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The control of carbon allocation, storage and usage is critical for plant growth and development and is exploited for both crop food production and CO2 capture. Potato tubers are natural carbon reserves in the form of starch that have evolved to allow propagation and survival over winter. They form from stolons, below ground, where they are protected from adverse environmental conditions and animal foraging. We show that BRANCHED1b (BRC1b) acts as a tuberization repressor in aerial axillary buds, which prevents buds from competing in sink strength with stolons. BRC1b loss of function leads to ectopic production of aerial tubers and reduced underground tuberization. In aerial axillary buds, BRC1b promotes dormancy, abscisic acid responses and a reduced number of plasmodesmata. This limits sucrose accumulation and access of the tuberigen protein SP6A. BRC1b also directly interacts with SP6A and blocks its tuber-inducing activity in aerial nodes. Altogether, these actions help promote tuberization underground., The work of P.C. was funded by BIO2014-57011-R (MINECO), BIO2017-84363-R (Spanish Ministry of Science and Innovation) (MCIN/AEI/10.13039/501100011033/) and FESF investing in your future. The work of S.P. was funded by BIO2015-73019-EXP (Spanish Ministry of Science and Innovation) (MCIN/AEI/10.13039/501100011033/), ERA-NET COSMIC EIG CONCERT-Japan (PCIN-2017-032) (Spanish Ministry of Science and Innovation) and European Union H2020 ‘ADAPT’ project. The work of R.T.-P. and J.C.O. was funded by CSIC-202020E079 (Spanish National Research Council). The work of V.W. was funded by BMBF (031B0191), DFG (SPP1530: WA3639/1-2, 2-1) and Max-Planck-Society. M.N. had an Excellence Severo Ochoa contract (MINECO, SEV-2013-0347). The CNB is a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712).

The long noncoding RNA (lncRNA)AUXIN-REGULATED PROMOTER LOOP(APOLO) recognizes a subset of target loci across theArabidopsis thalianagenome by forming RNA-DNA hybrids (R-loop) and modulating local three-dimensional chromatin conformation. Here we show thatAPOLOis involved in regulating the shade avoidance syndrome (SAS) by dynamically modulating the expression of key factors. In response to far-red (FR) light, the expression ofAPOLOanticorrelates with its targetBRANCHED1(BRC1), a master regulator of shoot branching inArabidopsis thaliana.APOLOderegulation results inBRC1transcriptional repression and an increase in the number of branches.APOLOtranscriptional accumulation fine-tunes the formation of a repressive chromatin loop encompassing theBRC1promoter, which normally occurs only in leaves as well as in a late response to FR treatment in axillary buds. In addition, our data reveal thatAPOLOparticipates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation ofYUCCA2(auxin synthesis),PIDandWAG2(auxin efflux). We found that direct application ofAPOLORNA to leaves results in a rapid increase in auxin accumulation that is associated with changes in the response of the plants to FR light. Collectively, our data support the view that lncRNAs coordinate the shade avoidance syndrome inArabidopsis thalianaand shed light on the potential of lncRNAs as bioactive exogenous molecules. Deploying exogenous RNAs that modulate plant-environment interactions are important new tools for sustainable agriculture.

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis (Arabidopsis thaliana) has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem elongates with the top leaf primordia developing into cauline leaves. Meristems in Arabidopsis initiate in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three Arabidopsis ecotypes and several Arabidopsis mutants with varied shoot architectures. Our results showed no negative correlation between cauline and rosette branch numbers in Arabidopsis, demonstrating that there is no tradeoff between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and Arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number of rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity, and temperature. Our data reveal different levels of plasticity in the regulation of branching at rosette and cauline nodes, and promote a framework for future branching analyses.

