**3. Influence of water deficiency on plant physiology: An overview**

Drought stress is a factor that occurs when little water is available in an appropriate thermo‐ dynamic state. The term "dry" indicates a period without appreciable precipitation during which the water content in the soil is reduced (Larcher, 2000). This situation can be exacer‐ bated when atmospheric conditions cause continuous water loss via transpiration or evapo‐ ration (Jaleel et al., 2009).

develops. The effects of SOM are connected with the hygroscopicity and high specific sur‐ face area of this type of material, which promotes increased water retention (Braida et al, 2011). In a long-term study, Franchini et al. (2009) reported that during the first four seasons after the adoption of aNTS, the soybean yield was similar or slightly lower than that ob‐ tained under conventional tillage (CT). However, from the fifth year onward, when the sys‐ tem had matured and consolidated, the soybean yield was higher under the NTS than the

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

**Figure 2.** Soybean yield in different management systems: conventional tillage (13 years); new no-tillage (3 years) and

Sowing according to agroclimatic zoning for each agricultural environment is another strat‐ egy to avoid productivity losses due to water restriction. The cultivar must be adapted to the region considering the climate and soil type that determine water retention. In regions with higher occurrences of drought, it is essential to cultivate material that is more tolerant to water restriction. Thus, when the chemical and physical conditions of soil are suitable, al‐ lowing good root development at depth, and the cultivar and sowing time are selected to minimize the effects of water restriction, it is possible to obtain a high productivity soybean

Overall, to achieve productivity under any conditions, it is essential that the process of pho‐ tosynthesis, which is responsible for all carbon assimilated for the production of biomass, has a minimum efficiency. Under water deficit conditions during the soybean cycle, photo‐

Drought stress is a factor that occurs when little water is available in an appropriate thermo‐ dynamic state. The term "dry" indicates a period without appreciable precipitation during

**3. Influence of water deficiency on plant physiology: An overview**

no-tillage consolidated (13 years), (adapted from Franchini et al., 2009).

synthesis is one of the main physiological processes affected.

CT (Figure 2).

Relationships

278

grain yield.

Depending on environmental conditions, different levels of water loss can occur. While wa‐ ter deficiency is a state caused by a moderate loss of water, which causes stomatal closure and reduction of gas exchange (Jaleel et al., 2009), desiccation is caused by much more ex‐ tensive losses of water, which can potentially lead to major disruption of cellular structure and metabolism and eventually to an interruption of enzyme-catalyzed reactions (Smirnoff, 1993, Jaleel et al. 2009).According to Jaleel et al. (2009), water stress is characterized by re‐ ductions in water content and leaf water potential and loss of cellular turgor, which decreas‐ es stomatal conductance and cellular expansion, consequently constraining plant growth. Severe water stress can result in impairment of photosynthesis, metabolic disorders and ulti‐ mately death of the plant. In addition, other factors, such as high temperatures, soil salinity, pathogen attack and mechanical damage, may also contribute to differences in the amount of water in plants by interfering with their development and reducing their productivity (Larcher, 2000).

Once successful stand establishment is achieved, one of the most sensitive physiological processes to drought stress is reduced cell expansion resulting from decreased turgor pres‐ sure (Raper & Kramer, 1987). As leaf water potential falls, cell and leaf expansion are affect‐ ed before photosynthesis. Bunce (1977) reported a linear relationship between soybean leaf elongation rate and turgor pressure. Decreasing leaf water potential to -0.80 MPa reduced leaf elongation rate by 40% relative to greater values of water potential. Consequently, leaf area and plant dry matter were reduced 60% and 65%, respectively. These results were sub‐ sequently confirmed in field experiments (Muchow et al., 1986). Thus, occurrence of water deficit during vegetative growth (emergence to R5) can reduce the leaf area indices (LAI) and the interceptation of photosynthetically active radiation by the total leaf area to levels insufficient for optimal crop growth rate (CGR) and yield. The effects of water stress on pho‐ tosynthetic rates of soybean leaves are readily detectable at leaf water potentials about -1.0 to -1.2 MPa (Raper& Kramer, 1987). The rate starts declining more rapidly as water potential falls below -1.8MPa. Plants suffering this level of drought would have greater reductions of CGR and yield because not only would LAI be reduced, but the net assimilation rate (photo‐ synthetic rate per unit LAI) would also be reduced. Drought stress effects on photosynthesis become irreversible once water potential falls below -1.6 MPa.

