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Size effects of chitooligomers on the growth and photosynthetic characteristics of wheat seedlings
Carbohydrate Polymers 138 (2016) 27–33
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Size effects of chitooligomers on the growth and photosynthetic characteristics of wheat seedlings Xiaoqian Zhang a,b , Kecheng Li a,∗ , Song Liu a , Ronge Xing a , Huahua Yu a , Xiaolin Chen a , Pengcheng Li a,∗ a b
Key Laborotory Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 29 September 2015 Received in revised form 18 November 2015 Accepted 19 November 2015 Available online 23 November 2015 Chemical compounds studied in this article: d-Glucosamine (PubChem CID: 439213) Chitobiose (PubChem CID: 90657429) Chitotriose (PubChem CID: 70702331) Chitotetraose (PubChem CID: 70702329) Chitopentaose (PubChem CID: 14055040) Chitohexaose (PubChem CID: 3081404) Chitoheptaose (PubChem CID: 3081414) Chitooctaose (PubChem CID: 24978517) Chlorophyll a (PubChem CID: 46936306) Chlorophyll b (PubChem CID: 6450186)
a b s t r a c t In this study, nine chitooligomers (COSs) including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow degrees of polymerization (DPs) (DP8–10, DP10–12) were prepared and applied to wheat seedlings to investigate the size effects of COSs on the growth and photosynthesis parameters of wheat seedlings. The results showed that the activities of COS were closely related to their DPs, and DP > 3 was necessary to insure a significant promotion effect on the growth and photosynthesis. Moreover, chitoheptaose exhibited the optimal activity compared with other COS samples. After 7 days of chitoheptaose treatment, the growth parameters of wheat seedlings could be significantly improved and the contents of soluble sugar, soluble protein and chlorophyll were increased by 59.4%, 22.0% and 20.3%, respectively. In addition, chitoheptaose could significantly enhance the net photosynthetic rate of wheat seedlings with the values of Fv/Fo, qP and Rfd increased by 11.0%, 18.6% and 14.7%, respectively, while NPQ was decreased obviously, which might resulted in the promotion of light utilization efficiency and the growth of wheat seedlings. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chitooligomers Size Growth Photosynthesis Chlorophyll fluorescence
1. Introduction Chitooligomers (COS) is a low molecular weight polymer which can be produced from either chitin itself or from chitosan, so it includes homo- or heterooligomers of N-acetyl d-glucosamine and d-glucosamine (Bahrke et al., 2002). Previous studies showed that COS possessed various potential applications in medicine and food fields due to its wide bioactivities, such as antitumor, Harish Prashanth & Tharanathan, 2005, antioxidative (Li, Liu, Xing, Qin, & Li, 2013), fat lowering (Muzzarelli, 1997), antiglycation (Zhang, Yu, Zhang, Zhao, & Dong, 2014), immunostimulatory (Peluso et al., 1994), antimicrobial (Choi et al., 2001; Mei, Dai, Yang, Xu, & Liang,
∗ Corresponding authors. E-mail addresses: [email protected] (K. Li), [email protected] (P. Li). http://dx.doi.org/10.1016/j.carbpol.2015.11.050 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
2015) and wound healing activities (Aam et al., 2010; Muzzarelli, 2009). It also had been recognized as a product to enhance plant growth, including stimulating growth of orchid tissue (Nge, Newa, Chnadrkrachnag, & Setvens, 2006), increasing the chlorophylls contents, enhancing the photosynthesis and enlarging the chloroplast of the Dendrobium orchid (Limpanavech et al., 2008), promoting mineral nutrient uptake of Phaseolus vulgaris (Chatelain, Pintado, & Vasconcelos, 2014) and alleviating salt stress to wheat seedlings (Zou et al., 2015). Therefore, COS has attracted increasing interest in the field of agriculture. The bioactivity of COS depends closely on its structure and physicochemical property. The degree of polymerization (DP), degree of deacetylation (DD), charge distribution and the oligomer structure pattern have important influence on its bioactivity (Aranaz, Harris, & Heras, 2010; Muzzarelli, 1996). Furthermore, the dosage, application method, pH and temperature of the medium
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and the presence or absence of an active unit would modify the activity of the molecule (Boonlertnirun, Boonraung, & Suvanasara, 2008; Kim & Rajapakse, 2005). Therefore, when investigating the influence of COS with a certain property on all kind of applications, all other parameters of COS should remain the same, and it would be of great importance in practical application. In particular, molecular size of COS is a very important factor in the study of structure–function relationship of COS, and thus the research on COS with single DP has attracted wide attention. According to previous studies, the COS with DP 4–7 shows strong inhibition of ascites cancer in BALB/c mice, while (GlcNAC)6 and (GlcN)6 exhibit particularly strong inhibiting effects for S-180 and MM156 solid tumor growth in syngenic mice (Suzuki et al., 1986). However, the DP effect of COS on antiangiogenic activity seems less clear, although the decamer does seem to be the most active in the experiment that was done (Wu et al., 2012). Moreover, it has been reported that the antibacterial activity of COS required structural essential with a DP of at least 5 and the inhibitory effect increased with increasing DP (Li et al., 2014). As for antioxidative activity, COS with low DP shows better effect of scavenging hydroxyl radical and reducing power than that with high DP, while the superoxide radical scavenging activity of all the tested COSs increased with DP increasing (Li, Xing, Liu, Li, et al., 2012). Therefore, it can be concluded that, depending on different bioactivities, the structure–function relationship between DP of COS and bioactivities could be different. In agriculture, the effect of chitosan on growth and photosynthetic characteristics of tobacco, rape, maize and soybean have been researched. The results reveal that chitosan could increase the content of chlorophyll, enhance the photosynthesis by increasing the value of Ci, Tr, Gs and Pn significantly and promote the growth of plants (Li et al., 2010). The molecular weight of chitosan is a vital factor to its activity. Chitosan with low molecular weight is reported to be more effective to promoting photosynthesis than that with high molecular weight (Khan, Prithiviraj, & Smith, 2002). Moreover, highly purified COS with single DP is of significance for studying bioactivity of COS. However, the high-resolution preparative separation of COS is difficult and there are few reports on it. So most of studies are assayed using heterogeneous COS, which brings difficulties to make comparisons between various studies and confirm which COS with well-defined DP plays a leading role in promoting the growth and photosynthesis. Therefore, in order to make it clear, COS with well-define DP is required and our laboratory has successfully prepared a pure fully deacetylated COS series (Li, Liu, Xing, Yu, et al., 2013; Li, Xing, Liu, Qin, et al., 2012). Accordingly, this work was conducted to use nine COSs including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP8–10, DP10–12) to investigated the DP effects of COSs on the growth and photosynthesis of wheat seedlings and find the optimal DP for the growth-promotion activity of COS.
