Flowering and dormancy in temperate perennials

by Maria C. Albani
Botanical Institute, University of Cologne, Germany.
Max Planck Institute for Plant Breeding Research, Cologne, Germany.

In most temperate environments one can see trees flowering very early in the spring. For most perennials flowering in the spring marks the event of floral emergence instead of the time of the induction of flowering and flower bud initiation as it is for many annual species.


A flowering cherry tree in Cologne, Germany, Spring 2018

Thus, trees can flower very early because most of them had initiated the flower buds already the previous year during the summer, autumn or winter. To survive the winter, many perennials also cease growth in the autumn and become dormant during the winter.  Environmental cues such as photoperiod and cold regulate growth cessation and bud dormancy release.  For example, short photoperiods in the autumn are required to induce growth cessation whereas prolonged cold is required to break bud dormancy.
A recent study in hybrid aspen, which is a cross between the European Populus tremula and the American aspen P. tremuliodes, highlights the role of photoperiod in setting the dormant state independently of growth cessation (Tylewicz et al., 2018). Short days block cellular communication through plasmodesmata closure and this process involves the phytohormone ABA. The authors created transgenic aspen with reduced ABA response, overexpression lines of the PDLP1 gene, which impairs trafficking via plasmodesmata, and DsRNAi lines of the chromatin remodelling factor PICKLE (PKL). These transgenics were used to demonstrate the ABA-dependent pathway for plasmodesmata closure and their role in bud dormancy. The authors also used grafting to show that closure of the plasmodesmata regulates the inability of the bud to grow. For this, they grafted scions of wild type and transgenics plants with reduced ABA response grown in short days (so that only scions of the transgenics will have open plasmodesmata) onto rootstocks of lines overexpressing the aspen FLOWERING LOCUS T 1 (FT1) gene. Under these conditions, buds of wild type scions did not reactivate growth whereas buds from scions of the transgenics that had compromised ABA response showed bud outgrowth. These results lead to the conclusion that plasmodesmata closure induced by short days blocks the FT1-derived growth promoting signals to access the meristem. The authors also suggested that re-opening of the plasmodesmata occurs slowly and only after exposure to low temperatures.

The study of Tylewicz et al., 2018 is not about flowering as it has been performed using juvenile/vegetative plants. It however raises interesting questions if one takes into account the flowering patterns in perennials. In P. deltoides trees grown in Starville (Mississippi, USA) it has been demonstrated that flower buds are initiated during the winter when plants are exposed to short day length and low temperatures. In this Populus species, flowering and the return to vegetative development is regulated by two paralogues of FT, FT1 and FT2 (Hsu et al., 2011).  FT1 expression was increased during the winter in many tissues including the reproductive buds, whereas FT2 trancripts were only  up-regulated in the leaves after the return to warm temperatures. These results suggested that FT1 regulates reproductive onset in response to winter temperatures whereas FT2 promotes vegetative growth after the winter in response to warm temperatures and long days.


Tylewicz S, Petterle A, Marttila S, Miskolczi P, Azeez A, Singh RK, Immanen J, Mähler N, Hvidsten TR, Eklund DM, Bowman JL, Helariutta Y, Bhalerao RP. 2018. Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication. Science 360(6385): 212-215. doi: 10.1126/science.aan8576.

Hsu CY, Adams JP, Kim H, No K, Ma C, Strauss SH, Drnevich J, Vandervelde L, Ellis JD, Rice BM, Wickett N, Gunter LE, Tuskan GA, Brunner AM, Page GP, Barakat A, Carlson JE, DePamphilis CW, Luthe DS, Yuceer C. 2011. FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar. Proceedings of the National Academy of Sciences, USA. 108(26):10756-61. doi: 10.1073/pnas.1104713108.

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TCP functions branching out

by Paula Elomaa
Department of Agricultural Sciences, Viikki Plant Science Centre, University of Helsinki, Finland

Since their discovery about 20 years ago, the TCP domain transcription factors have been shown to control diverse aspects of plant growth and development (reviewed in Nicolas and Cubas, 2015). The functions of class II TCP proteins, including the CINCINNATA and CYCLOIDEA/TEOSINTE BRANCHED1–like proteins, have been attributed to leaf development, floral symmetry patterns (zygomorphy) as well as outgrowth of lateral shoots. Many of these genes have been targeted during domestication and by adaptation under natural conditions. Emerging data emphasizes the importance of TCP proteins, and particularly their fine-tuned regulation, in integrating hormonal and environmental signals affecting development (Li et al., 2015; Nicolas and Cubas, 2015; 2016).

One of the founding members of the TCP protein family was the TEOSINTE BRANCHED1 (TB1) in maize found to suppress lateral branching, a major trait contributing to its domestication from teosinte (Doebley et al., 1997). Shoot branching control by TB1 orthologs is highly conserved among angiosperms, and a key trait also from an agronomic perspective (Nicolas and Cubas, 2015). A recent paper by Dixon et al. (2018) demonstrates a functional role for a TB1 ortholog of bread wheat (Triticum aestivum L.) in regulation of inflorescence architecture, providing potential to affect grain production. The inflorescence development in grasses involves complex branching events. While the indeterminate raceme in Arabidopsis elongates and develops individual pedicellate flowers in its axils, the basic unit in a grass inflorescence is the branched spikelet, a terminal unit capable of producing florets (Fig. 1A). In case of wheat, single spikelets develop in alternate phyllotaxis along the central rachis and each of them produce multiple florets. In their paper, Dixon et al. demonstrate that TB1 regulates the paired spikelet trait in wheat where two spikelets are formed in individual rachis nodes instead of a single one (Fig. 1B).

The highly-branched (hb) wheat line, analyzed in this paper, showed altered growth of lateral organs by developing multiple paired spikelets in their inflorescences as well as fewer tillers. The hb lines were delayed in their transition to reproductive development, and showed delayed inflorescence growth especially during early developmental stages (leaf stages L5-L7). However, the final length of the mature inflorescences was not altered. Analysis of the QTL region contributing to the paired spikelet trait revealed the presence of the TB1. The hb line showed increased (tetrasomic) dosage of chromosome 4D, and specifically the TB1 expression originating from the wheat D genome (TB-D1) was significantly upregulated both in hb tillers (including tiller buds) as well as in inflorescences during the stages when their growth was delayed. The dosage dependent TB-D1 regulation was confirmed by modifying the number of functional copies through crossings to tb-d1 mutant line, by analyzing the revertant phenotypes of hb plants as well as by overexpression of TB-D1 in transgenic plants. Intriguingly, Dixon et al. linked the TB1 function with regulation of flowering by showing that TB-D1 directly interacts with the major flowering regulator FT1, and that increased dosage of TB-D1 reduces the transcript levels of several meristem identity genes. The allelic diversity of TB1 in both B and D genomes was further associated with paired spikelet development in modern wheat cultivars.

