Effect of ethanol on plant growth

Plant Signal Behav. 2019; 14(12): 1681114.

ABSTRACT

With hydroponics culture, we monitored the response of the growth and nutrient accumulation of oilseed rape (Brassica napus L.) to five ethanol concentrations: 0 mL•L−1 (control), 0.0125 mL•L−1, 0.025 mL•L−1, 0.05 mL•L−1, and 0.25 mL•L−1, respectively. The results showed that a high concentration of exogenous ethanol (0.25 mL•L−1) significantly inhibited oilseed rape growth by 52.28%. However, the biomass of oilseed rape with a low concentration of exogenous ethanol (0.0125–0.05 mL•L−1) manipulation was raised by 16.62–44.08%. A similar result was found on the total nitrogen, phosphorus, and potassium of the oilseed rape. Results of micro-element determination showed that iron and zinc accumulation in oilseed rape was unchanged, while manganese and copper accumulation was increased first and then decreased with increasing ethanol concentration. This study provided a possibility for improving plant growth with low concentration ethanol application in oilseed rape planting.

KEYWORDS: Ethanol, oilseed rape, nutrient accumulation

Introduction

Oilseed rape (Brassica napus L.) is widely cultivated in the world as oil-food crop, and the yield of oilseed rape is seriously relying on the fertilizer supplied; however, the fertilizer utilization efficiency of oilseed rape was low, which results in waste of resources and brings great pressure to the local environment. Hence, it is very important to improve the ability of nutrient absorbing for oilseed rape. The absorbed nutrients not only promoted the growth of oilseed rape and produced more dry matter but also accumulated in the vegetative organs (especially in leaves) at the seedling stage, which has a critical impact on the yield because of its recycling to the grains. At the pod's mature stage, only a small part of nitrogen originates from the root absorbing, and the main nitrogen originates from leaves, stem, and root,1 which acts as “sink” at the seedling stage and “source” at the reproductive growth stage. As more as 88.5, 15.3, and 29.3 kg/hm2 of N, P2O5, and K2O in the leaves recycled after flowering, which guaranteed the formation of dry matter and satisfied the nutrient demand for grain at the reproductive growth stage.2 Because of the great effect of the biomass and nutrient accumulation at the seedling stage on the yield, some steps, which aim to promote the biomass and nutrient accumulation at the seedling stage, should be taken in the oilseed rape planting.

Ethanol, which was one of the products of anaerobic respiration in plants, plays an important role in the response of plants to the abiotic stress. There are higher rates of glycolysis and ethanol fermentation in hypoxic conditions, which provided more energy, limited the accumulation of lactate, and regulated the cytoplasmic pH, resulting in an increased hypoxic tolerance.3–6 The ethanol concentration in the roots of rice was decreased due to the inhibitor of alcohol dehydrogenase, resulting in reduced chilling tolerance, and this reduction of the chilling tolerance could be recovered with exogenously applied ethanol.7 Ethanol enhances high-salinity stress tolerance by detoxifying reactive oxygen species in Arabidopsis thaliana and rice.8 Not only ethanol increased the tolerance of abiotic stress, but it also has an impact on the growth of plants.9 However, the effects of different concentrations of ethanol on the biomass and nutrient accumulation of oilseed rape were not affirmed. Five treatments, which with five different concentrations of ethanol applied in the growth solution of oilseed rape, were set up in this study, in order to examine the effects of different concentrations of ethanol on (i) biomass of the root and shoot; (ii) root/shoot ratio and root architecture characteristics which have an important impact on the nutrient absorbing; and (iii) N, P, K, Fe, Mn, Cu, and Zn accumulation in oilseed rape at the seedling stage. Results from this study may provide some information that can be used to (i) determine whether ethanol has the potential of improving the oilseed rape growth and increasing the nutrient accumulation at the seedling stage and (ii) preliminarily judge the appropriate concentration of ethanol for oilseed rape.

