Deutsch
Department of Botany I - Plant-Physiology and Biophysics

Projects

Regulation of stomatal movement

The world-wide climate change is expected to induce extreme fluctuations in temperature and humidity. One strategy of plants to survive the related physiological variations is to precisely adapt their water use efficiency. A key element in this process is the control of the gas exchange via the stomata, formed by two guard cells. Thereby the open state of the stomatal pore is highly regulated by turgor driven volume changes of the guard cells. The fine tuning of these biological valves is triggered by several internal and external factors, which are initially detected by a multi-sensory network and then translated into the appropriate movement of the stomatal pore. Up to now only a few of the involved signal transduction pathways have been characterized in detail. However, overlaps and interferences between the individual pathways, when e.g. different signals act simultaneously or in short intervals, have so far rarely been investigated. To address this question we focused on the transcriptional analysis of stomatal guard cells. First we optimized our procedure to mechanically sample enriched intact guard cells that avoids the possible side-effects (mainly osmotic stress, loss of tugor, and/or exposure to potential fungal elicitors) of the commonly used protoplasting method. Therefore total leaves without petiole and major veins are mechanically disrupted by successive blender cycles. Guard cells represent the only living cells of the resulting epidermal fragments (Figure 1). These samples are then used for the subsequent analyses of expression patterns (by micro arrays or qPCR), metabolites or proteins. To identify genes enriched in guard cells (compared to total leaves) and genes responding to several stimuli that lead to stomatal closure, we conducted a series of microarray experiments (Figure 2). Thereby we could show that e.g. guard cells can directly detect drought via changes in the relative air humidity and that they are able to respond by autonomous induction of the stomatal closure (Figure 3). Nevertheless, the nature of the humidity sensing mechanism, its conversion into a chemical signal and finally into the mechanical stomatal movement is so far unknown and focus of our current research interest. We have started a bioinformatics approach to identify the interacting nodes between single signalling pathways. By use of marker genes and physiological treatments of the respective Arabidopsis mutants we will unravel the whole signal transduction network of stomatal closure step by step.

Our main methods:

We use physiological methods to determin the open state  of the stomata following application of several closing signals, and thus the appropriate time point for sampling (Figure 4 und 5).

Physiology

  1. measurement of stomatal movement via transpiration (gas exchange, IRGA)
  2. measurement of stomatal movement via leaf turgor (non-invasive pressure probe)
  3. preparation of intact guard cells
  4. ELISA-monitoring of the stress hormone ABA

Molecular biology/Expression analysis:

  1. standard methods of molecular biology
  2. preparation of low amount RNA
  3. quantitative real-time-PCR (qPCR)
  4. microarray analyses
scanning electron microscopy (SEM) of open and closed Arabidopsis thaliana stomata
From left to right: adult Arabidopsis plant, leaflets with major veins removed, mixer, epidermal fragment with only guard cells alive
Figure 1: Guard cell enrichement. Excised leaves of adult Arabidopsis plants areexcised and the major veins removed. The leaflets are treated in a household mixer filled with ice-water until only small epidermal fragments are left. In these preparations a vital stain reveals that guard cells only survive.
Experimental setup. Left: signals that lead to stomatal closure. Middle: open stoma and arrowhead to closed stoma. Right: monitored parameters.
Figure 2: Experimental setup: stomata are forced to close as a reaction to different (stress-) signals (left). The changes are monitored by different methods (upper right).Subsequently guard cell are enriched and analyzed by different methods (lower right). (Grafik P. Ache, 2017)
Left: ABA-induced stomatal closure in wild-type plant at dry air, leaf survives. Middle: Stomata of ABA-free mutant are unable to close; leaf wilts. Right: Mutant with guard cell restored ABA-synthesis, leaf survives.
Figure 3: Guard cell autonomous produced ABA is required and sufficient to trigger stomatal closure induced by low air-humidity. Excised leaves of water supplied Arabidopsis Col-0 plants close their stomata when subjected to dry air and survive. aba3-1 plants, unable to produce stress-induced ABA, wilt. Guard cell specific complementation of the ABA synthesis in the aba3-1 mutant restored the Col-0 survival phenotype.
Measuring methods: Left: gas exchange cuvette for measurement of water loss via stomata. Middle: pressure probe mounted on a leaf for measurement of the leaf turgor. Right: simultaneous measurement of gas exchange and leaf turgor.
Figure 4: Our Mesurements. To Measure the stomatal open state we mainly follow the gas exchange via the stomata. Thereby air humidity and CO2 content are monitored in an air stream before and after a cuvette containing leaf material. Alternatively we use a pressure probe to measure the leaf turgor that increases when stomata closure leads to a positive water balance or vice versa. Both measurements might by applied simultaneously. (Grafik: P. Ache, 2017)