Effect of sulphur application on lipid, RNA and fatty acid content
in developing seeds of rapeseed (
Brassica campestris
L.)
Altaf Ahmad, M.Z. Abdin *
Centre for Biotechnology,Faculty of Science,Hamdard Uni6ersity,New Delhi-110 062,India
Received 4 February 1999; received in revised form 11 August 1999; accepted 27 August 1999
Abstract
Changes in the contents of lipid, RNA and fatty acids were determined in the developing seeds of rapeseed (Brassica campestris
L. cv. Pusa Gold) grown with or without sulphur. In +S treatments, S was applied either as a single dose (T2) or the same dose
was split in two (T3) or three (T4) portions. Rapid accumulation of lipids started at 7 days after flowering (DAF) and continued
until 35 DAF. Application of S increased the lipid content in the seeds from the initial stage. The maximum increase was observed, when S was applied in three portions. There was a positive strong co-relation between S and lipid content in the seeds. RNA content increased from 7 to 35 DAF, followed by a decline until maturity. Application of S increased the RNA content in the seeds, compared to control (T1). Among the +S treatments, i.e. T2, T3and T4, the treatment T4resulted in the maximum
increase in RNA of developing seeds. The fatty acid composition of the oil changed substantially during seed development. S application in three portions increased the oleic acid (18:1) content, and decreased the erucic acid (22:1) content over other treatments. This leads to a reduced 22:1/18:1 ratio and thus, improves the quality of oil. The ratio of erucic acid to oleic acid (22:1/18:1) is closely related to the N:S ratio in the seeds. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Brassica campestrisL.; Fatty acids; Lipid; RNA; Sulphur; N:S ratio
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1. Introduction
Plant-derived lipids are used in a wide variety of cooking oils, cosmetics, detergents and lubricants, etc., and the production of oilseeds, therefore, involves ample economic interest. Rapeseed is an important oilseed crop having 40 – 50% oil in the seeds. Its meal, a protein supplement in the feed trade, competes with soybean meal. One of the major factors affecting growth, yield and quality of oil of rapeseed is the mineral nutrients, espe-cially sulphur [1 – 4]. Oilseed crops have a high demand of S; approximately 16 kg of S is required to produce one ton of seeds containing 91% dry matter [5,6]. Oilseeds not only respond to applied S, but their requirement for S is also the highest among crop plants, thus indicating a role of the
nutrient in oil biosynthesis [2,5 – 12]. Information about its role in the physiological and biochemical changes that occur in the developing seeds of rapeseed is meagre.
This study investigated the effect of S applica-tion on the changes in lipid, RNA and fatty acid content in rapeseed during the course of seed development.
2. Materials and methods
2.1. Plant material
The seeds of rapeseed (Brassica campestrisL. cv. Pusa Gold) were obtained from the National Re-search Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India. The crop was raised in the experimental field of Hamdard University, New Delhi, India according
* Corresponding author. Fax: +91-11-6988874.
E-mail address:[email protected] (M.Z. Abdin)
to the procedure described in our earlier paper [13]. The soil was sandy loam, with pH 7.2, and deficient in S (0.01%).
2.2. Treatment
All plots received 40 kg ha−1each of
phospho-rus and potassium at the time of sowing. S was applied at the rate of 40 kg ha−1as CaSO
4 either
in a single basal dose or in two or three portions. Nitrogen was applied as urea at the rate of 100 kg ha−1 in two or three portions. The first dose of
S and N was given at the time of sowing (basal), the second at 35 days after sowing (before flower-ing) and the third at 50 days after sowing (after flowering). The control treatment was made without S. Thus, there were four treatments: T1
(S0N50+50), T2 (S40N50+50), T3 (S20+20N50+50) and
T4 (S20+10+10N50+25+25). These treatments were
chosen on the basis of our earlier results in which we found a significant increase in the seed and oil yield of rapeseed with the treatment T4 [13]. The
experiments were conducted using a randomized block design with three replicates of each treat-ment. The plot size was 16 m2 (4×4m) with 10
rows, and a row to row distance of 30 cm. Four irrigations were given at different time intervals during the growth period of the crop. Weeding was carried out frequently. Two weeks after sow-ing, the seedlings were thinned to keep an intra-row spacing of 15 cm.
