Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays

Background and Aims

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs.

Methods

Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy.

Results

Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes.

Conclusions

The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.     

Probable involvement of cytoskeleton in stomatal-pore formation inAsplenium nidus L.

Summary Stomatal-pore formation in the fernAsplenium nidus L. commences in postcytokinetic guard cells at the mid-region of the ventral wall, before the deposition of any cellulosic wall material on it, by the local movement of the adjacent plasmalemmata apart from each other. In this way a rudimentary internal stomatal pore is formed. At this stage the ventral wall exhibits an undulated appearance and gives a positive reaction to aniline blue. Detailed study of postcytokinetic guard cells by electron microscopy, as well as after tubulin immunolabeling and actin staining, shows that stomatal pore initiation coincides with the initiation of the organization of the anticlinal microtubule bundles along the middle of the ventral wall and the colocalization of actin filaments at the same sites. Afterwards, the stomatal pore broadens towards the periclinal walls, a phenomenon keeping pace with the further bundling of the cytoskeletal elements beneath the plasmalemmata lining the middle of the ventral wall. At this stage the anticlinal microtubule bundles lining the stomatal pore are very prominent. The above findings, as well as the fact that treatments with antimicrotubule drugs inhibit the internal stomatal-pore formation, denote that the cortical cytoskeleton lining the ventral wall and particularly the microtubules are involved in this process. Afterwards, distinct local wall thickenings are deposited at the sites of junction of the mid-region of the ventral wall with the periclinal walls as well as at the junctions of the polar ventral-wall ends with the external periclinal wall. Along the middle-lamella region of the former wall thickenings the fore- and rear-chambers of the stomatal pore are formed. The final stomatal-pore opening is achieved by disruption of the expanded thin median periclinal wall region inherited from the guard cell mother cell and of the overlying cuticle, which covers the stomatal pore externally and internally. At the same time the fore-chamber of the stomatal pore broadens by a schizogenous opening towards the polar ventral-wall ends. The observations show that the stomatal-pore formation inA. nidus is a unique process, which is probably restricted to ferns.Abbreviations Af actin filament - GC guard cell - Mt microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - VW ventral wall     

A new callose function: Involvement in differentiation and function of fern stomatal complexes

Callose in polypodiaceous ferns performs multiple roles during stomatal development and function. This highly dynamic (1→3)-β-D-glucan, in cooperation with the cytoskeleton, is involved in: (a) stomatal pore formation, (b) deposition of local GC wall thickenings and (c) the mechanism of stomatal pore opening and closure. This behavior of callose, among others, probably relies on the particular mechanical properties as well as on the ability to form and degrade rapidly, to create a scaffold or to serve as a matrix for deposition of other cell wall materials and to produce fibrillar deposits in the periclinal GC walls, radially arranged around the stomatal pore. The local callose deposition in closing stomata is an immediate response of the external periclinal GC walls experiencing strong mechanical forces induced by the neighboring cells. The radial callose fibrils transiently co-exist with radial cellulose microfibrils and, like the latter, seem to be oriented via cortical MTs.Key words: callose, cytoskeleton, fern stomata, guard cell wall thickening, stomatal function, stomatal pore formationCallose represents a hemicellulosic matrix cell wall component, usually of temporal appearance, which is synthesized by callose synthases, enzymes localized in the plasmalemma and degraded by (1→3)-β-glucanases.^1–4 It consists of triple helices of a linear homopolymer of (1→3)-β-glucose residues.^5–7 The plant cell is able to form and degrade callose in a short time. On the surface of the plasmolyzed protoplast a thin callose surface film may arise within seconds.⁸ Callose is the only cell wall component that is implicated in a great variety of developmental plant processes, like cell plate formation,^9–11 microspore development,^12–14 trafficking through plasmodesmata,^15,16 formation and closure of sieve pores,¹⁶ response of the plant cells to multiple biotic and abiotic stresses,^4,5 establishment of distinct “cell cortex domains”,¹⁷ etc.Despite the widespread occurrence of callose, its general function(s) is (are) not well understood (reviewed in refs. ⁴ and ⁵). It may serve as: a matrix for deposition of other cell wall materials, as in developing cell plates;⁹ a cell wall-strengthening material, as in cotton seed hairs and growing pollen tubes;¹⁸ a sealing or plugging material at the plasma membrane of pit fields, plasmodesmata and sieve plate pores;¹⁶ a mechanical obstruction to growth of fungal hyphae or a special permeability barrier, as in pollen mother cell walls and muskmelon endosperm envelopes.^4,19,20 The degree of polymerization, age and thickness of callose deposits may cause variation in its physical properties.⁵Evidence accumulated so far showed that a significant number of ferns belonging to Polypodiales and some other fern classes forms intense callose deposits in the developing GC wall thickenings.^21–28 This phenomenon has not been observed in angiosperm stomata, although callose is deposited along the whole surface of the young VW and in the VW ends of differentiating and mature stomata (our unpublished data; reviewed in refs ²⁹ and ³⁰).Stomata are specialized epidermal bicellular structures (Fig. 1A) regulating gas exchange between the aerial plant organs and the external environment. Their appearance in the first land plants was crucial for their adaptation and survival in the terrestrial environment. The constituent GCs have the ability to undergo reversible changes in shape, leading to opening and closure of the stomatal pore (stomatal movement). The mechanism by which GCs change shape is based on: (a) the particular mechanical properties of GC walls owed to their particular shape, thickening, fine structure and chemical composition and (b) the reversible changes in vacuole volume, in response to environmental factors, through fairly complicated biochemical pathways.^30–33Open in a separate windowFigure 1(A) Diagrammatic representation of an elliptical stoma. (B–E) Diagram to show the process of stomatal pore formation in angiosperms (B and C) and Polypodiales ferns (D and E). The arrows in (B) indicate the forming stomatal pore. DW, dorsal wall; EPW, external periclinal wall; GC, guard cell; IPW, internal periclinal wall; ISP, internal stomatal pore; PE polar ventral wall end; VW, ventral wall.The present review is focused on the multiple-role of callose in differentiating and functioning fern stomata, as they are substantiated by the available information, including some unpublished data, and in particular in: stomatal pore formation, deposition of GC wall thickenings and opening and closure of the stomatal pore. The mode of deposition of fibrillar callose deposits in GC walls and the mechanism of their alignment are also considered.     

