The surfaces of aerial plant organs are covered by cuticle, and its primary function is to prevent uncontrolled water loss (Riederer and Schreiber, 2001). Plant cuticle is predominantly a mixture of waxes, including very-long-chain fatty acids and their derivatives, cyclic terpenoids, with cutin polymers providing a scaffold matrix, mainly C₁₆ and C₁₈ with or without hydroxy groups (Yeats and Rose, 2013). It is well known that the cuticular waxes, rather than the cutin matrix, are the barrier to non-stomatal water loss (Riederer and Schreiber, 1995; Jetter and Riederer, 2016). The diversity of cuticular wax components and their spatial arrangements results in a wide variety of cuticular transpiration barrier properties across species, organs, and developmental stages (Jetter and Riederer, 2016; Huang et al., 2017; Bourgault et al., 2020). Previous studies have focused on how the barrier properties of leaf or fruit cuticles affect water loss or chemical substrates (Riederer and Schreiber, 2001; Jetter and Riederer, 2016), whereas few studies have been conducted on the barrier properties of flower cuticles.

The thin cuticle covering the petal surface plays an important role in interactions with pollinators (Whitney et al., 2011). Previous studies reported that cuticular transpiration in fruit cuticular membranes was higher than that of leaf cuticles. It implied that it might not be necessary to form such efficient cuticular barriers to transpiration for fruit as the life time is usually shorter than leaf tissues (Schreiber and Riederer, 1996). Chemical analysis has indicated that the wax layer of Cosmos bipinnatus var. ‘Sensation Pinkie’ petals contains relatively high concentrations of C₂₂ and C₂₄ fatty acids and primary alcohols, which are much shorter than those in the wax layer of leaves. Therefore, the petal cuticle exhibits weaker transpiration barrier properties than the leaf cuticles (Buschhaus et al., 2015). Similarly, C-chain lengths of waxes in petal cuticles of ‘Movie star’ and ‘Tineke’ roses were generally between C₂₆ and C_29, but ranged from C₂₉ to C₃₃ in the leaf cuticles. The C-chain differences were thought to be one of the factors inducing higher transpiration rate for the petals compared to that for the leaves in roses (Cheng et al., 2019). A comparative study on cuticular wax chemical differences between flowers and other organs has also been conducted (Guo and Jetter, 2017). In addition, significant changes in the composition of the wax on snapdragon petals were found over 12days from the opening stage to senescence (Goodwin et al., 2003). However, more information about the chemical composition of petal cuticles and its contribution to barrier properties during petal development are still awaiting to be explored.

Oriental lily, a Lilium spp., is one of the most popular ornamental plants. Lilies are widely grown in temperate, subtropical, and tropical regions, and blooming from spring to early autumn. Oriental lily cut flowers usually comprise the flower head and the stem and keep some leaves. The lily flower head has two whorls of tepals (floral leaves) and each of the inner and outer whorls normally has three tepals (van Doorn and Han, 2011). The inner tepals are covered by the outer tepals during flower development. Lilies are widely planted by consumers for their elegant look and wide open, brightly colored tepals. The vase life of lily flowers varies with cultivar, cultivation, and post-harvest conditions. In addition, the shelf life of the cut flowers is impacted by rapid water loss or the deterioration of stem tissues after harvest (Torre and Fjeld, 2001; Cheng et al., 2020).

The present study aimed to characterize and compare the chemical composition of the cuticular waxes of lily tepals and leaves as well as to determine their potential contributions to limit cuticular transpiration from the tepals and leaves of three lily cultivars. The transpiration rate and chemical composition of the cuticular waxes (1) on the tepals at the green bud and open stages of the Lilium spp. cultivar ‘Casa Blanca’, and (2) on the leaves and tepals of two Lilium spp. cultivars ‘Huang Tianba’ and ‘Tiber’ were determined. These findings enabled comparisons of the cuticular wax chemicals on tepals at different development stages and organs, as well as link the transpiration barrier differences.

