*4.9. Correlation of Various Biochemical Parameters*

Pearson's correlation analysis was used to investigate the association between the physiological and biochemical characteristics of *C*. *capsularis* and *C*. *olitorius* under conditions of cold stress (Figure 8). Although most physiological and biochemical markers tested correlated significantly, some indices were more tightly related than others. Different physiological traits like photosynthetic pigment content (total chlorophyll) were observed to be positively correlated with different enzymatic and non-enzymatic antioxidants but negatively associated with reactive oxygen species (like H2O2 and MDA contents). At the same time, a strong positive significant positive correlation was observed between MDA and H2O2. It was observed that, in most cases, MDA and H2O2 made a high negative correlation with other enzymatic and non-enzymatic antioxidants. This high negative correlation suggests lipid peroxidation caused by cold stress was the main reason for decreasing nonenzymatic antioxidant activity and the occurrence of severe damage to jute varieties. In comparison, other enzymatic antioxidants such as SOD, POD, CAT, APx, and GSH exhibit a substantial positive connection with non-enzymatic antioxidants and physiological indices. Pearson correlation coefficient data supported the cluster analysis conclusion.

**Figure 8.** Pearson's correlation analysis of different physiological and biochemical traits under low-temperature stress.

#### **5. Discussion**

Like other tropical and subtropical plants, jute seedlings could be susceptible to chilling temperatures, and the injuries are results of their susceptibility. This circumstance shows dramatic reductions in the rates of many physiological characteristics when they are under chilling stress. Chlorophyll is a necessary and vital biomolecule in photosynthesis, serving as an absorber of light and a converter of light energy [30]. When plants are subjected to low-temperature stress, chlorophyll biosynthesis is impaired, resulting in a decrease in light harvesting [31]. It has been reported that plants with a high tolerance for cold keep a constant chlorophyll content, while plants with low cold tolerance experience a decrease in chlorophyll content [28]. The present study reported that except M33, all varieties significantly reduced the chlorophyll content. This may be due to a cold-induced increase in the activity of the chlorophyll degrading enzyme, chlorophyllase reported by Noreen, 2009 [32]. It was also observed that varieties Y49 showed stable or less decreased, whereas Y38 and T8 had a high level of changes compared to control. This indicates that leaf chlorophyll content was better protected in Y49 and M33 varieties, probably because of the high antioxidant enzyme activities.

Our study observed that varieties Y49 and M33 showed a less declining trend at chilling than other varieties compared to their control condition. In our research, nearly stable and small changes in chlorophyll and carotenoid contents in Y49 and M33 are consistent with *B*. *oleracea varacephalais* a plant with good cold tolerance reported by Atici et al. [33]. Like this, high chlorophyll stability index (CSI) under chilling stress in Y49 and M33 varieties are considered superior among the varieties studied. Due to their genetic heterogeneity and the difference in the method of defense, the examined varieties in our study behaved differently than at the same low temperature.

In response to cold stress, total chlorophyll, stable, or low decrease was noticed in Y49 (1.17%) and M33 (14.08%) while Y38 and T8 recorded the highest change as decreased. The lower fall in carotenoid concentration in response to cold stress compared to in chlorophyll *a* or *b* content may reflect the activity of xanthophyll cycle carotenoids in releasing thermal energy and protecting PSII reaction centers. It has been suggested that a decrease in chlorophyll biosynthesis in plants exposed to cold temperatures is partially due to the reduction of 5-aminolevulinic acid biosynthesis [34]. Reduced photosynthetic pigments diminish light absorption, and the greater decrease in carotenoid content than in chlorophyll *a* or *b* content in Y49 and M33 may be related to the release of heat energy and protection of PSII reaction centers by xanthophyll cycle carotenoids [35,36]. Numerous studies have established that low-temperature stress impairs photosynthesis, as evidenced by decreases in photosynthetic rate and pigment concentrations [37]

Proline is a well-established suitable osmolyte required to maintain osmotic balance and stabilize cellular structures in plants under a variety of abiotic conditions [38]. Plants protect their tissues from low-temperature damage by accumulating proline in the cells leading to a better osmotic adjustment by eliminating stress-induced excess H<sup>+</sup> and protecting enzymes from denaturation [39]. It was believed that more significant proline accumulation during stress conditions might account for a portion of plants' increased tolerance to cold stress conditions by mitigating the ROS-induced oxidative damage [40]. In this experiment, results showed that low-temperature stress significantly increased proline content where a high increasing trend was observed in Y49 and M33 by 67.84% and 60%, indicating that these two varieties improve the cold tolerance by scavenging ROS produced under stress condition. Whereas lower increase was observed in Y38 (11.39%) and T8 (7.89%), which reflected that these two have less radical scavenger activity. Moreover, in support of the outcomes of the present study, several other scholars reported that tolerant varieties accumulated a higher proline content than sensitive varieties during the low-temperature treatment period in seedling of sugarcane [41] and grafted watermelon [42].

