Stomatal Abundance Research Articles

An extremely low stomatal density mutant overcomes cooling limitations at supra-optimal temperature by adjusting stomatal size and leaf thickness.

The impact of global warming on transpiration and photosynthesis would compromise plant fitness, impacting on crop yields and ecosystem functioning. In this frame, we explored the performance of a set of Arabidopsis mutants carrying partial or total loss-of-function alleles of stomatal development genes and displaying distinct stomatal abundances. Using microscopy and non-invasive imaging techniques on this genotype collection, we examined anatomical leaf and stomatal traits, plant growth and development, and physiological performance at optimal (22°C) and supra-optimal (30°C) temperatures. All genotypes showed thermomorphogenetic responses but no signs of heat stress. Data analysis singled out an extremely low stomatal abundance mutant, spch-5. At 22°C, spch-5 had lower transpiration and warmer leaves than the wild type. However, at 30°C, this mutant developed larger stomata and thinner leaves, paralleled by a notable cooling capacity, similar to that of the wild type. Despite their low stomatal density (SD), spch-5 plants grown at 30°C showed no photosynthesis or growth penalties. The behavior of spch-5 at supra-optimal temperature exemplifies how the effect of very low stomatal numbers can be counteracted by a combination of larger stomata and thinner leaves. Furthermore, it provides a novel strategy for coping with high growth temperatures.

A Genetic Dissection of Natural Variation for Stomatal Abundance Traits in Arabidopsis.

Stomatal abundance varies widely across natural populations of Arabidopsis thaliana, and presumably affects plant performance because it influences water and CO2 exchange with the atmosphere and thence photosynthesis and transpiration. In order to determine the genetic basis of this natural variation, we have analyzed a recombinant inbred line (RIL) population derived from the wild accession Ll-0 and the reference strain Landsberg erecta (Ler), which show low and high stomatal abundance, respectively. Quantitative trait locus (QTL) analyses of stomatal index, stomatal density, and pavement cell density measured in the adaxial cotyledon epidermis, identified five loci. Three of the genomic regions affect all traits and were named MID (Modulator of Cell Index and Density) 1 to 3. MID2 is a large-effect QTL overlapping with ERECTA (ER), the er-1 allele from Ler increasing all trait values. Additional analyses of natural and induced loss-of-function er mutations in different genetic backgrounds revealed that ER dysfunctions have differential and opposite effects on the stomatal index in adaxial and abaxial cotyledon epidermis and confirmed that ER is the gene underlying MID2. Ll-0 alleles at MID1 and MID3 displayed moderate and positive effects on the various traits. Furthermore, detailed developmental studies tracking primary and satellite stomatal lineages show that MID3-Ll-0 allele promotes the spacing divisions that initiate satellite lineages, while the ER allele limits them. Finally, expression analyses suggest that ER and MID3 modulate satellization through partly different regulatory pathways. Our characterization of MID3 indicates that genetic modulation of satellization contributes to the variation for stomatal abundance in natural populations, and subsequently that this trait might be involved in plant adaptation.

The Tomato Genome Encodes SPCH, MUTE, and FAMA Candidates That Can Replace the Endogenous Functions of Their Arabidopsis Orthologs.

Stomatal abundance determines the maximum potential for gas exchange between the plant and the atmosphere. In Arabidopsis, it is set during organ development through complex genetic networks linking epidermal differentiation programs with environmental response circuits. Three related bHLH transcription factors, SPCH, MUTE, and FAMA, act as positive drivers of stomata differentiation. Mutant alleles of some of these genes sustain different stomatal numbers in the mature organs and have potential to modify plant performance under different environmental conditions. However, knowledge about stomatal genes in dicotyledoneous crops is scarce. In this work, we identified the Solanum lycopersicum putative orthologs of these three master regulators and assessed their functional orthology by their ability to complement Arabidopsis loss-of-function mutants, the epidermal phenotypes elicited by their conditional overexpression, and the expression patterns of their promoter regions in Arabidopsis. Our results indicate that the tomato proteins are functionally equivalent to their Arabidopsis counterparts and that the tomato putative promoter regions display temporal and spatial expression domains similar to those reported for the Arabidopsis genes. In vivo tracking of tomato stomatal lineages in developing cotyledons revealed cell division and differentiation histories similar to those of Arabidopsis. Interestingly, the S. lycopersicum genome harbors a FAMA-like gene, expressed in leaves but functionally distinct from the true FAMA orthologue. Thus, the basic program for stomatal development in S. lycopersicum uses key conserved genetic determinants. This opens the possibility of modifying stomatal abundance in tomato through previously tested Arabidopsis alleles conferring altered stomata abundance phenotypes that correlate with physiological traits related to water status, leaf cooling, or photosynthesis.