The microRNA156 (miR156)/SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE(SPL/SBP) regulatory hub is highly conserved among phylogenetically distinct species, but how it interconnects multiple pathways to converge to common integrators controlling shoot architecture is still unclear. Here, we demonstrated that the miR156/SlSBP15hub modulates tomato shoot branching (SB) by connecting phytohormones with important genetic pathways regulating both axillary bud (AB) development and outgrowth. We verified that plants overexpressing the miR156 (156-OE plants) display high SB, whereas plants overexpressing a miR156-resistant SlSBP15 alelle (rSBP15 plants) display arrested SB and are able to partially restore the wild-type (WT) phenotype in156-OE background. Although rSBP15 plants showed ABs smaller than MT, its activation is dependent on shoot apex-derived auxin transport inhibition. Additionally, hormonal measurements reveal that IAA and ABA concentrations were lower in 156-OE and higher in rSBP15-OE plants. SlSBP15 regulates AB development and outgrowth by inhibiting auxin transport and the activity ofGOBLET(GOB), and by interacting with BRANCHED1b (SlBRC1b) at the protein level to control abscisic acid (ABA) levels within ABs. Our data provide a new mechanism by which the miR156/SPL/SBPhub regulates SB, and suggest thatSlSBP15has potential applications in improving tomato architecture.

Shoot branching is an important trait that depends on the activity of axillary meristems and buds and their outgrowth into branches. It is remarkably plastic, being influenced by a number of external cues, such as light, temperature, soil nutrients, and mechanical manipulation. These are transduced into an internal hormone signaling network where auxin, cytokinins, and strigolactones play leading regulatory roles. Recently, sugars have also emerged as important signals promoting bud activation. These signals are in part integrated by the bud-specific growth repressor BRANCHED1 (BRC1).To understand how shoot branching is affected by particular growth conditions or in specific plant lines, it is necessary to count the number of branches and/or quantify other branch-related parameters. Here we describe how to perform such quantifications in Arabidopsis and in tomato.

Gravitropism is a finely regulated tropistic response based on the plant perception of directional cues. Such perception allows them to direct shoot growth upwards, above ground, and root growth downwards, into the soil, anchoring the plant to acquire water and nutrients. Gravity sensing occurs in specialized cells and depends on auxin distribution, regulated by influx/efflux carriers. Here we report that AtHB40, encoding a transcription factor of the homeodomain-leucine zipper I family, was expressed in the columella and the root tip. Athb40 mutants exhibited longer primary roots. Enhanced primary root elongation was in agreement with a higher number of cells in the transition zone and the induction of CYCLINB transcript levels. Moreover, athb40 mutants and AtHB40 overexpressors displayed enhanced and delayed gravitropistic responses, respectively. These phenotypes were associated with altered auxin distribution and deregulated expression of the auxin transporters LAX2, LAX3, and PIN2. Accordingly, lax2 and lax3 mutants also showed an altered gravitropistic response, and LAX3 was identified as a direct target of AtHB40. Furthermore, AtHB40 is induced by AtHB53 when the latter is upregulated by auxin. Altogether, these results indicate that AtHB40 modulates cell division and auxin distribution in the root tip thus altering primary root length and gravitropism.

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis thaliana has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem starts to elongate with the top leaf primordia developing into cauline leaves. Meristems in arabidopsis are initiated in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three arabidopsis ecotypes and several arabidopsis mutants with varied shoot architectures. Our results show that there is no negative correlation between cauline and rosette branch numbers in arabidopsis, demonstrating that there is no trade-off between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number or rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity and temperature. Our data reveal different plasticity in the regulation of branching at rosette and cauline nodes and promote a framework for future branching analyses.One sentence summaryDifferent plasticity of branching at cauline and rosette nodes of arabidopsis is revealed through detailed correlative analyses of branching under varied genetic and environmental contexts.

The effects of the phytohormone strigolactone (SL) and smoke-derived karrikins (KARs) on plants are generally distinct, despite the fact that they are perceived through very similar mechanisms. The homologous receptors DWARF14 (D14) and KARRIKIN-INSENSITIVE2 (KAI2), together with the F-box protein MORE AXILLARY GROWTH2 (MAX2), mediate SL and KAR responses, respectively, by targeting different SMAX1-LIKE (SMXL) family proteins for degradation. These mechanisms are putatively well-insulated, with D14-MAX2 targeting SMXL6, SMXL7, and SMXL8 and KAI2-MAX2 targeting SMAX1 and SMXL2 in

Shoot branching is a highly variable trait that evolves during plant development and is influenced by environmental and endogenous cues such as hormones. In particular, strigolactones (SLs) are hormones that play a key role in the control of shoot branching. Branch primordia, axillary buds formed in the leaf axils, display differential growth depending on their position in the plant and also respond to hormone signaling. In this chapter, we will describe how to quantify the degree of shoot branching in two plant model species, Arabidopsis and pea, commonly used to decipher the control of this complex trait. We will also propose several methods to perform treatments of SL or SL analogs, to investigate their bioactivity and effect on the shoot branching patterns of plants of different genotypes.