This work connects branching control with regulation of flowering, and demonstrates a fine-tuned regulatory link between these major developmental events. The established model by the authors propose that increased dosage of TB1 reduce the availability of FT to activate spikelet meristem identity genes, and facilitates inflorescence branching by modifying the temporal timing or rate of spikelet meristem maturation. As the number of spikelets determines the seed number and crop yield – keeping in mind the possible adverse effects due to altered sink-source relations – this work adds a valuable gene from the TCP family among the breeding targets, not only in wheat but potentially also in other cereals as discussed by Dixon and colleagues.


Dixon LE, Greenwood JR, Bencivenga S, Zhang P, Cockram J, Mellers G, Ramm K, Cavanagh C, Swain SM, Boden SA. 2018. TEOSINTE BRANHCED1 regulates inflorescence architecture and development in bread wheat (Triticum aestivum L.). The Plant Cell, doi: 10.1105/tpc.17.00961
Doebley J, Stec A, Hubbard L. 1997. The evolution of apical dominance in maize. Nature 386, 485-488. doi:10.1038/386485a0
Li S. 2015. The Arabidopsis thaliana TCP transcription factors: a broadening horizon beyond development. Plant Signaling & Behavior 10, e1044192-2. doi: 10.1080/15592324.2015.1044192
Nicolas M, Cubas P. 2015. The role of TCP transcription factors in shaping flower structure, leaf morphology, and plant architecture. In: Gonzalez DH, ed. Plant transcription factors. Evolutionary, structural, and functional aspects. Academic Press, Elsevier Inc. doi: 10.1016/B978-0-12-800854-6.00016-6
Nicolas M, Cubas P. 2016. TCP factors: new kids on the signaling block. Current Opinion in Plant Biology 33: 33-41. doi: 10.1016/j.pbi.2016.05.006

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A surprising role for ethylene in the regulation of petal cell shape

Beverley Glover
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK bjg26@cam.ac.uk

The different shapes of plant epidermal cells are always fascinating. One of the first experiences many students have of scanning electron microscopy is the excitement of seeing the amoeba-shaped cells of leaves, interspersed with stomata. Leaf epidermal cell shape is particularly intriguing because the amoeboid cells are so unexpected – something about the smooth flat surface of a leaf suggests that a much more regular arrangement of cells is likely. Recent work by Sapala et al. (2018) has suggested a function for these unusually shaped cells – in the dispersal of mechanical stresses as the cell grows. Accordingly, the differentiation of these cells can be thought of as a product of the need to minimise stress on the growing cell wall, and the pattern we observe may simply be the outcome of a series of mechanical compromises.

Petal epidermal cells, in contrast, have a very different shape. In most plant species they are regular at the base (loosely rounded, or hexagonal), but with significant expansion in the Z plane, perpendicular to the main surface of the tissue. This results in a conical growth form, and these cells are often called conical cells or conical-papillate cells (as they resemble short papillae). The function of this particular cell morphology has been very well studied, and they are known to play roles in light focusing (which enhances pigmentary colour), surface wettability and floral temperature (Whitney et al., 2011). Their most significant function is thought to be in providing grip to pollinating insects – a series of studies using mutant lines of Antirrhinum majus with flat petal epidermal cells revealed that bees preferred conical-celled flowers, but only when they were made difficult to handle (by being presented vertically, or made to move as if in the wind). The conical cells are thought to provide an opportunity for the pairs of cleft claws on the tarsae of bees to interlock with the petal surface, reducing energy expenditure and improving foraging efficiency (Whitney et al., 2009).

Figure 1. Beverley may2018 c

Fig. 1. Petal conical epidermal cells differ in size and shape in characteristic ways between closely related species. A. Scanning electron micrograph of petal of Veronica chamaedrys. B. Veronica officinalis. C. Veronica prostrate. D. Veronica spicata. All scale bars = 50 µm.

Since conical cells have an important function in pollination and therefore plant fitness, it is perhaps not surprising that they appear to be under tight developmental control. The size and shape of conical cells is subtly different in every plant species, and the detail of petal epidermal cell morphology can be diagnostic in species identification (Fig. 1). Although we have known for over 20 years that the outgrowth of petal epidermal cells into a conical form is regulated by MIXTA-like transcription factors from subgroup 9 of the MYB family (Noda et al., 1994), the detail of how specific parameters of conical cell shape and size are controlled is poorly understood.

In a recent paper van Es et al. (2018) reveal a surprising role for the plant growth regulator ethylene in the differentiation of petal epidermal cells. The authors set out to investigate the control of overall petal cell shape and size, observing that, unlike most vegetative tissues, ‘petals … have a morphology that requires differential regulation of cell proliferation and expansion in the basal and distal parts’. To better understand how this differential regulation of the primary drivers of development occur, they studied the three members of the TCP5-like transcription factor family in Arabidopsis. These proteins represent a sister group to the 5 members of the JAW subfamily of TCP proteins, and together these two groups form the CIN clade of the type II TCP family. Previous studies have shown that the TCP5-like proteins play a role in determining petal size and shape, and also in regulating petal epidermal cell shape (Huang and Irish, 2015).

van Es et al. (2018) began by describing the expression profiles of the three TCP5-like genes. TCP5 itself is expressed during cell elongation stages of petal development, TCP13 later in petal development, and TCP17 at a generally low level. Ectopic expression of TCP5 fused to GFP in the petal epidermis (using an L1-specific promoter) produced smaller petals, suggesting that the epidermis itself is regulating final shape and size of the whole petal. The conical petal epidermal cells of these transgenic lines were bigger and less regular than those of wild type plants. However, a very surprising result was that a tcp5 mutant line, and a tcp5 tcp13 tcp17 triple mutant line, showed similarly perturbed petal epidermal cells – although still loosely cone-shaped they were larger and less regular than wild type, and could not be easily distinguished from each other or from the transgenic line ectopically expressing TCP5.

