supervisor: prof. RNDr Lubomír Nátr, DrSc

In analogy to mechanics the plant organism exposition to unfavourable conditions is called stress and the effect of the exposition is called strain (Lewitt, 1980). Unique to living organisms are purposeful reactions of adaptation and hardening to stress, namely acclimation of organisms and adaptation of species. The thesis is aimed to the study of stress effects on plants and of plant acclimation and also to develop new methods for plant stress study.

The first section describes the changes in photosynthetic apparatus of winter wheat plants during the frost hardening detected by gas exchange measurements at 5°C. Also the relationships of changes in photosynthetic characteristics and of frost resistance during the acclimation in wheat genotypes are investigated.

Analysis of short term effects of water stress on the photosynthetic CO₂ uptake by wheat leaves was carried out in the second section. The aim was to obtain characteristics of water economy of wheat genotypes by measurements of stomatal and metabolic limitations to photosynthesis under water stress.

The effects of water stress on wheat genotypes was studied also during the cultivation in a greenhouse at varying experimental conditions.

The next two chapters present a development and application of the mathematical method for plant frost resistance evaluation from controlled freezing experiments and the mathematical method for sorting cereal cultivars by winter hardiness based on data on the winter survival. The two methods are implemented in programs for PC.

The last sections describes a method for automated root length and root branching measurements by computer image analysis and its application to study of low pH and Al^3- effects on two cultivars of winter wheat.

Plants from temperate climate are hardening to frost by growth at low temperatures above the freezing point. The elongation growth and dark respiration rate is more affected by the low temperature then the photosynthetic rate (Levitt, 1980). Lowered utilisation or transport of assimilates causes their accumulation in assimilation organs and the sink limitation of photosynthesis. Sink limitation can be detected by O₂ and CO₂ insensitivity of net photosynthetic rate.

Figure 1: Net photosynthetic rate of the 2nd leaf of winter wheat, cv. Mironovskaya 808 measured at 5°C and irradiance 500µmol PAR m^-2s^-1. Hardening of plants at 5°C, 200 µmol PAR m^-2s^-1 and 16 hours photoperiod lasted 7 days.

Figure 2: Induction of photosynthesis in nonacclimated wheat plants at low temperature at two levels of irradiance. Start of oscillation (detected from the derivative of induction curves and denoted by vertical lines) took place after fixation of 0.65 mmol m^-2 CO₂. Oscillation is probably induced by accumulation of nonreduced assimilates (phosphoglycerate) and subsequent depletion of inorganic phosphate inside the chloroplasts.

Plants grown at low temperatures do not exhibit sink limitation (Huner et al., 1986, Janáček and Prášil, 1992). Plants acclimated to low temperature are also less sensitive to photoinhibition and display increased photosynthetic capacity. Changes in photosynthetic apparatus during acclimation correlate well with the winter hardiness in cereal species (Öquist et al., 1993, Öquist a Huner, 1993). Maintenance of a high net photosynthetic rate by acclimation of photosynthetic apparatus is important for frost hardening of overwintering herbs (Huner et al., 1993).

Acclimation of plants to low temperature was studied by measurement of changes in photosynthetic assimilation of fully developed second leaf of winter wheat during the frost hardening. Pronounced changes in CO₂ (Fig. 1) and O₂ sensitivity of net photosynthetic rate, similar to changes after long term hardening of rye described in Huner et al. 1986, were detected already in the first week of hardening. The sink limitation of photosynthesis is indicated by the both CO₂ and O₂ insensitivity, an oscillation in net photosynthetic rate during the induction (Fig. 2) and after lowering the O₂ concentration in nonacclimated plants at low temperature. The sink limitation of photosynthesis appears to relax during the low temperature acclimation.

It is obvious from the above results that the short term acclimation can be used for study of changes in photosynthetic apparatus during the frost hardening instead of long lasting development of plants at low temperature. Correlation between frost hardiness and photosynthetic capacity among winter wheat genotypes was not detected. Positive results with winter wheat and rye genotypes described in (Öquist and Huner, 1993) can be explained by wider range of frost hardiness or that a longer period of hardening is needed to induce differences in photosynthetic capacity.

