Indian Journal of Experimental Biology Vol. 42, February 2004, pp. 197-201

Role of phenolics and boron in reproductive success in seasonally transient sterile Tecoma stans L.

S V S Chauhan *, Jolly Singh* & Satoshi Tahara**

*Department of Botany, School of Life Sciences, Dr B R Ambedkar Uni versity, Agra 282002, India

**Laboratory of Ecological Chemistry, Graduate School of Agric ulture, Hokkaido Uni versity , Sapporo 060-8589, Japan

Received 23 April 2003; revised I August 2003

Quantitati ve and qualitati ve analys is of phenolics and boron in sti gma o f transient sterile Tecoma stans L. during seedless (May-Jul y), parti all y seedbearing (August - November, April ) and seedbearing peri ods (December - March) was made. UV absorption profile of sti gmatic exudates indicated the presence o f simple phenolics. Total phenolics were higher in stigma during seedless peri od. Thin layer chromatographic analys is o f stigmatic extracts exhibited only three principal spots. Mass spectrophotometry showed the presence of deri vati ves of cinnamic ac id, namel y, caffeic ac id in these spots. Quanti ty of boron in stigma during seedless period was lowest but the difference with other periods was not significant. It was suggested that the accumul ation o f higher quantity of caffeic ac id in the stigma during seedless period due to hi gh temperatu re (40°-45°C) could lead to inhibiti on of pollen germination in vivo, thereby rendering the plants seedless . Thi s was confirmed by inhibit ion of in vitro po llen germinati on in the basal medium containing higher quantity of caffeic ac id.

Keywords: Boron, Caffeic ac id, Phenolics, Tecoma stans

Tecoma stans L. (Bignoni aceae). a native of central America, fl owers throughout the year but remains seedless in summer (May-August at 37°-45°C), partially seedbearing in the months of August­November and April (28°-37°C) and full y seedbearing during December-March( l7°-28°C) at Agra1

•

Morphological differences in the stigmatic structure as the cause of transient sterility in T. stans has been reported2

.3 . Pollen-pistil interaction is the most important phenomenon in seed formation4

. Phenolics play an impOitant role in pollen nutrition and in the selective promotion or inhibition of pollen grains on stigma5

. Deficiency of boron leads to accumulation of phenolics resulting to growth inhibition and such effects are reported in other ti ssues of higher plants, but no evidence as yet is available in pollen6

. In order to obtain a clear unders.tanding of pollen-pistil interaction, it is necessary to study not only the morphological but also the biochemical differences to establish the role of boron and phenolics in fruit formation .

Materials and Methods The present investigation was carried out on ten

Tecoma stans L. (Family: Bignoniaceae) plants from ten different sites of Agra during 1996-2000.

Analysis of stigmatic exudates- Ten stigmas from fresh open flowers at pre-anthesis stage from ten plants each from ten different sites were kept in

*Correspondent author : E-mail : svsc@ sancharnet.in

methyl alcohol for 10 min and then removed. The UV absorption profil e was determined wi th UV spectrophotometer at 400-200 nm. The absorption profile was also recorded after the addi tion of single drop of 2N aOH in the alcoholic washing fo llowing the procedure of Martin 7.

Detem1ination of phenolics-Quantitative estimation of total phenolics in stigma in ten replications from ten plants of ten different sites during seedless, partially seedbearing and seedbearing periods was made by the procedure of Bhatia et al 8

. The dry material (lg) was refluxed with 50 ml methyl alcohol for 4 hr in a soxhlet apparatus. Excess alcohol was evaporated at reduced pressure. The contents were then brought to a constant volume of I 00 ml by distilled water. To a suitable aliquot of this extract, 0.5 ml of Folin Dennis reagent was added and tube was shaken well fo r 3 min . To this, 1 ml solution of saturated Na2C03 was added. It was again shaken and volume was made to 15 ml by adding distilled water. It was kept in dark for 1 hr and absorbance was read at 725 nm. Total phenolics were calculated from standard curve deve loped for tannic acid.

Qualitative analysis of different phenolics in the stigmatic tissue was done by thin layer chromato­graphy (TLC) on silica gel plates of 60 F 254 of 0.25 rnm as described by Harbome9

. For visualization of spots, thymol and Gibb's reagents were used. Plates were exposed to UV -light at 365 and 254 nm to

198 INDIAN J EXP BIOL, FEBRUARY 2004

identify fluorescent and quenching compounds respectively, using their standards.

