Genetic variation and relationship among content of vitamins, pigments, and sugars in baby leaf lettuce

Abstract Baby leaf lettuce harvested approximately 30 days after planting is the primary component of spring mix salads, a popular type of packaged salads. Very little is known, however, about the content of vitamins, sugars, and pigments in young lettuce plants. Therefore, plants of 42 accessions harvested at baby leaf stage were analyzed for the contents of vitamin C, ß‐carotene, anthocyanins, chlorophylls, glucose, fructose, and sucrose. Significant differences among accessions were found for content of all seven compounds plus sucrose sweetness equivalency (SSE) and average vitamin load (AVLAC). “Floricos” was highest in all sugars, SSE and vitamin C; “Taiwan” was highest in ß‐carotene and AVLAC, and “Annapolis” and “Darkland” were highest for anthocyanins and chlorophyll contents, respectively. The lowest content of glucose and sucrose was found in iceberg “Salinas,” fructose in L. serriola accession UC96US23, vitamin C in PI 257288, and β‐carotene in “Solar.” The lowest relative sweetness (SSE) was calculated for UC96US23, followed by “Salinas,” while the lowest AVLAC was estimated for PI 257288. There were very strong, positive correlations among contents of the three sugars, and between β‐carotene and vitamin C, and β‐carotene and anthocyanins. Composition profiles of accessions presented in this study, together with identified associations between compounds, can be used by breeders, growers, and producers to select lettuces with desirable combinations of sugars, pigments, and vitamins. This information can help in development of new cultivars and breeding lines with desirable combination of traits, pleasing taste, and higher vitamin content.

Plants are an important source of phytochemicals (Dillard & German, 2000) and vitamins needed for proper functioning of human organisms and prevention of vitamin-related deficiencies, such as blindness (vitamin A), beriberi (vitamin B1), pellagra (vitamin B3), anemia (vitamin B6), scurvy (vitamin C), and rickets (vitamin D; Asensi-Fabado & Munné-Bosch, 2010). Vitamin C is also required for biosynthesis of collagen and certain hormones, and has a therapeutic potential in cancer and heart disease (Li & Schellhorn, 2007).
Three basic types of pigments that cause coloration of lettuce leaves are mainly chlorophylls (green color), anthocyanins (red-purple color), and carotenoids (yellow-orange color that is usually masked in photosynthetically active tissue). Consumption of plant pigments has a beneficial effect on human health (Khoo, Azlan, Tang, & Lim, 2017).
Epidemiological studies have shown positive associations between increased intake of carotenoids and decreased the risk of cancer (Tanaka, Shnimizu, & Moriwaki, 2012). Certain carotenoids, that are precursors for vitamin A biosynthesis, also have important roles in prevention of blindness due to age-related macular degeneration (Taylor & Ramsay, 2005). Anthocyanins are prominent phenolic compounds found abundantly in red-colored lettuce (Simko, Hayes, & Furbank, 2016;Sytar et al., 2018). Anthocyanin-rich lettuce demonstrated antidiabetic effects and may help in improving metabolic syndrome conditions of fatty liver and glucose metabolism (Cheng et al., 2014). Chlorophylls and their derivatives showed a positive effect as a cancer preventative agent (Ferruzzi & Blakeslee, 2007). The effect was attributed to chlorophylls' antioxidant and antimutagenic activity, mutagen trapping, modulation of xenobiotic metabolism, and induction of apoptosis (Ferruzzi & Blakeslee, 2007). Though lettuce is not a significant source of sugars (Mou, 2009) in a human diet, the presence of sugars in lettuce leaves substantially affects sensory perception of their taste (Chadwick, Gawthrop, Michelmore, Wagstaff, & Methven, 2016).
The content of bioactive compounds in lettuce is significantly influenced by a growing environment (Riga et al., 2019;Sytar et al., 2018) and the plant genotype (Mou, 2005;van Treuren, Eekelen, Wehrens, & Vos, 2018;Yang et al., 2018). The objective of the present study was to analyze the content of vitamin C, ß-carotene, anthocyanins, chlorophylls, glucose, fructose, and sucrose in baby leaf lettuce and to investigate relationships among contents of these compounds in 42 lettuce accessions. Most of the studies related to the content of vitamins, pigments, and sugars in lettuce were previously performed on plants harvested at full maturity (approximately 60-90 days after planting). Relatively little is known, however, about the content of these compounds in baby leaf lettuce, the product that became a highly popular part of a human diet in recent years.

