Effects of variety and chemical regulators on cold tolerance during maize germination

WANG Li-jun, ZHANG Ping, WANG Ruo-nan, WANG Pu, HUANG Shou-bing

College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, P.R.China

Abstract Maize growth and development is affected by low temperature (LT) especially at the early stages of development. To describe the response of different varieties to LT stress and determine an effective method to cope with LT stress, maize hybrids Zhengdan 958 (ZD 958) and Danyu 39 (DY 39) were planted and grown at 10 and 25°C, respectively. Effects of the chemicals potassium chloride (KCl), gibberellin (GA3), 2-diethylaminoethyl-3,4-dichlorophenylether (DCPTA), and all three combined chemicals (KGD) on coping with LT stress were tested by seed priming. The varieties performed signiflcantly different at 10°C.Compared to leaf, root growth was more severely affected by LT stress. Root/leaf ratio is likely a more reliable parameter to evaluate cold tolerance based on its close correlation with leaf malondialdehyde (MDA) content (R=–0.8). GA3 advanced seed germination by about 2 days compared with control treatment of water. GA3 and DCPTA both resulted in lower leaf MDA content and higher leaf and root area, and root/leaf ratio. KCl resulted in the highest evenness of plant height. KGD performed the best in increasing cold tolerance of maize morphologically and physiologically. Strategies to increase maize tolerance of cold stress, such as variety breeding or chemical selection, would increase maize yield especially at high-latitude regions and have great implications for food security.

Keywords: cold tolerance, maize, seedlings, root/shoot, chemical regulators

1. lntroduction

Despite the warming climate, low temperature (LT) stress remains one of the major abiotic factors severely affecting crop production worldwide, especially in the high-latitude regions (Sthapit and Witcombe 1998; Jhaet al. 2017).Maize (Zea maysL.) is one of the most important crops to ensure global food security (FAO 2012), but it is also a C4plant species that is sensitive to LT stress, particularly in the early growth stages (Maroccoet al. 2005; Presterlet al.2007). With rising temperatures, the maize growing area is expanding into some regions where the temperature is too low to grow maize before (Menget al. 2014), increasing the potential impacts of LT stress on maize production.

LT stress in the early maize growth stages retards seed germination, seedling emergence, and vegetative growth, negatively affecting morphogenesis, photosynthetic characteristics, and yield (Miedema 1982; Allen and Ort 2001; Hundet al. 2008). Seedling growth at suboptimal temperatures is limited by leaf and root extension (Miedema 1982). The shoot apex is below the soil surface before the 6-leaf stage (Stoneet al. 1999), and as a consequence,both shoot and root can be directly influenced by LT at the seedling stage. Under cold stress, the appearance rate of leaves almost stops (Riva-Rovedaet al. 2016), and cooling of the shoot apex results in delayed leaf development (Sowińskiet al. 2005; Rymenet al. 2007). In addition, LT stress causes maize roots to become swollen behind the tip, grow thicker,and reduces branching (Farooqet al. 2009). LT-induced root length was about 20% less than control, and the delay in development of photosynthesis was related to insufflcient growth of roots (Sowińskiet al. 2005). The effect of cold stress on the primary and lateral roots may be indirectly reflected in shoot elongation and leaf formation (Hundet al.2004). Higher root area/shoot area ratio and root length/leaf area ratio were found when seedlings were grown at suboptimal temperatures (Richneret al. 1997; Hundet al.2007). However, there are few studies of the relationship between root and shoot of maize under LT stress.

Inbred maize lines showed different responses to LT(Guanet al. 2009): LT-tolerant genotypes were highly resistant to LT stress and recovered faster from LT damage(Aguileraet al. 1999). In addition to innate cold tolerance,many chemicals have been used to reduce LT effects in maize (Waqaset al. 2017). Salicylic acid (SA, 0.5 mmol L–1) applied to the radicles increased LT tolerance of the aerial portion of maize (Kang and Saltveit 2002). Gibberellin(GA3, 0.1 mmol L–1) can increase seedling emergence and growth of maize at 10°C (Wanget al. 1996). Seed priming with calcium chloride (CaCl2) and potassium chloride (KCl)increased maize seedling tolerance to LT stress (Farooqet al. 2009). Exogenous application of compound plant growth regulators, such as PASP-KT-NAA, increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities under LT condition (Xuet al.2012). However, studies on compound regulators and comparative studies on the exogenous application of single and compound chemicals are limited in maize.

