Science in China Series C: Life Sciences © 2009
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Article
Response of the enzymes to nitrogen applications in cotton fiber (Gossypium hirsutum L.) and their relationships with fiber strength WANG YouHua1, FENG Ying1, XU NaiYin1,2, CHEN BingLin1, MA RongHui1 & ZHOU ZhiGuo†1 1 2
Key Laboratory of Crop Growth Regulation of the Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China; Institute of Industrial Crops, Jiangsu Academy of Agricultural Science, Nanjing 210014, China
To investigate the response of key enzymes to nitrogen (N) rates in cotton fiber and its relationship with fiber strength, experiments were conducted in 2005 and 2006 with cotton cultivars in Nanjing. Three N rates 0, 240 and 480 kgN/hm2, signifying optimum and excessive nitrogen application levels were applied.The activities and the gene expressions of the key enzymes were affected by N, and the characteristics of cellulose accumulation and fiber strength changed as the N rate varied. Beta-1,3-glucanase activity in cotton fiber declined from 9 DPA till boll opening, and the beta-1, 3-glucanase coding gene expression also followed a unimodal curve in 12—24 DPA. In 240 kgN/hm2 condition, the characteristics of enzyme activity and gene expression manner for sucrose synthase and beta-1,3-glucanase in developing cotton fiber were more favorable for forming a longer and more steady cellulose accumulation process, and for high strength fiber development. cotton (Gossypium hirsutum L.), nitrogen, fiber development, enzyme activity, gene expression, fiber strength
Cotton (Gossypium hirsutum L.) fiber quality is determined by its genetic background and the growth environment[1,2]. The genetic backgrounds of most cultivars have been optimized. Therefore, the influence of ecological factors is becoming more pronounced. As nitrogen (N) is one of the most important factors influencing fiber yield and quality, research concerning N effect on fiber quality are crucial. It was reported that either excessive or deficient N caused reduction in fiber strength[3], while optimal N application increased fiber quality[4]. However, most related research was concerned with the final fiber quality, with relatively limited investigation of the mechanisms of how N influences fiber quality development. The detailed physiological mechanism is remains uncertain. Fiber strength is one of the most important indices of fiber quality, and it is mainly determined by the cellulose synthesis process in which numerous enzymes are involved[5,6]. It is accepted that sucrose synthetase(SuSy) and beta-1,3-glucanase are two key enzymes[7,8] which
take part in cellulose synthesis, and long and steady cellulose deposition may result in high fiber strength[6]. As SuSy produces and transfers UDP-glucose (UDPG) to the subunit of cellulose synthetase for cellulose synthesis, the activity of SuSy is closely related to cellulose synthesis and the deposition rate[9], and subsequenlly related to fiber development. For example, in SuSy anti-sense transgenic cotton lines, fiber development was hindered due to the decrease of SuSy activity. Such environment factors as soil N influence SuSy activity. In developing rice seeds, SuSy activity was significantly higher in 100 kgN/hm2 than that in 50 kgN/hm2[11], and in developing wheat seeds, it was higher in 240 kgN/hm2 than that in 120 kgN/hm2. In corn Received July 14, 2008; accepted November 25, 2008 doi: 10.1007/s11427-009-0147-8 † Corresponding author (email:
[email protected]) Supported by the National Natural Science Foundation of China (Grant Nos. 30771277 and 30771279), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20060400944), and the State Key Laboratory Fund (Grant No. PPB08011)
Citation: WANG Y H, FENG Y, XU N Y, et al. Response of the enzymes to nitrogen applications in cotton fiber (Gossypium hirsutum L.) and their relationships with fiber strength. Sci China Ser C-Life Sci, 2009, 52(11): 1065-1072, doi: 10.1007/s11427-009-0147-8
leaves, SuSy activity was higher in 450 kgN/hm2 than in 0 kgN/hm2, with limited reports addressing cotton fiber cell development. Accompanied by cellulose synthesis, beta-1,3-glucan was also synthesized. In fiber cell, beta-1,3-glucanese hydrolyzes beta-1,3-glucan polymers by incising beta-1, 3-glycosidic bonds, which not only increase cell wall plasticity but also increase the UDPG supply for cellulose synthesis. High activity of beta-1,3-glucanese was observed as cellulose was quickly synthesized, with its activity also changed by ecological factors such as temperature. In low temperature conditions the activity of beta-1,3-glucanese increased and the gene expression decreased[15]. Soil N regulates enzyme activity at both the transcription level and the post-translation level. In wheat, soil N variation changes enzyme activity through regulating the gene expression, modulating mRNA splicing and modifying the enzyme precursor[16,17]. The objective of this paper was to elucidate the mechanism of the response of fiber strength development to N application by investigating the response of sucrose synthetase and beta-1, 3-glucanese to the soil N application rate, to help farmers in efficiently utilizing N fertilizers in high quality fiber production.
