Pursuing The Elusive Explanation For Leaf Shape

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Main Text

Introduction

No single shape defines the leaves of a plant. The shape of each leaf changes as it allometrically expands during development. The leaves displayed by a plant at successive nodes reflect the developmental and environmental context of each leaf from initiation onwards. If there were a true leaf shape phenotype representative of a particular genotype, it would include the ontogenetic trajectory of leaves from the primordium stage through the senescence of the mature organ, both the first-emerged juvenile leaves and subsequent adult leaves, the shapes of leaves found throughout a canopy, the year-to-year variations in leaves produced by long-lived perennials, and the leaf morphs associated with the innumerable environments a genotype might theoretically encounter.

It is within the above morphospace of intra-genotypic differences in leaf shape that evolution acts to create morphological diversity. Perhaps only a small subset of all possible leaf shapes have been measured for any single genotype, but a truly quantitative comparison of the diversity of leaf shapes observed among extant species — let alone the developmental and environmental plasticity of these leaves — remains barely examined. The molecular mechanisms underlying such morphological diversity are even more poorly understood. Yet, patterns of correlation among leaf shapes across extant species, temperature and precipitation, and between fossil leaves and the paleoclimate, provide tantalizing glimpses into hypotheses unifying the development, evolution, and environmental plasticity of leaf shape with the possible functions it might confer.

Here, we review our current understanding of the molecular pathways underlying leaf development, environmental plasticity, and the context of these phenomena within plant evolution and the paleorecord. We begin by comparing methods to measure leaf shape and what is known about the underlying quantitative genetic basis of leaf shape determination. We then describe intra-genotypic sources of leaf shape variance and the environmental inputs that modulate leaf shape over the lifetime of a plant. Juvenile-to-adult phase change in leaf shape (heteroblasty) is hastened by signaling through sugar (produced by photosynthesis) and the reduced activity of miR156 regulating SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes, whereas abrupt changes in morphology over the leaf series in response to the environment (heterophylly) are mediated by temperature and KNOTTED1-LIKE HOMEOBOX (KNOX) genes. We end by noting the correlation between leaf dissection and temperature/precipitation in the paleorecord, and that the direction of this correlation mirrors the environmental plasticity observed during the lifetime of a plant. An understanding of the changes in leaf shape over geologic time and plant evolution, combined with knowledge of molecular pathways regulating environmental plasticity and developmental timing during the lifetime of a single plant, promises to provide a functional context for leaf shape in response to unpredictable future climates.

Towards a Quantitative Synthesis of Leaf Shape

The measurement of leaf shape is an interesting modeling problem, in part because the shape of a leaf is in constant flux, as different regions expand at different rates (allometry). The allometric expansion of leaves was described as early as 1727, when Stephen Hales pricked a regular grid of points in a young fig leaf that subsequently deformed as it expanded [1]. Studies using this time-honored method recently demonstrated divergent patterns of leaf expansion among 75 core eudicot species that strongly correlate with miR396 and its targets, GROWTH-REGULATING FACTOR (GRF) genes [2]. Chimeric events in petals [3] and fluorescent particles 4 and 5 quantitatively reveal gradients of growth-regulating substances and tissue deformation. Such deformation, leading to leaf shape differences between species, can occur late in development [6]. Further, ultimatey leaf shape is impacted by the shoot apical meristem from which leaf primordia arise, and the genetic basis underlying this morphology has recently been described in maize and correlates with important agronomic traits [7].