2. Materials and methods 2.1. Materials Nine fully deacetylated COS fractions with well-defined DP were separated from the fully deacetylated COS mixture according to the method reported by our laboratory (Li, Liu, Xing, Yu, et al., 2013; Li, Xing, Liu, Qin, et al., 2012), including seven single COSs, chitobiose (≥98%), chitotriose (≥98%), chitotetraose (≥98%), chitopentaose (≥98%), chitohexaose (≥98%), chitoheptaose (≥93%), chitooctaose(≥90%) and two COSs with narrow DP which mainly included COS with DP8–10 (12.0%, 53.1%, 28.0%) and DP10–12 (18.4%, 49.4%, 22.3%), respectively. All other chemicals and reagents were of analytical grade.
2.2. Plant material and treatments The present study was conducted with wheat (Triticum aestivum L. Jimai 22) seeds. After being sterilized, wheat seeds were transferred to a Petri dish with moist gauze for germination at 25 ◦ C for 24 h in the dark. Then, 900 germinated seeds were individually transplanted to 30 Petri dishes with nylon mesh and each Petri dish contained 30 seeds. Wheat seedlings were cultivated in a growth incubator with a light intensity of 800 mol m−2 s−1 , a day/night cycle of 14 h/10 h at 25 ◦ C/15 ◦ C, respectively, and the relative humidity was controlled at 70%. When the second functional leaf of wheat seedlings was fully expanded, the experiments were randomly divided into ten groups, including a control group (sprayed with distilled water) and nine COS groups (sprayed with 15 mg/L chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10 and DP 10–12, separately), and each group contained three replicates in our experiment. The volume of distilled water or COS solutions sprayed on each sample was 45 mL. Additionally, the wheat seedlings were cultured with Hoagland solution and the solution was renewed every other day. 2.3. Growth parameters The growth of wheat seedlings were evaluated by shoot length, root length, fresh weight and dry weight. After 7 days of COS treatment, wheat seedlings of each group were harvested for determination of shoot length, root length, fresh weight (FW). Dry weight (DW) of samples was determined after being dried at 105 ◦ C for 3 h. 2.4. Determination of soluble sugar and soluble protein Soluble sugar content was measured by phenol-sulfuric acid reaction (Zou et al., 2015) and expressed as mg/g fresh weight. Soluble sugar concentration extracted from the fresh leaf samples was quantified colorimetrically at 485 nm using glucose as the standard. The content of soluble protein was measured from the second functional leaf following the method of Surendar, Devi, Ravi, Jeyakumar, and Velayudham (2013) and expressed as mg g–1 fresh weight. Soluble protein concentration was quantified colorimetrically at 595 nm using bovine serum albumin (BSA) as the standard. 2.5. Determination of chlorophyll contents Content of chlorophyll was determined by the method of Sedmak and Grossberg (1977). After 7 days of COS treatments, chlorophyll was extracted with 95% ethanol. Chlorophyll a (Chla), chlorophyll b (Chl-b) and total chlorophyll (a + b) content were determined with spectrophotometer at 665 nm and 649 nm. All the processes, from extraction to spectral measurement, were performed in dim light to avoid degradation of the chlorophylls in the samples. 2.6. Photosynthetic and chlorophyll fluorescence characteristics measurements Photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were measured with a portable photosynthesis system (L.MAN-LCProSD, BioScientific Ltd., UK). Atmospheric conditions consisted of 25 ± 2 ◦ C, gas flow rate of 200 mol s–1 , photosynthetic photon flux density of 800 mol m−2 s−1 and CO2 concentration of 395 ± 5 mol−2 s−1 . Before making measurements, the test leaves were held in the leaf chamber for 5 min or more to obtain a photosynthetic steady state.
X. Zhang et al. / Carbohydrate Polymers 138 (2016) 27–33
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Fig. 1. Size effects of COS on fresh weight of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
Chlorophyll fluorescence was measured using a portable fluorometer (PAM-2100, Walz, Germany). For each plant, fluorescence was analyzed from the upper surface of the second functional leaf. All parameters were measured after dark adaptation for 30 min, including the maximum quantum yield of PSII (Fv/Fm), quantum yield of PS II (PSII), maximum primary yield of photochemistry of PSII (Fv/Fo), photochemical quenching coefficient (qP), nonphotochemical quenching coefficient (NPQ) and variable chlorophyll fluorescence decrease ratio (Rfd). 2.7. Statistical analyses Each test was performed in triplicate, and each data was expressed as the means ± SD of three independent replicates. Statistical analyses were conducted using the SPSS 17.0 software and performed by one-way ANOVA followed by Duncan’s test (P < 0.05). 3. Results 3.1. Size effects of COS on the growth parameters of wheat seedlings As shown in Figs. 1–3, nine COSs including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) showed different impact on the fresh
weight, dry weight, shoot length and root length of wheat seedlings. Compared with the control, the exogenous COS including chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP8–10 and DP10–12 could increase the root fresh weight of wheat seedlings by 21.8%, 36.8%, 41.7%, 42.6%, 45.2%, 53.2%, 42.6%, 42.0% and 33.7%, respectively, and COSs (chitotetraose to chitoheptaose) increased shoot fresh weight by 13.1%, 14.6%, 18.8% and 27.9%, separately (P < 0.05) (Fig. 1). The results indicated that, among the nine COSs, chitoheptaose showed the optimal prompting effect on fresh weight. Dry weight and root length were also measured and had a consistent variation trend with fresh weight (P < 0.05). As Figs. 2 and 3 showed, COSs with DP > 3 could increased shoot dry weight, root dry weight and root length of wheat seedlings up to 24.5%–35.0%, 35.2%–64.8%, 13.4%–27.1%, respectively (P < 0.05), and chitoheptaose also showed the strongest activity. However, as showed in Fig. 3, there was no significant statistics difference on shoot length of wheat seedlings between nine COS groups and the control group (P > 0.05). 3.2. Size effects of COS on the soluble sugar and soluble protein contents of wheat seedlings Size effects of COS on the soluble sugar and soluble protein contents of wheat seedlings were shown in Figs. 4 and 5. The
Fig. 2. Size effects of COS on dry weight of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
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Fig. 3. Size effects of COS on length of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
increased in the order of chitoheptaose > chitotetraose, chitopentaose, chitohexaose, chitooctaose, DP 8–10 > DP 10–12 (P < 0.05), which indicated that chitoheptaose showed the most obvious promoting effect compared to other groups, while chitotetraose, chitopentaose, chitohexaose, chitooctaose and DP 8–10 exhibited similar effect. Moreover, the content of soluble protein also varied with a similar trend as the soluble sugar content. Chitoheptaose increased the content of soluble protein of the samples up to 22.0%, which were significantly higher than other COS groups similarly (P < 0.05). 3.3. Size effects of COS on the chlorophyll contents of wheat seedlings