Overexpression of a SDD1-Like Gene From Wild Tomato Decreases Stomatal Density and Enhances Dehydration Avoidance in Arabidopsis and Cultivated Tomato.

Stomata are microscopic valves formed by two guard cells flanking a pore, which are located on the epidermis of most aerial plant organs and are used for water and gas exchange between the plant and the atmosphere. The number, size and distribution of stomata are set during development in response to changing environmental conditions, allowing plants to minimize the impact of a stressful environment. In Arabidopsis, STOMATAL DENSITY AND DISTRIBUTION 1 (AtSDD1) negatively regulates stomatal density and optimizes transpiration and water use efficiency (WUE). Despite this, little is known about the function of AtSDD1 orthologs in crop species and their wild stress-tolerant relatives. In this study, SDD1-like from the stress-tolerant wild tomato Solanum chilense (SchSDD1-like) was identified through its close sequence relationship with SDD1-like from Solanum lycopersicum and AtSDD1. Both Solanum SDD1-like transcripts accumulated in high levels in young leaves, suggesting that they play a role in early leaf development. Arabidopsis sdd1-3 plants transformed with SchSDD1-like under a constitutive promoter showed a significant reduction in stomatal leaf density compared with untransformed sdd1-3 plants. Additionally, a leaf dehydration shock test demonstrated that the reduction in stomatal abundance of transgenic plants was sufficient to slow down dehydration. Overexpression of SchSDD1-like in cultivated tomato plants decreased the stomatal index and density of the cotyledons and leaves, and resulted in higher dehydration avoidance. Taken together, these results indicate that SchSDD1-like functions in a similar manner to AtSDD1 and suggest that Arabidopsis and tomatoes share this component of the stomatal development pathway that impinges on water status.

Similar geometric rules govern the distribution of veins and stomata in petals, sepals and leaves.

Investment in leaf veins (supplying xylem water) is balanced by stomatal abundance, such that sufficient water transport is provided for stomata to remain open when soil water is abundant. This coordination is mediated by a common dependence of vein and stomatal densities on cell size. Flowers may not conform to this same developmental pattern if they depend on water supplied by the phloem or have high rates of nonstomatal transpiration. We examined the relationships between veins, stomata and epidermal cells in leaves, sepals and petals of 27 angiosperms to determine whether common spacing rules applied to all tissues. Regression analysis found no evidence for different relationships within organ types. Both vein and stomatal densities were strongly associated with epidermal cell size within organs, but, for a given epidermal cell size, petals had fewer veins and stomata than sepals, which had fewer than leaves. Although our data support the concept of common scaling between veins and stomata in leaves and flowers, the large diversity in petal vein density suggests that, in some species, petal veins may be engaged in additional functions, such as the supply of water for high cuticular transpiration or for phloem delivery of water or carbohydrates.

