Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.     

Structure of the stomatal complex of the monocot Flagellaria indica

The structure of the mature stomatal complex of Flagellaria indica L. was studied since the Flagellariaceae is reported to be one of a handful of nongrass families with a grass-type stoma, and since relatively little is known about stomatal ultrastructure in monocots other than grasses. Both the grass guard cell and its nucleus are dumbbell-shaped, and the walls that separate adjacent grass guard cells are perforated. Electron and fluorescence microscopy reveal that the Flagellaria guard cell lacks these features. Instead, the Flagellaria guard cell is neither dumbbellnor kidney-shaped, its nucleus is roughly kidney-shaped, and the end walls are thickened and imperforate. Additional structural features of the stomatal apparatus of Flagellaria include: 1) the subsidiary cells have a protuberance that underlies the middle of the guard cell and that forms an additional and innermost aperture of the pore; 2) guard and subsidiary cell walls are thickened differentially and are layered; and 3) organelles in both cell types appear to be confined to specific domains. Although Flagellaria is closely related to grasses, it does not have a grass or dumbbell-shaped type of stomate. This suggests that the grass type of stomate may be less widespread than reported.     

The Developmental Basis of Stomatal Density and Flux

Equations for stomatal density and maximum theoretical stomatal conductance as functions of stomatal initiation rate, epidermal cell size, and stomatal size enable scaling from development to flux.Since the first published measurements of stomatal density by Johann Hedwig (1793) and Alexander von Humboldt (1798), the counting and measuring of stomata has been one of the most typical botanical activities, with an important role across fields of plant biology (Willmer and Fricker, 1996). Stomatal density (d) and size (s) are indicators of acclimation and adaptation to contrasting environments, and permit estimation of the theoretical anatomical maximum stomatal conductance (g_max; units: mol m⁻² s⁻¹; Brown and Escombe, 1900; Lawson et al., 1998; Franks and Beerling, 2009; Franks et al., 2009), which represents a first quantitative estimate of the anatomical constraint on maximum stomatal gas exchange. While decades of theory have focused on d and g_max, their basis in traits with a transparent relationship to epidermal development has not been expressed. We derived exact mathematical equations for d and g_max as functions of stomatal differentiation rate, also known as stomatal index (i, no. of stomata per no. of epidermal cells plus stomata), s, and epidermal cell size (e). These equations unify the quantitative understanding of epidermal development and maximum flux, revealing the developmental bases for d and g_max across genotypes or species, and enabling targeting of specific epidermal development traits in plant breeding for productivity.The genetic and developmental basis for high stomatal density and stomatal conductance is a research priority in plant physiology, agriculture, and paleobiology (Asl et al., 2011; Doheny-Adams et al., 2012; Dow et al., 2014; Franks et al., 2015; Roche, 2015; Wang et al., 2015b). Indeed, a higher g_max should benefit species under low CO₂, higher irradiance or nutrient supply, or under selection for high productivity or competition (Franks and Beerling, 2009; Taylor et al., 2012; Jones, 2014). Under the opposite conditions, a lower g_max would provide the potential benefits of reduced water loss and/or increased CO₂ gain relative to water loss (Franks and Beerling, 2009; Taylor et al., 2012; Jones, 2014; Franks et al., 2015). Decades of theory have focused on the basis of g_max in stomatal anatomy (Fig. 1, A–C). According to a classic formulation of g_max (Brown and Escombe, 1900) in a recently updated form,(1)where D (m² s⁻¹) represents the diffusivity in air of water or CO₂ (which differ by a factor of 1.6); ν the molar volume of air (m³ mol⁻¹); and d, a_max, and l, respectively, the stomatal density (pores m⁻²), the mean maximum area of a single stomatal pore (m²), and stomatal pore depth (m; Franks and Beerling, 2009; Franks et al., 2009). The most recent extensions of this equation incorporated basic assumptions about allometries among guard cell dimensions, which have become standard in the stomatal literature (e.g. Franks and Beerling, 2009; Franks et al., 2009; Taylor et al., 2012; Dow et al., 2014; McElwain et al., 2016) and enable the estimation of g_max as a function of d and s. In its simplest form:(2)where and such that b is a biophysical constant and m a morphological constant based on scaling factors representing the proportionality of stomatal length (L) and width (W), and pore length (p) and depth (l), with c = p/L, j = W/L, and h = l/W all treated as constant for the estimation of g_max (c, h, and j = 0.5 for nongrasses with kidney bean-shaped guard cells, or c = 0.5, h = 0.5, and j = 0.125 for grasses with their dumbbell-shaped guard cells; Franks and Beerling, 2009; McElwain et al., 2016), though these ratios can be allowed to vary for individual species or genotypes when more detailed information is available on stomatal dimensions (Franks and Farquhar, 2007; Franks et al., 2014).Open in a separate windowFigure 1.Anatomical variables determining maximum stomatal conductance (g_max). A to C, Stomatal dimensions (guard cell length, L; stomatal pore length, p; guard cell width, W; stomatal area, s; stomatal maximum pore area, a_max; stomatal depth, l) and epidermal development traits (epidermal cell area, e; stomatal index, i). D to F, The influence on stomatal density (d) and g_max of e and i: increasing i as from D to E would lead to higher d and g_max; reducing e as from E to F would lead to higher d and g_max. Larger s would also lead to lower d and g_max, though with a much smaller effect. Stomatal images after Beaulieu et al. (2008).The g_max estimated this way strongly predicted the operating stomatal conductance measured with leaf gas exchange systems (g_op) across Arabidopsis (Arabidopsis thaliana) genotypes under low CO₂, high humidity, and high red and blue light (Dow et al., 2014). However, across diverse species, the g_max values estimated by Equation 2 tend to be much higher than g_op (Feild et al., 2011; McElwain et al., 2016) for several reasons. First, for typical leaves transpiring even under the best conditions, the effective area of the stomatal pore (a’) is smaller than the anatomical maximum a_max, by an amount that varies across species, particularly