Molecular cloning and functional characterization in response to saline-alkali stress of the MhZEP gene in Arabidopsis thaliana.

Soil salinization is one of the major environmental factors that restrict plant growth and development. Zeaxanthin epoxidase (ZEP) functions in ABA biosynthesis and the xanthophyll cycle and has a vital role in plant responses to various environmental stresses. It was found by quantitative real-time PCR (qRT-PCR) that MhZEP responded to saline-alkali stress and showed the highest expression at 48 h of saline-alkali stress, which was 14.53-fold of 0 h. The MhZEP gene was cloned from the apple rootstock begonia (Malus halliana Koehne) and its protein physicochemical properties were analyzed. Subsequently, the functional characterization of MhZEP (ID: 103403091) was further investigated in Arabidopsis thaliana. The MhZEP contained a complete open reading frame with a length of 1998 bp, and encoded 665 amino acids with an isoelectric point of 7.18. Phylogenetic tree analysis showed that MhZEP was the most homologous and closely related to Glycine max. Compared with wild-type, transgenic plants grew better under saline-alkali stress and the MhZEP-OE line showed higher chlorophyll content, carotenoid content, enzyme activities (POD, SOD, CAT and APX) and K+ content, whereas they had lower chlorosis and Na+ content than the wild type (WT), which indicated that they had strong resistance to stress. The expression levels of saline-alkali stress-related genes in A. thaliana MhZEP-OE were examined by qRT-PCR, and it was found that the MhZEP improved the tolerance of A. thaliana to saline-alkali stress tolerance by regulating the expression of carotenoid synthesis genes (MhPSY, MhZDS, MhLYCB and MhVDE) and ABA biosynthesis genes (MhNCED5, MhABI1 and MhCYP707A2). And the potassium-sodium ratio in the cytoplasm was increased to maintain ionic homeostasis by modulating the expression of Na+ transporter genes (MhCHX15 and MhSOS1) and K+ transporter genes (MhHKT1;1, MhNHX1 and MhSKOR1). Moreover, the expression of H+-ATPase genes (MhAHA2 and MhAHA8) was increased to reduce the oxidative damage caused by saline-alkali stress. In summary, MhZEP acted as an essential role in plant resistance to saline-alkali stress, which lays the foundation for further studies on its function in apple.

Transcriptomic analysis reveals GA3 is involved in regulating flavonoid metabolism in grape development for facility cultivation.

Gibberellin, as one of the pivotal plant growth regulators, can improve fruit quality by altering fruit size and secondary metabolite content. Flavonoids are the most abundant secondary metabolites in grapes, which influence the color and quality of the fruit. However, the molecular mechanism of whether and how GA3 affects flavonoid metabolism has not been reported, especially for the 'Red globe' grape with delayed cultivation in Hexi corridor. In the present study, the 'Red globe' grape grown in delayed facilities was sprayed with 20, 40, 60, 80 and 100 mg/L GA3 at berries pea size (BPS), veraison (V) and berries ripe (BR), respectively. The results showed that the berry weight, soluble sugar content and secondary metabolite content (the flavonoid content, anthocyanin content and polyphenol content) at BR under 80 mg/L GA3 treatment were remarkably increased compared with other concentration treatments. Therefore, RNA sequencing (RNA-seq) was used to analyze the differentially expressed genes (DEGS) and pathways under 80 mg/L GA3 treatment at three periods. GO analysis showed that DEGs were closely related to transporter activity, cofactor binding, photosynthetic membrane, thylakoid, ribosome biogenesis and other items. The KEGG enrichment analysis found that the DEGs were mainly involved in flavonoid biosynthesis and phenylpropanoid biosynthesis, indicating GA3 exerted an impact on the color and quality of berries through these pathways. In conclusion, GA3 significantly increased the expression of genes related to flavonoid synthesis, enhanced the production of secondary metabolites, and improved fruit quality. In addition, these findings can provide a theoretical basis for GA3 to modulate the accumulation and metabolism of flavonoids in grape fruit.

Functional Identification of MhPYL4 Involved in Iron-Deficiency Stress in Malus Halliana Koehne.

The PYL protein family are crucial sensors of the core signals of abscisic acid (ABA) and significantly influence the plant's response to ABA-mediated abiotic stresses as well as its growth and development. However, research on the role of the MhPYL4 gene in iron (Fe) deficiency in apple trees is limited. Studies have shown that the MhPYL4 gene, when exposed to Fe-deficiency stress, exhibits more rapid transcriptional upregulation than other genes' quickly elevated transcription. However, the precise mechanism by which it alleviates this stress remains unclear. The MhPYL4 gene (ID:103432868), isolated from Malus halliana, was analyzed to elucidate its function. Arabidopsis plants engineered to overexpress the MhPYL4 gene exhibited increased leaf chlorosis and slower growth in response to Fe stress compared to the unmodified controls. The transgenic plants also exhibited elevated levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, as well as ferric chelate reductase (FCR) activities. Levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2-) were increased. In addition, these transgenic plants had lower concentrations of proline (Pro) and Fe2+, which indicated that their stress tolerance was reduced. Similarly, the overexpression of MhPYL4 in apple calli resulted in inhibited growth and increased susceptibility under Fe stress conditions. Physiological evaluations indicated that the overexpression of MhPYL4 in Arabidopsis reduced its Fe stress tolerance by inhibiting chlorophyll synthesis. In apple calli, it altered pH levels, antioxidant enzyme activity, and Fe-reducing capabilities under the same stress conditions. In summary, the elevated expression of the MhPYL4 gene reduced the tolerance of both Arabidopsis and apple calli to Fe stress, suggesting that MhPYL4 acts as a negative regulator in response to Fe deficiency.