Nitric oxide is involved in stomatal development by modulating the expression of stomatal regulator genes in Arabidopsis

As sessile organisms, plants require many flexible strategies to adapt to the environment. Although some environmental signaling pathways regulating stomatal development have been identified, how stomatal regulators are modulated by internal and external signals to determine the final stomatal abundance requires further exploration. In our studies, we found that nitric oxide (NO) promotes stomatal development with increased stomatal index as well as the relative number of meristemoids and guard mother cells [%(M+GMC)] in NO-treated wild-type Arabidopsis plants; this role of NO was further verified in the nox1 mutant, which exhibits higher NO levels, and the noa1 mutant, which exhibits low NO accumulation. To gain insight into the molecular mechanisms underlying the effect of NO, we further assayed the expression of genes involved in stomatal development and found that NO induces the expression of the master regulators SPCH, MUTE and SCRM2 to initiate stomatal development. In addition, MPK6 is also involved in NO-promoted stomatal development, as MPK6 expression was repressed in nox1 and NO-treated plants, and transgenic plants overexpressing MPK6 were less sensitive to SNP treatment in terms of changes in the%(M+GMC). Thus, our study shows that NO promotes the production of stomata by up-regulating the expression of SPCH, MUTE and SCRM2 and down-regulating MPK6 expression.

Fruit abscission in macadamia due to husk spot disease

Macadamia is indigenous to Australia. Only two species; M. integrifolia and M. tetraphylla and their interspecific hybrids are considered edible and they constitute the commercial macadamia industry. A fungal pathogen, Pseudocercospora macadamiae, causes husk spot in Australia, resulting in premature fruit abscission giving rise to nuts with low oil content. P. macadamiae is believed to have co-evolved with macadamia and the fungus infects the fruit pericarp (husk) via open stomata and grows intercellularly throughout the parenchymatous tissue where it causes accelerated abscission of immature and mature fruit. Depending on the time of infection, environmental conditions, and cultivar, the time to fruit abscission at the onset of husk spot symptoms may take up to 18 weeks. Factors that break the latency and influence fruit abscission due to P. macadamiae infection are unknown and may be involved in the production of ethylene by the husk tissue and/or abscisic acid by the fungus. Comparison of 18 macadamia genotypes revealed variations to accelerated fruit abscission from husk spot lesions on abscised fruit. This may be related to variation in stomatal abundance in the genotypes. The relationship between kernel maturation, in terms of oil content, fruit abscission in diseased and healthy husk is discussed.

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations.

Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention. This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO2. A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO2 concentrations ([CO2]) representing both modern (440 p.p.m. CO2) and ancient (1500 p.p.m. CO2) atmospheres. Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified. Densities and dimensions were unaffected by changes in [CO2], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO2]. The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO2] in bryophytes is in line with findings previously reported in other early lineages of vascular plants. These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO2 concentrations.

Stomatal Abundance and Distribution in <i>Prosopis strombulifera</i> Plants Growing under Different Iso-Osmotic Salt Treatments

Changes in several environmental parameters are thought to affect stomatal development. Under salt stress, plants can regulate their transpiration flux through a better control of the stomatal opening (as a short-term response) and through modifications of leaf anatomy (as a long-term response). We investigate how leaf micromorphology (stomatal abundance and distribution) of the halophyte Prosopis strombulifera (a spiny shrub particularly abundant in high-salinity areas of central Argentina) responds to different water status when plants are subjected to different salt treatments (NaCl, Na2SO4 and their iso-osmotic mixture). Different salt treatments on P. strombulifera plants influenced leaf micromorphological traits differently. In this study, Na2SO4-treated plants showed an increase in stomatal density (SD) and epidermal cell density (ECD) (with smaller stomata) at moderate and high salinity (-1.9 and -2.6 MPa), whereas in NaCl and NaCl + Na2SO4 treated plants, a decrease in these variables was observed. In Na2SO4-treated plants, transpiration was the highest at moderate and high salinity, with the highest content of ABA registered. A possible explanation is that, despite of these high ABA levels, there is no inhibition in stomatal opening, resulting in increased water loss, growth inhibition, and acceleration of senescence processes. We demonstrate that P. strombulifera responds to progressive salt stress by different salts changing the leaf development, particularly in Na2SO4-treated plants, leading to structural modifications in leaf size and micro-morphology of leaf cells.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity.

Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.

