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

Abstract

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 molecularbased 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.

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

“…diminutive stature and ease of laboratory culture make Callitriche an attractive model for eco-evolutionary investigations of diverse aspects of amphibious life in plants, including stomatal suppression induced by submergence. We recently described the dramatic suppression of stomatal development in submerged leaves of C. palustris (23). We also found that both aerial and The diversity of stomatal development regulation in Callitriche is related to the intrageneric diversity in lifestyles submerged leaves of this species differentiate stomata during a restricted developmental period, when the leaf primordia are ∼1 mm long (23).…”

“…S8 D and E). In C. palustris, cell proliferation occurred in the basal region of the leaf blade (23), as in C. japonica (SI Appendix, Fig. S8C), and SPCH orthologs were expressed in dividing cells in both species.…”

“…The Callitriche plants used in this study were originally collected from their wild habitats in Japan and maintained in our laboratory. We collected C. stagnalis, C. terrestris, and C. japonica specimens in Ibaraki Prefecture, Hyogo Prefecture, and Kanagawa Prefecture, respectively, and included them in our analysis together with the previously described C. palustris NH-1 strain (23). All plants were maintained in a growth chamber at 23°C under long-day conditions (16 h light/8 h darkness), with a light intensity of 60 μmol photons m −2 s −1 .…”

“…We recently described the dramatic suppression of stomatal development in submerged leaves of C. palustris (23). We also found that both aerial and The diversity of stomatal development regulation in Callitriche is related to the intrageneric diversity in lifestyles submerged leaves of this species differentiate stomata during a restricted developmental period, when the leaf primordia are ∼1 mm long (23). This contrasts strongly with the well-described process in Arabidopsis, in which the number of stomata continues to increase in even later stages of leaf development (25).…”

“…Although this plasticity in stomatal development has long been known for amphibious species from broad lineages ( 19 – 22 ), the molecular basis is still largely unexplored. To investigate this phenomenon, we previously described the process of leaf and stomatal development in the amphibious plant Callitriche palustris L. under highly reproducible conditions ( 23 ).…”