Proline metabolism influences the metabolic and/or signaling pathway in regulating plant stress responses. This study aimed to characterize the physiological significance of glutamate (Glu)-mediated proline metabolism in the drought stress responses, focusing on the hormonal regulatory pathway. The responses of cytosolic Ca²⁺ signaling, proline metabolism, and redox components to the exogenous application of Glu in well-watered or drought-stressed plants were interpreted in relation to endogenous hormone status and their signaling genes. Drought-enhanced level of abscisic acid (ABA) was concomitant with the accumulation of ROS and proline, as well as loss of reducing potential, which was assessed by measuring NAD(P)H/NAD(P)⁺ and GSH/GSSG ratios. Glu application to drought-stressed plants increased both salicylic acid (SA) and cytosolic Ca²⁺ levels, with the highest expression of calcium-dependent protein kinase (CPK5) and salicylic acid synthesis-related ICS1. The SA-enhanced CPK5 expression was closely associated with further enhancement of proline synthesis-related genes (P5CS1, P5CS2, and P5CR) expression and a reset of reducing potential with enhanced expression of redox regulating genes (TRXh5 and GRXC9) in a SA-mediated NPR1- and/or PR1-dependent manner. These results clearly indicate that Glu-activated interplay between SA- and CPK5-signaling as well as Glu-enhanced proline synthesis are crucial in the amelioration of drought stress in Brassica napus.

The antioxidant glutathione (GSH) mitigates adverse physio-metabolic effects and defends against abiotic types of stress, such as cadmium (Cd) stress. However, its function and role in resisting Cd phytotoxicity by leveraging plant antioxidant-scavenging, redox-regulating, and hormone-balancing systems have not been comprehensively and systematically demonstrated in the Cd-hyperaccumulating plant Brassica napus L. cv. Tammi (oilseed rape). In this study, the effects of exogenously applied GSH to the leaves of B. napus seedlings exposed to Cd (10 μM) were investigated. As a result, Cd stress alone significantly inhibited growth and increased the levels of reactive oxygen species (ROS) and the bioaccumulation of Cd in the seedlings compared with those in unstressed controls. Furthermore, Cd stress induced an imbalance in plant stress hormone levels and decreases in endogenous GSH levels and GSH redox ratios, which were correlated with reductions in ascorbate (AsA) and/or nicotinamide adenine dinucleotide phosphate (NADPH) redox states. However, the exogenous application of GSH to Cd-stressed B. napus seedlings reduced Cd-induced ROS levels and enhanced antioxidant-scavenging defenses and redox regulation by both increasing seedling AsA, GSH, and NADPH concentrations and rebalancing stress hormones, thereby enhancing Cd uptake and accumulation. These results demonstrate that GSH improved plant redox status by upregulating the AsA-GSH-NADPH cycle and reestablishing normal hormonal balance. This indicates that exogenously applied GSH can mitigate Cd phytotoxicity in B. napus and possibly other plants. Therefore, GSH can potentially be applied to Cd-polluted soil for plant remediation.

The aim of this study was to characterize hormonal crosstalk with the sugar signaling and metabolic pathway based on a time course analysis of drought intensity. Drought intensity-responsive changes in the assimilation of newly fixed carbon (C) into soluble sugar, the content of sugar and starch, and expression of genes involved in carbohydrate metabolism were interpreted as being linked to endogenous abscisic acid (ABA) and salicylic acid (SA) levels and their signaling genes. The ABA and SA levels in the drought-stressed leaves increased together during the early drought period (days 0–6), and additional ABA accumulation occurred with depressed SA during the late period (days 6–14). Although drought treatment decreased the assimilation of newly fixed C into soluble sugar, representing a 59.9%, 33.1%, and 62.9% reduction in ¹³C-glucose, ¹³C-fructose, and ¹³C-sucrose on day 14, respectively, the drought-responsive accumulation of soluble sugars was significant. During the early period, the drought-responsive accumulation of hexose and sucrose was concurrent with the upregulated expression of hexokinase 1 (HXK1), which, in turn, occurred parallel to the upregulation of ABA synthesis gene 9-sis-epoxycarotenoid dioxygenase (NCED3) and SA-related genes (isochorismate synthase 1 (ICS1) and non-expressor of pathogenesis-related gene (NPR1)). During the late period, hexose accumulation, sucrose phloem loading, and starch degradation were dominant, with a highly enhanced expression of the starch degradation-related genes β-amylase 1 (BAM1) and α-amylase 3 (AMY3), which were concomitant with the parallel enhancement of sucrose non-fermenting−1 (Snf1)-related protein kinase 2 (SnRK2).2 and ABA-responsive element binding 2 (AREB2) expression in an ABA-dependent manner. These results indicate that the drought-responsive accumulation of sugars (especially SA-mediated sucrose accumulation) is part of the acclamatory process during the early period. Conversely, ABA-responsive hexose accumulation and sucrose phloem loading represent severe drought symptoms during the late drought period.

