Method for improving heat tolerance of plant pollen by low-temperature pretreatment

Abstract

The present invention discloses a method for improving the heat tolerance of plant pollen by low-temperature pretreatment. During the flowering period of the plant, a short-term low-temperature pretreatment is carried out on the plant before high-temperature stress to improve the resistance of the plant pollen during the pollen development period under high-temperature stress. The results obtained by the present invention are helpful for taking preventive measures in production to improve the reproductive ability and yield of plants, especially autumn and winter crops, in high-temperature environments.

Description

Technical Field

[0001] The present invention relates to the technical field of plant propagation, and particularly to a method for improving the heat tolerance of plant pollen by low-temperature pretreatment. Background Art

[0002] Cruciferous plants have numerous types and are widely distributed throughout the world, including many economically important crops, and play a very important role in human production and life. In recent years, the greenhouse effect has led to frequent extreme high-temperature weather. Most cruciferous crops, such as Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis), usually bloom in spring. High temperature is one of the main environmental factors threatening the development of their pollen, resulting in a decrease in pollen fertility or even complete abortion, and causing a serious decline in seed yield and quality. In addition, Chinese cabbage is one of the indispensable vegetables on people's daily tables and is cultivated throughout the country, playing an important role in the annual supply of vegetables. Heat damage will seriously affect the quality.

[0003] Tropical and subtropical crops, such as rice, maize, cucumber, etc., are sensitive to cold stress due to the lack of cold acclimation during evolution, but have certain heat tolerance (Zhang et al., 2019). Crops growing in temperate climates, such as wheat, rapeseed, Chinese cabbage, etc., will experience low temperatures in winter and early spring. Therefore, during the long-term evolution process, these crops can usually tolerate certain cold stress, but are very sensitive to heat stress (Ritonga and Chen, 2020). Interestingly, high temperature and low temperature have some common physiological and biochemical reactions, such as changes in membrane fluidity, alterations in plant hormone levels, generation and accumulation of ROS, calcium, nitric oxide (NO) signals, mitogen-activated protein kinase (MAPK) signals, protein acylation and proteasome degradation, reprogramming of transcriptome or metabolome characteristics, and other stress-responsive signal cascades (Chinnusamy et al., 2007; Noctor et al., 2018). For example, cyclic nucleotide-gated ion channels 14 and 16 can not only promote the heat tolerance of rice and Arabidopsis seedlings at the seedling stage, but also improve their cold tolerance (Cui et al., 2020).

[0004] Plants are subjected to a variety of stresses with different natures, durations, and intensities throughout their life cycles. The physiological and biochemical responses of plants to abiotic and biotic stresses are similar (Ding et al., 2020). In recent years, with abnormal environmental changes and energy shortages, it has become an arduous task for agricultural scientists to deeply explore the resistance potential of plants themselves and cultivate crops that can adapt to various environmental stresses. With the improvement of scientific research levels, concepts and related research such as "plant stress memory" and "cross-stress tolerance" have emerged one after another (Liu et al., 2021). Cross-stress tolerance refers to the phenomenon that plants pre-exposed to a major stress can improve the efficiency and performance of these individuals (even their offspring) under minor stresses. The success of cross-stress priming is supported by hierarchical interactions of co-stress signaling pathways shared between stresses of different natures (Liu et al., 2021). Primary temperature sensing is thought to occur at the membrane through membrane fluidity and membrane-localized channels or receptors (Murata and Los, 1997). After sensing, secondary messengers are generated, including Ca 2+ and reactive oxygen species (ROS) (Yan et al., 2006). Environmental signals induce transient Ca 2+ influx into the cytoplasm (Finka et al., 2012; Ma et al., 2015), and repeated environmental signals can induce repeated Ca 2+ transients (Krebs et al., 2012). Appropriate amounts of reactive oxygen species can induce reactive oxygen species scavengers and other stress protection mechanisms (Prasad et al., 1994). Secondary Ca 2+Signals and ROS can trigger another signal event cascade, which may produce different signals and physiological responses according to the main stimuli (Zhang et al., 2019). For example, short-term heat shock treatment significantly enhanced the cold tolerance of non-heading Chinese cabbage seedlings, reduced the degree of membrane damage, increased the activities and contents of superoxide dismutase, catalase, and peroxidase, while the content of malondialdehyde decreased (Zheng et al., 2010). Short-term heat shock treatment in cucumber could improve its cold tolerance at the seedling stage, specifically manifested as an increase in the expression level of CsHSFA1d after heat shock. The cucumber lines overexpressing CsHSFA1d were more tolerant to cold stress than the wild type, while the lines with CsHSFA1d knocked down by RNA interference were more sensitive to cold stress. In addition, both overexpression of CsHSFA1d and heat shock pretreatment could increase the endogenous jasmonic acid (JA) content in cucumber seedlings after cold treatment. Exogenous application of JA restored the cold-sensitive phenotype of the CsHSFA1d knockdown line, and JA-induced degradation of CsJAZ5 would release CsICE1, which then activated the ICE-CBF-COR pathway (Qi et al., 2022). However, there are few studies on cold shock pretreatment improving the heat tolerance or other environmental stresses of plants at present.

