Physiological Mechanisms of Delaying Leaf Senescence in Maize Treated with Compound Mixtures of DCPTA and CCC

Wang Yong-chao, Gu Wan-rong,, Ye Le-fu,, Sun Yang, Li Li-jie, Zhang He, Li Jing,, and Wei Shi,*

1College of Agriculture, Northeast Agricultural University, Harbin 150030, China

2The Observation Experiment Station of Ministry of Agriculture for Crop Cultivation Science in Northeast Area, Harbin 150030, China

Physiological Mechanisms of Delaying Leaf Senescence in Maize Treated with Compound Mixtures of DCPTA and CCC

Wang Yong-chao1, Gu Wan-rong1,2, Ye Le-fu1,2, Sun Yang1, Li Li-jie1, Zhang He1, Li Jing1,2, and Wei Shi1,2*

1College of Agriculture, Northeast Agricultural University, Harbin 150030, China

2The Observation Experiment Station of Ministry of Agriculture for Crop Cultivation Science in Northeast Area, Harbin 150030, China

At the beginning of silking, maize production began to form, but leaves started senescence and photosynthetic capacity decreased at this time, all of those severely restricted the formation of the production. In order to study the effects of exogenous substances on the process of leaf senescence, 40 mg · L-1DCPTA and 20 mg · L-1CCC were mixed in the research. When the maize grew to the six expanded leaves stage, 10 mL compound mixtures (TR) were sprayed on both sides of leaves for per plant, and the control was treated with water (CK). Three plants were selected randomly for determination of physiological index at the 10, 20, 30, 40 and 50 days after silking. The results showed that TR could increase the chlorophyll content significantly, Fv/Fm, Fv/F0and Y(II) values of TR were higher than those of CK while F0values were opposite. Compared with CK, TR increased SOD and POD activity and soluble protein content, reduced MDA content. Correlation analysis showed that chlorophyll content had negative correlation with F0, and MDA content had negative correlation with other indexes. Compared with CK, TR reduced the negative correlation effect between chlorophyll content and MDA, increased the positive correlation effect between chlorophyll content and Fv/Fm, SOD, POD, soluble protein. The study provided theoretical and experimental evidence for the application of the compound mixtures of DCPTA and CCC to the production.

maize, plant growth regulator, chlorophyll fluorescence parameters, senescence, DCPTA

Introduction

With the increasing population, the demand for grain is growing, but the gradually reduced arable land limits the increase of grain yield. Now, the harvest index and the transformation of plant type of most crops have reached the limit, therefore, to find new ways of increasing yield have became an urgent problem (Spano et al., 2003). Photosynthesis is the main way of accumulating organic matter of plant. Organic matter which is produced by photosynthesis accounts for about 95% of the total plant dry matter weight (Dong et al., 1997). After silking, the contribution rate of photosynthesis to yield is more than 90% and it has an effect for yield formation (Yamori et al., 2010). Therefore, the means by increasing photosynthetic efficiency and extending photosynthetic time of growing late stage were becoming the important ways for increasing yield.

The silking stage is the key stage of yield formation. At this time, leaves begin to senescence and chlorophyll begin to be decomposed. As a result, it causes that the photosynthetic ability declines, which limits the accumulation of organic matter (Fischer, 2012). The main characteristic of leaf senescence is the disintegration of the photosynthetic system (Gunda et al., 2010). In addition, photosystem II (PSII) is considered as the primary site of injury of the photosynthetic apparatus in the aging process (Sharkey et al., 2010). The injury for PSII can cause a change in chlorophyll fluorescence. Therefore, chlorophyll fluorescence has been used as a reliable non-invasive and powerful method for evaluating the changes in function of PSII and for reflecting the primary photosynthetic processes (Hajiboland et al., 2010; Hazem et al., 2014). In the process of senescence, the damage of light system leads to an accumulation of excess reactive oxygen species and lipid peroxides. Plants have evolved complex enzymatic and nonenzymatic antioxidant defense systems to regulate cellular oxidative damage. It is well known that superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidases (APX) are important ROS scavenging enzymes, and they have been well studied in different plants (Gill et al., 2010; Balazs et al., 2012). Therefore, it has the important significance to study antioxidant enzyme activity in the process of leaf senescence.

Hormones and plant growth regulators play a crucial role in developmental processes as well as in the integration of environmental signals to plant development (Maes et al., 2011). Indeed, all the classical plant hormones have been described as playing a role in the regulation of leaf senescence (Prakash, 2013). For example, senescence is accelerated by ethylene, abscisic acid (ABA) and salicylic acid (SA), and delayed by auxin, gibberellic acid (GA) and cytokinins (CKs). Some studies showed compound regulator could increase GA3content and reduce ABA content, and these were conducive to delay the aging of cells and tissues, increase antioxidant enzyme activity and reduce MDA content in maize seeding (Gan, 2010; Shao et al., 2014). In addition, several reports had shown that abiotic stress could cause plant damage and senescence; plant growth regulators could alleviate damage and senescence. At the vegetative stage, severe heat stress can result in retarded growth, senescence, and even death (Wahid et al., 2007). Exogenous application of GABA had been implicated in some important roles in plant, such as a buffering mechanism in C and N metabolism, signal transduction (Kinnersley et al., 2000) and protection against oxidative stress (Harsh et al., 2014). As a result, GABA could alleviate the damage of heat stress. Drought stress can trigger an oxidative burst, accelerate the degradation of photosynthetic pigments and cell membrane damage, induce an array of antioxidant enzymes expression, and elicit membrane lipid accumulation (Zgallai et al., 2005; Ai et al., 2008). Exogenous application of silicon could alleviate the oxidative stress of wheat plant at later growth stages through modification of reactive species biosynthesis and transcriptional regulation of multiple defense pathways, such as antioxidant enzymes, ASC-GSH cycle, and flavonoid secondary metabolism (Ma et al., 2015).