The stress induced by water deficits in plants depends on the conditions provided by the environment, varying according to the intensity and duration of water deficits, the rate of water deficit induction and the stage of plant development when drought occurs (Pin‐ heiro & Chaves, 2011, Bertolli et al., 2012). Moreover, these factors can strongly influence the process of mitigation associated with acclimation to conditions after water shortages (Chaves et al., 2009). Acclimation responses in plants under water stress generally in‐ clude responses related to growth inhibition or leaf shedding, reducing the water con‐ sumption by these tissues and contributing to the maintenance of water balance and the assimilation of carbon (Chaves et al., 2009, Pinheiro & Chaves, 2011). Osmoregulatory el‐

ements produced in response to slow dehydration also have the function of maintaining metabolic activity, improving cell osmotic adjustment (Pang et al., 2011). The increase in sugar levels during leaf dehydration appears to be the result of coordinated regulation of the synthesis and translocation of sucrose, contributing to osmotic adjustment and ena‐ bling the maintenance of turgor in meristematic regions. These responses may eventually lead to restoration of cellular homeostasis, thereby increasing plant survival under stress (Chaves, 1991, Chaves et al., 2009).

A central question in plant biology is related to the problem of the optimization of CO2 fixa‐ tion in environments with limited water. In environments where there is water restriction caused by a lack of water from the soil or by a high atmospheric water demand plants tend to close their stomata to conserve water via reducing transpiration losses, which may limit the CO2 intake into the leaf for photosynthesis (Kaizer, 1987, Flexas et al., 2009, Pinheiro & Chaves, 2011). Control of the entry of CO2 and water loss is performed by continuously set‐ ting the opening of the stomata distributed across the leaves. This dynamic of opening and closing of stomata is heterogeneous and can be particularly enhanced under conditions of water stress, causing the patchy stomatal conductance phenomenon to occur (Mott & Buck‐ ley, 1998, Flexas et al., 2009).

The response of photosynthesis under water stress has been the focus of study and debate for decades, particularly with respect to what the most limiting factors for photosynthesis are (Lawlor & Cornic, 2002, Lawlor & Tezara, 2009, Flexas et al., 2009). However, there is still some controversy regarding the importance of the main physiological parameters and the time period over which they limit photosynthesis (Pinheiro & Chaves, 2011).

The decrease in CO2 diffusion from the atmosphere to the carboxylation site of Rubisco (ri‐ bulosebiphosphate carboxylase/oxygenase) is generally considered to be the main cause of reduced photosynthesis under conditions of mild and moderate water deficits (Grassi & Magnani, 2005, Chaves et al., 2003, 2009, Pinheiro & Chaves, 2011). Greater resistance to the diffusion of CO2 may be caused by both stomatal limitation and by lower conductance in the mesophyll (Flexas et al., 2009). The stomata act in regulating the entry of CO2 (an essential substrate for photosynthesis) into the leaves and the loss of water vapor to the atmosphere via transpiration. The control of gas exchange between the leaf and the atmosphere through the stomata is essential for the maintenance of photosynthetic activity and tissue hydration (Reynolds-Henne, et al. 2010, Aasamaa & Sõber, 2011). When the decrease in stomatal con‐ ductance is combined with high light levels, the leaves are subjected to an excessive amount of incident energy in relation to the amount of intercellular CO2 available for photosynthe‐ sis, and the rate of reductant energy production can therefore overlap with the rate of its consumption by the Calvin cycle. Under these circumstances, down-regulation of photosyn‐ thesis or even photoinhibition can become a powerful defense mechanism for plants (Pin‐ heiro & Chaves, 2011, Sanda et al. 2011). This protection can be achieved via thermal dissipation occurring in the light harvesting complex of the photochemical apparatus, in‐ volving the xanthophyll cycle (Demmig-Adams et al. 2006, Sarlikioti et al. 2010), and through alternative sinks for the excess energy, such as photorespiration (Lawlor & Cornic, 2002, Miyake, 2010) or the Mehler reaction, now referred to as the water-water cycle (Miyake, 2010). Because there is dependence on the fixation of CO2 and photochemical effi‐ ciency (ATP generation and NADPH) and vice-versa (Miyake et al., 2009), any stress or sit‐ uation that reduces the consumption of photochemical products through decreased carboxylation efficiency can generate excessive excitation energy (Baker et al., 2004, Sarlikio‐ ti et al., 2010). When the non radioactive de-excitation energy (NPQ) is insufficient to ad‐ dress this excess energy, alternative electron sinks, such as the water-water cycle and photorespiration, are activated, which is crucial for maintaining photosynthetic activity (Miyake et al., 2009, 2010, Lawlor & Tezara, 2009).

