Stomatal patterning is differently regulated in adaxial and abaxial epidermis in Arabidopsis.

Stomatal pores in leaves mediate CO2 uptake into the plant and water loss via transpiration. Most plants are hypostomatous with stomata present only in the lower leaf surface (abaxial epidermis). Many herbs, including the model plant Arabidopsis thaliana, have substantial numbers of stomata also on the upper (adaxial) leaf surface. Studies of stomatal development have mostly focused on abaxial stomata and very little is known of adaxial stomatal formation. We addressed the role of leaf number in determination of stomatal density and stomatal ratio, and studied adaxial and abaxial stomatal patterns in mutants deficient in known abaxial stomatal development regulators. We found that stomatal density in some genetic backgrounds varies between different fully expanded leaves and recommend using defined leaves for analyses of stomatal patterning. Our results indicate that stomatal development is at least partly independently regulated in adaxial and abaxial epidermis, as i) plants deficient in ABA biosynthesis and perception have increased stomatal ratios, ii) the epf1epf2, tmm and sdd1 mutants have reduced stomatal ratios, iii) erl2 mutants have increased adaxial but not abaxial stomatal index, and iv) stomatal precursors preferentially occur in abaxial epidermis. Further studies of adaxial stomata can reveal new insights into stomatal form and function.

Increased cuticular wax deposition does not change residual foliar transpiration.

The effect of contrasting environmental growth conditions (in vitro tissue culture, ex vitro acclimatisation, climate chamber, greenhouse and outdoor) on leaf development, cuticular wax composition, and foliar transpiration of detached leaves of the Populus × canescens clone 84 K were investigated. Our results show that total amounts of cuticular wax increased more than 10-fold when cultivated in different growth conditions, whereas qualitative wax composition did not change. With exception of plants directly taken from tissue culture showing rapid dehydration, rates of water loss (residual foliar transpiration) of intact but detached leaves were constant and independent from growth conditions and thus independent from increasing wax amounts. Since cuticular transpiration measured with isolated astomatous P. × canescens cuticles was identical to residual foliar transpiration rates of detached leaves, our results confirm that cuticular transpiration of P. × canescens leaves can be predicted with high accuracy from residual transpiration of detached leaves after stomatal closure. Our results convincingly show that more than 10-fold increased wax amounts in P. × canescens cuticles do not lead to decreased rates of residual (cuticular) transpiration.

Leaf hydraulic conductance is linked to leaf symmetry in bifacial, amphistomatic leaves of sunflower.

The hydraulic implications of stomatal positioning across leaf surfaces and the impact on internal water flow through amphistomatic leaves are not currently well understood. Amphistomaty potentially provides hydraulic efficiencies if the majority of hydraulic resistance in the leaf exists outside the xylem in the mesophyll. Such a scenario would mean that the same xylem network could equally supply a hypostomatic or amphistomatic leaf. Here we examine leaves of Helianthus annuus to determine whether amphistomaty in this species is associated with higher hydraulic efficiency compared with hypostomatic leaves. We identified asymmetry in the positioning of minor veins which were significantly closer to the abaxial than the adaxial leaf surface, combined with lower Kleaf when transpiration was driven through the adaxial rather than the abaxial surface. We also identified a degree of coordination in stomatal behaviour driven by leaf hydraulics, where the hydraulic conditions experienced by an individual leaf surface affected the stomatal behaviour on the opposite surface. We found no advantage to amphistomaty based on efficiencies in construction costs of the venous system, represented by vein density:stomatal density, only limited hydraulic independence between leaf surfaces. These results suggest that amphistomaty does not substantially increase whole-leaf hydraulic efficiency.

A role for SPEECHLESS in the integration of leaf stomatal patterning with the growth vs disease trade-off in poplar.

Occurrence of stomata on both leaf surfaces (amphistomaty) promotes higher stomatal conductance and photosynthesis while simultaneously increasing exposure to potential disease agents in black cottonwood (Populus trichocarpa). A genome-wide association study (GWAS) with 2.2M single nucleotide polymorphisms generated through whole-genome sequencing found 280 loci associated with variation in adaxial stomatal traits, implicating genes regulating stomatal development and behavior. Strikingly, numerous loci regulating plant growth and response to biotic and abiotic stresses were also identified. The most significant locus was a poplar homologue of SPEECHLESS (PtSPCH1). Individuals possessing PtSPCH1 alleles associated with greater adaxial stomatal density originated primarily from environments with shorter growing seasons (e.g. northern latitudes, high elevations) or with less precipitation. PtSPCH1 was expressed in developing leaves but not developing stem xylem. In developing leaves, RNA sequencing showed patterns of coordinated expression between PtSPCH1 and other GWAS-identified genes. The breadth of our GWAS results suggests that the evolution of amphistomaty is part of a larger, complex response in plants. Suites of genes underpin this response, retrieved through genetic association to adaxial stomata, and show coordinated expression during development. We propose that the occurrence of amphistomaty in P. trichocarpa involves PtSPCH1 and reflects selection for supporting rapid growth over investment in immunity.

Fossil evidence for open, Proteaceae-dominated heathlands and fire in the Late Cretaceous of Australia.