| Plant material and growth conditions
A set of 42 lettuce accessions evaluated in this study included 30 cultivars, six plant introductions, five breeding lines, and a single accession of Lactuca serriola L., the wild species closely related to cultivated lettuce (

| Quantification of compounds in lettuce leaves
The relative content of chlorophylls and anthocyanins was determined two days before harvest using SPAD-502 (Spectrum Technologies) and ACM-200 plus (Opti-Sciences) hand-held meters, respectively. These devices use light transmittance to provide good in situ estimates of relative contents of the two pigments (van den Berg & Perkins, 2005;Parry, Blonquist, & Bugbee, 2014). Chlorophylls and anthocyanins were measured on three leaves of similar age (avoiding youngest and oldest leaves) and size (circa 10 cm) per plant. The measuring clip was positioned about 1 cm from the edge of the leaf while ensuring that major veins were avoided. The content of chlorophylls is expressed in SPAD units; the content of anthocyanins is expressed in ACI (anthocyanins content index) units. For each plant, the averages of three measurements of chlorophylls and anthocyanins were recorded and used in statistical analyses.
All leaves of a plant (devoid of stem tissue) were harvested, split into three homogeneous samples, and used for analyses of sugars, vitamin C, and β-carotene. All laboratory analyses were performed by UC Davis Analytical Laboratory (https ://anlab. ucdav is.edu). Samples for analyses of soluble carbohydrates were dried at 55ºC, ground, and extracted with hot deionized water (Johansen, Glitsø, & Bach Knudsen, 1996) as follows: negative ion atmospheric-pressure chemical ionization, multiple ions scan, duration 2.5 min, curtain gas (CUR = 50), needle current (NC = −5), temperature (TEM = 400), gas 1 (GS1 = 50), gas 2 (GS2 = 40), declustering potential (DP = −40), focusing potential (FP = −200), and entrance potential (EP = −4). The amount of three sugars was calculated using a second order internal standard curve with 1/x weighting, plotting the ratio of analyte to internal standard concentration versus the ratio of analyte peak area to internal standard peak area. Each sugar had its own internal standard prepared from CAR10-1KT kit (MilliporeSigma). The content of glucose, fructose, and sucrose is reported in g per kg of fresh weight (g/kg FW).
Sucrose sweetness equivalency (SSE) was calculated by weighing the content of glucose, fructose, and sucrose by their relative sweetness. Relative sweetness is a dimensionless quantity based on a human perception relative to that perceived for the sweetness of sucrose. Thus, the relative sweetness of sucrose is 1.00, while the values for glucose and fructose are about 0.74 and 1.17, respectively (Joesten, Castellion, & Hogg, 2007).  The content for each compound is shown on the green-black-red scale, with green color indicating the minimal value and red color indicating the maximal value. Actual values for each compound and accession are shown in Table 1 of 12:1 from ß-carotene into retinol (NIH, 2019), it was shown that the conversion rate for green leafy vegetables is lower, ranging from 21:1 to 28:1 (Tang, 2010). Therefore, the content of ß-carotene was divided by 25 to get an approximate conversion into vita-