2.3. Sampling
Samples were collected at weekly intervals from 7 days after flowering (DAF) till harvest (59 DAF). The pods were removed from the plants, packed in polyethylene bags and brought to the laboratory. The seeds were separated from the pods and subjected to analyses.
2.4. Estimation of dry weight of seeds
For dry weight estimation the seeds were oven-dried at 60°C until constant weight was reached.
2.5. Estimation of lipid content
The lipid content was estimated by pulse NMR technique developed by Tiwari et al. [14].
2.6. Determination of fatty acid composition
The lipid was extracted with petroleum ether using Soxhlet [15]. The methyl esters of fatty acids present in the lipid were prepared by
esterification according to the method of
Morrison and Smith [16] with slight
modification. Oil (0.1 ml) was taken in air-tight, capped glass tubes. Sodium methoxide (0.5 ml of 0.5 N) in methanol was added. The contents of the tubes were mixed thoroughly and heated in boiling water for 10 min. The tubes were cooled
in cold water. Two drops of boron
trifluoride – methanol complex (solution 14% in methanol) were added to the solution in each tube, and then the tubes were heated in boiling water for 10 min. After cooling in cold water, 5 ml hexane was added to each tube and mixed thoroughly. After about 10 min the content of the tubes separated into two layers. The upper layer, containing methyl esters, was taken out and concentrated to 0.1 ml. The fatty acid
methyl esters were determined by gas
chromatography (model 8700, Perkin-Elmer,
USA) equipped with a flame ionization detector (FID). A 2 m long stainless steel packed column containing 3% OV-17 on chromosorb (WHP 100 – 200 mesh) was used. Helium was the carrier gas. Injector and detector temperatures were 240°C and the oven temperature was maintained at 215°C for 30 min. The carrier gas flow rate was 30 ml min−1. Individual fatty acid content
was calculated on the basis of the area under the chromatography peak, and then each fatty acid was expressed as a percentage of the total fatty acid content.
2.7. Determination of nitrogen and sulphur content
The N content was determined by the micro-Kjeldahl procedure [17], while that of S was by the method of Chesnin and Yien [18].
2.8. Determination of RNA content
2.9. Statistical analysis
The statistical analysis was done following the method of Nageswar Rao [21].
3. Results and discussion
3.1. Dry matter, lipid and RNA contents in the de6eloping seeds
Fig. 1 showed that the trend of dry matter accumulation in the seeds was similar in all the treatments. The increase in dry matter accumula-tion in the seeds started from 7 DAF and contin-ued till 42 DAF, thereafter no further increase was observed. S treatment (T2, T3 and T4) increased
the dry weight of the seed over −S treatment (T1)
right from the initial stage of seed development until maturity. The increase in the final seed weight with the application of S has also been observed in other studies [2,5 – 11]. The largest accumulation of dry matter in the seeds was ob-served for the T4 treatment (Fig. 1). The
explana-tion for the same is discussed in our previous paper [13].
Fig. 2 shows the lipid content in the developing seeds. Irrespective of treatments, lipid accumula-tion in the seeds commenced at 7 DAF and con-tinued until 35 DAF, beyond which no significant increase was observed. These findings agree with some earlier studies in rapeseed [22 – 24]. Applica-tion of S (T2, T3 and T4) increased the lipid
accumulation at all stages of seed development compared to −S treatment (T1). A reduction in
lipid content in −S plants was also observed
previously [23,25]. Among the +S treatments, the amount of lipid deposited in the seeds was
signifi-cantly (PB0.05) higher between 42 DAF and
maturity, when S was applied in three portions (T4), compared to the application of S in two
portions (T3) or as a single basal dose (T2).
RNA content increased from 7 to 35 DAF, followed by a decline until maturity (Fig. 3). This trend in the accumulation of RNA in developing seeds during the early stages of seed development is indicative of synthesis of enzymes and mem-brane proteins required for the synthesis and accu-mulation of oil. Application of S increased the
RNA content at all the growth stages over −S
treatment. The treatment T4resulted in the largest
increase in RNA content at all the developing stages of the seed. This treatment also reduced the loss of RNA at the late maturation stages of the seeds (Fig. 3). The increase in RNA content in the developing seeds under the influence of S is inter-esting and needs further experimentation to ex-plain the cause of the variations.
Fig. 1. Effect of S application on dry weight of developing seeds of rapeseed. Each point is the mean of five replications and the bars indicate S.E. HR, harvest.
