Glutathione and proline can coordinately make plants withstand the joint attack of metal(loid) and salinity stresses

Agricultural soils in the vicinity of extensive anthropogenic activities may exhibit salinity together with high levels of metals/metalloids (hereafter termed as “metal/s”) as co-stressors. Elevated concentrations of metals (such as As, Cd, Cr, Hg, Ni, and Pb) may affect photosynthetic apparatus, electron transport chain and chlorophyll biosynthesis, induce cellular damage, impair cellular redox homeostasis, and finally cause cellular metabolic arrest (Anjum et al., 2010, 2012a; Gill and Tuteja, 2010; Talukdar, 2012; Talukdar and Talukdar, 2014). Saline soil conditions, on the other hand, can cause osmotic stress that in turn can inhibit cell expansion and cell division, impact stomatal closure, induce cell turgor via lowering water potential, and alter the normal homeostasis of cells (Miller et al., 2010). However, the generation of osmotic stress through impaired plant water relations, and oxidative stress caused by uncontrolled generation of varied reactive oxygen species (ROS; such as such as -OH, H2O2, O−2) are common in plants exposed to high levels of salinity and/or metals (Benavides et al., 2005; Anjum et al., 2010, 2012a). 
 
Diverse plant taxa have been reported to adapt metabolically to salinity and exposure to metals by enhancing synthesis of sulfur (S)-rich peptides (such as glutathione, GSH) and low-molecular-weight nitrogenous and proteogenic amino acids/osmolytes (such as proline, Pro) (Khan et al., 2009; Anjum et al., 2010, 2012a; Talukdar, 2012; Kishor and Sreenivasulu, 2014; Talukdar and Talukdar, 2014). Nevertheless, both GSH and Pro share L-glutamate as a common biosynthesis precursor (Moat et al., 2003) (Figure ​(Figure1).1). However, very little or no effort has been made so far to dissect the intricacies of potential metabolic interrelationships between the GSH and Pro induction either under salinity/osmotic or metal stress conditions. 
 
 
 
Figure 1 
 
Schematic representation of the points of interrelationships in the major metabolic pathway of sulfur-rich peptide—glutathione (GSH) and nitrogenous and proteogenic amino acid—proline (Pro). 
 
 
 
Therefore, we discuss and interpret through this note the facts related with the mainstays (chemistry, biosynthesis, compartmentalization, significance) commonly and potentially shared by these two enigmatic compounds (GSH and Pro) in plants. The outcome of the present endeavor can be useful in designing future research aimed at sustainably alleviating isolated and/or joint impact of metal and salinity stresses in crop plants through exploiting the GSH and Pro metabolism.