The transpiration of cut flowers has been recently cared by researchers on different species and flower organs. As the occurrence of stomata, water loss occurred mainly through the leaf tissues in the intact cut flowers. When most of the stomata closed, leaves were still the main parts for water loss in roses, but not in carnation or chrysanthemum (Lin et al., 2020; Tsaniklidis et al., 2020; Fanourakis et al., 2021). These studies considered stomatal transpiration as the main pathway for water loss in cut flowers. However, the non-functional or non-stomatal transpiration, which has been widely reported to be the cuticular transpiration for leaves and fruit (Riederer and Schreiber, 2001), whereas relatively little is available for flowers. So far, only two studies reported the cuticular transpiration of flowers. The water permeability of intact petal of C. bipinnatus “Sensation” was 6.7×10⁻⁵ms⁻¹ (Buschhaus et al., 2015). Our previous study on rose petals found similar cuticular transpiration levels with 4.1×10⁻⁵ms⁻¹ for the ‘Movie star’ and 8.9×10⁻⁵ms⁻¹ for ‘Tineke’ (Cheng et al., 2019). Cut lily flowers contains flowering head with tepals, leaves, and stem (Figure 1; van Doorn and Han, 2011). Stomata occurred on the abaixal side of leaf and outer tepal surfaces of lily flowers (Supplementary Figure S1). The minimum conductance was used to indicate the cuticular transpiration or with non-functional stomata transpiration (Burghardt and Riederer, 2003). Leaf of lily flowers exhibited typical drying curves as reported previously (Supplementary Figure S2). The water loss of petals in lily flowers exhibited almost linearly following the time changes during the whole dehydration process (Supplementary Figures S3, S4). In addition, the stomata on lily tepals were shown covered by waxes or under closed status (Supplementary Figure S1). Therefore, the drying curve and water loss dynamic changes implied that the stomata of sepals might be non-functional.

The mean minimum conductance of the wax layer on the tepals of the green bud stage of the lily cultivar was higher than the tepals of the open flowers, which showed similar levels as reported results (Cheng et al., 2019). In a number of studies on leaf tissue transpiration, the reported water permeance ranges from 2.6×10⁻⁷ms⁻¹ for Zamioculcas zamiifolia (G.Lodd.) Engl. to 4×10⁻³ms⁻¹ for Ipomoea batatas (L.) Lam., with most values lower than 2×10⁻⁴ms⁻¹ (Schuster et al., 2017). The minimum water conductance of the cuticles on the green bud and open flower tepals and on the leaves of all three lily cultivars showed a similar level to that of most of the reported values. In addition, the minimum water conductance exhibited by the lily cuticles was higher in tepals than in leaves (Figure 3A). Taking water loss of petal and leaf cuticles in C. bipinnatus and rose cultivars (Buschhaus et al., 2015; Cheng et al., 2019) together, the wax layer on tepals and petals appears to be a less efficient barrier to transpiration compared to that on leaves. This might be related to the special functions of petals, which may have rapid cuticular transpiration together with volatilization of aroma to attract pollinators. On the other hand, flowers are relatively short-lived organs even when environmental conditions are favorable. Thus, it is probably not necessary for flowers to develop such a highly efficient transpiration barrier, compared to that needed on long-lived leaves. The cuticular wax loading on petals has been reported for only a few species, with values ranging from 2.7μgcm⁻² (C. bipinnatus; Buschhaus et al., 2015) to 37μgcm⁻² (T. officinale; Guo and Jetter, 2017). Only one study (on Antirrhinum majus L., the snapdragon) has examined the cuticular wax loading during petal development (Goodwin et al., 2003). By contrast, the accumulation of waxes on the tepals decreased substantially from the green bud to the open flower stage, for both the inner and outer tepals of the Lilium cultivar ‘Casa Blanca’. The overall tepal wax coverage was not significantly different between the cultivars ‘Huang Tianba’ and ‘Tiber’. However, wax coverage was higher on the tepals than on the leaves. As reported previously, wax coverage on the lily tepals exhibited cultivar-specific differences (Huang et al., 2017; Cheng et al., 2019). In addition, water permeance of the lily leaves was significantly lower than that of the tepals, supporting the assertion that there is no significant relationship between the cuticular water permeability and the total amount of wax (Riederer and Schreiber, 2001; Jetter and Riederer, 2016).