Ident ification of specific phenolic compounds present in the spots developed on TLC plates was done by mass spectrophotometry. The spots were eluted from the plates containing si lica gel in methyl alcohol after acid hydrolysis. Polin Dennis reagent and saturated Na2C0 3 were added and kept for 1 hr. Absorbance was read at 750 nm. Phenolics thus identified were further confirmed by TLC using their standards9

.

Determination of boron - Quantitative detem1ination of boron in ten replications from stigma of ten plants each from ten different sites was performed by the method as described by Reczynski and Ryczkowski 10

•

Powdered samples of stigma (50 mg) of various periods were kept in a silica dish, Calcium hydroxide (100 mg) was added and placed in a muffle furnace at 400°C for 4 hr. After cooling, 5 ml of IN H2S04 was added to each sample. It was heated to 80°C, and subsequently cooled to room temperature. A suitable volume of the sample was filtered and neutralized with IN NaOH and transfeJTed to 25 ml calibrated flask. To this solution, 1 ml of 0.2 M dihydroxy­benzoic acid (DHBA) and I ml of I N H2S04 were added. After 20 min, 2 ml of the reagent solution (prepared by mixing 200 ml of 1 mM DHBA in IN H2S04) was added to the flask and diluted with water to make up to 25 mi. Absorbance of anionic complex of boric acid with DHBA associated with cation was determined at 600 nm.

Effect of boron and caffeic acid on in vitro pollen germination- Effect of various concentrations of boron and caffeic acid on in vitro pollen germination and tube growth was studied by hanging drop technique 11 by supplementing them in sucrose solutions of various concentrations. The cultures were stored at room temperature (28° -30°C) in diffused laboratory light. All cultures were run in duplicate and random counts of lOO pollen grains were made to determine the percentage of germination. The length of 10 randomly selected pollen tubes was measured.

Data was statistically analyzed using Student t test for the significance.

Results and Discussion UV-absorption profile of stigmatic exudates- UY

spectrum of stigmatic exudates collected during seedless, partially seedbearing and seedbearing samples exhibited simi lar spectral characteristics (Fig. 1). However, the ab orbances at these wavelengths were different. As shown in Table I , absorbance at 33 J nm during seedless period was highest ( 1.47), while during seed bearing period it was lowest (0.63).

Upon addition of Shift's reagents in the methanolic extracts of stigma, the absorbance spectra exhibited bathochromic shifts towards higher wavelength.

SL

200.0 250.0 300.0 350.0 400.0

Fig. I - UV absorption profile of stigmat ic extracts during seedless(SL), partially seedbearing (PSB) and seedbearing(SB) periods

Table I - UV absorption profile of stigmatic exudates in methanol during seedless, partially seedbearing and seedbearing periods in Tecoma stans

Periods

Seedless Partially seedbearing Seedbearing

[Values are mean± SD of I 0 replicates]

Highest peaks of UV absorption* (nm) in MeOH

331.8( 1.47) 331 .8( 0.81) 33 1.2(0.62)

301.0 ( 1.17) 204.6( 1.81) 204.0( 1.51 )

291.4 (1.17) 209.6 (3.45)

*Values in paranthesis indicate absorbance

CHAUHAN et al.: ROLE OF PHENOLICS AND BORON IN REPRODUCTIVE SUCCESS 199

These results suggested the presence of different levels of simple phenolic compounds in the stigma during various periods7

.

Quantitative estimation of total phenolics- The quantity of total phenolics was highest in the stigma at all stages of development during seedless period (Table 2). Stigma at the time of pre-anthesis during seedless period contained highest quantity of total phenolics (4.3 mg/g) as compared to seedbearing period (1.70 mg/g) and partially seedbearing period (2.1 mg/g dry weight). Difference in the quantity of total phenolics was significant between various

Table 2-Quantity of total phenolics and boron in the sti gma of Tecoma stans during seedbearing, partially seedbeari ng and

seedless periods at different stages of developments

Periods

Seed bearing

Partially seed bearing

Seedless

[Values are mean± SO of 10 replicates]

Total phenolics(mg/g) of dry plant material (Boron (mg/g) of dry samples shown in parenthesis)

Stages of development* A B C

1.70 ± 0.16 1.90 ±0.71 1.50±0.50 (3.52 ± 1.02) (4 .14± 1.32) (3.82 ± 1.43)

2. 1 ± 1.01 2.4 ± 1.37 1.7 ±0.20

(3 .34 ± 0.68) (3.89 ± 1.00) (3 .54±0.2 1) 4.3 ± 1.02** 3.8 ± 1.36** 3.5 ±0.40** (3.2 1 ± 0.41) (3.66 ± 0.80) (3.2 1 ±0.41 )

*Stages of development, A: Pre-anthesis, B: Anthesis, C: Post­anthesis **Significant at P < 0.05

• • 4 • (~) ..... ... .. ...........