The mystery of why the loss of function and ectopically expressing lines produced the same phenotype was solved by a transcriptomic analysis, using wild type, the three lines described above, and an inducible epidermis-specific ectopic expression line. The authors discovered that genes encoding enzymes of ethylene synthesis (ACS2 and ACO2) were always down-regulated when the TCP5-like genes were up-regulated, and always up-regulated when the TCP5-like genes were mutated. Similarly, the activity of ethylene response factor genes (ERFs) was down-regulated in the ectopic expression lines and up-regulated in the mutant lines. To confirm these findings the authors showed that ethylene itself was present at higher concentrations in the inflorescences of mutant lines and at lower concentrations in an ectopic expression line. The hypothesis that ethylene was directly regulating petal epidermal cell shape was tested by inhibiting ethylene response using silver thiosulphate application in the mutant lines – this returned the petal epidermal cells to a normal size and shape. Finally, the authors showed that TCP5 binds directly to the ACS2 locus, suggesting a direct regulatory role for this transcription factor family in the ethylene response pathway of Arabidopsis petals.

So, why were the ectopic expression and mutant lines phenotypically so similar? The authors hypothesise that wild type petal epidermal cell shape is a product of wild type levels of ethylene production and perception. When the ethylene pathway is perturbed, in either direction, the tight developmental control of cell differentiation is lost and the epidermal cells grow in a less controlled way, producing larger and less regular shapes. In this scenario ethylene is not a specific regulator of any particular cell shape – less ethylene does not mean smaller cells and more ethylene larger cells, for example – but is instead a signal of ‘normal’, which allows tight regulatory control of cell shape. When perturbation of ethylene signalling tells the plant that all is not well, that tight regulatory control is lost, perhaps in part because the plant’s energies may switch to other activities downstream of ethylene signalling, such as induction of defence responses. It is surprising to find a hormone implicated in such a specific developmental process, but the idea that its role is as a signal of general well-being, allowing development to proceed in a coordinated fashion, fits well with recent developments in understanding plant hormone signalling. It will be interesting to see whether this deregulation of petal epidermal cell differentiation has consequences for pollinator attraction and plant fitness in an animal-pollinated system.

Huang T. and Irish V. 2015. Temporal control of plant organ growth by TCP transcription factors. Current Biology 25, 1765-1770. https://doi.org/10.1016/j.cub.2015.05.024
Noda K, Glover BJ, Linstead P and Martin C. 1994. Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature 369, 661-664. doi:10.1038/369661a0
Sapala A, Runions A, Routier-Kierzkowska A, Gupta M, Hong L, Hofhuis H, Verger S, Mosca G, Li C, Hay A, Hamant O, Roeder A, Tsiantis M, Prusinkiewicz P and Smith R. 2018. Why plants make puzzle cells and how their shape emerges. eLife 2018;7:e32794 DOI: 10.7554/eLife.32794
Van Es S, Sylveira S, Rocha D, Bimbo A, Martinelli A, Dornelas M, Angenent G and Immink R. 2018. Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis. The Plant Journal doi: 10.1111/tpj.13904
Whitney H, Chittka L, Bruce T and Glover BJ. 2009. Conical Epidermal Cells Allow Bees to Grip Flowers and Increase Foraging Efficiency. Current Biology 19, 1-6. https://doi.org/10.1016/j.cub.2009.04.051
Whitney H, Bennett KMV, Dorling MW, Sandbach L, Prince D, Chittka L and Glover BJ. 2011. Why do so many petals have conical epidermal cells? Annals of Botany 108, 609-616. doi:  10.1093/aob/mcr065

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ACME system allows in vivo quantification of cellular mechanical properties within developing plant organs

by Robert G. Franks

North Carolina State University, Raleigh, NC., USA

How organ size and shape are determined in developing organisms remains a key question of interest to developmental biologists. An understanding of the relationships between gene expression and the organ shape requires analyses of relationships between the subcellular, cellular, and organ levels and is often approached through mathematical modeling techniques (Boudon et al., 2015Heer and Martin 2017; Eder et al., 2017). One mechanism that links these multiple organizational scales is the biomechanics of the cells and tissues of the organ itself. In the developing plant it has become clear that the biomechanical properties of the cells, and particularly the cell walls, can be key determinants of both cell and organ shape. However, the relationships between gene expression and cell wall properties and organ shape currently are poorly understood.

The ACME system. Image kindly provided by Sarah Robinson (University of Bern)

In a new article Robinson et al. (2017) describe a new method to measure the mechanical properties of plant tissues. This method has several advantages over previously utilized methods; the measurements can be made in live tissues, have cellular scale resolution, and can be measured in the plane of growth. To measure the mechanical properties of an organ or tissue, the deformation of the tissue must be measured in response to a known, externally applied force. One method to achieve this is to use extensometers to apply a known force to a structure and then measure the deformation.  One disadvantage of this method is that the mechanical properties can only be measured at the tissue level. Robinson et al. have paired a micro-extensometer with a confocal microscope to measure the mechanical properties of the cells within an organ at single-cell resolution. They call this system Automated Confocal Micro-Extensometer or ACME. The authors have made available custom software and instructions for the 3D printing of custom parts required for outfitting a confocal microscope with the ACME system.

Robinson and colleagues use the ACME system to measure the changes in the mechanical properties of the individual cells of the developing hypocotyl in an Arabidopsis seedling upon the application of the plant hormone gibberellic acid that promotes growth in the hypocotyl (Robinson et al. 2017). Changes in the mechanical properties of the hypocotyl cells were found to occur in a gradient along the main axis of the hypocotyl in a pattern that correlated with the observed cellular growth patterns. The experimental determination of the mechanical properties of cells is critical for the successful construction of mathematical models of plant organ growth that are useful for understanding the complex interactions between the cellular and tissue scales that can significantly affect the shape of the mature organ.

The application of this technology to developing floral organs is promising. Although the relatively small size of the Arabidopsis flower may make it unsuitable for mounting on this system without modifications, species that generate larger flowers would provide suitable material for analysis of mechanical properties of cells of the developing floral organs. The quantitative biophysical data at cellular resolution that can be resolved with the ACME technology is well suited for developing mathematical models of floral organ size and shape determination.


Boudon F, Chopard J, Ali O, Gilles B, Hamant O, Boudaoud A, Traas J, Godin C. 2015. A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution. PLoS Computational Biology 11 (1), e1003950. https://doi.org/10.1371/journal.pcbi.1003950

Eder D, Aegerter C, Basler K. 2017. Forces controlling organ growth and size. Mechanisms of Development 144, 53–61. https://doi.org/10.1016/j.mod.2016.11.005

Heer NC, Martin AC. 2017. “Tension, contraction and tissue morphogenesis. Development  144 (23), 4249–60. 

Robinson S, Huflejt M, Barbier de Reuille P, Braybrook SA, Schorderet M, Reinhardt D, Kuhlemeier C. 2017. An automated confocal micro-extensometer enables in vivo quantification of mechanical properties with cellular resolution. The Plant Cell. https://doi.org/10.1105/tpc.17.00753.