DCPTA (2-diethylaminoethyl-3, 4-dichloro-phenylether) is one of the most representative tertiary compounds (Ren et al., 2003), which has been used in wheat and maize, especially in horticultural crops, such as cucumber and tomato (Dowens et al., 1998). DCPTA has various influences on physiological process in plants, which can regulate photosynthesis of the plants and the activity of antioxidant enzyme (Gausman et al., 1991). Some studies showed that DCPTA could speed up seedling growth, increase chlorophyll content, and improve photosynthesis in different plants, and these indicated that DCPTA participates in regulation of photosynthetic reaction (Gu et al., 2008; Zhou et al., 2004). Chlorocholine chloride (2-chloro-N, N, N-trimethylethanaminium chloride; CCC; CAS No. 50-29-3) is a growth retardant, with its mechanism based on the restraint ofgibberellins biosynthesis in plant tissues. It is well known that CCC induces changes in the growth rate of grasses and the morphogenesis of potato plants cultured in vitro (Luoanen et al., 2002). 500 mg · L-1CCC can increase the net photosynthetic rate (Pn), ratio of variable to maximum fluorescence (Fv/Fm), potential photochemical efficiency (Fv/F0), energy fluxes per excited cross-sections for absorption (ABS/ CS), trapping (TRo/CS) and electron transport (ETo/CS) of pistacia chinensis (Dong et al., 2012).

To evaluate the role of exogenously applied compound mixtures of DCPTA and CCC in the process of leaf senescence, we investigated the changes of chlorophyll fluorescence parameters in maize leaves treated with compound mixture and the effects of compound mixture on the oxidative response, that is, enzymatic (superoxide dismutase, peroxidase) and nonenzymatic antioxidant (soluble protein) systems and lipid peroxidant (malondialdehyde). In addition, the relationship between these physiological indexes and chlorophyll content was also studied in the experiment. The study provided a guiding role about the compound mixtures of DCPTA and CCC for the growth of maize seeding, at the same time, provided new ideas for the development of other active substances.

Materials and Methods

Experimental site and cultivar

The experiment was conducted at the Experimental Station of Northeast Agricultural University, Harbin (126°73'E, 45°73'N), Heilongjiang Province, China, from 2011 to 2012. The soil was a typical black soil (typical hapludoll in USDA soil taxonomy), characterized by a deep, high organic matter content. The soil fertility level was determined before experiment (Table 1). The climate was a temperate continental monsoon in the region. The rainfall was variable with greater distribution in July and August. Maize was planted at mid-April and harvested at the early October.

Table 1 Background data of productivity and soil fertility of experimental fields studied in 2011 and 2012

A high-yielding commercial cultivar, Zhengdan 958 (ZD 958) was used for two years experiments, provided by Beijing Doneed Co., Ltd. The seeds (percentage germination≥85%) were treated with Tebuconazole (triazole fungicide) by the Rainsun Agrochemical Company Ltd., Qingdao, Shandong. In the study area, the growth periods and active accumulated temperature of ZD958 were about 128 days and above 2 850℃, respectively.

Experiment design

Two treatments were set, application with water and compound mixtures under the plant density of 67 500 plants · hm-2. The plot area was 56 m2for every treatment, set up eight ridges, the length and weight of each ridge was 10 m and 0.7 m, respectively. A randomized block design with three replications was used for the study.

DCPTA (2-diethylaminminoethyl-3, 4-dichlorophenylether) and CCC (2-chloroethyltrimethylam monium chloride) were mixed together to be made compound mixtures which was liquid state. The concentrations of DCPTA and CCC were 40 mg · L-1and 20 mg · L-1, respectively. DCPTA was obtainedfrom Zheng Shi (Zhengzhou Zheng Shi Chemical Co., Ltd. China). CCC was obtained from Sigma (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). When the maize grew to the six expanded leaves stage, 10 mL compound mixtures were sprayed on both sides of leaves for per plant (TR), control plants were treated with water (CK) (in order to increase the adhesion, 0.02% Tween-20 was added to compound mixtures). The spraying time was 16: 00-19: 00 in the afternoon. During processing and after, growing environment was good and there hasnot inclement weather, illumination, temperature and humidity were all appropriate.