The binding of strigolactones to their receptor, the α/β hydrolase DWARF14 (D14), leads to the modulation of transcriptional activity by destabilization of specific transcriptional corepressors via proteasomal degradation. Subsequently, strigolactones also promote D14 degradation by the same pathway. Here we describe an innovative quantitative bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to the firefly luciferase, developed to identify new strigolactone analogs capable to activate the strigolactone signaling.

The binding of strigolactones to their receptor, the α/β hydrolase DWARF14 (D14), leads to the modulation of transcriptional activity by destabilization of specific transcriptional corepressors via proteasomal degradation. Subsequently, strigolactones also promote D14 degradation by the same pathway. Here we describe an innovative quantitative bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to the firefly luciferase, developed to identify new strigolactone analogs capable to activate the strigolactone signaling.

Shoot branching is a highly variable trait that evolves during plant development and is influenced by environmental and endogenous cues such as hormones. In particular, strigolactones (SLs) are hormones that play a key role in the control of shoot branching. Branch primordia, axillary buds formed in the leaf axils, display differential growth depending on their position in the plant and also respond to hormone signaling. In this chapter, we will describe how to quantify the degree of shoot branching in two plant model species, Arabidopsis and pea, commonly used to decipher the control of this complex trait. We will also propose several methods to perform treatments of SL or SL analogs, to investigate their bioactivity and effect on the shoot branching patterns of plants of different genotypes.

Protein-protein interactions (PPIs) represent an essential aspect of plant systems biology. Identification of key protein players and their interaction networks provide crucial insights into the regulation of plant developmental processes and into interactions of plants with their environment. Despite the great advance in the methods for the discovery and validation of PPIs, still several challenges remain. First, the PPI networks are usually highly dynamic, and the in vivo interactions are often transient and difficult to detect. Therefore, the properties of the PPIs under study need to be considered to select the most suitable technique, because each has its own advantages and limitations. Second, besides knowledge on the interacting partners of a protein of interest, characteristics of the interaction, such as the spatial or temporal dynamics, are highly important. Hence, multiple approaches have to be combined to obtain a comprehensive view on the PPI network present in a cell. Here, we present the progress in commonly used methods to detect and validate PPIs in plants with a special emphasis on the PPI features assessed in each approach and how they were or can be used for the study of plant interactions with their environment.

Plant shoot branching patterns determine leaf, flower and fruit production, and thus reproductive success and yield. Branch primordia, or axillary buds, arise in the axils of leaves and their decision to either grow or enter dormancy is coordinated at the whole plant level. Comparisons of transcriptional profiles of axillary buds entering dormancy have identified a shared set of responses that closely resemble a Low Energy Syndrome. This syndrome is aimed at saving carbon use to support essential maintenance functions, rather than additional growth, and involves growth arrest (thus dormancy), metabolic reprogramming and hormone signalling. This response is widely conserved in distantly related woody and herbaceous species, and not only underlies but also precedes the growth-to-dormancy transition induced in buds by different stimuli.

How do perennial plants adapt their growth to seasonal changes? A new study in the hybrid aspen reveals that, in short days, repression of a growth-promoting genetic pathway leads to upregulation of the BRANCHED1 genes, which in turn induce growth cessation.

