Application of histidine in promoting maturation starting and quality formation of tomato fruits

Abstract

The invention discloses an application of histidine (His) in promoting maturation start and quality formation of tomato fruits. The method comprises the following steps: irrigating tomato plants with an aqueous solution containing His, or injecting picked immature fruits with an MES buffer solution containing His to promote fruit maturation starting and turning red. Experimental results show that when tomato plants or tomato fruits in the in-vitro immature stage are treated with the His aqueous solution or the MES buffer solution with the specific concentration, tomato fruit maturation starting, coloring and softening can be promoted; according to the present invention, the tomato plants are subjected to His treatment, and the tomato plants are treated by using the 3-(2-Thienyl)-L-alanine (TIH) activating agent of the ATP-PRT biosynthesis pathway rate-limiting enzyme (ATP-PRT) and the adenosine triphosphate (ATP) substrate of the ATP-PRT so as to promote the maturation, the color turning and the softening of the fruits; his treatment and ATP treatment of the ATP-PRT substrate can promote in-vitro immature-stage tomato fruit maturation starting, reddening and seed maturation, and the effect of His can be significantly inhibited by an ethylene receptor inhibitor 1-MCP, which indicates that His can promote conversion of ethylene synthesis of immature-stage tomato fruits from a system I to a system II, and plays a role depending on an ethylene signal pathway.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of histidine in promoting the initiation of tomato fruit ripening and the formation of fruit quality. Background Art

[0002] Fruit is a unique organ of flowering plants. It has an attractive flavor, bright color, is sweet and delicious, and is also rich in vitamins, minerals, and other important nutrients. It is an essential part of the human diet and is beneficial to people's physical and mental health. The quality of fruits, such as aroma, color, texture, and nutrition, gradually forms during the ripening process. This process involves complex physiological and biochemical metabolic pathways such as the synthesis of aromatic compounds, pigment accumulation, and cell wall degradation, and is highly coordinated and precisely regulated by multiple factors and at multiple levels. Internationally, tomato has been recognized as a model plant for studying the ripening mechanism of climacteric fruits. Analyzing the physiological and molecular mechanisms of tomato fruit ripening not only helps to clarify the formation mechanism of fruit quality and provide a theoretical basis for fruit quality improvement, but also helps to efficiently regulate the ripening time of tomatoes and other climacteric fruits and manage their shelf life.

[0003] Currently, tomato fruits are harvested after the initiation of ripening (green mature stage), and usually ethylene treatment is used to promote their ripening or substances such as 1-MCP are used to delay their ripening to manage their shelf life. However, once tomato fruits initiate ripening, their ripening and senescence process is difficult to inhibit, and the controllable range is very small. For the initiation of tomato fruit ripening, systemic II ethylene synthesis is crucial (Chirinos X, Ying S, Rodrigues MA, Maza E, Djari A, Hu G, Liu M, Purgatto E, Fournier S, Regad F, Bouzayen M, Pirrello J. Transition to ripening in tomato requires hormone-controlled genetic reprogramming initiated in gel tissue. Plant Physiol, 2023, 191: 610 - 625.), which enables green mature fruits to ripen normally both in vitro and on the tree. However, the ethylene synthesis of immature tomato fruits is controlled by systemic I, so they cannot initiate ripening normally in vitro. If tomato fruits can be harvested when they are immature and ripened before the shelf life, their shelf life can be efficiently regulated. However, no substance that can promote the initiation of ripening of isolated immature tomato fruits has been found yet. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of histidine.

[0005] The new use provided by the present invention for histidine is the application of histidine in promoting the initiation of fruit ripening and quality formation.

[0006] Specifically, the application is that histidine is used to promote the initiation of ripening and color change of immature fruits in vitro.

[0007] In the said application, the fruit can be a climacteric fruit, specifically it can be a tomato.