[0005] In seed plants, reproductive development is an important process for forming seeds and completing the alternation of generations. Existing studies have shown that it is the most sensitive developmental process to environmental stresses such as temperature during the plant life cycle. Among them, male reproductive development (mainly anther and pollen development) is more sensitive to environmental stresses than female reproductive development (Begcy et al., 2019). Since environmental stresses have a serious impact on pollen development, it further affects the normal production of crop seeds and food and vegetables with seeds as products (De Storme and Geelen, 2014; Ghadirnezhad and Fallah, 2014). Plants respond to heat stress by regulating various physiological processes. Approaches such as creating heat-tolerant germplasms, cultivating new varieties, and applying chemical fertilizers can be used to improve the heat stress tolerance of plants, but these methods are either costly and time-consuming or have adverse effects on the environment (Lamaoui et al., 2018; Gebremedhin et al., 2019; Wang et al., 2019). Therefore, formulating economic, efficient, green, and environmentally friendly preventive measures is crucial for ensuring global food security. Chinese cabbage is a low-temperature-tolerant crop in autumn and winter. It contains rich cold-resistant gene resources and powerful physiological and biochemical reactions. For example, during the frost season in actual production, the temperature is extremely low. Chinese cabbage increases its ability to resist low temperature by producing more soluble sugars, and at the same time, it also makes the taste better. However, the current research on the effect of low-temperature pretreatment on improving its heat tolerance is still blank. Here, the present invention applies low-temperature pretreatment to improve the high-temperature tolerance of Chinese cabbage. Taking the tetrad stage, which is the most sensitive period to high temperature stress in Chinese cabbage and the period when the impact of instantaneous high temperature is the most serious, as an example, it is found that low-temperature pretreatment can significantly reduce the impact of high temperature on pollen development, increase the germination rate of pollen that develops and matures after being subjected to high temperature stress, and ultimately increase the seed setting rate of the plant, helping to alleviate the adverse effects of high temperature on seed production. The results of this study provide technical guidance for taking economic, efficient, green, and environmentally friendly preventive measures in production to improve the seed propagation ability and quality of Chinese cabbage in high-temperature environments.

[0006] References

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[0027] Zhang JY, Li XM, Lin HX, Chong K (2019) Crop Improvement Through Temperature Resilience. Annual Review of Plant Biology, 70:753-780. Summary of the Invention

[0028] The object of the present invention is to provide the application of low-temperature pretreatment in improving the heat tolerance of plants to solve the deficiencies of the prior art.

[0029] The present invention adopts the following technical solutions:

[0030] Method for improving heat tolerance of plant pollen by low-temperature pretreatment. During the flowering period of the plant, the plant is subjected to a short-term low-temperature pretreatment before high-temperature stress to improve the resistance of the plant pollen during the pollen development period to high-temperature stress.