The objectives of this study were to test the response of maize varieties to LT stress, investigate shoot and root meristem morphological changes under LT condition, and identify the effects of chemical regulators on alleviating LT damage. The widely adapted maize variety Zhengdan 958(ZD 958) and the regionally adapted variety Danyu 39 (DY 39)were tested for their responses to cold stress at germination stage. The former variety has adapted to environments in different regions of China, and the latter has adapted to environments in the Northeast China where cold stress is frequent before sowing maize (Liet al. 2009; Shiet al. 2010).

2. Materials and methods

2.1. Experimental design

This experiment was conducted in plant growth champers at two temperature levels of 10 and 25°C. Two trials were varietal and chemical trials, and each trial had flve replications. The low temperature of 10°C was supposed to be the minimum temperature for seedling emergence of maize; at 25°C, both roots and seedlings grow fast (Sánchezet al. 2014). Prior to the trial, maize seeds were disinfected with 1% (v/v) NaClO solution for 10 min, and then were washed clean with distilled water. The varietal trial had a completely randomized design, in which 50 seeds of two maize varieties, DY 39 and ZD 958 were evenly placed in a transparent germination box equipped with fllter paper and containing 10 mL distilled water, with six replicates at each temperature. The germination box had a transparent cover to avoid water evaporation, and its dimensions were 19 cm long, 13 cm wide, and 7 cm high. After placing seeds in the germination boxes, all seeds were transferred to the plant growth champers with constant temperature levels of 10 and 25°C, respectively. At each temperature level, the same photosynthetic photon flux density of 350 μmol m–2s–1,12 h light/12 darkness, and a relative humidity of 60% were used. The seeds were transferred to the new germination boxes in 2-day intervals until the end of the trial to avoid drying and bacterial contamination.

The chemical trial was also conducted using a completely randomized design with only the maize variety ZD 958 and four chemical regulators: 50 mg L–1potassium chloride (KCl),50 mg L–1gibberellin (GA3), 1 mg L–12-diethylaminoethyl-3,4-dichlorophenylether (DCPTA), and their combination(50 mg L–1KCl-50 mg L–1GA3-1 mg L–1DCPTA, KGD).ZD 958 has adapted widely to different regions of China,so study chemical effects on increasing cold tolerance of ZD 958 would be more valuable. Chemicals and their doses were determined by several prior experiments (data not shown) and previous relevant studies (Wanget al. 1996;Farooqet al. 2008a, b; Guet al. 2009, 2014). The disinfected seeds of ZD 958 were soaked for 12 h in aerated solutions of the four chemicals and distilled water was used as a control treatment (CK). The ratio of seed weight over solution volume was 1/5 g mL–1(Farooqet al. 2008a; Ahmadet al. 2016).After seed priming, the soaked seeds were dried under shade to a nearly constant weight, and then were incubated in germination boxes equipped with fllter paper and containing 10 mL H2O, under the same incubation conditions used in the varietal trial. Both trials lasted 11 days.

2.2. Sampling and data collection

In both trials, germination rate, evenness of plant height(Yanget al. 2006), leaf area (Montgomery 1991), root area, and leaf malondialdehyde (MDA) content (Heath and Packer 1968) were measured. The germinated seeds per germination box were counted daily. Germination rate was calculated as the proportion of germinated seeds over all the seeds used. Plant heights of flve randomly selected seedlings per germination box were measured at 10 days after sowing (DAS) to calculate the evenness of plant height as follows:

At 10 DAS, flve seedlings were randomly separated to measure leaf length and width. Leaf area was calculated as follows:

Leaf area=?(Leaf length×Maximum leaf width)×k(Montgomery 1991)

Where, k is 0.75 for expanded leaf, and 0.5 for unexpanded leaf.