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DELLA proteins restrict cell divisions by distinct mechanisms

by Jens Sundström
Swedish University of Agricultural Sciences

Breeding cereal crops with reduced stem length, in so-called semi-dwarf varieties, greatly contributed to yield increases associated with the green revolution. However, the semi-dwarf genotype was also associated with reduced inflorescence size. Now, researchers at the John Innes Centre in Norwich, UK, have demonstrated that these traits are regulated by distinct pathways (Serrano-Mislata et al., 2017).  This finding opens up new venues for breeding of semi-dwarf crops without compromising yields by reducing inflorescence size.

Breeding efforts between 1960 and 1985, largely carried out at international public goods institutions such as the International Maize and Wheat Improvement Centre in Mexico (CIMMYT), contributed to a massive increase in crop yields (Pingali, 2012). For instance, yields for wheat in many developing countries have increased almost 200% since the mid -1960s. One of the key traits introgressed in many high yielding varieties is the semi-dwarf genotype. Reduced stem elongation aids reduction of lodging and allows more resources to be allocated to other parts of the plant. Typically, varieties harbouring this trait are mutated in genes affecting responses to the plant hormone gibberellin (GA) (Daviere and Achard, 2013).

Jens blog Dec 2017 3

Fig. 1. Rye usually grow shoulder high. Field of rye-wheat in which the semi-dwarf genotype found in modern wheat varieties has been introgressed. The work by Serrano-Mislata et al. indicates that the semi-dwarf trait can be uncoupled from the reduction of inflorescence size.

Genetic analyses of GA response mutants, primarily carried out in the model species Arabidopsis thaliana have contributed to a working model for GA activity, in which GA acts as an “inhibitor of an inhibitor” (Harberd et al., 2009). DELLA-proteins, which belong to a sub-family of the plant specific GRAS family, act as key repressors of GA responses (Daviere and Achard, 2013). In the absence of GA, DELLA-proteins bind other transcription factors and inhibit their activity. In the presence of GA, the DELLA proteins are degraded and transcription of GA-responsive genes can occur. Plants with mutated DELLA-proteins have pleiotropic phenotypes; for example, reduced seed germination and reduced stem length.

In a recent report, Serrano-Mislata and co-workers (2017) demonstrated that DELLA proteins inhibit shoot growth by negatively regulating cell division rather than cell expansion. The authors provided evidence for this by expressing a stabilized form of DELLA proteins in either the internodes of a stem or in the apical segment of an inflorescence (Fig. 1).

In both cases, expression of the stabilized DELLA-protein resulted in fewer dividing cells as compared to the wild type. These results suggested that DELLA-proteins act as inhibitors of genes involved in cell-cycle regulation or cell division. To test this hypothesis, the authors performed a chromatin immunoprecipitation (ChIP) experiment that allowed them to identify promoters to which the DELLA proteins bind. One of the candidate genes identified in the ChIP experiment encodes a protein belonging to a family of cell cycle inhibitors. Next, the authors made a cross between a knock-out mutant of the cell cycle inhibitor and the line expressing the stabilized form of the DELLA-proteins. Interestingly, the resulting line retained the semi-dwarf phenotype, but the number of cells in the shoot apical meristem was similar to that in the wild type. Hence, cell division were inhibited in the stem internodes but unaffected in the shoot apical meristem, suggesting that DELLA proteins, at least in part, control growth through the activity of cell cycle inhibitors and that this regulation occurs through distinct pathways in different parts of the plant.

While the mechanistic and genetic insights revealed by Serrano-Mislata et al., (2017) are based on work done in Arabidopsis, their study also provides evidence for the presence of  conserved mechanisms in cereals. Hence, their findings may provide future tools for breeding high yielding semi-dwarf cereal varieties, without compromising growth in the seed-bearing parts of the plants.

Serrano-Mislata A, Bencivenga S, Bush M, Schiessl K, Boden S, Sablowski R. (2017). DELLA genes restrict inflorescence meristem function independently of plant height. Nature Plants 3(9):749-754. doi:10.1038/s41477-017-0003-y

Pingali PL. (2012). Green revolution: impacts, limits, and the path ahead. Proccedings of the Natural Academy of Sciences, USA 109(31):12302-12308. doi:10.1073/pnas.0912953109

Daviere JM and Achard P. (2013). Gibberellin signaling in plants. Development 140(6):1147-1151. doi: 10.1242/dev.087650

Harberd NP, Belfield E, Yasumura Y. (2009). The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: how an “inhibitor of an inhibitor” enables flexible response to fluctuating environments. The Plant Cell 21(5):1328-1339. https://doi.org/10.1105/tpc.109.066969

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Does gibberellin regulate the trade-off between flowering and runnering in strawberry?

by Timo Hytönen
Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

Strawberry is one of the most economically-important berry crops in the world. It is a rosette plant that reproduces both generatively and vegetatively through stolons called runners. There is a strong trade-off between flowering and runnering, but runners are important because fruit production is based on clonally propagated plants. In a diploid woodland strawberry, two classical mutants affecting flowering and runnering are known. Recessive mutations in Seasonal flowering locus (SFL) and Runnering locus (R) cause perpetual flowering and runnerless phenotypes, respectively (Figure 1; Brown and Wareing 1965). SFL encodes a major floral repressor, the woodland strawberry homolog of TERMINAL FLOWER1 (Koskela et al. 2012; Iwata et al. 2012), which mediates photoperiodic and temperature signals to control seasonal flowering (Rantanen et al. 2015). The molecular nature of R, however, has remained elusive.

TFL1mutantFigure 1. Classical mutations in woodland strawberry. Recessive mutations in SFL and R genes cause perpetual flowering and inability to produce runners, respectively (left), whereas the plant with dominant alleles (right) is seasonal flowering and produces runners.

Guttridge and Thompson (1964) showed that exogenous gibberellin (GA) treatment induces runner formation and suppresses flowering in a runnerless perpetual flowering mutant of woodland strawberry, indicating that GA may play a role in the trade-off between flowering and runnering. Now, over 50 years later, two studies provided molecular evidence for the role of GA in the control of axillary bud differentiation to runners or branch crowns. Tenreira et al. (2017) identified a gene encoding a GA biosynthetic enzyme, GA20-oxidase, as a plausible candidate for R (see also commentary by Lockhart 2017), and Caruana et al. (2017) reported a single functional DELLA protein that suppresses runner formation in woodland strawberry.

