**3. Effect of drought to photosynthesis**

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

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

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

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

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

sample chamber having higher

′) and the non-photochemical quenching *NPQ* = *FM* / *FM* ′−1, character‐

*qP* =(*FM* ′− *<sup>F</sup>* ′

*F*0 ′ )/ (*FM* ′− *F*<sup>0</sup>

94 Applied Photosynthesis - New Progress

PAR from the whole solar radiation.

= *F*<sup>0</sup> / (*FV* / *FM* + *F*<sup>0</sup> / *FM* ′

).

drought-stressed plants every hour during 24 h.

Photoacoustic spectrometer of special design with ~1 cm3

Drought stress is primarily affected to photosynthetic performance of plants. The long-term drought effect is expressed as reducing/delaying of a plant growth and development, prema‐ ture leaf senescence, and related reduction in a crop productivity [26, 27]. The dispute, what, mainly, limits photosynthesis under conditions of water deficiency: stomata closure or impairment of the metabolism is long enough [28, 29], but in the past decade, closure of stomata was perceived by experts as the predominant factor in mild and moderate drought stress [30].

The first response of a plant to onset of drought stress is the stomata closure and associated reduction of the relative water content of leaves and intracellular CO2 concentration, *C*<sup>i</sup> [3, 31]. This, in turn, causes decrease in a leaf turgor and a water potential [32]. In such a condition, gas-exchange analysis in plant leaves would be an informative technique for assessment of stomatal limitation to CO2 assimilation.

Non-stomatal mechanisms of the photosynthesis limitation under long-term or severe drought in the soil include changes in chlorophyll synthesis [33], structural changes in photosynthetic apparatus and depressing the Calvin cycle enzymes activities, which reduces crop yield [34] and decline in Rubisco activity [35, 36].

Short-term or mild drought-induced non-stomatal limitations to photosynthesis have smaller magnitude than stomatal ones. Closure of stomata and limited access of CO2 bring about reduced utilization of the energy of electron transport, and, accordingly, over-excitation of the plant photosynthetic apparatus. This, accordingly, increases the susceptibility of the system to photo-damage. Accumulation of singlet oxygen or superoxide radicals, when a dynamic balance between producing of such reactive substances and functioning of the plant antioxi‐ dant defense system is broken, may cause destruction of photosynthetic proteins and mem‐ brane lipids [37, 38].

Reduced rate of transpiration, especially at higher ambient temperatures, increases the heat accumulation and relevant increase in leaf temperature. The latter can also cause decline of the plant photosynthetic performance under drought [30].

A number of experiments have shown that the closure of stomata is controlled, mostly, by reducing soil water content, but not leaf water status. This suggests response of stomata to a chemical signal from roots, i.e. presence of abscisic acid produced by dehydrating roots, while a leaf water status is constant [39, 40]. The same time it means that the efficient way to control the stomatal conductance is to change the soil water content even preserving constant level of leaf water status.

Activity of the photosynthetic electron transport chain is rigidly regulated by the availability of CO2 in the chloroplast, limited by closure of stomata under drought stress [41]. Leaf dehydration leads to shrinking of cells and accordingly reducing of their volume. This causes an increase in the internal viscosity of the cell contents, and interaction between proteins and, consequently, their aggregation and denaturation [42].

Comparison of the results from different studies is quite difficult due to the essential variations in responses of the stomatal conductance and photosynthesis to changes of leaf water potential and relative water content in different genotypes [3]. It is considered as well established that drought-induced stomata closure declines the net photosynthesis in all plant species, though, with different magnitudes. That is why comparative studies of the photosynthetic parameters in different plant genotypes under drought stress may provide an important information concerning to the photosynthetic performance and adaptation potential of plants to moderate long-term drought.

