Introduction

Echeveria species are succulent species that belong to the Crassulaceae family which has a worldwide distribution with over 1,500 species with 33 genera and has been characterized due to its rosette succulent leaves (Jimeno et al. 2013). Because of its easy propagation and beauty of its leaf rosettes and colorful flowers, this genus has been growing in popularity among houseplant and botanical collectors. This genus has over 130 species which have diversity and has been found to be natives of Mexico to Southern America and Northern Argentina (Eggli and Taylor 2002).

Although succulent crops have a high market demand due to its water-sufficient trait (Sevilla et al. 2012), there are many questions as to what appropriate environmental conditions these ornamentals would thrive on and enhance the plant quality (Rowley 1978). Succulents under this genus are known for the development of gradient colors on the margins of the lower or mature leaves of the plant (Fischer and Schaufler 1981). The colors range from light pink to red and even deep red hues that are already close to brown or black. This change in color may be due to the presence of the anthocyanin pigments (Welch et al. 2008).

One of the primary environmental factors that affect the rate of plant development is temperature. It plays a predominant role in the control and proper growth of plants (Khodorva and Conti 2013). Different crop species respond to temperature for their phenological growth such their height, diameter, leaf structure, and color as well as completion of their reproductive stages (Hatfield and Prueger 2015).

A defined range of maximum and minimum temperatures for each species comes as a boundary for observable growth. It is deemed that there must be a certain level in which the plant will achieve its optimum growth (Hatfield et al. 2011). Results of researches suggest that the level of temperature is a key player in the assimilation of nutrients, hormones and even pigments that would affect the plants’ growth rate (Adams et al. 2001). Aside from growth parameters, studies of Rabino and Mancinelli (1986) revealed that temperature affected the total amount of anthocyanin in cabbage seedlings. Studies also showed that temperature affects the biosynthesis of anthocyanin in apples (Ubi et al. 2006), grapes (Yamane et al. 2006) and also in roses (Lo Piero et al. 2005).

In plants, the presence of anthocyanin usually adds beauty and colors to the ornamentals, however, in human health, anthocyanin has an important role and considered to be a source for dietary compounds and antioxidants (Devi et al. 2012). Thus, the study aimed to determine the effects of temperature levels on the growth, development, and quality as well as the anthocyanin content of two Echeveria species in controlled environments.

Materials and Methods

Planting materials

Two Echeveria species were chosen, namely E. agavoides and E. marcus species, for the conduct of the study. These succulent species were purchased from a succulent nursery farm in Anseong Province in South Korea.

E. agavoides possesses mainly of a greener tone leaf color while E. marcus has seen to be mainly on the blueish green leaf color. Healthy and disease-free succulents approximately sixty-days old grown from the said succulent nursery and were grown at an average temperature of 18°C (± 5 °C). Experimental plants t hat were c hosen were standardized in both maturity and size. Succulents were then transferred in the greenhouse of Sahmyook University, Seoul, South Korea at the start of the experiment.

Experimental design, treatments and growth conditions

The experiment was laid out in a completely randomized design having three treatments with four replications (six plants per replication), a total of seventy-two plants per species. Three temperature levels were 10°C, 20°C, and 30°C (± 1 °C). All experimental plants w ere placed i nside three plant growth chambers (KGC-175 VH, Koenic Ltd., South Korea). The relative humidity was set at 65%. There was a 14-hour light period and 10-hour dark period. The light condition was set at 75 μmol･m^-2s^-1.

Data gathered

Plant height and diameter

The succulents’ height and diameter data were obtained using a digital Vernier caliper (SR 44, Blue Bird Co., Japan). The p lant h eight was taken from t he b ase of t he soil to the highest part of the plant. The plant diameter was taken from the largest possible measurement from each opposing leaf tips. The data was taken at the termination of the study which was about 6 weeks.

Visual quality rating

After the exposure of succulents to their respective treatments, plants were subjected to the visual quality rating (VQR). Twelve (12) respondents or judges were asked to rate the representative plants per t reatment and case study. A modified visual score (Wang et al. 2005) was used with the corresponding visual grading (1 - 5) where, 1: very poor (leaves are chlorotic or irregular in color), 2: low (leaves are light green and no red color), 3: good (leaves are green with pinkish color), 4: very good (leaves are green, with light red color), 5: excellent (leaves are rich green with intense or distinguishable red color. Visual quality rating (VQR) is a sensory analysis, which has been widely used in horticultural crops such as fruits, vegetables, and ornamental crops. The use of visual quality rating has been adapted as a tool for measurement as a perception of the human eye (Boumaza et al. 2009).

