Variability in Epicuticular Wax in 35 Woody Plants in Linares, Northeast Mexico

Leaves contain waxy coating. Environmental conditions may strongly influence the quantity, composition, and morphology of the waxy coverings of leaf surfaces. Wax and cutin function as primary components of the leaf cuticle covering the leaf epidermal cells [1]. Waxes are composed of long-chain paraffins, alcohols, ketones, esters and free fatty acids in proportions determined by both genetic capability and environmental factors [2-4]. Epicuticular wax enhances the reflectance of visible and near infrared radiation from leaf surface thereby reducing net radiation and cuticular transpiration and seems to contribute drought resistance of plants [5-7]. These waxes also impart resistance to plants to absorption and penetration of foliarapplied herbicides [4,8,9]. It has been reported that leaves of mesquite (Prosopis spp.) develop a thick waxy cuticle [10,11]. An increase in wax accumulation was observed with leaf maturity in velvet mesquite (P. velutinu), while [12] observed most rapid wax accumulation on honey mesquite (P. glandulosa) with early leaf development and expansion.


Introduction
Leaves contain waxy coating. Environmental conditions may strongly influence the quantity, composition, and morphology of the waxy coverings of leaf surfaces. Wax and cutin function as primary components of the leaf cuticle covering the leaf epidermal cells [1]. Waxes are composed of long-chain paraffins, alcohols, ketones, esters and free fatty acids in proportions determined by both genetic capability and environmental factors [2][3][4]. Epicuticular wax enhances the reflectance of visible and near infrared radiation from leaf surface thereby reducing net radiation and cuticular transpiration and seems to contribute drought resistance of plants [5][6][7]. These waxes also impart resistance to plants to absorption and penetration of foliarapplied herbicides [4,8,9]. It has been reported that leaves of mesquite (Prosopis spp.) develop a thick waxy cuticle [10,11]. An increase in wax accumulation was observed with leaf maturity in velvet mesquite (P. velutinu), while [12] observed most rapid wax accumulation on honey mesquite (P. glandulosa) with early leaf development and expansion.
Researches undertaken on epicuticular wax under controlled condition have documented various factors such as, light level [13,14] photoperiod [15] temperature [14,16] and water stress [17][18][19] influencing the wax contents in leaves. Variations in wax properties can affect the cuticuar functions such as, regulation of gas exchange and transpiration, protection against pathogens, and absorption of foliar-applied chemicals as herbicides in agriculture. Few studies are available on variation in epicuticular wax characteristics among plants growing in the field. It has been reported by [20] that the composition and quantity of epicuticular waxes of shrubs in a semiarid environment showed seasonal variations in temperature and rainfall, and both cuticular and total transpiration appeared to be affected with changes in wax composition. Ecotypic variation in the quantity and composition of waxes on leaves of salt cedar (Tamarix pentandra Pall.) was considered as the basis of differences among populations in sensitivity to herbicide sprays [4]. Leaves of a cabbage cultivar (Brassica oleracea L. var. capitata), tolerant of foliar applications of the herbicide nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) were more heavily waxed than an intolerant cultivar [21]. The heavier wax deposit on leaves of the tolerant cultivar substantially decreased the rate and extent of herbicide penetration of the cuticle. The development of leaf cuticle of velvet mesquite was investigated [22] and detected well defined crystalline wax structures on even the youngest leaves. The amount of wax appeared to increase with leaf maturity, and a dendritic-shaped wax plate was formed in July in addition to the small, linear structure already present. The epicuticular wax structure of five Prosopis species, consisted of an aggregate coating of rods and dendritic platelets [23].

Materials and Methods
The study was under taken in thirty five woody trees species in the summer season (June 2015) at the experimental station of Facultad de Ciencias Forestales, Universidad Autonoma de Nuevo Leon, located in the municipality of Linares. Fresh leaves from different woody plant species were collected from the forest. Then leaflets were separated individually to complete a sub sample having an approximate surface area of 100 cm 2 which is determined by a leaf area meter. Sub-samples were gently rinsed in distilled water to remove foreign material, then air dried, and then placed in a beaker with 40 ml of analytical grade chloroform (99% pure) heated to 45°C. After 30s the chloroform was poured into preweighed foil pans which were then placed in a wellventilated laboratory and evaporated to dryness for 24 hours. Foil pans were then reweighed to quantify the amount of residual wax. Amount of wax for a field sample was the mean of 5 laboratory replications. Wax