[0031] Furthermore, the short-term low-temperature pretreatment of the plant before high-temperature stress is specifically operated as follows: 2-6 hours before high-temperature stress, the plant is subjected to a low-temperature pretreatment at 0-4 °C (simulating the temperature in the frost season in production practice) for 2-6 hours.

[0032] Furthermore, in the field, the low-temperature pretreatment is to continuously irrigate the roots of the plant with ice water or continuously spray ice water mist, keeping the field low-temperature and humid without waterlogging; indoors, the low-temperature pretreatment is to adjust the temperature control system to a low temperature.

[0033] Furthermore, the pollen development period of the plant is the early stage of pollen development.

[0034] Even further, the early stage of pollen development of the plant is the tetrad stage of pollen development.

[0035] Furthermore, the plant is a cruciferous crop in autumn and winter.

[0036] Even further, the cruciferous crops in autumn and winter include Chinese cabbage, cabbage or rape.

[0037] Beneficial effects of the present invention:

[0038] The present invention improves the resistance of plant pollen during the pollen development period, especially the early development stage, to high-temperature stress by subjecting the plant to a short-term low-temperature pretreatment before high-temperature stress during the flowering period of the plant. The results obtained by the present invention are helpful for taking preventive measures in production to improve the reproductive ability and yield of plants, especially autumn and winter crops, in a high-temperature environment.

[0039] The present invention is to subject the plant to a low-temperature pretreatment at 0-4 °C for 2-6 hours 2-6 hours before high-temperature stress. The method is convenient to apply, has strong operability, low cost, is green and environmentally friendly, and is safe and reliable. Description of the drawings

[0040] Figure 1 Effect of low-temperature pretreatment (0 °C) at different times on callose in the tetrad stage of pollen development of Chinese cabbage after high-temperature stress (38 °C, 24 h). The scale bar is 50 μm. 0, 2, 4, 6, 8, 10, 12 and 14 minutes represent the callose fluorescence quenching time. The shorter the quenching time, the less the callose content. The intensity of the fluorescence represents the amount of callose content. CK represents growth under normal conditions, HS represents high-temperature stress, and Cold_0.5, 1.0, 2.0, 4.0, 6.0, 8.0 h+HS represent low-temperature pretreatment for different times plus high-temperature stress.

[0041] Figure 2 Effect of low-temperature pretreatment (0°C) at different times on pollen viability and germination of Chinese cabbage after high-temperature stress (38°C, 24 h). The scale bar is 50 μm. CK represents growth under normal conditions, HS represents high-temperature stress, and Cold_0.5, 1.0, 2.0, 4.0, 6.0, 8.0 h+HS represent low-temperature pretreatment for different times plus high-temperature stress.

[0042] Figure 3 Effect of low-temperature pretreatment (0°C, 6 h) on pollen fertility of Chinese cabbage after high-temperature stress (38°C, 24 h). A, High-temperature stress had no significant effect on Chinese cabbage plants. The scale bar is 10 cm. B, After low-temperature pretreatment, the filament length and anther powder-dispersing ability after high-temperature stress could be increased. The scale bar is 500 μm. C, After low-temperature pretreatment, the seed-setting rate of Chinese cabbage pollen after stress could be improved. The scale bar is 1 cm. CK represents growth under normal conditions, HS represents high-temperature stress, and preCold+HS represents 0°C low-temperature pretreatment for 6 h plus high-temperature stress. D is the corresponding statistical analysis of C. After one-way ANOVA, multiple comparison tests were performed, and different letters (P<0.05) indicate that the differences between samples are statistically significant. Specific implementation manners

[0043] The present invention will be further explained below in conjunction with the embodiments and the drawings. The following embodiments are only used to illustrate the present invention, but do not limit the implementation scope of the present invention.