Fig. 4. Size effects of COS on soluble sugar content in leaves of Jimai-22. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
results showed that the exogenous COS treatment (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP 10–12) could increase the soluble sugar content of wheat seedlings by 34.4%, 36.7%, 44.0%, 59.4%, 33.0%, 33.7% and 27.2% (P < 0.05), respectively, while chitobiose and chitotriose had no significant effect on the soluble sugar contents compared with the control. The promoting effect of COSs on soluble sugar content
As is shown in Table 1, except chitobiose, all other COS samples could significantly increase the contents of Chl-a and total chlorophyll by 11.4%–20.1% and 10.6%–20.3%, separately, and COSs with DP > 4 could increase Chl-b contents by 9.1%-20.8% (P < 0.05). Furthermore, the total chlorophyll contents of COSs treatments increased in the order of chitoheptaose > chitopentaose, chitohexaose, chitooctaose, DP 8–10 > chitotetraose, DP 10–12. Thus chitoheptaose showed the best activity in increasing the chlorophyll contents, which increased the Chl-a and total chlorophyll contents up to 20.1% and 20.3%, respectively, but the Chl-b content had no obvious variation trend among all COS treatments. 3.4. Size effects of COS on the photosynthesis of wheat seedlings Table 2 showed the effects of nine COSs on photosynthetic rate and some related parameters of wheat seedlings. Compared with the control, all well-defined COSs except chitobiose could Table 1 Size effects of COS on chlorophyll contents of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
Fig. 5. Size effects of COS on soluble protein content in leaves of Jimai-22. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
DP
Chl-a (mg/g)
Chl-b (mg/g)
Chl(a + b) (mg/g)
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
0.942 ± 0.020e 1.000 ± 0.014d 1.005 ± 0.024cd 1.049 ± 0.027bcd 1.052 ± 0.011bc 1.070 ± 0.006b 1.131 ± 0.032a 1.077 ± 0.017b 1.059 ± 0.013bc 1.051 ± 0.013bcd
0.452 ± 0.004c 0.466 ± 0.009c 0.475 ± 0.013c 0.493 ± 0.011bc 0.515 ± 0.016ab 0.528 ± 0.010ab 0.546 ± 0.013a 0.531 ± 0.013ab 0.529 ± 0.017ab 0.519 ± 0.013ab
1.394 ± 0.032d 1.467 ± 0.040cd 1.480 ± 0.022c 1.542 ± 0.040bc 1.567 ± 0.044b 1.598 ± 0.041b 1.677 ± 0.028a 1.605 ± 0.042b 1.582 ± 0.023b 1.578 ± 0.022bc
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Table 2 Size effects of COS on photosynthesis of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05. DP
Ci (mol mol−1 )
Tr (mmol m−2 s−1 )
Gs (mmol m−2 s−1 )
Pn (mol m−2 s−1 )
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
275.66 ± 2.89f 278.74 ± 2.21ef 283.36 ± 2.87de 291.06 ± 2.58cd 294.14 ± 2.14bc 302.13 ± 1.70a 301.33 ± 2.96a 297.99 ± 1.57b 291.06 ± 1.68cd 286.44 ± 5.07cd
8.09 ± 0.10f 8.04 ± 0.06f 8.50 ± 0.06de 8.40 ± 0.07ef 9.18 ± 0.06c 9.21 ± 0.05c 10.22 ± 0.03a 9.83 ± 0.40b 8.80 ± 0.09d 8.19 ± 0.52de
0.82 ± 0.03c 0.81 ± 0.05c 0.83 ± 0.02c 0.87 ± 0.02c 1.08 ± 0.09b 1.05 ± 0.03b 1.30 ± 0.10a 1.07 ± 0.12b 0.99 ± 0.08b 0.91 ± 0.10bc
24.18 ± 0.55f 23.52 ± 0.50f 25.85 ± 0.47de 28.11 ± 0.60cd 28.80 ± 0.45bc 30.75 ± 1.23b 32.70 ± 0.66a 30.30 ± 1.32bc 27.71 ± 0.44cd 26.88 ± 1.19de
increase the value of Ci and Tr of wheat seedlings up to 2.8%–9.6% and 5.1%–26.3%, respectively, and COS with DP > 4 increase the value of Gs improved by 20.7%-58.5%, which lead to Pn higher than the control apparently (P < 0.05). The COS (chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP8–10 and DP10–12) could increase Pn by 6.9%, 16.3%, 19.1%, 27.2%, 35.2%, 25.3%, 14.6% and 7.9%, separately (P < 0.05), that is, chitoheptaose was the most effective among nine COS samples. Moreover, chitopentaose, chitohexaose and chitooctaose showed similar promoting effect on Pn, and chitobiose had no significant effect on photosynthesis of wheat seedlings. 3.5. Size effects of COS on the chlorophyll fluorescence parameters of wheat seedlings Table 3 illustrated the DP effects of COS on chlorophyll fluorescence parameter of wheat seedlings. All COS samples had no significant promotion effect on ФPSII and Fv/Fm values compared with the control (P > 0.05). However, COSs with DP > 3 (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP10–12) could significantly increased Fv/Fo, qP and Rfd up to 3.7%–11.0%, 3.7%–18.6%, 6.3%–14.7%, respectively, while NPQ was decreased by 18.6%–34.3% (P < 0.05), which indicated that COSs with DP > 3 improved the light utilization efficiency and chitoheptaose exhibited the optimal activity. After 7 days of chitoheptaose treatment, the value of Fv/Fo, qP and Rfd increased by 11.0%, 18.6% and 14.7%, separately. Therefore, the effect of COSs on chlorophyll fluorescence parameters was closely related to their DPs and a DP higher than 3 was required to insure the promotion effect on the chlorophyll fluorescence parameters of wheat seedlings. 4. Discussion DP is one of the most important parameters influencing the various properties of COSs. The effectiveness of COSs in various applications is dependent on their DP, and the optimal DP maybe exist differential among different plant species and different application. According to prior studies, chitin oligomers larger than
hexamer had strong elicitor activity for wheat leaves. However, oligomers larger than dimer or trimer had similar elicitor activity for tomato cells (Yamaguchi, Ito, & Shibuya, 2000). With respect to other applications, glucosamine trimer showed the highest hydroxyl radical scavenging activity among the COSs with DP2–12 while the high-DP oligomers from pentamer to heptamer had been reported to possess better functional characteristics in antibacterial and antitumor effect compared with those of relatively low DP (Jeon, Shahidi, & Kim, 2000; Li, Xing, Liu, Li, et al., 2012). It had been suggested that COS participated in the regulation of plant growth and development. The promotion effect of COS on soybean growing and nodulation in interaction with Bradyrhizobium had clearly revealed their high dependency on molecular weight (Cabrera et al., 2012). In addition, Talip et al. investigated the growth-promotion activity of two kinds of COSs with different molecular weights (3 kDa and 10 kDa) and found that COS of 3 kDa was more effective than COS of 10 kDa, which indicated that smaller molecular weight COS showed more potential as a plant growth promoter (Talip, Mahmud, Yacob, & Dahlan, 2010). However, the growth-promotion activity of COS which molecular weight was below 3 kDa remained unknown. The present study prepared nine COSs with molecular weight below 3 kDa, including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP8–10, DP10–12) to investigate which individual COS exhibited the optimal activity. Our results suggested that DP played an important role in the growth promotion activity of COS and DP > 3 might be essential for COS to exhibit significantly activity compared with the control. Furthermore, chitoheptaose showed the best growth promotion effect compared to all other COSs with single or narrow DPs (Figs. 1–3), revealing that the growth promotion activity of COS required an essential chemical structure. Moreover, the shoot length of all COSs groups had no significant difference compared with the control. However, the root length of all COS groups were increased obviously. Therefore, the effect of COS on root was more remarkable than that on shoot, which further led to the increasing of root cap ratio of wheat seedlings. This result could be supported by the previous works that found chitosan or oligomeric chitosan did not
