NO-measurements in stomatal guard cells

Nitric oxide (NO) measurements in stomatal guard cells

by Agurla S., Gayatri G., Raghavendra A. S. (2016)

  • Srinivas Agurla,
  • Gunja Gayatri,
  • Agepati S. Raghavendra,

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India


In Methods Mol Biol 1424: 49–56 –


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 coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops

Stomatal patterning shifts with ploidy level in wheat. Sample images of overall stomatal distribution along the adaxial epidermis (ac) and individual stomatal complexes (df) in Triticum baeoticum (2n; ad), T. araraticum (4n; be) and T. aestivumcv Cougar (6n; cf). Scale bar ac = 80 µm; df = 20 µm. Stomatal width (g) (ANOVA, F(2,81) = 169.5, P < 0.0001), area (h) (ANOVA, F(2,81) = 218.7, P < 0.0001), length (i) (ANOVA, F(2,73) = 80.29 p < 0.0001), density (j) (ANOVA, F(2,81) = 61.21 P < 0.0001), and conductance, gs (k) (ANOVA, F(2,25) = 7.494, P = 0.0028) are shown for all analysed wheat lines. Results of a posthoc Tukey test comparing sequential ploidy levels are indicated within each analysis, with an asterisk when significant at the p < 0.05 level or NS when not significant. For gk, data are shown as box plots (25th−75th percentile, horizontal line = median) with whiskers indicating maximum and minimum values, n = 6. l For each analysed wheat line, mean mesophyll porosity is plotted against mean stomatal conductance, gs, with ploidy level indicated for each point. Results of correlation analysis are presented (Pearson r2 value). Results for individual paired data are shown in Supplementary Fig. 2

Mesophyll porosity is modulated by the presence of functional stomata

Lundgren M. R., Mathers A., Baillie A. L., Dunn J., Wilson M. J., Hunt L., Pajor R., Fradera-Soler M., Rolfe S., Osborne C. P., Sturrock C. J., Gray J. E., Mooney S. J., Fleming A. J. (2019)

Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK


In Nature Communications10, Article number: 2825,

MicroCT imaging reveals variation in wheat leaf airspace with ploidy. Sample leaf images of Triticum urartu (2n), T. araraticum (4n) and T. aestivum cv Cougar (6n) in 3D renderings of tissue blocks (ac), transverse sections (df), longitudinal sections (gi), and paradermal sections (jl), with solid tissue in green and airspace in yellow. Mesophyll porosity (%) (mo) is plotted along leaf depth from adaxial to abaxial surfaces in the diploid (m), tetraploid (n), and hexaploid (o) lines, as indicated. T. baoeticum–dark blue; T. urartu–light blue; T. dicoccoides–dark orange; T. araraticum–light orange; T. aestivum (Crusoe)–dark green; T. aestivum (Cougar)–mid-green; T. aestivum (Shango)–light green. For clarity, only mean values of 6 replicated samples are presented in panels mo. Lines in ac indicate plane of section in gi, respectively, also indicated by vertical lines in jl. Horizontal lines in jl indicate plane of section for df, respectively. Image resolution = 2.75 µm. Scale bars al = 200 µm


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.

EPF overexpression arrests sub-stomatal cavity development and decreases mesophyll porosity in wheat. a Confocal overview of a TaEPF1 OE wheat leaf showing epidermal layer (purple), subtending mesophyll cells (green), a stomate (St) consisting of guard cells and associated subsidiary cells and, in the same file, an arrested stomatal precursor (Ap). Scale bar = 60 µm. bc Higher resolution images of (b) the stomate and (c) arrested stomatal precursor cell shown in a. Scale bars = 40 µm. dg microCT images of a wild-type (WT) (df) and a TaEPF1 OE leaf (eg) in a paradermal plane within the mesophyll directly subtending the epidermis (de) or deeper in the leaf (fg), with solid tissue in green and airspace in yellow. The larger airspaces in de indicate sub-stomatal cavities. Note fewer sub-stomatal cavities in the TaEPF1 OE leaf. Scale bars = 100 µm. hj In WT wheat and two independent lines of transgenic wheat overexpressing TaEPF1 (as indicated), (h) the density of stomata (n = 87), sub-stomatal cavities (n = 87) and arrested stomata precursor cells (n = 52 from 5 independent leaves); (i) mesophyll porosity as measured from microCT analysis (ANOVA, F(2,12) = 4.977, p = 0.027); and (j) stomatal conductance, gs, are shown (ANOVA, F(2,12) = 46.86, p < 0.0001). For i and j a posthoc Tukey analysis was performed (n = 5). Lines sharing the same letter are indistinguishable from each other at the p < 0.05 confidence limit. Data (hj) are shown as box plots (25th–75th percentile, horizontal line = median) with whiskers indicating maximum and minimum values

