Beyond stomatal development: SMF transcription factors as versatile toolkits for land plant evolution.

As master transcription factors of stomatal development, SPEECHLESS, MUTE, and FAMA, collectively termed SMFs, are primary targets of molecular genetic analyses in the model plant Arabidopsis thaliana. Studies in other model systems identified SMF orthologs as key players in evolutionary developmental biology studies on stomata. However, recent studies on the astomatous liverwort Marchantia polymorpha revealed that the functions of these genes are not limited to the stomatal development, but extend to other types of tissues, namely sporophytic setal and gametophytic epidermal tissues. These studies provide insightful examples of gene-regulatory network co-opting, and highlight SMFs and related transcription factors as general toolkits for novel trait evolution in land plant lineages. Here, we critically review recent literature on the SMF-like gene in M. polymorpha and discuss their implications for plant evolutionary biology.

Petal segmentation in CT images based on divide-and-conquer strategy.

Manual segmentation of the petals of flower computed tomography (CT) images is time-consuming and labor-intensive because the flower has many petals. In this study, we aim to obtain a three-dimensional (3D) structure of Camellia japonica flowers and propose a petal segmentation method using computer vision techniques. Petal segmentation on the slice images fails by simply applying the segmentation methods because the shape of the petals in CT images differs from that of the objects targeted by the latest instance segmentation methods. To overcome these challenges, we crop two-dimensional (2D) long rectangles from each slice image and apply the segmentation method to segment the petals on the images. Thanks to cropping, it is easier to segment the shape of the petals in the cropped images using the segmentation methods. We can also use the latest segmentation method for the task because the number of images used for training is augmented by cropping. Subsequently, the results are integrated into 3D to obtain 3D segmentation volume data. The experimental results show that the proposed method can segment petals on slice images with higher accuracy than the method without cropping. The 3D segmentation results were also obtained and visualized successfully.

Characterization of the Arabidopsis Mutant oligocellula6-D Reveals the Importance of Leaf Initiation in Determining the Final Leaf Size.

The leaf is a determinate organ with a final size under genetic control. Numerous factors that regulate the final leaf size have been identified in Arabidopsis thaliana; although most of these factors play their roles during the growth of leaf primordia, much less is known about leaf initiation and its effects on the final leaf size. In this study, we characterized oligocellula6-D (oli6-D), a semidominant mutant of A. thaliana with smaller leaves than the wild type (WT) due to its reduced leaf cell numbers. A time-course analysis showed that oli6-D had approximately 50% fewer leaf cells even immediately after leaf initiation; this difference was maintained throughout leaf development. Next-generation sequencing showed that oli6-D had chromosomal duplications involving 2-kb and 3-Mb regions of chromosomes 2 and 4, respectively. Several duplicated genes examined had approximately 2-fold higher expression levels, and at least one gene acquired a new intron/exon structure due to a chromosome fusion event. oli6-D showed reduced auxin responses in leaf primordia, primary roots and embryos, as well as reduced apical dominance and partial auxin-resistant root growth. CRISPR-associated protein-9-mediated genome editing enabled the removal of a 3-Mb duplicated segment, the largest targeted deletion in plants thus far. As a result, oli6-D restored the WT leaf phenotypes, demonstrating that oli6-D is a gain-of-function mutant. Our results suggest a new regulatory point of leaf size determination that functions at a very early stage of leaf development and is negatively regulated by one or more genes located in the duplicated chromosomal segments.

Precocious cell differentiation occurs in proliferating cells in leaf primordia in Arabidopsis angustifolia3 mutant.

During leaf development, the timing of transition from cell proliferation to expansion is an important factor in determining the final organ size. However, the regulatory system involved in this transition remains less understood. To get an insight into this system, we investigated the compensation phenomenon, in which the cell number decreases while the cell size increases in organs with determinate growth. Compensation is observed in several plant species suggesting coordination between cell proliferation and expansion. In this study, we examined an Arabidopsis mutant of ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING FACTOR 1, a positive regulator of cell proliferation, which exhibits the compensation. Though the AN3 role has been extensively investigated, the mechanism underlying excess cell expansion in the an3 mutant remains unknown. Focusing on the early stage of leaf development, we performed kinematic, cytological, biochemical, and transcriptome analyses, and found that the cell size had already increased during the proliferation phase, with active cell proliferation in the an3 mutant. Moreover, at this stage, chloroplasts, vacuoles, and xylem cells developed earlier than in the wild-type cells. Transcriptome data showed that photosynthetic activity and secondary cell wall biosynthesis were activated in an3 proliferating cells. These results indicated that precocious cell differentiation occurs in an3 cells. Therefore, we suggest a novel AN3 role in the suppression of cell expansion/differentiation during the cell proliferation phase.

Expression analyses of CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS in the one-leaf plant Monophyllaea glabra reveal neoteny evolution of shoot meristem.

The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

Decoding the leaf apical meristem of Guarea glabra Vahl (Meliaceae): insight into the evolution of indeterminate pinnate leaves.

In seed plants, growth of shoots and roots is indeterminate, while leaves are typically determinate organs that cease to grow after a certain developmental stage. This is due to the characteristics of the leaf meristem, where cell proliferation activity is retained only for a limited period. However, several plants exhibit indeterminacy in their leaves, exemplified by the pinnate compound leaves of Guarea and Chisocheton genera in the Meliaceae family. In these plants, the leaf meristem at the tip of the leaf retains meristematic activity and produces leaflets over years, resulting in a single leaf that resembles a twig. The molecular mechanism underlying the indeterminate leaf meristem of these plants has not been examined. In this research, we used Guarea glabra as a model to investigate the development of indeterminate pinnate leaves. Transcriptome analyses revealed that the gene expression profile in leaf apex tissue differed from that in the shoot apex. However, a class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) gene which is lost in Brassicaceae was highly expressed in both tissues. We established an in situ hybridisation system for this species using Technovit 9100 to analyse the spatial expression patterns of genes. We revealed that the leaf meristematic region of G. glabra expresses KNOX1, LEAFY and ANGUSTIFORIA3 simultaneously, suggesting the involvement of these genes in the indeterminacy of the leaf meristem.

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.

Cavity and entrance pore development in ant plant hypocotyls.

Some genera of Rubiaceae in South-eastern Asia are known as typical ant plants. They have large domatia, which form in well-developed hypocotyls in which ants nest. Previously, cavity formation processes were described; however, these reports were dependent on tissue sections of different individuals of different ages. No continuous time-course analyses were done because cavity formation occurs inside the thick tissues of highly swollen domatia. Here we observed cavity formation processes in ant plants by using X-ray computed tomography (CT) imaging and revealed previously overlooked features of cavity formation. Firstly, the cavity pore occurs at the hypocotyl base in not only gravity-dependent but also basal position-dependent manner. Secondly, the cavity forms prior to the start of short tunnel formation between the cavity and the pore. The cavity axis is parallel to the longitudinal axis of the hypocotyl; however, the short tunnel axis between the pore and cavity depends on gravity. Non-invasive CT scanning is a very powerful method to analyze deeply hidden morphogenic processes in organs.

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.

Correction: Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

[This corrects the article DOI: 10.1371/journal.pgen.1008873.].