ements produced in response to slow dehydration also have the function of maintaining metabolic activity, improving cell osmotic adjustment (Pang et al., 2011). The increase in sugar levels during leaf dehydration appears to be the result of coordinated regulation of the synthesis and translocation of sucrose, contributing to osmotic adjustment and ena‐ bling the maintenance of turgor in meristematic regions. These responses may eventually lead to restoration of cellular homeostasis, thereby increasing plant survival under stress

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

A central question in plant biology is related to the problem of the optimization of CO2 fixa‐ tion in environments with limited water. In environments where there is water restriction caused by a lack of water from the soil or by a high atmospheric water demand plants tend to close their stomata to conserve water via reducing transpiration losses, which may limit the CO2 intake into the leaf for photosynthesis (Kaizer, 1987, Flexas et al., 2009, Pinheiro & Chaves, 2011). Control of the entry of CO2 and water loss is performed by continuously set‐ ting the opening of the stomata distributed across the leaves. This dynamic of opening and closing of stomata is heterogeneous and can be particularly enhanced under conditions of water stress, causing the patchy stomatal conductance phenomenon to occur (Mott & Buck‐

The response of photosynthesis under water stress has been the focus of study and debate for decades, particularly with respect to what the most limiting factors for photosynthesis are (Lawlor & Cornic, 2002, Lawlor & Tezara, 2009, Flexas et al., 2009). However, there is still some controversy regarding the importance of the main physiological parameters and

The decrease in CO2 diffusion from the atmosphere to the carboxylation site of Rubisco (ri‐ bulosebiphosphate carboxylase/oxygenase) is generally considered to be the main cause of reduced photosynthesis under conditions of mild and moderate water deficits (Grassi & Magnani, 2005, Chaves et al., 2003, 2009, Pinheiro & Chaves, 2011). Greater resistance to the diffusion of CO2 may be caused by both stomatal limitation and by lower conductance in the mesophyll (Flexas et al., 2009). The stomata act in regulating the entry of CO2 (an essential substrate for photosynthesis) into the leaves and the loss of water vapor to the atmosphere via transpiration. The control of gas exchange between the leaf and the atmosphere through the stomata is essential for the maintenance of photosynthetic activity and tissue hydration (Reynolds-Henne, et al. 2010, Aasamaa & Sõber, 2011). When the decrease in stomatal con‐ ductance is combined with high light levels, the leaves are subjected to an excessive amount of incident energy in relation to the amount of intercellular CO2 available for photosynthe‐ sis, and the rate of reductant energy production can therefore overlap with the rate of its consumption by the Calvin cycle. Under these circumstances, down-regulation of photosyn‐ thesis or even photoinhibition can become a powerful defense mechanism for plants (Pin‐ heiro & Chaves, 2011, Sanda et al. 2011). This protection can be achieved via thermal dissipation occurring in the light harvesting complex of the photochemical apparatus, in‐ volving the xanthophyll cycle (Demmig-Adams et al. 2006, Sarlikioti et al. 2010), and through alternative sinks for the excess energy, such as photorespiration (Lawlor & Cornic, 2002, Miyake, 2010) or the Mehler reaction, now referred to as the water-water cycle

the time period over which they limit photosynthesis (Pinheiro & Chaves, 2011).