Abstract
A study has been undertaken on epicuticular wax on the leaves of 35 woody species in Linares, Northeast Mexico at the experimental station of Facultad de Ciencias Forestales, Universidad Autonoma de Nuevo Leon, located in the municipality of Linares. Considerable variation in wax accumulation was found among species showing prominent interspecific variation. Wax load varied from 11.18 to 702.04 µg/cm 2 among species studied during summer. Few species selected with high epicuticular wax viz, Forestiera angustifolia (702.04 µg/cm 2 ), Diospyros texana (607.65 µg/cm 2 ), Bernardia myricifolia (437.53 µg/cm 2 ), Leucophylum leucocephala (388.50 µg/cm 2 ), during summer which could well be adapted under semi-arid environments for their efficiency in the reflection of radiation load, reduced transpiration, gas exchange and probably impart drought resistance. The large variations in epicuticular wax could be related to their physiological functions such as transpiration, gas exchange, water relations etc.

Results and Discussion
The present study indicated the presence of large variations in the wax contents among the different woody tree species studied. The results obtained are presented below. Table 1 gives the summary of Kwalis analysis. Table 2 presents the epicuticular wax content in 35 woody species. The species showed significant variation in epicuticular wax contents among these 35 species. These variations are seen in Figure 1. Most of the tree species has shown a large variation in the wax contents. The wax contents in these thirty five woody species ranged from 11.18 to 702.04 µg/cm 2 . On the basis of total epicuticular wax contents species are selected and categorized into high, medium and low wax content species.   [24] demonstrated variability in wax content among different species of Prosopis and in different seasons.
The large variation in epicuticular wax observed in the present study could be related to physiological function and adaptation of the species to varying environments. With respect to the physic-chemical and physiological functions it has been reported that epicuticular wax enhances the reflectance of visible and near infrared radiation from leaf surface, which in turn reduce net radiation and cuticular transpiration and seem to contribute drought resistance of plants [5][6][7]. The presence of wax impart resistance of plants to absorption and penetration of foliar-applied herbicides [4,8,9]. We selected few species with high epicuticular wax viz, Forestiera angustifolia (702.04 µg/cm 2 ), Diospyros texana (607.65 µg/cm 2 ), Bernardia myricifolia (437.53 µg/cm 2 ), Leucophylum leucocephala (388.50 µg/cm 2 ), during summer which could well be adapted under semi-arid environments for their efficiency in the reflection of radiation load, reduced transpiration, gas exchange and probably impart drought resistance. They could impart resistance to insect attack and herbicide penetration as reported by few authors [4,8,9].
Few studies are available on variation in epicuticular wax characteristics among plants growing in the field. The large variations in epicuticular wax could be related to their physiological functions such as transpiration, gas exchange, water relations etc. The result of epicuticular wax of the species reported in the present study was undertaken in the summer. The wax content was reported to vary in different environments [13,14], photoperiod [15], temperature [14,16] and water stress [17][18][19]. It has been reported by [20] that the composition and quantity of epicuticular waxes of shrubs in a semiarid environment showed seasonal variations in temperature and rainfall, and both cuticular and total transpiration appeared to be affected with changes in wax composition. We claim this is the first research ever dealt with the analysis of epicuticular wax in a large number of woody species.

Conclusion
It is well documented that epicuticular wax on leaf surface play an important role in the physiological function and adaptation of trees in an ecosisystem with respect to loss of transpiration owing to the reflection of solar radiation, and other aspects such as gas exchange, water stress, herbicide resistance etc. The present study show a large variations in the contents of epicuticular wax thereby giving opportunity to select species for future research with respect to the adaptation of these species to semiarid environment. The species selected with high epicuticular wax contents viz., Forestiera angustifolia (702.04 µg/cm 2 ), Diospyros texana (607.65 µg/cm 2 ), Bernardia myricifolia (437.53 µg/ cm 2 ), Leucophylum leucocephala (388.50 µg/cm 2 ) may be well adapted to the semi-arid conditions. Future research needs to be directed on these selected species with special reference to their physiological function and adaptation to water stress.