Overexpression of Avocado (Persea americana Mill.) PaRAP2.1 Promotes Fatty Acid Accumulation in Arabidopsis thaliana

Fatty acids in avocado fruit (Persea americana Mill.) are vital composition affecting flavour and nutritive value. Hence, horticulturalists are interested in illustrating the functions of transcription factors on fatty acid accumulation in avocado fruit. In the present study, the APETALA2/ethylene-responsive transcription factor gene, PaRAP2.1, was cloned from avocado mesocarp, and the subcellular localization demonstrated that PaRAP2.1 was located in the cytoplasm and nucleus. The PaRAP2.1 was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation. Furthermore, PaRAP2.1 were functionally verified its effect on fatty acid biosynthesis. Histological analyses of lipid droplets displayed that the striking difference in the lipid droplets in the mature seeds between PaRAP2.1-overexpressing transgenic and wild-type Arabidopsis thaliana lines were revealed based on confocal microscopy images. Subsequently, fatty acid analyses of PaRAP2.1-overexpressing Arabidopsis thaliana lines displayed the significantly higher contents of fatty acids than those in the wild-type plants. Meanwhile, expression amount of ten genes involving in fatty acid biosynthesis dramatically up-regulated in the mature seeds of PaRAP2.1-overexpressing lines than those of wild-type plants. These results provide a theoretical basis for future research in regard to the function of PaRAP2.1 on fatty acid biosynthesis.


Introduction
Fatty acids are important components in plant (Ge et al., 2017(Ge et al., , 2018. The fatty acid biosynthesis has been studied extensively, and these have expounded transcription factors that regulate fatty aicd biosynthesis in plants (Ge et al., 2021a(Ge et al., , 2021b. Members of many transcription factor families involving APETALA2/ethylene responsive factor (AP2/ERF) superfamily have been found to be involved in regulation in fatty acid biosynthesis in plants (Yeap et al., 2017). The AP2/ERF transcription factors are a multifarious superfamily expressed in plants, and AP2/ERF members have the conserved DNA binding domain, namely the AP2 domain, that binds to the gene's promoter region to regulate expression (Zhang et al., 2020). They are classified into three separate groups: ERF, RAV, and AP2 families according to the repeat number in AP2 domain (Zhang & Li, 2018). Currently, with studies in dicotyledonous plants such as Ricinus communis (Xu et al., 2013), Ziziphus jujuba (Zhang & Li, 2018), Arabidopsis thaliana (Xie et al., 2019), Dimocarpus longan (Zhang et al., 2020), monocotyledonous plants such as Phyllostachys edulis (Wu et al., 2015), and gymnosperm such as Taxus chinensis (Zhang et al., 2019), we present a more in-depth knowledge of the functions and classification of AP2/ERF members.
In our previous study, the 137 PaAP2/ERF genes were identified in avocado, and then the expression patterns of them in five developmental stages of avocado mesocarp were presented according to transcriptome data (Ge et al., 2021a). Subsequently, two PaAP2/ERF genes (PaWRI2 and PaWRI1) belonging to AP2 subfamily and eight PaAP2/ERF genes (PaRAP2.1,PaERF023, belonging to ERF subfamily were highly transcribed during five developmental stages of avocado mesocarp, which might regulate the accumulation of fatty acids in the avocado mesocarp (Ge et al., 2021a). Furthermore, the PaWRI1, a AP2 subfamily member, was selected to carry out the transgenic functional analysis, and the result implied that PaWRI1 might contribute to fatty acid accumulation (Ge et al., 2021a). Similarly, most of the genes governing fatty acid synthesis are found to be regulated by WRI1 in many plants (Kong & Ma, 2019).
However, neither one of PaAP2/ERF genes belonging to ERF subfamily has been found to modulate the vital genes participating in the fatty acid biosynthesis until now. In our previous study, the eight PaAP2/ERF genes belonging to ERF subfamily were considered to take part in fatty acid biosynthesis in avocado mesocarp, and the PaRAP2.1 was more abundantly transcribed than other sever genes (Ge et al., 2021a). Therefore, in this study, we first chose PaRAP2.1, and performed the cloning and subcellular localization of PaRAP2.1. Second, to further exploit potential function of PaRAP2.1 on fatty acid accumulation, PaRAP2.1-overexpressing transgenic A. thaliana were developed, after which gene expression, lipid droplet observation, and targeted fatty acids detection of the transgenic A. thaliana and wild-type (WT) lines were carried out to analyse the contents of fatty acids. The data enriches our understanding in regard to the functions of PaRAP2.1 on fatty acid biosynthesis in the avocado mesocarp.

Plant Materials and Growth Conditions
Avocado fruits (cultivar 'Hass') were collected from six 10-year-old trees in September 2018 at the Chinese Academy of Tropical Agricultural Sciences. Arabidopsis thaliana wild-type Col-0 seeds were disinfected surfaces with 70% ethanol for 30 s and 15% sodium hypochlorite for 15 min, and then rinsed with distilled water three times for 20 s. Then, the seeds were removed moisture from the surface, and placed on Murashige and Skoog medium.