Tenreira et al. (2017) identified FvGA20ox4 as a candidate gene for R by genetic mapping and whole-genome sequencing of a pooled mutant sample. They found a 9-bp deletion in the second exon of the gene and showed by enzyme assays that only non-mutated FvGA20ox4 was able to convert GA12 to GA20 which is the precursor of active GA1. Furthermore, in situ hybridization experiments showed FvGA20ox4 expression in axillary meristems. Together with previous growth regulator experiments (e.g. Guttridge and Thompson 1964; Hytönen et al. 2009), these new data provide strong evidence for FvGA20ox4 being the R gene. However, the role of four other GA20-oxidase encoding genes in axillary bud differentiation remains unresolved. Mutant complementation or targeted mutagenesis of FvGA20ox4 is still required to obtain a final proof.

In another recent study, Caruana et al. (2017) performed a mutagenesis screen in a runnerless woodland strawberry. They found a mutant that continuously produced runners and, using a mapping-by-sequencing strategy, they identified a gene encoding a DELLA growth repressor FvRGA1, as the prime candidate. Next, they generated an inducible dominant negative version of the corresponding DELLA protein and showed that it was able to suppress the formation and elongation of runners in woodland strawberry indicating that a single DELLA protein controls axillary bud fate.

Does GA regulate the trade-off between flowering and runnering in strawberry then? The studies discussed here provide solid evidence for a role of the GA pathway in the control of axillary bud fate, and based on the presented evidence the following working model can be proposed: FvGA20ox4 likely encodes a rate-limiting enzyme of the GA biosynthetic pathway in the axillary bud. In the presence of an active GA20ox enzyme, GA20 is produced and further converted to GA1 by GA3-oxidases; GA1 then causes the degradation of FvRGA1 leading to runner growth, whereas the reduction of GA1 level leads to the accumulation of this DELLA protein and the differentiation of axillary buds to branch crowns. GA also indirectly affects flowering by controlling the number of shoots capable of producing an inflorescence (Tenreira et al. 2017; Caruana et al. 2017), but additional signals are required for floral induction in apical meristems of the crowns.


Brown T, Wareing PF. 1965. The genetical control of the everbearing habit and three other characters in varieties of Fragaria vesca. Euphytica 14: 97-112. https://doi.org/10.1007/BF00032819 

Caruana JC, Sittmann JW, Wang W, Liu Z. 2017. Suppressor of Runnerless encodes a DELLA protein that controls runner formation for asexual reproduction in strawberry. Molecular Plant http://dx.doi.org/10.1016/j.molp.2017.11.001

Guttridge CG, Thompson PA. 1964. The effect of gibberellins on growth and flowering of Fragaria and Duchesnea. Journal of Experimental Botany 15: 631–646. https://doi.org/10.1093/jxb/15.3.631.

Hytönen T, Elomaa P, Moritz T, Junttila O. 2009. Gibberellin mediates daylength controlled differentiation of vegetative meristems in strawberry (Fragaria x ananassa Duch.). BMC Plant Biology 9:18. doi:  10.1186/1471-2229-9-18

Iwata H, Gaston A, Remay A, Thouroude T, Jeauffre J, Kawamura K, Oyant LHS, Araki T, Denoyes B, Foucher  F. 2012. The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry. Plant Journal 69: 116–125. http://doi.org/10.1111/j.1365-313X.2011.04776.x

Koskela E, Mouhu K, Albani MC, Kurokura T, Rantanen M, Sargent D, Battey NH, Coupland G, Elomaa P, Hytönen T. 2012. Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry, Fragaria vesca. Plant Physiology 159: 1043-1054. http://doi.org/10.1111/tpj.12809

Lockhart J. 2017. Flowering versus runnering: uncovering the protein behind a trait that matters in strawberry. Plant Cell 29: 2080-2081. https://doi.org/10.1105/tpc.17.00709

Rantanen M, Kurokura T, Jiang P, Mouhu K, Hytönen T. 2015. Strawberry homolog of TERMINAL FLOWER1 integrates photoperiod and temperature signals to inhibit flowering. Plant Journal 82: 163-173. http://doi.org/10.1111/tpj.12809

Tenreira T, Lange MJP, Lange T, Bres C, Labadie M, Monfort A, Hernould M, Rothan C, Denoyes B. 2017. A specific gibberellin 20-oxidase dictates the flowering-runnering decision in diploid strawberry. Plant Cell 29: 2168-2182. https://doi.org/10.1105/tpc.16.00949

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Enhancing the possibilities of promoter research

Rainer Melzer
School of Biology and Environmental Science, University College Dublin

Developmental regulatory genes have played a pivotal role during the evolution and domestication of plants. From a molecular genetics perspective, our understanding of those regulators is largely driven by the analysis of full loss-of-function mutants. This has provided profound insights into gene regulatory circuits governing developmental processes. However, from the analysis of evolutionary and domestication processes it is also evident that in many cases not loss-of-function mutations but variations in gene expression patterns and expression strength caused phenotypic change (Meyer and Purugganan, 2013). This is a least partly due to the pleiotropic functions of many developmental regulators: complete loss-of-function mutations often yield so dramatic phenotypes that the fitness of the plant is severely impaired, hence the relevance of null mutants during evolution and domestication is limited.

From the analysis of a few exemplary cases we know that the modularity of promoter architectures is one important component of pleiotropic gene functions. For example, APETALA3, a transcription factor coding gene that controls petal as well as stamen development, has specific enhancer regions that are required for AP3 expression in stamens (Hill et al., 1998). Studying promoter functions at the molecular level is therefore extremely valuable for our understanding of evolutionary and domestication processes. Detailed promoter studies are usually quite laborious, however, and are largely limited to a few genetic model plants. Promoter studies often encompass the identification of putative enhancer elements using in silico approaches and the expression of a reporter gene under control of a mutated promoter version lacking those elements (or containing multiples of them). Altered expression pattern of the reporter gene informs about the function of putative enhancer elements (Hernandez-Garcia and Finer, 2014). Although this approach has been very successful, in silico predictions do not always identify critical promoter elements (Hong et al., 2003). In many cases, a more unbiased approach to study promoter functions might be at least equally promising.


Traditional breeding generated a large variation in tomato fruit size. The method presented by Rodríguez-Leal et al. (2017) has the potential to modify and fine-tune quantitative traits in just a few generations.

