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Project Article | Xuzhou Academy of Agricultural Sciences Reveals Significant Effects of Long-Term Fixed Fertilization on Soil Properties and Sweet Potato Yield

Release time:

2025-02-13

Xinjiang wild apple (Malus sieversii) is an establishment species of wild fruit forests in the Tianshan Mountains and is also a key species in international biodiversity conservation. Due to its retention of the original apple genes, it serves as a genetic resource for the future genetic improvement and artificial breeding of cultivated apple varieties. Seed germination and dormancy, as important mechanisms for plant growth and reproduction, require a deep understanding of the role of abscisic acid (ABA) receptors in seed dormancy and germination in Malus species, providing a theoretical foundation for cultivating apple varieties with specific dormancy traits.

Recently, Associate Professor Ye Chunxiu from Xinjiang Agricultural University published a research paper titled Construction of a Membrane Yeast Two-Hybrid Library and Screening of MsPYR1-Like Interacting Proteins in Malus sieversii in the journal Molecular Biotechnology, revealing the potential role of MsPYR1-like in seed dormancy and germination in Malus species.

Nanjing Pronet Biotech provided plant hormone testing services for this research!

Research Overview

To explore the biological effects of the ABA receptor MsPYR1-like in Xinjiang wild apple seeds, particularly its role in seed dormancy and germination, the pyrabactin resistance 1-like (PYR1-like) gene, associated with ABA receptors, was cloned from wild apple (Malus sieversii) seeds. Bioinformatics analysis of the 206 amino acid protein encoded by this gene revealed a typical domain of the PYR/PYL/RCAR family. The analysis indicated that MsPYR1-like shares high sequence similarity with PYR1-like proteins in apples and other plants, suggesting that this family is highly conserved across species.

Figure 1: Bioinformatics analysis of the Malus MsPYR1-like gene. Multiple sequence alignment of MsPYR1-like amino acid sequences.

Physicochemical properties of the MsPYR1-like protein were analyzed, and its secondary and tertiary structures were predicted using bioinformatics tools. The conserved domain of the MsPYR1-like protein includes a hydrophobic ligand-binding site, characteristic of the PYR/PYL/RCAR protein family. The protein has 33 potential phosphorylation sites, including 14 serine, 15 threonine, and 4 tyrosine sites. The secondary structure of the MsPYR1-like protein mainly consists of α-helix (37.38%), extended strands (15.05%), β-turns (6.80%), and random coils (40.78%), with the random coils dominating, suggesting the structure may have instability. A comparison with the tertiary structure of tomato PYR1-like (SlPYL1) revealed 76.04% sequence identity and a GMQE score of 0.84, indicating that the two proteins may have similar biological functions.

Figure 2: Gene sequence analysis of MsPYR1-like and prediction of the conserved domain, secondary and tertiary structures, and phosphorylation sites of the protein.

The MsPYR1-like gene was cloned into an expression vector tagged with green fluorescent protein (GFP) for subcellular localization analysis in tobacco leaves via Agrobacterium-mediated transient transformation. The MsPYR1-like protein was observed in the nucleus and cell membrane, which is different from the subcellular localization of proteins such as AtPYR1, suggesting that MsPYR1-like may have unique functions.

Figure 3: Subcellular localization of the MsPYR1-like protein observed in tobacco leaves via Agrobacterium-mediated transient transformation.

Long-distance PCR (LD-PCR) technology was used to amplify the first-strand cDNA into a three-frame double-stranded cDNA, followed by homogenization and fragment screening. The double-stranded cDNA was recombined with the pPR3-N vector to construct a yeast membrane two-hybrid library, and the library’s capacity, average insert length, and recombination rate were evaluated. The library quality and applicability were ensured, laying the foundation for subsequent yeast two-hybrid screening experiments.

Figure 4: Construction of the MsPYR1-like yeast membrane two-hybrid library.

The bait vector pBT3-SUC-PYR1 containing the MsPYR1-like gene was co-transformed into yeast strains with the library vector. A total of 28 candidate proteins interacting with MsPYR1-like were identified from the library. These interacting proteins are functional components of the ABA signaling pathway, such as PP2C, revealing the complex regulatory mechanisms of MsPYR1-like in ABA signaling. A comparison with functionally similar genes in other plants,such as strawberry FaPYR1 and Arabidopsis PYL11/RCAR5, revealed both the common and unique features of the MsPYR1-like gene, providing important insights into its role in Xinjiang wild apple.

Figure 5: Screening of proteins interacting with MsPYR1-like using the pPR3-N yeast membrane two-hybrid (MYTH) library.

The MsPYR1-like gene was cloned into an overexpression vector and transformed into Arabidopsis via Agrobacterium-mediated transformation to analyze its effect on seed germination. Seed germination was observed to be inhibited, suggesting that MsPYR1-like may play a negative regulatory role in seed germination. This further supports the role of MsPYR1-like in regulating seed dormancy and germination.

Figure 6: Phenotypic analysis of seed germination in Arabidopsis overexpressing MsPYR1-like and its interacting genes in the Xinjiang wild apple transcriptome.

GO functional enrichment analysis of MsPYR1-like interacting genes revealed that these genes are related to the structure and function of ribosomes, the composition of cytoplasmic small ribosomal subunits, and protein-nucleic acid complexes, participating in cytoplasmic translation and ribonucleoprotein complex biosynthesis. These genes also respond to abscisic acid and are involved in the negative regulation of seed maturation, germination, and seedling development. KEGG pathway analysis indicated that these genes are involved in translation, genetic information processing, ribosomal function, oxidative phosphorylation, and molecular chaperone and protein folding, sorting, and degradation pathways. Using the STRING database and Cytoscape software, the protein-protein interaction network of MsPYR1-like was predicted, revealing its potential role in ABA signaling.

Figure 7: KEGG pathway analysis of MsPYR1-like interacting genes.

Figure 8: Protein-protein interaction network of MsPYR1-like with other proteins.

Research Findings

In conclusion, this article provides new insights into the role of MsPYR1-like in seed dormancy and germination in Malus species and lays the foundation for further studies on ABA signaling pathways. These findings help to reveal the molecular mechanisms underlying seed dormancy release and have potential applications in breeding apple varieties with specific dormancy traits.

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