PHYSIOLOGICAL FUNCTION OF NON-GLANDULAR TRICHOMES IN RED CLOVER ( TRIFOLIUM PRATENSE L . )

The objective of the trial was to study the physiological function and potential ecological significance of non-glandular trichomes in red clover based on their relation to the leaf transpiration intensity. The transpiration rate, relative water content and water deficit of the leaves were recorded in field-grown, high and slightly pubescent genotypes. According to results, the presence of nonglandular epidermal trichomes on the leaf surface of red clover was a micromorphological trait related to the regulation of leaf transpiration in stressful conditions. Non-glandular trichomes significantly enhanced water retention of the leaf tissue at high leaf water deficit. In the environmental conditions, limiting leaf evaporation, the pubescent genotypes displayed higher levels of transpiration. It can be suggested that the non-glandular trichomes in red clover are morphological structures supporting the function of leaf stoma apparatus. A positive relation between the degree of pubescence and resistance to powdery mildew was established under natural conditions.


Introduction
A great diversity of morpho-types has been observed in red clover with regard to the degree of pubescence of stems, petioles and leaves with non-glandular hairs (trichomes).The density of this type of trichomes on generative stems was a main trait of distinctness, homogeneity and stability of red clover cultivars.Nonglandular trichomes in red clover were with unknown physiological function.Their formation was confirmed by natural selection.It was found that it was a mechanism of resistance to potato leafhopper (Kusmiyati, 1995;Taylor, 2008) and air pollution with harmful gases (Sharma, 1977).In these cases, non-glandular trichomes functioned as a mechanical barrier, filter and isolation of leaf surface.In many plant species, leaf pubescence increased radiation reflectance and created the microlayer of more humid air around the leaf tissue, thus reducing leaf temperature, transpiration and water use (Gausman and Cardenas, 1973;Clawson et al., 1986;Peter and Shanower, 1998).In red clover, such function of nonglandular trichomes was presumable (Bowley and Lackie, 1989).Abaxial and adaxial trichome densities in genus Trifolium were also investigated as characteristics of epidermal leaf surface which can influence the water capture and thereby increase the sensitivity of plants to fungal diseases, as according to the results such hypothesis was rejected (Bradley et al., 2003).
In some cases, breeding was conducted against pubescence of red clover with non-glandular trichomes as they made drying difficult and caused dusting of hay (Taylor, 2008;Hejduk and Knot, 2010).However, in terms of that, red clover had many other fields of use, and its persistence was associated primarily with resistance to stress, and the breeding against the traits of increasing stability and longevity of individual plants can be defined as unfounded.
The objective of the trial was to study the physiological function and potential ecological significance of non-glandular trichomes in red clover based on their relation to the leaf transpiration intensity.

Material and Methods
The study was conducted by measuring the transpiration rate (mg/min/g), relative water content (%) and water deficit (%, 100 -relative water content) of the leaves from field-grown, high pubescent (nonglandular-haired) red clover half-sibs families E3 and E4, and the slightly pubescent 'Nika 11' cultivar (control).The maternal plant of family E3 originated from wild growing Bulgarian ecotype, and of E4 -from synthetic population including Canadian germplasm.They were selected according to productivity and resistance to powdery mildew (Erysiphe polygoni) in the previous period (2008)(2009)(2010).The families and the control cultivar were sown in the spring in 2012, in randomized rows using the length of 5 m, with four replications at the Experimental Station of Soybean, Northern Bulgaria (latitude 43°24' N, longitude 25°32' E, altitude 144 m).
The experiment was conducted during June-September in 2012 and 2013.Seven measurements were conducted, each measurement was preceded by a 10day period without rainfall.The intensity of transpiration, the relative water content and water deficit of leaf were determined by a weighting method (Georgiev, 2004).In the afternoon hours of the day, between 1 pm and 2 pm, 12 compound leaves of the same physiological age of each accession were weighed immediately after harvesting; after 5-minute exposition to direct sunlight; after 24-hour staying in the water in a state of full turgor; and in dry conditions after drying to a constant weight for 3-4 hours at a temperature of 60°C.The degree of pubescence of adaxial and abaxial leaf surfaces and petioles was determined under binocular, by five-grade scale, according to the methodology of UPOV (1-lacking 5-very high).It was determined in the second cut for both experimental years.The intensity of infestation by powdery mildew (Erysiphe polygoni) in natural background of infestation was assessed in September in both experimental years.The susceptibility of red clover genotypes to powdery mildew was evaluated by a fourscope system, depending on the percentage of leaves with typical symptoms (Segarra et al., 2009).Based on the highest scope during that period, the genotypes were rated as (1) resistant -leaves remained healthy, (2) slightly sensitive -15% of the leaves had spots, (3) moderately sensitive -up to 30% of the leaves had symptoms, (4) severely sensitive -up to 45% of the leaves had symptoms.
The data were analysed by ANOVA.Regarding the traits -intensity of transpiration and leaf water deficit, the average values for the populations were compared using Least Significant Difference at 5% level of probability (LSD 5%).Z test was used for evaluation of the differences between the average rating assessments of the genotypes according to the degree of pubescence.