1 Materials and methods 1.1
Experimental design
Field experiments were carried out at the Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu province, China (118º50′E, 32º02′N) in 2005 and 2006. The experimental site soil was a yellow-brown loam with 2.5% organic matter, 11.0 g/kg total N, 70.1% available N, 12.7 mg/kg available P and 80.4 mg/kg available K present at a depth of 20 cm. Cotton cultivar AC-33B (the average fiber strength was 32 cN/tex) and KC-1 (the average fiber strength was 32 cN/tex) were planted. Treatments were set at 0 kgN/hm2 (zero N supplied), 240 kgN/hm2 (optimum N supplied), 480 kgN/hm2 (excessive N supplied) accor-
ding to previous research[18] and they were respectively designated as N0, N1 and N2. Cotton was sown in a nursery bed on April 20 and the seedlings were transplanted to the field on May 20. Each plot was 8.4 m wide and 6.3 m long. Within each plot, a spacing of 90 cm between rows and a spacing of 28 cm between plants was used, and 3 replicates for each treatment were randomly assigned in the field. In the trials, numerous flowers in the first and second fruiting positions in 6—8 sympodial branches were labeled at the anthesis day, and 15—20 bolls were collectede every 7 d from 10 DPA (days post anthesis) to 38 DPA. Developing cotton fibers were excised from the seeds with a scalpel, the detached fibers were promptly soaked inliquid nitrogen and then stored in a −70℃ refrigerator for further measurements. Matured fiber was collected from 10—15 labeled bolls which were of similar size after being opened, then air dried and ginned for fiber strength quantification. 1.2 Gene expression analysis by reverse transcription PCR (RT-PCR) The total RNA was extracted according to the modified hot-boric acid method[19]. Primers for RT-PCRs were synthesized by Yingjun Co., Ltd. (Shanghai). The optimal quantity of mRNA and cycles for RT-PCR analysis was determined by quantity and cycle number degressive reactions in the pre-experiment. The program for RT-PCRs was as follows: mixture incubated at 42℃ for 1 h, 5 min at 94℃ for template denaturing, then followed by optimal cycles (Table 1) at 94℃ for 30 s and optimal elongation temperature (Table 1) for 30 s, and a final 7 min incubation at 72℃ for completion extension. Constitutive gene EF1α was used as a control[20]. Electrophoresis results were analyzed by the software Quantity One. 1.3
Enzyme activity assay
The activity of SuSy was quantified by fructose and UDPG colorimetry[21], and beta-1,3-glucanase activity was determined with the Laminarin method[21].
Table 1 Primers, annealing temperature, optimal cycles and the product length for each gene Gene Sucrose synthase (U73588) beta-1,3-glucanase (D88416) EF1α (AJ223969) 1066
Primer forward: 5′-AGAACCCAAAGTTGCGTGAG-3′ reverse: 5′-ACCGTTACAGGTTGCGAATG-3′ forward: 5′-GAGGACATACAAAGCCTCGCA-3′ reverse: 5′-AGGTTGTAGTATTCCAAGCCT-3′ forward: 5′-AGACCACCAAGTACTACTGCAC-3′ reverse: 5′-CCACCAATCTTGTACACATGC-3′
Tm / ℃
cycles
Length / bp
58.0
27
305
58.0
29
455
55.0
26
496
WANG Y H, et al. Sci China Ser C-Life Sci | Nov. 2009 | vol. 52 | no. 11 | 1065-1072
1.4
Cellulose content quantification
To estimate the cellulose content, samples (1 g DW) were hot extracted with the Fibertec 1020 system (Foss Tecator AB). Samples and 150 mL 1.25% H2SO4 were boiled together for 30 min before being rinsed thrice with 100 mL distilled water. After that, they were boiled with 150 mL 1.25% NaOH for 30 min and were again rinsed thrice with 100 mL distilled water. The treated samples were dried in the oven at 130℃ for 2 h, and weighed. Finally samples were placed in a Muffle Furnace at 500℃ for 4 h, before being weighed again. The cellulose content was calculated in terms of the weight decrease of the sample fibers. 1.5
Fiber strength measurement
The fiber strength for 3.2 mm spacing (cN/tex) was determined by Y162 A (Taichang textile instrument Co., Ltd., China) and measurements were repeated 6 times for each sample.