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Using the biomass-ratio and idiosyncratic hypotheses to predict mixed-species litter decomposition

Background and Aims

Microsporogenesis leading to monosulcate pollen grains has already been described for a wide range of monocot species. However, a detailed study of additional callose deposition after the completion of the cleavage walls has been neglected so far. The study of additional callose deposition in monosulcate pollen grain has gained importance since a correlation between additional callose deposition and aperture location has recently been revealed.

Methods

Microsporogenesis is described for 30 species belonging to eight families of the monocots: Acoraceae, Amaryllidaceae, Alstroemeriaceae, Asparagaceae, Butomaceae, Commelinaceae, Liliaceae and Xanthorrhoeaceae.

Key Results

Five different microsporogenesis pathways are associated with monosulcate pollen grain. They differ in the type of cytokinesis, tetrad shape, and the presence and shape of additional callose deposition. Four of them present additional callose deposition.

Conclusions

In all these different microsporogenesis pathways, aperture location seems to be linked to the last point of callose deposition.     

Non-lignified helical cell wall thickenings in root cortical cells of Aspleniaceae (Polypodiales): histology and taxonomical significance

Background and Aims

Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae.

Methods

Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed.

Key Results

The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species.

Conclusions

This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.     

The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species

Background and Aims

In flowering plants, microsporogenesis is accompanied by various types of cytoplasmic partitioning (cytokinesis). Patterns of male cytokinesis are suspected to play a role in the diversity of aperture patterns found in pollen grains of angiosperms. The relationships between intersporal wall formation, tetrad shape and pollen aperture pattern ontogeny are studied.

Methods

A comparative analysis of meiosis and aperture distribution was performed within tetrads in two triporate eudicot species with contrasting aperture arrangements within their tetrads [Epilobium roseum (Onagraceae) and Paranomus reflexus (Proteaceae)].

Key Results and Conclusions

Intersporal wall formation is a two-step process in both species. Cytokinesis is first achieved by the formation of naked centripetal cell plates. These naked cell plates are then covered by additional thick, localized callose deposits that differ in location between the two species. Apertures are finally formed in areas in which additional callose is deposited on the cell plates. The recorded variation in tetrad shape is correlated with variations in aperture pattern, demonstrating the role of cell partitioning in aperture pattern ontogeny.     