Field management

Fertilizer application followed high yield practice with a base fertilizer gift of 75 kg N · hm-2, 75 kg P2O5· hm-2, 90 kg K2O · hm-2, and a top dressing with 150 kg · hm-2of urea at seven expanded leaves stage and 75 kg hm-2(46%) N at tassel stage (VT). Soils were plowed and harrowed when their mellowness was considered physically acceptable. Maize was sown on April 28, 2011and on April 29, 2012. By a mechanical ditcher, seeding furrows (5 cm deep and 0.7 cm wide) were directly formed. With manual planting cultivation methods, two seeds were sown into the prepared furrow per 21 cm spacing for the plant density (67 500 plants · m-2) treatment. The seeds were covered with moist soil from both sides of the furrow and compacted soil with feet.

With manual thinning out the maize plant, one plant was remained per sowing point at the two-leaf stage. Other management practices, including insect and weed control were conducted according to local agronomic practices unless otherwise indicated.

Data collection

Date were collected for chlorophyll content, chlorophyll fluorescence parameters, SOD and POD activity, MDA and soluble protein content in 2011 and 2012. From silking stage, three plants were randomly selected and listed for measuring chlorophyll fluorescence parameters in each trial plot. At 10, 20, 30, 40 and 50 days after silking, three plants were randomly selected for determination of physiological indices respectively in each trial plot. Ear leaf of plant was cut, treated with liquid nitrogen and stored in -80℃.

Chlorophyll content

Ear leaf that had been removed the veins was cut, mixed and weighed 0.2 g. The leaves were soaked for 72 h at 4℃ by 10 mL 80% acetone in dark. The absorbance values of extracting solution were determined with UV-1601 UV-spectrophotometer by colorimetric method at wavelength of 649 nm and 665 nm (Porra, 2002).

Chlorophyll fluorescence parameters

Chlorophyll fluorescence parameters of the middle part of ear leaves were determined with PAM-2500 chlorophyll fluorescence analyzer (WALZ, Germany) between 9:00 and 12:00 in sunny. After a 20 min dark adaptation period, the initial (F0) and maximum fluorescence (Fm) were determined. Light intensity was set 600 μmolm ·-2· s-1for determining maximum fluorescence (Fm'), initial fluorescence (F0'), steadystate fluorescence (Fs). Maximal photochemical efficiency of PSII Fv/Fm=(Fm-F0)/Fm, potential photochemical efficiency Fv/F0= (Fv/Fm)/(1-Fv/Fm), actual photochemical efficiency of PSII in the light Y(II)=(Fm'-Fs)/Fm' (Demming-Adams et al., 1996).

SOD and POD activities

SOD activity was determined according to Giannopolitis's method (Giannopolitis, 1997). 20 uL enzyme solution was drawn and mixed with 3 mL SOD enzyme solution (pH 7.8 phosphate buffer 1.5 mL, 750 mol · L-1NBT 0.3 mL, 130 mmol · L-1Met 0.3 mL, 20 mol · L-1FD 0.3 mL, 100 mol · L-1EDTA-Na20.3 mL, distilled water 0.3 mL). Control and enzyme solution were placed for 30 min in 4 000 lx light. The blank was placed in dark for zero, compared in 560 nm. POD activity was determined according to Hernandez's method (Hernandez, 2000). 20 uL enzyme solution was drawn and mixed with 3 mL POD enzyme solution (1.4 uL guaiacol, 0.85 uL 30% H2O2and 0.1 mol · L-1pH 6.0 phosphate buffer). The absorbancevalues were recorded once every 30 s in 470 nm.

MDA and soluble protein content 2 mL enzyme solution was drawn and mixed with 0.67% TBA 2 mL, than water-bath heating for 30 min in 100℃, centrifuged after cooling down. The supernatant was determined respectively in 450 nm, 532 nm and 600 nm (Zhang, 2011). Soluble protein concentration was measured with coomassie brilliant blue G-250 staining (Li and Chen, 1998).

Date analysis

Experimental data were expressed as mean with standard error. The average of two-year data was used in the correlation and path coefficient analyses. Statistical analysis was performed using SPSS 15.0 and Excel 2007 and all means were carried out using LSD Fischer test at a significance level of p＜0.05. Microsoft Excel 2007 was used to chart.

Results

Chlorophyll content in ear leaf

The chlorophyll content of ear leaf showed a decreasing trend. The chlorophyll content of TR was significantly higher than that of CK in 2011 and similar changes were observed from 30 to 50 days after silking in 2012. Though there was no significantly difference between 10 and 20 days, compared with CK, the chlorophyll content of TR improved 4.76% and 4.03%, respectively. Two-year experiments were with the consistent tendency of chlorophyll content (Fig. 1).

Fig. 1 Chlorophyll content (A) in maize plant leaves treated (TR) or not (CK) with compound mixtures2011 and 2012 represent planting years. CK represents leaves treated with water, TR represents leaves treated with 40 mg · L-1DCPTA+20 mg · L-1CCC. Bars are means ± standard error of three replicates. Different letters above columns indicate significant differences (p＜0.05) between TR and CK by Least Significant Difference (LSD) test.