Hunter’s Lab

The color quality of leaves was determined using the Hunter’s Lab. This was gathered with the use of a hand-held spectrophotometer (Konica Minolta Spectrophotometer CM2600d, Japan) which determines three color coordinates namely the L* a* b* color space to indicate lightness hue and saturation of colors. Lightness is indicated by the L* while the chromaticity coordinates are represented by a* and b* values. The lightness of the color is represented by the L* color value with a 0 to 100 value range. A higher positive value would indicate a lighter color and a lower value indicates a darker color. Positive a* values indicate the red direction while a negative a* value indicates the green direction. On the other hand, a positive b* value indicates a yellow direction while a negative value indicates a blue direction.

One leaf of each plant was tagged to trace color changes. The color value was measured by choosing the area within the tagged leaf which were located at 1cm from the margin of the top leaf surface (adaxial) and the underside of the leaf (abaxial).

Anthocyanin analysis

A modified quantitative method for anthocyanin (Fuleki and Francis 1968) was used in this study by gathering 1 inch from the tip of a tagged succulent leaf. One-gram fresh-cut leaf samples were macerated using a mortar and pestle. The macerated sample was added with 1 ml of 95 % ethanol and 1.5 N HCl (85:15) which served as the extracting solvent. The mixed solution was transferred to a separate container. Samples were then centrifuged at 13,000 rpm at 4°C using the Micro Refrigerated Centrifuge Smart R17 (Hanil Science Co. Ltd., Seoul, South Korea). Samples were then stored and refrigerated overnight at 4°C to solidify the pulp residues at the bottom of the tubes after the centrifuge process. This procedure was made in order for the residues to remain at the base at the tube and easy extraction of the liquid extract alone.

Samples were taken out of the refrigerator and were fluids from the tubes without plant residues were placed in a microplate that was then analyzed for a full-spectrum UV/Vis absorbance at 535nm using the Fluostar Optima Microplate Reader (BMG Labtech, Ortenberg, Germany). A solution was placed at the primary or first plate entry.

Image analysis

Photos were taken using a digital single-lens reflex camera (Canon 750D, Japan) with the same aperture, brightness, and contrast at the same distance with a pixel size of 1080 p. Individual images were cropped to show the succulents alone without the pots and were processed using the Image-Pro Premier ver. 9.3 (Media Cybernetics, Inc., USA). Smart segmentation was applied to individual representative images to determine the ratio of the colors green and red pigments. Colored overlays of identified colors were presented as bases for color identification.

Statistical analysis

Data gathering was done every two weeks for a month. Aside from the Hunter’s Lab and anthocyanin content analysis, growth and development parameters were also collected. Statistical analyses were conducted using Statistical Product and Service Solutions for Windows, version 16.0 (SPSS Inc., Japan). The data were analyzed using analysis of variance (ANOVA), and the differences between the means were tested using Duncan’s multiple range test (p < 0.05).

Results

Plant height and diameter

Based on the statistical analysis, plant height and diameter was highly affected by temperature levels for both species. Data for plant height is shown in Table 1 while plant diameter is shown in Table 2.

Results revealed that succulent plant species of E. agavoides that were subjected under high temperatures of 20°C and 30°C which had 42.36 mm and 42.33 mm. These temperature levels did not significantly differ from each other. Shortest plants were observed from the lowest temperature level of 10°C which gave 41.72 mm. Different results were observed with those of E. marcus wherein the lowest and highest temperatures of 10°C and 30°C gave the shortest plants with 40.30 mm and 41.81 mm. Tallest plants were observed from those of 20°C having 42.54 mm which significantly differed from other temperature levels.

In these results, it may be noted that despite belonging to the same family, E. agavoides has a higher tolerance for high temperatures. While E. marcus plant height may be hindered by too low or too high temperatures.

E. agavoides species plant diameter was significantly affected by different temperature levels. Results showed that plants that were exposed to 20°C and 30°C were not significantly different from each other having a plant diameter of 94.46 mm and 93.40 mm, respectively. Shortest plant diameter was measured from those plants subjected to 10°C with 79.42 mm. For E. marcus, it was observed that those grown under 20°C highly gave the largest plants with 79.94 mm. Results also suggested that a low temperature of 10°C and a high temperature of 30°C were not significantly different from each other which had 73.72 mm and 72.53 mm, respectively.

A similar trend can be observed for both parameters of each succulent species. Although with different significant levels, it was also noted that 20°C was consistent with the results for growth parameters. It may be said that E. agavoides is sensitive to colder temperature while E. marcus may is sensitive to both lower temperature and too high temperatures.

Visual quality rating

The results for the visual quality rating of Echeveria species in response to temperature levels are shown in Table 3. Results revealed that temperature levels highly affected the visual quality rating of Echeveria species.