0 .... -.. ' .. ..... ~·

..... ..... ~ ...... '

1 • • • 2 - • 3

SL PSB SB

Fig. 2-Thin layer chromatogram developed with Thymol Reagent

periods at all stages, but the difference was insignificant between the stages of development during the same period.

Qualitative analysis of phenolics by TLC- TLC spectrum in thymol reagent during different periods showed the presence of five distinct spots (Fig. 2). However, only 4 spots were considered as thymol positive compounds. Spot 1 was present during all the three periods, but was largest during seedless period. Similarly, spots 2 and 3 were also present during al l the three periods, but were much larger during seedless period. It was interesting to note that spot 4 was conspicuously present only during seedless period.

Detection with Gibb's reagent showed only 3 principal reproducible spots (Fig. 3) . Spots 1 and 3 were present during all the three periods and spot l was the largest during seedless period. It was interesting to note that spot 4 was distinctly present only during seedless period.

TLC plate exposed to UV light at 365 nm showed the presence of only 3 principal spots indicating the presence of fluorescent compounds (Fig. 4). The spot 1 during seedless period was the largest, while spot 2 detected by thymol and Gibb's reagents failed to show any colour reaction under UV light. Spot 3 was small , while spot 4 was blue and was largest during seedless period. However, at other periods, spot 4 was present only in traces (Fig. 4). Thus, spots 1,3 and 4 are the principal ones and belong to simple phenolic compounds. The large size of these spots during

4

0 0 , ..... , .......

• II • 3

SL PSB SB

Fig. 3 - TLC chromatogram developed with Gibbs Reagent

200 INDIAN J EXP BlOL, FEBRUARY 2004

seedless period indicated the presence of principal compounds in higher quantity as compared to other periods. These results also confirmed the presence of simple phenolics in the stigma9

·13 and these may be the

derivatives of hydroxy-cinnamic acid9. Analysis of

compounds eluted from TLC plates by mass spectrometry, suggested that phenolic compound(s) in the stigmatic extract contained caffeic acid.

Quantitative estimation of boron - Boron in the stigma at pre-anthesis, anthesis and post-anthesis stages of development during seedbearing and partiall y seedbearing periods was more or less similar, but slightly higher in the sti gma (Table 2) during seedbearing period (3.52-4.14 mg/g) as compared to partially seedbearing period (3.34-3.89 mg/g) and lowest during seed less period (3 .21-3.66 mg/g). However, thi s difference during all the three peri ods was insignificant.

E.ffect of boron and caffeic acid on in vitro pollen germination - The pollen cultured by hanging drop technique" in various concentrations of sucrose solutions (10, 15 and 20%) supplemented with different concentrations of boron and caffeic acid ex hibited some interest ing results (Table 3). However, Table 3 shows the results of 10% sucrose solution on ly exhibi ting optimum pollen germination (57%) as well as longest pollen tubes (590.21 J.!m) as compared to other concentrations (15 and 20% ). Therefore, for studying the effect of boron and caffeic acid only I 0% sucrose solution was further used as the basal

3

SL PSB SB

Fig. 4-Fluorescent spots detected on thin layer chromatogram in UV of 365 nm

medium. Addition of boric (100 mgll) acid to the basal medium enhanced both pollen germination (62%) and tube length (790.12 J.!m). However, addition of higher quantity of boric acid (150 and 200 mg/1 ) failed to enhance germination and tube elongation. It was interesting to note that addition of lower concentrations of caffeic acid (5 and 10 mg/1 ) in the basal medium enhanced germination and tube growth. On the other hand, increasing the quantity of caffeic acid in the basal medium reduced germinc:tion and tube growth and addition of 50 mg/1 caffeic acid in the basal medium completely mhibited pollen germination (Table 3).