RNA Extraction and cDNA Synthesis
The total RNA was extracted from avocado mesocarps and A. thaliana seeds. The mRNA was extracted from total RNA using poly-T oligo-attached magnetic beads. The first-strand cDNA was synthesized based on the sequence of the extracted RNA. The concentration of cDNA was diluted to 12.5 ng/µL.

Construction of PaRAP2.1 Transient Expression Vector and Subcellular Localization
The vector plasmids sequenced correctly were transformed into Agrobacterium, spread on the plates including 25 mg/L kanamycin and 25 mg/L rifamycin. The monoclonal shaking bacteria were selected to grow overnight, the bacterial solutions were collected, and then resuspended in infiltration medium. Agrobacterium solutions containing vectors were blended in proportion. The liquid mixtures were transfused into leaves of tobacco for 28 days. After 3 days, the leaves of tobacco were scanned through confocal scanning microscope.

Vector Construction and Plant Transformations
To generate the PaRAP2.1-overexpressing (OE) construct, the full-length PaRAP2.1 CDSs was amplified and transferred into the pCAMBIA1300 vector including the 35S promoter. We introduced the recombinant plasmids into Agrobacterium tumefaciens (GV3101). The floral dip method was used for genetic transformation of wild-type A. thaliana. Hygromycin-resistant plants were screened from transformed seeds, and then the T 1 generation were obtained. T 1 seeds were sown, and finally T 3 transgenic A. thaliana plants were obtained.

Quantitative Real Time PCR of Gene Expression
The eight genes participating in fatty acid biosynthesis expressed in the seeds of the WT and PaRAP2.1-OE A. thaliana lines were chosen for qRT-PCR, and AtActin7 was used as an endogenous control for normalizing data ( Table 1). The qRT-PCR amplification process was described by Ge et al. (2019). Relative gene expression levels were calculated with the 2 −∆∆Ct method (Livak & Schmittgen, 2001). For each sample, the qRT-PCR analysis was completed with three biological replicates and two technical replicates.

Analysis of Fatty Acid Compositions by Gas Chromatography-Mass Spectrometry
The fatty acid compositions of the A. thaliana seeds from the WT and PaRAP2.1-OE plants were determined by gas chromatography-mass spectrometry (GC-MS) as described by Ge et al. (2019). The oils extracted from the seeds of the WT and PaRAP2.1-OE (20 L) were saponified at 80 °C (30 min). After cooling, the solutions were mingled with 3 mL BF 3 -MeOH (14%) and incubated at 75 °C (30 min) to generate fatty acid methyl esters (FAMEs). The analyses were performed through an Agilent 7890B-7000B GC-MS with a DB-5MS column.The FAMEs were identified by comparing the retention times of the peaks with those of commercial standards and comparing the respective ion chromatograms with those in the NIST 2011 library. Methyl nonadecanoate was added as an internal standard and the FAMEs were quantified based on the calibration curves for the standards (R 2 ≥ 0.995). The FAME contents (mg/100 g fresh weight) are herein presented as the mean ± standard deviation of three biological replicates, each with two technical replicates.

Histological Analyses
To visualize the lipid droplets in the mature seeds from the WT and PaRAP2.1-OE A. thaliana plants, the method of sample handling and lipid droplet observation was described by Ge et al. (2019).

Cloning and Subcellular Localization Analysis of PaRAP2.1
Using the melon cDNA as a template, the fragment of PaRAP2.1 was amplified and analyzed. Band 1 was about 1131 bp according to the DNA marker (DL2000) (Figure 1), which was consistent with the anticipative result of the present study. Through laser scanning microscopy, it was observed that PaRAP2.1 was located in the nucleus and cytoplasm of tobacco leaves. However, as shown in Figure 2, the green fluorescent signal of PaRAP2.1-GFP was mainly concentrated on the plasma membrane. These results indicated that the PaRAP2.1 might be a transcription factor that played a role in the nucleus and cytoplasm. However, subcellular localization shows that BnWRI1, the same AP2/ERF transcription factor, is only distributed in the nucleus (Wu et al., 2014;Li et al., 2015). jas.ccsenet.

The Eff
The leaves fragment w controls, i phenotypic stage ( Fig  PaWRI1-

Conclusion
The functional analyses of transgenic A. thaliana lines overexpressing PaRAP2.1 illustrated the effects of the encoded transcription factors on fatty acid biosynthesis. The data suggested that PaRAP2.1 might be conducive to fatty acid biosynthesis. The results described herein may help to demonstrate the involvement of PaRAP2.1 in the fatty acid biosynthetic pathways in plants. The produced data offer worthy clues in regard to the biological functions of AP2/ERF transcription factors in plants.