| Statistical analysis
The content of each compound in every accession was subjected
Correlations that were significant using both Pearson and The highest SSE was estimated for "Floricos" (20.0 g SE /kg FW) that has the highest contents of all three sugars (Table S1, Figure   F I G U R E 3 Differences in sucrose sweetness equivalency (SSE) and average vitamin load (AVL AC ) among 42 lettuce accessions harvested at baby leaf stage. Analysis of means (ANOM) was performed to identify accessions with quantities significantly different from the overall mean. Values that are outside of light blue areas are significantly different (p < .05) from the overall means that are indicated as horizontal and vertical red lines. SSE was calculated from the content of glucose, fructose, and sucrose per kg of FW and multiplied by relative sweetness of sugars. AVL AC value indicates percent of Recommended Daily Allowance (RDA) of vitamin A and vitamin C that is obtained from 1 kg FW of lettuce. Detailed information regarding calculations of SSE and AVL AC is provided in material and methods. Yellow-and green-colored arrows mark quadrants with high and low relative sweetness and AVL AC , respectively. For clarity of the figure, names are shown only for the accessions whose values are significantly different from the overall mean at both traits or have the lowest or the highest value of the trait S3), while the lowest SSE (0.5 g SE /kg FW) was estimated for the L. serriola accession UC96US23 with very low contents of sugars.
Though the relative sweetness of fructose (1.17) is higher than those of sucrose (1.00), or glucose (0.74) (Joesten et al., 2007), glucose was found to be the sugar whose content was most highly correlated with the perception of lettuce sweetness (Chadwick et al., 2016). That study found, however, that the liking of lettuce taste was not determined solely by the content of sugars, but the ratio between sweet (sugars) and bitter (sesquiterpenoid lactone) compounds. High content of sugars was pleasing for the palate, while high content of sesquiterpenoid lactone compounds (such as 8-deoxylactucin-15-sulfate) was undesirable (Chadwick et al., 2016). Because neither the tasting evaluations nor the analyses of compounds related to bitter taste were performed in the present study, it is not possible to determine whether the cultivar with the highest SSE ("Floricos") was also the most liked. It is expected, however, that UC96US23 taste would be generally disliked, as this wild species was the least sweet (estimate based on SSE) and also contains the bitter-tasting compounds (Chadwick et al., 2016).
AVL AC ranged from 108% RDA/kg FW (PI 257288) to 495% RDA/ kg FW ("Taiwan"). The high AVL AC value calculated for "Taiwan" was mostly due to the very high RAE of vitamin A (765% RDA/kg FW), though the content of vitamin C was also significantly higher (225% RDA/kg FW) than the overall mean (149% RDA/kg FW). For plant breeders, growers, and producers, it is important to know about both the nutritional value (e.g., AVL AC ) and the taste attributes (such as SSE) of accessions. When significant differences (as compared to the overall mean) in SSE and AVL AC were used for grouping of accessions, four of them ("Parris Island Cos," "Balady Cairo," "Solar," and breeding line SM13-R3) had high SSE and low AVL AC , five of them had low SSE and high AVL AC ("Taiwan," "Annapolis," "Valmaine," accessions PI 491086, and UC96US23), and one of them had both low SSE and AVL AC ("Merlot") ( Figure 3, Table S1). None of the accessions was classified as having both high SSE and AVL AC , though "Floricos" seems to be closest to this group.
In addition to taste and nutritional quality, visual perception of leaf color is another important factor affecting consumers preferences. The color of lettuce leaves is predominantly determined by the amount and the ratio of chlorophylls and anthocyanins (Simko I, 2019, unpublished results). Red-colored lettuces, such as "Annapolis," "Eruption," and "Merlot," contained the highest amounts of anthocyanins ( Figures S2 and S3). These cultivars likely also had significantly higher contents of flavonoids and phenolic compounds than green-colored lettuces (Sytar et al., 2018), though such analyses were not performed at this time. Significantly high (as compared to the overall mean) levels of chlorophylls were found in two very dark green cultivars ("Darkland" and "Parris Island Cos") ( Figures S2-S4). Opposite, very light green-colored "La Brillante" had very low contents of both chlorophylls and anthocyanins ( Figures   S2 and S3). These data confirmed a strong relationship between the content of two pigments and visual appearance of lettuce color (Gazula, Kleinhenz, Scheerens, & Ling, 2007;Simko et al., 2016).
High heritability previously detected for the contents of β-carotene, chlorophylls (Cassetari et al., 2015), and anthocyanins (Mamo et al., 2019) indicates that new cultivars and breeding lines with desirable combination of traits could be developed.

| CON CLUS IONS
Results of this study show large differences in the content of sugars, vitamins, and pigments in lettuce accessions harvested at baby leaf stage. The highest content of glucose, fructose, sucrose, and vitamin C per unit of fresh weight was detected in "Floricos," β-carotene in "Taiwan," and anthocyanins and chlorophylls in "Annapolis" and "Darkland," respectively. In contrast, the lowest content of glucose and sucrose was found in iceberg "Salinas," fructose in L. serriola accession UC96US23, vitamin C in dark green romaine PI 257288, and β-carotene in light green romaine "Solar." Very strong, positive correlations were identified among contents of the three sugars, and between β-carotene and vitamin C, and β-carotene and anthocyanins. Tests in additional environmental conditions are needed to identify the magnitude of genotype × environment interaction on the content of these compounds in baby leaf lettuce. Composition profiles of accessions together with associations between compounds identified in this study can be used by breeders, growers, and producers to select lettuces with desirable combinations of sugars, pigments, and vitamins. More detailed studies are needed to determine heritability of other traits (such as sugars, sesquiterpenoid lactone compounds, and vitamins) that affect the taste and the nutritional quality of baby leaf lettuce.

ACK N OWLED G EM ENTS
The author would like to thank L. Rosental for excellent technical assistance in various phases of this research and to Dr. Dirk Holstege for providing detailed description of methods used by the analytical laboratory. The mentioning of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA).

CO N FLI C T O F I NTE R E S T
The author has declared that there is no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.