A Leaf Lamina Compression Method for Estimating Turgor Pressure

A portable wiltmeter instrument to estimate leaf turgor pressure according to an adaptation of the flattening method was developed. In the instrument, a flexible inflating membrane presses the leaf against a flattening plate having small orifices surrounded by a finely engraved network of obtuse indentations through which air flow is delivered. During a measurement, as the compression builds up, the leaf is progressively molded against the flattening plate, and as a consequence, the air flow (x) crossing the plate is reduced toward zero. The smallest leaf compression (p0) that blocks the air passage is an estimate of the leaf turgor. Wiltmeter measurements were compared with pressure probe measurements of cell turgor pressure in detached leaves of lettuce (Lactuca sativa L.), kale (Brassica oleracea L. var. Acephala), and chicory (Chichorium endivia L.), which were allowed to suffer diverse levels of wilting caused by transpiration. Such observed wiltmeter readings were a little lower than the cell turgor pressure measured with a pressure probe; the regression coefficients between these methods were: 1.156 for lettuce, 1.13 for kale, and 1.036 for chicory. This portable quantitative procedure to measure leaf firmness has potentially valuable applications related to postharvest and field plant physiology studies. Leaf cell turgor pressure is a water status variable related to firmness, growth, and with the consumer perception of wilting and freshness. Turgor pressure measurements are usually made at a laboratory using laborious procedures. With the pressure probe technique, for example, cell turgor pressure is estimated after impaling the probe capillary into plant cells within plant tissues (Hüsken et al., 1978; Nonami et al., 1987). Typically a pressure probe is made of an oil-filled microcapillary connected to a coupling chamber having a pressure transducer. In a measurement, after the capillary is impaled into a cell, the lowviscosity oil transmits cell fluid pressure to the pressure transducer. Next, using a micrometrical piston system (Nonami et al., 1987) or using a thermoelastic pressurization system (Pessoa and Calbo, 2004), the oil/water meniscus is returned to the preimpalement position and, at this point, the transduced cell turgor pressure is measured. In the field, plant water status is frequently accessed using the pressure chamber (Scholander et al., 1964). The variable measured by this instrument, however, is not turgor pressure but the complementary air pressure, which is needed to extract sap water out of the petiole of leaves subjected to an air pressure ramp. In other words, the pressure chamber measures the leaf water tension state, which in several applications can be taken as an estimate of the leaf water potential (Boyer, 1985). The pressure chamber method, however, is invasive, and it involves measurements that have to be made in detached leaves. Additionally, these measurements became very time-consuming when used to estimate leaf turgor pressure, because the procedure is indirect and requires a series of points relating applied air pressure with extracted sap volume of the individual leaves, in which turgor pressure is being estimated (Calbo and Moraes, 1997). A specific portable instrument to measure leaf turgor firmness index was developed by Heathcote (Heathcote et al., 1979). In this instrument, a leaf resting over a cavity is pressed by a central rod and the deformation read with a micrometer is taken as a cell turgordependent index. This portable leaf strain probe, however, is sensitive to leaf thickness and leaf venation, even when measuring different leaves from a single plant (Turner and Sobrado, 1983). More recently, a leaf patch clamp pressure probe for field use was developed (Zimmermann et al., 2008). In this instrument, an electric pressure signal is generated as a function of the changes in leaf volume and turgor pressure. For measurements, the leaf is clamped between two planar pads; the first pad is a rigid support and the second one is a soft silicone sensor pad, in which an encased pressure transducer reads a signal that is always a fraction of the applied leaf compression. According to the authors, this instrument enables continuous data acquisition from leaves with different thicknesses. The leaf cell turgor pressure response in the patch clamp is approached with aid of an intricate mathematical approximation applied to a data set that presents a delayed time line response that can be as large as 4 h on sunny days. For convex-shaped organs such as many fruits, a flattening method is being used to estimate the turgor-dependent pressure firmness for several applications, including some new fruit firmness half-life determinations (Caron et al., 2003; Kluge et al., 1999; Nizio et al., 2008). The flattening method was developed initially to model a grape berry as if it were a thin-walled balloon filled with pressurized water (Berstein and Lustig, 1981; Bernstein and Lustig, 1985). According to this model, an external force applied with a transparent plate flattens a fruit surface area in which the value is equal to the turgor pressure multiplied by the fruit flattened area. More recently (Lintilhac and Outwater, 1998; Lintilhac et al., 2000), an analogous thin-walled balloon approach was used to measure epidermal cells under the microscope with a procedure named ball tonometry. Using a more general plant physiology reasoning, the flattening procedure was extended to estimate cell turgor pressure of other convex fruits and vegetables covered by soft dermal tissues (Calbo and Calbo, 1989; Calbo