Table 3 Size effects of COS on chlorophyll fluorescence parameters of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05. DP
ФPSII
Fv/Fm
Fv/Fo
qP
NPQ
Rfd
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
0.60 ± 0.02c 0.61 ± 0.02bc 0.61 ± 0.03bc 0.62 ± 0.02bc 0.63 ± 0.02bc 0.64 ± 0.01bc 0.66 ± 0.01a 0.65 ± 0.03ab 0.63 ± 0.01bc 0.62 ± 0.01bc
0.82 ± 0.00b 0.82 ± 0.00b 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab 0.84 ± 0.00a 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab
4.62 ± 0.05e 4.71 ± 0.04de 4.70 ± 0.07de 4.79 ± 0.02cd 4.85 ± 0.02bc 4.91 ± 0.03bc 5.08 ± 0.03a 4.94 ± 0.04b 4.88 ± 0.03bc 4.84 ± 0.03bc
0.729 ± 0.007f 0.728 ± 0.012f 0.735 ± 0.010f 0.756 ± 0.003de 0.764 ± 0.007de 0.775 ± 0.011cd 0.809 ± 0.013a 0.793 ± 0.009bc 0.784 ± 0.004bc 0.769 ± 0.014de
0.70 ± 0.03a 0.62 ± 0.02b 0.60 ± 0.04b 0.57 ± 0.01bc 0.51 ± 0.02cd 0.48 ± 0.01d 0.46 ± 0.03d 0.47 ± 0.02d 0.48 ± 0.03d 0.51 ± 0.03cd
4.29 ± 0.05e 4.22 ± 0.03e 4.44 ± 0.03d 4.56 ± 0.06cd 4.66 ± 0.04c 4.90 ± 0.04b 5.09 ± 0.08a 4.92 ± 0.03b 4.87 ± 0.04b 4.82 ± 0.02b
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affect shoot height of rice and soybean (Boonlertnirun et al., 2008; Khan et al., 2002). The root system not only anchored the above ground part of the plant but also acted as a highly elaborate absorptive organ enabling nutrients to be absorbed and transported to the rest of the plant (González-Pérez et al., 2012). Well-developed root system was necessary for crop growth and increase in production. COS with DP > 3 could control the shoot growth in order to insure the root growth, which is favorable to the accumulation of biomass. Under normal physiological conditions, the major part of light absorbed by the photosynthetic pigments was used for photosynthetic quantum conversion. So photosynthetic pigment content directly influenced the light absorption, transmission, distribution between the PSI and PSII and energy conversion. Moreover, it had been reported that COS with molecular weight of 2 kDa (DP 8–16) could increase the content of chlorophyll of peanut and coffee and the increased chlorophylls helped the leaves of rice to be greener (Dzung, Khanh, & Dzung, 2011). The present study demonstrated that COSs with DP > 3 could increase the contents of chlorophyll and further proved that chitoheptaose was the best (Table 1). Photosynthesis was the primary process for crops to form their grain yields. Net photosynthetic rates (Pn), stomatal conductance (Gs), and transpirationrates (Tr) of wheat could be used as the potential selection markers to assess their cultivar performances (Subrahmanyam, Subash, Haris, & Sikka, 2006; Khan et al., 2002). Li et al. (2010) reported that treatments of a COS mixture (DP 2–10) were beneficial to increase photosynthetic parameters of B. napus L. In agreement with this study, our results (Table 2) indicated that COSs with well-defined DP (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP 10–12) could improve the values of Gs, Tr, Ci and Pn. It seemed that the promotion effect of COS on photosynthesis also needed an essential chemical structure. A DP of at least 4 was required to induce the improvement of photosynthesis. Moreover, chitoheptaose exhibited the most efficient activity. Chlorophyll fluorescence was a reaction of the photosynthesis apparatus in which excess light energy that was not consumed in the photochemical reactions or converted to heat was radiated from the plant as fluorescent light. As fluorescence signals originated only from the plants, chlorophyll fluorescence parameters had been successfully used to evaluate the functionality of photosynthetic apparatus and monitor the growth of plants (Mehta, Jajoo, Mathur, & Bharti, 2010). It had been reported that, with the foliar anatomical study, chitosan could increase significantly the chloroplast diameter of Dendrobium orchid in young leaves and old leaves, and the reduction of ycf2 gene expression was detected in the young leaves after 12–48 h of chitosan treatment, which indicated that chloroplast was one of the target sites for chitosan action in Dendrobium and the change in chloroplast size maybe one of the factors led to stimulate improvement of photosynthetic efficiency (Limpanavech et al., 2008). In present study, we suggested (Table 3) DP > 3 was necessary for COS to significant enhancement in chlorophyll fluorescence. The reason may be that COS reduced the gene expression and enlarged the main photosynthesis apparatus-chloroplast, and chitoheptaose exhibited the optimal activity in inducing the enlargement of chloroplast (Limpanavech et al., 2008). It had reported that strongly cationic COS could target anionic pectin of the plant cell wall and displace calcium ions. Then the disruption of calcium bound pectin of the plant cell wall by deacetylated chitosan could be perceived and interpreted by plant cells as a signal commanding the strongest possible response (Cabrera, Boland, Cambier, Frettinger, & Van Cutsem, 2010). The optimal effect of chitoheptaose on growth and photosynthesis of wheat seedlings maybe achieved by the manner of effecting the concentration of calcium in cytoplasm, but the exact growth-promotion mechanism of COS was not clear and needed to be further studied.
5. Conclusion The present study focused on the effect of seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) on the growth and photosynthetic characteristics of wheat seedlings. It was concluded as follows: (i) five single COSs (chitotetraose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) could enhance significantly the growth and photosynthesis of wheat seedlings. (ii) The effect of COS on the growth and photosynthesis of wheat seedlings was closely related to its DP. Generally, a DP higher than 3 is required for COS to induce a biological response on growth and photosynthesis promotion. (iii) Chitopentaose, chitohexaose, chitoheptaose, chitooctaose and DP 8–10 showed to be more effective in growth promotion than other COS samples (chitobiose, chitotriose, chitotetraose, DP 10–12), and chitohexaose exhibited to be optimal among the nine samples. These results are conducive to reveal the growth-promotion mechanism of COS and expand its application in agriculture fields.
Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 41406086), the Public Science and Technology Research Funds Projects of Ocean (Nos. 201305016-2, 2014050382), Shangdong Provincial Science and Technology Major Project, China (Grant No. 2015ZDZX05003) and Qingdao Minsheng Science and Technology Plan (14-2-3-47-nsh).
