Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Spatially-resolved localization and chemical speciation of nickel and zinc in Noccaea tymphaea and Bornmuellera emarginata.

Hyperaccumulator plants present the ideal model system for studying the physiological regulation of the essential (and potentially toxic) transition elements nickel and zinc. This study used synchrotron X-ray Fluorescence Microscopy (XFM) elemental imaging and spatially resolved X-ray Absorption Spectroscopy (XAS) to elucidate elemental localization and chemical speciation of nickel and zinc in the hyperaccumulators Noccaea tymphaea and Bornmuellera emarginata (synonym Leptoplax emarginata). The results show that in the leaves of N. tymphaea nickel and zinc have contrasting localization, and whereas nickel is present in vacuoles of epidermal cells, zinc occurs mainly in the mesophyll cells. In the seeds Ni and Zn are similarly localized and strongly enriched in the cotyledons in N. tymphaea. Nickel is strongly enriched in the tip of the radicle of B. emarginata. Noccaea tymphaea has an Fe-rich provascular strand network in the cotyledons of the seed. The chemical speciation of Ni in the seeds of N. tymphaea is unequivocally associated with carboxylic acids, whereas Zn is present as the phytate complex. The spatially resolved spectroscopy did not reveal any spatial variation in chemical speciation of Ni and Zn within the N. tymphaea seed. The dissimilar ecophysiological behaviour of Ni and Zn in N. tymphaea and B. emarginata raises questions about the evolution of hyperaccumulation in these species.

Mesophyll porosity is modulated by the presence of functional stomata.

The formation of stomata and leaf mesophyll airspace must be coordinated to establish an efficient and robust network that facilitates gas exchange for photosynthesis, however the mechanism by which this coordinated development occurs remains unclear. Here, we combine microCT and gas exchange analyses with measures of stomatal size and patterning in a range of wild, domesticated and transgenic lines of wheat and Arabidopsis to show that mesophyll airspace formation is linked to stomatal function in both monocots and eudicots. Our results support the hypothesis that gas flux via stomatal pores influences the degree and spatial patterning of mesophyll airspace formation, and indicate that this relationship has been selected for during the evolution of modern wheat. We propose that the coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops, providing a target for future improvement.

Leaf economics spectrum in rice: leaf anatomical, biochemical, and physiological trait trade-offs.

The leaf economics spectrum (LES) is an ecophysiological concept describing the trade-offs of leaf structural and physiological traits, and has been widely investigated on multiple scales. However, the effects of the breeding process on the LES in crops, as well as the mechanisms of the trait trade-offs underlying the LES, have not been thoroughly elucidated to date. In this study, a dataset that included leaf anatomical, biochemical, and functional traits was constructed to evaluate the trait covariations and trade-offs in domesticated species, namely rice (Oryza species). The slopes and intercepts of the major bivariate correlations of the leaf traits in rice were significantly different from the global LES dataset (Glopnet), which is based on multiple non-crop species in natural ecosystems, although the general patterns were similar. The photosynthetic traits responded differently to leaf structural and biochemical changes, and mesophyll conductance was the most sensitive to leaf nitrogen (N) status. A further analysis revealed that the relative limitation of mesophyll conductance declined with leaf N content; however, the limitation of the biochemistry increased relative to leaf N content. These findings indicate that breeding selection and high-resource agricultural environments lead crops to deviate from the leaf trait covariation in wild species, and future breeding to increase the photosynthesis of rice should primarily focus on improvement of the efficiency of photosynthetic enzymes.

Analyses of Aloe Polysaccharides Using Carbohydrate Microarray Profiling.

Background: As the popularity of Aloe vera extracts continues to rise, a desire to fully understand the individual polymer components of the leaf mesophyll, their relation to one another, and the effects they have on the human body are increasing. Polysaccharides present in the leaf mesophyll have been identified as the components responsible for the biological activities of A. vera, and they have been widely studied in the past decades. However, the commonly used methods do not provide the desired platform to conduct large comparative studies of polysaccharide compositions, as most of them require a complete or near-complete fractionation of the polymers. Objective: The objective for this study was to assess whether carbohydrate microarrays could be used for the high-throughput analysis of cell wall polysaccharides in aloe leaf mesophyll. Methods: The method we chose is known as comprehensive microarray polymer profiling (CoMPP) and combines the high-throughput capacity of microarray technology with the specificity of molecular probes. Results: Preliminary findings showed that CoMPP can successfully be used for high-throughput screening of aloe leaf mesophyll tissue. Seventeen species of Aloe and closely related genera were analyzed, and a clear difference in the polysaccharide compositions of the mesophyll tissues was seen. Conclusions: These preliminary data suggest that the polysaccharides vary between species and that true species of Aloe may differ from segregate genera.