Pericarps retained in the tree canopy and stomatal abundance are components of resistance to husk spot caused by Pseudocercospora macadamiae in macadamia

Pseudocercospora macadamiae Beilharz, Mayers and Pascoe infects macadamia fruit via stomata causing husk spot disease. Information on the variability of fruit stomatal abundance, its association with diseased fruit pericarps (sticktights) that are retained in the tree canopy, and its influence on the husk spot intensity (incidence, severity and lesion number) among macadamia genotypes is lacking. We examined a total of 230 macadamia trees comprising 19 cultivars, 56 wild germplasm accessions and 40 breeding progeny, for the prevalence of sticktights and husk spot intensity over three production seasons. We observed a strong association between the prevalence of sticktights and disease intensity indicating its usefulness as a predictor of husk spot and as a useful phenotypic trait for husk spot resistance selection in breeding programmes. Similarly, stomatal abundance varied among macadamia genotypes, and a significant linear relationship (P < 0.001; 93%) was observed between fruit stomatal abundance and husk spot for all the macadamia genotypes analysed, confirming the utility of that trait for disease resistance screening. The genotypes were grouped into disease resistance groups. Correlations between fruit stomatal abundance, disease intensity and prevalence of sticktights revealed that the numbers of sticktights, and relative stomatal abundance were the main factors influencing the intensity of husk spot among macadamia genotypes. This is the first comprehensive study of natural variation of stomatal abundance in Macadamia species that reveals genetic variation, and provides relevant relationships with disease intensity and the prevalence of sticktights. The phenotypic plant traits indentified in this study may serve as selection tools for disease resistance screening in macadamia breeding programmes.

Natural variation in stomatal abundance of Arabidopsis thaliana includes cryptic diversity for different developmental processes.

Background and AimsCurrent understanding of stomatal development in Arabidopsis thaliana is based on mutations producing aberrant, often lethal phenotypes. The aim was to discover if naturally occurring viable phenotypes would be useful for studying stomatal development in a species that enables further molecular analysis.MethodsNatural variation in stomatal abundance of A. thaliana was explored in two collections comprising 62 wild accessions by surveying adaxial epidermal cell-type proportion (stomatal index) and density (stomatal and pavement cell density) traits in cotyledons and first leaves. Organ size variation was studied in a subset of accessions. For all traits, maternal effects derived from different laboratory environments were evaluated. In four selected accessions, distinct stomatal initiation processes were quantitatively analysed.Key Results and ConclusionsSubstantial genetic variation was found for all six stomatal abundance-related traits, which were weakly or not affected by laboratory maternal environments. Correlation analyses revealed overall relationships among all traits. Within each organ, stomatal density highly correlated with the other traits, suggesting common genetic bases. Each trait correlated between organs, supporting supra-organ control of stomatal abundance. Clustering analyses identified accessions with uncommon phenotypic patterns, suggesting differences among genetic programmes controlling the various traits. Variation was also found in organ size, which negatively correlated with cell densities in both organs and with stomatal index in the cotyledon. Relative proportions of primary and satellite lineages varied among the accessions analysed, indicating that distinct developmental components contribute to natural diversity in stomatal abundance. Accessions with similar stomatal indices showed different lineage class ratios, revealing hidden developmental phenotypes and showing that genetic determinants of primary and satellite lineage initiation combine in several ways. This first systematic, comprehensive natural variation survey for stomatal abundance in A. thaliana reveals cryptic developmental genetic variation, and provides relevant relationships amongst stomatal traits and extreme or uncommon accessions as resources for the genetic dissection of stomatal development.

Hydraulics of Asteroxylon mackei, an early Devonian vascular plant, and the early evolution of water transport tissue in terrestrial plants