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Size effects of chitooligomers on the growth and photosynthetic characteristics of wheat seedlings Xiaoqian Zhang a,b , Kecheng Li a,∗ , Song Liu a , Ronge Xing a , Huahua Yu a , Xiaolin Chen a , Pengcheng Li a,∗ a b
Key Laborotory Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 29 September 2015 Received in revised form 18 November 2015 Accepted 19 November 2015 Available online 23 November 2015 Chemical compounds studied in this article: d-Glucosamine (PubChem CID: 439213) Chitobiose (PubChem CID: 90657429) Chitotriose (PubChem CID: 70702331) Chitotetraose (PubChem CID: 70702329) Chitopentaose (PubChem CID: 14055040) Chitohexaose (PubChem CID: 3081404) Chitoheptaose (PubChem CID: 3081414) Chitooctaose (PubChem CID: 24978517) Chlorophyll a (PubChem CID: 46936306) Chlorophyll b (PubChem CID: 6450186)
a b s t r a c t In this study, nine chitooligomers (COSs) including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow degrees of polymerization (DPs) (DP8–10, DP10–12) were prepared and applied to wheat seedlings to investigate the size effects of COSs on the growth and photosynthesis parameters of wheat seedlings. The results showed that the activities of COS were closely related to their DPs, and DP > 3 was necessary to insure a significant promotion effect on the growth and photosynthesis. Moreover, chitoheptaose exhibited the optimal activity compared with other COS samples. After 7 days of chitoheptaose treatment, the growth parameters of wheat seedlings could be significantly improved and the contents of soluble sugar, soluble protein and chlorophyll were increased by 59.4%, 22.0% and 20.3%, respectively. In addition, chitoheptaose could significantly enhance the net photosynthetic rate of wheat seedlings with the values of Fv/Fo, qP and Rfd increased by 11.0%, 18.6% and 14.7%, respectively, while NPQ was decreased obviously, which might resulted in the promotion of light utilization efficiency and the growth of wheat seedlings. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chitooligomers Size Growth Photosynthesis Chlorophyll fluorescence
1. Introduction Chitooligomers (COS) is a low molecular weight polymer which can be produced from either chitin itself or from chitosan, so it includes homo- or heterooligomers of N-acetyl d-glucosamine and d-glucosamine (Bahrke et al., 2002). Previous studies showed that COS possessed various potential applications in medicine and food fields due to its wide bioactivities, such as antitumor, Harish Prashanth & Tharanathan, 2005, antioxidative (Li, Liu, Xing, Qin, & Li, 2013), fat lowering (Muzzarelli, 1997), antiglycation (Zhang, Yu, Zhang, Zhao, & Dong, 2014), immunostimulatory (Peluso et al., 1994), antimicrobial (Choi et al., 2001; Mei, Dai, Yang, Xu, & Liang,
∗ Corresponding authors. E-mail addresses: [email protected] (K. Li), [email protected] (P. Li). http://dx.doi.org/10.1016/j.carbpol.2015.11.050 0144-8617/© 2015 Elsevier Ltd. All rights reserved.
2015) and wound healing activities (Aam et al., 2010; Muzzarelli, 2009). It also had been recognized as a product to enhance plant growth, including stimulating growth of orchid tissue (Nge, Newa, Chnadrkrachnag, & Setvens, 2006), increasing the chlorophylls contents, enhancing the photosynthesis and enlarging the chloroplast of the Dendrobium orchid (Limpanavech et al., 2008), promoting mineral nutrient uptake of Phaseolus vulgaris (Chatelain, Pintado, & Vasconcelos, 2014) and alleviating salt stress to wheat seedlings (Zou et al., 2015). Therefore, COS has attracted increasing interest in the field of agriculture. The bioactivity of COS depends closely on its structure and physicochemical property. The degree of polymerization (DP), degree of deacetylation (DD), charge distribution and the oligomer structure pattern have important influence on its bioactivity (Aranaz, Harris, & Heras, 2010; Muzzarelli, 1996). Furthermore, the dosage, application method, pH and temperature of the medium
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and the presence or absence of an active unit would modify the activity of the molecule (Boonlertnirun, Boonraung, & Suvanasara, 2008; Kim & Rajapakse, 2005). Therefore, when investigating the influence of COS with a certain property on all kind of applications, all other parameters of COS should remain the same, and it would be of great importance in practical application. In particular, molecular size of COS is a very important factor in the study of structure–function relationship of COS, and thus the research on COS with single DP has attracted wide attention. According to previous studies, the COS with DP 4–7 shows strong inhibition of ascites cancer in BALB/c mice, while (GlcNAC)6 and (GlcN)6 exhibit particularly strong inhibiting effects for S-180 and MM156 solid tumor growth in syngenic mice (Suzuki et al., 1986). However, the DP effect of COS on antiangiogenic activity seems less clear, although the decamer does seem to be the most active in the experiment that was done (Wu et al., 2012). Moreover, it has been reported that the antibacterial activity of COS required structural essential with a DP of at least 5 and the inhibitory effect increased with increasing DP (Li et al., 2014). As for antioxidative activity, COS with low DP shows better effect of scavenging hydroxyl radical and reducing power than that with high DP, while the superoxide radical scavenging activity of all the tested COSs increased with DP increasing (Li, Xing, Liu, Li, et al., 2012). Therefore, it can be concluded that, depending on different bioactivities, the structure–function relationship between DP of COS and bioactivities could be different. In agriculture, the effect of chitosan on growth and photosynthetic characteristics of tobacco, rape, maize and soybean have been researched. The results reveal that chitosan could increase the content of chlorophyll, enhance the photosynthesis by increasing the value of Ci, Tr, Gs and Pn significantly and promote the growth of plants (Li et al., 2010). The molecular weight of chitosan is a vital factor to its activity. Chitosan with low molecular weight is reported to be more effective to promoting photosynthesis than that with high molecular weight (Khan, Prithiviraj, & Smith, 2002). Moreover, highly purified COS with single DP is of significance for studying bioactivity of COS. However, the high-resolution preparative separation of COS is difficult and there are few reports on it. So most of studies are assayed using heterogeneous COS, which brings difficulties to make comparisons between various studies and confirm which COS with well-defined DP plays a leading role in promoting the growth and photosynthesis. Therefore, in order to make it clear, COS with well-define DP is required and our laboratory has successfully prepared a pure fully deacetylated COS series (Li, Liu, Xing, Yu, et al., 2013; Li, Xing, Liu, Qin, et al., 2012). Accordingly, this work was conducted to use nine COSs including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP8–10, DP10–12) to investigated the DP effects of COSs on the growth and photosynthesis of wheat seedlings and find the optimal DP for the growth-promotion activity of COS.
2. Materials and methods 2.1. Materials Nine fully deacetylated COS fractions with well-defined DP were separated from the fully deacetylated COS mixture according to the method reported by our laboratory (Li, Liu, Xing, Yu, et al., 2013; Li, Xing, Liu, Qin, et al., 2012), including seven single COSs, chitobiose (≥98%), chitotriose (≥98%), chitotetraose (≥98%), chitopentaose (≥98%), chitohexaose (≥98%), chitoheptaose (≥93%), chitooctaose(≥90%) and two COSs with narrow DP which mainly included COS with DP8–10 (12.0%, 53.1%, 28.0%) and DP10–12 (18.4%, 49.4%, 22.3%), respectively. All other chemicals and reagents were of analytical grade.
