The diversity of stomatal development regulation in Callitriche is related to the intrageneric diversity in lifestyles.

Stomata, the gas exchange structures of plants, are formed by the division and differentiation of stem cells, or meristemoids. Although diverse patterns of meristemoid behavior have been observed among different lineages of land plants, the ecological significance and diversification processes of these different patterns are not well understood. Here we describe an intrageneric diversity in the patterns of meristemoid division within the ecologically diverse genus Callitriche (Plantaginaceae). Meristemoids underwent a series of divisions before differentiating into stomata in the terrestrial species of Callitriche, but these divisions did not occur in amphibious species, which can grow in both air and water, in which meristemoids differentiated directly into stomata. These findings imply the adaptive significance of diversity in meristemoid division. Molecular genetic analyses showed that the different expression times of the stomatal key transcription factors SPEECHLESS and MUTE, which maintain and terminate the meristemoid division, respectively, underlie the different division patterns of meristemoids. Unlike terrestrial species, amphibious species prematurely expressed MUTE immediately after expressing SPEECHLESS, which corresponded to their early termination of stomatal division. By linking morphological, ecological, and genetic elements of stomatal development, this study provides significant insight that should aid ecological evolutionary developmental biology investigations of stomata.

Experimental validation of the mechanism of stomatal development diversification.

Stomata are the structures responsible for gas exchange in plants. The established framework for stomatal development is based on the model plant Arabidopsis, but diverse patterns of stomatal development have been observed in other plant lineages and species. The molecular mechanisms behind these diversified patterns are still poorly understood. We recently proposed a model for the molecular mechanisms of the diversification of stomatal development based on the genus Callitriche (Plantaginaceae), according to which a temporal shift in the expression of key stomatal transcription factors SPEECHLESS and MUTE leads to changes in the behavior of meristemoids (stomatal precursor cells). In the present study, we genetically manipulated Arabidopsis to test this model. By altering the timing of MUTE expression, we successfully generated Arabidopsis plants with early differentiation or prolonged divisions of meristemoids, as predicted by the model. The epidermal morphology of the generated lines resembled that of species with prolonged or no meristemoid divisions. Thus, the evolutionary process can be reproduced by varying the SPEECHLESS to MUTE transition. We also observed unexpected phenotypes, which indicated the participation of additional factors in the evolution of the patterns observed in nature. This study provides novel experimental insights into the diversification of meristemoid behaviors.

Landscapes of facilitation: how self-organized patchiness of aquatic macrophytes promotes diversity in streams.

Spatial heterogeneity plays a crucial role in the coexistence of species. Despite recognition of the importance of self-organization in creating environmental heterogeneity in otherwise uniform landscapes, the effects of such self-organized pattern formation in promoting coexistence through facilitation are still unknown. In this study, we investigated the effects of pattern formation on species interactions and community spatial structure in ecosystems with limited underlying environmental heterogeneity, using self-organized patchiness of the aquatic macrophyte Callitriche platycarpa in streams as a model system. Our theoretical model predicted that pattern formation in aquatic vegetation - due to feedback interactions between plant growth, water flow and sedimentation processes - could promote species coexistence, by creating heterogeneous flow conditions inside and around the plant patches. The spatial plant patterns predicted by our model agreed with field observations at the reach scale in naturally vegetated rivers, where we found a significant spatial aggregation of two macrophyte species around C. platycarpa. Field transplantation experiments showed that C. platycarpa had a positive effect on the growth of both beneficiary species, and the intensity of this facilitative effect was correlated with the heterogeneous hydrodynamic conditions created within and around C. platycarpa patches. Our results emphasize the importance of self-organized patchiness in promoting species coexistence by creating a landscape of facilitation, where new niches and facilitative effects arise in different locations. Understanding the interplay between competition and facilitation is therefore essential for successful management of biodiversity in many ecosystems.

Callitriche cophocarpa biomass as a potential low-cost biosorbent for trivalent chromium.

The present study focused on the use of the dry mass of the macrophyte Callitriche cophocarpa as an effective biosorbent for chromium removal from concentrated solutions, typical for industrial effluents. In order to evaluate the usability of C. cophocarpa as the Cr(III) sorbent, its detailed physicochemical characterization has been performed as well as the preliminary adsorption studies. The biosorbent was characterized by specific surface area (SSA), porosity, total organic carbon (TOC), inorganic content as well as the cation exchange capacity (CEC), dominant exchangeable cations and anion exchange capacity (AEC), point of zero charge (pHpzc) and buffering capacity. The effect of the initial chromium concentration, solution pH and co-existing anions on the sorption effectiveness have been investigated. Based on theoretical isotherm models, the maximum adsorption capacity of the dry C. cophocarpa has been determined as 77.1 mg Cr(III)/g. Finally, the strength of Cr-binding onto the plant biomass has been evaluated using the BCR extraction method, stating that chromium was strongly and - under environmental conditions - irreversibly bound to the plant biomass.

Do chromium-resistant bacterial symbionts of hyperaccumulator Callitriche cophocarpa support their host in phytobial remediation of water?