as the actual pore geometry usually deviates from simplified cylindrical geometry (Franks and Farquhar, 2007). Second, as guard cells close under adverse conditions, a’ declines (Fanourakis et al., 2015). Third, there may be a substantial contribution of diffusion resistances in the intercellular airspaces, especially in the case of a partly cutinized substomatal chamber (Roth-Nebelsick, 2007; Feild et al., 2011). Fourth, leaf surface features such as hairs or papillae surrounding the stomata, or encryption of stomata, may affect the diffusion through stomata, and especially will influence the boundary layer, which in addition to stomatal conductance determines overall diffusional conductance and therefore gas exchange (Kenzo et al., 2008; Hassiotou et al., 2009; Maricle et al., 2009). Clearly, much more research is needed to establish models that include all the factors that determine the anatomical influence of stomata on gas exchange rates and to validate these against a wide diversity of plants, yet the anatomical maximum defined as in Equations 1 and 2 is a strong constraint: g_max correlates across diverse species with g_op and light-saturated photosynthetic rate (McElwain et al., 2016), and scales up, in combination with leaf area allocation, to the determination of ecosystem net primary productivity (Wang et al., 2015a). The anatomical g_max is therefore a theoretical value estimating the maximum stomatal diffusion capacity, and like other theoretical physiological variables, such as photosynthetic parameters including the maximum carboxylation rate (V_cmax), it cannot be reached in practice, but is useful for generating hypotheses regarding the capacity for stomatal diffusion in various domains, such as comparisons of genotypes or species, functional types, or trends in evolutionary time (Franks and Beerling, 2009; Doheny-Adams et al., 2012; Taylor et al., 2012; McElwain et al., 2016; de Boer et al., 2016).Despite the well-recognized importance of both d and g_max, there has been limited understanding of their genetic and developmental basis and their relationships to other epidermal traits. Ever since the seminal work of E.J. Salisbury early last century, d has been known to be positively associated with stomatal initiation rate, also known as stomatal index (i = no. of stomata per no. of epidermal cells plus stomata; Salisbury, 1927; Wengier and Bergmann, 2012), and negatively with mean epidermal cell area (e), as increases in e would space stomata apart (Fig. 1, D–F). Studies of plants of different species (Beaulieu et al., 2008; Brodribb et al., 2013) or of given species grown in different irradiance and vapor pressure deficit treatments (Carins Murphy et al., 2012, 2014) found that d related negatively to e. Further, a negative relationship of d with s within plant canopies or across species has been found numerous times and sometimes attributed to a “general association” or “trade-off” (e.g. Weiss, 1865; Grubb et al., 1975; Tichá, 1982; Hetherington and Woodward, 2003; Sack et al., 2003; Franks and Beerling, 2009; Brodribb et al., 2013; Wang et al., 2015a; de Boer et al., 2016). Yet, while numerous correlational studies within and across species have confirmed these relationships, their formal mathematical basis has remained unclear.To directly link g_max, and thus stomatal flux, to underlying epidermal development traits, we derived new equations for d and g_max as functions of e, i, and s, where e and s are projected cell areas (units: m²).As defined by Salisbury (1927), i is the number of stomata (n_s) divided by the sum of n_s and the number of epidermal cells (n_e):(3)Stomatal density (d) is related to n_s, n_e, s, and e as:(4)where area is that of the whole leaf (units: m²). Equation 4 can be rearranged as(4a)The ratio n_e/n_s can be expressed in terms of i by rearranging Equation 3:(5)Applying Equation 5 to Equation 4a gives(6)This equation gives d as a function of e, i, and s—traits with a transparent relationship to development, all being related to epidermal cell differentiation and expansion. Equation 6 can be applied to Equation 2 to give g_max as a function of e, i, and s:(7)These expressions rely on mean values for e, s, and i, so their accuracy may be affected by variation of these variables within leaves, or by variation in sampling methods as there exists no standard measurement protocol (see “Materials and Methods”). We tested the correctness of the derivation of Equation 6 and its applicability to real measurements of d, e, i, and s for abaxial leaf surfaces compiled from the published literature for 141 values from 81 species from 28 angiosperm families (“Supplemental Data”). We further checked for quantitative consistency between g_max as estimated from e, s, and i (Eq. 7) and the literature standard estimate of g_max from d and s (Eq. 2) using the same dataset. In both cases we found extremely tight correspondence (Fig. 2). Considering relationships within individual plant families for which ≥ 6 points were available showed similarly tight correspondence (R² = 0.96–1.0, P < 0.001, n = 6–30 for Betulaceae, Ericaceae, Fabaceae, Fagaceae, Orchidaceae, Rosaceae, and Sapindaceae; slopes and intercepts did not differ at P < 0.05 among families or from 1.0 and 0, respectively).Open in a separate windowFigure 2.Developmental basis for maximum stomatal flux variables: the estimation of abaxial leaf stomatal density (d; A) and theoretical maximum stomatal conductance (g_max; B) as functions of epidermal cell area (e), stomatal index (i), and stomatal area (s). A, Values estimated using Equation 6 plotted against reported values of d; B, values estimated using Equation 7 plotted against values estimated using Equation 2 with inputs of s and d as standard in the current literature. Data were compiled from single or mean values for leaves from published papers for seedlings (circles) and adults (squares) of 54 European woody species; four species of annual herbs of genus Gomphrena (Amaranthaceae; triangles); 22 species of genus Stanhopea, Orchidaceae (diamonds); and one grass species (Paspalum dilatatum, Poaceae; hexagon). The lines are ordinary least squares regressions fitted to the data with fixed zero intercept. The high R² values indicate the correctness of the derivation, its applicability to real measurements, and the quality of the measurements.These equations clarify precise geometric linkages among stomatal flux, anatomy, and development. We propose five examples of potentially powerful applications of these relationships to inform fundamental research across plant development, physiology, paleobiology, and crop science.