2.2. Plant material and treatments The present study was conducted with wheat (Triticum aestivum L. Jimai 22) seeds. After being sterilized, wheat seeds were transferred to a Petri dish with moist gauze for germination at 25 ◦ C for 24 h in the dark. Then, 900 germinated seeds were individually transplanted to 30 Petri dishes with nylon mesh and each Petri dish contained 30 seeds. Wheat seedlings were cultivated in a growth incubator with a light intensity of 800 mol m−2 s−1 , a day/night cycle of 14 h/10 h at 25 ◦ C/15 ◦ C, respectively, and the relative humidity was controlled at 70%. When the second functional leaf of wheat seedlings was fully expanded, the experiments were randomly divided into ten groups, including a control group (sprayed with distilled water) and nine COS groups (sprayed with 15 mg/L chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10 and DP 10–12, separately), and each group contained three replicates in our experiment. The volume of distilled water or COS solutions sprayed on each sample was 45 mL. Additionally, the wheat seedlings were cultured with Hoagland solution and the solution was renewed every other day. 2.3. Growth parameters The growth of wheat seedlings were evaluated by shoot length, root length, fresh weight and dry weight. After 7 days of COS treatment, wheat seedlings of each group were harvested for determination of shoot length, root length, fresh weight (FW). Dry weight (DW) of samples was determined after being dried at 105 ◦ C for 3 h. 2.4. Determination of soluble sugar and soluble protein Soluble sugar content was measured by phenol-sulfuric acid reaction (Zou et al., 2015) and expressed as mg/g fresh weight. Soluble sugar concentration extracted from the fresh leaf samples was quantified colorimetrically at 485 nm using glucose as the standard. The content of soluble protein was measured from the second functional leaf following the method of Surendar, Devi, Ravi, Jeyakumar, and Velayudham (2013) and expressed as mg g–1 fresh weight. Soluble protein concentration was quantified colorimetrically at 595 nm using bovine serum albumin (BSA) as the standard. 2.5. Determination of chlorophyll contents Content of chlorophyll was determined by the method of Sedmak and Grossberg (1977). After 7 days of COS treatments, chlorophyll was extracted with 95% ethanol. Chlorophyll a (Chla), chlorophyll b (Chl-b) and total chlorophyll (a + b) content were determined with spectrophotometer at 665 nm and 649 nm. All the processes, from extraction to spectral measurement, were performed in dim light to avoid degradation of the chlorophylls in the samples. 2.6. Photosynthetic and chlorophyll fluorescence characteristics measurements Photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were measured with a portable photosynthesis system (L.MAN-LCProSD, BioScientific Ltd., UK). Atmospheric conditions consisted of 25 ± 2 ◦ C, gas flow rate of 200 mol s–1 , photosynthetic photon flux density of 800 mol m−2 s−1 and CO2 concentration of 395 ± 5 mol−2 s−1 . Before making measurements, the test leaves were held in the leaf chamber for 5 min or more to obtain a photosynthetic steady state.
X. Zhang et al. / Carbohydrate Polymers 138 (2016) 27–33
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Fig. 1. Size effects of COS on fresh weight of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
Chlorophyll fluorescence was measured using a portable fluorometer (PAM-2100, Walz, Germany). For each plant, fluorescence was analyzed from the upper surface of the second functional leaf. All parameters were measured after dark adaptation for 30 min, including the maximum quantum yield of PSII (Fv/Fm), quantum yield of PS II (PSII), maximum primary yield of photochemistry of PSII (Fv/Fo), photochemical quenching coefficient (qP), nonphotochemical quenching coefficient (NPQ) and variable chlorophyll fluorescence decrease ratio (Rfd). 2.7. Statistical analyses Each test was performed in triplicate, and each data was expressed as the means ± SD of three independent replicates. Statistical analyses were conducted using the SPSS 17.0 software and performed by one-way ANOVA followed by Duncan’s test (P < 0.05). 3. Results 3.1. Size effects of COS on the growth parameters of wheat seedlings As shown in Figs. 1–3, nine COSs including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) showed different impact on the fresh
weight, dry weight, shoot length and root length of wheat seedlings. Compared with the control, the exogenous COS including chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP8–10 and DP10–12 could increase the root fresh weight of wheat seedlings by 21.8%, 36.8%, 41.7%, 42.6%, 45.2%, 53.2%, 42.6%, 42.0% and 33.7%, respectively, and COSs (chitotetraose to chitoheptaose) increased shoot fresh weight by 13.1%, 14.6%, 18.8% and 27.9%, separately (P < 0.05) (Fig. 1). The results indicated that, among the nine COSs, chitoheptaose showed the optimal prompting effect on fresh weight. Dry weight and root length were also measured and had a consistent variation trend with fresh weight (P < 0.05). As Figs. 2 and 3 showed, COSs with DP > 3 could increased shoot dry weight, root dry weight and root length of wheat seedlings up to 24.5%–35.0%, 35.2%–64.8%, 13.4%–27.1%, respectively (P < 0.05), and chitoheptaose also showed the strongest activity. However, as showed in Fig. 3, there was no significant statistics difference on shoot length of wheat seedlings between nine COS groups and the control group (P > 0.05). 3.2. Size effects of COS on the soluble sugar and soluble protein contents of wheat seedlings Size effects of COS on the soluble sugar and soluble protein contents of wheat seedlings were shown in Figs. 4 and 5. The
Fig. 2. Size effects of COS on dry weight of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
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Fig. 3. Size effects of COS on length of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
increased in the order of chitoheptaose > chitotetraose, chitopentaose, chitohexaose, chitooctaose, DP 8–10 > DP 10–12 (P < 0.05), which indicated that chitoheptaose showed the most obvious promoting effect compared to other groups, while chitotetraose, chitopentaose, chitohexaose, chitooctaose and DP 8–10 exhibited similar effect. Moreover, the content of soluble protein also varied with a similar trend as the soluble sugar content. Chitoheptaose increased the content of soluble protein of the samples up to 22.0%, which were significantly higher than other COS groups similarly (P < 0.05). 3.3. Size effects of COS on the chlorophyll contents of wheat seedlings
Fig. 4. Size effects of COS on soluble sugar content in leaves of Jimai-22. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
results showed that the exogenous COS treatment (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP 10–12) could increase the soluble sugar content of wheat seedlings by 34.4%, 36.7%, 44.0%, 59.4%, 33.0%, 33.7% and 27.2% (P < 0.05), respectively, while chitobiose and chitotriose had no significant effect on the soluble sugar contents compared with the control. The promoting effect of COSs on soluble sugar content
As is shown in Table 1, except chitobiose, all other COS samples could significantly increase the contents of Chl-a and total chlorophyll by 11.4%–20.1% and 10.6%–20.3%, separately, and COSs with DP > 4 could increase Chl-b contents by 9.1%-20.8% (P < 0.05). Furthermore, the total chlorophyll contents of COSs treatments increased in the order of chitoheptaose > chitopentaose, chitohexaose, chitooctaose, DP 8–10 > chitotetraose, DP 10–12. Thus chitoheptaose showed the best activity in increasing the chlorophyll contents, which increased the Chl-a and total chlorophyll contents up to 20.1% and 20.3%, respectively, but the Chl-b content had no obvious variation trend among all COS treatments. 3.4. Size effects of COS on the photosynthesis of wheat seedlings Table 2 showed the effects of nine COSs on photosynthetic rate and some related parameters of wheat seedlings. Compared with the control, all well-defined COSs except chitobiose could Table 1 Size effects of COS on chlorophyll contents of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
Fig. 5. Size effects of COS on soluble protein content in leaves of Jimai-22. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05.