After measuring the leaf area, MDA content of leaves was measured using the thiobarbituric acid (TBA) colorimetric technique (Heath and Packer 1968). Maize leaf segments(0.3 g) were ground in an ice-cold mortar fllled with 5.0 mL of 50 mmol L–1pH 7.8 phosphate buffer (including 2%polyvinylpyrrolidone) as an extraction buffer. The extracts were centrifuged at 4°C for 15 min at 10 000×g. The resulting supernatant (1 mL) was collected and mixed with 2 mL of 0.5% TBA in a centrifuge tube. The tubes were boiled in the water for 20 min, ice cooled, and then centrifuged for 10 min at 3 000×g. The supernatant was collected in a cuvette and its absorbance at 450, 532, and 600 nm was read with a spectrometer. The MDA content was calculated as:

MDA (nmol g–1FW)=1 000×6.45(OD532–OD600)–0.56OD450

Roots of flve plants per treatment were scanned with EPSON PREFCTION V700, and the root images were analyzed with WinRHIZO Pro Software (version 2009c,Regent Instruments Inc., Candan) to measure root area.

2.3. Statistics

One-way analysis of variance (ANOVA) of plant height evenness, root area, leaf area, root area/leaf area (root/leaf) ratio, and MDA content were performed in SAS 9.3(SAS Institute, Carey, NC, USA). The differences were compared with the least signiflcant difference (LSD) test,and were considered statistically signiflcant whenP＜0.05.The correlations of root area, leaf area, root /leaf ratio, and MDA content were calculated with procedure of correlation in SAS 9.3.

3. Results

3.1. Effects of variety on cold resistance

The germination rate of ZD 958 was remarkably higher than that of DY 39 at 25°C, especially during the flrst half of the germination test (Fig. 1). It took 4 days for ZD 958 to reach its highest germination level of approximately 100% at 25°C,whereas DY 39 needed at least 1 wk to reach its highest germination level (approximately 90%). Compared to the treatment of 25°C, seeds started to germinate at 8 DAS for both varieties at 10°C, and germination rate of DY 39 was higher than that of ZD 958 in the following period.

The evenness of plant height was higher in DY 39 than that in ZD 958 at both temperatures, and the difference was signiflcant at 10°C (Fig. 2).

DY 39 was larger than ZD 958 in both root area and leaf area at both temperatures at 10 DAS (Fig. 3-A and B). The difference was not signiflcant in the root area at 25°C, but was signiflcant at 10°C. Leaf area was signiflcantly different between the varieties at both 10 and 25°C. ZD 958 had a slightly larger root/leaf ratio at 25°C than DY 39, but root/leaf ratio was signiflcantly smaller in ZD 958 at 10°C. Averaging the two varieties, root area was reduced from approximately 54 cm2at 25°C to 40 cm2at 10°C, leaf area was reduced from 24 to 21 cm2, and root/leaf ratio was reduced from 223 to 183% (Fig. 3-C).

Fig. 1 Effects of variety and champer temperature on germination rate of maize seeds. DY 39, Danyu 39; ZD 958, Zhengdan 958.Bars mean SE.

Leaf MDA content at 10 DAS was nearly at the same level between DY 39 and ZD 958 (3.7vs. 4.2 nmol g–1FW) at 25°C (Fig. 4). The MDA content of ZD 958 was signiflcantly higher than that of DY 39 at 10°C.

3.2. Effects of chemicals on resistance to cold

Chemicals used to soak seeds prior to the seed germination test had no signiflcant effect on the germination rate at 25°C, but dramatically increased seed germination at 10°C(Fig. 5). The germination rate reached 100% at 7 DAS in the treatment of KGD at the low temperature of 10°C, but was less than 20% in other treatments. At the end of the germination test, the germination rates of DCPTA, KCl, and water were approximately 60%.

Plant height evenness of chemical-soaked seeds was higher than that of water-soaked seeds at both 10 and 25°C,except in the treatment of KCl at 25°C (Fig. 6). Treatment with KGD had the highest values of 27.7 at 25°C, and treatments of GA3and KCl had the highest values (28.6 and 28.2) at 10°C.

Fig. 2 Effects of variety and champer temperature on evenness of maize plant height at 10 days after sowing. DY 39, Danyu 39;ZD 958, Zhengdan 958. Differences between values with the same letter are not signiflcant at P=0.05, comparisons are within the same temperature only. Bars mean SD.