Materials and methods

Plant growth and nutritional status

Seeds of oilseed rape (814, Brassica napus L.) were vernalized in 4°C refrigerator one night and then germinated in a culture dish with quartz sand and vermiculite (1:3). After sprouted 1 week, the seedlings were transferred into a hydroponics box, and the size of the box is 22 cm × 15 cm × 7 cm. Hydroponic experiments were conducted in a growth chamber (light intensity of 400 μmol/(m2.s), day/night temperature of 28°C and 22°C, and 60% relative humidity, respectively). Plants were supplied with modified Hoagland nutrient solution containing 0.5 mM K2SO4, 0.6 mM MgSO4 · 7H2O, 0.3 mM KH2PO4, 0.5 mM CaCl2 · 2H2O, 1 µM H3BO3, 0.5 µM MnSO4·H2O, 0.5 µM ZnSO4 · 7H2O, 0.2 µM CuSO4 · 5H2O, 0.07 µM Na2MoO4 · 2H2O, and 0.1 mM Na-Fe-EDTA. The nutrient solution was aired continuously and renewed every 3 d.

Ethanol manipulation and sampling

After 4 d of transplanting, oilseed rape plants were treated with different concentrations of ethanol (0, 0.0125, 0.025, 0.05, and 0.25 mL•L−1, ethanol concentration was 95%), and there were three repeats per treatment. After 3 weeks of ethanol treating, the growth of oilseed rape was pictured, and the seedlings were harvested, which separated from root to shoot. The biomass; root/shoot ratio; root characters; and nitrogen (N), phosphorus (P), potassium (K), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) content and accumulation were measured and calculated.

Determination of biomass and root/shoot ratio

The roots and shoots of oilseed rape were dried at 105°C for 0.5 h and then dried at 70°C to a constant weight to get biomass. The root/shoot ratio was calculated by dry weight of root/dry weight of shoot.

Assay of root characters

The roots of the seedlings (fresh sample) were gently washed using a banister brush, and then, the root system was scanned to get a digital image using a scanner (Epson V700, Japan). The total root length, total root surface area, root volume, and the number of root tips were determined by WinRHIZOTM2003b software (Regent Instruments, QC, Canada). Later, the shoots and roots were dried at 70°C to a constant weight, weighed to get biomass, and then ground to fine powder for storage.

Measurements of N, P, K, and other nutrient content and accumulation

The dry samples were ground to fine powder, and then, 0.2 g of plant materials was used to determine the N content by a modified Kjeldahl digestion method. The remaining digests were used for analyses of P content (molybdivanadate method) by automated colorimetry and K content by a flame photometer. The content of Zn, Mn, Fe, and Cu was determined by microwave digestion and inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer 5100 PC, Norwalk, CT, USA).

The accumulation of nutrient (total nutrient) = nutrient content × biomass.

Statistical analysis

The statistical significance of differences between treatments was analyzed using IBM SPSS Statistics 22.0. Data were compared by two-way analysis of variance (ANOVA) and Tukey’s HSD post hoc test at a significance level of p < 0.05. The data in the tables are presented as mean ± standard deviation (SD). Figures were constructed using GraphPad Prism 6.0.

Results

Effects of exogenous ethanol on the biomass of oilseed rape

The exogenous ethanol, which the concentration was below 0.05 mL•L−1, stimulated the growth of oilseed rape; however, the 0.25 mL•L−1 exogenous ethanol inhibited the growth of oilseed rape (Figure 1). With 0.0125–0.05 mL•L−1 exogenous ethanol treatment, the biomass of oilseed rape was significantly increased by 16.60–43.77%; however, the biomass of oilseed rape severely decreased by 52.45% with 0.25 mL•L−1 exogenous ethanol treatment. The biomass of 0.05 mL•L−1 was significantly higher than that of other treatments. There was no significant difference between 0.0125 and 0.025 mL•L−1 (Figure 2).

