Molecular Plant Breeding 2025, Vol.16, No.3, 202-210 http://genbreedpublisher.com/index.php/mpb 205 the accumulation of glucose and fructose in tomato fruit. These results show that relieving the inhibition of sucrose decomposition or reducing sugar consumption can effectively increase the sweetness of fruit. For golden pitaya, it is possible to consider identifying sucrose invertase inhibitor analogs in its genome and editing them to make them functionally deficient to enhance the sugar accumulation of the pulp. Another important target gene is the gene related to sugar transport and signaling pathways. Source-sink allocation in plants is often regulated by sugar signals. For example, calcium-dependent protein kinases SlCDPK27 and SlCDPK26 in tomatoes were found to act as “sugar brakes”: they reduce the conversion of sucrose to hexose by phosphorylating sucrose synthase. After knocking out these two genes, the glucose and fructose content of tomato fruits increased by up to 30%, significantly improving sweetness without reducing yield (Zhang et al., 2024). This suggests that in golden pitaya, it is also possible to consider targeting signal factors that negatively regulate sugar accumulation, such as similar CDPKs or other kinases/transcription factors, to increase sugar content. 3.2 Genes related to organic acid metabolism Moderately reducing organic acid content and increasing the sugar-acid ratio are another effective strategy to improve flavor. Especially for some fruits with excessive or unbalanced sourness, reducing acidity through gene editing can significantly improve edibility. Genetic factors that affect fruit acidity mainly include: enzymes that regulate organic acid synthesis, transporters that promote organic acid accumulation, and proton pumps that control vacuolar acidification. Recent studies have used CRISPR/Cas9 to mutate the CitPH4 gene (encoding a MYB-type transcription factor that regulates the vacuolar proton pump) of citrus, and found that the citric acid content decreased significantly and the sour taste of the fruit was greatly reduced (Miao et al., 2024). In golden pitaya, if there are similar key factors that regulate vacuolar pH (such as transcription factors such as PH4 or PH5), they can also be used as editing targets to reduce acidity. In addition, enzyme-encoding genes directly involved in the synthesis and decomposition pathways of organic acids are also potential targets. For example, the production of citric acid involves citrate synthase, and the removal of malic acid involves malic enzyme and citrate lyase. In kiwifruit, researchers used CRISPR/Cas9 to knock out the AcNAC1 gene encoding the vacuolar NAC transcription factor, which positively regulates the accumulation of organic acids. The citric acid content of the mutant kiwifruit dropped dramatically, and the sour taste was significantly reduced (Ling et al., 2024). This case shows that by targeting transcription factors that regulate organic acid metabolism, the acidity of the fruit can also be effectively changed. If there are similar transcription factors that promote acid accumulation (such as NAC and MYB) in golden pitaya, it can be considered to knock them out to reduce acidity and enhance sweet taste. 3.3 Genes related to aroma compound metabolism Unlike sugar acid, most aromatic volatiles are secondary metabolites, and a few key steps are often controlled by specific genes. Therefore, there is a large space for regulation through gene editing. When selecting aroma improvement target genes, two ideas need to be considered: one is to remove bad odor components, and the other is to increase pleasant aroma components. The mutation of the TomLOXC gene in tomatoes leads to a reduction in some green volatiles and a prominent fruity aroma, which is considered to help improve flavor. There are also multiple LOX family genes in golden pitaya. Members that are highly expressed during the fruit ripening period can be selected as editing objects to reduce the grassy smell (Wu et al., 2022). In terms of increasing pleasant aroma components, key enzymes that limit related metabolism can be targeted. For example, the FaFAD1 gene in strawberries controls the presence or absence of γ-decalactone (with a strong fruity aroma) (Oh et al., 2021). Another category that deserves attention is the enzymes that degrade aromatic substances. The low content of aromatic substances in some fruits is not due to insufficient synthesis, but to excessive degradation. For example, the presence of esterases in strawberries will decompose ester aromas and weaken the aroma concentration. Recent studies have shown that inhibiting a carboxylesterase FanCXE1 in strawberry fruits can increase the retention of ester volatiles, thereby enhancing the fruity aroma (Martínez-Rivas et al., 2022). Inspired by this, if a significantly expressed volatile degrading enzyme gene is found in golden pitaya, it may be considered to make it functionally deficient or reduce its expression to increase the accumulation of aroma substances.
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