Legume Research

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Legume Research, volume 46 issue 12 (december 2023) : 1604-1609

Effect of Exogenous Abscisic Acid (ABA), a Potential Growth Regulator on Physiological Response to Chilling Stress of Adzuki Bean (Vigna angularis) at Flowering Stage

Xiang Hong-Tao1,2,*, Li Wan2,3, Liu Jia2, He Ning2, Wang Xue-Yang2, Li Bo2,3, Xie Hong-Chang1, Wang De-Ming1
1Suihua Branch, Heilongjiang Academy of Agricultural Machinery Sciences, Suihua 152054, Heilongjiang, China.
2Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China.
3Institute of Crop Cultivation and Tillage, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China.
  • Submitted05-04-2023|

  • Accepted11-09-2023|

  • First Online 22-12-2023|

  • doi 10.18805/LRF-748

Cite article:- Hong-Tao Xiang, Wan Li, Jia Liu, Ning He, Xue-Yang Wang, Bo Li, Hong-Chang Xie, De-Ming Wang (2023). Effect of Exogenous Abscisic Acid (ABA), a Potential Growth Regulator on Physiological Response to Chilling Stress of Adzuki Bean (Vigna angularis) at Flowering Stage . Legume Research. 46(12): 1604-1609. doi: 10.18805/LRF-748.
Background: Low temperatures during flowering severely affect crop growth and yield. 

Methods: This experiment was conducted under potted conditions with LXD 4 and TJH as test materials and pre-sprayed with exogenous ABA at a concentration of 20 mg·L-1 at flowering stage, respectively and then with an average of 15°C condition for 5 days.

Result: The results showed that foliar spraying exogenous ABA effectively inhibited the increase of MDA content and relatively significantly improved the photosynthetic parameters such as Gs and Tr and alleviated the loss of yield factors caused by chilling stress. Compared with spraying water, spraying exogenous ABA can significantly increase the yield per pot of LXD 4 by 6.72~17.77% and TJH by 6.41~37.04% under low temperature conditions. In summary, foliar spraying exogenous ABA ameliorated the effects of chilling stress on the physiological characteristics of adzuki bean leaves and improved yield as a result of improved antioxidant defense mechanisms which impeded lipid peroxidation. Thus, we conclude that foliar spraying exogenous ABA could decelerate the damages caused by chilling stress to some extent.
Adzuki bean (Vigna angularis) is widely planted in China, it is planted in almost different regions of the country, especially in the Northeast region. Adzuki bean has the biological characteristics of light, temperature and infertile, but does not tolerate low temperature and is sensitive to chilling (Donnelly, 2021). In recent years, low temperatures have occurred frequently in spring and summer in Northeast China, which has a great impact on the production of adzuki bean. Sudden cold damage has become an important abiotic adversity stress that restricts adzuki bean production in Heilongjiang province (Xiang et al., 2019a).

Plant endogenous hormones regulate plant growth and response to stress (Klingler et al., 2010). ABA, an endogenous hormone discovered and identified in the 1960s, plays an important role in plant stress tolerance and resistance, under low temperature stress, a significant increase in the leaf abscisic acid content (at least 2.5-fold) was measured in plants (Pruvot et al., 1996). Exogenous treatment with abscisic acid (ABA) resulted in an improvement in growth and survival of nonacclimated, chilled seedlings (Anderson et al., 1994). Exogenous hormones regulate the physiological metabolism of plants by changing the levels of endogenous hormones. Exogenous ABA has multiple pathways in resisting low temperature stress. The impact of low temperature on crop yield is obvious and in severe cases it can even lead to production failure. This study proposes that exogenous ABA treatment can improve the yield and antioxidant defense capacity of adzuki bean by enhancing the cold tolerance at flowering stage. The main research purpose is to confirm that exogenous ABA treatment can improve the cold tolerance of adzuki bean.
Plant materials
 
The cold-tolerant and cold-sensitive adzuki bean varieties, LXD 4 and TJH were used as experimental materials. Exogenous ABA was provided from China National Practical Bean Technology System, purchased from Sigma Company. The experiment was carried out at the potted planting field of the Institute of Crop Cultivation and Tillage, Heilongjiang Academy of Agricultural Sciences (45.57°N and 126.37°E) in Harbin, Heilongjiang province, northeastern China.
 