Chlorophyll fluorescence parameters in ear leaf

With the increase of days after silking, F0values of ear leaf showed an upward trend, Fv/Fm, Fv/F0and Y(II) were downward trend. F0values represented for fluorescence yield, which was completely open in the reacting centre of photosystem II (PSII) and it could measure the degree of permanent damage for PSII in leaves. Compared with CK, TR reduced F0values and the rate of increase (Fig. 2-A). Fv/Fmrepresented for maximal photochemical efficiency of PSII, and its values would significantly reduce when stress or senescence occurred. Fv/Fmvalues were increased by TR; TR reduced the rate of decline in the late growing stage (Fig. 2-B). Fv/F0represented for potential photochemical efficiency of PSII. TR of Fv/F0was higher than that of CK and Fv/F0values of TR declined more slowly than those of CK in 40 to 50 days after silking (Fig. 2-C). Y(II) was actual quantum yield of PSII in light, which represented for the actual photosynthetic capacity. TR significantly increased actual quantum yield of PSII (Fig. 2-D), made ear leaves keep relatively highphotosynthetic capacity in late growing stage and those provided a guarantee for yield formation. Two years experiments showed the same trend for chlorophyll fluorescence parameters.

Fig. 2 Primary fluorescence, Fo(A), maximum quantum efficiency of PSII photochemistry; Fv/Fm, potential photochemical efficiency; Fv/Fo(C), actual quantum yield of PSII in light; Y(ⅠⅠ) (D), in maize plant leaves treated (TR) or not (CK) with compound mixtures2011 and 2012 represent planting year. CK represents leaves that treated with water, TR represents leaves treated with 40 mg · L-1DCPTA+20 mg · L-1CCC. Bars are means ± standard error of three replicates. Different letters above columns indicate significant differences (p＜0.05) between TR and CK by Least Significant Difference (LSD) test.

Senescence physiology in ear leaf

The active oxygen metabolism was imbalance in the process of leaf senescence; hazardous substances such as MDA were continuous accumulation, as a result, the function of cells was affected. In the early senescence, plants would clear the harmful substances by improving the activity of antioxidant enzymes, such as reactive oxygen species. SOD and POD were the main antioxidant enzymes which played an important role in retarding the process of senescence. With the increase of days after silking, MDA content of ear leaf gradually increased, and the rate appeared a tendency to slow down after. Two-year experiments showed the same trend. Compared with CK, TR reduced the accumulation of MDA content, and it declined 28.09% and 39.81%, respectively in 40 days after silking of 2011 and 2012 years (Fig. 3-A). Different trends occurred in SOD and POD activities with the increase of days after silking. SOD and POD showed reverse 'V'shape and 'M' shape respectively, and two years showed the same trend. From 30 to 50 days after silking, SOD and POD activity were significantly increased by TR. Though there was no significant difference between TR and CK, enzymatic activity of TR was higher than those of CK (Fig. 3-B, C). With the increase of days after silking, the content of soluble protein showed a downward trend after the first rose in ear leaf and the extent of the decline rose in 40 to 50 days after silking. Soluble protein content of TR was higher than CK, and the difference was significant without 20 days in 2011 and 50 days in 2012 after silking (Fig. 3-D).

Fig. 3 Malonaldehyde content, MDA (A), superoxide dismutase; SOD (B), peroxidase; POD (C), soluble protein content; (D), in maize plant leaves treated (TR) or not (CK) with compound mixtures2011 and 2012 represent planting years. CK represents leaves treated with water, TR represents leaves reated with 40 mg · L-1DCPTA+20 mg · L-1CCC. Bars are means ± standard error of three replicates. Different letters above columns indicate significant differences (p＜0.05) between TR and CK by Least Significant Difference (LSD) test.

Relationship between chlorophyll content and chlorophyll fluorescence parameters

In order to study the relationship between chlorophyll content and chlorophyll fluorescence parameters of TR and CK in the process of leaf senescence, the research analyzed the changed trend of F0, Fv/Fm, Fv/F0and Y(II) with the decline of chlorophyll content(Fig. 4). Fv/Fm, Fv/F and Y(II) declined with the reduction of chlorophyll content; in addition, the declining rate of TR was faster than that of CK. F0values rose with the reducing of chlorophyll content, the declining rate of TR was slower than that of CK. The results showed that Fv/Fm, Fv/F0and Y(II) of TR declined faster than those of CK when chlorophyll content reduced same degree, and F0was converse. Two-year experiments showed the same trend.

Fig. 4 Relationship between chlorophyll content and chlorophyll fluorescence parameters(A), relationship between chlorophyll content and Fo; (B), relationship between chlorophyll content and Fv/Fm; (C), relationship between chlorophyll content and Fv/Fo; (D), relationship between chlorophyll content and Y(II). 2011 and 2012 represent planting years. CK represent leaves treated with water; TR represents leaves treated with 40 mg · L-1DCPTA+20 mg · L-1CCC. Dashed and solid lines represent the linear trend of CK and TR, respectively. yCKand yTRrepresent linear regression equation of CK and TR, respectively.