The present study aimed to assess the nitrogen (N) use efficiency of acidified pig slurry for regrowth yield and its environmental impacts on perennial ryegrass swards. The pH of digested pig slurry was adjusted to 5.0 or 7.0 by the addition of sulfuric acid and untreated as a control. The pig slurry urea of each treatment was labeled with 15N urea and applied at a rate of 200 kg N/ha immediately after cutting. Soil and herbage samples were collected at 7, 14, and 56 d of regrowth. The flux of pig slurry-N to regrowth yield and soil N mineralization were analyzed, and N losses via NH3, N2O emission and NO3- leaching were also estimated. The pH level of the applied slurry did not have a significant effect on herbage yield or N content of herbage at the end of regrowth, whereas the amount of N derived from pig slurry urea (NdfSU) was higher in both herbage and soils in pH-controlled plots. The NH4+-N content and the amount of N derived from slurry urea into soil NH4+ fraction (NdfSU-NH4+) was significantly higher in in the pH 5 plot, whereas NO3- and NdfSU-NO3- were lower than in control plots over the entire regrowth period. Nitrification of NH4+-N was delayed in soil amended with acidified slurry. Compared to non-pH-controlled pig slurry (i.e. control plots), application of acidified slurry reduced NH3 emissions by 78.1%, N2O emissions by 78.9% and NO3- leaching by 17.81% over the course of the experiment. Our results suggest that pig slurry acidification may represent an effective means of minimizing hazardous environmental impacts without depressing regrowth yield.

Drought alters the level of endogenous reactive oxygen species (ROS) and hormonal status, which are both involved in the regulation of stress responses. To investigate the interplay between ROS and hormones in proline metabolism, rapeseed (Brassica napus L.) plants were exposed to drought or exogenous H₂O₂ (Exo-H₂O₂) treatment for 10 days. During the first 5 days, the enhanced H₂O₂ concentrations in drought treatment were associated with the activation of superoxide dismutase (SOD) and NADPH oxidase, with enhanced ABA and SA levels, while that in Exo-H₂O₂ treatment was mainly associated with SA-responsive POX. During the latter 5 days, ABA-dependent ROS accumulation was predominant with an upregulated oxidative signal-inducible gene (OXI1) and MAPK6, leading to the activation of ABA synthesis and the signaling genes (NCED3 and MYC2). During the first 5 days, the enhanced levels of P5C and proline were concomitant with SA-dependent NDR1-mediated signaling in both drought and Exo-H₂O₂ treatments. In the latter 5 days of drought treatment, a distinct enhancement in P5CR and ProDH expression led to higher proline accumulation compared to Exo-H₂O₂ treatment. These results indicate that SA-mediated P5C synthesis is highly activated under lower endogenous H₂O₂ levels, and ABA-mediated OXI1-dependent proline accumulation mainly occurs with an increasing ROS level, leading to ProDH activation as a hypersensitive response to ROS and proline overproduction under severe stress.

The leaf senescence process is characterized by the degradation of macromolecules in mature leaves and the remobilization of degradation products via phloem transport. The phytohormone ethylene mediates leaf senescence. This study aimed to investigate the ethephon-induced ethylene effects on starch degradation and sucrose remobilization through their interactive regulation with other hormones. Ethephon (2-chloroethylphosphonic acid) was used as an ethylene-generating agent. Endogenous hormonal status, carbohydrate compounds, starch degradation-related gene expression, sucrose transporter gene expression, and phloem sucrose loading were compared between the ethephon-treated plants and controls. Foliar ethephon spray enhanced the endogenous ethylene concentration and accelerated leaf senescence, as evidenced by reduced chlorophyll content and enhanced expression of the senescence-related gene SAG12. Ethephon-enhanced ethylene prominently enhanced the endogenous abscisic acid (ABA) level. accompanied with upregulation of ABA synthesis gene 9-cis-epoxycarotenoid dioxygenase (NCED3), ABA receptor gene pyrabactin resistance 1 (PYR1), and ABA signaling genes sucrose non-fermenting 1 (Snf1)-related protein kinase 2 (SnRK2), ABA-responsive element binding 2 (AREB2), and basic-helix-loop-helix (bHLH) transcription factor (MYC2).) Ethephon treatment decreased starch content by enhancing expression of the starch degradation-related genes α-amylase 3 (AMY3) and β-amylase 1 (BAM1), resulting in an increase in sucrose content in phloem exudates with enhanced expression of sucrose transporters, SUT1, SUT4, and SWEET11. These results suggest that a synergistic interaction between ethylene and ABA might account for sucrose accumulation, mainly due to starch degradation in mature leaves and sucrose phloem loading in the ethephon-induced senescent leaves.