Experimental design
 
Adzuki bean seeds were planted in each resin pot (diameter of 30 cm and height of 25 cm) filled with about 16 kg of soil. There are six treatments in this experiment (Three replication), as shown in Table 1 and the temperature change during the day is shown in Fig 1. When plants to grow to flowering stage (51 days after sowing), foliar spraying of exogenous ABA (concentration of 20 mg·L-1 with a spraying rate of 225 L·hm-2), was conducted and the pots were kept into the artificial climate room with different low temperature for chilling treatment. The duration is 1, 2, 3, 4 and 5 days, respectively. Each treatment was sampled separately and immediately placed in liquid nitrogen and then stored in a -80°C refrigerator for the determination of physiological indicators.

Table 1: Test design scheme.



Fig 1: Changes in temperature during one day.


 
Measurement items and methods
 
Determination of MDA content
 
The content of MDA (malondialdehyde) was determined using the thiobarbituric acid (TBA) method (Dhindsa et al., 1981).
 
Content of soluble sugar, soluble protein and free proline
 
Glucose equivalents were used to determine soluble sugar content to the anthrone-sulfuric acid method described by Khosrowshahi et al., 2020).

The soluble protein content was determined according to a method introduced by Chaturvedi et al., (2015).

or the free proline assay, leaf samples (0.5 g) were homogenized in 10 mL of 3% sulfosalicylic and the homogenate was filtered through filter paper. Extract (2 mL) was added to 2 mL of glacial acetic acid and 2 mL of acid ninhydrin and the mixture was bathed in water of 100°C for 30 min. After cooling down, 4 mL of methylbenzene was added with agitation. Absorbance of the red methylbenzene supernatant was taken at 520 nm with a spectrophotometer.
 
Gas-exchange parameters
 
Net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci) and stomatal conductance (Gs) were measured on the third fully expanded trifoliate leaves from the main apex using a portable photosynthesis system (Li-Cor 6400, Li-Cor Inc., Nebraska, USA). Plants were measured under PPFD of 1,000 µmol(photon) m-2·s-1, CO2 concentration of 500 mmol mol-1, 25±3°C and 80% humidity.
 
Statistical analysis
 
Microsoft Excel 2013 and SPSS 25.0 was used to analyze the one-way ANOVA of all the collected data. Duncan test (p<0.05) was used to evaluate the difference within treatments and the significant differences among different materials were determined.
Effect of exogenous ABA on adzuki bean yield under chilling stress
 
15°C during flowering stage can significantly affect the yield of adzuki beans. For LXD 4, the yield shows CK>T2 at each time and the result of significant analysis showed that CK was higher than T2 from 2d to 5d. In addition, with the extension of the treatment time, the greater the degree of yield loss, T2 of LXD 4, compare with 1d, the yield at 5d decreased by 10.30 g per pot, a decrease of 59.81%. For TJH, the yield shows that, CK was consistently higher than T2, significantly. Under low temperature conditions, spraying exogenous ABA can inhibit production reduction. For LXD 4, T1 was significantly higher than T2 by 6.72%, 7.69%, 10.85%, 9.92% and 17.77% during treatment from 1d to 5d. For TJH, T1 was significantly higher than T2 by 6.41%, 15.67%, 20.47% and 37.04% from 2d to 5d (Table 2).

Table 2: Effect of exogenous ABA on the yield of adzuki beans under chilling stress at flowering stage (g.pot-1).



