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 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.

To characterize cultivar variation in resistance gene (R-gene)-mediated calcium signaling and hormonal regulation in effector-triggered immunity (ETI) and disease susceptibility, Xanthomonas campestris pv. campestris (Xcc) was inoculated in two Brassica napus cultivars (cvs. Capitol and Mosa). At 14 days post inoculation (DPI) with Xcc, there was a necrotic lesion in cv. Mosa along with the significant accumulation of H₂O₂ and malondialdehyde (MDA), whereas no visual symptom was observed in cv. Capitol. The cultivar variations in the R-gene expressions were found in response to Xcc. ZAR1 is a coiled-coil-nucleotide binding site-leucine-rich repeat (CC-NB-LRR)-type R-gene that is significantly induced in cv. Capitol, whereas toll/interleukin-1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NB-LRR)-type R-gene, TAO1, is significantly upregulated in cv. Mosa Xcc-inoculated plants. The defense-related gene’s non-race-specific disease resistance 1 (NDR1) and mitogen-activated protein kinase 6 (MAPK6) were enhanced, whereas calcium-dependent protein kinase (CDPK5) and calcium-sensing protein 60g (CBP60g) were depressed in cv. Capitol Xcc inoculated plants, and opposite results were found in cv. Mosa. The calcium-sensing receptor (CAS), calmodulin (CaM), expression was induced in both the cultivars. However, the CAS induction rate was much higher in cv. Mosa than in cv. Capitol in response to Xcc. The phytohormone salicylic acid (SA) and jasmonic acid (JA) levels were significantly higher in cv. Capitol along with the enhanced SA receptors (NPR3 and NPR4) and JA synthesis and signaling-related gene expression (LOX2, PDF1.2), whereas the JA level was significantly lower in cv. Mosa Xcc inoculated plants. The SA synthesis and signaling-related genes (ICS1, NPR1) and SA were present at higher levels in cv. Mosa; additionally, the SA level present was much higher in the susceptible cultivar (cv. Mosa) than in the resistant cultivar (cv. Capitol) in response to Xcc. These results indicate that ZAR1 mediated the coordinated action of SA and JA synthesis and signaling to confirm ETI, whereas TAO1 enhanced the synthesis of SA through CAS and CBP60g to antagonize JA synthesis and signaling to cause disease susceptibility in the Brassica napus–Xcc pathosystem.