Presence of m?i n peaks in UV spectrum of stigmatic extracts at 331 and 204 nm and their bathocromic shifts suggest the presence of simple phenolic compounds7 and these, may be the derivat ives of hydroxy-cinnamic acid9

. TLC analysis of the extracts showed the presence of multiple spots. UV fluorescence of these spots further suggest the

f . I h 1. . h . 9 ,? M presence o s1mp e p eno tcs 111 t e sttgma · -. ass spectrometric analysis of the compounds from TLC plates revealed that they contained the derivative of hydroxy-cinnamic acid namely, caffeic acid. The quantitative analysis indicated that pheno lics in the stigma during seedless period were present in far larger quantity as compared to other two periods. Analysis of total phenoli cs in stigma, during seedles;;; period (May to Jul y at 40°-45°C) showed the presence of hi gher quantity of total phenoli cs as compared to that during other periods.

Phenolics play an important role in pollen nutrition and in selective promotion or inhibition of pollen germination on stigma5

. The germinating pollen grains secrete enzymes on the stigma and release sugars from phenolic glycosides. Free sugar provides proper nutrition for the growth of pollen tube13

.

Table 3-Effect of boric ac id and caffeic acid on i11 vitro pollen germinati on using I 0% of sucrose

[Values are mean"± SD of ten repli .~at i on s]

Treatment Cone. Sucro,;e ( 10%) (mg/1 ) PG (%) TL (p.m)

Control 57 590.2 I ± 12.23 Boric acid 100 62 790. 12 ± 31.32

150 42 427 .34 ± 17.06 200 39 307.7 1 ± 2 1.04

Caffeic acid 5 60 578.16 ± 14. 13 10 55 506.79 ± 17.10 25 15 124.52 ± 18.9 1 50 0 0.00

PG: pollen germination ; and TL: tube length

CHAUHAN et al.: ROLE OF PHENOLICS AND BORON IN REPRODUCTIVE SUCCESS 201

Production of phenolic compounds is highly regulated 14

and there is an additional level of control in stimulation of synthesis by appropriate environmental influence and phenolics accumulate as a result of stress 15

•

Another part of present investigation is the quantitative analysis of boron in the stigmatic tissue of Tecoma stans during different periods. The quantity of boron in the stigma during different periods was more or less equal, but was highest duting seedbeating period as compared to that in two other periods. Interestingly, the quantity of phenolics during the seedbearing period was lowest. On the other hand, the quantity of boron was lowest during seedless period with highest quantity of phenolics. However, this diff~rence in the quantity of boron during different penods was insignificant. Boron deficiency is known to lead to the accumulation of phenolics resulting in growth inhibition in other tissues of higher plants, but such effects are not yet repotted in pollen6

. However, boron deficient stigma of Vitis vinifera fail to support pollen germination 16

• Therefore, it seems quite likely that accumulation of phenolics (caffeic acid) in the stigma of Tecoma stans plants during seedless period is largely due to significant increase in the temperature during this period ( 40° -45°C) as suggested by Harborne 15

• It was further confirmed by in vitro pollen germination studies clearly indicating that addition of larger quantities of caffeic acid in the basal medium showing optimum pollen germination inhibited both germination and tube growth at room temperature (28°-38°C). Thus, accumulation of caffeic acid in the stigmatic tissue during seedless period (May to July) inhibited in vivo pollen germination on stigmatic surface rendering the plants seedless. This is supported by the fact that there is only 2-5% in vivo pollen germination with small pollen tubes on the stigmatic surface of pistils collected from T.stans plants during seedless period 1•

Acknowledgement One of the authors (SVS) is grateful to Japan

society of Promotion of Science, Japan for the award of fellowship.

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Tecoma swns in relat ion to fruit formation . ln Proceeding of International symposium on pollination in tropics, edited by G K Veeresh, R U Shanker and K N Ganeshaiah, (International Union fo r the study of social insects - Indian chapter, Uni versity of Agriculture Sciences, Bangalore) 1993, 54.

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10 Reczynski W & Ryczkowski M, Boron distribution in the developing ovule of Clivia miniata Rye!, Plant Celllncompat Newslett, 26 (1994) 65.

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12 Martin F & Teleck L, The stigmatic secretion on the sweet potato, Am J Bot, 58 (1971) 317.

13 Vasill K, The histology and physiology of pollen germination and pollen tube growth on the stigma and style , in Fertilization in higher plants, edited by H F Linskens (North Holl ~· nd Pub!. Co. Amsterdam) 1974, I 05.

14 Murphy T M & Thompson W F, Detection of induced gene products in Molecular plant development (Prentice Hall , Engleward Cliffs, New Jersey, USA) 1988,54.

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16 Gartel W, Micronutrients- Their significance in vein nutrition with special regard to boron deficiency and toxicity , Wemberg Keller, 21 ( 1974) 435 .