Salicylic Acid Acts Upstream of Hydrogen Sulfide inAlleviating LowTemperature Combined with Low Light Stress inPepper Seedlings: Evidence from a Single Stress Endpoint

Low temperature combined with low light stress is a major environmental constraint limiting the yield and quality of greenhouse-grown pepper. Although Salicylic acid (SA), and hydrogen sulfide (H 2 S) have been implicated in pepper stress responses, their upstream/downstream hierarchy and the physiological mechanisms through which they interact under low temperature combined with low light stress remain to be directly defined. In particular, whether SA acts upstream of H 2 S and whether SA-mediated protection depends on endogenous H 2 S are not yet resolved. Seedlings of pepper ( Capsicum annuum L.) cultivar ‘Hangjiao No. 2’ were treated with exogenous SA, the H 2 S donor sodium hydrosulfide (NaHS), the SA biosynthesis inhibitor 2-aminoindan-2-phosphonic acid (AIP), and the H 2 S scavenger hypotaurine (HT) to systematically examine the signal hierarchy between SA and H 2 S under low temperature combined with low light stress conditions as well as their regulatory effects on photosynthetic characteristics, antioxidant activities, osmotic adjustments, membrane integrity, and endogenous hormone homeostasis. This design directly tests the SA–H 2 S hierarchy rather than only assessing the effect of an H 2 S donor. The study results showed that the combined stress simultaneously activated SA and H 2 S biosynthesis. Exogenous SA further promoted H 2 S accumulation and upregulated the enzyme protein content of L-cysteine desulfhydrase (LCD), a key enzyme in H 2 S biosynthesis, whereas NaHS did not affect the SA content or the enzyme protein content of isochorismate synthase (ICS), a key enzyme in SA biosynthesis, while AIP suppressed H 2 S production. Accordingly, SA positively regulates H 2 S generation by activating LCD. Exogenously applied SA and NaHS alleviated the stress-induced degradation of photosynthetic pigments, decrease in photosynthetic rates, membrane lipid peroxidation, reactive oxygen species (ROS) accumulation, and hormone imbalance, while restoring antioxidant enzyme activities and the redox homeostasis of the ascorbate–glutathione cycle. When endogenous H 2 S was scavenged by HT, the ameliorative effects of SA on photosynthesis, membrane stability, ROS scavenging, and hormone homeostasis were almost abolished. By contrast, when SA synthesis was inhibited by AIP, exogenous H 2 S still partially maintained stress tolerance-related functions. In summary, under the single stress endpoint examined in this study (day 7), SA is positioned upstream of H 2 S: SA promotes endogenous H 2 S production, and H 2 S is required for several SA-mediated protective effects. H 2 S, as an indispensable downstream signaling molecule, contributes to photosynthetic protection, osmotic adjustments, membrane stability, antioxidant defense, and hormone network remodeling. These findings support a model in which the SA–H 2 S signaling cascade acts as a central regulatory pathway in the pepper response to low temperature combined with low light stress. However, because all measurements were performed at a single time point, the temporal order of SA and H 2 S signaling remains to be established by time-course experiments.