Analyzing the terminal morphology of leaves yields insights into the quantitative genetic mechanisms by which leaf shape changes during evolution. Simply measuring length and width captures a substantial amount of shape variation in maize leaves, which quantitative trait loci (QTL) analysis reveals is mostly comprised of small, additive effects in many genes [8]. For more sophisticated leaf shape analyses, a common approach is to place equidistant points along the contour (pseudo-landmarks), anchored by homologous points found in every leaf (typically the tip and base of the petiole; Figure 1A). Such an approach has been used to describe evolution of Anitrrhinum leaf morphology as traversal through a morphospace [9] and to demonstrate that the entire leaf shape of Arabidopsis is a derived feature [10]. Alternatively, rather than using a limited number of landmarks, an outline can be thought of as a wave connecting back into itself, forming a closed contour to which a Fourier analysis can be applied (Figure 1B). This approach, known as Elliptical Fourier Descriptors 11, 12 and 13, treats shape as a decomposed harmonic series and has been applied to tomato and grapevine, revealing that, like in maize, leaf shape is polygenic, mostly additive, and highly heritable 14 and 15. Although not possible for all leaves, if homologous points can be found in every leaf sampled, a simple landmark analysis is a powerful morphometric approach, as has been used in grapevine and Potentilla ( Figure 1C) 16 and 17. The selection of morphometric method is important, as each method measures different shape features, biasing results (Figure 1D,E). The current difficulty in modeling macro-evolutionary differences between leaves, or comparing results between studies using a common method, remains a major hurdle hampering a truly global, comparative analysis of leaf shape.

Recently, morphometric studies in leaves have turned to the last remaining major source of shape variance: changes in leaf shape at successive nodes, or heteroblasty (Figure 2). Heteroblasty is the change in features of lateral organs arising from successive nodes and reflects the temporal development of the meristem as it transitions from juvenile to adult vegetative phases, up through and including the transition to reproductive fate. Pathways regulating flowering time can have roles in vegetative development independent of their roles in flowering [18]. However, natural variation in the cis regulation of the floral repressor FLC alters leaflet number among Cardamine hirsuta accessions, linking early flowering to precocious leaflet formation in juvenile leaves [19]. If instead natural variation in flowering time is used as a random effect in models to statistically eliminate its effects (rather than making it an object of study), heteroblastic changes in tomato leaf shape are lowly heritable and are similar between tomato and its wild relatives [20]. Using discriminant analyses in grapevine leaves, a similar conclusion is reached: across Vitis spp., changes in leaf shape across successive nodes are highly conserved, so much so that node position can be predicted separately from species identity [17].

Such an interpretation, which suggests that differences in leaf shape between species and the heteroblastic series are independent and affect different shape features of the leaf, is consistent with recent molecular insights suggesting that different pathways regulate these phenomena, which we discuss next.

Heteroblasty: Light, Sugar, and mir156

Goebel initially hypothesized that juvenile leaves represent arrested leaf development due to limiting photosynthate. He reasoned that the first-produced leaves on many plants are often irregular in shape, consistent with shade producing similarly shaped leaves and seemingly prolonging the production of juvenile leaf shapes. Later in development, more leaves provide more photosynthate, creating a fully developed, adult leaf [21]. Heteroblasty affects many leaf morphology features in addition to shape and complexity. In Arabidopsis thaliana, the presence of abaxial trichomes was used as a marker of heteroblasty [22], a phenotype that overlaps with leaf polarity regulated by adaxial−abaxial regulators, such as KANADI [23]. But the initial conflation of adaxial−abaxial identity with heteroblasty in Arabidopsis is fortuitous: tasiR-ARFs (trans-acting short interfering RNAs that regulate AUXIN RESPONSE FACTORs) regulate both heteroblastic 24 and 25 as well as adaxial−abaxial identity pathways 26, 27 and 28.

Antagonism between another class of small RNAs — miR156 and miR172 — which target SPL (SQUAMOSA PROMOTER BINDING-LIKE) and AP2 (APETALA2) genes, respectively, more directly regulates juvenile-to-adult phase transitions and the overall transition to reproductive fate 29 and 30. The effects of these antagonistic small RNAs on developmental transitions is perhaps most dramatic in maize, where the activity of miR172 downregulating the AP2-like glossy15 gene [31] regulates juvenile-to-adult phase transition of epidermal cell features, such as epicuticular waxes [32], and overexpression of miR156 in the Corngrass1 mutant confers species-level, neotenic-like changes in development, prolonging juvenility by targeting teosinte glume architecture1 and other transcripts [33].

The antagonistic relationship between miR156 and miR172 regulating heteroblastic development is nearly universal among flowering plants and occurs in many profoundly heteroblastic species, including Eucalyptus [34], Quercus acutissima (sawtooth oak), Hedera helix (English ivy), and the transition from juvenile bipinnate compound leaves to adult phyllodes in Acacia species ( Figure 2) [35].