Majority of terrestrial plants protect themselves against water stress by avoidance of tissue dehydration. Dehydration can be avoided either by lowering the water outflow - by stomatal closure, leaf rolling, leaf abscission, reduced growth and shortened ontogenesis, or by ensuring the water supply - by osmotic adjustment, increased root/shoot ratio (Levitt, 1980). Genetically fixed pattern of plant response to water stress, or plant water stress strategy, is manifested for example in different levels of osmotic adjustment in wheat (Morgan, 1984) or barley (Blum, 1989) genotypes.

The portion of decrease in net photosynthetic rate caused by stomatal closure can be estimated as coefficient of stomatal limitation of photosynthesis, calculated as control coefficient of stomata (Woodrow et al., 1990).

The effect of water stress and of CO₂ concentration on the stomatal limitation of photosynthesis was calculated from net photosynthesis rate and transpiration rate measured in open gasometric system at the concentration of CO₂ from 100 to 800 mmol mol^-1, irradiance 400 mmol m^-2s^-1 PAR and leaf temperature 22.5-23.5°C. Osmotic potential of the nutrient solution was lowered by polyethyleneglycol with aproximate molecular mass 8000.

Figure 3: Coefficient of stomatal limitation of three winter wheat cultivars (Chlumecka 12, Mexico 50-B21, Zdar) at water stress simulated by adding PEG 8000 into nutrient solution. Letter c is for control, s for stress.

Coefficient of stomatal limitation depends linearly on the osmotic potential of nutrient solution down to -1 MPa. The slope of regression line decreases with increasing CO₂ concentration. The differences in the coefficient between wheat genotypes are not significant.

Linear increase in coefficient of stomatal limitation with water potential of nutrient solution decreasing down to -1 MPa is caused by more pronounced effect of water stress on stomata than on biochemical processes in mesophyll (Fig. 3). The coefficient of stomatal limitation decreased uniformly in the whole range of stress with increasing CO₂ concentration. The coefficient of stomatal limitation may be used as a simple measure of ratio of photosynthetic limitations by stomata and mesophyll.

The aim of this part of thesis was detection of genetically based differences in reaction to water stress between wheat cultivars by gas exchange measurements at variable conditions.

Plants of winter wheat (cv. Mironovská 808, Karlik, Viginta, Chlumecká 12, Vala) were grown in the greenhouse with coarse temperature control (20 to 35 °C). Water stress by lowering soil matrix potential to -0.06 MPa was imposed on plants when the elongation growth of the flag leaf was completed. The gas exchange rates were measured by portable photosynthesis measurement instrument LI6000 (Licor, USA). The net photosynthetic rate and stomatal conductivity were calculated by a new method corrected for water adsorption in the instrument.

The effect of temperature and irradiance on the net photosynthetic rate had to be corrected by regression analysis to obtain differences between genotypes. Linear dependence of net photosynthetic rate on irradiance, temperature and square of temperature was significant.

Early cultivars had higher net photosynthetic rate. There was no difference in the net photosynthetic rate between treatments.

Table 1: Mean values of net photosynthetic rate (µmol m^-2s^-1).

Table 2: Mean values of stomatal sensitivity coefficient.

The coefficient of stomatal limitation was lower in stress variant. There was no difference between genotypes in the stomatal limitation coefficient.(Tab. 2).

Genotypic differences in the plant water economy between wheat genotypes were not detected by means of the stomatal limitation coefficient.

The values of the stomatal coefficient measured under different conditions cannot be compared directly because of strong dependence of the stomatal conductivity on the relative air humidity.

The frost hardiness of plant can be characterised by the critical temperature (CT₅₀), the freezing temperature at which the frost injury is half (50%) of the maximal injury. If the injury is measured by the electrolyte leakage, the maximum and the minimum of relative electrolyte leakage depends on experimental conditions, the size of the sample and the time of leakage (Prášil a Zámečník, 1990). CT₅₀ was previously calculated by logit or probit analysis after transformation of data to interval 0-100% (Zhu a Liu, 1987) using apriori knowledge of the minimal and maximal response. My method is based on simultaneous estimation of CT₅₀, minimal and maximal response by nonlinear regression of measured data by s-shaped function.

The aim of the work was to develop the method for calculation of CT₅₀ by nonlinear regression with logistic function, the method for testing differences between obtained values and to implement both methods user friendly on PC.

Figure 4: Freezing temperature effect on the electrolyte leakage from plant tissue.