[0044] I. Materials and methods

[0045] Plant materials and treatments

[0046] (1) Chinese cabbage variety 'Byq97-02' (Brassica campestris L.ssp.chinensis Makino, syn. B.rapa ssp.chinensis) was sown in a substrate composed of peat, vermiculite and perlite mixed according to a mass ratio of 3:2:1. The flower pots were watered thoroughly and cultured in a greenhouse with a relative humidity of 65% and a maximum light intensity of 300 μmol·m -2 ·s -1 to the flowering stage, and the light cycle and temperature of day / night were 16 h / 8 h and 22°C / 18°C respectively.

[0047] (2) For low temperature treatment, the cabbage plants grown to the flowering stage under normal conditions (above (1)) were marked (i.e., different pollen development stages were marked, the flowers of the cabbage inflorescence were removed, and the five-level flower buds were left for testing. The five typical stages of pollen development are: pollen mother cell stage, tetrad stage, mononucleate stage, binucleate stage, and trinucleate mature stage (i.e., pollen mature stage). According to the size of the flower buds and microscopic observation, the tetrad stage can be marked in the present invention) and then transferred to an ultra-low temperature incubator (Ningbo Southeast Instrument Co., Ltd.) for low temperature pretreatment, the condition was 0°C for 0, 0.5, 1, 2, 4, 6, and 8 hours, respectively, and other conditions remained unchanged; after the low temperature pretreatment was completed, the cabbage plants were transferred to high temperature conditions (38°C for 16 hours during the day / 28°C for 8 hours at night) for treatment for 24 hours, and other conditions remained unchanged; after the high temperature treatment was completed, the cabbage plants were transferred to grow under normal conditions. Cabbage plants sown at the same time and grown under normal conditions were used as controls.

[0048] Phenotypic observation

[0049] The flower buds of the pollen development tetrad stage of the treated and untreated cabbage plants (control cabbage plants) were taken (the flower buds were sampled immediately after the high temperature treatment of the pollen development tetrad stage was completed) and placed in a 1.5 ml centrifuge tube. 1.0 ml of Karl Fischer fixative with a volume ratio of ethanol: chloroform: acetic acid = 3:2:1 was added and fixed at room temperature for 3 hours. After that, rinse three times with 0.2M phosphate buffer, pH 7.2, add 0.1w / v% aniline blue solution (solvent is 0.2M phosphate buffer, pH 7.2) to cover the flower buds, and stain in the dark at 4°C refrigerator for 3 days. Then, use tweezers to pick out the flower buds, put them on a slide, gently squeeze out the tetrads, remove the remaining impurities, cover with a coverslip, and observe with a fluorescence microscope (Nikon, Eclipse 90i, Japan). First, find the target sample in the white field and take a picture, and then take pictures under fluorescence conditions (microscope built-in) at 0, 2, 4, 6, 8, 10, 12 and 14 minutes (callose quenching test).

[0050] Mature pollen (pollen released from the anthers of mature buds after high temperature treatment at the tetrad stage of pollen development and recovery to maturity under normal conditions) of the treated and untreated Chinese cabbage plants (control Chinese cabbage plants) were placed in 30 μL of a culture medium (15 wt% sucrose, 0.4 mmol·L -1 HBO3, 0.4mmol·L -1After culturing in Ca(NO3)2 and 0.1 wt% agar (adjusted to pH 5.8 with 2M NaOH) at 20 °C in the dark for 3 h, 30 μL of Alexander dye solution (for detecting pollen viability) was dropped respectively and stained for 30 min at room temperature in the dark. Then a cover glass was covered, and photographs were taken and observed under a microscope (Nikon, Eclpse 90i, Japan).

[0051] Pollination experiment

[0052] Using each treated and untreated Chinese cabbage plant (control Chinese cabbage plant) as the male parent (pollen from the anthers of the mature stage buds that had recovered under normal conditions after the high-temperature treatment of the flower bud at the tetrad stage of pollen development), and the untreated Chinese cabbage plant as the female parent, hybridization was carried out, and the silique setting was observed about 20 days later.

