Transcriptome Comparison of Strawberry Ripen Fruit Harvested/Collected from Plants Grown under Three Different Temperature Treatments

Special Article: Fruit Science and Viticulture

Ann Agric Crop Sci. 2023; 8(3): 1137.

Transcriptome Comparison of Strawberry Ripen Fruit Harvested/Collected from Plants Grown under Three Different Temperature Treatments

Bao YX²; Jia YD²; Liu XJ²; Zhu YR²; Tong YN¹*; Wang Y²*; Liu LL¹; Yang LF¹; Zuo ZJ³

1Tianjin Academy of Agricultural Sciences, Tianjin 300192, China

2Nankai university, Tianjin 300350, China

3Zhejiang Provincial Key Laboratory of Forest Aromatic Plants-based Healthcare Functions, Zhejiang A&F University, Hangzhou 311300, China

*Corresponding author: Tong YN Tianjin Academy of Agricultural Sciences, No.268 Baidi Road, Nankai District, Tianjin 300192, China. Tel: +86 13802002722; Fax: +86022-82939032 Wang Y, Nankai University, No.94 Weijin Road, Nankai District, Tianjin 300350, China. Tel: +86 13821811751 Email: tongyana03@163.com; wangyong@nankai.edu.cn

Received: June 13, 2023 Accepted: July 13, 2023 Published: July 20, 2023

Abstract

In this study, the runner plants of strawberry cv. Ssanta was used as the material, and three different daily temperature combinations were designed, which were CK (22°C/22°C), DHT (day high temperature, 30°C/22°C) and NLT (night low temperature, 22°C/8°C), respectively. The results showed that DHT accelerated fruit ripening process, increased the single fruit weight and the horizontal and vertical diameter, increased the sucrose, SSC content of the fruit, reduced the TA content, and improved the fruit quality. The results of qRT-PCR showed that after DHT, the expression of sucrose metabolism related genes (Fa SPS, Fa SUT2 and Fa SUT3) was significantly increased, but the expression of citrate synthesis related genes (Fa PDHE, Fa PEPC, Fa PK, Fa CS) was not affected, while NLT treatment showed the opposite trend. In order to further understand the effect of temperature on gene expression in strawberry fruit, transcriptome sequencing and differential expression analysis were carried out. The results showed that temperature treatment had an effect on multiple metabolic pathways in the fruit, including TCA cycle, starch and sucrose metabolism, fatty acid metabolism, amino acid biosynthesis, etc.

Keywords: Cultivate strawberry; Temperature control; Sugar acid metabolism; Transcriptome analysis

Abbreviations: CK: Suitable for Plant Growth; DHT: Day High Temperature; NLT: Night Low Temperature; SSC: Soluble Solids Content; TA: Titratable Acidity; DEGs: Differentially Expressed Genes; CS: Citrate Synthase; PEPC: Phosphoenolpyruvate Carboxylase; Aco: Acetyl-CoA; PDHE: Pyruvate Dehydrogenase

Introduction

Strawberry (Fragaria × ananassa Duch.) is cultivated and consumed worldwide. Most commercially important cultivars of strawberry are octaploids (2n=8x=56) [1] and there are two flowering types: one is an annual Short Day and Low Temperature-type (SDLT) (seasonal strawberry), and the other is the perpetual (everbearing) day-neutral or Long Day and High Temperature-type (LDHT) [2]. The SDLT type is also known as single-cropping or June-bearing type [3]. Because of a pronounced interaction of photoperiod and temperature, floral initiation also takes place in most SDLT cultivars under long day conditions if the temperature is low. Therefore, temperature is considered to be one of the most important factors that influence the strawberry production and fruit quality [4,5,22].

For quality fruit production, strawberry requires cool and moist climate [6] with 25/12° C (day/night) optimum temperature [7]. However, it is also reported that a DIF (difference between daytime temperature and night temperature) of 12°C (31°C/19°C, day/night temperature) was most suitable for the growth, especially for the sugar content accumulation of strawberry cultivar Hong Yan [22]. The importance of DIF has been emphasized also for other fruit production [8,9].

In the present study, we grew strawberry plants after blooming at continuous 22°C (without a DIF) as the reference (CK), and compared with those grown at a DIF of 8°C (30°C/22°C, day/night temperature, as DHT treatment) and with those grown at a DIF of 14°C (22°C/8°C, day/night temperature, as NLT treatment). After measurement of the fruit phenotype, sugar/acid ratio and sucrose content, we detected the expression of genes related to sucrose and titrate metabolism, and then we performed a transcriptome analysis and comparison, in order to explore genes involved in the influence of strawberry fruit quality, which could contribute to the further elucidation of mechanisms under which the fruit quality control by hinger or lower temperature or by temperature difference between daytime and night.

Materials and Methods

Plant Growth and Temperature Treatments

Runner plants of strawberry cv.Ssanta were grown in plastic pots (1.65 L) filled with commercial potting media (Peat: vermiculite: perlite 1:1:1) and kept under ambient environmental conditions in a glasshouse until the appearance of the initial flowers. Irrigation and supplement of liquid fertilizer were regularly carried out during the growing period.

90 healthy plants with uniform size were taken and divided into three groups for the temperature treatments in the condition-controlled growth chambers, which have a photoperiod of 16 hours dark and 8 hours light. Three different daily temperature combinations were designed as CK (22°C/22°C), which was suitable for plant growth, DHT (day high temperature, 30°C/22°C) and NLT (night low temperature, 22°C/8°C), respectively. The plants were irrigated and fertilized regularly to maintain their healthy growth.

The temperature treatments were continued for around 8 weeks. Ripen fruits were harvested on the 30th day of the experiment and every 5 days afterwards and stored at 4°C or at -80°C after deep frizzed in liquid N2.

The 45d harvested fruits were then analyzed for their physical properties, fruit quality parameters and extraction of RNA for RT-PCR essay and transcriptome analysis.

Each laboratory analytical test (described below) was performed with at least three biologic repeats.

Fruit Physical Property and Quality Parameter Determination

The weight of each sample was measured by using an electronic balance with an accuracy of 0.01g. The size of the fruits was measured with the Vernier Caliper. After the fruit was picked, ground into juice, centrifuged, and 300μL supernatant was taken for determination. The SSC content of each fruit was measured three times and the average value was the SSC content of the fruit. The original juice was diluted 50 times, and 300μL was taken for determination. The average value was TA content after repeated determination for three times. Soluble Solids Content (SSC) and content of Titratable Acidity (TA) were determined by using a refractometer (ATAGO, Ai Tan, Japan, PAL-1).

Measurement of Sucrose

Sucrose content of the fresh ripen strawberry fruits was determined by using the testing kit from Plant Sucrose Content Assay Kit (Solarbio, BC2465). The process was performed according to the user´s instruction.

RNA Extraction and Purification

Total RNA was extracted by using the Rapid RNA Extracting Kit from. Fresh harvested ripen fruits were sliced and submerged in liquid N2, then crashed into powder and extracted in the lysing solution containing protease K. Contaminated DNA was removed by passing a specific little column and purified RNA was deposited in alcohol and finally dissolved in RNase-free water. RNA samples were used directly or stored in the fridge, after quality and quantity check.

Real time RT-PCR

Purified total RNA was used to conduct the quantitative analysis of gene expression by using TB Green Premix Ex Taq, with actin gene as the reference. The primer pares designed by using Primer 5 and used in the present study are listed in Table 1.