2 Results 2.1 The response of key enzymes (SuSy and beta-1, 3-glucanese) to N rate (1) Sucrose synthetase. The expression of SuSy was
very high in 7—21 DPA, declining from 24 DPA. This trend was very similar in the two cultivars (Figure 1A). The transcript quantity of SuSy in N1 condition was the highest and the duration of the high expression stage was the longest. As SuSy produces UDPG and directly submits it to cellulose synthetase for cellulose synthesis[6], the longer duration of the high SuSy expression stage provided more UDPG and provided a longer period for high rate cellulose synthesis in N1 than in N0 and N2. The course of SuSy activity followed a typical unimodal curve, with a peak value around 31 DPA. Trends in each of the two years were similar (Figure 2). The major difference among N rates was the peak value of SuSy activity. It was the highest in N1, followed by N2 and N0. The difference was more pronounced before the peak emergence than after. The course of SuSy activity were expressed by its gene expression manner as they were both highest in N1 and very high in the early stage of fiber development (Figure 1A and B). (2) beta-1,3-glucanase. The period of beta-1,3glucanese transcripts in 12—24 DPA was also a unimodal curve and the peak emerged at around 18 DPA, remaining at a high level. The trends in the two cultivars
Figure 1 A, The time-course expression of SuSy at different N rates. AC-33B, Cotton cultivar AC-33B; KC-1, cotton cultivar KC-1; EF1α, constitutive expression gene in fiber cells as a control; DPA, day post anthesis. B, Quantitive analysis of the response of SuSy expression at different N rates (2006). N1, 240 kgN/hm2; N2, 480 kgN/hm2; N0, 0 kgN/hm2. WANG Y H, et al. Sci China Ser C-Life Sci | Nov. 2009 | vol. 52 | no. 11 | 1065-1072
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were similar (Figure 3A). In the N1 rate, the transcripts in the early stage (9—12 DPA) were the lowest, after 12 DPA they quickly enhanced and attained the highest level of the 3 N rates. Synthesis of beta-1,3-glucan consumed UDPG with the synthesis of cellulose, while beta-1,3-glucanase hydrolyzed beta-1,3-glucan which produced added UDPG to increase cellulose synthesis rate and loosened cells wall to facilitate cell expansion and cellulose deposition[2]. The highest expression of the gene in the mass cellulose deposition stage (18—24
DPA) in the N1 rate would benefit high strength fiber development. The activity of beta-1,3-glucanase decreased throughout the entire fiber development stage rapidly declining in the 10—24 DPA (Figure 4). During both of the two years, the trends in the two cultivars were similar, and the major difference in the different N rates was the value of the enzyme activity. It was highest in N1, and followed by N2 and then in N0. The enzyme activity was in accord with the gene expression (Figures 3A and B).
Figure 2 Dynamic of SuSy activity at different N rates (2005—2006). N1, 240 kgN/hm2; N2, 480 kgN/hm2; N0, 0 kgN/hm2.