It has been proved that soluble sugars play a critical role in the process of cold tolerance. Soluble sugars protect plant cells from cold stress-induced damage in a variety of ways, including as osmoprotectants, nutrition, and by reacting with the lipid bilayer [43]. In our study, varieties Y49 and M33 recorded high soluble sugar accumulation, where other varieties displayed a decreasing trend compared to the control. The highest reduction was displayed in Y1 (31.84%), followed by T8 and Y38 with 30.94% and 32.84 % respectively. Thus, more accumulated varieties attained low-temperature tolerance by increasing membrane cryostability. Increased membrane cryostability is required for freezing tolerance since membrane instability is the main source of plant injury [44]. Considerable research indicated that cold-tolerant varieties accumulated higher soluble sugar, especially sucrose, which decreased significantly in the susceptible varieties [45,46].

It has been reported that cold stress increased uncontrolled ROS production, resulting in lipid peroxidation, protein degradation, DNA degradation, and mutation. Lastly, it affected cellular metabolism and physiology, impairing the plant's membrane stability. Normally, the breakdown of unsaturated fatty acids produced MDA as the primary product in biological membranes. Likewise, greater H2O2 accumulation in many cell compartments, including chloroplasts, mitochondria, and apoplastic space, relates to oxidative damage in plants under cold stress [47]. Both MDA and H2O2 are useful indicators for detecting and monitoring oxidative stress in plants [48,49]. In the current study's treatment groups, lipid peroxidation was enhanced and triggered cell-produced MDA accumulation. It has been shown that cold stress increases lipid peroxidation and H2O2 concentrations, but lesser accumulation observed in tolerant varieties suggests protection against oxidative damage through a better regulating mechanism to control the synthesis of more MDA and H2O2 [47]. Our research showed that at low-temperature stress, MDA levels and H2O2 increased more pronouncedly in the Y1, Y38, and T8 varieties than other varieties compared to control, indicating that the damage caused by cold injury in these varieties was more severe than others. Among the tested varieties, less augmented MDA levels and H2O2 were noticed in Y49 and M33 varieties, indicating a greater ROS scavenging mechanism effectiveness and higher tolerance to cold stress. The current research findings were similar to those previously reported on sugarcane seedlings; tolerant seedlings showed the lower MDA content while susceptible seedlings demonstrated higher MDA levels [41]. It also noted that MDA content was lower in the treatment groups while proline content was more elevated, and other radical scavenging enzymes were observed [50]. The enzymatic antioxidant system may prevent the degradation of polyunsaturated fatty acids. Thus, increased proline under cold stress helps adjust osmotic levels by reducing the MDA content and improving the cell membrane.

Due to the increased electron leakage to molecular oxygen, unfavorable conditions promote the generation of reactive oxygen species (ROS) such as H2O2 (hydrogen peroxide), O2 <sup>−</sup> (superoxide), and OH<sup>−</sup> (hydroxyl) radicals (Arora et al., 2002). An increase in ROS accumulation under abiotic stress parallels increased lipid peroxidation. To mitigate H2O2 induced oxidative damage and lipid peroxidation caused by the accumulation of MDA, plants increase their defense mechanisms against ROS by enhancing the ability of variety components both enzymatic and non-enzymatic to detoxify ROS [51]. In our study, a significant increase of SOD, CAT, POD, and APx were observed in the varieties of Y49 and T8 compared to other varieties and control conditions. It has been discovered that increasing enzyme activity results in a decrease in MDA and H2O2 concentrations below those found in control plants [48]. SOD and CAT serve as the initial line of defense for plants' antioxidative machinery. They prevent the production of more hazardous reactive oxygen species (ROS) and play a vital role in intracellular H2O2 signaling [52]. In the initial stage, SOD catalyzes the dismutation of O2 − to H2O2 and O2 molecules. Hydrogen peroxide is less toxic than superoxide radicals. On the other hand, POD enzymes catalyze the conversion of H2O2 to H2O and O2. Then, H2O2 is detoxified by APx, POD, and CAT in different organelles and antioxidant cycles [51]. CAT is an antioxidant enzyme with a high capacity for rapidly H2O2 scavenging and is more engaged in H2O2 detoxification (removes H2O2 by breaking it down to create H2O and oxygen and oxidizes H+ donors via peroxide consumption), which is required for cold stress tolerance [53,54]. In the current investigation, varieties (Y49 and M33) with a relatively high CAT activity accumulated less H2O2 and MDA than varieties with a relatively low CAT activity and vice versa. In comparison, following 24 h of low-temperature treatment at 5 ◦C, the activity of CAT increased up to 2.3-fold in cold-treated leaves compared to control leaves. Similar results were also obtained from different studies; Zhang et al. stated that cold-tolerant banana varieties demonstrated a significant increase in CAT activity under cold stress [55] and winter-type wheat revealed much higher CAT activity than the spring type soybean plants when exposed to cold [56].