[0053] Statistical analysis

[0054] These experiments were confirmed by three biological and technical replicates. Statistical analysis and chart drawing were performed using Graphpad prism 9.0 (GraphPad Software, San Diego, USA) software. The data were expressed as mean ± SD, and further evaluated using analysis of variance to determine significance. At a 95% confidence level, the differences were considered significant (p < 0.05).

[0055] II. Results and analysis

[0056] An increase in temperature has an adverse effect on plant development and seed production. By performing low-temperature pretreatment on Chinese cabbage plants during the flowering period and then applying high-temperature stress, immediately after the treatment, flower buds at the tetrad stage of pollen development were taken for callose quenching experiments. It was found that the callose signal in the tetrads weakened after high-temperature stress, but a certain period of low-temperature pretreatment (2 - 6 h of low-temperature pretreatment) could significantly increase the callose signal in the tetrads after high-temperature stress ( Figure 1 ).

[0057] Taking mature pollen germinated in vitro after high-temperature stress, high-temperature stress after low-temperature pretreatment, and control normal conditions restored to the mature stage, and staining with Alexander dye, it was found that 2 - 6 h of low-temperature pretreatment could improve the viability and germination rate of pollen after high-temperature stress, and the effect of 6 h of low-temperature pretreatment was the best ( Figure 2 ).

[0058] Taking 6 hours of low-temperature pretreatment as an example for detailed observation and analysis, it was found that the filaments of the plants subjected to high-temperature stress after low-temperature pretreatment were significantly elongated, and the pollen dispersal ability of the anthers was significantly increased. Using the Chinese cabbage plants after high-temperature stress and after low-temperature pretreatment followed by high-temperature stress as the male parent, and the untreated Chinese cabbage plants as the female parent for hybridization, and the self-crossing between the untreated Chinese cabbage plants as the control, it was found that low-temperature pretreatment could significantly improve the pollen seed setting rate after high-temperature stress( Figure 3 ).

[0059] The above results indicate that low-temperature pretreatment before high-temperature stress can stimulate the resistance potential of Chinese cabbage itself and improve the resistance of Chinese cabbage plants to subsequent stress.

[0060] III. Summary

[0061] In the research of the present invention, we utilized the cold tolerance advantage of Chinese cabbage itself and the similar physiological and biochemical reaction mechanisms of plants in response to high and low temperature stresses. By performing a short-term low-temperature pretreatment on Chinese cabbage plants before they were subjected to high-temperature stress, the resistance mechanism of Chinese cabbage was stimulated, and the fertility of Chinese cabbage pollen (at the early development stage, represented by the tetrad stage) under high-temperature stress could be significantly improved. The results obtained in the present invention are helpful to provide technical guidance for taking economic, efficient, green, and environmentally friendly preventive measures in production to improve the seed production ability and quality of Chinese cabbage in high-temperature environments.

[0062] The above-described embodiments have elaborated on the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the principle scope of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for improving the heat resistance of plant pollen by low temperature pretreatment, characterized in that: During the flowering period of the plants, the plants are subjected to a short low-temperature pretreatment before high-temperature stress to improve the resistance of the plant pollen under high-temperature stress during the pollen development period; the plant pollen is cabbage pollen; the plants are subjected to a short low-temperature pretreatment before high-temperature stress, and the specific operation is: 2-6 hours before high-temperature stress, the plants are subjected to a low-temperature pretreatment of 0-4°C for 2-6 hours.

2. The method for improving the heat resistance of plant pollen by low temperature pretreatment according to claim 1, characterized in that: In the field, the low-temperature pretreatment is to continuously irrigate the roots of the plants with ice water or spray ice water mist to keep the field low temperature and moist without water accumulation; indoors, the low-temperature pretreatment is to adjust the temperature control system to a low temperature.

3. The method for improving the heat resistance of plant pollen by low temperature pretreatment according to claim 1, characterized in that: The plant pollen development stage is the early stage of plant pollen development.

4. The method for improving the heat resistance of plant pollen by low temperature pretreatment according to claim 3, characterized in that: The early stage of plant pollen development is the tetrad stage of plant pollen development.

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