Figure 3 A, The expression of beta-1,3-glucanase in different N rates (2006). DPA, day post anthesis. N1, 240 kgN/hm2; N2, 480 kgN/hm2; N0, 0 kgN/hm2. B, Quantity dynamics of beta-1,3-glucanase transcripts at different N rates (2006). N1, 240 kgN/hm2; N2, 480 kgN/hm2; N0, 0 kgN/hm2. 1068
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2.2
Fiber cell cellulose depostion
The cellulose content period followed a typical “S” curve which with the Logistic equation[5] (model, Table 2), All of the cellulose content was more than 80%. It was reported that when the cellulose content was higher than 80%, fiber strength was positively correlated to the duration of the mass cellulose synthesis stage[2], and negatively correlated to the value of the maximum cellulose deposition rate. In N1, the duration of the mass cellulose synthesis rate (T, Table 2) was the longest, which with the longer and higher gene expression and enzyme activity of SuSy in N1. The cellulose synthesis rate (%/d) is derived by re-
gressing the Logistic equations of the cellulose content. It also follows a unimodal curve, with the peak value emerging at around 20 DPA (Figure 5). In the stage before the peak emerged, the cellulose synthesis rate (%/d) on each day in N1 was the lowest while at N0 was the highest. In the stage after the peak emerged, it declined most rapidly in N0 and most slowly in N1. This suggests that the cellulose synthesis in N1 was the most tempered while in N0 was the most acute, the trends between the cultivars in the two years were similar. The tempered cellulose synthesis in N1 might be due to the longest duration and the highest expression of SuSy in 7—24 DPA, and also might be due to the highest expression of the beta-1,3-glucanase coding gene after 15 DPA, which
Table 2 Cellulose content dynamics at different N rates (2005—2006)a) Year
Cultivar
N rate (kgN/hm2)
Symbol
Model
2005
AC-33B
0
N0
Y=85.0447/(1+29.1068e−0.2216t)
KC-1
2006
AC-33B
240
N1
480
N2
0
T (d)
Vmax %/d
0.9882**
5
12
4.7113
−0.1739t
0.9757
**
6
15
3.9378
−0.1801t
0.9938**
6
14
4.0820
−0.2216t
0.9877
**
5
12
4.7775
0.9928
**
Y=90.5860/(1+31.0346e
Y=90.6605/(1+27.2905e
Y=86.2394/(1+32.8489e
) ) )
−0.1750t
240
N1
Y=93.7508/(1+29.6262e
6
15
4.1016
480
N2
Y=91.7655/(1+30.2617e−0.1795t)
0.9961**
6
14
4.1184
0
N0
Y=85.8171/(1+27.9744e−0.1754t)
240 KC-1
N0
n
R2
N1
)
0.9857**
6
15
3.7639
−0.1525t
0.9891**
6
17
3.4907
−0.1623t
**
6
16
3.7328
0.9930**
6
16
3.4899
Y=91.5592/(1+23.2990e
)
480
N2
Y=91.9813/(1+30.1597e
0
N0
Y=83.2014/(1+17.8425e−0.1678t)
240
N1
480
N2
)
0.9988
−0.1380t
0.9903
**
6
19
3.2926
−0.1451t
0.9914**
6
18
3.3166
Y=95.4597/(1+19.6074e Y=91.4429/(1+16.1439e
) )
a) AC-33B, cotton cultivar 33B; KC-1, cotton cultivar KC-1; Y, cellulose content (%); n=5, P0.05＝0.6584, P0.01＝0.8413; n=6, P0.05＝0.5693, P0.01＝ 0.7653; **, significantly different at P＝0.01; T, the duration at the mass cellulose synthesis stage (d); Vmax, the maximum cellulose deposition rate (%/d).
Figure 4
Dynamics of beta-1,3-glucanase activity at different N rates (2005—2006). N1, 240 kgN/hm2; N2, 480 kgN/hm2; N0, 0 kgN/hm2.
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led to added UDPG supplication for cellulose synthesis. 2.3
Fiber strength
The response of fiber strength development to N rate (Table 3) suggested that, the strength of the fiber developed at different N rates was significantly different from 24 DPA, it was highest in N1 and lowest in N0, while it was not significant before 17 DPA. Data showed that it was the longest duration and the most tempered for mass cellulose synthesis at the N1 rate, while it was the most acute in N0, and this might be the reason that the strength of the fiber developed in the N1 condition was the highest and the lowest in N0.