Callitriche cophocarpa Sendtn. is a macrophyte widely distributed in aquatic systems of the temperate climate zone and a known hyperaccumulator of chromium. Ten pure symbiotic bacterial isolates of C. cophocarpa were obtained and identified. Three of the isolates showed the highest resistance to Cr(VI): Microbacterium sp. (Ct1), Aeromonas sp. (Ct3) and Acinetobacter sp. (Ct6). Acinetobacter sp. (Ct6) was able to survive up to a concentration of 104 mg/L (2 mM). The isolates were also able to effectively detoxify Cr(VI) by reducing it to Cr(III). We tested whether inoculation of plants with a consortium consisting of Ct1, Ct3 and Ct6 affects: (1) the phytoextraction of chromium from leachates, (2) the physiological state of plants after Cr(VI) treatment. The solutions were landfill leachates and contained 10.7 mg/L of Cr(VI) - an amount 530 times exceeding the legal limits. We influenced the plants with Cr in two steps, each lasting for 10 days, first using mature shoots and then apical ones. The highest Cr content concomitant with the highest bioconcentration factor (BCF) were found in the inoculated plants: 1274 and 119 mg/kg dry mass (d.m.), respectively. The physiological status of the plants was assessed by biometric tests and advanced chlorophyll fluorescence analyses. The photosynthetic activity of mature shoots was influenced by Cr(VI) more negatively than that of young apical shoots. The inoculation with the bacterial consortium significantly reduced the negative effect of Cr(VI) on mature organs. In some cases the inoculated mature plants exhibited photosynthetic activity that was even higher than in the control plants. The results unequivocally show a beneficial effect of C. cophocarpa inoculation with the tested isolates resulting in a significant improvement of the phytoremediation properties of this aquatic chromium hyperaccumulator.

Callitriche as a potential model system for evolutionary studies on the dorsiventral distribution of stomata.

Controlling the distribution of stomata is crucial for the adaptation of plants to new, or changing environments. While many plant species produce stomata predominantly on the abaxial leaf surface (hypostomy), some produce stomata on both surfaces (amphistomy), and the remaining few produce them only on the adaxial surface (hyperstomy). Various selective pressures have driven the evolution of these three modes of stomatal distribution. Despite recent advances in our understanding of stomatal development and dorsiventral leaf polarity, the genetic basis for the evolution of different stomatal distributions is still unclear. Here, we propose the genus Callitriche as a new model system to investigate patterns in the evolution of stomatal distribution. Callitriche comprises species with diverse lifestyles, including terrestrial, amphibious, and obligately aquatic plants. We found that species in this genus cover all three modes of dorsiventral stomatal distribution, making it a desirable model for comparative and evolutionary analyses on distribution modes. We further characterized the genetic basis of the different distribution modes, focusing on the stomatal key transcription factor SPEECHLESS. Future research using the promising model system Callitriche would open a new direction for evolutionary developmental biology studies on stomata.

The complete chloroplast genome sequence of Callitriche palustris (Plantaginaceae).

Callitriche palustris L. is an annual aquatic or marsh plant, wide spread in temperate regions throughout the world. In present study, we sequenced, assembled and annotated the complete chloroplast (cp) genome of C. palustris. The length of C. palustris complete cp genome was 150,138 bp, with a typical quadripartite structure comprising a pair of inverted repeat regions (IRs; 25,667 bp), a large single copy region (LSC; 81,432 bp) and a small single copy region (SSC; 17,372 bp). The whole cp genome contained 134 genes, including 89 protein-coding genes (PCGs), 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The maximum likelihood (ML) phylogenetic analysis indicated that C. palustris was a member of Plantaginaceae, but the relationships between subfamilies and tribes need more samplings. This cp genome would provide a valuable genetic resource for C. palustris' phylogenetic study.

Terrestrial Morphotypes of Aquatic Plants Display Improved Seed Germination to Deal with Dry or Low-Rainfall Periods.

In temporary ponds, seed germination largely determines how well aquatic plant assemblages recover after dry periods. Some aquatic plants have terrestrial morphotypes that can produce seeds even in dry years. Here, we performed an experiment to compare germination patterns for seeds produced by aquatic and terrestrial morphotypes of Ranunculus peltatus subsp. saniculifolius over the course of five inundation events. During the first inundation event, percent germination was higher for terrestrial morphotype seeds (36.1%) than for aquatic morphotype seeds (6.1%). Seed germination peaked for both groups during the second inundation event (terrestrial morphotype: 47%; aquatic morphotype: 34%). Even after all five events, some viable seeds had not yet germinated (terrestrial morphotype: 0.6%; aquatic morphotype: 5%). We also compared germination patterns for the two morphotypes in Callitriche brutia: the percent germination was higher for terrestrial morphotype seeds (79.5%) than for aquatic morphotype seeds (41.9%). Both aquatic plant species use two complementary strategies to ensure population persistence despite the unpredictable conditions of temporary ponds. First, plants can produce seeds with different dormancy periods that germinate during different inundation periods. Second, plants can produce terrestrial morphotypes, which generate more seeds during dry periods, allowing for re-establishment when conditions are once again favorable.