A recent paper by Rodríguez-Leal et al. (2017) presents a method that will substantially facilitate such an unbiased molecular genetic analysis. Using tomato fruit size as a model system, the authors target the promoter of Solanum lycopersicum CLAVATA3 (SlCLV3), a gene know to be involved in fruit size regulation, using CRISPR-Cas9. However, unlike more conventional CRISPR-Cas9 approaches that aim to generate full loss-of-function mutants, the authors designed not just one but eight guide RNAs that spanned a 2 kb range of the putative promoter region of SlCLV3. Because of variations in guide RNA directed cleavage activities and subsequent repair processes, many types of mutations can be induced, ranging from large promoter deletions to inversions, small deletions and single nucleotide substitutions (Rodríguez-Leal et al., 2017).

As the analysis of the mutant plants proved difficult because different mutations are introduced in the two alleles of the target gene, the authors devised a genetic screen in which they crossed a wild-type plant with a transgenic plant that expressed the eight guide RNAs and Cas9, and also carried a full loss-of-function allele of SlCLV3. In the resulting progeny, mutations were induced by CRISPR-Cas9 in the wild-type allele. In this set-up, the phenotypic effects of even mutations causing only subtle phenotypic changes are relatively easy to detect as the second SlCLV3 allele is a null allele. Thus, the authors essentially developed an elegant yet simple procedure to randomly mutagenize one particular locus and screen for phenotypic consequences of the mutations. Indeed, using this system, it was possible to create an allelic series of 14 SlCLV3 mutant alleles that showed a continuous variation in carpel number (thus leading to a variation in fruit size) (Rodríguez-Leal et al., 2017).

This method is potentially of huge importance for dissection promoter functions. It may be possible to quickly generate a large number of random mutations and analyse their phenotypic effect in a streamlined way. This will prove to be very powerful to untangle the functional elements in promoters. In turn, our understanding of how master regulators of development may have contributed to morphological evolution may be substantially increased. For example, it has been proposed that alterations in the expression of floral homeotic transcription factors contributed to floral diversity (reviewed by Theißen and Melzer, 2007). The approach presented by Rodríguez-Leal et al. (2017) constitutes a promising avenue to test whether and how promoter mutations can induce such phenotypic changes. Theoretically, a large number of mutant alleles can be generated from one transgenic plant. Thus, the approach may also be suitable for phylogenetically informative non-model plants that are difficult to transform.
Last but not least, plant domestication and crop improvement also often proceeds via variations in quantitative traits. Rodríguez-Leal et al. (2017) demonstrated that a substantial variation in tomato fruit size can be obtained in just a few generations, bypassing years of breeding efforts. It will be interesting to see to which extent the same method can be applied to other quantitative traits in crops.


Hernandez-Garcia CM, Finer JJ. 2014. Identification and validation of promoters and cis-acting regulatory elements. Plant Science 217, 109-119. https://doi.org/10.1016/j.plantsci.2013.12.007

Hill TA, Day CD, Zondlo SC, Thackeray AG, Irish VF. 1998. Discrete spatial and temporal cis-acting elements regulate transcription of the Arabidopsis floral homeotic gene APETALA3. Development 125, 1711-1721.

Hong RL, Hamaguchi L, Busch MA, Weigel D. 2003. Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. The Plant Cell 15, 1296-1309. https://doi.org/10.1105/tpc.009548

Meyer RS, Purugganan MD. 2013. Evolution of crop species: genetics of domestication and diversification. Nature Reviews Genetics 14, 840-852. https://doi.org/10.1038/nrg3605

Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett ME, Lippman ZB. 2017. Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470-480. https://doi.org/10.1016/j.cell.2017.08.030

Theißen G, Melzer R. 2007. Molecular mechanisms underlying origin and diversification of the angiosperm flower. Annals of Botany 100, 603-619. https://doi-org./10.1093/aob/mcm143

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Untangling complexity: shedding a new light on LEAFY and APETALA1 interactions

by Leonie Verhage and Francois Parcy
Institut de Biosciences et Biotechnologies de Grenoble (France)

Ever since their discovery almost 30 years ago, the transcription factors LEAFY (LFY) and APETALA1 (AP1) (together with its paralog CAULIFLOWER (CAL)) have been extensively studied for their roles in floral transition. Early genetic and molecular experiments indicated that LFY and AP1/CAL were partly redundant and partly complementary in the process of floral initiation, and numerous subsequent studies fit this model (see Denay et al., 2017 and Wils and Kaufmann, 2017 for recent reviews). However, combining a set of new experiments with published datasets, Goslin and colleagues manage to stir up the prevalent view (Goslin et al., 2017).


Scanning electron micrograph of an ap1 cal mutant. Floral meristems are transformed into proliferative inflorescence meristems. This mutant background was used by Goslin and colleagues. (Image courtesy of Marie le Masson and Christine Lancelon Pin)

To unravel the redundancy of LFY and AP1/CAL, the authors utilized a mutant line harboring a 35S:LFY-GR construct in an ap1/cal background (Wagner et al., 1999). With this line, they performed induction experiments and microarray analysis, in the same way as was previously performed with 35S:AP1-GR in the ap1/cal background (Kaufmann et al., 2010), to make the datasets comparable. This allowed them to compare the downstream genes that are regulated by LFY in the absence of AP1/CAL to genes that are regulated by AP1 in the presence of LFY.

Among the many things uncovered by these analyses, a few were expected and many completely unanticipated.

As already reported by Winter et al., 2015, there is a large overlap between the genes that are differentially regulated upon induction of LFY-GR or AP1-GR. It is likely that this represents true redundancy, where LFY and AP1 can regulate genes in the same way, independent of each other. However, due to a lack of experiments where AP1 is induced in the absence of LFY, it cannot be excluded that this set of genes can be regulated by LFY alone, or by LFY and AP1 together.

More surprisingly, many direct targets of LFY were found to be down-regulated, whereas most of the well-known targets are induced (such as the floral organ identity genes or the LATE MERISTEM IDENTITY genes).

Interestingly, a subset of genes showed differential expression in ap1 cal upon AP1 induction but not upon induction of LFY. By comparing these genes with previously published ChIP-seq data of LFY, the authors could identify a set of genes to which LFY is able to bind, but that are not differentially regulated in absence of AP1. This was the case for APETALA3 (AP3) and AGAMOUS (AG), consistent with a previous report showing that AP1 can act on these genes (Ng and Yanofsky, 2001). Hence, for regulation of these B- and C- type floral organ identity genes, LFY and AP1 appear to act interdependently.