[0008] The present invention also provides a method for promoting the initiation of fruit ripening and quality formation, including: irrigating and treating plants with an aqueous solution containing histidine (His) to promote the initiation of fruit ripening, color change and softening;

[0009] Or, injecting and treating immature fruits after picking with a MES buffer solution containing His to promote the initiation of fruit ripening and turning red through the ethylene signal;

[0010] The fruit can be a climacteric fruit, specifically it can be a tomato;

[0011] Specifically, irrigate with an aqueous solution containing histidine once at 10 DPA and 15 DPA of the plants;

[0012] Specifically, the immature fruit in vitro is the immature fruit at 26 DPA;

[0013] The concentration of His in the aqueous solution and the MES buffer solution is 1 - 150 μmol / L -1 Specifically, it can be 10 - 150 μmol / L -1 More specifically, it can be 100 μmol / L -1 .

[0014] The experimental results of the present invention show that treating tomato plants or immature tomato fruits in vitro with a His aqueous solution or a MES buffer solution at a specific concentration can promote the initiation of tomato fruit ripening, color change and softening; Treating tomato plants with His, an activator 3-(2-Thienyl)-L-alanine (TIH) of the rate-limiting enzyme (ATP phosphoribosyl transferase, ATP-PRT) in the His biosynthetic pathway, and adenosine triphosphate (ATP), the substrate of ATP-PRT, can all promote the ripening color change and softening of fruits; Treating with His and the substrate ATP of ATP-PRT can both promote the initiation of ripening, turning red and seed maturation of immature tomato fruits in vitro, and the effect of His will be significantly inhibited by the ethylene receptor inhibitor 1-MCP, which indicates that His can promote the transformation of ethylene synthesis in immature tomato fruits from system I to system II and plays a role depending on the ethylene signal pathway. Description of the Drawings

[0015] Figure 1 Effects of treating tomato plants with different concentrations of His on the initiation of fruit ripening in Example 1 of the present invention. Among them, (A) is the fruit phenotype at 38 DPA (38 days after anthesis); (B) is the fruit color-breaking time. Note: The scale bar in the figure is 1 cm; the same letter mark indicates no significant difference, and different letter marks indicate significant differences (Duncan method, P < 0.05).

[0016] Figure 2 Determination of related indexes of fruit ripening initiation and quality formation in tomato plants treated with His in Example 2 of the present invention. Among them, (A) is the fruit phenotype at 32 DPA; (B) is the fruit ripening process from 32 to 41 DPA; (C) is the fruit color at 32 DPA; (D) is the single fruit weight at 35 DPA; (E) is the pericarp hardness at 32 DPA; (F) is the number of seeds in a single fruit at 35 DPA; (G) is the dry weight of a single seed at 35 DPA. Note: The scale bar in the figure is 1 cm; the same letter mark indicates no significant difference, and different letter marks indicate significant differences (Duncan method, P < 0.05).

[0017] Figure 3 Effects of ATP and His treatments on the initiation of ripening of detached immature tomato fruits in Example 3 of the present invention. (A) Ripening process map of detached 26 DPA tomato fruits from 0 to 11 d after harvest and (B) population map at 11 d after harvest; (C) Ripening process map of detached 26 DPA tomato fruits from 0 to 12 d after harvest; (D) Color-breaking time of fruits treated with ATP, His, and (E) His, His, and 1-MCP combination treatments. Note: The scale bar in the figure is 1 cm; n.s. indicates no significant difference, and ** indicates P < 0.01 (independent samples T-test). Detailed implementation manners

[0018] The present invention will be further described in detail below in combination with specific implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0019] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0020] In the following examples, ATP (A832633), TIH (T832877), and 1-MCP (M875517) were purchased from Shanghai Macklin Biochemical Co., Ltd., L-His (CH6131) was purchased from Beijing Coolaber Technology Co., Ltd., and ethylene gas was purchased from Beijing Huayuan Gas Co., Ltd. MES buffer (100 mmol L -1 , pH 5.5, BN20389) was purchased from Beijing Bairuiji Biotechnology Co., Ltd.