Analysis of the chlorophyll fluorescence and photosynthesis in plant leaves has revealed that in conditions favorable for photosynthesis, i.e. lack of environment stresses, at low light intensities, etc. when alternative mechanisms of light energy utilization did not required, the quantum efficiency of photochemistry is tightly linked with quantum efficiency of CO2 fixation [9], and the photosynthesis rate is not sensitive to mild under drought stress [10, 43]. In this condition, photorespiration increases and its magnitude depends on the light intensity [44]. In a number of researches, the reduction in ФPSII has been observed under long-term drought, which has been attributed, mostly, to reducing of photochemistry and, in less extent, to dissipative processes in the plant photosynthetic apparatus. However, in some other research‐ es, the increasing ФPSII has been observed in plants exposed to moderate long-term drought [12, 13]. Such contradiction in behavior of ФPSII may be explained by a heterogeneity of the photosynthetic performance across the leaf blade [14, 45]. Thus, simultaneous analysis of chlorophyll fluorescence and photosynthesis in plant leaves may reveal mechanisms and magnitude of protective changes in plants under drought stress, and correlations between changes in chlorophyll fluorescence parameters and morpho-physiological indicators, traditionally used for estimation of drought tolerance of plants, may be used as an effective instrument for monitoring of plants in the field.

### **4. Comparative measurements of ФPSII in different cotton genotypes**

The operating quantum efficiency of photochemistry, ФPSII, has been determined simultane‐ ously in well-watered and moderately drought-stressed plants of three genotypes of cotton cultivated in Uzbekistan with the aim of estimating the magnitude of the effect of drought on the photosynthetic performance and monitoring its changes during a key period of the ontogenesis—in flowering and maturing stages from last July to last September [12, 46]. Figure 1 shows the results of this experiment. The dates of measurements are shown on the Xaxis. Stressed plants of all cotton genotypes display higher values of ФPSII in comparison with well-watered plants. Moreover, in the drought-tolerant plants of Navbakhor, this increase was maximal (up to 15% over the most period of measurements), while in Gulsara characterized by lower drought tolerance, it was minimal (approximately 2%). And, in Liniya-49 having an

Monitoring of the Drought Tolerance of Various Cotton Genotypes Using Chlorophyll Fluorescence http://dx.doi.org/10.5772/62232 97

dehydration leads to shrinking of cells and accordingly reducing of their volume. This causes an increase in the internal viscosity of the cell contents, and interaction between proteins and,

Comparison of the results from different studies is quite difficult due to the essential variations in responses of the stomatal conductance and photosynthesis to changes of leaf water potential and relative water content in different genotypes [3]. It is considered as well established that drought-induced stomata closure declines the net photosynthesis in all plant species, though, with different magnitudes. That is why comparative studies of the photosynthetic parameters in different plant genotypes under drought stress may provide an important information concerning to the photosynthetic performance and adaptation potential of plants to moderate

Analysis of the chlorophyll fluorescence and photosynthesis in plant leaves has revealed that in conditions favorable for photosynthesis, i.e. lack of environment stresses, at low light intensities, etc. when alternative mechanisms of light energy utilization did not required, the quantum efficiency of photochemistry is tightly linked with quantum efficiency of CO2 fixation [9], and the photosynthesis rate is not sensitive to mild under drought stress [10, 43]. In this condition, photorespiration increases and its magnitude depends on the light intensity [44]. In a number of researches, the reduction in ФPSII has been observed under long-term drought, which has been attributed, mostly, to reducing of photochemistry and, in less extent, to dissipative processes in the plant photosynthetic apparatus. However, in some other research‐ es, the increasing ФPSII has been observed in plants exposed to moderate long-term drought [12, 13]. Such contradiction in behavior of ФPSII may be explained by a heterogeneity of the photosynthetic performance across the leaf blade [14, 45]. Thus, simultaneous analysis of chlorophyll fluorescence and photosynthesis in plant leaves may reveal mechanisms and magnitude of protective changes in plants under drought stress, and correlations between changes in chlorophyll fluorescence parameters and morpho-physiological indicators, traditionally used for estimation of drought tolerance of plants, may be used as an effective

**4. Comparative measurements of ФPSII in different cotton genotypes**

The operating quantum efficiency of photochemistry, ФPSII, has been determined simultane‐ ously in well-watered and moderately drought-stressed plants of three genotypes of cotton cultivated in Uzbekistan with the aim of estimating the magnitude of the effect of drought on the photosynthetic performance and monitoring its changes during a key period of the ontogenesis—in flowering and maturing stages from last July to last September [12, 46]. Figure 1 shows the results of this experiment. The dates of measurements are shown on the Xaxis. Stressed plants of all cotton genotypes display higher values of ФPSII in comparison with well-watered plants. Moreover, in the drought-tolerant plants of Navbakhor, this increase was maximal (up to 15% over the most period of measurements), while in Gulsara characterized by lower drought tolerance, it was minimal (approximately 2%). And, in Liniya-49 having an

consequently, their aggregation and denaturation [42].

instrument for monitoring of plants in the field.

long-term drought.

