PHYSIOLOGICAL FUNCTION OF NON-GLANDULAR TRICHOMES IN RED CLOVER (TRIFOLIUM PRATENSE L.) Acad. G. Bonchev Str., Bldg. 21, 1113 Sofia, Bulgaria

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1 Journal of Agricultural Sciences Vol. 58, No. 3, 2013 Pages DOI: /JAS N UDC: Preliminary communication PHYSIOLOGICAL FUNCTION OF NON-GLANDULAR TRICHOMES IN RED CLOVER (TRIFOLIUM PRATENSE L.) Galina K. Naydenova 1* and Georgi I. Georgiev 2 1 Experimental Station of Soybean, 61 Ruski, 5200 Pavlikeni, Bulgaria 2 Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bldg. 21, 1113 Sofia, Bulgaria Abstract: 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 and resistance to powdery mildew was established under natural conditions. Key words: red clover, non-glandular trichomes, intensity of leaf transpiration. Introduction A great diversity of morpho-types has been observed in red clover with regard to the degree of 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 increased radiation reflectance and created the * Corresponding author: gmvg@abv.bg

2 218 Galina K. Naydenova and Georgi I. Georgiev 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 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 ( ). 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 N, longitude E, altitude 144 m). The experiment was conducted during June September in 2012 and Seven measurements were conducted, each measurement was preceded by a 10- day 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 of adaxial and abaxial leaf surfaces and petioles was determined under binocular, by

3 Physiological function of non-glandular trichomes in red clover 219 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. Results and Discussion Families E3 and E4 fell within a homogeneous group according to the degree of 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. Nika 11 (Control) E3 E4 Z min for degree of Genotypes Degree of scope Direction* Degree of scope Direction Degree of scope Direction E3 compared to Е4 Е3 and E4 compared to Nika 11 (Control) Adaxial trichomes <Z 0.05 <Z 0.05 Abaxial trichomes >Z 0.05 <Z 0.05 Petiole trichomes <Z 0.05 <Z 0.05 *1 lodged, 2 semi-straight, 3 straight. Significant differences in the intensity of leaf transpiration of heavily and slightly pubescent phenotypes were observed in five of the seven measurements.

4 220 Galina K. Naydenova and Georgi I. Georgiev 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 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 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 with nonglandular trichomes with drought resistance made trichomes an important micromorphological trait. Table 2. Intensity of leaf transpiration, leaf water deficit and meteorological conditions during the experiment. Measurement Date 11 July 28 Aug. 10 Sept. 26 June 30 July 18 Aug. 5 Sept Intensity of transpiration (mg/min/g) Nika 11 Control b 2.24 b 7.01 a b 3.09 b E a 0.89 a b a 2.14 a E ab 1.75 ab 8.36 ab a 2.44 ab LSD P n.s. < 0.05 = 0.05 < 0.05 n.s. < < 0.05 Leaf water deficit (%) Nika 11 Control E E LSD P n.s. n.s. n.s. n.s. n.s. <0.05 n.s. Meteorological conditions Temperature at 2 pm ( C) Relative humidity at 2 pm (%) Wind speed (m/s) Rainfalls in previous 1- month period (mm) Means with the same letter are not significantly different according to LSD test at P 0.05.

5 Physiological function of non-glandular trichomes in red clover 221 The 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 with non-glandular trichomes on the sensitivity of leaf epidermal tissue to this pathogen. Table 3. Susceptibility of heavily pubescent red clover E3 and E4 and of slightly pubescent cultivar Nika 11 to powdery mildew (Erysiphe polygoni). Genotypes Annual rating (1 4) * September 2012 September 2013 Nika 11 (Control) 3 3 E3 1 1 E4 1 1 * 1 resistant; 2 slightly susceptible; 3 moderately susceptible; 4 severely susceptible. 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