Plant phenotype under different ethanol treatments.

The biomass of oilseed rape with different ethanol treatments.

The effects of exogenous ethanol on the root and shoot biomass showed similar results as the effects of exogenous ethanol on the whole plant biomass. The root and shoot biomass of 0.05 mL•L−1 was significantly higher than that of other treatments. And there was no significant difference between 0.0125 and 0.025 mL•L−1. With 0.0125–0.05 mL•L−1 exogenous ethanol treatment, the root and shoot biomass of oilseed rape increased by 17.95–66.67% and 16.37–39.82%, respectively. The root and shoot biomass of 0.25 mL•L−1 severely decreased by 33.33% and 55.75% compared with control. The biomass increment, which is because of the exogenous ethanol treatment, of the shoot was higher than that of the root, but the value of the weight gain rate of the shoot was lower than that of the root (Figure 2).

Effects of exogenous ethanol on the root/shoot ratio and root characters of oilseed rape

There was no significant difference among 0.125 mL•L−1, 0.025 mL•L−1, and control on the root/shoot ratio. The root/shoot ratio of 0.05 mL•L−1 was significantly increased by 23.53% than the control and 0.125 mL•L−1, and there was no significant difference between 0.05 and 0.025 mL•L−1. The root/shoot ratio of 0.25 mL•L−1 was significantly higher than that of other treatments. And the root/shoot ratio of 0.25 mL•L−1 was 52.94% higher than the control (Figure 3(a)). It indicated that the exogenous ethanol promoted the root/shoot ratio of oilseed rape under high concentration.

The root/shoot ratios and root characters of oilseed rape with different ethanol treatments.

Exogenous ethanol, which the concentration was below 0.05 mL•L−1, has little effect on the root length of oilseed rape, and there was no significant difference among control, 0.125 mL•L−1, 0.025 mL•L−1, and 0.05 mL•L−1. However, the root length was significantly decreased by 66.27% with the exogenous ethanol concentration reached 0.25 mL•L−1 (Figure 3(b)).

At 0.125 or 0.025 mL•L−1, exogenous ethanol supplied has little effect on the root surface area of oilseed rape. At 0.05 mL•L−1, exogenous ethanol supplied significantly promoted the root surface area by 24.15%. At 0.25 mL•L−1, exogenous ethanol supplied significantly inhibited the root surface area by 49.68% (Figure 3(c)). The root volume of oilseed rape has no significant change with exogenous ethanol supplied which the concentration was below 0.25 mL•L−1. However, the root volume of oilseed rape significantly decreased by 27.27% with the exogenous ethanol concentration reached 0.25 mL•L−1 (Figure 3(d)). Exogenous ethanol, which the concentration was below 0.05 mL•L−1, severely stimulated the root tips of oilseed rape, and the number of root tips significantly increased with the increase of ethanol concentration. However, the root tips significantly decreased with the exogenous ethanol concentration reached 0.25 mL•L−1 (Figure 3(e)).

Differences of N, P, and K content and accumulation in oilseed rape among the ethanol treatments and control

There was no significant difference among 0.125 mL•L−1, 0.025 mL•L−1, 0.05 mL•L−1, and the control on the N content of the root. And the root N content showed no significant difference between 0.25 mL•L−1 and the control, but the root N content of 0.125, 0.025, and 0.05 mL•L−1 was significantly higher than that of control. The shoot N content of 0.125, 0.025, and 0.05 mL•L−1 was significantly higher than control, and there was no significant difference between 0.25 mL•L−1 and control (Figure 4(a)). The N accumulation in the root, shoot, and whole plant of 0.125, 0.025, and 0.05 mL•L−1 was also significantly more than the control and increased by 27.75–83.79%, 32.22–68.84%, and 31.68–70.63%, respectively. The N accumulation in the root, shoot, and whole plant of 0.25 mL•L−1 was significantly decreased by 36.27%, 52.82%, and 50.84% than that of control, respectively (Figure 4(b)).