Can the developmental ‘clock’ regulating heteroblastic changes through miR156 and miR172 be separated from the proximate mechanisms by which morphological changes are produced in leaves? Elegant work in Arabidopsis thaliana and the compound-leaved species Cardamine hirsuta demonstrates that as SPL protein levels increase during juvenile-to-adult phase transition, they complex with and titrate away TCPs (TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORs) from a complex they form with CUCs (CUP-SHAPED COTYLEDONs) that prevents functional CUC dimers from forming. The increasing levels of CUC then proceed to increase the number of serrations and leaflets as the heteroblastic series progresses [36].

TCPs, important transcription factors mediating cell proliferation in developing leaves 37 and 38, seem to be specifically associated with SPLs and heteroblastic development. Transcriptional studies show TCP activity in conjunction with light regulates heteroblastic development in the basal eudicot Gevuina avellana (Proteaceae) under forest canopy [39] and correlates with heteroblastic development (and not the shade avoidance response) in the shoot apex of Solanum lycopersicum (tomato) [40], suggesting that TCP-mediated licensing of CUC activity is widespread throughout the eudicots (Figure 3A).

Although miR156 is sufficient to regulate heteroblastic transitions, what is the ultimate signal controlling developmental timing? Inspired by Goebel’s original hypothesis that the nutritional status of the shoot mediates changes in the morphology of leaves at successive nodes [21] and subsequent experiments in the water fern Marsilea 41, 42 and 43 and flowering plants 44, 45, 46 and 47 demonstrating that sugar affects leaf morphology, two papers have formally established a mechanistic relationship between increasing sugar and the repression of miR156 activity hastening the heteroblastic series 48 and 49. These classic experiments use seedling defoliation, photosynthetic mutants, and mutations in HEXOKINASE1 signaling to demonstrate the ability of sugar to act as a heteroblastic signaling molecule (Figure 3A).

miR156 and tasiRNA regulation of developmental timing is conserved in the convergent, leaf-like phyllids of the moss Physcomitrella patens [50]. But even beyond the land plants, and similar to or separate from a miR156 mechanism, Goebel’s hypothesis that the nutritional status of photosynthetic organisms mediates transitions in developmental timing may explain heteroblastic-like phenomena in distantly related lineages to the land plants, including the branched whorls of the chlorophyte Acetabularia acetabulum [51] and the dissected fronds of the brown alga Laminaria digitata ( Figure 2) [52].

Heterophylly: Temperature, Plasticity, and KNOX Genes

TCPs, SPLs, and miR156 are broadly conserved and seem to universally regulate heteroblastic transitions in the land plants, but their role in mediating differences in leaf morphology between species seems less clear. Although in some leguminous lineages LEAFY (LFY) activity modulates leaf complexity [53], the molecular pathway principally responsible for modulating evolutionary difference in leaf form involves the KNOX (KNOTTED1-LIKE HOMEOBOX) genes, which specify meristematic indeterminacy [54] and are negatively regulated by ARP (ASYMMETRIC LEAVES1, ROUGH SHEATH2, PHANTASTICA) proteins 55 and 56 to allow lateral organ differentiation to occur. KNOX activity is often sufficient to confer increased leaf complexity by reactivating indeterminacy within differentiated leaf primordia to produce leaflets and increase leaf dissection 57 and 58. The interaction between KNOX and ARP activity is predictive of compound leaf morphology in primordia across the flowering plants 6 and 59. Changes in leaf complexity are labile, and KNOX pathways have been found to increase leaf complexity in Galapagean wild tomatoes [60] and are necessary for leaf complexity in Cardamine hirsuta as well [61].

Changes in leaf complexity and dissection occur not only between species over evolutionary time, but also during the lifetime of an organism. In contrast to heteroblasty, which is developmentally programmed changes in the morphology and phenotype of lateral organs linked to juvenile-to-adult and reproductive phase change, heterophylly produces dramatic, and often abrupt, changes in leaf morphology in response to the environment [62]. The most well known examples of heterophylly are arguably found in aquatic plants, in which submerged leaves are often highly dissected and exhibit repressed laminar outgrowth compared with aerial leaves [63]. Classic work suggested that heterophylly in some species is mediated by light and limiting amounts of photosynthate [21].