The most surprising result, however, was the presence of genes that are differentially expressed upon LFY or AP1 induction, but in different directions. Apparently, besides acting redundantly or interdependently, LFY and AP1 can also act antagonistically. Notably, this turned out to be the case for several genes involved in inflorescence meristem identity, including TERMINAL FLOWER1 (TFL1). Contrary to the longstanding belief that AP1 and LFY are both repressors of TFL1, only AP1 repressed TFL1, whereas LFY actually activates this gene. It is not completely clear why LFY would up-regulate a gene that inhibits floral meristem identity. The authors speculate that it might be a way to better define the floral transition, so that it occurs only when AP1 is expressed high enough to overcome TFL1.

Goslin et al.  paper is a nice example of how to combine new experiments and existing datasets in a time with ever growing amounts of genome-wide data, with a surprising outcome. Two transcription factors that were long thought to function similarly in initiation of flower formation suddenly turn out to have a much more intriguing relationship, posing many new questions. When LFY and AP1 act together, the biochemical basis of their interaction is elusive. They might be part of the same regulatory complex, especially since their binding sites have been reported to be adjacent (Winter et al., 2015), but a direct interaction between the two proteins has not been observed. Analysis by targeted proteomics has uncovered AP1 interactors in floral tissue (Smaczniak et al., 2012), but has never been analyzed in earlier tissue in which LFY is expressed. Another question is how LFY and AP1 sometimes work together, and sometimes do not, sometimes activate and other times repress. One possibility is that there might be spatio-temporal differences in expression of interaction partners of LFY and AP1 (see also the previous Flowering Highlight on Spatially resolved floral transcriptome profiling by Aalt-Jan van Dijk). Altogether, there is still a lot to be understood about these two ‘very well known’ regulators!


Denay G, Chahtane H, Tichtinsky G, Parcy F. 2017. A flower is born: an update on Arabidopsis floral meristem formation. Current Opinion in Plant Biology 35, 15–22. https://doi.org/10.1016/j.pbi.2016.09.003

Goslin K, Zheng B, Serrano-Mislata A, et al. 2017. Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation. Plant Physiology 174, 1097–1109. https://doi.org/10.1104/pp.17.00098

Kaufmann K, Wellmer F, Muiño JM, et al. 2010. Orchestration of floral initiation by APETALA1. Science 328, 85–89.  https://doi.org/10.1126/science.1185244

Ng M, Yanofsky MF. 2001. Activation of the Arabidopsis B class homeotic genes by APETALA1. The Plant Cell 13, 739–753. https://doi.org/10.1105/tpc.13.4.739

Smaczniak C, Immink RGH, Muiño JM, et al. 2012. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proceedings of the National Academy of Sciences 109, 1560–1565. https://doi.org/10.1073/pnas.1112871109

Wagner D, Sablowski RW, Meyerowitz EM. 1999. Transcriptional activation of APETALA1 by LEAFY. Science 285, 582–584.  https://doi.org/10.1126/science.285.5427.582

Wils CR, Kaufmann K. 2017. Gene-regulatory networks controlling inflorescence and flower development in Arabidopsis thaliana. BBA – Gene Regulatory Mechanisms 1860, 95–105. https://doi.org/10.1016/j.bbagrm.2016.07.014

Winter CM, Yamaguchi N, Wu M-F, Wagner D. 2015. Transcriptional programs regulated by both LEAFY and APETALA1 at the time of flower formation. Physiologia Plantarum 155, 55–73. https://doi.org/10.1111/ppl.12357

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Spatially resolved floral transcriptome profiling

by Alt-Jan Van Dijk
Plant Research International, Wageningen University

Progress in sequencing technology has had a clear impact on flowering research. For example, ChIP-seq has been applied to study molecular aspects of transcriptional regulation of flowering. Also, RNA-seq data for different stages of flower development have been generated in various species (e.g. Singh et al., 2013, Wang et al., 2014, Mantegazza et al., 2014, Han et al., 2017). However, what is still lacking to a large extent is the high-resolution characterization of spatial variation in gene expression. This holds true for plant transcriptomes in general but also specifically for flower development.

A paper by Giacomello et al., 2017 presents an approach to profile plant transcriptomes with high spatial resolution. To do so, it employs semi-randomized primers with barcodes, which indicate the position of a primer on an ~1,000 spot array. Each spot is 100 μm in diameter and provides spatial resolution. After fixation and permeabilization of tissue sections on the array, polyadenylated transcripts are captured by the primers. The transcripts are then converted into cDNA and analyzed by sequencing.

van dijk june 2017

Schematic overview of the approach to profile spatially resolved floral transcriptomes. (Left) Floral tissue is positioned on top of the array surface. (Middle) Transcripts bind to barcoded primers, enabling to recover with high resolution the spatial location from which transcripts originated. Here, brown circles indicate spots on the array, and colours indicate expression of different genes in different locations. (Right) The resulting high-resolution spatial transcriptome was analysed using a model to connect spatial location (i.e. location on the array, indicated here by brown circles) to gene network expression (purple diagram).

Amongst others, Giacomello and colleagues applied their approach to analyze Arabidopsis inflorescence tissue. Comparison with the AtGenExpress Development dataset (Schmid et al., 2005) for five broadly defined tissue domains (stem, meristem, flowers of stage 9, 11 and 12) revealed a reasonable overlap. Note that, of course, the key difference between AtGenExpress and the data by Giacomello et al. is that this new data provides a much higher spatial resolution. Some of the patterns provided for individual genes are rather convincing. For example, they observed ubiquitous expression of the housekeeping gene TUB2, whereas the floral organ identity genes showed expression specifically in flowers. In spots under stamens, markedly higher expression was observed for AP3, PI, and AG than for AP1 and AP2, in agreement with the known expression patterns of these genes. When going through results from individual replicates, for example for AP3 and PI, there also seems to be quite a bit of variation between the replicates. Whether this is evidence of true biological variation or still might indicate technical variation, is not clear to me.

Be that as it may, this study not only presents high resolution data on spatial transcriptomes in floral tissues. In addition, it demonstrates how such data can be analysed. To do so, two key steps are taken. First, not just gene expression levels are used as the variable of interest, but ‘pathway scores’ which reflect the expression level of groups of genes that constitute pathways. Second, the influence of location on expression of the pathways is analysed by not just comparing each pair of locations to each other. Instead, a model is built to analyze the influence that various factors have on expression levels. These factors involve in particular the spatial location, both at the tissue level and at the level of the different spots on the array. One of the reported findings is the enrichment of the stamen filament development pathway in floral stage 11, and that the pollen exine formation pathway was altered in floral stages 10 and 11. Note that these stages indeed produce exine, one of the major constituents of the pollen wall that is deposited on the pre-pollen cells.