Wheat plant selection for high yields entailed improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions.

Assessment of photosynthetic traits and temperature tolerance was performed on field-grown modern genotype (MG), and the local landrace (LR) of wheat (Triticum aestivum L.) as well as the wild relative species (Aegilops cylindrica Host.). The comparison was based on measurements of the gas exchange (A/ci, light and temperature response curves), slow and fast chlorophyll fluorescence kinetics, and some growth and leaf parameters. In MG, we observed the highest CO2 assimilation rate [Formula: see text] electron transport rate (Jmax) and maximum carboxylation rate [Formula: see text]. The Aegilops leaves had substantially lower values of all photosynthetic parameters; this fact correlated with its lower biomass production. The mesophyll conductance was almost the same in Aegilops and MG, despite the significant differences in leaf phenotype. In contrary, in LR with a higher dry mass per leaf area, the half mesophyll conductance (gm) values indicated more limited CO2 diffusion. In Aegilops, we found much lower carboxylation capacity; this can be attributed mainly to thin leaves and lower Rubisco activity. The difference in CO2 assimilation rate between MG and others was diminished because of its higher mitochondrial respiration activity indicating more intense metabolism. Assessment of temperature response showed lower temperature optimum and a narrow ecological valence (i.e., the range determining the tolerance limits of a species to an environmental factor) in Aegilops. In addition, analysis of photosynthetic thermostability identified the LR as the most sensitive. Our results support the idea that the selection for high yields was accompanied by the increase of photosynthetic productivity through unintentional improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions.

Examining the contribution of cell wall polysaccharides to the mechanical properties of apple parenchyma tissue using exogenous enzymes.

The viscoelastic mechanical properties of water-rich plant tissues are fundamental for many aspects of organ physiology and plant functioning. These properties are determined partly by the water in cellular vacuole and partly by the mechanical properties of the cell wall, the latter varying according to the composition and organization of its polysaccharides. In this study, relationships between the viscoelastic properties of apple cortex parenchyma tissue and cell wall pectin, hemicelluloses, and cellulose structures were studied by infusing the tissue with selected sets of purified enzymes in a controlled osmoticum. The results showed that tissue elasticity and viscosity were related, and controlled to variable extents by all the targeted polysaccharides. Among them, pectic homogalacturonan domains, crystalline cellulose, and fucosylated xyloglucan were revealed as being of prime importance in determining the viscoelastic mechanical properties of apple cortex tissue.

Physiological and structural tradeoffs underlying the leaf economics spectrum.

The leaf economics spectrum (LES) represents a suite of intercorrelated leaf traits concerning construction costs per unit leaf area, nutrient concentrations, and rates of carbon fixation and tissue turnover. Although broad trade-offs among leaf structural and physiological traits have been demonstrated, we still do not have a comprehensive view of the fundamental constraints underlying the LES trade-offs. Here, we investigated physiological and structural mechanisms underpinning the LES by analysing a novel data compilation incorporating rarely considered traits such as the dry mass fraction in cell walls, nitrogen allocation, mesophyll CO2 diffusion and associated anatomical traits for hundreds of species covering major growth forms. The analysis demonstrates that cell wall constituents are major components of leaf dry mass (18-70%), especially in leaves with high leaf mass per unit area (LMA) and long lifespan. A greater fraction of leaf mass in cell walls is typically associated with a lower fraction of leaf nitrogen (N) invested in photosynthetic proteins; and lower within-leaf CO2 diffusion rates, as a result of thicker mesophyll cell walls. The costs associated with greater investments in cell walls underpin the LES: long leaf lifespans are achieved via higher LMA and in turn by higher cell wall mass fraction, but this inevitably reduces the efficiency of photosynthesis.

Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages.

Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 µm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 µm) and at their periphery, numerous small LDs (<0.5 µm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species.

Leaf Mass per Area (LMA) and Its Relationship with Leaf Structure and Anatomy in 34 Mediterranean Woody Species along a Water Availability Gradient.