Recent work in the amphibious plant Rorippa aquatica (Brassicaceae) confirms this view, and also demonstrates an important contribution of temperature in inducing leaf dissection in this heterophyllous species ( Figure 3B) [64]. Remarkably, plants swapped between temperatures can produce leaves with the distal half of their morphology reflecting the first condition and the proximal half of their shape reflecting the second, showing that aquatic heterophylly in this species is continuously specified during leaf development and that these morphological changes can be specified very abruptly. The expression of class I KNOX genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) predict the morphology of leaves, whether induced by submergence, cold, or low light intensity. The expression of CUP-SHAPED COTYLEDONS3 (CUC3), which confers leaf dissection and forms a positive feedback loop of regulation with KNOX gene activity [65], and GA activity, which KNOX pathways inhibit [66], also track heterophyllous changes in leaf shape.

In the above case of aquatic heterophylly, the plasticity in leaf morphology is immediate and abrupt, changing with the environmental stimulus, suggesting that KNOX activity modulates morphological changes across the leaf series through plasticity, rather than a strictly heteroblastic mechanism. Another example of plasticity in plants is the shade-avoidance response. In response to a reduced ratio of red to far-red wavelengths of light, due to shading by other plants, phytochrome proteins signal a syndrome of plastic responses, including increased leaf area and internode lengths to overgrow competitors 67, 68 and 69. In tomato, leaf complexity increases as well, suggesting that morphological patterning has been altered in leaf primordia, and like heterophylly, changes in leaf morphology can change mid-leaf series in plants swapped between light conditions [40]. Micro-dissection of the first initiated leaf primordium (P1) and the shoot apical meristem and the incipient leaf (SAM + P0) indicates that increased expression of the tomato class I KNOX gene LeT6 and the CUC family member GOBLET [70] mediate the increases in leaf complexity [40]. The transcripts regulated by heteroblasty (including the TCP member Lanceolate) [71] largely do not overlap with those altered by the shade avoidance response in the shoot apical meristem, as shown by a transcriptomic analysis performed on leaf primordia and the meristem at the same developmental stage but over a time course. These results indicate that distinct molecular pathways regulate heteroblasty vs. plasticity, at least with respect to the shade-avoidance response (Figure 3B).

Class I KNOX genes are not the only homeobox genes associated with specifying both leaf complexity and environmental response. Recently, homologs of the class I homeodomain leucine zipper (HD-ZIP I) gene LATE MERISTEM IDENTITY1 (LMI1), originally described as a LFY target with LFY-independent roles in leaf serration formation [72], have been described as underlying the phenotype of the REDUCED LEAF COMPLEXITY (RCO) mutation in Cardamine hirsuta [73]. Cis-regulatory variation in this locus has independently led to evolutionary differences in the depth of marginal serrations between Capsella species [74]. Like the temperature-dependent heterophylly in Rorippa aquatica, lobing in Capsella becomes more pronounced under colder temperatures. This temperature-dependent change in morphology is concomitant with increased RCO expression due to cis regulation ( Figure 3B).

Whereas the regulation of heteroblasty — through miR156 activity — through the accumulation of photosynthate and the nutritional status of the plant seems to be widespread and conserved, our knowledge of plasticity is more limited, perhaps because the number of environmental conditions a plant may encounter are limitless and likely change depending on the species. However, it is remarkable that KNOX genes (and other homeobox genes like RCO) are regulated by temperature and changes in light quality. Not only that, the direction of change itself is not hardwired — increasing in response to foliar shade in tomato but decreasing in response to low light in Rorippa aquatica. The available evidence suggests a tentative model where heteroblasty, through miR156, SPL, and TCP activity, is sensitive to light intensity and sugar, whereas KNOX genes and other homeobox genes mediate plasticity and heterophyllic responses in response to light quality and especially temperature ( Figure 3C). The downstream output of these distinct environmental inputs and pathways is the CUC genes, which mediate leaf serration and lobing, potentially explaining leaf dissection as a common environmental response in leaves affected by many factors but distinct pathways.