As shown in Table 3, it is clear that chilling stress increased the hundred-grain weight. For LXD 4, T2 was significantly heavier than CK by 3.23%, 8.00%, 16.30%, 8.06% and 4.30% during treatment from 1d to 5d. For TJH, T2 was significantly heavier than CK by 19.24%, 10.82%, 12.04%, 16.71% and 10.88% during treatment from 1d to 5d. Spraying exogenous ABA reduced the hundred-grain weight of adzuki bean. For LXD 4, T1 was lighter than T2 by 0.73%, 0.83%, 1.38%, 1.97% and 1.24% during treatment from 1 to 5 days. For TJH, T1 was higher than T2 by 3.44%, 1.64%, 1.35%, 5.48% and 2.15% during treatment from 1 to 5 days.

Table 3: Effect of exogenous ABA on the hundred-grain weight of adzuki beans under chilling stress at flowering stage (g).



Effect of exogenous ABA on MDA content in adzuki beans leaves under chilling stress
 
As shown in Fig 2, the MDA content showed an increasing trend after low temperature treatment during the flowering stage. Compared with CK, T2 of LXD 4 significantly increased by 19.68 nmol·g-1, 25.45 nmol·g-1, 30.00 nmol·g-1, 39.89 nmol·g-1 and 40.03 nmol·g-1. T2 treatment of TJH significantly increased by 14.17 nmol·g-1, 24.42 nmol·g-1, 30.48 nmol·g-1, 39.79 nmol·g-1 and 43.91 nmol·g-1. Exogenous ABA can significantly inhibit the increase of MDA content under low temperature conditions. The analysis of variance showed that after treatment for 1 to 5 days, both LXD 4 and TJH showed that T1 was significantly lower than T2.

Fig 2: Effect of exogenous ABA on MDA content of adzuki beans leaves under chilling stress at flowering stage.


 
Effect of exogenous ABA on photosynthesis in adzuki beans leaves under chilling stress
 
It can be seen from Fig 3 that, with the extension of the stress time, compared with CK, Pn, Gs, Ci and Tr in T2 treatment reduced significantly both of LXD 4 and TJH. At the same time, under natural environment, spraying exogenous ABA has the effect of increasing Pn, Gs, Ci and Tr, especially Pn, Gs and Tr can be significantly increased (Fig 3A, 3B, 3C and 3D).

Under low temperature conditions, spraying with exogenous ABA can improve the photosynthesis. As shown in Fig 4A, compared with T2, T1 treatment of LXD 4 increased by 4.84%, 14.02%, 12.01%, 21.31% and 18.39% and the ANOVA results showed that there was a significant difference at the 4th and 5th day. TJH showed that, T1 was significantly higher than T2 by 14.43%, 9.19%, 21.18%, 10.93% and 16.39%.

With the Gs, it can be seen from Fig 3B, compared with T2, T1 treatment of LXD 4 significantly increased by 45.84%, 19.46%, 57.66% and 20.56% from 2d to 5d. TJH showed that T1 was significantly higher than T2 by 17.39%, 4.11% and 42.23% from 3d to 5d.

As shown in Fig 3C, compared with T2, T1 of LXD 4 increased by 5.67%, 8.30%, 9.77%, 19.54% and 13.95%, the analysis of variance showed that T1 was significantly higher than T2 at each time. For TJH, T1 was significantly higher than T2 by 6.90%, 10.58% and 11.47% during treatment from 3 to 5 days.

t can be seen from Fig 3D that, spraying exogenous ABA can inhibit the reduce of Tr, compared with T2, T1 of LXD 4 was significantly higher than T2 by 19.54%, 20.33%, 28.17% and 140.35% from 1 to 4 days. For TJH, T1 was significantly higher than T2 by 47.05%, 72.28% and 20.91% from 3 to 5 days.

Fig 3: Effect of exogenous ABA on net photosynthetic rate (Pn) (A), stomatal conductance (Gs) (B), intercellular CO2 concentration (Ci) (C) and transpiration (Tr) (D) of adzuki beans leaves under chilling stress at flowering stage.


 
Effect of exogenous ABA on the content of osmotic regulation substances in adzuki beans leaves under chilling stress
 
Under the normal temperature condition, soluble sugar, soluble protein and proline content in adzuki bean leaves showed a stable change trend, but the content of these substances in leaves had an upward trend with the increase of chilling time intensity. As shown in Fig 4, the soluble sugar, soluble protein and proline contents in T2 were always significantly higher than CK.

