**4. Discussion**

Soil contamination with chromium (Cr) has become a serious concern for rice researchers due to its direct entry into food chains and severe health issues globally [48]. Thus, our prime focus was to investigate latent approaches to diminish Cr-accumulation and its associated phytotoxicity. For this purpose, we utilized seed priming with spermine (SPM) in minimizing the Cr-tempted toxic effects by targeting the seed germination indices, growth and biomass, photosynthetic apparatus, soluble protein levels, nutrients balance, oxidative damages, antioxidant enzymes' defense systems, phytohormone production and modifications in cellular ultrastructure. Our findings showed that the seed germination parameters, i.e., GE, GP, VI, GI and MGT, were significantly reduced by Cr exposure in both rice cultivars (Table 1) which ultimately minimized the plant development, biomass (fresh and dry weight, shoot or root lengths) (Table 1, Figure S1). Comparatively, seed primed SPM caused readjustments of seed germination indices, plant growth and biomass attributes under Cr toxicity (Tables 1 and S2; Figure S1). These alterations were more substantial in YLY689 (tolerant variety) than CY927 (sensitive variety). Recent studies revealed that Cr-stress inhibited the rates of seed germination indices [1]. This may be due to the alteration in seed coat structure and reactions with ions, electrons or intact radicals. Consequently, sugar supply to the seeds was compromised [49,50]; although SPM applications aided the conversion of starch hydrolysis into soluble sugar that improved the respiration of seeds and starch metabolism, and consequently enhanced plant growth and biomass production [51].

Furthermore, Cr also inhibited the biomass of rice seedling [1,11], maize [22], and *Brassica napus* [7] seedlings that might be associated with a disturbance of photosynthetic systems and nutrients imbalance. As predicted, Cr stress significantly reduced the chlorophyll pigments (Figure 1a–d), *Fv*/*Fm* and *Fm* values (Figure 1e–h), gas exchange parameters (Table S2) and nutrient accumulation (Table S3) in both rice varieties, especially CY927. The possible reason is that Cr caused inhibition inside the production of chlorophyll biosynthetic enzymes (δ-amino-levulinic acid dehydratase and protochlorophyllide reductase), which led to the impairment of chlorophyll biosynthetic pathways [52], while SPM priming mitigated Cr stress on both cultivars, and precisely in YLY689. Potentially, seed primed SPM boosted the plant growth traits (Table 1) by eliminating Cr accumulation via maintaining the nutrient uptake balance (Table S3), cell cycle and cell division, which led to the improvement in chlorophyll pigments (Figure 1a–d) and gas exchange indices (Table S2) under Cr exposure. Most probably, SPM application enhanced the levels of photosynthetic apparatus by regulating the nutrient uptake which might have modulated the expression of genes associated with metal homeostasis, lessening the Cr accretion under heavy metal stress [53,54]. It has been reported that photosynthetic efficiency maintains the level of total soluble sugar (TSS) and total soluble protein (TTP) that might be stimulating the metabolites' induction, and further helps to detoxify the Cr stress under Cr-induced toxicity [55,56]. Our upshots specified that the TSS and TSP are directly associated with the photosynthetic contents (Figure 2b,c) and the rates of gas exchange indices (Table S2). Seed priming with SPM might have improved the photosynthetic and biosynthetic enzyme activities (i.e., δ-aminolaevulinic acid and protochlorophyllide reductase) that regulated the photosynthetic biosynthesis pathways [57] and subsequently improved the TSS and TSP. Our results revealed that seed primed SPM minimized the Cr-indulged oxidative damages and minimized the electrolyte leakage.

In our current investigations, Cr exposure enhanced the extra accumulation of H2O2 and O2 •− (Figure 2f–i), MDA contents (Figure 2d,e), electrolyte leakage (Figure 2a), particularly in CY927, and caused oxidative or membrane damages and lipid peroxidation as noticed in rice seedlings [57,58], Indian mustard [58] and tomato [59]. Seeds primed with SPM may have improved the ROS hemostasis by decreasing over-production of ROS (Figure 2d–i) that revealed the role of SPM in mitigating oxidative damages and subsequently improving photosynthetic performance. Probably, SPM improved the catabolism, maintained the cellular metabolism, and inhibited the overproduction of free radicals which

controlled the level of MDA contents, electrolyte leakage, and ROS over-generation. Additionally, seed primed SPM treatment provided stability to cellular membranes by reducing the MDA contents (Figure 2d,e) and electrolyte leakage (Figure 2a) in rice seedlings under Cr-induced stress, thus enhancing the photosynthetic apparatus, plant growth and biomass production, specifically in YLY689. SPM capability in reducing ROS (H2O2 and O2 •−) over production was verified by histochemical straining, which matches recent observations under conditions of metal stress [21,60].

