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.

P-MIRU, a Polarized Multispectral Imaging System, Reveals Reflection Information on the Biological Surface.

Reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term 'reflectome', cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Although previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multispectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multispectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. The P-MIRU system extends our visual ability and unveils information on biological surfaces.

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.

First records of non-native species Callitrichedeflexa (Plantaginaceae), which was previously misidentified as C.terrestris in Japan.

Background: The cosmopolitan genus Callitriche (Plantaginaceae) is a clade of small herbaceous plants that encompasses terrestrial and aquatic species. In Japan, six Callitriche species have been identified: four native and two naturalised species. Callitricheterrestris, a naturalised terrestrial species, was first reported in 1984 in Kanagawa Prefecture and it is thriving today. New information: We report the presence of a new naturalised terrestrial species, Callitrichedeflexa, which has been previously misidentified as C.terrestris because of its similar morphology. Callitrichedeflexa can be distinguished from C.terrestris through genetic differences and distinct morphological traits, such as longer pedicels. Re-examination of herbarium specimens in the Kanagawa Prefectural Museum of Natural History confirmed that most of the specimens labelled as C.terrestris, including voucher specimens from the original report, were indeed C.terrestris, but a few were C.deflexa. We also noted that the plants referred to as "C.terrestris" in our previous developmental studies should be corrected to C.deflexa.

WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus.

Plants can regenerate their bodies via de novo establishment of shoot apical meristems (SAMs) from pluripotent callus. Only a small fraction of callus cells is eventually specified into SAMs but the molecular mechanisms underlying fate specification remain obscure. The expression of WUSCHEL (WUS) is an early hallmark of SAM fate acquisition. Here, we show that a WUS paralog, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), negatively regulates SAM formation from callus in Arabidopsis thaliana. WOX13 promotes non-meristematic cell fate via transcriptional repression of WUS and other SAM regulators and activation of cell wall modifiers. Our Quartz-Seq2-based single cell transcriptome revealed that WOX13 plays key roles in determining cellular identity of callus cell population. We propose that reciprocal inhibition between WUS and WOX13 mediates critical cell fate determination in pluripotent cell population, which has a major impact on regeneration efficiency.

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.

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.