Model-based analysis of avoidance of ozone stress by stomatal closure in Siebold's beech (Fagus crenata)

Background and Aims

Resistance of plants to ozone stress can be classified as either avoidance or tolerance. Avoidance of ozone stress may be explained by decreased stomatal conductance during ozone exposure because stomata are the principal interface for entry of ozone into plants. In this study, a coupled photosynthesis–stomatal model was modified to test whether the presence of ozone can induce avoidance of ozone stress by stomatal closure.

Methods

The response of Siebold''s beech (Fagus crenata), a representative deciduous tree species, to ozone was studied in a free-air ozone exposure experiment in Japan. Photosynthesis and stomatal conductance were measured under ambient and elevated ozone. An optimization model of stomata involving water, CO₂ and ozone flux was tested using the leaf gas exchange data.

Key Results

The data suggest that there are two phases in the avoidance of ozone stress via stomatal closure for Siebold''s beech: (1) in early summer ozone influx is efficiently limited by a reduction in stomatal conductance, without any clear effect on photosynthetic capacity; and (2) in late summer and autumn the efficiency of ozone stress avoidance was decreased because the decrease in stomatal conductance was small and accompanied by an ozone-induced decline of photosynthetic capacity.

Conclusions

Ozone-induced stomatal closure in Siebold''s beech during early summer reduces ozone influx and allows the maximum photosynthetic capacity to be reached, but is not sufficient in older leaves to protect the photosynthetic system.     

Closure of plasmodesmata in maize (Zea mays) at low temperature: a new mechanism for inhibition of photosynthesis

Background and Aims

Photosynthesis is one of the processes most susceptible to low-temperature inhibition in maize, a tropical C4 crop not yet fully adapted to a temperate climate. C4 photosynthesis relies on symplasmic exchange of large amounts of photosynthetic intermediates between Kranz mesophyll (KMS) and bundle sheath (BS) cells. The aim of this study was to test the hypothesis that the slowing of maize photosynthesis at low temperature is related to ultrastructural changes in the plasmodesmata between KM and BS as well as BS and vascular parenchyma (VP) cells.

Methods

Chilling-tolerant (CT) KW 1074 and chilling-sensitive (CS) CM 109 maize (Zea mays) lines were studied. The effect of moderate chilling (14 °C) on the rate of photosynthesis, photosynthate transport kinetics, and the ultrastructure of plasmodesmata linking the KMS, BS and VP cells were analysed. Additionally, the accumulation of callose and calreticulin was studied by the immunogold method.

Key Results

Chilling inhibited photosynthesis, photosynthate transfer to the phloem and photosynthate export from leaves in both lines. This inhibition was reversible upon cessation of chilling in the CT line but irreversible in the CS line. Simultaneously to physiological changes, chilling induced swelling of the sphincters of plasmodesmata linking KMS and BS cells and a decreased lumen of the cytoplasmic sleeve of plasmodesmata at the BS/VP interface in the CS line but not in the CT line. Accumulation of calreticulin, which occurred near the neck region of the closed plasmodesmata was observed after just 4 h of chilling and over-accumulation of callose at the KMS/BS and BS/VP interfaces occurred after 28 h of chilling.

Conclusions

Stronger chilling sensitivity of the CM 109 maize line compared with the KW 1074 line, shown by decreased photosynthesis and assimilate export from a leaf, is related to changes in the ultrastructure of leaf plasmodesmata at low temperature. The chain of reactions to chilling is likely to include calreticulin action resulting in rapid and efficient closure of the plasmodesmata at both KMS/BS and BS/VP interfaces. Callose deposition in a leaf was a secondary effect of chilling.     

Plasticity of stomatal distribution pattern and stem tracheid dimensions in Podocarpus lambertii: an ecological study

Background and Aims

Leaf and wood plasticity are key elements in the survival of widely distributed plant species. Little is known, however, about variation in stomatal distribution in the leaf epidermis and its correlation with the dimensions of conducting cells in wood. This study aimed at testing the hypothesis that Podocarpus lambertii, a conifer tree, possesses a well-defined pattern of stomatal distribution, and that this pattern can vary together with the dimensions of stem tracheids as a possible strategy to survive in climatically different sites.