INTRODUCTION
Agricultural soils in the vicinity of extensive anthropogenic activities may exhibit salinity together with high levels of metals/metalloids (hereafter termed as "metal/s") as co-stressors. Elevated concentrations of metals (such as As, Cd, Cr, Hg, Ni, and Pb) may affect photosynthetic apparatus, electron transport chain and chlorophyll biosynthesis, induce cellular damage, impair cellular redox homeostasis, and finally cause cellular metabolic arrest (Anjum et al., 2010(Anjum et al., , 2012aGill and Tuteja, 2010;Talukdar, 2012;Talukdar and Talukdar, 2014). Saline soil conditions, on the other hand, can cause osmotic stress that in turn can inhibit cell expansion and cell division, impact stomatal closure, induce cell turgor via lowering water potential, and alter the normal homeostasis of cells (Miller et al., 2010). However, the generation of osmotic stress through impaired plant water relations, and oxidative stress caused by uncontrolled generation of varied reactive oxygen species (ROS; such as such as -OH, H 2 O 2 , O − 2 ) are common in plants exposed to high levels of salinity and/or metals (Benavides et al., 2005;Anjum et al., 2010Anjum et al., , 2012a. Diverse plant taxa have been reported to adapt metabolically to salinity and exposure to metals by enhancing synthesis of sulfur (S)-rich peptides (such as glutathione, GSH) and low-molecularweight nitrogenous and proteogenic amino acids/osmolytes (such as proline, Pro) (Khan et al., 2009;Anjum et al., 2010Anjum et al., , 2012aTalukdar, 2012;Kishor and Sreenivasulu, 2014;Talukdar and Talukdar, 2014). Nevertheless, both GSH and Pro share L-glutamate as a common biosynthesis precursor (Moat et al., 2003) (Figure 1). However, very little or no effort has been made so far to dissect the intricacies of potential metabolic interrelationships between the GSH and Pro induction either under salinity/osmotic or metal stress conditions. Therefore, we discuss and interpret through this note the facts related with the mainstays (chemistry, biosynthesis, compartmentalization, significance) commonly and potentially shared by these two enigmatic compounds (GSH and Pro) in plants. The outcome of the present endeavor can be useful in designing future research aimed at sustainably alleviating isolated and/or joint impact of metal and salinity stresses in crop plants through exploiting the GSH and Pro metabolism.
GSH and Pro may occur in the concentrations of few mM (2-3 mM) in various plant tissues (Noctor et al., 2002;Kishor et al., 2005). The GSH and Pro levels of plant tissues are indicators of the S (reduced) (Hubberten et al., 2012) and nitrogen (N) (Sánchez et al., 2001) nutritional status of the plant respectively. GSH and Pro have also been reported to act as sources of (reduced)-S (Anjum et al., 2010) and N (reviewed by Kishor and Sreenivasulu, 2014), respectively, under stress conditions. Additionally, their status may presumably be improved through enhancing L-glutamate level via N and S nutrition, respectively (Anjum et al., 2012b). Moreover, modulation of biosynthesis of GSH (Bartoli et al., 2009) and Pro (Abraham et al., 2003) is reportedly light dependent. In particular, GSH levels may depend on growth and photosynthetically active photon flux density at low light intensities (up to ca. 100 μmol m −2 s −1 ) (Ogawa et al., 2004). GSH (Son et al., 2014) and Pro (Sivakumar et al., 2001) can negatively/positively modulate the photosynthesis functions by influencing the activity of ribulose-1,5-bisphosphate oxygenase, an enzyme involved in the first major step of carbon fixation. Moreover, an increased intracellular ROS-availability can shift the reduced GSH toward a more oxidized GSH (i.e., GSSG) status (Anjum et al., 2010(Anjum et al., , 2012aNoctor et al., 2012). In contrast, increased status of cellular H 2 O 2 (or exogenous H 2 O 2 ) can increase Pro level by modulating the ex-novo synthesis of Pro (Matysik et al., 2002). Oxidation of Pro generates NADP/NADPH cycling or redox balance (Kishor et al., 2005) that in turn may regulate the reduction of GSSG to GSH via GSH reductase (Anjum et al., 2010(Anjum et al., , 2012aNoctor et al., 2012). Interaction of Pro (Iqbal et al., 2014) and GSH (Mhamdi et al., 2010;Ghanta et al., 2014) with a number of defenserelated phytohormones (such as ethylene, jasmonic acid and salicylic acid) and/or their analogs has also been reported to modulate plant stress tolerance.
A differential coordination of other components of ascorbate (AsA)-GSH pathway (enzymes such as ascorbate peroxidase, GSH reductase, GSH peroxidase, GSH sulfo-transferase, monodehydroascorbate reductase, dehydroascorbate reductase and catalase; and non-enzymes such as AsA) with GSH (Khan et al., 2009;Anjum et al., 2012aAnjum et al., , 2014Talukdar, 2012;Talukdar and Talukdar, 2014) and Pro (Omidi, 2010;Hossain et al., 2011;Anjum et al., 2014;Hasanuzzaman et al., 2014) was also reported to control plant tolerance to abiotic stress factors including the metal and salinity stress. Nevertheless, the status and responses of GSH and Pro together have been little explored in the same plant under similar stress conditions (Siripornadulsil et al., 2002;Hossain et al., 2011;Anjum et al., 2014;Hasanuzzaman et al., 2014). Notably, these studies helped to infer that there exists a close relation between GSH and Pro, and that exogenous and/or synthesized/ stress-caused elevated Pro can protect plants against the metal and salinity-stress impacts by safe-guarding the activity of previous enzymatic components, improving the cellular redox environment via decreasing H 2 O 2 level and maintaining an increased level of reduced GSH and GSSG/GSH ratio.
Though an increased cellular GSH status is indicative of a plant's capacity to tolerate different stress pressures (Khan et al., 2009;Anjum et al., 2010Anjum et al., , 2012aTalukdar, 2012;Noctor et al., 2012;Talukdar and Talukdar, 2014), it is debatable whether accumulation of Pro is a plant response to abiotic stresses or it is associated with stress tolerance (Sorkheh et al., 2012;Kishor and Sreenivasulu, 2014). Also, elevated GSH is not always correlated with enhanced tolerance to stresses such as metals (Xiang et al., 2001; reviewed by Anjum et al., 2012a). Despite previous facts, as versatile redox buffers, Pro (Kishor and Sreenivasulu, 2014) and GSH (Anjum et al., 2010(Anjum et al., , 2012aNoctor et al., 2012) have been extensively evidenced to protect cellular metabolism against a range of abiotic stresses.
The causal relationships of Pro accumulation and significance of GSH metabolism with enhanced tolerance to single stress factor (either metal or salinity) have been reported extensively in separate studies using natural variants, mutants or transgenic plants (Matysik et al., 2002;Anjum et al., 2010Anjum et al., , 2012aNoctor et al., 2012;Kishor and Sreenivasulu, 2014). However, significance of the potential "metabolic interrelationships" between GSH and Pro with reference to the plant's adaptive responses to prevailing multiple stressors has not been fully appreciated and the molecular insights of these relationships have yet to be developed.
Nevertheless, owing to the facts that: (a) deficiency of S and N has become extensive in agricultural soils on the globe (reviewed by Anjum et al., 2012b); (b) plant's S requirement and S metabolism are closely related to N nutrition, and the N metabolism is strongly affected by the plant's S status (Fazili et al., 2008;Anjum et al., 2012b); and (c) both GSH (Kopriva and Rennenberg, 2004;Anjum et al., 2012b) and Pro (Sánchez et al., 2001;Rais et al., 2013) are closely related to these nutrients, integrated efforts should be made to work-out the coordinated role of S and N in the GSH and Pro metabolic pathways, develop more insights into their biochemistry/physiology and molecular biology and understand potential interrelationships among different components of these pathways.