[0021] Preparation of reagent mother liquors in the following examples:

[0022] ATP (100 mmol L -1 ) mother liquor: Weigh 1.01 g of the drug and dissolve it in 20 mL of distilled water. After filtering and sterilizing with a 0.22-μm sterile filter membrane, it was aliquoted into 1.5-mL sterile centrifuge tubes and stored at -20 °C.

[0023] His (50 mmol L -1 ) mother liquor: Weigh 0.39 g of the drug and dissolve it in 50 mL of distilled water. After filtering and sterilizing with a 0.22-μm sterile filter membrane, it was aliquoted into 5-mL sterile centrifuge tubes and stored at -20 °C.

[0024] TIH (50 mmol L -1 ) mother liquor: Weigh 0.17 g of the drug and dissolve it in 20 mL of distilled water. After filtering and sterilizing with a 0.22-μm sterile filter membrane, it was aliquoted into 1.5-mL sterile centrifuge tubes and stored at -20 °C.

[0025] Example 1. Screening of the concentration of histidine treatment

[0026] Tomato plants were treated by irrigation with 1, 10, 100, 150, and 200 μmol L -1 His at 10 - 15 DPA (irrigation once at 10 DPA and once at 15 DPA), about 100 mL per plant each time, for a total of 2 irrigations. Each treatment group included 12 tomato plants, and tomato plants without His treatment were used as the negative control.

[0027] Figure 1 To study the effect of treating tomato plants with different concentrations of His on the initiation of fruit ripening, where (A) is the fruit phenotype at 38 DPA; (B) is the fruit color-breaking time.

[0028] As can be seen from Figure 1 , 100 μmol L -1 His treatment had the best promoting effect on the initiation of tomato fruit ripening and color change. Therefore, this concentration was selected for subsequent studies.

[0029] Example 2. Effect of treating tomato plants with histidine on the initiation of fruit ripening and quality formation

[0030] 100 μmol L -1 The activator TIH of the rate-limiting enzyme ATP-PRT in the His and His biosynthetic pathways and the substrate ATP of ATP-PRT were used to treat tomato plants at 10-15 DPA (referring to watering once at 10 DPA and 15 DPA), with each plant watered with about 100 mL each time, for a total of 2 waterings. Each treatment group included 12 tomato plants, and tomato plants that were not treated with these metabolites were used as negative controls. The color of the fruit was measured using a colorimeter NR60CP + (Shenzhen Sanenshi Technology Co., Ltd.) The larger the L value, the brighter it is, and the smaller the L value, the darker it is; a positive a value indicates a tendency towards red, and a negative a value indicates a tendency towards green; a positive b value indicates a tendency towards yellow, and a negative b value indicates a tendency towards blue. The determination of fruit hardness was carried out according to the instructions using a fruit hardness tester FT-327 (Italy TR Company).

[0031] Figure 2 The results show that the fruit ripening and quality formation-related indicators of His-treated tomato plants were initiated and determined, including: (A) fruit phenotype at 32 DPA; (B) fruit ripening process at 32-41 DPA; (C) fruit color at 32 DPA; (D) single fruit weight at 35 DPA; (E) fruit skin hardness at 32 DPA; (F) number of seeds in a single fruit at 35 DPA; and (G) dry weight of a single seed at 35 DPA.

[0032] Depend on Figure 2 It can be seen that His treatment, ATP-PRT activator TIH treatment, and ATP-PRT substrate ATP treatment all significantly advanced the onset of tomato fruit ripening, promoted the color change and softening of tomato fruits, and His treatment could also promote seed production.

[0033] Example 3: Effect of histidine treatment on the initiation of ripening of isolated immature tomato fruits

[0034] Unripe tomato fruits at 26 DPA were harvested and placed in plastic baskets for 4 h. About 200 μL of 100 μmol L -1 His or ATP-PRT substrate ATP in MES buffer (10 mmol L -1 The fruit injected with MES buffer was used as negative control, with 9 fruits in each group. Plastic wrap was covered on the plastic basket to keep it moist and placed in a constant light room (25°C, 16h light / day). The above operation was repeated 3 days later, and the fruit ripening time was observed and recorded every day.