The chemical composition of petal cuticles differs among different flowers. The wax layers on the petals of Solanum tuberosum L. (potato) and A. majus (snapdragon) flowers contained relatively high concentrations of n-alkanes and methyl-branched alkanes (Goodwin et al., 2003; Guo and Jetter, 2017). Similarly, the major cuticular wax components of Vicia faba L. (faba bean) and Rubus idaeus L. (raspberry) petals were n-alkanes (Griffiths et al., 1999, 2000). Primary alcohols (21%) and n-alkanes (29%) were found in similar amounts in the wax layer of T. officinale (Guo et al., 2017). Our current study found that the waxes on petals of the rose cultivars ‘Movie star’ and ‘Tineke’ were dominated by n-alkanes (46.8 and 64.3%) and n-alkenes (47.3 and 20.2%; Cheng et al., 2019). Similarly, in the present study, n-alkanes were found to be the major components of the lily tepal waxes (Figures 4, 6). However, primary alcohols and alkyl esters were the predominant components in petal waxes of C. bipinnatus and Petunia hybrida Vilm. W115 (Petunia) flowers, whereas n-alkanes and triterpenoids occurred at low levels (King et al., 2007; Buschhaus et al., 2015). Minor amounts of branched n-alkanes or n-alkenes were found in the tepal waxes of the lily cultivars ‘Huang Tianba’ and ‘Tiber’, and sterols were found in the tepal waxes of open lily flowers (Figures 4, 6).

It has been proposed that the hydrophobic barrier of the cuticle is mainly determined by the chemical composition and chain-length distribution of the very-long-chain aliphatics and their structural arrangements (Riederer and Schreiber, 1995). The accumulation of very-long-chain components in the wax layer shift from C₁₆ and C₁₈ to longer than C₂₀ carbon chains during the development of leaves in Arabidopsis (Jenks et al., 1995; Busta et al., 2017) and Prunus laurocerasus (Jetter and Schäffer, 2001). In maize, the predominant aliphatic components in the leaf cuticle were C_21, C₂₃, and C₂₅ n-alkanes at early stages of development but shifted to chain lengths over C₂₈ in later development (Bourgault et al., 2020). Similarly, in the present study, the fatty acid fraction in the tepal waxes of the green bud stage mainly contained C₂₀ and C₂₂, whereas they ranged from C₂₀ to C₂₆ in the open flower tepal cuticles (Figure 5; Supplementary Tables S1 and S2). The n-alkanes with chain lengths over C₃₁ and alkyl esters over C₃₆ were detected only in the cuticles on the tepals of open flowers (Figure 5). A shift to longer hydrocarbon chain lengths is due to the bio-synthesis of waxes regulated by a series of genes and enzymes, as well as their functional properties during developmental stages (Busta et al., 2017).

Previous studies indicated that C-chains of cuticular waxes accumulating in petal waxes are shorter compared to those in leaf waxes. C. bipinnatus petal and leaf waxes are dominated by primary alcohols and fatty acids with carbon chain lengths of C₂₂ and C₂₄, and C₂₈ and C₃₀, respectively (Buschhaus et al., 2015). The waxes on the flowers and leaves of T. officinale contained branched C₂₇ to C₃₁, and non-branched C₂₇ to C₃₃ n-alkanes (Guo and Jetter, 2017). Alcohol esters with a predominance of C₂₂ to C₂₈, and C₂₈ to C₃₂ were found in P. hybrida flower and leaf waxes, respectively (King et al., 2007). In Arabidopsis, n-alkanes with a chain length of C₂₉ dominate the aliphatic pattern in petal waxes, whereas a chain length of C₃₁ is dominant in the leaf waxes (Jenks et al., 1995; Shi et al., 2011). In rose cultivars ‘Movie star’ and ‘Tineke’, the predominant chain lengths are C₃₁ and C₃₃ n-alkanes in the leaf waxes, whereas C₂₇ and C₂₉ n-alkanes and n-alkenes dominate the petal waxes (Cheng et al., 2019). In the present study, the chain lengths of the n-alkanes ranged from C₂₂ to C₃₃, with C₂₇ and C₂₉ chain lengths predominating in the tepal waxes, whereas chain lengths of C₃₁ and C₂₉ were dominant in the leaf waxes of the ‘Huang Tianba’ and ‘Tiber’ cultivars, respectively (Figure 7). These findings corroborated the typical chain-length distributions of the aliphatic component, being shorter in petal waxes compared to that in leaf waxes (Buschhaus et al., 2015; Jetter and Riederer, 2016).