Methods

Leaves and wood were sampled from trees growing in a cold, wet site in south-eastern Brazil and in a warm, dry site in north-eastern Brazil. Stomata were thoroughly mapped in leaves from each study site to determine a spatial sampling strategy. Stomatal density, stomatal index and guard cell length were then sampled in three regions of the leaf: near the midrib, near the leaf margin and in between the two. This sampling strategy was used to test for a pattern and its possible variation between study sites. Wood and stomata data were analysed together via principal component analysis.

Key Results

The following distribution pattern was found in the south-eastern leaves: the stomatal index was up to 25 % higher in the central leaf region, between the midrib and the leaf margin, than in the adjacent regions. The inverse pattern was found in the north-eastern leaves, in which the stomatal index was 10 % higher near the midrib and the leaf margin. This change in pattern was accompanied by smaller tracheid lumen diameter and length.

Conclusions

Podocarpus lambertii individuals in sites with higher temperature and lower water availability jointly regulate stomatal distribution in leaves and tracheid dimensions in wood. The observed stomatal distribution pattern and variation appear to be closely related to the placement of conducting tissue in the mesophyll.     

An easy method for cutting and fluorescent staining of thin roots

Background and Aims

Cutting plant material is essential for observing internal structures and may be difficult for various reasons. Most fixation agents such as aldehydes, as well as embedding resins, do not allow subsequent use of fluorescent staining and make material too soft to make good-quality hand-sections. Moreover, cutting thin roots can be very difficult and time consuming. A new, fast and effective method to provide good-quality sections and fluorescent staining of fresh or fixed root samples, including those of very thin roots (such as Arabidopsis or Noccaea), is described here.

Methods

To overcome the above-mentioned difficulties the following procedure is proposed: fixation in methanol (when fresh material cannot be used) followed by en bloc staining with toluidine blue, embedding in 6 % agarose, preparation of free-hand sections of embedded material, staining with fluorescent dye, and observation in a microscope under UV light.

Key Results

Despite eventual slight deformation of primary cell walls (depending on the species and root developmental stage), this method allows effective observation of different structures such as ontogenetic changes of cells along the root axis, e.g. development of xylem elements, deposition of Casparian bands and suberin lamellae in endodermis or exodermis or peri-endodermal thickenings in Noccaea roots.

Conclusions

This method provides good-quality sections and allows relatively rapid detection of cell-wall modifications. Also important is the possibility of using this method for free-hand cutting of extremely thin roots such as those of Arabidopsis.     

Release of developmental constraints on tetrad shape is confirmed in inaperturate pollen of Potamogeton

Background and Aims

Microsporogenesis in monocots is often characterized by successive cytokinesis with centrifugal cell plate formation. Pollen grains in monocots are predominantly monosulcate, but variation occurs, including the lack of apertures. The aperture pattern can be determined by microsporogenesis features such as the tetrad shape and the last sites of callose deposition among the microspores. Potamogeton belongs to the early divergent Potamogetonaceae and possesses inaperturate pollen, a type of pollen for which it has been suggested that there is a release of the constraint on tetrad shape. This study aimed to investigate the microsporogenesis and the ultrastructure of pollen wall in species of Potamogeton in order to better understand the relationship between microsporogenesis features and the inaperturate condition.

Methods

The microsporogenesis was investigated using both light and epifluorescence microscopy. The ultrastructure of the pollen grain was studied using transmission electron microscopy.

Key Results

The cytokinesis is successive and formation of the intersporal callose wall is achieved by centrifugal cell plates, as a one-step process. The microspore tetrads were tetragonal, decussate, T-shaped and linear, except in P. pusillus, which showed less variation. This species also showed a callose ring in the microsporocyte, and some rhomboidal tetrads. In the mature pollen, the thickening observed in a broad area of the intine was here interpreted as an artefact.

Conclusions

The data support the view that there is a correlation between the inaperturate pollen production and the release of constraint on tetrad shape. However, in P. pusillus the tetrad shape may be constrained by a callose ring. It is also suggested that the lack of apertures in the pollen of Potamogeton may be due to the lack of specific sites on which callose deposition is completed. Moreover, inaperturate pollen of Potamogeton would be better classified as omniaperturate.Key words: Alismatales, callose, microsporogenesis, pollen aperture, Potamogeton illinoensis, P. polygonus, P. pusillus, tetrad shape     

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family

Background and Aims

Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene.

