**2. Materials and methods**

for cotton production in the past has lead to an excessive soil salinization and to the exhaus‐ tion of its largest water resource—the Aral Sea. Therefore, revealing the adaptation potential of local agricultural crops to water deficit and creating their drought-tolerant genotypes are an important task: this would allow, in particular, to obtain higher cotton yield and quality in conditions of limited water resources and to improve local environment by stopping desertifi‐

Creating drought-tolerant genotypes of agricultural crops is complicated because the lack of systematic knowledge on physiological parameters reflecting the genetic potential for improved productivity under conditions of water deficiency. The effect of drought stress on the photosynthetic performance and drought-induced morpho-physiological, biochemical and biophysical changes in various plant species have been extensively studied; stomatal and non-stomatal limitations to photosynthesis, their role and possible mechanisms have been suggested [3]. These studies have shown that photosynthetic performance is very informative

Nowadays, the methods of chlorophyll fluorescence control along with the classical measure‐ ments of photosynthesis based on gas-exchange analysis are widely used by agronomists in monitoring of crops and their response to environmental stresses [4]. Revealing physical characteristics of chlorophyll fluorescence in plant leaves and employing achievements in laser physics, optoelectronics and computer technologies allowed developing a variety of efficient experimental methods and easy to use devices for measuring such key fluorescence parame‐ ters, as a maximal (saturated) and a minimal (dark) fluorescence, a prompt and a delayed fluorescence, a kinetics of induction of chlorophyll fluorescence and their relationship with quantitative indicators of photosynthesis in plants [5, 6]. These methods are fast, noninvasive and estimate the photosynthetic performance of plants even under mid-day solar radiation, and portable devices commercially manufactured on their basis determine the parameters of plant photosynthetic performance with multiple replication of measurements and recording the results in a memory for subsequent statistical processing using relevant computer pro‐

Here, the results of long-term effect's study of drought on the chlorophyll fluorescence and morpho-physiological indicators of cotton plants grown under field conditions are described. Literature on researches concerning to mechanisms of stress effect of drought on photosyn‐ thesis in plants are analyzed. The long-term effect of drought on cotton plants has been studied during the key period of their ontogenesis — in flowering and maturing stages from last July to last September by simultaneously measuring indicated parameters in well-watered and moderately drought-stressed plants. Correlations between the chlorophyll fluorescence and morpho-physiological indicators (leaf blade area and thickness, relative water content and transpiration) have been defined in three genotypes of cotton with different degrees of drought

Comparative measurements of the operating quantum efficiency of photochemistry in Photosystem II, ФPSII, and its changes during the day time in well-watered and moderately drought-stressed plants have shown that in contrary to the widely accepted idea on tight links between ФPSII and the quantum efficiency of CO2 uptake [9], and decline of photosynthesis in

and sensitive indicator of stress effects of drought in plants.

cation of the region.

92 Applied Photosynthesis - New Progress

grams [7, 8].

tolerance.

Three local genotypes of cotton (*Gossypium hirsutum L.*), Navbakhor, Liniya-49, and Gulsara were grown on the two levels of irrigation: under well-watered and moderately droughtstressed conditions [17] at the experimental cotton station of the Institute of Genetics and Plant Experimental Biology, Uzbekistan Academy of Sciences, Tashkent (41°10´N, 69°07´E, 400 m above sea level), in 2013–2014. All plants were sown on 10th April with the scheme of 90 cm (distance between rows) × 20 cm (distance between plants) × 1 (amount of plants per hole). Thousand plants of each genotype and water treatment were grown in 4 rows, 250 plants each. During the entire period of ontogenesis, well-watered plants were irrigated 5 times: 1–before flowering, 3–during flowering-maturing, and 1–in maturing stages, and the drought-stressed plants–3 times: in the scheme 1—2—0. Thus, moderate drought stress was induced in the most sensitive stage of cotton plants—in mass flowering-maturing period. During this period, rainfall did not occur. All other growth conditions, including content of nutrients in soil, were the same.

