Under the natural light regime in the outside growth garden, the clone of R. hippuris
used in this study produced distinct submerged and aerial leaves. The measured values for leaf size are equivalent with those reported for specimens collected in natural habitats of the study species (Kadono, 1994
). Therefore, we judged that leaf characteristics of the clone were more or less equivalent to those found in natural habitats, and we used these values as those typical of submerged and aerial leaves of the study species when interpreting the results of the growth experiments.
The results clearly showed that R/FR is one of the major determinants of heterophyllous leaf formation of R. hippuris
. Higher and lower R/FR caused responses of the leaf characters more typical of those of submerged and aerial leaves, respectively, under both aerial and submerged conditions. Similar responses of heterophyllous amphibious plants have been reported in submerged leaves of Marsilea vestila
) and Hippuris vulgaris
(Bodkin et al., 1980
). Our results showed that the responses occur in leaves not only under water but also under aerial conditions. The observed strong responses to R/FR are in agreement with the idea that heterophyllous plants perceive R/FR as an index of depth (Spence, 1981
; Smith, 1982
), although underwater light quality in natural conditions can be highly variable (Smith, 1994
Although R. hiipuris
responded to R/FR under aerial and submerged conditions, differences of character values between the conditions at a given R/FR value were often great. The responses were greater under submerged conditions for leaf, leaf-tip and cell shapes than those under aerial conditions, and vice versa for stomata density. These observations suggested that not only R/FR but also other cues provided by submergence, such as osmotic stress and CO2
concentration, were operating in determining leaf morphology (Wells and Pigliucci, 2000
Four traits, i.e. leaf L/W, leaf-tip index, and upper and lower cell shapes, showed either quantitative or threshold-type responses depending on R/FR: steep responses between R/FR of 0·95 and 4·6 and a weak or no response to more extreme R/FR, such as 0·0013 and 791·3. The R/FR of daylight is approx. 1–1·1, but can decrease to 0·1 under vegetation, depending on canopy density (Smith, 1982
). Assuming R/FR is 1 at the water surface, the expected depth in pure water at which R/FR = 4·6 is about 1·5 m. Measurements of R/FR in natural lakes (Smith, 1982
; Chambers and Spence, 1984
) have suggested that an R/FR of 4·6 corresponded to that at a water depth of 1–1·5 m. The range of R/FR at which R. hippuris
showed high sensitivity of the four traits corresponds to that which is experienced by the plants in natural conditions. Stomata densities showed quantitative responses to R/FR of 4·6 and lower, and they exceeded the natural range of stomata densities at R/FR of 0·0013 under aerial conditions. The response to the extremely low R/FR may be a by-product of the underlying regulatory mechanism of stomata density.
Underwater leaves of R. hippuris
responded to blue-light intensity in all of the six analysed traits except for leaf-tip index. High blue light caused a shift of trait values toward those of typical aerial leaves. The results suggested that the response to blue-light intensity is important in determining leaf developmental fate near the water surface. A quantitative response to the three levels of blue-light intensity investigated is observed at R/FR below 0·9, which is close to the value expected at the water surface in natural conditions. The responses to blue light were weak or absent at higher R/FR (1·7; expected at about 50 cm depth under pure water with an R/FR of 1·0 at the surface), suggesting a dominance of R/FR over blue light as a developmental cue below the water surface. At low R/FR (0·5), the value expected under aerial conditions, even weak blue light was effective for producing aerial-type leaves under submerged conditions, and blue light was found to be necessary for this response, especially regarding cell shapes and stomata densities. In the aquatic fern Marsilea quadrifolia
, it has been reported that application of blue light induced the development of aerial-type leaves (Lin and Yang, 1999
). Our study indicated that blue-light intensity can be a critical cue of heterophyllous leaf development also for aquatic seed plants.
In conclusion, our experiments clearly showed that R/FR and blue-light intensity provide quantitative cues for R. hippuris
to determine water depth, especially near the water surface. The utilization of these quantitative cues is likely to make it possible for R. hippuris
to prepare aerial leaves at the shoot apical meristem just beneath the water surface prior to its emergence from the water. Dosage-dependent regulation of leaf morphogenesis is expected to be important, especially in the habitats of many amphibious plants where plants experience water-level fluctuation. Further study will be required to determine whether the response to light quality found in this study is a general one among other heterophyllous plants. Furthermore, we need to compare the response to light quality between plants that grow in terrestrial and aquatic habitats. Blue light and R/FR are known to be major cues of the morphogenesis of many terrestrial plants, because they inform about the degree of shade by surrounding vegetation (Franklin, 2008
). Interestingly, the correlation of R/FR with the level of shade is reversed between terrestrial and aquatic conditions, i.e. low R/FR and high R/FR are indicative of low PAR levels in aerial and underwater conditions, respectively. It has been reported recently that stomata density of Arabidopsis thaliana
decreased in response to low R/FR (Boccalandro et al., 2009
), and the direction of the response was opposite of what we found here. Comparative studies on the mechanisms underlying these responses should help to explain the evolution of these opposite responses to the same environmental cue.