The control of branch outgrowth is critical for plant fitness, stress resilience and crop yield. The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in this process as it integrates signals that inhibit axillary bud growth to control shoot branching. Despite the remarkable activity of BRC1 as a potent growth inhibitor, the mechanisms by which it promotes and maintains bud dormancy are still largely unknown. Here we combine ChIP-seq, transcriptomic and systems biology approaches to characterise the BRC1-regulated gene network. We identify a group of BRC1 direct target genes encoding transcription factors (BTFs) that orchestrate, together with BRC1, an intricate transcriptional network enriched in abscisic acid signalling components. The BRC1 network is enriched in feed-forward loops and feed-back loops, robust against noise and mutation, reversible in response to stimuli, and stable once established. This knowledge is fundamental to adapt plant architecture and crop production to ever-changing environmental conditions. Dutch Scientific Organization NWO-JSTP 833.13.008 Ministerio de Economía y Competitividad BIO2014-57011-R Ministerio de Economía y Competitividad BIO2017-84363-R Ministerio de Economía y Competitividad BIO2017-84066-R

The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.

In the last decade strigolactones have been recognized as a novel type of plant hormones. They are involved in the control of key developmental processes such as lateral shoot outgrowth and leaf and root development, among others. In addition, strigolactones modulate plant responses to abiotic stresses like phosphate starvation and drought. Here we summarize the current knowledge of the widely conserved functions of strigolactones in the control of plant development and stress responses as well as some of their reported species-specific roles. In addition, we will review their known genetic and functional interactions with other phytohormones. The newly discovered activities of strigolactones as plant hormones raise the possibility of using these compounds and their signalling pathways as tools to optimise species of agronomical importance.

The biological activity of natural and novel strigolactone D-lactam analogues is assessed using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase., Strigolactones (SLs) are plant hormones with various functions in development, responses to stress, and interactions with (micro)organisms in the rhizosphere, including with seeds of parasitic plants. Their perception for hormonal functions requires an α,β-hydrolase belonging to the D14 clade in higher plants; perception of host-produced SLs by parasitic seeds relies on similar but phylogenetically distinct proteins (D14-like). D14 and D14-like proteins are peculiar receptors, because they cleave SLs before undergoing a conformational change that elicits downstream events. Structure–activity relationship data show that the butenolide D-ring is crucial for bioactivity. We applied a bioisosteric approach to the structure of SLs by synthetizing analogues and mimics of natural SLs in which the D-ring was changed from a butenolide to a lactam and then evaluating their bioactivity. This was done by using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase, in parallel with the quantification of germination-inducing activity on parasitic seeds. The results obtained showed that the in planta bioassay is robust and quantitative, and thus can be confidently added to the SL-survey toolbox. The results also showed that modification of the butenolide ring into a lactam one significantly hampers the biological activity exhibited by SLs possessing a canonical lactonic D-ring.

Shoot branching is a major determinant of plant architecture and is regulated by both endogenous and environmental factors. BRANCHED1 (BRC1) is a central local regulator that integrates signals controlling shoot branching. So far, the regulation of BRC1 activity at the protein level is still largely unknown. In this study, we demonstrated that TIE1 (TCP interactor containing EAR motif protein 1), a repressor previously identified as an important factor in the control of leaf development, also regulates shoot branching by repressing BRC1 activity. TIE1 is predominantly expressed in young axillary buds. The gain-of-function mutant tie1-D produced more branches and the overexpression of TIE1 recapitulated the increased branching of tie1-D, while disruption of TIE1 resulted in lower bud activity and fewer branches. We also demonstrated that the TIE1 protein interacts with BRC1 in vitro and in vivo. Expression of BRC1 fused with the C-terminus of the TIE1 protein in wild type caused excessive branching similar to that observed in tie1-D and brc1 loss-of-function mutants. Transcriptome analyses revealed that TIE1 regulated about 30% of the BRC1-dependent genes, including the BRC1 direct targets HB21, HB40 and HB53. These results indicate that TIE1 acts as a positive regulator of shoot branching by directly repressing BRC1 activity. Thus, our results reveal that TIE1 is an important shoot branching regulator, and provide new insights in the post-transcriptional regulation of the TCP transcription factor BRC1.