For E. agavoides, 10°C and 20°C were not significantly different from each other with the highest visual quality rating of 3.67 and 3.75, respectively, and these ratings are described as very good quality. The results for 10°C and 20°C, however, did not significantly differ from each other. These were followed by those treated with 30°C with a visual quality rating of 2.08 described as a low quality which was significantly lower compared to the two previously mentioned temperature levels.

For E. marcus, succulents that were exposed to 20°C had a rating of 3.75 followed by those treated under 10°C which had a visual quality rating of 3.67. The lowest visual quality rating was observed from in those grown under 30°C with 2.42 described as low quality.

Hunter’s Lab

Statistical results of the analysis showed that only L* value was significantly affected by the different temperature levels. Results of the Hunter’s Lab of Echeveria species are shown on two separate tables below.

Succulent plants that were grown under 20°C gave the highest value for the brightness of color or hunter L* value with 47.36. These were then followed by those plants exposed with 10°C with 40.31 for hunter L* value and 30°C with an L* hunter value of 37.95. However, these two treatments did not significantly differ from each other for E. agavoides species in an adaxial portion of the leaves. Hunter a* was significantly affected by the temperature which had a similar trend to those of L* value. However, b* values were not significantly different from each other (Table 4).

On the abaxial portion of the tagged leaves, the highest hunter L* value was taken from those of 10°C and 20°C which did not significantly differed from each other. These were then followed by those succulents grown under 30°C with a Hunter L* value of 35.72 which means that it had a darker color value compared to other temperature levels.

For E. marcus, similar results were also observed whereby only hunter L* values were significantly affected by the temperature levels (Table 5). Results revealed that low and high temperatures had a higher lightness value with 42.12 for 10°C and 40.51 for 30°C which did not significantly differ with each other for the top portions of the leaves. For the bottom portions, 10°C and 20°C did not significantly differ from each other with 34.34 and 34.25 hunter *L values. Hunter a* was significantly affected by the temperature which had a similar trend to those of L* value. However, b* values were not significantly different from each other.

Anthocyanin analysis

Results of the anthocyanin analysis of Echeveria species in response to different temperature levels are shown in Table 6. The average anthocyanin content was significantly affected by temperature levels for both E. agavoides and E. marcus species.

Results showed that exposure of E. agavoides plants to low temperatures of 10°C had the highest anthocyanin content with an amount of 0.92 μg/g FW. This result, however, was significantly the same with plants that were subjected to 20°C with 0.89 μg/g FW. The high temperature of 30°C gave the lowest anthocyanin content among temperature levels with an amount of 0.54 μg/g FW which significantly differed from the other temperature levels.

Succulent plants of E. marcus were also significantly affected with the use of different temperature levels on its anthocyanin content. E. marcus species which were grown under 20°C had the highest anthocyanin content amounting to 0.63 μg/g FW which was significantly higher compared with those of 10°C and 30°C with more or less the same average amount of 0.49 μg/g FW. Statistical analysis also revealed that the use of 10°C was significantly different from those results of 30°C. Thus, the use of too low temperatures or too high temperatures is found to reduce the anthocyanin content of the species.

It may be evident that higher anthocyanin content is taken from those with E. agavoides compared to those of E. marcus by a few percentages. This may be due to the fact that E. agavoides may be more sensitive to temperature levels, thus more production of the anthocyanin has been taken. On the other hand, E. marcus has been known as a succulent that is more tolerant of cooler or hotter climates.

Image analysis

The image analysis was done to determine the ratio of red and green pixels and to quantify these data from a raw image taken at the termination of the study. The image was subjected to the smart segmentation and histogram of the image.

Smart segmentation of images coupled with the original images for three temperature levels and their corresponding histogram results is presented on Fig. 1 for E. agavoides and Fig. 2 for E. marcus.

The results for smart segmentation for the red and green pixels including the ratio of pixels in a raw image is shown in T able 7 . Results s uggest t hat the smart segmentation results of green and red pixel ratio were significantly affected by the different temperature levels for both Echeveria species.

E. agavoides succulents subjected to 20°C gave the highest red pixel count of 936.6 which accounts to 33.45% of the t otal p ixel i n an image. However, these results did not significantly differ from those of 10°C with a 532.9-pixel count which accounts for 30.31% of the total pixel. The lowest recorded number of a pixel was found in the plant that was exposed to 30°C which has 37.3-pixel count accounting for only 0.04% of the total image. This means that there are more prominent green pixels compared to the red ones. An increase of green pixels was noticed when there was a l ower temperature f or succulents.