(Chaves, 1991, Chaves et al., 2009).

Relationships

280

ley, 1998, Flexas et al., 2009).

Part of the reduction of photosynthesis can be attributed to non-stomatal effects at the chloroplast level, and electron transport and photophosphorylation are the main targets of inhibition (Sanda et al., 2011). The reduction in ATP synthesis that is an initial re‐ sponse to water deficits can lead to reduction of the capacity for Ribulose-1,5-*bis*phos‐ phate (RuBP) regeneration, reducing potential photosynthesis (Lawlor & Tezara, 2009, Pinheiro & Chaves, 2011). Furthermore, Rubisco activity may be impaired by Rubisco ac‐ tivase activity and the reduction of ATP. Lawlor & Tezara (2009) claim that inhibitors such as RuBP analogues bind to the active site of Rubisco, decreasing its activity, espe‐ cially when the concentration of RuBP is under saturated due to water deficiency. The regulation and restoration of Rubisco are mediated by Rubisco activase and require a high rate of ATP/ADP conversion. Thus, because phosphorylation is reduced under wa‐ ter stress, the activity of Rubisco is depressed (Parry et al., 2002).

In addition to solar energy, water availability and air temperature are elements that show seasonal variation (Rossato et al. 2009) and can affect photochemical activity. High temperatures may also affect photochemical activity directly through the inactivation of photosystem II and structural disorganization of the thylakoids or indirectly by the re‐ duction of the activity of the Calvin cycle (Zhang & Sharkey, 2009). Plants subjected to water deficits usually exhibit reduced transpiration and therefore show a low capacity for dissipation of latent heat, increasing the temperature of the leaves. This condition can decrease the fixation of CO2 because under increasing temperatures, Rubisco activity tends to be reduced (Kumar et al., 2009).

#### **3.1. Effects of water stress on photosynthesis in soybean plants**

Studies on the soybean crop have been focused on describing the impacts caused by the imposition of water stress on physiological parameters, particularly on photosynthesis in different genotypes (Desclaux & Roumet, 1996, Kron et al., 2008, Firmano et al., 2009, Ca‐ tuchi et al., 2011, Bertolli et al., 2012, Catuchi et al., 2012). In the cultivars 'New Tanba‐ kuro' and 'Tamanisiki' (Ohashi et al. 2009), 'CD 202' and 'CD 226RR' (Catuchi et al., 2011), 'BR 16' and 'Embrapa 48' (Catuchi et al., 2012) and 'MG/BR-46' (Conquista) (Stolf-Moreira et al., 2011) and wildtype plants (Kao & Tsai, 1998), it has been observed that the photo‐ synthetic rate, stomatal conductance and transpiration were reduced by water deficits. Moreover, there was a reduction in the intercellular CO2 concentration (Ci) observed in the cultivars 'BR 16' (Catuchi et al., 2012) and 'Embrapa 48' and wildtype species. Addi‐ tionally, in the cultivars 'New Tanbakuro' and 'Tamanisiki', Ci was altered slightly under

early water stress treatments, while for the cultivars 'MG/BR-46' (Conquista) and 'BR 16' (Stolf-Moreira et al., 2011), Ci increased as the water deficit progressed, indicating differ‐ ent physiological responses for different soybean cultivars. Furthermore, an increase in the intrinsic efficiency of water use was observed when the cultivars 'CD 202', 'CD 226RR', BR 16 and EMBRAPA 48 were subject to episodes of water restriction (Catuchi et al., 2011 and 2012). According to Manavalan et al. (2009), this increase may indicate bet‐ ter control of water loss via transpiration, contributing to the productivity of soybean. However, it is important to take into account that the studies discussed herein used dif‐ ferent methods of water stress induction, which could interfere with making more relia‐ ble and suitable comparisons among cultivars (Bertolli et al., 2012).

