Most plant species require all the vegetative (shoot, stem, and root) and reproductive (flower, fruit, and seed) organs to complete their life cycle. These plants produce numerous branches through the growth of the shoot apical meristem (SAM) and root apical meristem (RAM) (Weigel and Jurgens 2002). However, the morphology of W. globosa does not fit traditional botanical descriptions. Our study revealed that W. globosa normally budded new fronds from its unique meristematic area by vegetative propagation. The meristematic area of W. globosa was located at the base of the RpM and was a collection of poorly differentiated cells with the ability to divide; there were no morphologically strict divisions in the meristem area. The dividing cells may perform different functions than the SAM in Arabidopsis thaliana, including expression of some key genes involved in SAM activity and the distribution of auxin and cytokinin (Zadnikova et al. 2014; Maugarny-Calès et al. 2018).
The new generations produced by vegetative propagation were called DFs or new buds as in a budding yeast and were released horizontally from the RpM. Usually, several individuals at different developmental stages coexisted in a single W. globosa frond. A single Wolffia frond can produce 11 daughter fronds on average and live for about 17 days on average (Bernard and Bernard 1990). Each bud began to senescence on the 10th day of survival and the average life span was about 17 days (Bernard and Bernard 1990). Our study also confirmed the rapid propagation of Wolffia from the structural perspective. This reproductive strategy enabled rapid accumulation of biomass in Wolffia, which roughly doubled in 48 hours, and allows its wide distribution in various habitats around the world (Ziegler et al. 2013). Wolffia seldom flower under natural conditions, and no seed has been reported so far. However, Wolffia flowers have been reported in the laboratory; the causes of its reduction of sexual reproduction could be revealed in the future.
The reduction of the root in Wolffia is one of the most striking examples of structural reduction in the plant kingdom. Duckweeds include five genera; members of Wolffia and Wolffiella are rootless, while members of Spirodela, Landoltia, and Lemna produce either a single or few roots (Cao et al. 2016). There is a reduction of the number of roots from Spirodela to Lemna and they disappear entirely in Wolffia. Early studies suggested that duckweeds did not use their roots to acquire nutrients, and instead acquire nutrients through their fronds (Gorham 1941; Muhonen et al.1983; Ice and Couch 1987; Meijer and Sutton 1987).
Echlin et al. (1981) found that most absorption of ions occurred in the root tip region of Lemna minor, and observed a Casparian band structure in the endodermis of the root tip. They suggested that the root of L. minor can not only absorb nutrients and water but also transport these to the frond. Kim (2007) carried out a detailed study of root development of S. polyrhiza and found a large number of plasmodesmata between the cells of the root. They therefore concluded that the transport of metabolites between the root and frond may rely on the symplastic pathway. The root of duckweeds plays an important role in absorbing nutrients and maintaining their floating lifestyle. For example, the density of cells in the root tip of S. polyrhiza is high, such that it forms a pendulum-like structure to cope with water fluctuations and stabilize the plant on the water surface. However, for the rootless Wolffia, White and Wise (1998) suggested they stay afloat and upright not by buoyancy but by surface tension. In their opinion, if buoyancy kept Wolffia plants at the water’s surface, then they would sink late in the day as their starch content reached a maximum. In our study, the dorsal part of W. globosa was always above the water, and it was difficult to submerge the plants or turn them over. In addition, most of the chloroplasts, which were filled with starch grains, were concentrated at the dorsal side. Previous studies have shown that dormant individuals of Wolffia were full of starch grains and sank in the water. We speculate that the content of starch grains affects the stable floating of Wolffia. Furthermore, the loss of the nutrient uptake and stabilization functions of the root in Wolffia may have allowed them to lose this organ. Phylogenetic analysis using different molecular markers has confirmed that duckweeds comprise a single monophyletic clade (Tippery et al. 2015), suggesting that rootlessness has a single evolutionary origin in Lemnoideae.
We also propose that Wolffia is a suitable model to study structural reduction in angiosperms and to explore the cause of rootlessness. First, Wolffia is easy to cultivate, completes its life cycle in the lab, and reproduces quickly. Second, Wolffia plants can be genetically transformed, as can the rooted Spirodela and Lemna, allowing us to conduct genetic studies (Cantó-Pastor et al. 2015; Heenatigala et al. 2018; Yang et al. 2018b). Wolffia species are the smallest flowering plants in the world, in both size and morphological structures, containing one leaf, one stamen and one gynoecium, which represent the core elements for angiosperms to complete their life cycle. Our study indicates that the simplicity of this species has been overestimated, because Wolffia has all the same organelles as other angiosperms at the ultrastructural level. Hillman (1961) pointed out that although the gross morphology and vegetative reproduction of Lemnaceae are somewhat unusual, their anatomy, particularly the prominent air spaces and reduced vascular structures, resembles that of many aquatic angiosperms. Anderson et al. (1973) also pointed out that although Wolffia lacks vascular tissue, the range of tissue and cell types appears as heterogeneous as in most leaves and varies considerably from meristematic to mature chlorenchymous tissue. Not only is it suitable to study structural reduction, but Wolffia would also be an ideal system to explore fundamental processes of angiosperm development that cannot be addressed using other model plants.