GSH can be produced in both the cytosol and the chloroplast of the plant's leaves, which is the primary component of plants' non-enzymatic antioxidant system. ROS detoxification in the chloroplast is known to be primarily carried out by the ascorbate–GSH cycle due to its high reductive potential and electron donor properties; GSH could scavenge H2O2, or react non-enzymatically with 1O2, O2 •−, and •OH and protects the various biomolecules by forming adducts (glutathiolated) or reducing them in the presence of ROS

or organic free radicals [57]. However, GSH is a strong antioxidant in its own right; its key role is to renew another hydrophilic antioxidant, ascorbic acid, mainly through the Asc-GSH cycle. The APx directly reduces H2O2 into H2O and O2, utilizing ascorbic acid (AA) as a reducing agent. Many researchers reported that increased APx activity can reduce ROS levels and promote resistance to oxidative stress, whereas reduced APx activity can decrease the cold tolerance of plants [58]. In our investigation, Y49 and M33 accumulated high GSH by 78.66% and 45.65%, whereas APx activity increased by 44.35% and 71.78%, respectively compared to control. This resulted in higher efficiency of the H2O-H2O and ascorbate-glutathione cycles. On the other hand, APx is more likely to be responsible for fine-tuning ROS in the signaling pathway, whereas CAT may be accountable for removing excess ROS during stress [59,60]. On the other hand, much reduced APx content was observed in Y38 and T8; as a result, these varieties cannot neutralize ROS under cold stress and showed susceptibility to cold stress. Phenols (flavonoids, polyphenols) are a large class of specialized metabolites found in plant tissue that exhibit antioxidant activity due to their structure (aromatic ring with −OH or −OCH3 substituents) [61]. They have a high capacity for electron or hydrogen atom donation due to their quick stabilization of generated phenol radicals. Additionally, by trapping lipid alkoxy radicals, they will directly capture 1O2 and reduce lipid peroxidation [62]. This procedure is helpful for avoiding chilling harm and cell collapse during periods of cold stress [63]. According to our results (Figure 4a,b), low temperature significantly increases total flavonoid in Y49 (23.97%) and M33 (4.94%), and polyphenols content increased 17.72% and 32.95% for Y49 and M33, respectively. In contrast, other varieties showed decreasing trend compared to that of the control. For flavonoids highest decrease was recorded in Y1, Y38, and T8 (15.49%, 18.16%, 9.24%, respectively), whereas polyphenols in Y38 (22.62%) were followed by Y1 (11.75%), and (12.02%). It has been reported that increased phenolic levels have been shown to contribute to ROS detoxification, enhance phenolic compound accumulation in plant cell walls, and increase cell wall thickness, showing that these compounds have a role in stress tolerance at low temperatures [61]. The findings of this study were similar to previous publications where the synthesis of phenolic compounds in plant tissue was seen under abiotic stress [64–66].

Therefore, plants were resistant and adapted to low temperatures by alleviating oxidative stress caused by low temperature and thus protecting the photosynthetic system [67,68]. Current experimental results revealed that different antioxidant and non-enzymatic antioxidant activities at low-temperature stress had been changed; those are relevant to cold tolerance. It was noted that the synergistic interactions of the SOD-POD-CAT-APx system were found to be efficient in preventing oxidative damage in jute plants exposed to cold stresses. Overall, the results indicated that cold indices, PCA, and cluster heat map generated a wide range of variability and could be used as credible approaches for screening jute varieties and identifying tolerant varieties based on physiological and biochemical performance under cold stress. From both of PCA and heat map, it was observed that all varieties were placed in three separate groups. In cluster heat map analysis, Group I (Y49 and M33 varieties) was low-temperature tolerance (MSTI 1.26) with improved physiological and biochemical traits such as total chlorophyll, soluble sugar, proline, and different enzymatic and non-enzymatic antioxidants activities. Whereas Group-II (Y38, T8, and Y1) was low-temperature susceptible (MSTI 0.93) by displayed low level activates of different antioxidants and osmolytes, thus these groups were identified as low-temperature susceptible varieties. Groups-III (M18, W57) with MSTI 0.98 were moderately tolerant or susceptible due to intermediate physiological and biochemical activities. Many researchers revealed that cluster analysis could be a promising tool to screen the desirable varieties based on the similarity [69]. The study thus explained the cold-tolerant mechanisms in jute and verified the cold tolerance level of selected varieties.
