Long-term seasonal changes in evaporative demand and its association with micro-meteorological elements were examined using a supersonic pan-evaporimeter in a greenhouse from June 6, 2009 to December 31, 2011. Daily pan evaporation rate was strongly correlated with the reference evapotranspiration rate, which could be widely used as an index of evaporative demand. Cucumber plants (Cucumis sativus L.) were grown during different seasons (spring, summer, autumn, and winter), and the effects of evaporative demand on transpiration and leaf conductance (leaf gas exchange characteristics) were analyzed on an hourly basis. A positive linear relationship between the leaf gas exchange characteristics and evaporative demand was observed; however, the impact of evaporative demand on the gas exchange characteristics differed between seasons. Furthermore, an excessive plant evaporative demand was observed during the daytime in the summer season.

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For the purpose of definite classification of Viola eizanensis and Viola chaerophylloides f. sieboldiana, which have similar leaf morphology, the characteristic flavonoids in their leaves were analyzed by UV-DAD, LC-ESI/MS and NMR. The HPLC chromatograph patterns of methanol extracts were significantly different from each other. Two characteristic flavonoids were isolated and identified. One was apigenin 8-C-rhamnosyl - (1 → 2) - glucoside (vitexin-2″ -O-rhamnoside) from the leaves of both Viola species, and the other was acacetin 7-O- (4-O-acetylrhamnosyl) (1 → 6) - glucoside only from the leaves of Viola chaerophylloides f. sieboldiana. As the HPLC chromatograph patterns of the methanol extracts of both species were not affected by the season and growing regions, the leaf flavonoid analysis in combination of morphology may ensure an easy and more reliable method for the definite classification of the Viola species. These results will also contribute to the accumulation of information as medicinal plants.

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