Nonlinear regression is calculated by standard Newton-Marquardt algorithm using direct calculation of derivatives of loss function up to order two. Minimal or maximal value of injury can be set constant in the program (it is known previously that I_min=100%, I_max=0% for plants survival, I_max=0 for growth). The test of heterogeneity of CT₅₀ among group of tests is based on the test of Scheffe, comparison of couples on the Student test. Examples are in (Janáček and Prášil, 1991), and at fig. 4.

Overwintering is influenced by number of factors, as frost, soil shifts and flooding, so the severity of winter or winterhardiness of plant genotypes cannot be characterised by single physical quantity. Luckily, it is possible to compare both winterhardiness of different genotypes and severity of different winters relatively easily, because the interaction between severity of winter and winterhardiness of genotypes is not significant (Prášil et al., 1989). A method similar to two-factorial analysis of variance can be used. Let the probability of plant survival be the logistic function f

of the difference of the hardiness of genotype and severity of winter. The corresponding regression relation is

,

where y_ij is relative survival, a_i coefficient of winterhardiness of i^th genotype, b_j coefficient of severity of the j^th winter, e_ij experimental error and x_ij error of the relative survival (with binomial distribution).

Regression model is solved by linearisation and iterative solution of resulting normal equations. Tens of experiments with hundreds of genotypes can be solved simultaneously with my implementation of the method on PC. Method can be used even in cases, when the sortiment of cultivars is gradually changing. The method is used in Research institute of crop production, Prague, for evaluation of winterhardiness of cereal genotypes (Prášil et al., 1994).

Length of the root system is very sensitive to soil stresses, as aluminium toxicity at low pH (Foy, 1983). Counting of intercepts with test line system is much more effective than manual measurement of roots by the ruler (Newman, 1966). Automated length measurement by computer image analysis (Harris a Campbell, 1989) is by one to two thirds quicker than line intercept method (Farrell et al., 1993), moreover it is possible to count root tips at the same time (Zoon a van Tierderen, 1990).

Aim of the work was do develop a method for evaluation of effect of low pH with aluminium ions on cereal genotypes by root measurements using commercial system for computer image analysis LUCIA D (Laboratory Imaging, ČR). Image processing consisted in enhancement of thin roots, segmentation by thresholding and skeletonisation (Fig. 5). The total root length and the number of root tips were measured automatically.

ab

Figure 5: Digital image processing a) the original image with automatically detected tips, b) skeletonised image.

Measurements of wheat genotypes cv. Atlas and Zdar, in cooperation with ing. Bláha, RICP Prague, shown more pronounced decrease in both the total length and number of tips in more sensitive cultivar Zdar after treatment with low pH and Al ions.

It seems recommendable to complete the data by measurements of the lengths of the main axes, so that other interesting characteristics, as the mean length of secondary roots, can be calculated.

While frost can be characterised by the single quantity, the freezing temperature, the water stress or overwintering is influenced by various soil and climatic conditions as well as the status of the plant under study. Water stress is typical for wider range of climates and some degree of water shortage prevail for main part of the life span in majority of plants. Winter is not a resting period for overwintering herbs, as is for conifers, and the acclimation of photosynthetic apparatus in overwintering herbs is necessary for overwintering.

Study of low temperature acclimation of photosynthesis requires measurements of steady state values the of net photosynthetic rate as well as the time courses of net photosynthetic rate during transients in experimental conditions.

Apriori information, for example that the CO₂ dependence of net photosynthetic rate is hyperbolic (Farquhar et al., 1980 ), or that the temperature dependence of net photosynthetic rate is unimodal, is necessary for construction of reliable regression models.