The use of Callitriche cophocarpa Sendtn. for the reclamation of Cr-contaminated freshwater habitat: benefits and limitations.

This work is the first attempt to evaluate suitability of Callitriche cophocarpa Sendtn. (water-starwort) to remove Cr under real-world conditions. Our earlier laboratory-scale studies demonstrated outstanding hyperaccumulation properties of this aquatic higher plant (macrophyte) toward chromium in solution. We introduced C. cophocarpa plants into the watershed with sediments heavily polluted (on average 1400 mg/kg d.w. of Cr) by a tannery. The plants grew vigorously and exhibited no physiological or anatomical disorders. Based on chemical fractionations of bottom sediments, we found low Cr bioavailability. The element was strongly associated with the sediments and could be classified into the following fractions (%): oxidizable III (68.2) > residual IV (28.8) > reducible II (1.6) > exchangeable I (1.4). Despite this, Cr content in plant organs at the contaminated sites was 33 up to 83 times greater than in the control leaf/stem and roots, respectively. Altering redox potential during, i.e., sediment deposition on land may change chemical forms of bound metals in a solid phase, and thus further increase Cr phytoextraction by plants. With this in mind, we concluded that the species, being an outstanding Cr accumulator under laboratory conditions, can be useful in the reclamation of Cr-polluted sediments under controlled, oxidizing conditions.

Dimorphic Leaf Development of the Aquatic Plant Callitriche palustris L. Through Differential Cell Division and Expansion.

Heterophylly, or phenotypic plasticity in leaf form, is a remarkable feature of amphibious plants. When the shoots of these plants grow underwater, they often develop surprisingly different leaves from those that emerge in air. Among aquatic plants, it is typical for two or more distinct leaf development processes to be observed in the same individual exposed to different environments. Here, we analyze the developmental processes of heterophylly in the amphibious plant Callitriche palustris L. (Plantaginaceae). First, we reliably cultured this species under laboratory conditions and established a laboratory strain. We also established a framework for molecular-based developmental analyses, such as whole-mount in situ hybridization. We observed several developmental features of aerial and submerged leaves, including changes in form, stomata and vein formation, and transition of the meristematic zone. Then we defined developmental stages for C. palustris leaves. We found that in early stages, aerial and submerged leaf primordia had similar forms, but became discriminable through cell divisions with differential direction, and later became highly distinct via extensive cell elongation in submerged leaf primordia.

Accumulation patterns of Cr in Callitriche organs--qualitative and quantitative analysis.

The aims of this study were both the qualitative and quantitative analysis of chromium accumulation in the shoots of Callitriche cophocarpa. This globally distributed, submersed macrophyte exhibits outstanding Cr phytoremediation capacity in an aquatic environment. Cr was applied separately for 7 days at two stable forms as Cr(VI) and Cr(III), known from their diverse physicochemical properties and toxicities. The maps of Cr depositions in young leaves, mature leaves, and stems were obtained by micro X-ray fluorescence spectroscopy (µXRF). The detailed analysis of XRF maps was done based on Image-Pro PLUS (Media Cybernetics) software. Cr was accumulated either in trichomes or vascular bundles in respect to the element speciation and the plant organ. The concentration of Cr significantly increased in the following order: Cr(VI) mature leaves < Cr(VI) young leaves = Cr(VI) stems < Cr(III) young leaves ≤ Cr(III) mature leaves ≤ Cr(III) stems. The observed differences in distribution and accumulation of Cr were correlated with the different reduction potential of Cr(VI) by particular plant organs. The reduction of Cr(VI) is considered the main detoxification mechanism of the highly toxic Cr(VI) form. The unique L-band electron resonance spectrometer (L-band EPR) was applied to follow the reduction of Cr(VI) to Cr(III) in the studied material.

Seasonal acclimation in the photosynthetic and respiratory temperature responses of three submerged freshwater macrophyte species.

• Investigations of seasonal temperature acclimation in gas exchange are few and only exist for terrestrial and marine plants. Here we report on results obtained for three freshwater macrophyte species (Callitriche obtusangula, Potamogeton pectinatus and Potamogeton perfoliatus). • We collected plants from the field at monthly intervals and measured photosynthetic and respiratory temperature-response curves. Fitted and calculated parameters were derived from the curves and a simple model was used to evaluate the acclimative capacity to seasonal variation in water temperature. • For all species, optimum temperatures for gross photosynthesis showed little temporal variation. In addition, the shape of the temperature-response curves at suboptimal temperature was not optimized to temporal differences in water temperature. The only consistent seasonal trend in gas exchange was a gradual decrease in photosynthetic and respiratory capacity over time. • Our measurements and model predictions did not point to an acclimative seasonal response in the thermal dependence of oxygen exchange. Hence, we conclude that either other processes constrain the plants' response, or temporal variation in water temperature is less important than seasonal loss of photosynthetic capacity.