Relationship between chlorophyll content and senescence

In order to study the relationship between decline of chlorophyll and senescent indexes of leaf, the research analyzed the changed trend of MDA, soluble protein content, SOD and POD activity along with the decline of chlorophyll content (Fig. 5). The results showed that MDA content appeared upward trend, and SOD, POD activities and soluble protein content appeared downward trend with the decline of chlorophyll content. When the chlorophyll content decreased,the rising rate of MDA content about TR was slower than that of CK; the declining rate of soluble protein content about TR was faster than that of CK. The result indicated MDA content of TR rose slower than that of CK when chlorophyll content reduced same degree, and soluble protein content of TR was faster than that of CK. Two years experiments had the same trend. The relationship between chlorophyll content and activity of SOD and POD was opposite in two years experiments. With the chlorophyll content declined, the declining rates of SOD and POD activities about TR was faster than those of CK in 2012 year, and the declining rate of TR was similar with that of CK in 2011 year. In summary, the closer relationship between senescent indexes and chlorophyll content appeared by TR.

Fig. 5 Relationship between chlorophyll content and senescence physiology(A), relationship between chlorophyll content and MDA content; (B), relationship between chlorophyll content and SOD activity; (C), relationship between chlorophyll content and POD activity; (D), relationship between chlorophyll content and soluble protein content. 2011 and 2012 represent planting year. CK represent leaves treated with water, TR represents leaves treated with 40 mg · L-1DCPTA+20 mg · L-1CCC. Dashed and solid lines represent the linear trend of CK and TR, respectively. yCKand yTRrepresent linear regression equation of CK and TR, respectively.

Correlation analysis of the various indexes

Table 2 showed that chlorophyll content of CK had extremely significant negative correlation with F0 and MDA content, and it had extremely significant positive correlation with Fv/Fm, Fv/F0, Y(II) and SOD activity, while it had been uncorrelated with POD activity and soluble protein. TR showed the same trend with CK, but the chlorophyll content of TR had significant positive correlation with soluble protein content and the correlative coefficient was 0.84*. Compared with CK, TR reduced the correlation between chlorophyll fluorescence parameters and SOD activity, and the correlation between MDA content with chlorophyll fluorescence parameters. Therefore, TR could mitigate the extent of leaf senescence by decreasing the reduced degree of the chlorophyll fluorescence parameters. The comparison of various senescent indexes showed the correlation of SOD, POD, MDA and soluble protein was improved under TR. The more closer of the correlation between these index, the faster of the physiological response of plant in the process of leaf senescence.

Path coefficient analysis between chlorophyll content and various indexes

The correlation analysis only reflected the relationship of various indexes, not really reflected direct or intrinsic effect between various index and chlorophyll content. Path coefficient analysis not only studied the direct effect of independent variables on dependent variable, but also compared their relative importance by stepwise regression. Direct path coefficient and indirect path coefficients were obtained by calculation of correlation coefficient and decomposition. The results of path coefficient analysis showed that the absolute value ranking of direct path coefficient was Y(II)＞F0＞Fv/Fm＞Fv/F0, MDA＞SOD＞POD＞soluble protein under CK. TR was Fv/F0＞Fv/Fm＞F0＞Y(II), soluble protein＞POD＞SOD＞MDA, and the larger absolute value represented the greater effect on chlorophyll content. The above results showed TR mainly affected the direct effect of Fv/F0, Y(II), soluble protein and MDA content with chlorophyll, thus, these affected chlorophyll content (Table 3).

Table 2 Correlation analysis of the various indicators

In addition to the direct effect, various indexes also affected chlorophyll content by indirect effects of each other. Under CK, the total indirect effect of Fv/Fm, Fv/ F0, MDA, POD, SOD and soluble protein was more than direct effect, and the situation of TR was F0, Fv/Fm, Y(II), SOD, POD and MDA. The reasons of indirect effect was more than direct effect were that various indexes of CK had larger indirect effects to chlorophyll content by Y(II), and TR did this by Fv/F0. After the superposed of these direct and indirect effects of various indexes each other, path coefficient between F0and MDA was negative number, and this result showed that these two indexes had negative effects on chlorophyll content, both years had the same trend (Table 3).

Table 3 Path coefficient analysis between chlorophyll content and various indicators

Discussion

Chlorophyll content

Chlorophyll is a class of the important pigment related with photosynthesis; therefore, it is essential to the growth of plants. It has been documented that the large remobilization for the early stage of leaf senescence is mainly attributed by the degradation of stromal proteins (especially Rubisco) accumulated in chloroplasts (Avice et al., 2014). In the aging process, chlorophyll content reduced gradually. As the result, photosynthetic capacity decreased. 5-aminolevuliniccacid (ALA) was found to be accumulated in leaf tissues in the presence of different C-labeled protochlorophyllide and levulinic acid (Richter et al., 2010). Recently, some studies had been made in improving plant growth and yield using ALA as an exogenous agent in different crop plants. It was well established that ALA improved chlorophyllcontent, but there was still a large knowledge gap to fill regarding the mechanism of action of ALA and what type of genes were activated/inactivated during chlorophyll synthesis (Nudrat et al., 2013). Exogenous spermidine (Spd) pretreatment could increase the content of chla, chlb and chl (a+b) under drought stress (Li et al., 2015). DCPTA could increase the content of chla, chlb in maize and soybean (James et al., 1991), but the further study showed that the increase of chlorophyll content after the treatment was the result of the volume increase of chlorophyll, not the increasing synthesis of chla and chlb (Shen et al., 1999). Be similar with DCPTA, the research showed that CCC could make the leaf color deepen, leaf thickness increase, chlorophyll content rise, photosynthesis increase. The research showed compound mixtures could significantly improved chlorophyll content of ear leaf after silking and reduced the reductive rate of chlorophyll content. The regulation of DCPTA and CCC on chlorophyll content may be carried out by the different metabolic pathways; diverse regulatory pathways were more stable and better than single pathway.