The chlorophyll fluorescence was measured in attached leaves by using portable chlorophyll fluorometer Mini-PAM (Walz, Effeltrich, Germany) allowing up to 3000 measurements in the field without battery recharging [7]. The Mini-PAM fluorometer measures the chlorophyll fluorescence parameters even under mid-day solar radiation by means of simultaneous application of a CW measuring light and saturating light flashes. Measurements were carried out in the early morning, from 7.00 to 8.00, on the third, matured leaves with 10-fold replication. In most of the experiments, the operating quantum efficiency of primary photochemistry, *ΦPSII* = *FV* ′ / *FM* ′=(*FM* ′− *FS* ′) / *FM* ′ (*FM* ′—is a maximum and *FS* ′—a steady state levels of fluores‐ cence at any arbitrary moment of a leaf illumination [18]), was determined as an indicator of the photosynthetic performance. For calculation of this parameter, measurements of a dark fluorescence and, consequently, dark adaptation of leaves were not a need [19, 20], which essentially simplified field experiments. The maximum fluorescence was measured at appli‐ cation of saturating light flashes with duration 0.8 s and photosynthetic photon flux density (PPFD) 8000 μmol m-2 s-1. However, in some experiments, the photochemical quenching factor *qP* =(*FM* ′− *<sup>F</sup>* ′ )/ (*FM* ′− *F*<sup>0</sup> ′) and the non-photochemical quenching *NPQ* = *FM* / *FM* ′−1, character‐ izing efficiencies of photochemical utilization and non-photochemical losses of the absorbed light energy accordingly, were also determined. The electron transport rate *ETR* =*ΦPSII* ×*PAR* ×0.5×*α* was controlled as an indicator of activity of the photosynthetic electron transport chain; here photosynthetic active radiation (PAR) is the solar radiation intensity in spectral range 400–750 nm expressed as PPFD in μmol m-2 s-1, and α is leaf absorption. In general, it is assumed that α = 0.85 and a ratio *PSII* :*PSI* =1:1. PAR intensities were controlled by portable luxometer Yu-116 with a dielectric multilayer filter filtering out PAR from the whole solar radiation.

The gas-exchange measurements were carried out using photosynthesis analyzer LI-6400 (Licor, USA) at temperature 24°C [21]. The curves of CO2 response were measured in leaves of both water treatments by means of gradual lowering of the external CO2 concentration, from 400 μmol mol-1 to 0 μmol mol-1 at PPFD 1000 μmol m-2 s-1, and the light response curves—at ambient СО<sup>2</sup> concentration with step-by-step increasing of PPFD from 0 μmol m-2 s-1 to 2000 μmol m-2 s-1. The light and CO2 responses of the chlorophyll fluorescence and the photosyn‐ thesis were measured after adaptation of leaves to each value of PPFD and CO2 concentration during 15 min. The operating values of the minimum fluorescence under continuous illumi‐ nation during the measurements, *F′*0, were calculated according to [22] using the equation *F*0 ′ = *F*<sup>0</sup> / (*FV* / *FM* + *F*<sup>0</sup> / *FM* ′ ).

Relative water content and transpiration of plant leaves were determined by their weighting [23]. In addition, a leaf thickness and a leaf blade area were also measured in each cotton genotype. For estimation of the magnitude and diurnal variations of photoinhibition, the values of ФPSII have been consistently measured simultaneously in both well-watered and drought-stressed plants every hour during 24 h.

Photoacoustic spectrometer of special design with ~1 cm3 sample chamber having higher sensitivity at low (10–250 Hz) frequencies of light modulation [24] has been used for measuring photoacoustic characteristics of plant leaves. The sources of a CW measuring light and saturating light flashes of the spectrometer were a semiconductor LED (650 nm, 20 mW) and a halogen lamp (400–700 nm, 20 W) with a mechanical chopper, respectively. Intensity of the measuring light was supported as 50–100 μmol m-2 s-1 and intensity of the saturating flashes did not exceed 2500 μmol m-2 s-1. The photobaric component was selected from the total photoacoustic waves generated in a plant leaf at application of low-frequency (10 Hz) modulated light by recording quadrature signal from a lock-in amplifier [25].