To cope with the Cr-triggered oxidative damage, plants stimulate their antioxidative defense mechanisms as indicated by the upregulation in enzymatic activities such as SOD, APX, POD and CAT in both rice cultivars (Figure 4a–h). Similar outcomes were documented in a recent investigation that plants increase their antioxidative defense mechanism to scavenge Cr-induced cellular oxidative damage [1]. Seed priming with SPM further stimulated the antioxidative enzyme activities and reduced the ROS or MDA accumulation, which revealed that SPM boosted the antioxidants' defense mechanisms and reduced the oxidative damage, thus maintaining ROS homeostasis in rice seedlings. Previous studies documented that PAs regulate antioxidant activities to eliminate the ROS extra generation, and hence maintain the redox balance, and thus improve the growth traits of wheat [21], *Brassica juncea* [61], and mung bean [20] under heavy metals stress. Expectedly, the sole treatment of Cr significantly restricted the accumulation of nutrients (Na, K, P, Ca, Mn, Zn, Cu and Fe) which eventually leads to lowered biomass production. The excessive Cr accumulation in plant parts may limit the accessibility of nutrients and disturb their hemostasis [1,7]. SPM applications displayed the capably to improve the balance of nutrients by lowering the Cr accretion via a vacuole sequestering approach and enhanced the tolerance capacity of YLY689 against Cr stress than CY927. Likewise, earlier investigations supported our results that the reduction in Cr-facilitation can improve the nutrients' bioavailability within the tissues of *Raphanus sativus* [62] and maize [22].

It has been reported that phytohormones, viz., SA and JA interactions with PAs including SPM and spermidine (SPD), modulated plant physiological attributes, and enhanced antioxidant enzyme activities in scavenging the extra accumulation of ROS under adverse environmental stresses [63–66]. Under heavy metal stress conditions, SA levels were enhanced inside plant parts that helped to reduce the extra accumulation of ROS [67,68]. Remarkably, seeds primed with SPM significantly improved the endogenous levels of SA rather than JA under Cr toxicity in both rice varieties, especially in YLY689 as compared to CY927 (Figure 5b,c). Hereafter, we targeted the gene expression of SA-related genes (*PR1*, *PR2*, and *NPR1*) to analyze the genetic responses under Cr stress (Figure 5d–f). We verified that Cr significantly stimulated the endogenous SA levels that clarified the potential roles of SA in alleviating Cr toxicity in rice seedlings. Nevertheless, Cr displayed no significant differences in the levels of endogenous JA (Figure 5b). This might be due to the fact that Cr stress, in addition to the consequent synthesis of JA, stimulated the lipases to deliver a substrate for lipoxygenase [33,69]. Still, the molecular basis of this phenomenon needed thorough exploration.

Notably, Cr-alone treatments enhanced the endogenous abscisic acid (ABA) levels, while seed priming with SPM significantly lowered them (Figure 5a). Conceivably, PAs may regulate the ABA levels to modulate the signal transduction pathways, and specific protein's activation and repression, which supplementarily controls the cell growth or cell death [70]. Therefore, PAs interact with phytohormones to maintain the plant's growth attributes, stomatal conductance, and chlorophyll contents to alleviate the nastiest effects of multiple toxicities [66].

The ultrastructural analysis revealed that Cr alone treatments severely damaged the cellular organelles (Figure 6A–H) and CY927 demonstrated more damaging effects than YLY689. The extra accumulation of Cr within cell organs ruptured the chloroplast structure by disturbing the granule thylakoid development, swollen mitochondria and damages to thylakoid membrane, as supported by earlier studies [71]. The Cr-induced chloroplast damage led to the reduction in chlorophyll contents which halted the photosynthetic rate and gas exchange parameters [71]. Conversely, seed primed SPM applications reestablished the structures of granule thylakoids, stroma, chloroplasts, thylakoid membrane, cell wall, vacuoles and mitochondria under Cr stress, specifically in YLY689 (Figure 6d,h). Possibly, SPM repaired the cellular ultrastructure (chloroplast and thylakoids) that helps to reduce the overproduction of ROS and improve the photosynthetic apparatus, thus leading to higher plant development and biomass against heavy metals' exposure [72].