PREMISE OF THE STUDY: The origin of biomes is of great interest globally. Molecular phylogenetic and pollen evidence suggest that several plant lineages that now characterize open, burnt habitats of the sclerophyll biome, became established during the Late Cretaceous of Australia. However, whether this biome itself dates to that time is problematic, fundamentally because of the near-absence of relevant, appropriately aged, terrestrial plant macro- or mesofossils. METHODS: We recovered, identified, and interpreted the ecological significance of fossil pollen, foliar and other remains from a section of core drilled in central Australia, which we dated as Late Campanian-Maastrichtian. KEY RESULTS: The sediments contain plant fossils that indicate nutrient-limited, open, sclerophyllous vegetation and abundant charcoal as evidence of fire. Most interestingly, >30 pollen taxa and at least 12 foliage taxa are attributable to the important Gondwanan family Proteaceae, including several minute, amphistomatic, and sclerophyllous foliage forms consistent with subfamily Proteoideae. Microfossils, including an abundance of Sphagnales and other wetland taxa, provided strong evidence of a fenland setting. The local vegetation also included diverse Ericaceae and Liliales, as well as a range of ferns and gymnosperms. CONCLUSIONS: The fossils provide strong evidence in support of hypotheses of great antiquity for fire and open vegetation in Australia, point to extraordinary persistence of Proteaceae that are now emblematic of the Mediterranean-type climate southwestern Australian biodiversity hotspot and raise the profile of open habitats as centers of ancient lineages.

Association genetics, geography and ecophysiology link stomatal patterning in Populus trichocarpa with carbon gain and disease resistance trade-offs.

Stomata are essential for diffusive entry of gases to support photosynthesis, but may also expose internal leaf tissues to pathogens. To uncover trade-offs in range-wide adaptation relating to stomata, we investigated the underlying genetics of stomatal traits and linked variability in these traits with geoclimate, ecophysiology, condensed foliar tannins and pathogen susceptibility in black cottonwood (Populus trichocarpa). Upper (adaxial) and lower (abaxial) leaf stomatal traits were measured from 454 accessions collected throughout much of the species range. We calculated broad-sense heritability (H(2) ) of stomatal traits and, using SNP data from a 34K Populus SNP array, performed a genome-wide association studies (GWAS) to uncover genes underlying stomatal trait variation. H(2) values for stomatal traits were moderate (average H(2) = 0.33). GWAS identified genes associated primarily with adaxial stomata, including polarity genes (PHABULOSA), stomatal development genes (BRASSINOSTEROID-INSENSITIVE 2) and disease/wound-response genes (GLUTAMATE-CYSTEINE LIGASE). Stomatal traits correlated with latitude, gas exchange, condensed tannins and leaf rust (Melampsora) infection. Latitudinal trends of greater adaxial stomata numbers and guard cell pore size corresponded with higher stomatal conductance (gs ) and photosynthesis (Amax ), faster shoot elongation, lower foliar tannins and greater Melampsora susceptibility. This suggests an evolutionary trade-off related to differing selection pressures across the species range. In northern environments, more adaxial stomata and larger pore sizes reflect selection for rapid carbon gain and growth. By contrast, southern genotypes have fewer adaxial stomata, smaller pore sizes and higher levels of condensed tannins, possibly linked to greater pressure from natural leaf pathogens, which are less significant in northern ecosystems.

Simultaneous and Independent Abaxial and Adaxial Gas Exchange Measurements.

Stomata can be distributed exclusively on the abaxial or adaxial leaf surface, but they are most commonly found on both leaf surfaces. Variations in stomatal arrangement, patterning, and the impact on photosynthesis can be measured using an infrared gas exchange system. However, when using standard gas exchange techniques, both surfaces are measured together and averaged to provide leaf-level values. Employing an innovative gas exchange apparatus with two infrared gas analyzers, separate gaseous flux from both leaf surfaces can be quantified simultaneously and independently. Here, we provide examples of typical measurements that can be performed using a "split chamber" gas exchange system.

A comparative study of the distribution and density of stomata in the British flora.

The distribution of stomata over both leaf surfaces may affect both the photosynthetic rate and water use efficiency of species, implying that species with different photosynthetic and water requirements may also have different stomatal distributions. A database containing data on the distribution of stomata on the leaves of 469 British plant species was used to look for relationships between stomatal distribution (including both location on the leaf and density) and both habitat and morphological variables. Statistical models were applied to the data that minimized any effects that phylogenetic constraints may have had on the data. Hypostomaty is common in woody species, species which typically occur in shaded habitats and species with large or glabrous leaves. Amphistomaty, however, predominates in species which occur in non-shaded habitats, species with small, dissected or hairy leaves, and in annual species. Amphistomaty, therefore, tends to occur in species where CO2 may be limiting photosynthesis (unshaded environments), or where there are structures to prevent water loss from the leaf (e.g. hairs). Hypostomaty, however, occurs in slow-growing species (e.g. trees), species with leaves which have large boundary layers (large or entire leaves) and in species where CO2 is unlikely to limit photosynthesis (shaded habitats).