A surprising role for ethylene in the regulation of petal cell shapeBeverley GloverDepartment of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UKbjg26@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 minimize 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).

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 are 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 >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 occurs, they studied the three members of the TCP5-like

doi:10.1093/jxb/erz063

transcription factor family in Arabidopsis. These proteins represent a sister group to the five 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 green fluorescent protein (GFP) in the petal epidermis (using an L1-specific promoter) produced smaller petals, suggesting that the epidermis itself is regulating the 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 those of the 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 the 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.

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

Similarly, the activity of ethylene response factor genes (ERF genes) 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 the 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 hypothesize 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 co-ordinated 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.

ReferencesHuang T, Irish VF. 2015. Temporal control of plant organ growth by TCP transcription factors. Current Biology 25, 1765–1770.Noda K, Glover BJ, Linstead P, Martin C. 1994. Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature 369, 661–664.Sapala A, Runions A, Routier-Kierzkowska A, et al. 2018. Why plants make puzzle cells and how their shape emerges. eLife 7, e32794van Es S, Sylveira S, Rocha D, Bimbo A, Martinelli A, Dornelas M, Angenent G, Immink R. 2018. Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis. The Plant Journal 94, 867–879.Whitney HM, Bennett KM, Dorling M, Sandbach L, Prince D, Chittka L, Glover BJ. 2011. Why do so many petals have conical epidermal cells? Annals of Botany 108, 609–616.Whitney HM, Chittka L, Bruce TJ, Glover BJ. 2009. Conical epidermal cells allow bees to grip flowers and increase foraging efficiency. Current Biology 19, 948–953.