*2.6. DEGs Related to the Biosynthesis of Unsaturated Fatty Acids and Fatty Acids Metabolism*

An increase in polyunsaturated fatty acids has also been reported to play a crucial role in the chilling tolerance of plants [33]. In this study, ten DEGs were mapped to the pathways related to the biosynthesis of unsaturated fatty acids and fatty acids metabolism. Interestingly, all of these DEGs were annotated as endoplasmic reticulum omega-6 fatty acid desaturase (FAD2) and were all up-regulated in "Variegatum" (Table 5). Since the microsomal enzyme FAD2 principally acts on the desaturation of C18:1 to C18:2 [34], we deduce that "Variegatum" strongly accumulates C18:2 in leaf as a protective molecule under cold conditions. Besides, we also detected nine GDSL-Lipase involved in lipid biosynthesis in plants. All the GDSL genes were down regulated in "Variegatum" (Table 5), suggesting a probable opposite function of GDSL and FAD2 genes during cold endurance in "Variegatum".

**Table 5.** Key DEGs related to the enriched KEGG pathways involved in the cold responses in variegated *P. tobira.*



**Table 5.** *Cont*.

#### *2.7. Disturbance of Protein Processing in Endoplasmic Reticulum under Cold Conditions*

The endoplasmic reticulum is a subcellular compartment where proteins and lipids are folded with the help of chaperones. The enrichment of this pathway (Figure 6C) indicates a disturbance of proteins and lipids synthesis under cold conditions. Nine DEGs, all being heat shock proteins (HSP) were detected within this pathway. Notably, we observed that all the small HSP genes (15–22 kDa) were down-regulated while the high molecular weight HSP genes (70–90 kDa) were up-regulated in the variegated leaves (Table 5). This result highlights the weight dependent roles of HSP genes for a stout cold response in variegated *P. tobira*.

### *2.8. DEGs in the Phenylalanine Metabolism*

In this important pathway, we found three DEGs including two POD genes (*c68309.graph\_c0* and *c74970.graph\_c1*) and *c55523.graph\_c0* annotated as an aminotransferase TAT2. Interestingly, all these genes were up-regulated in "Variegatum", showing that they contribute positively to the enhanced cold response (Table 5). More importantly, the activation of these genes correlates well with the strong enzymatic activity of POD detected through our biochemical assay in "Variegatum" when the temperature reached 10 ◦C (Figure 4A).

#### **3. Discussion**

#### *3.1. Characteristics of Leaf Variegation in P. tobira*

Leaf variegated plants have green/white (or yellow) sectors and cells in the green sectors contain normal appearing chloroplasts, while cells in the white sectors have impaired chloroplast biogenesis and lack photosynthetic pigments [12]. Moreover, it has been shown that leaf variegated plants accumulate high levels of ROS [29,30]. Although these mechanisms are commonly found in variegated plants, a recent study of the rice *z3* mutant leaves showed a new mechanism of variegation, which was caused by an unbalanced distribution of citrate in a transverse pattern in leaf tissues [34]. In our study, we also noted a defected chloroplast development in the yellow sector, reduced chlorophyll content and a high level of ROS in the variegated cultivar (Figures 2 and 3). We also observed an abundance of starch granules in the yellow sector as compared to the green sector, suggesting that the yellow sectors are nutrient sinks because they are unable to perform photosynthesis. Similar

