ABIOTIC STRESS GENE 1 mediates aroma volatiles accumulation by activating MdLOX1a in apple

Fruit aroma is an important organoleptic quality, which influences consumer preference and market competitiveness. Aroma compound synthesis pathways in plants have been widely identified of which the lipoxygenase pathway is crucial for fatty acid catabolism to form esters in apple. However, the regulatory mechanism of this pathway remains elusive. In this study, linear regression analysis and transgene verification revealed that the lipoxygenase MdLOX1a participates in ester biosynthesis. Yeast one-hybrid library screening indicated that a novel abiotic stress gene, MdASG1 (ABIOTIC STRESS GENE 1), was a positive regulator of the MdLOX1a promoter and ester production based on yeast one-hybrid and dual-luciferase assays, and correlation analysis among eight apple cultivars. Overexpression of MdASG1 in apple and tomato stimulated the lipoxygenase pathway and increased the fatty acid-derived volatile content, whereas the latter was decreased by MdASG1 silencing. Furthermore, MdASG1 overexpression enhanced the salt-stress tolerance of tomato and apple ‘Orin’ calli accompanied by a higher content of fatty acid-derived volatiles compared with that of non-stressed transgenic tomato fruit. Collectively, these findings indicate that MdASG1 activates MdLOX1a expression and participates in the lipoxygenase pathway, subsequently increasing the accumulation of aroma compounds especially under moderate salt stress treatment. The results also provide insight into the regulation of aroma production, and the potential strategy of prudent development and utilization of saline-alkali land to produce high-quality fruit, thereby reducing pressure on arable land and ensuring national food security. One-sentence Summary MdASG1 directly activates MdLOX1a expression to promote aroma volatiles accumulation especially under moderate salt stress.


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Plant volatile organic compounds are secondary metabolites that play important roles 43 in biotic and abiotic stress responses, and act as signals to attract or repel pests, confer 44 resistance to pathogens, and participate in seed dispersal (Rodriguez et al., 2013). Many  (Dimick and Hoskin, 1983;Song, 2007). Twenty types of volatile compounds are 125 characteristic of the apple fruit aroma, which include trans-2-hexenal, hexanol, butyl acetate, hexyl acetate, and 2-methyl butyl acetate (Dixon and Hewett, 2000). With 127 ripening of the fruit, the abundance of esters increases significantly (Rowan et al., 1999; 128 Echeverr´a et al., 2004). In 'Golden Delicious' apple, esters account for 80% of the 129 total volatile aroma components (López et al., 2010). In 'Golden Delicious', 23 130 functional LOXs have been identified of which MdLOX1a and MdLOX5e might be 131 involved in volatile component production (Vogt et al., 2013). LOX genes play a crucial 132 role in the lipoxygenase pathway. However, little information is available on the 133 regulation of LOXs in apple.

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In this study, we selected a ripening-related gene, MdLOX1a, to investigate ester provide new insights into the regulation of aroma compound production and a potential 142 strategy to develop and utilize saline-alkali land to produce high-quality fruit, thereby 143 reducing pressure on arable land and ensuring national food security.

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MdLOX1a is involved in ester formation in apple and phylogenetic analysis of We sampled apple fruit at four developmental and ripening stages (Fig. 1A) for gas 148 chromatography-mass spectrometry (GC-MS) analysis. With progression of ripening, 149 large amounts of esters were produced. In ripe fruit, the ester content attained about 14 150 µg·g −1 fresh weight, which was almost seven times that of immature fruit at 57 days 151 after full bloom (DAFB) (Fig. 1B). The lipoxygenase pathway is one pathway for the 152 synthesis of esters and the crucial participating enzyme is lipoxygenase. In apple, eight 153 groups of LOXs are involved in the lipoxygenase pathway (Vogt et al., 2013). To clarify 154 the key lipoxygenase genes in fruit ripening, we analyzed eight lipoxygenase genes 155 from each group by quantitative real-time PCR analysis during fruit development and 156 ripening (Fig. 1C, Supplemental Fig. S1). As the fruit matured, the transcript level of  To analyze the function of MdLOX1a in volatile aroma biosynthesis, we generated 187 MdLOX1a-overexpressing transgenic 'Orin' calli (Fig. 2, A and B). The content of fatty 188 acid-derived volatiles, including esters, was significantly increased compared with that 189 of the wild type (WT) (Fig. 2C). In addition, the corresponding synthetic genes in the 190 lipoxygenase pathway were up-regulated (Fig. 2D). Taken together, these results  Interestingly, binding was not observed in the absence of the m6 region (Fig. 3D).

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Therefore, we concluded that the specific binding motif of MdASG1 was located in the 215 m6 region. Addition of a cold probe weakened the binding. When the binding sites were 216 mutated, binding was eliminated (Fig. 3E). A dual-luciferase reporter assay, performed   Tomato fruit required an additional 10 mL saturated NaCl to extract volatile compounds.  Table S4.  Supplemental Table S2.  Supplemental Table S2.  Supplemental Table S3.

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Fluorescence detection of reactive oxygen species 520 Reactive oxygen species were detected with fluorescent probes using a previously  Table S2). Agrobacterium

Statistical analysis
Student's t-test ( * P < 0.05, ** P < 0.01) was used to determine the significance of  The authors declare no conflict of interests.