[0035] Harvest immature tomato fruits at 26 DPA and place them in plastic baskets. Let them stand for 4 h. Ethylene treatment group: After injecting about 200 μL of MES buffer (10 mmol L -1 pH 5.5) above the fruit stigma with a 1 mL sterile syringe, place them in a sealed plastic box and treat them with 50 ppm ethylene for 4 h; His treatment group: After injecting about 200 μL of MES buffer containing 100 μmol L -1 His above the fruit stigma with a 1 mL sterile syringe, place them in a sealed plastic box and let them stand for 4 h; His + 1-MCP treatment group: After injecting about 200 μL of MES buffer containing 100 μmol L -1 His above the fruit stigma with a 1 mL sterile syringe, place them in a sealed plastic box and treat them with 20 ppm 1-MCP for 12 h; Negative control group: After injecting about 200 μL of MES buffer above the fruit stigma with a 1 mL sterile syringe, place them in a sealed plastic box and let them stand for 12 h. There are 9 fruits in each group. Cover the plastic basket with plastic wrap to keep it moist and place it in a constant light greenhouse (25 °C, 16 h light / day). Repeat the above operation after 3 days and observe and record the fruit ripening time every day.

[0036] Figure 3 Effects of ATP and His treatments on the initiation of ripening of isolated immature tomato fruits. (A) Ripening process of isolated 26 DPA tomato fruits from 0 - 11 d after harvest and (B) population map at 11 d after harvest; (C) Ripening process of isolated 26 DPA tomato fruits from 0 - 12 d after harvest; (D) Color break time of fruits treated with ATP, His, and (E) His, His and 1-MCP combination treatments.

[0037] It can be seen from Figure 3 that immature tomato fruits cannot initiate ripening normally under in vitro conditions, and ethylene treatment cannot promote the initiation of their ripening either. This indicates that the ethylene synthesis of the fruits at this time is controlled by system I. The reason why it cannot initiate ripening is not only that the ethylene release amount is small, but also the ethylene signal transduction and the response of downstream ripening genes are inhibited. However, both His treatment and ATP-PRT substrate ATP treatment can promote the initiation of ripening, turning red and seed maturation of isolated immature tomato fruits, and the effect of His will be significantly inhibited by the ethylene receptor inhibitor 1-MCP. This shows that His can promote the transformation of ethylene synthesis in immature tomato fruits from system I to system II and plays a role depending on the ethylene signal pathway.

[0038] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that further improvements can be made to the present invention. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of histidine in promoting the initiation of fruit ripening and quality formation.

2. The use according to claim 1, wherein, the use is that histidine is used to promote the initiation of ripening and color change of immature fruits in vitro.

3. The use according to claim 1 or 2, wherein, the fruit is a climacteric fruit.

4. The use according to claim 3, wherein, the fruit is tomato.

5. A method for promoting the initiation of fruit ripening and quality formation, comprising: irrigating and treating plants with an aqueous solution containing histidine to promote the initiation of fruit ripening, color change and softening.

6. A method for promoting the initiation of fruit ripening and quality formation, comprising: treating harvested immature fruits with MES buffer solution containing histidine to promote the initiation of fruit ripening and red color change through ethylene signal.

7. The method according to claim 5 or 6, wherein, the fruit is a climacteric fruit, specifically tomato; specifically, irrigate with an aqueous solution containing histidine once at 10 DPA and 15 DPA of the plant; specifically, the immature fruit in vitro is the immature fruit at 26 DPA.

8. The method according to claim 5 or 6, wherein, The concentration of histidine in the aqueous solution and MES buffer is 1 - 150 μmol L -1 .

9. The method according to claim 5 or 6, wherein, The concentration of histidine in the aqueous solution and MES buffer is 10 - 150 μmol / L -1 .

10. The method according to claim 5 or 6, wherein, The concentration of histidine in the aqueous solution and MES buffer is 100 μmol L -1 .

Images