Averaged across chemical treatments, the root area,leaf area, and root/leaf ratio were signiflcantly larger in the treatment of 25°C than that in 10°C at 10 DAS (Table 1).Root area ranged from 53.6 cm2in the treatment of KGD to 50.4 cm2in the treatment of KCl at 25°C, and from 41.8 to 27.5 cm2at 10°C. The range of leaf area among chemical treatments was much smaller than that of root area.Difference in the root/leaf ratio among chemical treatments was not signiflcant at 25°C, but was signiflcant at 10°C. In the treatments of KGD, GA3, and DCPTA, the root/leaf ratio was signiflcantly larger than that in the control treatment.

Leaf MDA content was signiflcantly lower in the chemical treatments than that in the control at both temperatures(Fig. 7). In particular, the MDA content was reduced from approximately 10 nmol g–1FW in the treatment of water to 2.5 nmol g–1FW in the treatment of KGD at 10°C. On average across treatments of chemicals, the MDA content at 10°C was 5.4 nmol g–1FW, about twice that at 25°C.

The leaf MDA contents in the chemical trials were plotted against root area, leaf area, and root/leaf ratio (Fig. 8).Negative relationships were found, except between MDA and root/leaf ratio at 25°C. At 10°C, the correlation was weak between MDA and leaf area (R=–0.57), but was stronger between MDA and root area (R=–0.76), and was signiflcantly different between MDA and root/leaf ratio.

4. Discussion

4.1. Effects of maize variety on cold tolerance

Fig. 3 Effects of variety and champer temperature on root area (A), leaf area (B), and root area/leaf area (root/leaf ratio, C) at 10 days after sowing. DY 39, Danyu 39; ZD 958, Zhengdan 958. Differences between values with the same letter are not signiflcant,comparisons are within the same temperature only. Bars mean SD.

Fig. 4 Effects of variety and champer temperature on leaf malondialdehyde (MDA) content of maize seedling at 10 days after sowing. DY 39, Danyu 39; ZD 958, Zhengdan 958.Difference between values with the same letter are not signiflcant(P＜0.05), comparisons are within the same temperature only.Bars mean SD.

Fig. 5 Effects of champer temperature and chemical regulator on germination rate of maize seeds. KGD, the mixture of KCl,GA3, and DCPTA; GA3, gibberellin; DCPTA, 2-diethylaminoethyl-3,4-dichlorophenylether; KCl, potassium chloride. Bars mean SD.

Fig. 6 Effect of chemicals on evenness of maize seedling plant height. KGD, the mixture of KCl, GA3, and DCPTA; GA3,gibberellin; DCPTA, 2-diethylaminoethyl-3,4-dichlorophenylether;KCl, potassium chloride. Differences between values with the same letter are not signiflcant at P＜0.05, comparisons are within the same temperature only. Bars mean SD.

This study further conflrmed that LT can greatly hinder seed germination and seedling growth of maize, consistent with flndings of Jhaet al. (2017). LT stress reduces leaf chlorophyll content, photosynthesis, activity of antioxidant enzymes, and stability of membranes in many crops (Nie and Baker 1991; Allenet al. 2001; Xuet al. 2012), reducing dry matter production and yield (Nayyaret al. 2007). The negative impacts of LT stress on maize is dependent on the variety (Richneret al. 1997; Leeet al. 2002). In the present study, the decreases in root area, leaf area, and root/leaf ratio by LT were greater in ZD 958 than that in DY 39.These results indicated that DY 39 was more resistant to low temperature than ZD 958 at early developmental stages, suggesting that effects of variety on coping with cold stress depend on the regions where the variety comes from. According to the present results, maize varieties adapted to cold regions likely have stronger resistance to cold stress at least during seedling emergence. Variety ZD 958 comes from the North China Plain, and can adapt to different regions of China (Liet al. 2009), but cold resistance is likely not the most important trait of this variety. Cold resistance of maize at seedling emergence is inheritable(Revillaet al. 2000). Our results indicated that selection of, or breeding for, an LT resistant maize variety is feasible(Fenzaet al. 2017).