Leaf Shape in Ancient, Present, and Future Climates: Concerted Evolution and Plasticity

Distinct pathways have evolved to mediate heteroblastic changes in leaf shape in response to photosynthesis and plastic, heterophyllous responses to changes in temperature and light quality (Figure 3C). Such exquisite integration of environmental and developmental inputs, coupled with changes in leaf shape across evolution (Figure 2), demonstrates the sensitivity of leaf shape to the environment. As the molecular pathways and morphometric features underpinning environmentally responsive changes in leaf shape continue to be resolved, the functions such changes confer to the disparate environments plants have — and will — confront can be examined.

Relationships between leaf dissection and paleoclimates provide the most sweeping evidence of the responsiveness of leaf dissection to temperature and precipitation (Figure 4A) [75]. Entire leaves are associated with tropical and sub-tropical climates and leaf serration can be used as a reliable index of the paleoclimate (Figure 4B) 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 and 91. The relationship between shape and climate — such that larger, entire leaves are associated with warmer, wetter environments — has been confirmed in extant populations [92] and occurs within the context of phylogenetic effects on leaf shape [93]. The relationship between leaf shape and climate can evolve repeatedly in response to similar environments, as documented in Viburnum [94].

Notably, plastic responses in leaf shape track correlations found in the fossil record. Transplant experiments demonstrate increased leaf dissection in Acer rubrum in temperate compared with sub-tropical climates [95]. In grapevine, leaves measured from the same vines across multiple seasons show trends mirroring the paleorecord, such that a more pronounced sinus is associated with the colder, drier growing season [96]. We also note that the increased leaf dissection observed at colder temperatures in Rorippa aquatica [64] and Capsella [74] are consistent with those found over geologic time. That plasticity within the lifetime of a single plant, extant species distributions, and correlations with the paleoclimate follow each other suggests an intimate relationship between leaf morphology and climate. Whether the molecular pathways we have described — especially KNOX and RCO — mediate such phenomena remains to be determined (Figures 3 and 4).

The hypotheses concerning the possible functions of dissected leaves are many and depend on the scale being discussed. Paleoclimate correlations are focused on marginal serrations [97], which have been hypothesized as a constraint arising in thinner leaves more reliant on major veins 82 and 98, an adaptive feature allowing early season growth in deciduous forests [99], sites of increased transpiration [100], or that arise from hydathodes to maintain optimal leaf turgor [101]. Similar plasticity in annuals observed in cold temperatures involves more than just serrations and cannot be explained by features exclusive to woody eudicots alone 64 and 74. Leaf dissection may simply reduce leaf area and affect canopy light interception while also minimizing the distance between photosynthetic tissue and veins 102 and 103. The potentially reduced leaf area in dissected leaves also facilitates heat transfer, reducing the boundary layer effect [104], but leaf shape likely also affects boundary layers independent of size 105 and 106. Leaf dissection may even reflect developmental constraints related to packing in buds [107]. The possible functions of compound leaves have centered on the associated costs of producing a branch with many leaves vs. a complex leaf with many leaflets, and the premium put on vertical growth and ‘branch shedding’ [108]. While hypotheses about the function of leaf shape are fascinating, they are numerous and cannot be generalized. Focusing on changes in shape common to evolutionary transitions into new environments, plastic responses in model organisms and patterns observed in the paleorecord may help narrow the field of relevant hypotheses to arrive at a more robust understanding of leaf morphology.

Conclusions

Integrating natural variation in leaf shape with molecular pathways has always been a central goal of evolutionary developmental biology. The realization that distinct pathways are modulated by specific environmental inputs, though, raises the tantalizing possibility that a synthesis explaining historical patterns observed in the paleorecord, conserved plastic responses in extant plants, and the extensive developmental genetic theory of leaves may be achieved. Such a synthesis will require an increased physiological focus among developmental biologists and ecologists incorporating molecular approaches into their functional studies of leaves. If leaf shape is a functional response of plants to changes in the environment, then understanding its adaptive value — and how to molecularly manipulate it — will prove to be invaluable in breeding the next generation of crops and sustainably maintaining biodiversity and crop yield in future climates.