• (1) An expansion of available data on stomatal differentiation. Measurements of i can be technically challenging given the need to resolve all epidermal pavement cells in an image, but Equation 6 can be rearranged to allow estimation of i from measurements of d, e, and s, greatly expanding the data availability for this important developmental trait:(8)
• (2) Analysis of the developmental and genetic drivers of d and g_max across genotypes of a given species or across phylogenetically diverse species; i.e. quantifying how much of the variation in d and g_max arises due to differences in i, e, or s. Thus, the developmental basis for observed shifts in g_max in response to climate, CO₂, and lifeform evolution can be inferred using Equations 6 and 7.
• (3) Clarifying the quantitative role of shifts in genome and cell sizes (i.e. e and s) on g_max. The question of the role and impact of cell size is especially important given the strong developmental plasticity and evolutionary lability of cell size, and its relationship to other traits. For example, within some lineages, epidermal cell size correlates positively with genome size and leaf size and/or negatively with venation density (Beaulieu et al., 2008; Brodribb et al., 2013).
• (4) Resolving the coordinated shifts of stomatal traits in fossils and experimental plants, thereby improving inferences concerning shifts in response to global temperature and atmospheric CO₂. Previous studies of adaptation and acclimation in response to CO₂ have tended to quantify d and/or i (e.g. Beerling et al., 1998; Royer, 2001) and/or more rarely s and e (e.g. Ogaya et al., 2011; Haworth et al., 2014), and assumed that a shift in any one of these traits was an important marker of adaptation. Equations 6 and 7 allow estimation of the quantitative dependency of shifts in d and g_max on other variables.
• (5) Prediction of how each trait should be adjusted, through breeding or genetic manipulation, to optimize productivity through changes in g_max. Equations 6 and 7 clarify the separate roles of e, i, and s in determining higher g_max. Given the increasing resolution of the genetic basis for these traits in model species (e.g. Ferris et al., 2002; Delgado et al., 2011), these traits can be made specific targets for breeding for higher g_max and thereby for productivity. Other traits would also need to be targeted (e.g. hydraulic and photosynthetic traits) to enable higher productivity above and beyond the potential cost of constructing, maintaining, and operating additional stomatal apparatus (Assmann and Zeiger, 1987).