This paper represents the next step in the application of sequencing technology to study flowering. It is interesting to see how more and more data relevant for the study of flowers and their development is being generated using sequencing-related techniques. Time course data has been available for a while. Given the increased resolution with which spatial aspects of transcriptome expression can be measured, an important next step will be to measure the expression with both high temporal and high spatial resolution. In addition, it will be exciting to see if applications of sequencing such as ChIP-seq also can be given high spatial resolution.


Giacomello S, Salmén F, Terebieniec BK, et al. 2017.  Spatially resolved transcriptome profiling in model plant species. Nature Plants 3:17061. doi: 10.1038/nplants.2017.61

Han Y,  WanH,  Cheng T,  Wang J,  Yang W, Pan H, Zhang Q.  2017. Comparative RNA-seq analysis of transcriptome dynamics during petal development in Rosa chinensis. Scientific Reports 7: 43382. doi:10.1038/srep43382

Mantegazza O, Gregis V, Chiara M, Selva C, Leo G, Horner DS, and Kater MM. 2014. Gene coexpression patterns during early development of the native Arabidopsis reproductive meristem: novel candidate developmental regulators and patterns of functional redundancy. Plant Journal 79:861-877. doi:10.1111/tpj.12585

Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU.  2005. A gene expression map of Arabidopsis thaliana development. Nature Genetics  37, 501. doi:10.1038/ng1543

Singh VK, Garg R, Jain M. 2013. A global view of transcriptome dynamics during flower development in chickpea by deep sequencing. Plant Journal 11, 691. doi: 10.1111/pbi.12059

Wang H, You C, Chang F, Wang Y, Wang L,Qi J, Ma H. 2014. Alternative splicing during Arabidopsis flower development results in constitutive and stage-regulated isoforms. Frontiers in Genetics 5: doi:25. 10.3389/fgene.2014.00025

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A miraculous mirabilis: the gymnosperm Welwitschia provides new insights into the origin of flowers

by Frank Wellmer
Smurfit Institute of Genetics, Trinity College Dublin, Ireland

The specification of male and female reproductive organs in gymnosperms and angiosperms is thought to be remarkably similar and to depend on the activities of B and C class MADS domain transcription factors. When B and C class factors are co-expressed, male organs are formed, while C class activity alone leads to the development of female organs. It has been shown that the expression of gymnosperm B and C class genes can rescue the developmental defects of floral mutants, in which the homologous organ identity genes are disrupted (Zhang et al., 2004). Thus, it appears that the biochemical activities of the corresponding transcription factors have remained largely unchanged since the two groups of seed bearing plants diverged around 150 million years ago.

Despite these similarities, it is not known how in angiosperms male and female organs became part of the same reproductive unit (i.e. the flower), while in gymnosperms they are separated in unisexual structures (e.g., male and female cones). It has been proposed that changes in the expression patterns of B and/or C function genes, leading to partially overlapping domains of expression and activity, were crucial to the origin of bisexual flowers. However, how these changes may have been brought about is currently not known. To address this question, detailed knowledge on the regulation of B and C class genes in both gymnosperms and angiosperms is required. While over the past 25 years, the regulation of floral organ identity genes has been extensively studied especially in the model angiosperm Arabidopsis thaliana, little is known about how the expression of orthologous genes in gymnosperms is controlled.


A female Welwitschia mirabilis in the Namibian desert by Thomas Schoch

Using Welwitschia mirabilis, a recent study addressed this knowledge gap and provided molecular evidence for the regulation of B class genes by the plant-specific transcription factor LEAFY (LFY) (Moyroud et al., 2017). LFY has been shown previously to be pivotal for triggering the expression of floral organ identity genes during early flower development in Arabidopsis (Busch et al., 1999; Parcy et al., 1998).

In contrast to extant angiosperms, which possess a single LFY gene, gymnosperms typically have two LFY paralogs, one that is LFY-like and one termed NEEDLY (NDLY) or NDLY-like (Frohlich and Parker, 2000). It thus appears that early on during their evolution, angiosperms lost the NDLY-like gene and retained only the gene that is LFY-like. It is attractive to speculate that this change in the complement of known key regulators of B and C class genes may have been a crucial step in the evolution of flowers. However, what are the functions of the LFY and NDLY transcription factors in gymnosperms and how similar are they to those of LFY in angiosperms?

To answer these questions, Moyroud and colleagues first characterized the expression of LFY, NDLY as well as of likely B and C class genes in developing male cones of Welwitschia. They found that the expression of both LFY and NDLY precedes or parallels that of the organ identity genes as would be expected if the corresponding transcription factors were involved, as LFY in angiosperms, in activating the expression of B and C class genes. Furthermore, they observed that at later stages of male cone development the expression of LFY and B class genes was noticeably different to that of NDLY and the C class gene under study. Thus, based on the expression patterns of these genes alone, it can be hypothesized that LFY may control B class genes, while NDLY might be involved in the control of C class gene activity. In support of this idea, Moyroud and colleagues showed, using advanced biochemical and biophysical techniques, that LFY and NDLY have overlapping but distinct sets of binding sites. They further demonstrated that LFY from Welwitschia as well as from other gymnosperms can bind to putative regulatory elements in the promoters of B class genes.

Thus, it appears that LFY in both gymnosperms and angiosperms plays a key role in the control of these organ identity genes. While the molecular activities of NDLY need to be further characterized, the study by Moyroud et al., (2017) led to the attractive and testable hypothesis that this transcription factor may not share all functions with its paralog LFY and might control C class gene activity in gymnosperms.


Busch MA, Bomblies K, Weigel D. 1999. Activation of a floral homeotic gene in Arabidopsis. Science 285, 585-587. https://doi.org/10.1126/science.285.5427.585
Frohlich MW, Parker DS. 2000. The mostly male theory of flower evolutionary origins: from genes to fossils. Systematic Botany 25, 155-170. http://dx.doi.org/10.2307/2666635
Moyroud E, Monniaux M, Thevenon E, Dumas R, Scutt CP, Frohlich MW, Parcy F. 2017. A link between LEAFY and B-gene homologues in Welwitschia mirabilis sheds light on ancestral mechanisms prefiguring floral development. New Phytologist DOI: https://doi.org/10.1111/nph.14483
Parcy F, Nilsson O, Busch MA, Lee I, Weigel D. 1998. A genetic framework for floral patterning. Nature 395, 561-566. https://doi.org/10.1038/26903
Zhang P, Tan HT, Pwee KH, Kumar PP. 2004. Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms. The Plant Journal 37, 566-577. https://doi.org/10.1046/j.1365-313X.2003.01983.x

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