Compared to shoot growth, low temperature seems to affect root growth more severely (Hundet al. 2007). In the present study, the extent to which root area decreased under LT condition was much greater than that of leaf area(Fig. 3; Table 1), indicating that root growth can probably reflect LT resistance of maize more accurately than shoot growth (Hundet al. 2008).

4.2. Effects of chemical regulator on cold tolerance

MDA content is an indicator of biological activity that can be used to evaluate maize response to environmental stress(Heath and Packer 1968; Gill and Tuteja 2010). The more severely the crops are damaged by environmental stress,the higher the MDA content in the crops is. In the present study, LT resulted in more MDA contents in leaves of maize seedlings and in the CK treatment of water. MDA content reflected the capacity of maize variety and the effect of chemical regulators on coping with LT stress. With this in mind, the obvious correlation between MDA content and root/leaf ratio indicate that root/leaf ratio is likely a parameter that can better represent LT resistance in maize (Fig. 8).This hypothesis should be verifled in future research.

Table 1 Effects of chemical regulators and temperature on leaf area and root area at the 3-leaf stage of maize seedlings1)

Fig. 7 Effects of chemicals on malondialdehyde (MDA)content in leaves at 3-leaf stage of maize seedlings grown at 25 and 10°C. KGD, the mixture of KCl, GA3, and DCPTA; GA3,gibberellin; DCPTA, 2-diethylaminoethyl-3,4-dichlorophenylether;KCl, potassium chloride. Differences between values with the same letter are not signiflcant at P＜0.05, comparisons are within the same temperature only. Bars mean SD.

Fig. 8 Scatter plot of malondialdehyde (MDA) in leaves against root area, leaf area, and root area/leaf area ratio at 3-leaf stage of maize seedlings grown at 25 and 10°C. Values were derived from flve chemical treatments. ＊, signiflcant at P＜0.05; ns, not signiflcant.

The chemicals used in the present study increased shoot and root growth, and reduced leaf MDA content of maize at cold conditions through soaking seeds before sowing. Effects of GA3and KCl on coping with cold stress at the maize seedling stage were consistent with flndings of Wanget al. (1996) and Farooqet al. (2008b). These two chemicals increased the evenness of plant height(Fig. 6), but did not greatly accelerate seed germination(Fig. 5). GA3and KCl reduced leaf MDA content at the LT condition, but their positive effects were lower than that of KGD and DCPTA (Fig. 7). KCl even reduced root and leaf area under LT stress (Table 1). Previous studies indicated that DCPTA can enhance growth and development of many crops by foliar application (Hayman and Yokoyama 1990;Keithlyet al. 1990, 1991), but these studies did not focus on LT stress. Our results showed that, for the flrst time,seed priming with DCPTA increased leaf and root area and reduced leaf MDA content at the LT condition, suggesting that DCPTA can increase cold tolerance of maize at early stages of development. Previous studies indicated that seed priming with GA3can promote seed respiration and starch degradation under cold conditions due to increased amylase activities, which increase germination rate (Liet al.2013). Seed priming with the chemicals used in the present study was reported to increase activation of antioxidants and soluble sugar content in the crop leaves under cold stress (Farooqet al. 2008a; Guet al. 2009; Liet al. 2013).Compared to the single chemicals used, KGD (mixed compound of KCl, GA3, and DCPTA) accelerated seed germination fastest (Fig. 5), and resulted in the largest root and leaf area (Table 1) and the lowest MDA content (Fig. 7)at the LT condition. These results suggested that application of mixed speciflc chemicals likely enhanced cold tolerance of crops, providing a feasible strategy to cope with LT stress in crop production.

5. Conclusion

Low temperature (LT) stress decreased maize seed germination, inhibited shoot and root growth, and increased MDA content in the leaves, interrupting the function of the cell membrane system. Compared to the aboveground parts, LT stress had a larger effect on root extension. Root area/shoot area ratio represents cold tolerance of maize more accurately, and can be considered as a more reliable parameter for selecting or breeding cold-tolerance maize varieties. Strategies including application of cold-tolerance variety and mixed chemicals can increase maize yield or expand maize growing area where LT stress exists at the maize sowing stage around the world.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31701361, 31401331) and the earmarked fund for China Agriculture Research System(CARS-02-26).