Reduction of net photosynthesis in soybean plants can be induced by both stomatal and non-stomatal factors (of both biochemical and photochemical origin). When a crop is sub‐ jected to a water deficit, the plants can reduce their stomatal conductance (gs), limiting the entry of CO2 into the substomatal chambers and thus reducing the diffusion of carbon to the site of carboxylation, resulting in significant decreases in carbon assimilation (Yu et al., 2004). Furthermore, Flexas et al. (2006a) report that the effects of water stress on the initial activity of Rubisco may be reproduced by induction of stomatal closure, independent of the reduction in the relative water content in the leaves of soybean plants. Thus, we can expect a lower regulation of photochemical and biochemical processes when the availability of CO2 is the most limiting component for photosynthesis in plants under severe water stress (Flexas et al. 2006b).

Although Pankovic et al. (1999) report that the content of Rubisco in soybean leaves increas‐ es as plants acclimate to water stress, other authors have observed that under the imposition of water deficits in pine, tobacco and soybean, there is reduced transcription of the subunits of this enzyme (Pelloux et al., 2001, Kawaguchi, et al., 2003, Majumdar et al., 1991, respec‐ tively). Reduction of the Rubisco activity in soybean plants under drought stress can be in‐ duced by reducing the content of the enzyme itself and possibly through increases in strongly binding inhibitors, as reported by Flexas et al. (2006a). When stomatal closure oc‐ curs for a period of several days, another mechanism involving gene expression can operate, resulting in a decrease in the total amount of Rubisco and/or an increase in the content of inhibitors that bind strongly to this enzyme (Flexas et al., 2006a).

Moreover, when the availability of CO2 and biochemical activity are reduced due to wa‐ ter deficits, the excess reductants in the photochemical apparatus must be dissipated as heat or drained through alternative electron sinks (Miyake et al., 2009, 2010) to reduce photoinhibition and the production of reactive oxygen species (ROS). Studies have re‐ vealed that PSII of soybean plants is resistant to moderate water stress (Kirova et al., 2008), and the potential quantum efficiency of PSII (Fv / Fm) and electron transport rate (ETR) are not altered by the imposition of water stress (Ohashi et al., 2006). Bertolli et al. (2012) reported that the decrease in the ETR was more sensitive than the decrease of Fv / Fm when the relative water content declined in soybean plants (cv. CD202), supporting the idea that the potential photochemical efficiency would not be readily affected by wa‐ ter deficiency. However, the reduction of the ETR could be due to a lower energy (ATP / NADPH) demand being required for carbon fixation metabolism under water deficit. Be‐ cause the stomatal conductance was decreased as water deficiency progressed, the reduc‐ tion of CO2 diffusion from the substomatic chamber to the active site of Rubisco may have influenced the efficiency of the Calvin cycle, which, in turn, may have reduced the consumption of ATP / NADPH from the photochemical apparatus, reducing the efficien‐ cy of the electron transport chain (Bertolli et al., 2012).

Depending on the intensity and duration of drought stress, metabolic limitations are fre‐ quently observed to be correlated with decreases in ATP, which reduces the capacity for RuBP turnover (Parry et al., 2002). This reduction in ATP synthesis is due to the decrease in electron transport and photophosphorylation caused by the reduction of the reactions asso‐ ciated with the chloroplast membranes (Catuchi et al., 2011). These membranes may suffer structural damage caused by decreasing the content of free water and ROS overproduction (Lawlor & Cornic, 2002), reducing the efficiency of the photochemical apparatus. Kao & Tsai (1998) also reported that the amount of quinone (QA) in the reduced state in a wild type soybean plant under water stress is greater than in plants irrigated under high light. The in‐ crease in the reduced state of QA is associated with the amount of inactivated and damaged D1 protein in the PSII reaction centers, indicating susceptibility of the photochemical appa‐ ratus to water deficits.