Leaf mass per area (LMA) is a morphological trait widely used as a good indicator of plant functioning (i.e. photosynthetic and respiratory rates, chemical composition, resistance to herbivory, etc.). The LMA can be broken down into the leaf density (LD) and leaf volume to area ratio (LVA or thickness), which in turn are determined by anatomical tissues and chemical composition. The aim of this study is to understand the anatomical and chemical characteristics related to LMA variation in species growing in the field along a water availability gradient. We determined LMA and its components (LD, LVA and anatomical tissues) for 34 Mediterranean (20 evergreen and 14 deciduous) woody species. Variation in LMA was due to variation in both LD and LVA. For both deciduous and evergreen species LVA variation was strongly and positively related with mesophyll volume per area (VA or thickness), but for evergreen species positive relationships of LVA with the VA of epidermis, vascular plus sclerenchyma tissues and air spaces were found as well. The leaf carbon concentration was positively related with mesophyll VA in deciduous species, and with VA of vascular plus sclerenchymatic tissues in evergreens. Species occurring at the sites with lower water availability were generally characterised by a high LMA and LD.

A Simplified and Rapid Method for the Isolation and Transfection of Arabidopsis Leaf Mesophyll Protoplasts for Large-Scale Applications.

Arabidopsis leaf mesophyll protoplasts constitute an important and versatile tool for conducting cell-based experiments to analyze the functions of distinct signaling pathways and cellular machineries using proteomic, biochemical, cellular, genetic, and genomic approaches. Thus, the methods for protoplast isolation and transfection have been gradually improved to achieve efficient expression of genes of interest. Although many well-established protocols have been extensively tested, their successful application is sometimes limited to researchers with a high degree of skill and experience in protoplasts handling. Here we present a detailed method for the isolation and transfection of Arabidopsis mesophyll protoplasts, in which many of the time-consuming and critical steps present in the current protocols have been simplified. The method described is fast, simple, and leads to high yields of competent protoplasts allowing large-scale applications.

Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass.

BACKGROUND: Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. RESULTS: The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. CONCLUSIONS: In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants.

Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: a fluorescence microscopy analysis.

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15 µm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides.

[Effects of local induction of ipt-gene in roots on cytokinins content in leaf cells tobacco plants].

Identification of cytokinins in differentiated leaf cells has received little attention. We have carried out immunohistochemical localization of cytokinins in leaves of transgenic tobacco plants in which the level of increased due to induced in their roots the expression of ipt-gene controlling cytokinin synthesis. Immuno-labeling of cytokinins with the help of antibodies raised against zeatin riboside was characteristic of mesophyll cells. The label was localized in cytoplasm adjacent to cell walls and was absent in vacuoles. Immunohistochemical staining also revealed the presence of cytokinins in guard cells. Induction of cytokinin synthesis enhanced the immunohistochemical staining of both mesophyll cells and guard cells, which was accompanied by elevated stomatal conductance. The possibility of a direct effect of cytokinins on stomatal conductance and their indirect influence through photosynthesis in the mesophyll cells is discussed.

[Ultrastructure and pigment composition of Sagittaria sagittifolia L. leaves].

Comparative analysis of the cellular ultrastructure and pigment content of both submerged and above-water Sagittaria sagittifolia leaves with transmission electron microscopic and biochemical methods were realized. Essential distinctions of S. sagittifolia ultrastructure of photosynthesizing cells in submerged leaves were revealed during the comparison with those in mesophyll cells of above-water leaves. The changes of chloroplast ultrastructure in submerged leaves are examined as the adaptative signs of photosynthesizing cells under influence of altered environment.

To feed or not to feed: plant factors located in the epidermis, mesophyll, and sieve elements influence pea aphid's ability to feed on legume species.

The pea aphid (Acyrthosiphon pisum Harris), a legume specialist, encompasses at least 11 genetically distinct sympatric host races. Each host race shows a preference for a certain legume species. Six pea aphid clones from three host races were used to localize plant factors influencing aphid probing and feeding behavior on four legume species. Aphid performance was tested by measuring survival and growth. The location of plant factors influencing aphid probing and feeding was determined using the electrical penetration graph (EPG) technique. Every aphid clone performed best on the plant species from which it was originally collected, as well as on Vicia faba. On other plant species, clones showed intermediate or poor performance. The most important plant factors influencing aphid probing and feeding behavior were localized in the epidermis and sieve elements. Repetitive puncturing of sieve elements might be relevant for establishing phloem feeding, since feeding periods appear nearly exclusively after these repetitive sieve element punctures. A combination of plant factors influences the behavior of pea aphid host races on different legume species and likely contributes to the maintenance of these races.