Succulent species of E. marcus pixel count and percentage of red and green pixel ratio was significantly affected by temperature levels. Among temperature levels that were used to grow E. marcus plants, the highest pixel count was taken from those that were subjected to 20°C with a pixel count of 632.5 which accounts for 39.46%. This result was not significantly different from those of 10°C with a red pixel count of 388.0 which accounts for 29.43%. The lowest amount of pixel count of 52.9 only was taken from those succulents that were grown under 30°C which accounts for 0.94% of the total segmented image.

It was noted that the green pixel increases when the temperature is too low or when too high for E. marcus. However, higher temperatures would significantly increase green pixel compared to lower temperatures. Table 7

Discussion

Many environmental factors act singly or interact to affect plant productivity (Haferkamp 1988) thus, basic knowledge on the changes or manipulation of this environmental factor must be taken to advantage to provide an optimum and conducive growing conditions for the crop. Temperature is considered one of the vital component as an environmental factor for plant growth and development.

Based on the study, results revealed that for E. agavoides, succulents grown in 10°C had the shortest and smallest in diameter compared to those of 20°C and 30°C. A review done by Hatfield and Prueger (2015) stated that the rate of phenological development in warm temperatures, especially in controlled environment studies, has found to increase height, weight, diameter, and other growth parameters. This is because most plant species require a higher optimum temperature for vegetative development such as corn (Warrington and Kanemasu 1982), sweet orange (Ribeiro et al. 2012) and pineapple (Friend and Lydon 1979) among others. Studies of Gent and Enoch (1983), using a mathematical model, revealed that increased temperature or warm environments coupled with nonstructural carbohydrate has a corresponding increased dry matter. They added that with reaching the high optimum temperature levels will also increase rates of photosynthesis, growth and maintains respiration. This was the same case in Pineapple wherein the use of 30/20°C presented higher carbon metabolism, photosynthetic rates, higher shoot growth and increased root growth rates compared to lower temperature levels. Went (1953) further discussed the effects of temperatures on plant growth of which he stated that very low and very high temperatures affect the physio-chemical process involved in its development and at times may cause injury effects.

According to Pennisi et al. (2016), there are significant negative effects that may harm plants in low temperatures including damaged foliage, wilted stature, produced misshapen new growth, discolored foliage and had a portion or the whole plant dies. They also added that the effects of low temperature may also be unseen by the naked eye but may manifest in the later part of growth and development through delayed flowering or stunted growth. This may be due the interruption and damage along pathways of water, nutrients and other important caused by freezing of plant cells. On the other hand, high temperatures or heat stress, plants are said to go through three mechanisms including excessive membrane fluidity, disruption of protein function and turnover, and metabolic imbalances (Farrell 2015). With these mechanisms occurring in the plant, the net photosynthesis is first to be inhibited (Allakhverdiev et al. 2008). In the case of E. agavoides, low temperature at 10°C prompted delayed or stunted growth in succulents.

Results showed that growing E. agavoides in temperatures lower than 20°C had the highest anthocyanin content. It is an established theory that temperature affects the gene expression of enzymes involved in producing anthocyanin (Christie et al. 1994;Leyva et al. 1995;Shvarts et al. 1997). Rehman et al. (2017) reported that exposure to high temperatures of Malus profusion induced anthocyanin inhibition and activated its degradation. Proponents of the study explain that exposure to high temperatures of 33 - 25°C increased the expression of anthocyanin repressors MYBs and MpMYB15 and reduced MpVHA-B1 and MpVHA-B2 transcriptors which are involved in the vascular transportation of anthocyanin. These were evident in the studies of Mori et al. (2007) which suggested that the exposure of grapes in high temperature of a maximum of 35°C reduced the total anthocyanin content to less than half of the control (20 - 25°C).

In the studies of Lo Piero et al. (2005) with red oranges, it was concluded that low temperatures could also reduce the expression of anthocyanin, especially during long exposures. On the other hand, Solecka et al. (1999) reported that anthocyanins have increased when subjecting oilseed rape leaves to low temperatures which were explained to be involved in the process of protecting mesophyll cells against cold environments. This may be the reason for which there was a high anthocyanin content for plants treated at 10°C that was comparable to those of 20°C in E. agavoides.

The anthocyanin content in plants are generally the reason for the change in color in plants and have been specifically studied in ornamental plants as changes of color in the foliage or inflorescence can increase visual quality. The use of Hunter’s Lab is able to differentiate the lightness of the color and the difference between two opposing colors such as that or red/green and yellow/blue (Konica Minolta 2018). Results showed that there was no significant difference between a* and b* values both adaxial and abaxial leaf. This may be because the difference was exiguous between the hues, however, this was not true to the intensity (lightness or deepness) of the colors which was found to be significantly different between temperature levels. Studies of Shisa and Takano (1964) suggested that the effects of temperature on the epidermal cells have affected the lightness or deepness of red coloration of rose flower petals.