The core of plant physiology is a set of functional solutions to a tradeoff between CO(2) acquisition and water loss. To provide an important evolutionary perspective on how the earliest land plants met this tradeoff, we constructed a mathematical model (constrained geometrically with measurements of fossils) of the hydraulic resistance of Asteroxylon, an Early Devonian plant. The model results illuminate the water transport physiology of one of the earliest vascular plants. Results show that Asteroxylon's vascular system contains cells with low hydraulic resistances; these resistances are low because cells were covered by scalariform pits, elliptical structures that permit individual cells to have large areas for water to pass from one cell to another. Asteroxylon could move a large amount of water quickly given its large pit areas; however, this would have left these plants particularly vulnerable to damage from excessive evapotranspiration. These results highlight a repeated pattern in plant evolution, wherein the evolution of highly conductive vascular tissue precedes the appearance of adaptations to increase water transport safety. Quantitative insight into the vascular transport of Asteroxylon also allows us to reflect on the quality of CO(2) proxy estimates based on early land plant fossils. Because Asteroxylon's vascular tissue lacked any safety features to prevent permanent damage, it probably used stomatal abundance and behavior to prevent desiccation. If correct, low stomatal frequencies in Asteroxylon reflect the need to limit evapotranspiration, rather than adaptation to high CO(2) concentrations in the atmosphere. More broadly, methods to reveal and understand water transport in extinct plants have a clear use in testing and bolstering fossil plant-based paleoclimate proxies.

A 300-million-year record of atmospheric carbon dioxide from fossil plant cuticles.

To understand better the link between atmospheric CO2 concentrations and climate over geological time, records of past CO2 are reconstructed from geochemical proxies. Although these records have provided us with a broad picture of CO2 variation throughout the Phanerozoic eon (the past 544 Myr), inconsistencies and gaps remain that still need to be resolved. Here I present a continuous 300-Myr record of stomatal abundance from fossil leaves of four genera of plants that are closely related to the present-day Ginkgo tree. Using the known relationship between leaf stomatal abundance and growing season CO2 concentrations, I reconstruct past atmospheric CO2 concentrations. For the past 300 Myr, only two intervals of low CO2 (<1,000 p.p.m.v.) are inferred, both of which coincide with known ice ages in Neogene (1-8 Myr) and early Permian (275-290 Myr) times. But for most of the Mesozoic era (65-250 Myr), CO2 levels were high (1,000-2,000 p.p.m.v.), with transient excursions to even higher CO2 (>2,000 p.p.m.v.) concentrations. These results are consistent with some reconstructions of past CO2 (refs 1, 2) and palaeotemperature records, but suggest that CO2 reconstructions based on carbon isotope proxies may be compromised by episodic outbursts of isotopically light methane. These results support the role of water vapour, methane and CO2 in greenhouse climate warming over the past 300 Myr.

Documenting forest stand invasion: fossil stomata and pollen in forest hollows

Stomata and pollen from forest hollow sediments in the Great Lakes were evaluated for their ability to document stand-scale forest invasion. In surface sediments of 19 forest hollows, stomate presence is related most closely to tree presence within 10-20 m for eastern hemlock (Tsuga canadensis (L.) Carr.), pine (Pinus spp. L.), balsam fir (Abies balsamea (L.) Mill.), and northern white cedar (Thuja occidentalis L.). Although the abundance of hemlock pollen in surface sediments is positively related to the abundance of hemlock trees within 100 m, it does not consistently reflect the presence of hemlock trees within this distance. Pollen and stomata preserved in forest hollow sediments from northwestern Wisconsin were used to document two stages of stand invasion by eastern hemlock. First, hemlock stomata initially appear approximately 2400 14C years BP, representing the initial colonization of the forest stand. Hemlock pollen also first appears at this time but in such low abundance that the presence of hemlock trees at the site cannot be known with certainty. Second, hemlock pollen percentages increase sharply around 350 14C years BP, approximately 2000 years following initial colonization, and represent an expansion of the local hemlock population not reflected by stomatal and needle abundance.

Internal Leaf Surface and Stomatal Abundance in Arctic and Alpine Populations of Oxyria Digyna

Internal leaf surface is greater in alpine populations of Oxyria digyna than in arctic populations grown under identical conditions. Outlines of lower epidermal cells of leaves vary among 10 arctic and alpine populations. Numbers of stomata, as well as epidermal cells, per unit area in the upper epidermis increase from north to south. Such a trend is not found in the lower epidermis. An alpine population from Mt. Washington has many more stomata per unit area on the lower epidermis than the upper, suggesting a closer affinity to the European alpine populations and to arctic populations than to its western North American and alpine conditions may be due to the development of ecological races which differ in structure and metabolic potential