Chlorophyll fluorescence

In the process of leaf senescence, not only the chlorophyll content declined, but also photosynthetic system was also destroyed (Per et al., 2013). Fv/Fmvalue under optimal growth conditions was around 0.85 in many plants, but it markedly declined during stress (Bjokman et al., 1987). Under drought stress, compared with the control, Fv/Fmvalues were increased in Me-JA and COR-treated plants, and the difference was significant (Wu et al., 2012). With regard to the chlorophyll fluorescence parameters, we found that application of 40 mg · L-1DCPTA on maize seedling caused the increase in Fm, Fv/Fm, and Fv/Fo. The results showed that the light transformation efficiency increased in the reaction centre of PSII and the primary reaction of the photosynthesis was promoted and promoted the excited energy transformation from LHC II to PSII after spraying optimum concentration DCPTA on plants (Gu et al., 2014). 500 mg · L-1CCC increased Fv/Fmand Fv/Fovalues in Pistacia chinensis leaves, and compared with control, the significant was difference (Dong et al., 2012). Exogenous 1 mmol · L-1GSH treatment and excess-S could improve photosynthesis and growth under salt stress (Fatma et al., 2014; Matsufuji et al., 2013). The study was similar to previous studies, compound mixtures could increase Fv/Fmand Fv/F0values and reduce F0 values. Previous studies had showed that DCPTA and CCC could improve chlorophyll fluorescence characteristics in different degrees, but these studies focused on the regulation under stress conditions. Our study focused on the effects of chlorophyll fluorescence characteristics by compound mixtures in the process of leaf senescence. Senescence is a program of plants' own processes, rather than being caused by external stimuli, therefore, the regulation mechanism might differ from adversity. In our study, chlorophyll fluorescence parameters and chlorophyll content were highly significant correlation, so the compound mixtures of DCPTA and CCC might affect chlorophyll fluorescence parameters by affecting chlorophyll content.

Antioxidant enzymes

In the process of leaf senescence, chlorophyll resolved continually, and chlorophyll fluorescence characteristics were destroyed. As the result, light system is damaged. Excessive light energy could cause the accumulation of H2O2and MDA. For removing harmful substances, the plants would improve their antioxidant enzyme activity, thereby reducing damage. Accumulation of enzymatic and nonenzymatic antioxidants had a substantial role in plant growth and developmental events by regulating ROS production under non stress as well as stress conditions (Ashraf, 2009; Akram et al., 2012; Perveen et al., 2010). The activities of antioxidant enzymes such as POD and SOD increased in ALA-treated pakchoi leaf tissues. It also has been demonstrated that exogenous spermidine (Spd) was effective in enhancing the activity ofperoxidase to lessen in this way the oxidative stress (Roychoudhury et al., 2011). Exogenously applied spermidine could increase SOD, POD, CAT activity and reduce MDA content under drought stress in creeping bentgrass (Zhou et al., 2015). Other studies had shown that DCPTA could reduce the accumulation of MDA and increase SOD and POD activities (Ma et al., 2011). Our studies were similar to previous studies, compound mixtures could increase SOD and POD activities in maize leaves; increase the content of soluble protein; and reduce the accumulation of MDA. POD activity appeared upward trend at 30 to 40 days after silking, which was different from previous studies. A possible reason was that the gene that could increase POD activity was started, and intermediate products of POD catalyzed corresponding substrate oxidation caused oxidation and decomposition of chlorophyll. In these studies, the declining rate of chlorophyll content was faster than other phases at 30 to 40 days after silking, and the direct effect of POD on the chlorophyll content was negative in path coefficient analysis. Above results showed it had a certain relationship between POD activity and chlorophyll breakdown.

Conclusions

Leaves began to aging and chlorophyll content decreased with the increase of days after silking. The chlorophyll content of TR was higher than that of CK. TR reduced the amount of decomposition of chlorophyll and increased the time for photosynthesis.

F0values rose; Fv/Fm, Fv/F0and Y(II) declined with the increase of days after silking. TR was lower than CK in F0values and higher in Fv/Fm, Fv/F0and Y(II). TR could alleviate the damage that caused by senescence of excess light energy to leaves by increasing the utilization of light energy.

In the process of leaf senescence, reactive oxygen species increased, thus causing damage to leaves. Compared with CK, TR could increase SOD and POD activities, reduced the damage of reactive oxygen species to leaves, thus reducing MDA accumulation.

TR increased the correlation between chlorophyll content and POD, soluble protein and reduced the correlation between chlorophyll content and MDA content.

Path coefficient analyses showed that TR mainly affected the direct effects among chlorophyll and Fv/F0, Y(II), soluble protein and MDA content, thus affecting the chlorophyll content.

