Nitric oxide-induced synthesis of hydrogen sulfide alleviates osmotic stress in wheat seedlings through sustaining antioxidant enzymes, osmolyte accumulation and cysteine homeostasis

Highlights

• Nitric oxide (NO) and hydrogen sulfide (H₂S) induce tolerance to osmotic stress.

• NO induces activities of H₂S and Cys -synthesizing enzymes.

• NO maintains H₂S level and Cys homeostasis under osmotic stress.

• Together with H₂S, NO activates the defense system of plants against osmotic stress.

Abstract

Nitric oxide (NO) and hydrogen sulfide (H₂S) have been shown to act as signaling molecules in various physiological processes, play significant roles in plant cellular processes, and also mediate responses to both biotic and abiotic stresses in plants. The present investigation was carried out to test the effect of exogenous NO on endogenous synthesis of H₂S in osmotic-stressed wheat (Triticum aestivum L.) seedlings. The results show that application of NO to wheat seedlings, suffered from PEG8000-induced osmotic stress, considerably enhanced the activities of H₂S-synthesizing enzymes l-cysteine desulfhydrase (LCD) and d-cysteine desulfhydrase (DCD) leading to enhanced level of endogenous H₂S content. At the same time exogenous NO also enhanced the activity of cysteine (Cys)-synthesizing enzyme O-acetylserine(thiol)lyase (OAS-TL) and maintained Cys homeostasis under osmotic stress. NO and H₂S together markedly improved the activities of antioxidant enzymes viz. ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT). Furthermore, NO and H₂S caused additional accumulation of osmolytes proline (Pro) and glycine betaine (GB), all these collectively resulted in the protection of plants against osmotic stress-induced oxidative stress. On the other hand, NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] and H₂S scavenger HT (hypotaurine) invalidated the effect of NO on endogenous H₂S levels and Cys homeostasis which resulted in weak protection against osmotic stress. Application of N-ethylmaleimide (NEM) suppressed GR activity and caused an increase in oxidative stress. We concluded that NO in association with endogenous H₂S activates the defense system to the level required to counter osmotic stress and maintains normal functioning of cellular machinery.

Introduction

The existence of hydrogen sulfide (H₂S), a colourless gas with a characteristic unpleasant smell of rotten egg, has been substantiated for its toxicity with fatal effects and root cause for life destructions and extinctions on the planet earth over millions of years [1], [2]. However, in recent years H₂S has emerged as a third gaseous transmitter after nitric oxide (NO) and carbon monoxide [3], [4]. Strong evidence of H₂S emission from plants was reported by Wilson et al. [5], when they quantified H₂S from leaves of cucumber, pumpkin, cantaloupe, corn, soybean and cotton. Rennenberg [6] further confirmed the release of H₂S by higher plants. In plants, H₂S is considered to be a by-product of cysteine (Cys) degradation. The enzymes l-cysteine desulfhydrase (LCD; EC 4.4.1.1) and d-cysteine desulfhydrase (DCD; EC 4.4.1.15) desulfurylate L-Cys and D-Cys, respectively into H₂S, ammonia, and pyruvate [7], [8]. However, O-acetylserine(thiol)lyase (OAS-TL; EC 2.5.1.47), a cysteine synthase-like protein synthesizes Cys from O-acetylserine (OAS) and sulfide (Fig. 6). Thus, Cys is the key sulfur containing precursor of various biomolecules including antioxidants and other defense compounds [9], [10].

Information on the role of H₂S as signaling molecule in plants is limited compared to those in animals. However, several studies showed that H₂S plays crucial role in a plethora of plant cellular processes including seed germination, root morphogenesis, photosynthesis, flower senescence and guard cell signaling [11], [12], [13], [14]. Exogenous application of sodium hydrosulfide (NaHS), an H₂S donor, has been shown to mitigate adverse effects of various abiotic stresses [14], [15], [16], [17], [18], [19], functions as a potent antioxidant [20] and signaling molecule [21], [22]. Since, Cys is the source molecule of H₂S and other defense molecules, therefore maintenance of Cys homeostasis is vital for normal functioning of cellular system under stressful conditions.

Synthesis of NO in plants is mainly carried out by nitrate reductase (NR)-catalyzed reduction of nitrite (NO₂⁻) and _l-arginine-dependent nitric oxide synthase-like activity [23], [24], [25], [26], [27]. NO has been shown to regulate several functions including seed germination, adventitious root formation, flowering, stomatal closure and senescence [28], [29] and exhibit antioxidant and cytoprotective effects with DNA, lipids, proteins and chlorophyll [30], [31]. In addition, NO mediates responses to both biotic and abiotic stresses in plants [32], [33], acts as a second messenger in signaling cascades involving cytosolic Ca²⁺, cyclic adenosine diphosphate ribose (cADPR), salicylic, jasmonic and abscisic acids, hydrogen peroxide (H₂O₂), cyclic guanosine 5′-monophosphate (cGMP) and mitogen activated protein kinase (MAPK) [34], [35], [36], [37], [38]. A significant number of studies show that exogenous application of NO elevates activities of antioxidant enzymes [31], [39], [40], [41] and gives protection to the plants against various abiotic stresses [14], [31], [32], [42].

Manifestation of osmotic stress is one of the primary effects of several abiotic stresses including drought. Osmotic stress affects various physiological processes and causes stomatal closure, turgor loss, reduced photosynthetic activity and suppressed carbon assimilation [43], [44]. In plants, stressful conditions create an imbalance between production and scavenging of reactive oxygen species (ROS) that results in over production of ROS. Excessive accumulation of ROS creates oxidative stress and results in the oxidation of lipids, proteins and nucleic acids [44], [45] leading to reduced carbon assimilation and crop yield. Being sessile in nature, plants have no choice to escape stress-induced detrimental effects, the character which makes plants a unique example with the inherent quality of not to quit under any stressful conditions. Plants resist osmotic stress by accumulating osmolytes such as proline (Pro) and glycinebetaine (GB). These osmolytes stabilize the quaternary structure of proteins, maintain membrane stability and protect plants against various abiotic stresses through osmotic adjustment [46], [47], [48], [49]. To cope with the detrimental effects of oxidative stress plants are equipped with a defense system, orchestrated by various antioxidant enzymes such as ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT). Timely and precise activation of these defense systems prior to the onset of damage is of prime importance for the endurance of plants under stressful conditions. Therefore, precise response to stress stimulus is vital and it is transmitted by a network of signaling molecules.

Although, in recent years the role of NO and H₂S in abiotic stress tolerance of plants gained much attention, little or vague information is available on the effect of exogenous NO on endogenous synthesis of H₂S and Cys homeostasis under osmotic stress. Considering the important roles of NO and H₂S in plants, the present work was planned to test the effect of NO on the synthesis of H₂S and maintenance of Cys homeostasis and their role in the tolerance of wheat to osmotic stress.