**1. Introduction**

Leaf variegation has been observed in many species of higher plants [1–3] and this special attractive trait has become a focus of plant breeding as it increases the economic value of ornamental plants [4]. There are two categories of leaf variegation in plants: structural-related variegation and pigment-related variegation [5,6]. Two different types of structural variegation have been described, including the air-space type and epidermis type of variegation, which play adaptive roles to varying light conditions [7]. Pigment-leaf variegation is most common in ornamental plants because of the chlorophyll-deficiency [6]. It is marked by the existence of sections that contain abnormal plastids [5]. The leaf color variegation in plants are divided into several types based on color classification for instance green, yellow and albino (white) sectors on leaves [1,2,8,9].

Nuclear and plastid mutations or changes in expression of several genes which contribute to chloroplast biogenesis and chlorophyll biosynthesis induce the leaf variegation [10,11]. The white sectors of variegated leaves lack photosynthetic activity, therefore, leaf variegation may affect photosynthetic efficiency [12]. Previously, a transcriptome study of the *Arabidopsis* white-green variegated mutant *immutans* (*im*) and an *Arabidopsis FtsH2* mutant line (*var2*) revealed that the genes related to photosynthesis were down-regulated in the white sectors of leaves [13,14]. Furthermore, the chlorophyll-deficient leaf-mutant also showed the expressional repression of transcriptional factors *GLK1*, *Ftsz* and *MinD* that regulate chloroplast development and division [15]. Recently, a mutation in the transcription factor mitochondrial transcription termination factor (*mTERF*) has been found to induce colorlessness in leaves of variegated fig [3]. Although these studies have provided a deep understanding of the variegation mechanism in plants, the advantage of this trait for the good fitness or for the plant physiology is still poorly understood.

Several potential physiological advantages of variegation have been proposed in plants. For example, it was reported that leaf variegation is involved in plant defense from enemies including aposematic coloration, mimicry of dead or infested plants, masquerade and camouflage [16–19]. It can also play physiological roles such as improved water or gas transport [20], mitigation of UV radiation [21] and thermoregulation [22]. Investigations led on forest trees displaying variegated leaves hinted that the trait might be a strategy to prevent the attack of herbivores [23,24]. Later, studies by Mwafongo et al. [25] on leaf variegation patterns in *Ledebouria revoluta* highlighted two possible functions including the photoprotection role and the aposematic role. Very recently, Shelef et al. [22] demonstrated that under lower temperatures, variegated wild type *Silybum marianum* leaves were significantly warmer than all-green mutants, conferring cold stress tolerance. These studies showed that variegation in plants is not just a color mutation but has some physiological advantages.

*Pittosporum tobira* (Thunb.) Aiton belonging to the family Pittosporaceae originated from East Asia and at present is being widely cultivated as an ornamental flowering plant in temperate and subtropical regions around the world [26]. Typically, *P. tobira* plants are about 2–3 m high with thick, rubbery and dark green colored leaves. The fragrant flowers of *P. tobira* have been well studied for their antimicrobial and anti-oxidant activities [27,28]. Importantly, some cultivars exhibit leaf variegation with yellowish or creamy white leaf margins and green interior, which have a greater aesthetic appeal and ornamental value compared to the typical all-green *P. tobira*. These particular variegated cultivars are spread to temperate regions. However, besides the aesthetic advantage, the intrinsic physiological importance of leaf variegation for *P. tobira* is unknown.

In the present work, we studied two *P. tobira* cultivars namely, "Variegatum" and "Green Pittosporum" with distinct leaf coloration features. To thoroughly understand the role of leaf variegation in *P. tobira* under cold condition, we investigated the physio-biochemical characteristics at different temperature gradients and profiled leaf transcriptome of the two cultivars under cold stress. Our findings elucidate the leaf variegation mechanism in *P. tobira* and provide novel insights into the thermo-protective function of this important trait.

#### **2. Results**

## *2.1. Characteristics of Variegated Leaves in Pittosporum Tobira*

A naturally occurring leaf variegated cultivar of *Pittosporum tobira* named "Variegatum" was collected from Pingdingshan, Henan province in China. The cultivar "Variegatum" bears yellowish margins and green interior leaves, whereas, the typical cultivar "Green Pittosporum" exhibits dark green colored leaves (Figure 1). The phenotypic characteristics such as leaf thickness and shape were found to be similar for both cultivars except for the variegation. It is well documented that the leaves of variegated plants having green/yellow sectors have impaired chloroplast biogenesis, less photosynthetic pigments in the yellow sectors and also accumulate excessive levels of reactive oxygen species (ROS) [29,30]. To verify these observations in *P. tobira*, we analyzed the chloroplast ultrastructure in the yellow sector compared to the green sector of the variegated leaf. As shown in Figure 2A,B, the green sector contained well-developed chloroplasts with stacked grana. In contrast, in the white sector of the leaf, plastids did not contain stacked grana but contained large starch granules and many plastoglobuli (Figure 2C,D). Next, we assessed the photosynthetic parameters

and malonaldehyde (MDA) in both cultivars in August when the ambient temperature is around 20 ◦C (Figure 1). The net photosynthetic rate (*Pn*), the intercellular CO<sup>2</sup> concentration (*Ci*) and the transpiration (*Tr*) rate were found similar between leaves from both cultivars (Figure 3A–C), showing that the photosynthetic efficiency is not significantly impaired in "Variegatum" as compared to "Green Pittosporum". Next, we compared the content of photosynthesis-related pigments such as total chlorophyll (chlT) and carotenoids (Ca) in both leaf types. The results revealed that the chlT contents were significantly lower (*p* < 0.05) in "Variegatum" compared to the "Green Pittosporum" (Figure 3D), while Ca was higher in "Variegatum" compared to the "Green Pittosporum" (Figure 3E), indicating that the yellowish phenotype in "Variegatum" is underlined by a reduced chlorophyll content and a stronger accumulation of carotenoids. We further measured the MDA content, which is associated with lipid peroxidation via an increased generation of ROS [31]. The MDA was significantly (*p* < 0.01) and highly accumulated in "Variegatum" leaves compared to "Green Pittosporum" leaves (Figure 3F), implying a high level of ROS in the variegated leaves.

Taken together, our results showed that leaf variegation trait in *P. tobira* is associated with defected chloroplast biogenesis in the yellow sector, reduced chlorophyll content, strong accumulation of carotenoids and high level of ROS.

**Figure 1.** Overview of the experiment design and phenotypes of the two *Pittosporum tobira* cultivars, namely "Variegatum" with green/yellowish variegated leaf and "Green Pittisporum" with complete dark green leaf. Leaf samples were harvested at different dates following decrease of ambient temperature. The bar = 2 cm.

**Figure 2.** Chloroplast ultrastructure of the green (**A**,**B**) and yellow (**C**,**D**) sectors in variegated leaves of *Pittosporum tobira* cultivar "Variegatum". C = chloroplast; P = plastid; SG = starch granule; G = grana; V = vacuole, PL = plastoglobuli.

**Figure 3.** Physio-biochemical comparison of leaf from variegated "Variegatum" and non-variegated "Green Pittosporum" cultivars. (**A**) net photosynthetic rate (*Pn*), (**B**) intercellular CO<sup>2</sup> concentration (*Ci*), (**C**) transpiration rate (*Tr*), (**D**) total chlorophyll content (ChlT), (**E**) carotenoids content (Ca) and (**F**) malonaldehyde content (MDA). \*, \*\* above the bars represent significant difference between the two cultivars at *p* < 0.05 and *p* < 0.001, respectively, using Tukey's honestly significant difference (HSD) test.