Effects of ethanol treatments on N, P, K accumulation in oilseed rape.

The root P content of 0.25 mL•L−1 was significantly higher than other treatments due to the concentration effect. There was no significant difference in root P content among 0.0125 mL•L−1, 0.025 mL•L−1, 0.05 mL•L−1, and control. In the shoot, the P content of 0.05 mL•L−1 was significantly higher than control, but no significant difference from other treatments (Figure 4(c)). In the root, there was no significant difference in the P accumulation between 0.0125 mL•L−1, 0.25 mL•L−1, and the control; however, the P accumulation of 0.025 and 0.05 mL•L−1 were significantly more by 34.24% and 58.54% than that of control, respectively. In the shoot, the P accumulation of 0.0125, 0.025, and 0.05 mL•L−1 was significantly more by 52.34–101.24% than that of control, but there was no significant difference between 0.0125 and 0.025 mL•L−1. The P accumulation of 0.25 mL•L−1 was significantly decreased by 43.38% than control. In the whole plant, the 0.05 mL•L−1 showed the most P accumulation, which was 1.60 g•plant−1 and 85.71% more than control. The P accumulation of 0.0125 and 0.025 mL•L−1 had no significant difference but significantly more by 40.14% and 52.31% than that of control. The P accumulation of 0.25 mL•L−1 showed a significant decrease, which was 30.58% less than control (Figure 4(d)).

Except 0.0125 mL•L−1, the K content in root of 0.025, 0.05, and 0.25 mL•L−1 was significantly decreased compared with control. With 0.0125–0.05 mL•L−1 exogenous ethanol treatment, the K content in shoot showed no significant change but significantly decreased with 0.25 ml•L−1 exogenous ethanol treatment (Figure 4(e)). With 0.0125–0.05 mL•L−1 exogenous ethanol treatment, the K accumulation in oilseed rape was significantly increased by 22.78–42.93% but significantly decreased by 61.91% with 0.25 mL•L−1 exogenous ethanol treatment (Figure 4(f)).

Furthermore, the exogenous ethanol treatment has a slight effect on the allocation of N and K in the plants; there was 84.51–88.41% of N and 84.25–88.54% of K allocated in the shoot of the plant. The 67–69.21% of P was allocated in the shoot of the plant in the control, 0.0125 mL•L−1, 0.025 mL•L−1, and 0.05 mL•L−1; however, only 51.93% of P was allocated in the shoot in the 0.25 mL•L−1.

Fe, Zn, Mn, and Cu accumulation in the ethanol treatments

High concentration of exogenous ethanol treatment (0.25 mL•L−1) has a little effect on the Fe and Zn contents in the root of oilseed rape but significantly promoted the Fe and Zn contents in the shoot (Figure 5(a,c)). Low concentration of exogenous ethanol treatment (0.0125, 0.025, and 0.05 mL•L−1) reduced the Fe and Zn contents in the root of oilseed rape but has a little effect on the Fe and Zn contents in the shoot of oilseed rape. The exogenous ethanol, which the concentration was below 0.25 mL•L−1, has little effect on the Fe and Zn accumulation which indicated that exogenous ethanol has little effect on the Fe and Zn uptake of oilseed rape (Figure 5(b,d)).

Effects of ethanol treatments on microelement accumulation in oilseed rape.

The exogenous ethanol treatment has little impact on the Mn content of oilseed rape (Figure 5(e)). The Mn accumulation in the root of the oilseed rape of 0.0125 mL•L−1 was significantly higher by 56.73% than that of control, and other ethanol treatments showed no significant difference compared with control. The Mn accumulation in the shoot of oilseed rape of 0.25 mL•L−1 was significantly lower by 29.37% than that of control, and other ethanol treatments showed no significant difference compared with control (Figure 5(f)). For the whole plant, the Mn accumulation of 0.0125 mL•L−1 was significantly higher by 41.40% than that of control, and the Mn accumulation of 0.25 mL•L−1 was significantly lower by 26.25% than control, and the other ethanol treatments showed no significant difference compared with control.