In contrast to the chemical analysis of petal waxes, few studies have investigated the transpiration barrier properties of petal cuticles. A study on water permeability of petal waxes has been conducted for C. bipinnatus (Buschhaus et al., 2015). As mentioned above, the relatively high concentrations of C₂₂ and C₂₄ carbon chain components in C. bipinnatus petal waxes are thought to create a less efficient barrier to transpiration compared to that of the leaf cuticles. Similar results have been found for rose petal waxes in the ‘Movie star’ and ‘Tineke’ cultivars, which accumulated mainly C₂₆ and C₂₉, resulting in higher cuticular transpiration compared to that in leaves with a predominance of C₃₁ (Cheng et al., 2019). Patwari et al. (2019) found that the lack of very-long-chain wax esters induced a higher cuticular permeability in wsd1 mutants in Arabidopsis. Taken previous results together with present study on lily, C-chain lengths distribution in cuticular wax might play a pivotal role in limiting non-stomatal transpiration in plant tissues. Flowers that accumulate very long chain of aliphatics might exhibit slower weight loss and longer shelf life during marketing.

In conclusion, cuticular transpiration of lily flower green bud tepals was significantly higher than that of the open flower tepals and was significantly higher in the tepals than in the leaves. The most abundant aliphatic compounds in the tepal waxes of the lily cultivars ‘Casa Blanca’ and ‘Huang Tianba’ were n-alkanes, whereas large amounts of primary alcohols were detected in the tepal waxes of the ‘Tiber’ cultivar. In addition, there was a predominance of relatively short carbon chain aliphatics in the tepal waxes of the green bud stage, compared to the chain lengths in tepal waxes of the open flower stage, and similar differences between the tepal and leaf waxes were found. These findings suggest that the less efficient transpiration barrier properties of the tepal waxes are probably related to the accumulation of relatively short-chain aliphatics. Furthermore, cuticular transpiration and the barrier properties of the cuticular waxes of lily cultivars exhibited organ- and cultivar-specific differences. These findings provide further insight to link the cuticular chemicals with functional properties as well as with the physiological adaptations of plant tissues. The findings of this study also contribute to the cultivar selection for flowers, in which accumulation of very long chain of aliphatics might exhibit slower weight loss and longer shelf life during marketing. The cuticular barrier properties are varied in organ-, cultivar-, and species differences, which are largely affected by the accumulation of chemical compositions and arrangement of cuticle (Riederer and Schreiber, 2001; Huang et al., 2017). In addition, the growing conditions have also been widely reported to be one of the most important factors in influencing the transpiration changes. Therefore, selecting appropriate cultivar, setting appropriate growing conditions including water and light availability, temperature and humidity control, etc., will largely influence the water loss of flowers. Combining post-harvest treatments such as nano-silver solution with sugars to spray on flowers forms thin aqueous membrane, slowing down the rapid post-harvest changes (Naing and Kim, 2020). Therefore, caring the transpiration barrier properties will be helpful for the development of techniques with the potential to extend the shelf life of cut flowers based on the transpiration barrier properties of the cuticular waxes after harvest.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

HH and GC designed the project and prepared the manuscript. GC and LW performed most of the experiments. HW and XY took part in part of the experiments. NZ, XW, and LH helped to make interpretation of the results. All authors contributed to the article and approved the submitted version.

This work was supported by the Characteristic Innovation of Project of Education Department of Guangdong Province (Natural Science 2020KTSCX053), the Common Technical Innovation Team of Guangdong Province on Preservation and Logistics of Agricultural Products (2019KJ145), the Foundation of Guangxi Key Laboratory of Fruits and Vegetables Storage-Processing Technology (2021-01), and the special fund for scientific innovation strategy-construction of high level Academy of Agriculture Science of Guangdong Academy of Agricultural Sciences (R2019QD-012).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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