conclusions were previously reported in different species including variegated *Arabidopsis* [1,35,36], tobacco [37], begonia [6] and fig [3]. However, the photosynthetic efficiency was not obviously affected in "Variegatum" (Figure 3), contrasting with the reports that leaf variegation affects photosynthetic efficiency [12]. In fact, the yellowish area on "Variegatum" leaves is located on the margin and has a very low surface coverage. So, an explanation to this observation can be that the green part of the leaf is large enough to ensure the photosynthetic activity. Leaf variegation has been attributed to a deficiency or a significant reduction of photosynthetic pigments including carotenoids. In the *Arabidopsis* white-green variegated mutant *immutans* (*im*), an inhibition of carotenoids formation was observed [38]. Similarly, the white section in leaf of variegated *Epipremnum aureum* contains 10-fold less carotenoids than the green section [39]. In *Cyclamen purpurascens*, the light green leaf stripes were found with reduced carotenoids and chlorophyll contents [40]. In green/yellow patterns variegated species, similar observations were also noticed in *Aucuba japonica* [41] and *Coleus bluemei* [42]. Intriguingly, we observed a higher concentration of carotenoids in the variegated leaves of *P. tobira* as compared to the complete green leaves (Figure 3), a phenomenon which has not yet been reported in variegated plants. Since carotenoids function as accessory light-harvesting pigments, broadening the spectral range over which light can support photosynthesis in plants [43], we deduce that the high carotenoids content in "Variegatum" may compensate the reduced chlorophyll to maintain similar photosynthetic activity as in leaves of "Green Pittosporum".

#### *3.2. Protective Role of Leaf Variegation in P. tobira under Cold Condition*

The natural occurrence of variegation in plants suggests that the trait might play some adaptive functions beyond their aesthetic value [32]. It has been suggested that leaf variegation plays several physiological and ecological functions such as defense from enemies, adaptations to light, temperature, etc. [16–25]. We tested the hypothesis that leaf variegation plays a low temperature protective function in *P. tobira*, which is an ornamental shrub widely grown in temperate climate and therefore is annually subjected to cold stress. It is well known that increased activities of antioxidant enzymes such as POD, CAT, SOD under abiotic stress conditions including drought, salt, chilling, heat, etc., promote enhanced stress tolerance in plants [44]. Our results demonstrated that "Variegatum" has much more efficient ROS-scavenging machinery compared to "Green Pittosporum" and accumulates less MDA, an indicator of limited cellular membrane damage due to lipid peroxidation. Hence, "Variegatum" better tolerates low temperature stress than "Green Pittosporum" (Figure 4). We further sequenced the transcriptomes of both leaf types under cold condition (10 ◦C). Differential gene expression (DEG) analysis resulted in 309 DEGs between the two cultivars, enriched in biological pathways related to the biosynthesis of unsaturated fatty acids, sesquiterpenoid and triterpenoid biosynthesis, fatty acid metabolism, phenylalanine metabolism and protein processing in endoplasmic reticulum, which may be crucial pathways involved in cold stress alleviation (Figure 6).

Cell membrane structure, integrity and fluidity are affected by lipid composition and the degree of fatty acid (FA) desaturation in plants [45]. It has been documented that changes in unsaturated fatty acids content can improve plant tolerance to environmental stresses such as cold, heat and drought [46–51], since modification of membrane fluidity results in an environment suitable for the function of critical integral proteins, such as the photosynthetic machinery, during stresses [52]. In this study, we detected ten FAD2 genes all significantly up-regulated in "Variegatum" leaves under cold condition (Table 5). The microsomal enzyme FAD2 principally acts on the desaturation of C18:1 (monounsaturated FA) to C18:2 (polyunsaturated FA) [53], suggesting that "Variegatum" tends to increase polyunsaturated FA (PUFA) level, a mechanism to maintain cell membrane fluidity under low temperature [54,55]. This skill of adjusting membrane fluidity by varying the unsaturated fatty acid content is characteristic of cold-responsive plants [52]. Cold acclimating potato (*Solanum commersonii*) was found to accumulate linoleic acid (18:2) in the membrane glycerolipids of the leaves, whereas commercial, non-acclimating potato (*Solanum tuberosum*) did not show this trait during cold stress [56]. Our findings are in perfect accordance with reports of Liu et al. [51], who showed that over-expression of

tomato FAD2 gene alleviates the photoinhibition of photosystems 2 and 1 and improves tolerance under chilling stress. Similar observations were reported in various plants such as cotton [57], *A. thaliana* [58], *Olea europaea* [59], *Synechocystis* sp. [60], etc., under low temperature conditions.