Under low temperature condition, spraying with exogenous ABA can further increase the content of osmotic regulation substances. As shown in Fig 4A, compared with T2, T1 treatment of LXD 4 significantly increased than T2 by 1.07% and 8.93% at 1d and 2d. TJH showed that, T1 was significantly higher than T2 at each time. With the soluble protein, it can be seen from Fig 4B that, compared with T2, T1 treatment of LXD 4 increased by 1.74%, 6.00%, 10.12%, 16.71% and 17.40%, the analysis of variance showed that after treatment 3 to 5 days, it showed that T1 was significantly higher than T2. TJH showed that, when treated from 4 to 5 days, T1 was significantly higher than T2 by 2.53% and 4.05%. The increasing effect of exogenous ABA on proline is very obvious. As shown in Fig 4C, compared with T2, T1 was always significantly higher than T2 both of LXD4 and TJH.

Fig 4: Effect of exogenous ABA on osmotic regulation substances content of adzuki beans leaves under chilling stress at flowering stage.



Photosynthesis is a determinant of crop productivity and an important indicator of crop response to environmental stress. Photosynthesis is most sensitive to chilling stress (Allen et al., 2001) and chilling stress significantly inhibits Pn, resulting in a decrease in yield (Strauss et al., 2007). This study also found that low temperature led to a significant decrease in Pn and at the same time, Gs, Ci and Tr were also significantly reduced. We believe that this is because low temperature causes ROS metabolism disorder, which affects the structure and activity of photosynthetic organs, which in turn affects the capture, transformation and distribution of light energy in leaves. This view is also consistent with (Ayub et al., 2011).

When encountering adversity stress, plants will automatically turn on the protection system to resist the threat of adversity factors and then try to maintain normal physiological and metabolic activities and avoid damage (Xiang et al., 2019b). Plants subjected to low temperature stress will turn on a series of stress self-protection mechanisms and these protection systems coordinate organically with each other to minimize damage (Xiang et al., 2019a). Exogenous ABA has the function of regulating the stress resistance physiology of plants and has multiple pathways in resisting low temperature stress. The main stress response is to increase the content of osmotic regulators to regulate the content of MDA decreased. This study found that, exogenous ABA can effectively change the physiological indicators of stress resistance in adzuki bean under low temperature conditions. The application of exogenous ABA can significantly increase the content of osmotic adjustment substances, further reduce the content of MDA (Fig 2).

We can also see from the results of this study that spraying of exogenous ABA can significantly increase the yield per pot of adzuki bean (Table 2) after low temperature stress and effectively alleviate the impact of low temperature on yield.
The flowering stage is the crucial stage for yield formation and we found that low temperature during this stage causes a significant reduction of yield of adzuki bean. At the same time, the MDA content in adzuki beans leaves was increased. The content of soluble sugar, soluble protein and proline are increased. The photosynthetic parameters such as Pn, Gs, Ci and Tr were reduced in chilling condition. Exogenous ABA has the function of resisting low temperature and reducing crop damage. The results of this study indicated that, it can effectively increase the content of soluble sugar, soluble protein and proline and promote photosynthesis such as Gs and Tr and then improve the resistance of adzuki beans to low temperature stress and maintain normal physiological activities. Chilling during the flowering period caused changes in yield factors and low temperature caused a significant decrease in the grain weight per pot. Spraying exogenous ABA can significantly increase the yield per pot of LXD 4 by 6.72~17.77% and TJH by 6.41~37.04% under low temperature conditions. Thus, we conclude that exogenous ABA solution application at 20 mg´L-1 at flowering stage can enhance the adzuki bean production under chilling stress.
The authors thank the Heilongjiang Key R and D Program Project (GA21B009-14) and China Agriculture Research System (CARS-08-G8) Project for the financial support.
All authors declare that they have no conflicts of interest.

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