Ai L, Li Z H, Xie Z X, et al. 2008. Coronatine alleviates polyethylene glycol-induced water stress in two rice (Oryza sativa L.) cultivars. J Agron Crop Sci, 194, 360-368.

Akram NA, Ashraf M, Al-Qurainy F. 2012. Aminolevulinic acid induced regulation in some key physiological attributes and activities of antioxidant enzymes in sunflower (Helianthus annuus L.) under saline regimes. Sci Hortic, 142, 143-148.

Ashraf M. 2009. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol Adv,27: 84-93.

Avice JC, Etienne P.2014. Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L). J Exp Bot,65: 3813-3824.

Balazs Varga, Tibor Janda, Emese Laszlo, et al. 2012. Influence of abiotic stresses on the antioxidant enzyme activity of cereals. Acta Physiol Plant,34: 849-858.

Bjokman O, Demming B.1987. Photon yield of O2evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta, 70, 489-504.

Buchanan-Wollaston V. 1997. The molecular biology of leaf senescence. J Exp Bot,48: 181-1991.

Demmig A B, Adams W W, Baker D H, et al. 1996. Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiol Plant, 98, 253-264.

Dong Q, Wang J, Pang M, et al. 2012. Effects of Growth Regulations on Photosynthetic and Physiological Indices and Chlorophyll Fluorescence Parameters of Pistacia chinensis. Acta Bot Boreal,32: 484-490.

Dong S T, Gao R Q, Hu C H, et al. 1997. Study of canopy photosynthesis property and high yield potential after anthesis in maize. Acta Agron Sin,23: 318-325.

Dowens B P, Crowell D N. 1998. Cytokinin regulates the expression ofa soybean β-expansingene by a post-transcriptional mechanism. Plant Mol Biol,37: 437-444.

Fatma M, Asgher M, Masood A, et al. 2014. Excess sulfur supplementation improves photosynthesis and growth in mustard under salt stress through increased production of glutathione. Environ Exp Bot,107: 55-63.

Fischer A M. 2012. The complex regulation of senescence. Crit Rev Plant Sci,31: 124-147.

Gan SS.2010. The Hormonal Regulation of Senescence. Plant Hormones, 597-617.

Gausman H W, Quisenberry J E, Yokoyama H. 1991. Introduction to effects of plant biochemical regulators. In: Gausman H W. Plant biochemical regulator, Marcel Dekker. Inc., New York. pp. 1-16.

Giannopolitis C N, Ries S K. 1977. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol,59: 309-314.

Gill S S, Tuteja N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem,48(12): 909-930.

Gunda S E, Titus F A, Mosisa W, et al. 2010. Photosynthesis and leafnitrogen dynamics during leaf senescence of tropical maize cultivars in hydroponics in relation to N efficiency in the field. Plant Soil,330: 313-328.

Gu W R, Li Z H, Zhai Z X. 2008. Regulation of tertiary amine bioregulator on photosynthesis and chlorophyll fluorescence parameters of corn leaves. Acta Agriculture Boreali-Sinica, 23: 85-89.

Gu W R, Meng Y, Zhang J B, et al. 2014. Regulation of foliar application DCPTA on growth and development of maize seedling leaves in Heilongjiang Province. Journal of Northeast Agricultural University,21: 1-11.

Hajiboland R, Aliasgharzadeh N, Laiegh S F, et al. 2010. Colonization woth arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum lycopersicum L.) plants. Plant Soil,331: 313-327.

Harsh N, Ramanpreet K, Simranjit K, et al. 2014. γ-Aminobutyric acid (GABA) imparts partial protection from heat stress injury to rice seedlings by improving leaf turgor and upregulating osmoprotectants and antioxidants. J Plant Growth Regul,33: 408-419.

Hazem M K, Gert S, Richard J L. 2014. Frequently asked questions about in vivo chlorophyll fluorescence: practical issues. Photosynth Res,122: 121-158.

Hernandez J A, Jimenez A, Mullineaux P, et al. 2000. Tolerance of pea (Pisum sativum L.) to long-term salt stress is associated with induction of antioxidant defenses. Plant Cell Environ,23: 853-862

James H, Keithly J H, Yokoyama H. 1991. Regulation of crop growth and yield by tertiary amine bioregulators. In: Gausman H W. Plant biochemical regulator. Marcel Dekker. Inc., New York. pp: 223-246.

Kinnersley AM, Lin F.2000. Receptor modifiers indicate that γ-aminobutyric acid (GABA) is a potential modulator of ion transport in plants. Plant Growth Regul,32: 65-76.

Li H S, Chen C L. 1998. Principle and technology of physiological and biochemical experiments in plants. Huazhong Agricultural University Press, Wuhan. (in Chinese)

Li Z, Zhou H, Peng Y, et al. 2015. Exogenously applied spermidine improves drought tolerance in creeping bentgrass associated with changes in antioxidant defense, endogenous polyamines and phytohormones. Plant Growth Regul,76: 71-82.

Luoanen J, Rikala R, Aphalo P J. 2002. Effect of CCC and daminozide on growth of silver birch container seedlings during three years after spraying. New Forest,23: 71-80.