Fig. 3. Effect of S application on RNA content in the developing seeds of rapeseed. Each point is the mean of five replications and the bars indicate S.E. HR, harvest
3.2. Compositional changes in fatty acids during the de6elopment of seeds
Fig. 4 shows the compositional changes in fatty acids during seed development. The changes in the five fatty acids, palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3) and erucic acid (22:1) are described here, because they comprised over 90% of the total lipid in develop-ing seeds of rapeseed.
Palmitic acid (16:0) content was 13, 14, 17 and 18% at 7 DAF in the treatments T1, T2, T3and T4,
respectively, which declined gradually at subse-quent stages of seed development, and was only 2, 4, 6 and 4%, respectively in the oil of mature seeds. This indicated that +S treatments (T2, T3
and T4) increased the palmitic acid content by
1 – 7% at 7 DAF and 2 – 4% at maturity of the seeds over the −S treatment (T1).
Oleic acid (18:1) was the major fatty acid at the initial stages (7 – 14 DAF) of seed development, accounting for 51, 52, 44 and 35% at 7 DAF in the treatments T1, T2, T3and T4, respectively. Its level
declined gradually at the subsequent stages of seed development; and it was only 11, 16, 19 and 30% in the oil of mature seeds from treatments T1, T2,
T3and T4, respectively. However, in the treatment
T4, oleic acid content at initial stage (7 DAF) of
the seed development and in the mature seeds was not significantly different statistically.
Linoleic acid (18:2) content was 4, 4, 17.4 and 27% at 7 DAF and 12, 12, 17 and 17% in the mature seeds with the treatments T1, T2, T3 and
T4, respectively.
Linolenic acid (18:3) content increased slightly in the oil during seed development. It ranged between 4 – 9, 5 – 10, 7 – 11 and 11 – 15%, respec-tively, under the treatments T1, T2, T3 and T4.
Erucic acid (22:1) content began increasing right from 7 DAF and increased gradually until the seeds matured. S treatments greatly decreased its content in the oil of mature seeds. In the treatment
T1, the content was 6% at 7 DAF and reached
51% in the oil of mature seeds. In the treatments T2, T3 and T4, the initial content was 5, 8 and
11%, respectively, while the final content was 49, 47 and 42%, respectively.
These findings show that major changes occur in the oleic acid and erucic acid content during seed development. The pattern of change of con-tent of erucic acid may be consiscon-tent with the chain elongation of oleic acid to give erucic acid. Fig. 4. Effect of S application on fatty acid composition in the
Supply of S in portions during seed development reduced the conversion of oleic acid (18:1) to erucic acid (22:1), leading to the reduced 22:1/18:1 ratio and, thus, improved the quality of the oil. Among the +S treatments, the application of T4
caused the largest decrease in the 22:1/18:1 ratio at 21 DAF onwards (Fig. 5).
Fig. 7. Relationship between N/S ratio and 22:1/18:1 ratio in the developing seeds of rapeseed.
Fig. 5. Effect of S application on 22:1/18:1 ratio in the lipid of developing seeds of rapeseed. Each point is the mean of five replications and the bars indicate S.E. HR, harvest.
3.3. S content and N:S ratio in the de6eloping seeds
In general, sulphur content in the developing seeds increased until 35 DAF, and thereafter be-came constant till harvest. Application of S in-creased S content in the developing seeds over controls (T1). The highest seed-S was found with
the application of the T4 treatment. It is
interest-ing to note that the accumulation of S in the developing seeds followed the same trend as that of lipid accumulation and that S content corre-lated positively (r2=0.887) with lipid content in
the seeds (Fig. 6). This indicates that S might play a significant role in oil biosynthesis in the rape-seed. The N:S ratio in the developing seed
de-creased with the application of +S treatments
(T2, T3, T4) over control (T1) and was positively
correlated with 22:1/18:1 ratio (r2=0.803) (Fig.
7).
Acknowledgements
The authors are thankful to Technology Mis-sion on Oilseeds and Pulses (TMO&P), Ministry of Agriculture, Government of India, for provid-ing financial assistance to carry out this study. The authors are also thankful to Professor M. Iqbal, Head of the Department of Botany, Hamdard University for providing help and encouragement Fig. 6. Relationship between S and lipid content in the
and Mohd. Israr, Research Scholar, for technical help during this study.
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