• Blum, A.: Osmotic adjustment and growth of barley genotypes under drought stress. -Crop. Sci. 29, 230-233, 1989.
• Farrell, R. E., Walley, F. L., Lukey, A. P., Germida, J. J.: Manual and digital line-intercept method of measuring root length: a comparison. -Agronomy Journal. 85, 1233-1237, 1993.
• Farquhar, G. D., Caemerer, S. von, Berry, J. A.: A biochemical model of photosynthetic CO₂ assimilation in leaves of C₃ species. - Planta 149, 78-90, 1980.
• Foy, C.D.: The physiology of plant adaptation to mineral stress. -Iowa State Journal of Research. 57, 1007-1017, 1983.
• Harris, G. A., Campbell, G. S.: Automated quantification of roots using a simple image analyzer. -Agron. J. 81, 935-938, 1989.
• Huner, N.P.A., Migus, W., Tollenaar, M.: Leaf CO₂ exchange rates in winter rye grown at cold-hardening and nonhardening temperatures. -Can.J.Plant Sci. 66, 443-452, 1986.
• Huner, N.P.A., Öquist, G, Hurry, V.M., Krol, M., Falk, S., Griffith, M.: Photosynthesis, photoinhibition and low temperature acclimation in cold tolerant plants. -Photosynt. res. 37, 19-39, 1993.
• Janáček, J., Prášil, I.: Quantification of Plant frost injury by nonlinear fitting of an S-Shaped function. -Cryo-letters 12, 47-52, 1991.
• Janáček, J., Prášil, I.: Stimulation of net photosynthetic rate of two wheat cultivars by 1kPa O₂ during the low temperature acclimation. -Photosynthetica 26, 341-346, 1992.
• Lewitt, J.: Responses of plants to environmental stresses. -2^nd ed., Academic Press, London,, 1980.
• Morgan, J. M.: Osmoregulation as a selection criterion for drought tolerance in wheat-Aust. J. Agric. Res. 34, 607-614, 1984.
• Newman, E.I.: A method of estimating the total length of root in a sample. -J.Appl.Ecol. 3: 139-145, 1966.
• Öquist, G., Huner, N.P.A.: Cold-hardening-induced resistance to photoinhibition of photosynthesis in winter rye is dependent upon an increased capacity for photosynthesis. -Planta 189, 150-156, 1993.
• Öquist, G., Hurry, V.M., Huner, N.P.A.: Low-temperature effects on photosynthesis and correlation with freezing tolerance in spring and winter cultivars of wheat and rye. -Plant Physiol. 101, 245-250, 1993.
• Woodrow, I.E., Ball, J.T., Berry, J.A.: Control of photosynthetic carbon dioxide fixation by the boundary layer,stomata and ribulose 1,5-biphosphate carboxylase/oxygenase. -Plant Cell Env. 13, 339-347, 1990.
• Zhu, G.H., Liu, Z.Q.: Determination of median lethal temperature using logistic function. - in “Plant cold hardiness:, P.H.Li, ed. Alan R. Liss, New York, 291-298, 1987.
• Zoon, F.C., van Tienderen, P.H., 1990: A rapid quantitative measurement of root length and root branching by microcomputer image analysis. -Plant and Soil 126, 301-308, 1990.

souhrn dizertační práce v oboru fyziologie rostlin

Rychlost čisté fotosyntézy a stomatální vodivost rostlin ozimé pšenice byla měřena během aklimace k nízké teplotě a při vodním stresu. Aklimace fotosyntetického aparátu plně vyvinutého listu ozimé pšenice k nízké teplotě vedla kodstranění limitace fotosyntézy spotřebou asimilátů, projevující se sníženou citlivostí rychlosti čisté fotosyntézy ke změnám v koncentraci CO₂ a O₂.

Analýza výměny plynů pomocí koeficientu stomatální limitace u rostlin pšenice při vodním stresu navozeném buď roztoky polyetylénglykolu v laboratorních podmínkách nebo omezením zálivky ve skleníku neprokázala rozdíly mezi genotypy pšenice ve způsobu hospodaření s vodou. Roztoky PEG8000 s vodním potenciálem do -1 MPa i kultivace v půdě s matričním potenciálem do -0.06 MPa působily spíše zavírání stomat než zpomalování metabolismu fixace oxidu uhličitého.

Dále jsou v práci popsány původní metody pro výpočet kritické teploty poškození rostlin mrazem pomocí nelineární regrese logistickou funkcí a pro porovnávání zimovzdornosti kultivarů obilovin na základě údajů o vyzimování. První metoda je příkladem hodnocení stresu, dobře charakterizovaného jednou veličinou (teplotou mrznutí), druhá je příkladem hodnocení mnohasložkového stresu.

Měření celkové délky kořenového systému a počítání kořenových špiček pomocí počítačové analýzy obrazu bylo použito k porovnání odolnosti dvou genotypů pšenice k nízkému pH a k hlinitým iontům. Nižší tolerance se projevila výraznějším poklesem celkové délky i větvení.

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Jiri Janacek