Plant shoot systems give rise to characteristic above-ground plant architectures. Shoots are formed from axillary meristems and buds, whose growth and development is modulated by systemic and local signals. These cues convey information about nutrient and water availability, light quality, sink/source organ activity and other variables that determine the timeliness and competence to maintain development of new shoots. This information is translated into a local response, in meristems and buds, of growth or quiescence. Although some key genes involved in the onset of bud latency have been identified, the gene regulatory networks (GRNs) controlled by these genes are not well defined. Moreover, it has not been determined whether bud dormancy induced by environmental cues, such as a low red-to-far-red light ratio, shares genetic mechanisms with bud latency induced by other causes, such as apical dominance or a short-day photoperiod. Furthermore, the evolution and conservation of these GRNs throughout angiosperms is not well established. We have reanalyzed public transcriptomic datasets that compare quiescent and active axillary buds of Arabidopsis, with datasets of axillary buds of the woody species Vitis vinifera (grapevine) and apical buds of Populus tremula x Populus alba (poplar) during the bud growth-to-dormancy transition. Our aim was to identify potentially common GRNs induced during the process that leads to bud para-, eco- and endodormancy. In Arabidopsis buds that are entering eco- or paradormancy, we have identified four induced interrelated GRNs that correspond to a carbon (C) starvation syndrome, typical of tissues undergoing low C supply. This response is also detectable in poplar and grapevine buds before and during the transition to dormancy. In all eukaryotes, C-limiting conditions are coupled to growth arrest and latency like that observed in dormant axillary buds. Bud dormancy might thus be partly a consequence of the underlying C starvation syndrome triggered by environmental and endogenous cues that anticipate or signal conditions unfavorable for sustained shoot growth.

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development., This work was supported by grants from the Spanish Ministry of Economy and Competitivness, Plan Estatal de I+D+I and European Social Fund to JF, IA, and PC (CGL2007-66516, CGL2013-49097-C2-2-P and BIO2014-57011-R), “Juan de la Cierva” program co-financed by the European Social Fund to MB (JCI-2010-07374) and Proyecto Intramural CSIC 201430E023 to JF.

Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.

Significance Shoot-branching patterns affect key aspects of plant life and are important targets for crop breeding. However, we are still ignorant of the genetic mechanisms controlling locally an important decision during branch development: whether the axillary bud grows out to give a lateral shoot or remains dormant. Here we show that the TEOSINTE BRANCHED1, CYCLOIDEA, PCF (TCP) transcriptional regulator BRANCHED1 (BRC1), which acts inside axillary buds, binds and activates three genes encoding Homeodomain leucine zipper (HD-ZIP) transcription factors. These factors, together with BRC1, trigger a cascade leading to local abscisic acid (ABA) accumulation and response, essential for bud dormancy under light-limiting conditions. This finding demonstrates a direct relationship between BRC1 and ABA signaling and places ABA downstream of BRC1 in the control of axillary bud dormancy.

The TCP transcription factors govern key plant developmental processes and have profound effects on the growth patterns of meristems and organs, partly explained by direct transcriptional control of cell cycle genes. This view is nevertheless incomplete, as accumulated evidence indicates that TCPs also act through other mechanisms, such as the regulation of hormone activity. Several TCP factors not only act as mediators of hormone-induced changes in cell proliferation, but also as modulators, or even key players, of hormone synthesis, transport and signal transduction. This adds another layer of complexity to the role of TCPs in plant development.

In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways conserved widely across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogues, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts that encode two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued to evolve under a strong purifying selection that was consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this difference does not reflect relaxation but rather positive selective pressure for adaptation.

A major focus of evolutionary developmental (evo-devo) studies is to determine the genetic basis of variation in organismal form and function, both of which are fundamental to biological diversification. Pioneering work on metazoan and flowering plant systems has revealed conserved sets of genes that underlie the bauplan of organisms derived from a common ancestor. However, the extent to which variation in the developmental genetic toolkit mirrors variation at the phenotypic level is an active area of research. Here we explore evidence from the angiosperm evo-devo literature supporting the frugal use of genes and genetic pathways in the evolution of developmental patterning. In particular, these examples highlight the importance of genetic pleiotropy in different developmental modules, thus reducing the number of genes required in growth and development, and the reuse of particular genes in the parallel evolution of ecologically important traits.

Hybridization between species can lead to introgression of genes from one species to another, providing a potential mechanism for preserving and recombining key traits during evolution. To determine the molecular basis of such transfers, we analyzed a natural polymorphism for flower-head development in Senecio. We show that the polymorphism arose by introgression of a cluster of regulatory genes, the RAY locus, from the diploid species S. squalidus into the tetraploid S. vulgaris. The RAY genes are expressed in the peripheral regions of the inflorescence meristem, where they promote flower asymmetry and lead to an increase in the rate of outcrossing. Our results highlight how key morphological and ecological traits controlled by regulatory genes may be gained, lost, and regained during evolution.

TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors are a family of plant-specific proteins that contain a TCP domain, a noncanonical basic helix–loop–helix structure involved in DNA binding, and protein–protein interactions. Some TCP proteins have important functions in plant development, such as control of leaf shape and flower symmetry, and the suppression of lateral shoot branching. The TCP family is divided into two classes, class I and class II. Class II is further subdivided into subclasses CINCINNATA (CIN) and CYCLOIDEA/TEOSINTE BRANCHED1 (CYC/TB1). Whereas early studies proposed antagonistic roles for class I and II proteins on the control of cell proliferation, here we describe recent work which supports the belief that both protein types are functionally interconnected and in some cases contribute to some functions during plant development. We outline the role of class I and CIN genes in the modulation of meristem gene expression, to control simple and compound leaf maturation. In addition, we review the roles of class I and class II CIN and CYC / TB1 genes in flower development, and comment on recent advances in our understanding of the up- and downstream pathways of CYC / TB1 genes in the control of lateral shoot branching. Finally, we discuss how these TCP transcription factors are integrated in the hormone-signaling pathways that control these developmental processes.

The Teosinte branched1/Cincinnata/proliferating cell factor (TCP) family is a group of genes encoding plant-specific transcription factors that share the so-called TCP domain, a 59-amino-acid stretch predicted to form a noncanonical basic helix–loop–helix (bHLH) structure. The TCP domain allows DNA binding and facilitates protein–protein interactions. TCP genes have been found across the plant kingdom, from green algae to eudicots. Gene duplication and diversification of the family members led to an increase in family size, so that more than 20 TCP genes are found in eudicots species. Two major clades (class I and II) of TCP proteins can be distinguished with slightly different TCP domains. In this chapter, we summarize recent work analyzing the evolution of the TCP family in angiosperms, as well as the biochemical role of TCP proteins as transcriptional regulators. We highlight the role of particular residues of the TCP domain in protein–DNA and protein–protein interactions, and discuss the involvement of other protein regions in their posttranslational regulation and function.

Shoot branching patterns depend on a key developmental decision: whether axillary buds grow out to give a branch or whether they remain dormant in the axils of leaves. This decision is controlled by endogenous and environmental stimuli mediated by hormonal signals. Although genes involved in the long-distance signaling of this process have been identified, the genes responding inside the buds to cause growth arrest remained unknown in Arabidopsis thaliana. Here, we describe an Arabidopsis gene encoding a TCP transcription factor closely related to teosinte branched1 (tb1) from maize (Zea mays), BRANCHED1 (BRC1), which represents a key point at which signals controlling branching are integrated within axillary buds. BRC1 is expressed in developing buds, where it arrests bud development. BRC1 downregulation leads to branch outgrowth. BRC1 responds to developmental and environmental stimuli controlling branching and mediates the response to these stimuli. Mutant and expression analyses suggest that BRC1 is downstream of the MORE AXILLARY GROWTH pathway and that it is required for auxin-induced apical dominance. Therefore, BRC1 acts inside the buds as an integrator of signals controlling bud outgrowth and translates them into a response of cell growth arrest. The conservation of BRC1/tb1 function among distantly related angiosperm species suggests that a single ancestral mechanism of branching control integration evolved before the radiation of flowering plants.

The flowers of the primitive angiosperm plants were radially symmetrical (actinomorphic). Flowers with bilateral symmetry (zygomorphic) evolved in several clades independently as an adaptation to specialized methods of pollination and played an important role in the diversification of flowering plants. In the model species Antirrhinum majus (snapdragon), the related genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) are key in the development of this trait. This raises the question of whether they played a role in the evolution of floral bilateral symmetry. To address this, the evolution of CYC in relation to the evolution of zygomorphy is being investigated. Phylogenetic and functional analyses of CYC-like genes are being carried out in groups either closely related to Antirhinum or in families where zygomorphy evolved as an independent event. In addition, the origin of zygomorphy is being studied by comparing the function of CYC-like genes in species with zygomorphic flowers with their function in species with radially symmetrical flowers.