Results and Discussion
Families E3 and E4 fell within a homogeneous group according to the degree of pubescence with abaxial trichomes and were significantly different with regard to the density of trichomes on the adaxial leaf surface and petioles -it is higher in E3 (P<0.05,Table 1).Disintegration according to the trait in the studied families was very low, which confirmed the thesis of two-locus dominant-recessive model of its inheritance (Bowley and Lackie, 1989).
Table1.Density and direction of non-glandular trichomes in genotypes of red clover.Significant differences in the intensity of leaf transpiration of heavily and slightly pubescent phenotypes were observed in five of the seven measurements.
The rate of transpiration in leaves in E3 was lower than that of the control cultivar in all measurements conducted at leaf water deficit of over 40% (Table 2).The other family -E4 had an identical performance, but its differences compared to the control cultivar were insignificant in most measurements, which correlated to the slight pubescence of adaxial leaf surface.Data from the first and second year of plant life were unidirectional, which gave a reason to assume that, in conditions of severe drought, leaf epidermal characteristics (or the pubescence with nonglandular trichomes in this case) had greater influence on the rate of leaf transpiration, compared to the volume of the root system, which varied depending on age and genotypic effects.It should be noted that, in a severe drought, the leaf of clover was closed along the axis of the middle leaf and the abaxial leaf surface, which was more pubescent (Table 1), because it was the surface exposed to sunshine.In terms of lower drought resistance of red clover and that of leguminous grasses, the function to increase yield and quality of the grass swards in summer and autumn was assigned.In addition, the relation of the pubescence with nonglandular trichomes with drought resistance made trichomes an important micromorphological trait.The pubescence effect on water retention of leaves was insignificant when the plants were under moderate water stress (water deficit = 30-35%) -measurements 1 and 5.The fourth measurement showed that the leaf epidermal surface was influenced by factors that caused closing of the stoma and slowing the transpiration -high air humidity (75%), relatively low daily temperature (23.2 o C), very low leaf water deficit (16-19%) and moderate to strong wind (6 m/s).In this measurement the pubescent phenotypes transpired more intensively, and the difference in comparison with the control genotype was significant for E3 (P<0.05).It can be assumed that in this case trichomes isolated the leaf surface and stoma apparatus from the direct influence of the mentioned factors and affected the gradient of water potential in the leaf/atmosphere system, providing a constant level of transpiration.The presented data gave reason to form the hypothesis that the nonglandular trichomes in red clover were an element of compensating or providing a mechanism in terms of the function of stoma apparatus.
Red clover was distinguished with very good aftermath ability, and the infestation by powdery mildew (Erysiphe polygoni) in late summer and autumn can greatly reduce the yield and quality of aftermath, and winter survival of plants.The maternal genotypes of families E3 and E4 were also selected according to resistance to powdery mildew.In both experimental years the pubescent phenotypes in natural background of infestation show resistance to the disease while the control cultivar was moderately sensitive (Table 3).This gave reason to investigate the effect of pubescence with non-glandular trichomes on the sensitivity of leaf epidermal tissue to this pathogen.

Conclusion
According to the results, the non-glandular epidermal trichomes on the leaf surface of red clover were a micromorphological trait related to the regulation of leaf transpiration in stressful conditions.At high leaf water deficit, non-glandular trichomes significantly enhanced water retention of the leaf tissue.In the environmental conditions, limiting leaf evaporation, the pubescent genotypes displayed higher levels of transpiration.It can be suggested that the non-glandular trichomes in red clover are morphological structures supporting the function of leaf stoma apparatus.A relation of high degree of pubescence with resistance to powdery mildew was observed under natural background infestation and this relation should be further investigated.

Table 2 .
Intensity of leaf transpiration, leaf water deficit and meteorological conditions during the experiment.