DP
Chl-a (mg/g)
Chl-b (mg/g)
Chl(a + b) (mg/g)
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
0.942 ± 0.020e 1.000 ± 0.014d 1.005 ± 0.024cd 1.049 ± 0.027bcd 1.052 ± 0.011bc 1.070 ± 0.006b 1.131 ± 0.032a 1.077 ± 0.017b 1.059 ± 0.013bc 1.051 ± 0.013bcd
0.452 ± 0.004c 0.466 ± 0.009c 0.475 ± 0.013c 0.493 ± 0.011bc 0.515 ± 0.016ab 0.528 ± 0.010ab 0.546 ± 0.013a 0.531 ± 0.013ab 0.529 ± 0.017ab 0.519 ± 0.013ab
1.394 ± 0.032d 1.467 ± 0.040cd 1.480 ± 0.022c 1.542 ± 0.040bc 1.567 ± 0.044b 1.598 ± 0.041b 1.677 ± 0.028a 1.605 ± 0.042b 1.582 ± 0.023b 1.578 ± 0.022bc
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Table 2 Size effects of COS on photosynthesis of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05. DP
Ci (mol mol−1 )
Tr (mmol m−2 s−1 )
Gs (mmol m−2 s−1 )
Pn (mol m−2 s−1 )
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
275.66 ± 2.89f 278.74 ± 2.21ef 283.36 ± 2.87de 291.06 ± 2.58cd 294.14 ± 2.14bc 302.13 ± 1.70a 301.33 ± 2.96a 297.99 ± 1.57b 291.06 ± 1.68cd 286.44 ± 5.07cd
8.09 ± 0.10f 8.04 ± 0.06f 8.50 ± 0.06de 8.40 ± 0.07ef 9.18 ± 0.06c 9.21 ± 0.05c 10.22 ± 0.03a 9.83 ± 0.40b 8.80 ± 0.09d 8.19 ± 0.52de
0.82 ± 0.03c 0.81 ± 0.05c 0.83 ± 0.02c 0.87 ± 0.02c 1.08 ± 0.09b 1.05 ± 0.03b 1.30 ± 0.10a 1.07 ± 0.12b 0.99 ± 0.08b 0.91 ± 0.10bc
24.18 ± 0.55f 23.52 ± 0.50f 25.85 ± 0.47de 28.11 ± 0.60cd 28.80 ± 0.45bc 30.75 ± 1.23b 32.70 ± 0.66a 30.30 ± 1.32bc 27.71 ± 0.44cd 26.88 ± 1.19de
increase the value of Ci and Tr of wheat seedlings up to 2.8%–9.6% and 5.1%–26.3%, respectively, and COS with DP > 4 increase the value of Gs improved by 20.7%-58.5%, which lead to Pn higher than the control apparently (P < 0.05). The COS (chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP8–10 and DP10–12) could increase Pn by 6.9%, 16.3%, 19.1%, 27.2%, 35.2%, 25.3%, 14.6% and 7.9%, separately (P < 0.05), that is, chitoheptaose was the most effective among nine COS samples. Moreover, chitopentaose, chitohexaose and chitooctaose showed similar promoting effect on Pn, and chitobiose had no significant effect on photosynthesis of wheat seedlings. 3.5. Size effects of COS on the chlorophyll fluorescence parameters of wheat seedlings Table 3 illustrated the DP effects of COS on chlorophyll fluorescence parameter of wheat seedlings. All COS samples had no significant promotion effect on ФPSII and Fv/Fm values compared with the control (P > 0.05). However, COSs with DP > 3 (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP10–12) could significantly increased Fv/Fo, qP and Rfd up to 3.7%–11.0%, 3.7%–18.6%, 6.3%–14.7%, respectively, while NPQ was decreased by 18.6%–34.3% (P < 0.05), which indicated that COSs with DP > 3 improved the light utilization efficiency and chitoheptaose exhibited the optimal activity. After 7 days of chitoheptaose treatment, the value of Fv/Fo, qP and Rfd increased by 11.0%, 18.6% and 14.7%, separately. Therefore, the effect of COSs on chlorophyll fluorescence parameters was closely related to their DPs and a DP higher than 3 was required to insure the promotion effect on the chlorophyll fluorescence parameters of wheat seedlings. 4. Discussion DP is one of the most important parameters influencing the various properties of COSs. The effectiveness of COSs in various applications is dependent on their DP, and the optimal DP maybe exist differential among different plant species and different application. According to prior studies, chitin oligomers larger than
hexamer had strong elicitor activity for wheat leaves. However, oligomers larger than dimer or trimer had similar elicitor activity for tomato cells (Yamaguchi, Ito, & Shibuya, 2000). With respect to other applications, glucosamine trimer showed the highest hydroxyl radical scavenging activity among the COSs with DP2–12 while the high-DP oligomers from pentamer to heptamer had been reported to possess better functional characteristics in antibacterial and antitumor effect compared with those of relatively low DP (Jeon, Shahidi, & Kim, 2000; Li, Xing, Liu, Li, et al., 2012). It had been suggested that COS participated in the regulation of plant growth and development. The promotion effect of COS on soybean growing and nodulation in interaction with Bradyrhizobium had clearly revealed their high dependency on molecular weight (Cabrera et al., 2012). In addition, Talip et al. investigated the growth-promotion activity of two kinds of COSs with different molecular weights (3 kDa and 10 kDa) and found that COS of 3 kDa was more effective than COS of 10 kDa, which indicated that smaller molecular weight COS showed more potential as a plant growth promoter (Talip, Mahmud, Yacob, & Dahlan, 2010). However, the growth-promotion activity of COS which molecular weight was below 3 kDa remained unknown. The present study prepared nine COSs with molecular weight below 3 kDa, including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP8–10, DP10–12) to investigate which individual COS exhibited the optimal activity. Our results suggested that DP played an important role in the growth promotion activity of COS and DP > 3 might be essential for COS to exhibit significantly activity compared with the control. Furthermore, chitoheptaose showed the best growth promotion effect compared to all other COSs with single or narrow DPs (Figs. 1–3), revealing that the growth promotion activity of COS required an essential chemical structure. Moreover, the shoot length of all COSs groups had no significant difference compared with the control. However, the root length of all COS groups were increased obviously. Therefore, the effect of COS on root was more remarkable than that on shoot, which further led to the increasing of root cap ratio of wheat seedlings. This result could be supported by the previous works that found chitosan or oligomeric chitosan did not
Table 3 Size effects of COS on chlorophyll fluorescence parameters of wheat seedlings. Values are the mean ± SD of three replicates. Different letters indicate significant differences at P < 0.05. DP
ФPSII
Fv/Fm
Fv/Fo
qP
NPQ
Rfd
CK DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP8–10 DP10–12
0.60 ± 0.02c 0.61 ± 0.02bc 0.61 ± 0.03bc 0.62 ± 0.02bc 0.63 ± 0.02bc 0.64 ± 0.01bc 0.66 ± 0.01a 0.65 ± 0.03ab 0.63 ± 0.01bc 0.62 ± 0.01bc
0.82 ± 0.00b 0.82 ± 0.00b 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab 0.84 ± 0.00a 0.83 ± 0.01ab 0.83 ± 0.01ab 0.83 ± 0.01ab