96 Applied Photosynthesis - New Progress

**Figure 1.** The changes in ФPSII in leaves of three genotypes of cotton: Navbakhor (a), Liniya-49—(b) and Gulsara—(c) growing in well-watered (•) and moderately drought-stressed (○) conditions during a long period of their ontogene‐ sis.

intermediate degree of drought tolerance had intermediate values for differences in ФPSII. Irrigation of the drought-stressed plants on 10th September shortened this difference, though, with different extent in different genotypes.

Measurements of morpho-physiological indicators in plants of all genotypes have demon‐ strated considerable reduction in leaf relative water content and of leaf blade expansion and increase in leaf thickness under long-term drought stress. These changes are presented in Table 1. It is seen that in the most drought-tolerant cotton genotype Navbakhor, these changes are maximal, and in Gulsara having lower drought tolerance, these are minimal. Correlations between ФPSII and these morpho-physiological indicators have been defined in all three genotypes, but with different extent. The last may be attributed to the possibility of other protective reactions in plants affected to long-term drought stress [47].

Leaf transpiration was lower in drought-stressed plants than in well-watered plants of all genotypes for 5–15% (not shown), which may be considered as typical for the field-grown cotton plants [48]. However, diurnal changes in transpiration of plants were much more than differences between two treatments, therefore reliable correlations between changes in the


**Table 1.** Morpho-physiological indicators of the well-watered and moderately drought-stressed cotton genotypes.

transpiration and the chlorophyll fluorescence parameters under drought stress were not established.

For determination of changes in the photosynthetic performance of plants under drought stress and kinetics of photoinhibition over the day, the quantum efficiency of photochemistry has been measured hourly during 24 h. Figure 2 shows such dependencies measured in wellwatered and drought-stressed plants of Navbakhor. As shown in previous figure, in the drought-stressed plants, ФPSII is higher than in well-watered plants during all the day, includ‐ ing a night time. In addition, decline of ФPSII in mid-day in the drought-stressed plant is smaller but occurs for longer time [12]. Such a photoinhibitory depression of the primary photochem‐ istry under high-intensity solar radiation is characterized by various components with different relaxation periods [49, 50]. Obviously, adaptive changes in the structure and func‐

**Figure 2.** Diurnal changes in ФPSII measured in leaves of the cotton genotype Navbakhor grown in well-watered (•) and moderately drought-stressed (○) conditions in the field.

tioning of the plant photosynthetic apparatus under moderate long-term drought may bring about depressing, mainly short-period, components of photoinhibition and its long-period components will dominate in drought-stressed plants [51]. Such changes in the proportion of different components of photoinhibition results in decreasing of the amplitude and reshaping of the form of diurnal changes ФPSII as it is shown in Figure 2. It should be noted that difference in values of ФPSII measured in well-watered (0.34) and drought-stressed (0.48) plants at midday, 0.14, is considerably higher than those in other periods of the day. This fact may be considered as enhancing of photorespiration that may contribute in ФPSII only as a prompt component.

Therefore, protective response of cotton plants to drought stress expressed in photosynthetic indicators is the increase in quantum efficiency of primary photochemistry, in morphology is the increase of leaf thickness with decreasing leaf blade expansion and in physiology is the reduce in transpiration. If reduce in the leaf blade expansion and transpiration may be explained logically by considerations of minimizing the moisture loss [47], increase of ФPSII looks as somehow contradictory with the literature data: at the onset of drought stress, the plant should response by reducing photosynthesis to protect the photosynthetic apparatus [52]. At constant values of efficiency of alternative ways of energy utilization, this has to bring about lower quantum efficiency of photochemistry. Then, the excessive energy of absorbed light may be utilized by enhancing the activity of an alternative channel—photorespiration. Lastly, in C3 plants could be significant, particularly in cotton, which typical growth conditions are associated with higher temperatures and water deficiency. At present, protective role of photorespiration under environmental stresses are poorly studied and published researches on this matter is very minor [53].

transpiration and the chlorophyll fluorescence parameters under drought stress were not