Membrane fatty acid composition is, to a great extent, determined by the activities of complexly regulated integral fatty acid desaturases and lipases [52]. GDSL-lipase participates in fatty acid catabolism and studies have shown that the linoleic acid and other PUFAs contents are significantly decreased when GDSL genes are over-expressed [61–64]. Here, we detected nine GDSL-lipase genes all down-regulated in "Variegatum" under low temperature stress (Table 5), denoting a strategy to keep the high level of PUFA for the maintenance of cell membrane stability. We deduce that down-regulation of GDSL genes and up-regulation of FAD2 genes is therefore an integrated and efficient mechanism to cope with cold stress in *P. tobira* cv. "Variegatum".

Another group of genes detected within the DEGs between "Variegatum" and "Green Pittosporum" under cold condition are heat shock proteins (HSP) (Table 5). HSPs are molecular chaperones that are constantly present in cells to correctly fold proteins involved in routine cellular processes such as translocation, cell-signaling and metabolism [65]. However, HSPs become abundant in most organisms in response to protein denaturation caused by environmental, metabolic and pathological stresses [66]. For example, *Arabidopsis*, grape, rice, *Brassicas* increase the production of HSPs to augment survival in cold environments [67–70]. On the other hand, it was reported that a complex coordination of HSPs underlines cold tolerance in plants [65]. In fact, some HSPs are either up- or down-regulated when heat shock factors (HSFs) bind to their promoter regions [71,72]. This suggests that not all HSPs positively participate in cold or stress tolerance in plants. Each group of these HSPs has a unique mechanism [65]. In our study, we observed that small HSPs were all down-regulated while high molecular weight HSP genes were up-regulated in "Variegatum", pointing out an opposite function of HSPs for cold response in *P. tobira* with respect to their molecular weights. For now, a clear explanation for this phenomenon is yet to be found, hence, an in-depth investigation of the role of HSP genes and their relation with the significantly altered HSF transcription factors under cold condition in *P. tobira* is necessary in order to clarify this intriguing finding.

Our transcriptome analysis also unveiled several peroxidase genes from the phenyalanine pathway as well as some cytochrome *P450* genes from the sesquiterpenoid and triterpenoid biosynthesis as candidate genes, which positively contribute to the enhanced cold responses in "Variegatum" (Table 5). Peroxidase genes have been extensively studied in plants for their ROS-scavenging activity under various biotic and abiotic stresses, including chilling [73–75]. Similarly, Liu et al. [76] recently investigated the prominent biological pathways engaged in wild banana tolerance to chilling. They observed significant changes in the sesquiterpenoid and triterpenoid biosynthesis, particularly cytochrome *P450* genes, a finding that supports well the results of our study.

Taken together, we showed that leaf variegation in *P. tobira* is associated to defected chloroplast development, reduced chlorophyll content, high content of carotenoids and a high level of ROS. The results of transcriptome analysis were consistent with the enzymatic activity under cold conditions. These results pointed out that the leaf variegation trait plays low temperature protective effect in *P. tobira* by inducing a strong ROS-scavenging activity through catalase and peroxidase enzymes, inducing heat shock proteins for cellular homeostasis and, more importantly, by maintaining high levels of PUFA for cell membrane stability and fluidity through a coordinated up-regulation of FAD2 and down-regulation of GDSL-lipase genes. The modulation of the expression levels of these key genes may be orchestrated by transcription factors from the families of NAC, WRKY, HSF and AP2/ERF. A proposed schematic model for the stronger cold response in "Variegatum" is summarized in Figure 8.

**Figure 8.** A schematic model of the proposed mechanism underlying the strong cold response in *P. tobira* cv. "Variegatum".
