The quantitative measurement of nitric oxide (NO) in plant cells acquired great importance, in view of the multifaceted function and involvement of NO as a signal in various plant processes.
Monitoring of NO in guard cells is quite simple because of the large size of guard cells and ease of observing the detached epidermis under microscope. Stomatal guard cells therefore provide an excellent model system to study the components of signal transduction.
The levels and functions of NO in relation to stomatal closure can be monitored, with the help of an inverted fluorescence or confocal microscope. We can measure the NO in guard cells by using flouroprobes like 4,5-diamino fluorescein diacetate (DAF-2DA). This fluorescent dye, DAF-2DA, is cell permeable and after entry into the cell, the diacetate group is removed by the cellular esterases.
The resulting DAF-2 form is membrane impermeable and reacts with NO to generate the highly fluorescent triazole (DAF-2T), with excitation and emission wavelengths of 488 and 530 nm, respectively. If time-course measurements are needed, the epidermis can be adhered to a cover-glass or glass slide and left in a small petri dishes.
Fluorescence can then be monitored at required time intervals; with a precaution that excitation is done minimally, only when a fluorescent image is acquired.
The present method description is for the epidermis of Arabidopsis thaliana and Pisum sativum and should work with most of the other dicotyledonous plants.
The formation of stomata and leaf mesophyll airspace must be coordinated to establish an efficient and robust network that facilitates gas exchange for photosynthesis, however the mechanism by which this coordinated development occurs remains unclear. Here, we combine microCT and gas exchange analyses with measures of stomatal size and patterning in a range of wild, domesticated and transgenic lines of wheat and Arabidopsis to show that mesophyll airspace formation is linked to stomatal function in both monocots and eudicots.
Our results support the hypothesis that gas flux via stomatal pores influences the degree and spatial patterning of mesophyll airspace formation, and indicate that this relationship has been selected for during the evolution of modern wheat. We propose that the coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops, providing a target for future improvement.
Previous studies have showed that UV-B can stimulate closure as well as opening of stomata. However, the mechanism of this complex effect of UV-B is not clear. The purpose of this paper is to investigate the role and the interrelationship of H2O2 and NO in UV-B-induced stomatal closure in broad bean (Vicia faba L.). By epidermal strip bioassay and laser-scanning confocal microscopy, we observed that UV-B-induced stomatal closure could be largely prevented not only by NO scavenger c-PTIO or NO synthase (NOS) inhibitor L-NAME, but also by ascorbic acid (ASC, an important reducing substrate for H2O2 removal) or catalase (CAT, the H2O2 scavenger), and that UV-B-induced NO and H2O2 production in guard cells preceded UV-B-induced stomatal closure. These results indicate that UV-B radiation induces stomatal closure by promoting NO and H2O2 production. In addition, c-PTIO, L-NAME, ASC and CAT treatments could effectively inhibit not only UV-B-induced NO production, but also UV-B-induced H2O2 production. Exogenous H2O2-induced NO production and stomatal closure were partly abolished by c-PTIO and L-NAME. Similarly, exogenous NO donor sodium nitroprusside-induced H2O2 production and stomatal closure were also partly reversed by ASC and CAT. These results show a causal and interdependent relationship between NO and H2O2 during UV-B-regulated stomatal movement. Furthermore, the L-NAME data also indicate that the NO in guard cells of Vicia faba is probably produced by a NOS-like enzyme.
Stomatal and nonstomatal limitations of photosynthesis in mung bean (Phaseolus radiatus L.) leaves under the combination of 0.35 W/m(2) UV-B radiation and 0.4% NaCl stress were studied. Separated or combined treatments of enhanced UV-B radiation and NaCl stress all resulted in a decrease in net photosynthetic rate, stomatal conductance, photosynthetic ability, efficiency of CO(2) carboxylation and Rubisco content, and the extent of those decreases enhanced obviously with the increase of treatment days. Intercellular CO(2)concentration tended to decrease in short period, followed by a gradual increase and to be higher than the control on the seventh day after treatment under NaCl stress alone, while a tendency of gradual increase was observed under enhanced UV-B radiation alone or two stresses combined and the concentration was higher than the control after three day of treatments. By contrast, the tendency of stomatal limitation value was just reverse to the intercellular CO(2) concentration, but the stomatal limitation value was always higher than control under all stress conditions (did not include that on the fifth day under the combined stresses). As compared with an individual stress the effect of combined stress on the above parameters was more serious. These results indicate that under all stress conditions the inhibition of photosynthesis in mung bean leaves is the results of both stomatal and nonstomatal limitations. And the stomatal limitation is dominant in short period, nonstomatal limitation becomes the dominant one in longer period. The decrease in Rubisco content leads to nonstomatal limitation of photosynthesis under all stresses.
The adaptation of Camellia rusticana, an evergreen broad-leaved shrub found in areas of heavy snowfall in Japan, to heavy snowfall environments, and the mechanisms by which it is damaged in winter above the snow, were investigated.
The stomatal response and photosynthetic characteristics of C. rusticana were compared to those of Camellia japonica found in areas of light snowfall. In field conditions, the mean net photosynthesis of C. rusticana at photon flux density (PFD) over 200 μmol m−2s−1 (Pn(>200). was 50% larger than that of C. japonica, but in both light saturated and CO2 saturated conditions, the O2 evolution rate (Pc) of C. rusticana was not different from that of C. japonica.
Mean leaf conductance at PFD over 200 μmol m−2s−1 (gl(>200)) was about 100% larger than that of C. japonica in the field. The Pn(>200)) was 50% ratio of C. rusticana was 37% higher than that of C. japonica which suggests that C. rusticana‘s larger Pn(>200) can be explained by its larger gl(>200).
When C. rusticana trees wintering underneath the snow were projected above it, the leaves of these plants showed serious drought within five days in non-freezing conditions. Their Pc and the maximum stomatal conductance decreased by half and did not recover.
The leaves of C. rusticana showed larger gl(>200) and a less sensitive stomatal response to the decrease of leaf water potential than that of C. japonica.
The stomata characteristics of C. rusticana caused larger net photosynthesis than that of C. japonica during the no snow period, and caused the need for snow cover in winter as protector from winter drought.