**Morpho-physiological indicators Water treatment Cotton genotypes**

Relative water content, % Well-watered 79.4 78.8 77.4

Leaf blade area, m2 Well-watered 71.1 77.7 80.9

Relative leaf thickness, g m-2 Well-watered 0.853 0.974 0.987

**Table 1.** Morpho-physiological indicators of the well-watered and moderately drought-stressed cotton genotypes.

**Navbahor Liniya-49 Gulsara**

Drought-stressed 72.5 74.4 74.3 Percentage of the difference 8.7% 5.6% 5.0%

Drought-stressed 63.1 73.0 77.1 Percentage of the difference 11.3% 6.1% 4.7%

Drought-stressed 0.981 1.09 1.052 Percentage of the difference 15.0% 11.9% 6.6%

For determination of changes in the photosynthetic performance of plants under drought stress and kinetics of photoinhibition over the day, the quantum efficiency of photochemistry has been measured hourly during 24 h. Figure 2 shows such dependencies measured in wellwatered and drought-stressed plants of Navbakhor. As shown in previous figure, in the drought-stressed plants, ФPSII is higher than in well-watered plants during all the day, includ‐ ing a night time. In addition, decline of ФPSII in mid-day in the drought-stressed plant is smaller but occurs for longer time [12]. Such a photoinhibitory depression of the primary photochem‐ istry under high-intensity solar radiation is characterized by various components with different relaxation periods [49, 50]. Obviously, adaptive changes in the structure and func‐

established.

98 Applied Photosynthesis - New Progress

1.0

0.8

0.6

ФPSII

0.4

0.2

0.0

and moderately drought-stressed (○) conditions in the field.

0 4 8 12

Time of the day (h)

**Figure 2.** Diurnal changes in ФPSII measured in leaves of the cotton genotype Navbakhor grown in well-watered (•)

16 20 24

Thus, cotton genotypes with different degrees of drought-tolerance studied displayed specific changes in the chlorophyll fluorescence parameters, as well as in morpho-physiological indicators under long-term drought stress. Diurnal curves of ФPSII variations in well-watered and moderately drought-stressed plants provide information on the magnitude and different time components of photoinhibition developed under high-intensity solar radiation.

Photoacoustic waves generated in plant leaves at application of modulated light have been studied for precise control of the photosynthetic performance and quantitative estimation of the photosynthetic oxygen evolution. Photobaric component of the photoacoustic waves related to photosynthetic evolution of oxygen has been measured in the photoacoustic cell of special design with a small measuring chamber (~1 cm3 ) in lock-in amplifier by selecting quadrature signal at low frequencies [15, 54]. Figure 3 shows kinetics of changes of the photoacoustic signal from the well-watered (relative water content 100%) and short-term dehydrated (relative water content 65%) leaves of the cotton genotype Navbakhor, generated at application of low frequency (10 Hz) measuring light. It is shown from the figure that the steady-state photoacoustic signal considerably declines at application of additional CW light of high intensity (~2500 μmol m-2 s-1) to plant leaf, which saturates photosynthetic oxygen evolution process and, accordingly, excludes periodic changes of pressure in the measuring chamber, which is the photobaric wave. Therefore, relative change in the photoacoustic signal (ratio of amplitude of change to the total photoacoustic signal) may be used as a measure of

**Figure 3.** Induction curves of the photoacoustic signal generated in leaves of the cotton genotype Navbakhor with rela‐ tive water content 100% (a) and 65% (b). Arrows up and down show switching on and off, respectively, the measuring (dashed arrows) and saturating (bolt arrows) lights.

the photosynthetic oxygen evolution. In experiments, before measuring photoacoustic signals, the plant leaves were adapted to dark for 10 min. After reaching the steady-state photoacoustic signal, the saturating CW light was applied, which causes decrease in the photoacoustic signal for 0.82 (Figure 3a) in the well-watered leaf and for 0.50 (Figure 3b) in the dehydrated leaf. Thus, the photoacoustic measurements have shown that photosynthetic oxygen evolution in plant leaves depresses in short time water deficiency: decrease in the relative water content for 45% causes decrease of photosynthetic activity 1.5 times. Simultaneous measurements of ФPSII in these two samples displayed decline of the operative quantum efficiencies of photo‐ chemistry in the same ratio (0.75:0.51). However, the advantage of photoacoustic measure‐ ments is evident in the case of significant level of photorespiration in plant leaves, when direct correlation between ФPSII and the net photosynthesis is disturbed (see the next section).