The high concentration of exogenous ethanol (0.25 mL•L−1) was significantly promoting the Cu content in the root and shoot of oilseed rape (Figure 5(g)). The exogenous ethanol with 0.025 and 0.05 mL•L−1 was significantly promoting the Cu content in the root but not in the shoot compared with control. There was no significant difference between 0.0125 mL•L−1 and control on the Cu content. The exogenous ethanol treatment has little impact on the Cu accumulation in the shoot of oilseed rape, but the Cu accumulation in the root was significantly increased by 40.18–127.36% with 0.0125–0.05 mL•L−1 ethanol; there was no significant difference between 0.25 mL•L−1 and the control on the Cu accumulation in the root of oilseed rape (Figure 5(h)). For the whole plant, the Cu accumulation was significantly increased by 38.28–72.62% with 0.0125–0.05 mL•L−1 ethanol, and there was no significant difference between 0.25 mL•L−1 and control.

Besides, the distribution of Fe, Zn, Mn, and Cu in the root and shoot of oilseed rape was different. There were 86.85–91.17% of Fe, 43.65–50.02% of Zn, 60.83–67.06% of Mn, and 33.00–46.02% of Cu distributed in the roots of oilseed rape.

Discussion

The effect of ethanol on plant growth was shown to depend mainly on ethanol concentrations and other stress conditions.7,8,10 Data from our experiments supported observations from oilseed rape that low ethanol concentrations (0.0125–0.05 mL•L−1) enhanced plant growth (Figures 1 and 2). The stimulation of photosynthesis and stomatal conductance which is due to the ethanol treatment can be explained by the increase of dry matter accumulation.11 Also, some researchers suggest that ethanol not only enhances crop biomass but also enhances high-salintiy stress tolerance in Arabidopsis thaliana.12 In this research, through cultured oilseed rape in ethanol nutrient solution instead of spray leaf manipulation, our findings indicated that low concentration of ethanol enhances shoot and root dry matter, especially accelerate root growth by increasing root branching, which results in raised root/shoot ratio (Figure 3). Good root configuration was beneficial to plant nutrient uptake and then accelerated plant biomass accumulation.13 In addition, ethanol induced an increase in ATPase activity in plasma membranes,7 which was beneficial for nutrient absorbing. These may be the reasons for the increase of N, P, and K accumulation. High concentrations of ethanol (0.25 mL•L−1) negatively affected plant growth (Figures 1 and 2), which was consistent with the previous study in A. thaliana.12 The root length, root surface area, root volume, and root tips were significantly decreased with 0.25 mL•L−1 ethanol treatment compared with control, resulting in decreased N, P, and K accumulation (Figures 3 and 4). However, the effect of ethanol on the accumulation of Fe, Zn, Mn, and Cu was different with N, P, and K, which may be because of different absorption mechanisms. Due to this, the accumulation of biomass and nutrient in early stage is severely to the yield increase for oilseed rape; thus, these data support the idea that adjustments of the frequency and amount of ethanol application to plants are useful to enhance plant growth in the agricultural field.

Conclusion

1. Low concentration of ethanol (0.0125–0.05 mL•L−1) stimulated the root tips and promoted biomass and N P K accumulation of oilseed rape at the seedling stage, and the 0.05 mL•L−1 is the best concentration of ethanol for oilseed rape growing. The high concentration of ethanol, which was 0.25 mL•L−1, severely inhibited the growth of oilseed rape.

2. Exogenous ethanol treatment has little effect on the Fe and Zn accumulation of oilseed rape but promoted the Cu accumulation. The exogenous ethanol with the concentration 0.0125 mL•L−1 stimulated Mn accumulation.