Methods

Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4·1b10).

Key Results

Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides.

Conclusions

This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.Key words: Bryophyte, callose, callose synthase, exine development, moss, Physcomitrella patens, spores, sporogenesis     

Plant responsiveness to root-root communication of stress cues

Background and Aims

Phenotypic plasticity is based on the organism''s ability to perceive, integrate and respond to multiple signals and cues informative of environmental opportunities and perils. A growing body of evidence demonstrates that plants are able to adapt to imminent threats by perceiving cues emitted from their damaged neighbours. Here, the hypothesis was tested that unstressed plants are able to perceive and respond to stress cues emitted from their drought- and osmotically stressed neighbours and to induce stress responses in additional unstressed plants.

Methods

Split-root Pisum sativum, Cynodon dactylon, Digitaria sanguinalis and Stenotaphrum secundatum plants were subjected to osmotic stress or drought while sharing one of their rooting volumes with an unstressed neighbour, which in turn shared its other rooting volume with additional unstressed neighbours. Following the kinetics of stomatal aperture allowed testing for stress responses in both the stressed plants and their unstressed neighbours.

Key Results

In both P. sativum plants and the three wild clonal grasses, infliction of osmotic stress or drought caused stomatal closure in both the stressed plants and in their unstressed neighbours. While both continuous osmotic stress and drought induced prolonged stomatal closure and limited acclimation in stressed plants, their unstressed neighbours habituated to the stress cues and opened their stomata 3–24 h after the beginning of stress induction.

Conclusions

The results demonstrate a novel type of plant communication, by which plants might be able to increase their readiness to probable future osmotic and drought stresses. Further work is underway to decipher the identity and mode of operation of the involved communication vectors and to assess the potential ecological costs and benefits of emitting and perceiving drought and osmotic stress cues under various ecological scenarios.     

Differences in the response sensitivity of stomatal index to atmospheric CO2 among four genera of Cupressaceae conifers

Background and Aims

The inverse relationship between stomatal density (SD: number of stomata per mm² leaf area) and atmospheric concentration of CO₂ ([CO₂]) permits the use of plants as proxies of palaeo-atmospheric CO₂. Many stomatal reconstructions of palaeo-[CO₂] are based upon multiple fossil species. However, it is unclear how plants respond to [CO₂] across genus, family or ecotype in terms of SD or stomatal index (SI: ratio of stomata to epidermal cells). This study analysed the stomatal numbers of conifers from the ancient family Cupressaceae, in order to examine the nature of the SI–[CO₂] relationship, and potential implications for stomatal reconstructions of palaeo-[CO₂].

Methods

Stomatal frequency measurements were taken from historical herbarium specimens of Athrotaxis cupressoides, Tetraclinis articulata and four Callitris species, and live A. cupressoides grown under CO₂-enrichment (370, 470, 570 and 670 p.p.m. CO₂).

Key Results

T. articulata, C. columnaris and C. rhomboidea displayed significant reductions in SI with rising [CO₂]; by contrast, A. cupressoides, C. preissii and C. oblonga show no response in SI. However, A. cupressoides does reduce SI to increases in [CO₂] above current ambient (approx. 380 p.p.m. CO₂). This dataset suggests that a shared consistent SI–[CO₂] relationship is not apparent across the genus Callitris.

Conclusions

The present findings suggest that it is not possible to generalize how conifer species respond to fluctuations in [CO₂] based upon taxonomic relatedness or habitat. This apparent lack of a consistent response, in conjunction with high variability in SI, indicates that reconstructions of absolute palaeo-[CO₂] based at the genus level, or upon multiple species for discrete intervals of time are not as reliable as those based on a single or multiple temporally overlapping species.     

A stomatal optimization theory to describe the effects of atmospheric CO2 on leaf photosynthesis and transpiration

Background and Aims

Global climate models predict decreases in leaf stomatal conductance and transpiration due to increases in atmospheric CO₂. The consequences of these reductions are increases in soil moisture availability and continental scale run-off at decadal time-scales. Thus, a theory explaining the differential sensitivity of stomata to changing atmospheric CO₂ and other environmental conditions must be identified. Here, these responses are investigated using optimality theory applied to stomatal conductance.