Maes L, Zhang Y, Reed D W, et al. 2011. Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants. New Phytol, 189, 176-189.

Ma D Y, Sun D X, Wang C Y, et al. 2015. Silicon application alleviates drought stress in wheat through transcriptional regulation of multiple antioxidant defense pathways. J Plant Growth Regul, DOI 10.1007/ s00344-015-9500-2.

Ma Q H, Tan X H, Liang L, et al. 2011. Effects of plant growth regulators DA-6 and DCPTA on reactive oxygen species and related physiological. Food Science,32: 296-299.

Matsufuji Y, Yamamoto K, Yamauchi K, et al. 2013. Novel physiological roles for glutathione in sequestering acetaldehyde to confer acetaldehyde tolerance in Saccharomycescerevisiae. Appl Microbiol Biotechno,197: 297-303.

Nudrat A A, Muhammad A. 2013. Regulation in plant stress tolerance by a potential plant growth regulator, 5-Aminolevulinic Acid. J Plant Growth Regul,32: 663-679.

Per LG, Andrea C, Luca B, Karin K. 2013. Plant senescence and crop productivity. Plant Mol Biol,82: 603-622.

Perveen S, Shahbaz M, Ashraf M. 2012. Regulation in gas exchange and quantum yield of photosystem II (PSII) in salt-stressed and nonstressed wheat plants raised from seed treated with triacontanol. Pak J Bot,42: 307-308.

Ping H E, Mitsuru O, Masako T, et al. 2002. Changes of photosyntheticcharacteristics in relation to leaf senescence in two maize hybrids with different senescent appearance. Photosynthetica,40: 547-552.

Porra R J. 2002. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophyll a and b. Photosynthesis Research,73: 149-156.

Prakash P. Kumar. 2013. Regulation of biotic and abiotic stress responses by plant hormones. Plant Cell Rep,32: 943.

Roychoudhury A, Basu S, Sengupta D N. 2011. Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance. J Plant Physiol,168: 317-328.

Richter A, Peter E, Lorenzen S, et al. 2010. Rapid dark repression of 5-aminolevulinic acid synthesis in green barley leaves. Plant Cell Physiol,51: 670-681.

Shao R X, Li J, Chen J H, et al. 2014. Effects of compound regulator seed dressing on endogenous hormones and lipid peroxidation of membrane system during aging process of maize. Journal of Nuclear Agricultural Sciences,28: 1142-1147. (in Chinese)

Sharkey T D, Zhang R. 2010. High temperature effects on electron and proton circuits of photosynthesis. J Integr Plant Biol,52: 712-722.

Shen M S, Lin Y J, Chen M C. 1999. Studies on influence of DTA-6 regulation on leaf cells ultrastructure in Stevia rebaudiana Bertoni. Sugar Crops of China 4, 1-3.

Spano G, Di Fonzo N, Perrotta C, et al. 2003. Physiological characterization of stay green mutants in durum wheat. Journal of Experimental Botany,54: 1415-1420.

Wahid A, Gelani S, Ashraf M, et al. 2007. Heat tolerance in plants: an overview. Environ Exp Bot,61: 199-223.

Wu H L, Wu X L, Li Z H, et al. 2012. Physiological evaluation of drought stress tolerance and recovery in cauliflower (Brassica oleracea L.) seedlings treated with methyl jasmonate and coronatine. J Plant Growth Regul,31: 113-123.

Yamori W, Noguchi K O, Hikosaka K, et al. 2010. Phenotypic plasticity in photosynthetic temperature acclimation among crop species with different cold tolerances. Plant Physiol,52: 388-399.

Zgallai H, Steppe K, Lemeur R. 2005. Photosynthetic, physiological and biochemical responses of tomato plants to polyethylene glycolinduced water deficit. J Integr Plant Biol,47: 1470-1478.

Zhang Z L. 2001. Experimental guide of plant physiology. Higher Education Press, Beijing.

Zhou L, Hui Z, Yan P, et al. 2015. Exogenously applied spermidine improves drought tolerance in creeping bentgrass associated with changes in antioxidant defense, endogenous polyamines and phytohormones. Plant Growth Regul,76: 71-82.

Zhou T, Hu Y J, Zhou X M. 2004. Effect of DTA-6 on seedling photosynthesis and growth of wild barely. Pratacul Science,21(4): 31-34.

Zhu X C, Song F B, Liu S Q, et al. 2011. Effects of arbuscular mycorrhizal fungus on photosynthesis and water status of maize under high temperature stress. Plant Soil,346: 189-199.

S512.1

A

1006-8104(2015)-03-0001-15

Received 1 June 2015

Supported by the National Natural Science Foundation of Youth Science Foundation (31201164); Northeast Agricultural University Doctor Started Foundation (2012RCB01); China Postdoctoral Foundation (2012M511434); Project of National Non-profit Institute Fund (BSRF201405)

Wang Yong-chao (1987-), male, Ph. D, engaged in the research of maize cultivation physiology and stress regulation. E-mail: wangyongchao723@163. com

*Corresponding author. Wei Shi, professor, supervisor of Ph. D student, engaged in the research of crop yield and physiology, the macro agriculture. E-mail: weishi5608@163.com