4.62 ± 0.05e 4.71 ± 0.04de 4.70 ± 0.07de 4.79 ± 0.02cd 4.85 ± 0.02bc 4.91 ± 0.03bc 5.08 ± 0.03a 4.94 ± 0.04b 4.88 ± 0.03bc 4.84 ± 0.03bc
0.729 ± 0.007f 0.728 ± 0.012f 0.735 ± 0.010f 0.756 ± 0.003de 0.764 ± 0.007de 0.775 ± 0.011cd 0.809 ± 0.013a 0.793 ± 0.009bc 0.784 ± 0.004bc 0.769 ± 0.014de
0.70 ± 0.03a 0.62 ± 0.02b 0.60 ± 0.04b 0.57 ± 0.01bc 0.51 ± 0.02cd 0.48 ± 0.01d 0.46 ± 0.03d 0.47 ± 0.02d 0.48 ± 0.03d 0.51 ± 0.03cd
4.29 ± 0.05e 4.22 ± 0.03e 4.44 ± 0.03d 4.56 ± 0.06cd 4.66 ± 0.04c 4.90 ± 0.04b 5.09 ± 0.08a 4.92 ± 0.03b 4.87 ± 0.04b 4.82 ± 0.02b
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X. Zhang et al. / Carbohydrate Polymers 138 (2016) 27–33
affect shoot height of rice and soybean (Boonlertnirun et al., 2008; Khan et al., 2002). The root system not only anchored the above ground part of the plant but also acted as a highly elaborate absorptive organ enabling nutrients to be absorbed and transported to the rest of the plant (González-Pérez et al., 2012). Well-developed root system was necessary for crop growth and increase in production. COS with DP > 3 could control the shoot growth in order to insure the root growth, which is favorable to the accumulation of biomass. Under normal physiological conditions, the major part of light absorbed by the photosynthetic pigments was used for photosynthetic quantum conversion. So photosynthetic pigment content directly influenced the light absorption, transmission, distribution between the PSI and PSII and energy conversion. Moreover, it had been reported that COS with molecular weight of 2 kDa (DP 8–16) could increase the content of chlorophyll of peanut and coffee and the increased chlorophylls helped the leaves of rice to be greener (Dzung, Khanh, & Dzung, 2011). The present study demonstrated that COSs with DP > 3 could increase the contents of chlorophyll and further proved that chitoheptaose was the best (Table 1). Photosynthesis was the primary process for crops to form their grain yields. Net photosynthetic rates (Pn), stomatal conductance (Gs), and transpirationrates (Tr) of wheat could be used as the potential selection markers to assess their cultivar performances (Subrahmanyam, Subash, Haris, & Sikka, 2006; Khan et al., 2002). Li et al. (2010) reported that treatments of a COS mixture (DP 2–10) were beneficial to increase photosynthetic parameters of B. napus L. In agreement with this study, our results (Table 2) indicated that COSs with well-defined DP (chitotetraose, chitopentaose, chitohexaose, chitoheptaose, chitooctaose, DP 8–10, DP 10–12) could improve the values of Gs, Tr, Ci and Pn. It seemed that the promotion effect of COS on photosynthesis also needed an essential chemical structure. A DP of at least 4 was required to induce the improvement of photosynthesis. Moreover, chitoheptaose exhibited the most efficient activity. Chlorophyll fluorescence was a reaction of the photosynthesis apparatus in which excess light energy that was not consumed in the photochemical reactions or converted to heat was radiated from the plant as fluorescent light. As fluorescence signals originated only from the plants, chlorophyll fluorescence parameters had been successfully used to evaluate the functionality of photosynthetic apparatus and monitor the growth of plants (Mehta, Jajoo, Mathur, & Bharti, 2010). It had been reported that, with the foliar anatomical study, chitosan could increase significantly the chloroplast diameter of Dendrobium orchid in young leaves and old leaves, and the reduction of ycf2 gene expression was detected in the young leaves after 12–48 h of chitosan treatment, which indicated that chloroplast was one of the target sites for chitosan action in Dendrobium and the change in chloroplast size maybe one of the factors led to stimulate improvement of photosynthetic efficiency (Limpanavech et al., 2008). In present study, we suggested (Table 3) DP > 3 was necessary for COS to significant enhancement in chlorophyll fluorescence. The reason may be that COS reduced the gene expression and enlarged the main photosynthesis apparatus-chloroplast, and chitoheptaose exhibited the optimal activity in inducing the enlargement of chloroplast (Limpanavech et al., 2008). It had reported that strongly cationic COS could target anionic pectin of the plant cell wall and displace calcium ions. Then the disruption of calcium bound pectin of the plant cell wall by deacetylated chitosan could be perceived and interpreted by plant cells as a signal commanding the strongest possible response (Cabrera, Boland, Cambier, Frettinger, & Van Cutsem, 2010). The optimal effect of chitoheptaose on growth and photosynthesis of wheat seedlings maybe achieved by the manner of effecting the concentration of calcium in cytoplasm, but the exact growth-promotion mechanism of COS was not clear and needed to be further studied.
5. Conclusion The present study focused on the effect of seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) on the growth and photosynthetic characteristics of wheat seedlings. It was concluded as follows: (i) five single COSs (chitotetraose to chitooctaose) and two COS fractions with narrow DPs (DP 8–10, DP 10–12) could enhance significantly the growth and photosynthesis of wheat seedlings. (ii) The effect of COS on the growth and photosynthesis of wheat seedlings was closely related to its DP. Generally, a DP higher than 3 is required for COS to induce a biological response on growth and photosynthesis promotion. (iii) Chitopentaose, chitohexaose, chitoheptaose, chitooctaose and DP 8–10 showed to be more effective in growth promotion than other COS samples (chitobiose, chitotriose, chitotetraose, DP 10–12), and chitohexaose exhibited to be optimal among the nine samples. These results are conducive to reveal the growth-promotion mechanism of COS and expand its application in agriculture fields.
Acknowledgements The study was supported by the National Natural Science Foundation of China (No. 41406086), the Public Science and Technology Research Funds Projects of Ocean (Nos. 201305016-2, 2014050382), Shangdong Provincial Science and Technology Major Project, China (Grant No. 2015ZDZX05003) and Qingdao Minsheng Science and Technology Plan (14-2-3-47-nsh).
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