Methods

An analytical model for stomatal conductance is proposed based on: (a) Fickian mass transfer of CO₂ and H₂O through stomata; (b) a biochemical photosynthesis model that relates intercellular CO₂ to net photosynthesis; and (c) a stomatal model based on optimization for maximizing carbon gains when water losses represent a cost. Comparisons between the optimization-based model and empirical relationships widely used in climate models were made using an extensive gas exchange dataset collected in a maturing pine (Pinus taeda) forest under ambient and enriched atmospheric CO₂.

Key Results and Conclusion

In this interpretation, it is proposed that an individual leaf optimally and autonomously regulates stomatal opening on short-term (approx. 10-min time-scale) rather than on daily or longer time-scales. The derived equations are analytical with explicit expressions for conductance, photosynthesis and intercellular CO₂, thereby making the approach useful for climate models. Using a gas exchange dataset collected in a pine forest, it is shown that (a) the cost of unit water loss λ (a measure of marginal water-use efficiency) increases with atmospheric CO₂; (b) the new formulation correctly predicts the condition under which CO₂-enriched atmosphere will cause increasing assimilation and decreasing stomatal conductance.     

Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning

Background and Aims

Angiosperm stomata consistently possess a pair of guard cells, but differ between taxa in the patterning and developmental origin of neighbour cells. Developmental studies of phylogenetically pivotal taxa are essential as comparative yardsticks for understanding the evolution of stomatal development.

Methods

We present a novel ultrastructural study of developing stomata in leaves of Amborella (Amborellales), Nymphaea and Cabomba (Nymphaeales), and Austrobaileya and Schisandra (Austrobaileyales), representing the three earliest-divergent lineages of extant angiosperms (the ANITA-grade).

Key Results

Alternative developmental pathways occur in early-divergent angiosperms, resulting partly from differences in pre-patterning and partly from the presence or absence of highly polarized (asymmetric) mitoses in the stomatal cell lineage. Amplifying divisions are absent from ANITA-grade taxa, indicating that ostensible similarities with the stomatal patterning of Arabidopsis are superficial. In Amborella, ‘squared’ pre-patterning occurs in intercostal regions, with groups of four protodermal cells typically arranged in a rectangle; most guard-mother cells are formed by asymmetric division of a precursor cell (the mesoperigenous condition) and are typically triangular or trapezoidal. In contrast, water-lily stomata are always perigenous (lacking asymmetric divisions). Austrobaileya has occasional ‘giant’ stomata.

Conclusions

Similar mature stomatal phenotypes can result from contrasting morphogenetic factors, although the results suggest that paracytic stomata are invariably the product of at least one asymmetric division. Loss of asymmetric divisions in stomatal development could be a significant factor in land plant evolution, with implications for the diversity of key structural and physiological pathways.     

Stomatal vs. genome size in angiosperms: the somatic tail wagging the genomic dog?

Background and Aims

Genome size is a function, and the product, of cell volume. As such it is contingent on ecological circumstance. The nature of ‘this ecological circumstance’ is, however, hotly debated. Here, we investigate for angiosperms whether stomatal size may be this ‘missing link’: the primary determinant of genome size. Stomata are crucial for photosynthesis and their size affects functional efficiency.

Methods

Stomatal and leaf characteristics were measured for 1442 species from Argentina, Iran, Spain and the UK and, using PCA, some emergent ecological and taxonomic patterns identified. Subsequently, an assessment of the relationship between genome-size values obtained from the Plant DNA C-values database and measurements of stomatal size was carried out.

Key Results

Stomatal size is an ecologically important attribute. It varies with life-history (woody species < herbaceous species < vernal geophytes) and contributes to ecologically and physiologically important axes of leaf specialization. Moreover, it is positively correlated with genome size across a wide range of major taxa.

Conclusions

Stomatal size predicts genome size within angiosperms. Correlation is not, however, proof of causality and here our interpretation is hampered by unexpected deficiencies in the scientific literature. Firstly, there are discrepancies between our own observations and established ideas about the ecological significance of stomatal size; very large stomata, theoretically facilitating photosynthesis in deep shade, were, in this study (and in other studies), primarily associated with vernal geophytes of unshaded habitats. Secondly, the lower size limit at which stomata can function efficiently, and the ecological circumstances under which these minute stomata might occur, have not been satisfactorally resolved. Thus, our hypothesis, that the optimization of stomatal size for functional efficiency is a major ecological determinant of genome size, remains unproven.