication, likely contributing to the compact, high-yielding varieties commonly cultivated. In addition to timing and placement of flowers, branching patterns are principle components of plant architecture. Overexpression of AtTFL1 in Arabidopsis produces a highly branched vegetative inflorescence (Ratcliffe et al., 1998). Overexpression of the rose TFL1 homolog KSN resulted in nonflowering shoots, preserving apical dominance and reducing axillary bud outgrowth (Randoux et al., 2014); lateral branching in rice requires the FT ortholog Hd3a to form a floral activation complex at the axillary meristem (Tsuji et al., 2015). We demonstrate that GhSFT and GhSP together control branching patterns in cotton. Although GhSFT regulates flowering and advances the transition to determinacy in all meristems, GhSP is required to maintain indeterminate growth in monopodial and sympodial branches. When GhSP was silenced, flowering was uncoupled from photoperiod, growth terminated and all meristems converted to floral fates. These findings suggest that GhSP maintains all monopodial branching in an indeterminate state, and GhSP must be dynamically altered with each new sympodial unit. Furthermore, basal GhSFT expression is always present and is sufficient to stimulate flowering in TX701 in noninductive long days once GhSP is reduced (Fig. 4h). Thus, when GhSP is reduced by silencing, it likely dips below a critical threshold and determinate growth prevails with GhSFT activity in the same tissues. Taken together, these findings indicate that relative changes in SFT and SP guide cotton meristems between monopodial and sympodial programs. In addition to influencing photoperiodic flowering, flower placement and branching architecture, GhSP and GhSFT expression levels influence leaf marginal meristems and cambial meristems. As most evident in TX701 and consistent with GhSFT and GhSP transcripts measured under inductive and noninductive photoperiods (Fig. 2), GhSFT overexpression, GhSP silencing or short-day photoperiods, converted deeply lobed leaves to lanceolate. Cotton stem expansion was reduced with high GhSFT or low GhSP levels, and this was particularly prominent in GhSPsilenced plants where stems were herbaceous rather than woody. A role for SP in cambial meristems has not been reported in any other woody perennial. Our findings emphasize that quantitative relations between SFT and SP control diverse shoot architectures (Fig. S6). Reducing SP expression has been exploited to induce precocious flowering in trees with extended juvenile phases, such as in apple (Malus domestica) and pear (Pyrus communis; Freiman et al., 2012; Kotoda et al., 2006; Yamagishi et al., 2014). Overexpressing the apple FT orthologs from a virus vector had no impact on flowering or branching in apple whereas delivering the Arabidopsis FT from the same virus stimulated precocious flowering (Yamagishi et al., 2011, 2014). Similarly, overexpressing AtFT from dclcrv resulted in aberrant flowers (McGarry et al., 2013), but this was not observed with GhSFT (Fig. 8). Poplar (Populus trichocarpa) transformed with an RNAi construct targeting the SP ortholog CEN demonstrated earlier onset of flowering, but maintained indeterminate vegetative meristems (Mohamed et al., 2010). We show that dynamic variations in cotton SFT and SP levels rewire networks, guiding meristems between