6 222 Galina K. Naydenova and Georgi I. Georgiev 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 with resistance to powdery mildew was observed under natural background infestation and this relation should be further investigated. References Bowley, S., Lackie, S. (1989): Genetics of nonglandular stem trichomes in red clover (Trifolium pratense L.) Journal of Heredity 80(6): Bradley, D., Gilbert, G., Parker, I. (2003): Susceptibility of clover species to fungal infection: the interaction of leaf surface traits and environment. American Journal of Botany 90(6): Clawson, K.L., Specht, J.E., Blad, B.L., Garay, A.F. (1986): Water use efficiency in soybean density isolines - a calculation procedure for estimating daily values. Agronomy Journal 78: Gausman, H., Cardenas, R. (1973): Light reflectance by leaflets of pubescent, normal, and glabrous soybean lines. Agronomy Journal 65: Georgiev, G. (2004): Environmental plant physiology, Sofia. Hejduk, S., Knot, P. (2010): Effect of provenance and ploidity of red clover varieties on productivity, persistence and growth pattern in mixture with grasses. Plant Soil and Environment 56(3): Kusmiyati, F. (1995): Pubescence in red clover: its inheritance and its relationship to potato leafhopper resistance. McGill University, Montreal, Canada. Peter, J., Shanower, T. (1998): Plant glandular trichomes chemical factories with many potential uses. Resonance 3(3): Segarra, G., Reis, M., Casanova, E., Trillas, M. (2009): Control of powdery mildew (Erysiphe polygoni) in tomato by foliar applications of compost tea. Journal of Plant Pathology 91 (3): Sharma, G. (1977): Cuticular feature as indicator of environmental pollution. Environmental Pollution 5: Sharma, G., Butler, J. (1974): Environmental pollution: Leaf cuticular patterns in Trifolium pratense L. Annals of Botany 39(5): Taylor, N. (2008): A century of clover breeding developments in the United States. Crop Science 48:1-13. UPOV (2001): Guidelines for the conduct of tests for distinctness, uniformity and stability TG/5/7. Red clover (Trifolium pratense L.). Received: October 5, 2013 Accepted: December 2, 2013

7 Physiological function of non-glandular trichomes in red clover 223 FIZIOLOŠKA FUNKCIJA NEŽLEZDANIH TRIHOMA KOD CRVENE DETELINE (TRIFOLIUM PRATENSE L.) Galina K. Naydenova 1* i Georgi I. Georgiev 2 1 Ogledna stanica za soju, 61 Ruski, 5200 Pavlikeni, Bugarska 2 Institut za fiziologiju i genetiku biljaka, Bugarska akademija nauka, Acad. G. Bonchev 21, 1113 Sofija, Bugarska R e z i m e Cilj ovog ogleda je bio proučavanje fiziološke funkcije i potencijalnog ekološkog značaja nežlezdanih trihoma kod crvene deteline na osnovu njihovog odnosa prema intenzitetu transpiracije lista. Intenzitet transpiracije, relativni sadržaj vode i deficit vode u listovima su praćeni kod genotipova sa izraženim i slabo prisutnim trihomima, gajenim u poljskim uslovima. Prema rezultatima, prisustvo nežlezdanih epidermalnih trihoma na površini listova crvene deteline je mikromorfološka osobina koja je povezana sa regulacijom transpiracije lista u uslovima stresa. Nežlezdani trihomi značajno povećavaju zadržavanje vode u tkivu lista pri visokom deficitu vode. U uslovima spoljašnje sredine, ograničavanjem isparavanja sa površine lista, maljavi genotipovi su ispoljili veći intenzitet transpiracije. Na osnovu toga može se pretpostaviti da su nežlezdani trihomi kod crvene deteline morfološke strukture koje potpomažu funkciju stominog aparata lista. Ustanovljen je pozitivan odnos između stepena zastupljenosti trihoma i otpornosti na pepelnicu u prirodnim uslovima. Ključne reči: crvena detelina, nežlezdani trihomi, intenzitet transpiracije lista. Primljeno: 5. oktobra Odobreno: 2. decembra * Autor za kontakt: gmvg@abv.bg