METHODS
Vicia faba L., minor cv. Fuego grown in 2L plastic containers in soil in a climate chamber for 4 weeks and then water supply was
completely stopped for following 11d. Soil water content (SWC) was measured using ThetaProbe (Delta-T, UK). Shoot water
potential (SWP) was estimated by pressure chamber technique (Model 3005, Soil Moisture Equipment, USA). The same chamber
was used for xylem sap sampling from shoot and also from intact root systems in soil. Sap pH was measured by pH microelectrode
(Microelectrodes Inc., Bedford, MA, USA). H+ sensitive fluorescent probe Oregon green 488 dextran (25 µM) was used for
apoplastic pH measurement in leaves with the Leica inverted microscope (DMI6000B and DFC 360FX camera) as described in
Geilfus and Muhling (2013). ABA content was estimated by mass spektrometry after extraction and UPLC separation.
APOPLASTIC PH CHANGES IN VICIA FABA UNDER DROUGHT
Thirupathi Karuppanapandian1, Christoph-Martin Geilfus2, Karl Hermann Mühling2,
Ondřej Novák3, Vít Gloser1
1Department of Experimental Biology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
2Institute of Plant Nutrition and Soil Science, Christian Albrechts University, Hermann-Rodewald-Str. 2, 24118 Kiel, Germany
3Laboratory of Growth Regulators, Palacký University, Olomouc, Czech Republic
The apoplastic space plays an important role in the response of plants to the environment. Plants
experiencing soil water deficits frequently generate a high apoplastic pH and this was proposed as
part of sequence in signaling drought and other stresses. Leaf apoplastic pH can have an effect on
leaf growth and stomata functioning (Wilkinson and Davies, 1997). However little is known about
dynamics of the pH change of leaf apoplast in plants under stress. This is also due to methodological
limitations of pH measurements inside of intact plant leaves. Non-invasive optical method of direct
in-situ pH measurement (Geilfus and Mühling, 2013) can help to clarify role of pH in plant response
to drought.
Does pH change in xylem and leaf apoplast
contribute to early regulation of stomatal
aperture when soil water availability declines?
Can pH changes in leaf apoplast be triggered by
pH changes in xylem sap?
REFERENCES
Geilfus G-M, Mühling K-H (2013) New Phytol. 197: 1117-1129
Wilkinson S, Davies WJ (1997) Plant Physiol. 113: 559-573
Significant decline of soil water content (SWC) (A)
was accompanied by decrease of transpiration
rate (B) of V. faba. Shoot water potential (C) of
plants, however, significantly decreased first on
day 8. This indicates fast early response of
stomatal regulation to soil drying that does not
involve changes in shoot water potential but some
other mechanism. Plants were grown and
examined under controlled environmental
conditions. Shown are means ± SE of 5 replicates.
Significant increase of pH in leaf apoplast (D) was
already detectable after 2 days of drying. Colour
windows show detected signal of indicator in leaf
apoplast on days 1, 6 and 11. pH of shoot xylem
sap and root xylem sap also significantly changed
with progressive drying of soil. Shoot xylem sap
pH (E) increased significantly after 5 days of
drying. In the same time root xylem sap pH (F)
significantly acidified. This clearly indicates that
pH in both leaf apoplast and xylem sap respond
sensitively to soil drying but they are independent.
Means ± SE of 4 replicates.
Analysis of factors affecting transpiration rate. Massive decrease in transpiration rate in the
early phase of drying was not related to the decline of leaf water potential (A) or increase of
leaf apoplastic pH (B). We also did not find any relationship with sap osmolality (C) or ABA
content in leaf biomass (D) in the first stage of drying. Other signalling mechanism(s) is,
therefore, involved in the early sensitive response of stomata to drying soil.
ACKNOWLEDGEMENTS
This work was financially supported by “Employment of Best Young Scientists for International Cooperation Empowerment”
(CZ.1.07/2.3.00/30.0037) co-financed from European Social Fund and the state budget of the Czech Republic.
responds sensitively
to water availability
in soil
changes are
independent of
pH changes in
xylem sap
is not the modulator
of the early stomatal
response to drought
pH of leaf
apoplast