Funding Statement

This work was supported by the opening Foundation of the State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences [Y20160015].

Acknowledgments

We gratefully acknowledge the funding support from the Natural Science Foundation of Hunan Province (2019JJ50248 and 2019JJ50242); Scientific Research Foundation of Hunan Provincial Education Department (17K042); The opening Foundation of the State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences (Y20160015).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

References

1. Rossato L, Lainé P, Ourry A.. Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein patterns. J Exp Bot. 2001;52(361):1–7. doi: 10.1093/jexbot/52.361.1655. [PubMed] [CrossRef] [Google Scholar]

2. Liu XW, Lu JX, Li XS, Pu RY, Liu B. Study on characteristics of dry matter and nutrient accumulation and transportation in leaves of winter oilseed rape. Plant Nutr Fert Sci. 2011;17:956–963. [Google Scholar]

3. Roberts JKM, Andrade FH, Anderson IC. Further evidence that cytoplasmic acidosis is a determinant of flooding intolerance in plants. Plant Physiol. 1985;77:492–494. doi: 10.1104/pp.77.2.492. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

4. Ricard B, Toai TV, Chourey P, Saglio P. Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant. Plant Physiol. 1998;116:1323–1331. doi: 10.1104/pp.116.4.1323. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

5. Roberts JKM, Callis J, Wemmer D, Walbot V, Jardetzky O. Mechanisms of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under anoxia. Proc Natl Acad Sci USA. 1984b;81:3368–3372. doi: 10.1073/pnas.81.11.3379. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

6. Roberts JKM, Callis J, Jardetzky O, Walbot V, Freeling M. Cytoplasmic acidosis as a determinant of flooding intolerance in plants. Proc Natl Acad Sci USA. 1984a;81:6029–6033. doi: 10.1073/pnas.81.19.6029. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

7. Kato-Noguchi H. Low temperature acclimation mediated by ethanol production is essential for chilling tolerance in rice roots. Plant Signal Behav. 2008;3:202–203. doi: 10.4161/psb.3.3.5542. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

8. Nguyen HM, Sako K, Matsui A, Suzuki Y, Mostofa MG, Ha CV, Tanaka M, Tran LP, Habu Y, Seki M. Ethanol enhances high-salinity stress tolerance by detoxifying reactive oxygen species in Arabidopsis thaliana and rice. Front Plant Sci. 2017:8. doi: 10.3389/fpls.2017.01001. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

9. Kato-Noguchi H, Kugimiya T. Effects of ethanol on growth of rice seedlings. Plant Growth Regul. 2001;35:93–96. doi: 10.1023/A:1013850707053. [CrossRef] [Google Scholar]

10. Weiss K, Kroschewski B, Auerbach H. Effects of air exposure, temperature and additives on fermentation characteristics, yeast count, aerobic stability and volatile organic compounds in corn silage. J Dairy Sci. 2016;99:8053–8069. doi: 10.3168/jds.2015-10323. [PubMed] [CrossRef] [Google Scholar]

11. Zhao Y, Tan H, Chen LM.. Comparison of the effects of foliar application of methanol and ethanol on the growth and photosynthetic characteristics in brassica napus l. J Yangzhou University (Agricultural and Life Science Edition). 2013;34(3):39––43.. [Google Scholar]

12. Sako K, Sunaoshi Y, Tanaka M, Matsui A, Seki M. The duration of ethanol-induced high-salinity stress tolerance in Arabidopsis thaliana. Plant Signal Behav. 2018;13:e1500065. [PMC free article] [PubMed] [Google Scholar]

13. Mu XH, Chen FJ, Wu QP, Chen QW, Wang JF, Yuan LX, Mi GH. Gentic improvement of root growth increases maize yield via enhanced post-silking nitrogen uptake. Eur J Agron. 2015;63:55–61. [Google Scholar]

Articles from Plant Signaling & Behavior are provided here courtesy of Taylor & Francis