and Nery, 1995; Calbo et al., 1995) composed of thin-walled poliedric cells that cover the internal plant cellular structure made out of parenchymatous cells having deformable intercellular air volumes. Accordingly, for some regular cellular lattices, it was demonstrated that there is a simple mathematical relation between the flattening pressure and cell turgor (Calbo and Nery, 2001). In these lattice models, the flattening pressure and the cell turgor pressure are related by a cell compression ratio, whose magnitude ranges from zero to one depending on remaining intercellular air volume fraction during mechanical axial compression assays. A first attempt to develop a portable instrument to measure the leaf turgor using the flattening method involved a setup having a plain rigid base over which the leaf was compressed by a piston flattening plate, whose surface was finely indented around a few small air flow outlet orifices (Calbo, 1991). For a measurement, the leaf was progressively compressed by this piston and the reduction of applied air flow, filtered between the leaf and the flattening plate, was used as a criterion of leaf flattening that was used to estimate the leaf turgor. The instrument was simple and quantitative, but the piston borders caused leaf deformation marks, especially in thicker leafs, and these marks were considered to be a potential cause of leaf turgor underestimation in thick leaves. In this communication, a portable instrument that makes use of the flattening method to estimate the leaf turgor pressure status, without causing leaf indentation marks, is presented for possible use in postharvest and field-oriented plant physiology studies. This wiltmeter instrument performance was then considered mainly with reference to the pressure probe Received for publication 3 Sept. 2009. Accepted for publication 9 Dec. 2009. We are indebted to Mr. João Batista Gomes from Embrapa Vegetables for building preliminary models of the portable instrument used to measure leaf firmness. To whom reprint requests should be addressed; e-mail [email protected]. 418 HORTSCIENCE VOL. 45(3) MARCH 2010 method to measure cell turgor in leaves of vegetable crops. Materials and Methods The instrument in Figure 1 enables the use of a leaf lamina compression method adaptation to measure the leaf turgor-dependent firmness pressure (Calbo and Pessoa, 2009). This wiltmeter was assembled with a flattening plate [Fig. 1 (1)] having a few (greater than five) nearly centralized orifices [Fig. 1 (2)]; an air flow source; a flowmeter [Fig. 1 (4)]; and a membrane hydraulic leaf compressing mechanism. The air flow source was composed of an air compressor [Fig. 1 (9)], an air escape pressure regulator [Fig. 1 (10)], and an inlet air flow restriction [Fig. 1 (11)]. The membrane hydraulic leaf compressing mechanism, on the other hand, was made with a sandwiched membrane [Fig. 1 (6)] fixed at the instrument base and works with water introduction to press the leaf against the flattening plate while this applied pressure is measured in a manometer [Fig. 1 (7)]. The air flow source delivers an air inlet pressure (Dp0) of 6 kPa. Of this pressure, while the instrument is open, 4 kPa is dissipated at the inlet air restriction [Fig. 1 (11)] R1 and 2.0 kPa at the flow meter restriction [Fig. 1 (3)] R2. At this condition, the flowmeter reading is 200 mm for an air flow of 90 mL min. For practical use, this inlet air pressure is obtained by simple adjustment of the pressure regulator knob [Fig. 1 (10)] at 200 mm in the flowmeter manometer of the opened wiltmeter. Measurement involves fixing the leaf [Fig. 1 (5)] with the screw nut [Fig. 1 (12)] and subjecting the leaf to a progressive compression against the flattening plate [Fig. 1 (1)] with the syringe [Fig. 1 (8)]. As the applied pressure increases, the leaf is progressively molded against the flattening plate while the air flow is attenuated down to zero in this air compression ramp-up assay. Theoretical considerations. A quadratic model [Eq. (1)] generates an approximation for the nonlinear relation between the applied leaf compression (p) and the air flow (x) for x values close to zero (Fig. 2). p = p0 A x + B x [1] In this equation, p is the applied pressure, x is the air flow, p0 is the estimated applied pressure at the intercept (x0 = 0), whereas A Fig. 1. Scheme of a wiltmeter instrument to measure leaf turgor pressure using the flattening method implemented with aid of an air flow attenuation procedure. The system is composed of a flattening plate with a slightly granular base (1) having centralized microair inlet orifices (2); an air restriction (3) flow meter in which air flow is read in a U tube manometer (4) where the progress of the leaf (5) flattening is followed; a flexible membrane fixed in the instrument base (6) is the element used to compress the leaf against the flattening plate while pressure, read in the manometer (7), is being applied with a waterfilled syringe (8). The air flow needed for this flattening attenuation mechanism is fed by an air compressor (9) coupled to a pressure regulator (10) and an inlet air restriction (11). During the measurement, the leaf remains clamped with aid of a bolt screw nut (12), while the spring (13) eases instrument opening and leaf freeing by forcing the flattening plate movement around the axle (14). Fig. 2. Typical curves relating applied leaf compression versus the attenuated or filtrated air flow between a kale leaf and the flattening plate obtained during a wiltmeter measurement. (A) Applied leaf compression (p) versus read air flow (x). (B) Linearization using the inverse of the applied leaf pressure (1/p) versus