Evaluation of anti-bacterial and anti-oxidant potential of andrographolide and echiodinin isolated from callus culture of Andrographis paniculata Nees.

OBJECTIVE: To evaluate the anti-bacterial and anti-oxidant activity of andrographolide (AND) and echiodinin (ECH) of Andrographis paniculata. METHODS: In this study, an attempt has been made to demonstrate the anti-microbial and anti-oxidant activity of isolated AND and ECH by broth micro-dilution method and 2,2-diphenyl-2-picryl-hydrazyl (DPPH) assay, respectively. Structure elucidation was determined by electro-spray ionization-MSD, NMR ((1)H and (13)C) and IR spectra. RESULTS: AND was effective against most of the strains tested including Mycobacterium smegmatis, showing broad spectrum of growth inhibition activity with Minimum inhibitory concentration values against Staphylococcus aureus (100 µg/mL), Streptococcus thermophilus (350 µg/mL) Bacillus subtilis (100 µg/mL), Escherichia coli (50 µg/mL), Mycobacterium smegmatis (200 µg/mL), Klebsiella pneumonia (100 µg/mL), and Pseudomonas aeruginosa (200 µg/mL). ECH showed specific anti-bacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa at a concentration higher than 225 µg/mL. Both AND and ECH were not effective against the two yeast strains, Candida albicans and Saccharomyces cerevisiae tested in this study. CONCLUSION: This preliminary study showed promising anti-bacterial activity and moderate free radical scavenging activity of AND and ECH, and it may provide the scientific rationale for its popular folklore medicines.

Ultradian variation of isoprene emission, photosynthesis, mesophyll conductance, and optimum temperature sensitivity for isoprene emission in water-stressed Eucalyptus citriodora saplings.

Water availability is a major limiting factor on plant growth and productivity. Considering that Eucalyptus spp. are among the few plant species able to produce both isoprene and monoterpenes, experiments were designed to investigate the response of isoprene emission and isoprenoid concentrations in Eucalyptus citriodora saplings exposed to decreasing fraction of transpirable soil water (FTSW). In particular, this study aimed to assess: (a) the kinetic of water stress-induced variations in photosynthesis, isoprene emission, and leaf isoprenoid concentrations during progressive soil water shortage as a function of FTSW; (b) the ultradian control of isoprene emission and photosynthesis under limited soil water availability; and (c) the optimum temperature sensitivity of isoprene emission and photosynthesis under severe water stress. The optimum temperature for isoprene emission did not change under progressive soil water deficit. However, water stress induced a reallocation of carbon through the MEP/DOXP pathway resulting in a qualitative change of the stored isoprenoids. The ultradian trend of isoprene emission was also unaffected under water stress, and a similar ultradian trend of stomatal and mesophyll conductances was also observed, highlighting a tight coordination between diffusion limitations to photosynthesis during water stress. The kinetics of photosynthetic parameters and isoprene emission in response to decreasing FTSW in E. citriodora are strikingly similar to those measured in other plant functional types. These findings may be useful to refine the algorithms employed in process-based models aiming to precisely up-scale carbon assimilation and isoprenoid emissions at regional and global scales.

Leaf rolling allows quantification of mRNA abundance in mesophyll cells of sorghum.

In the leaves of most C(4) plants, mesophyll (M) and bundle sheath (BS) cells develop and maintain highly differentiated biochemical networks. Separation and analysis of M and BS cells has greatly influenced our understanding of the C(4) pathway. A number of approaches including mechanical separation, digestion with cell wall-degrading cocktails, laser-capture microdissection, and leaf rolling have been used to isolate these cell types. Although leaf rolling is conceptually and practically the simplest method, to date it has only been used to assess the metabolite content of M cells from C(4) leaves of maize. This study reports an adapted leaf-rolling method for the isolation of high-quality RNA from M cells of sorghum. Analysis of leaf cell structure, RNA integrity, and transcript abundance of marker genes demonstrated that the sap collected by leaf rolling was from M cells and had no significant contamination. It was concluded that leaf rolling is a fast, cheap, and efficient method of measuring transcript abundance in M cells of sorghum.