We have isolated and characterized AtREM1, the Arabidopsis ortholog of the cauliflower (Brassica oleracea) BoREM1. AtREM1 belongs to a large gene family of more than 20 members in Arabidopsis. The deduced AtREM1 protein contains several repeats of a B3-related domain, and it could represent a new class of regulatory proteins only found in plants. Expression of AtREM1 is developmentally regulated, being first localized in a few central cells of vegetative apical meristems, and later expanding to the whole inflorescence meristem, as well as primordia and organs of third and fourth floral whorls. This specific expression pattern suggests a role in the organization of reproductive meristems, as well as during flower organ development., This work was supported by Comisión Interministerial de Ciencia y Tecnologia (Spain; grant no. AGF98-0206). Support to research activity at Centro Nacional de Biotecnologı́a is provided through specific agreement between Consejo Superior de Investigaciones Cientificas and Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria. J.M.F.-Z. was funded by a predoctoral fellowship from Dirección General de Investigación Cientı́fica y Tecnológica (Spain). B.F.-C. was a recipient of a postdoctoral fellowship from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (Spain), and P.C. and J.A.J. are recipients of postdoctoral Ministerio de Educación y Ciencia (Spain) contracts.

(Abstract) The inflorescence stem of the flowering plant Arabidopsis thaliana (thale cress) is an excellent model system to investigate plant vascular tissue patterning and development. Plant vasculature is a complex conducting tissue arranged in strands called vascular bundles, formed by xylem (tissue that carries water) and phloem (tissue that carries photosynthates and signaling molecules). Xylem and phloem are originated from cell division of the meristematic cells of the vascular cambium. In Arabidopsis the flowering stem elongates about three weeks after germination. At this stage it is possible to visualize defects in its development and morphology. Here we describe a protocol to embed in plastic (resin) stem segments either freshly dissected from living plants or previously assayed for β-glucuronidase. This protocol provides an excellent cellular morphology ideal to visualize stem cell types including those of vascular bundles using high-resolution light microscopy.

Plants interpret a decrease in the red to far-red light ratio (R:FR) as a sign of impending shading by neighboring vegetation. This triggers a set of developmental responses known as shade avoidance syndrome. One of these responses is reduced branching through suppression of axillary bud outgrowth. The Arabidopsis thaliana gene BRANCHED1 (BRC1), expressed in axillary buds, is required for branch suppression in response to shade. Unlike wild-type plants, brc1 mutants develop several branches after a shade treatment. BRC1 transcription is positively regulated 4 h after exposure to low R:FR. Consistently, BRC1 is negatively regulated by phytochrome B. Transcriptional profiling of wild-type and brc1 buds of plants treated with simulated shade has revealed groups of genes whose mRNA levels are dependent on BRC1, among them a set of upregulated abscisic acid response genes and two networks of cell cycle– and ribosome-related downregulated genes. The downregulated genes have promoters enriched in TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) binding sites, suggesting that they could be transcriptionally regulated by TCP factors. Some of these genes respond to BRC1 in seedlings and buds, supporting their close relationship with BRC1 activity. This response may allow the rapid adaptation of plants to fluctuations in the ratio of R:FR light.

Flowers can be classified into two basic types according to their symmetry: regular flowers have more than one plane of symmetry and irregular flowers have only a single plane of symmetry. The irregular condition is thought to have evolved many times independently from the regular one: most commonly through the appearance of asymmetry along the dorso-ventral axis of the flower. In most cases, the irregular condition is associated with a particular type of inflorescence architecture. To understand the molecular mechanism and evolutionary origin of irregular flowers, we have been investigating genes controlling asymmetry inAntirrhinum. Several mutations have been described inAntirrhinum, a species with irregular flowers, that reduce or eliminate asymmetry along the dorso-ventral axis. We describe the nature of these mutations and how they may be used to analyse the molecular mechanisms underlying floral evolution.

In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways conserved widely across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogues, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts that encode two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued to evolve under a strong purifying selection that was consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this difference does not reflect relaxation but rather positive selective pressure for adaptation.