Protocol Stomatal Movement in Leaves Responses of Plant Cells to Changes in Their Environment Stomatal Movements in Leaves Preparation for this lab session Read the protocol carefully so that you will be completely familiarized with the experimental procedures and goals before you come to the lab Prepare your lab notebook by writing down the sequence of experiments you will perform and the reason for each experiment and plan a tentative time schedule for the experiments Also make notes of any particular questions you would like to discuss Objectives of Today s Lab 1 Dissect leaf epidermis and mount onto culture dishes 2 Careful handling and proper use of a microscope 3 Observe and quantify opening and closing of stomata in response to the following physiological stimuli light dark fungal toxin fusicoccin and coapplication of malate and abscisic acid 4 Prepare experimental materials for the next lab class on Arabidopsis root hair growth Background and Significance The transport of inorganic and organic ions across the plasma membrane and organelle membranes of higher plants by ion channels electrogenic pumps and cotransporters is essential to vital processes such as osmoregulation growth development signal transduction and the storage of solutes Recently there has been renewed interest in ion transport mechanisms across membranes of higher plants These investigations have been triggered in part by the recognition that a molecular analysis of ion transport components is important for the understanding of how intrinsic and environmental signals are translated into physiological responses It has become apparent that osmoregulatory functions of different cell types and initial events in stimulus response coupling in plants are dependent on membrane receptor and ion transport mechanisms We are only now beginning to gain insight into the molecular basis of these membrane associated mechanisms that regulate and coordinate the transport of inorganic and organic ions in response to changing of external signals Ion transport osmoregulation and movements 1 Protocol Stomatal Movement in Leaves Transport of solutes into and out of specialized cells and tissues is of prime importance for numerous processes during the life cycle of higher plants These processes include vital functions such as stomatal movements ion uptake by roots cell elongation during growth germination involvement of Ca 2 in the initiation of developmental responses and transport of photosynthetically synthesized substances to storage organs The most visible phenomena involving ion transport are the movements of plant organs such as roots in response to the gravitational field and leaves in response to directional changes of light incidence Plant movements are driven by osmotic regulation of specialized cells Recently such cells have received considerable attention as model systems for mediator induced ion transport as they experience large signaldependent changes in their solute concentration in relatively short periods of time Opening and Closing of Stomata Similarly stomata on which we will focus in this lab class constitute a simple system consisting of two guard cells surrounding gas exchange pores in the epidermis of leaves By the opening and closing of stomatal pores guard cells regulate the gas exchange for photosynthesis between the leaf and the atmosphere while minimizing water loss from the plant to the atmosphere Guard cells respond dynamically to multiple stimuli by means of ion transport Opening of stomatal pores is elicited by light low CO 2 concentrations and humidity which induce an increase in the K salt content of guard cells The main counter ion to K is the organic anion malate and in some species Cl While K and Cl ions are taken up by transport via the plasma membrane plasmalemma malate is synthesized by degradation of osmotically inactive starch by glycolysis Subsequently K salts cross the vacuolar membrane tonoplast and are sequestered in the vacuole Osmotic movement of water into the guard cells leads to an increase in volume and turgor which drive the opening of the gas exchange pores due to mechanical constraints of the cell walls Stomatal closing is mediated by release of K salts and by reconversion of malate to osmotically inactive starch Guard cells possess a variety of mechanisms which modulate ion transport in response to signals such as blue light red light CO 2 and the hormones abscisic acid and auxin In this lab class we will study the opening and closing of stomatal pores in response to physiological signals such as light darkness and the fungal toxins fusicoccin FC and the plant hormone abscisic acid Experimental Procedures 2 Protocol Stomatal Movement in Leaves 1 2 3 Planting Broad Beans Plant Vicia faba broad bean plants long pod 5 weeks and 4 weeks before the lab class date Plant two pots of seeds per group of 2 students one pot five weeks prior second pot 4 weeks prior to lab class Use 3 4 Long Pod broad bean seeds per pot Plant the seeds so that they are completely covered by the soil approximately 1 cm deep into soil The plants should be grown and maintained at the UCSD greenhouses at the field station by the gardener The lab course instructor Diann Johnson should initiate planting and check on condition of plants one week before lab class Watering Vicia faba bean plants are not very drought resistant and dry up or are overwatered easily Water the plants on every other day A recommended schedule is AM Monday AM Wednesday and PM Friday The pots should be deep enough or the pots should be placed in trays to allow retaining of drained water Exact amount of water used varies depending on green house humidity and temperature how many plants are in the same pot and how big the pot is Optimal temperature is 20 C If there are too many plants or grown large plants in the same tray it may be necessary to check the moisture level in the soil on Sunday Dip with finger about one inch into the soil and if the soil is dry a sparse amount of water should be added to avoid wilting of the plants Resume the normal watering schedule on Monday morning Epidermal Strip Isolation The youngest almost fully expanded leaves from 4 to 5 weekold plants are excised from the plants Epidermal strips can be prepared from leaves by stripping the leaves manually with a pair of forceps Submerge the leaf completely in cold water 0 5 cm deep in a large petri dish 20 cm diameter with the lower side of the epidermis