3 Discussion It is a common phenomenon that one gene’s expression
does not match with the dynamics of its enzyme activity. In our research, the expression of SuSy, as well as the beta-1,3-glucanase coding gene did not match with the dynamics of its enzyme activity very well. This might be because there were many steps, such as mRNA translocation, ribosome binding, zymogen activation in the process of going from gene transcripts to the active enzyme. SuSy is a key enzyme in fiber cell development which produces and delivers UDPG to cellulose synthetase for cellulose synthesis. Soil N alters SuSy (U73588) expression and the enzyme activity. In the N1 condition, in the mass cellulose synthesis stage, SuSy activity increased most and the gene expression was the most tempered, which resulted in a higher and more stable UDPG supplication condition for rapid and tem-
Figure 5 Cellulose synthesis rate at different N rates (2006). DPA, day post anthesis; N1, 240 kgN/ hm; N2, 480 kgN/hm; N0, 0 kgN/hm. Table 3
Fiber strength development at different N rates (2005—2006)a)
Year
Cultivar
N rate kgN/hm2
Symbol
2005
AC-33B
0
KC-1
2006
AC-33B
KC-1
Fiber strength (CN/tex) 17 DPA
24 DPA
31 DPA
N0
14.54bA
20.46bB
25.00cA
38 DPA 27.14dD
BO 29.60cdBC
240
N1
16.98abA
24.85aA
30.83abA
32.65aAB
34.59aA
480
N2
15.97abA
22.21bAB
27.87bcA
30.02bcBCD
31.96bcABC
0
N0
15.31abA
21.44bB
24.86cA
28.51cdCD
28.93dC
240
N1
18.10aA
25.00aA
31.67aA
33.43aA
34.50aA
480
N2
15.28abA
21.58bB
28.02bcA
31.63abABC
33.28abAB
0
N0
15.68bA
20.72cB
25.22cC
28.54cCD
29.98cdB
240
N1
19.13aA
25.67aA
31.01aAB
33.61aAB
34.97aA
480
N2
17.57abA
23.37bAB
28.15bBC
31.41bABC
32.40bAB
0
N0
15.56bA
20.50cB
25.62cC
27.67cD
28.90dB
240
N1
18.88aA
26.43aA
32.24aA
34.18aA
35.49aA
480
N2
16.59bA
22.49bcAB
28.54bBC
30.73bBC
31.66bcAB
a) AC-33B, the cotton cultivar AC-33B; KC-1, the cotton cultivar KC-1; BO, boll open; a, b, c and A, B, C means significant at P=0.01 and P=0.05 level; DPA, day post anthesis. 1070
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pered cellulose synthesis than that in the two other N conditions. Beta-1,3-glucanese is essential for processes in the plant anti-pathogen pathway, while in developing fiber cells, it works as a cellulose synthesis promoter by hydrolysis of beta-1,3-glucan to provide added UDPG for cellulose synthesis. Our research indicated that soil N altered the gene expression and the dynamics of enzyme activity. In the 240 kgN/hm2 condition, Beta-1,3glucanase activity was the highest among the three N rates, and transcripts of its coding gene were the most from 18 DPA on , which promoted cellulose synthesis and fiber strength development more than in the two other N conditions. The fiber strength developed in N1 was the highest while in N0 it was the lowest. Before 17 DPA, the strength of the developing fiber at different N rates was close, while after 24 DPA, it was significantly different at N rates, which suggests that the 17—24 DPA stage is a vital period for fiber strength development. This result is consistent with the fact that 15—25 DPA is the stage when fiber elongation and second cell wall thickening overlapped and it is the time window when mass cellulose begins to be synthesized in fiber development[6,9]. In the N1 condition, either the expression of the gene or the dynamics of the enzyme activity for SuSy and beta-1,3-glucanese were more optimized than that in N0 or N2, making the mass cellulose synthesis in N1 more stable and longer lasting (Table 2 and Figure 5), matched with the request for high strength fiber development[1,2]. In the N1 condition, fiber strength increased most rapidly and finally the fiber strength was the highest. Compared with N1, in N0, the SuSy coding gene ex1
Bradow J M, Bauer P J, Oscar H, et al. Quantitation of cotton fi-
pression and its enzyme activity was lower, especially in the period before 31 DPA. At the same time, the activity and the transcripts of the beta-1,3-glucanese coding gene were lower, especially in the period after 15 DPA, which made the duration of the mass cellulose synthesis stage shorter and the maximum cellulose synthesis rate (%/d) higher. As a high value for the maximum cellulose synthesis rate (%/d) is negative to fiber strength formation[2], the strength of the fiber developed in N0 was the least. These results indicated that soil N not only influences fiber enzyme activity, but also influences gene expression. The mechanism of how soil N/plant N influences gene transcription and the post-translational modification of the key genes involved in fiber development awaits further investigation.
4 Conclusion The strength of the fiber developed at different N rates was markedly different, which was highly related to the difference of the activity and gene expression of the key enzymes (SuSy and beta-1,3-glucanese) involved in fiber development. The strength of the fiber developed in the N1(240 kgN/hm2) condition was the highest, which might be due to the fact that both the transcripts and the enzyme activity were higher and maintained a high level for a longer time in the mass cellulose synthesis stage (17—24 d), which made a more and steady UDPG supplication for cellulose synthesis and then caused a more steady and longer duration of the cellulose synthesis stage. While in the N0 (0 kgN/hm2) or N2 (480 kgN/hm2) conditions, the enzymes were comparatively less active and resulted in lower fiber strengths. 6
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