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.

Developmental progression of stomatal differentiation and mesophyll airspace formation. af Confocal images of leaf 3 of wheat (6n) seedlings taken either at the distal tip region (ac) or proximal base (df). Images are from the epidermis (ad) or subtending mesophyll (be), with cell walls false-coloured and overlaid in (cf). A mature stomata is visible in a, with two immature stomata in d. A large airspace subtends the stomata in a (indicated by asterisks in bc). Small airspaces (asterisks) are visible in ef at cell junctions. gl Confocal images of Arabidopsis leaves at maturity (gi) or early development (jl). Images are from the epidermis (gj) or subtending mesophyll (hk) with cell wall false-coloured and overlaid in il. A mature stomata is visible in centre (g), with numerous stomata at various developmental stages in j. A relatively large airspace (asterisks) is visible below the central stomate (h), whereas some very small airspaces (asterisks) are distributed within the immature mesophyll (kl) at cell junctions. Scale bar cf = 20 µm; il = 25 µm
Mesophyll porosity is modulated by gas exchange through stomatal pores. ad 3D microCT renderings of tissue blocks (resolution = 2.75 μm; samples = 1.1 mm2) and (eh) exemplar SEM images of stomata from leaves of Arabidopsis EPF2-OECol-0focl1-1and epf1epf2 lines (scale bars = 10 μm). Means and standard deviation are shown for (i) stomatal density (ANOVA, F(3,11) = 19.17, p < 0.0001), (j) palisade mesophyll porosity (ANOVA, F(3,20) = 6.329, p = 0.0034) and (k) stomatal conductance, gs (ANOVA, F(3,20) = 26.22, p < 0.0001) for the Arabidopsis lines in ad, with n = 6 except for stomatal density where n = 4 (focl-1), n = 2 (EPF2-OE) and n = 3 (epf1epf2). Col-0 data are as in ref. 13. Lines indicated with the same letter cannot be distinguished from each other at the p < 0.05 confidence limit (posthoc Tukey). l Palisade mesophyll porosity is plotted against stomatal conductance, gs, for individual leaf samples from the four Arabidopsis lines, as indicated. Linear regression results are presented

UV-B induced stomatal closure

The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B induced stomatal closure in broad bean

by He J.-M., Xu H., She X.-P., Song X.-G., Zhao W.-M. (2005)

Jun-Min He ACD , Hua Xu C , Xiao-Ping She C , Xi-Gui Song C and Wen-Ming Zhao A– Author Affiliations 

A Center of Bioinformation, School of Life Sciences and Technology,

B Xi’an Jiaotong University, Xi’an 710049, People’s Republic of China.

C School of Life Sciences, Shaanxi Normal University, Xi’an 710062, People’s Republic of China


In Funct Plant Biol 32: 237–247 – –


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

[Stomatal and nonstomatal limitations of photosynthesis in mung bean leaves under the combination of enhanced UV-B radiation and NaCl stress]

by He J.-M., She X._P., Liu C., Zhao W. M. (2004) – in Chinese –

Center of Bioinformation, School of Life Sciences and Technology, Xi’an Jiaotong University, Xi’an 710049, China


 Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30: 53–58 – PMID: 15583409 –


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.

Stomatal response to a heavy snowfall

Adaptation of stomatal response of Camellia rusticana to a heavy snowfall environment: Winter drought and net photosynthesis

Kume A., Tanaka C. (1996)

Atsushi Kume, Chikako Tanaka,

Department of Biology, School of Education, Waseda University, Tokyo, Japan


In Ecological Research 11: 207-216 –


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.