By linking leaf epidermal anatomy and development with physiological flux, these equations allow scaling from the differentiation and expansion of epidermal cells and stomata to plant productivity. Given ongoing improvement of models for the influence of the anatomy and dynamic behavior of stomata and of internal and external leaf tissues on gas exchange, consideration of these important traits in terms of their development will have potential applications across the widest range of fields in plant biology and earth system science.     

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal     

The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response?

This paper examines the dependence of whole leaf hydraulic conductance to liquid water (K(L)) on irradiance when measured with a high pressure flowmeter (HPFM). During HPFM measurements, water is perfused into leaves faster than it evaporates hence water infiltrates leaf air spaces and must pass through stomates in the liquid state. Since stomates open and close under high versus low irradiance, respectively, the possibility exists that K(L) might change with irradiance if stomates close tightly enough to restrict water movement. However, the dependence of K(L) on irradiance could be due to a direct effect of irradiance on the hydraulic properties of other tissues in the leaf. In the present study, K(L) increased with irradiance for 6 of the 11 species tested. Whole leaf conductance to water vapour, g(L), was used as a proxy for stomatal aperture and the time-course of changes in K(L) and g(L) was studied during the transition from low to high irradiance and from high to low irradiance. Experiments showed that in some species K(L) changes were not paralleled by g(L) changes. Measurements were also done after perfusion of leaves with ABA which inhibited the g(L) response to irradiance. These leaves showed the same K(L) response to irradiance as control leaves. These experimental results and theoretical calculations suggest that the irradiance dependence of K(L) is more consistent with an effect on extravascular (and/or vascular) tissues rather than stomatal aperture. Irradiance-mediated stimulation of aquaporins or hydrogel effects in leaf tracheids may be involved.     

Sex differences in the rate of fatigue development and recovery

Background

Many musculoskeltal injuries in the workplace have been attributed to the repetitive loading of muscle and soft tissues. It is not disputed that muscular fatigue is a risk factor for musculoskeltal injury, however the disparity between gender with respect to muscular fatigability and rate of recovery is not well understood. Current health and safety guidelines do not account for sex differences in fatiguability and may be predisposing one gender to greater risk. The purpose of this study was to quantify the sex differences in fatigue development and recovery rate of lower and upper body musculature after repeated bouts of sustained isometric contractions.

Methods

Twenty-seven healthy males (n = 12) and females (n = 15) underwent bilateral localized fatigue of either the knee extensors (male: n = 8; female: n = 8), elbow flexors (male: n = 8; female: n = 10), or both muscle groups. The fatigue protocol consisted of ten 30-second sub-maximal isometric contractions. The changes in maximum voluntary contraction (MVC), electrically evoked twitches, and motor unit activation (MUA) were assessed along with the ability to control the sustained contractions (SLP) during the fatigue protocol using a mixed four-factor repeated measures ANOVA (gender × side × muscle × time) design with significance set at p < 0.05.

Results

There was a significant loss of MVC, MUA, and evoked twitch amplitude from pre- to post-fatigue in both the arms and legs. Males had greater relative loss of isometric force, a higher rate of fatigue development, and were less capable of maintaining the fatiguing contractions in the legs when compared to the females.

Conclusion

The nature of the induced fatigue was a combination of central and peripheral fatigue that did not fully recover over a 45-minute period. The results appear to reflect sex differences that are peripheral, and partially support the muscle mass hypothesis for explaining differences in muscular fatigue.

     

Climatic sensitivity of species’ vegetative and reproductive phenology in a Hawaiian montane wet forest

Understanding how tropical tree phenology (i.e., the timing and amount of seed and leaf production) responds to climate is vital for predicting how climate change may alter ecological functioning of tropical forests. We examined the effects of temperature, rainfall, and photosynthetically active radiation (PAR) on seed phenology of four dominant species and community-level leaf phenology in a montane wet forest on the island of Hawaiʻi using monthly data collected over ~ 6 years. We expected that species phenologies would be better explained by variation in temperature and PAR than rainfall because rainfall at this site is not limiting. The best-fit model for all four species included temperature, rainfall, and PAR. For three species, including two foundational species of Hawaiian forests (Acacia koa and Metrosideros polymorpha), seed production declined with increasing maximum temperatures and increased with rainfall. Relationships with PAR were the most variable across all four species. Community-level leaf litterfall decreased with minimum temperatures, increased with rainfall, and showed a peak at PAR of ~ 400 μmol/m²s⁻¹. There was considerable variation in monthly seed and leaf production not explained by climatic factors, and there was some evidence for a mediating effect of daylength. Thus, the impact of future climate change on this forest will depend on how climate change interacts with other factors such as daylength, biotic, and/or evolutionary constraints. Our results nonetheless provide insight into how climate change may affect different species in unique ways with potential consequences for shifts in species distributions and community composition.     

Discovery of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent and selective PI3Kδ inhibitors

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.