Abstract

   Soluble sugars and organic acids constitute the primary flavor
   determinants in fruits and elucidating their metabolic mechanisms
   provides crucial theoretical foundations for fruit breeding practices
   and food industry development. Through integrated physiological and
   transcriptomic analysis of pomegranate varieties ‘Sharp Velvet’ with
   high acid content and ‘Azadi’ with low acid content, this study
   demonstrated that the differences in flavor between the two varieties
   were mainly caused by differences in citric acid content rather than in
   soluble sugar content. Transcriptome profiling identified 11 candidate
   genes involved in sugar and acid metabolism, including three genes
   associated with soluble sugar metabolism (FBA1, SS, and SWEET16) and
   eight genes linked to organic acid metabolism (ADH1, GABP1, GABP2,
   GABP3, GABP4, ICL, ME1, and PDC4). These data indicated that
   differences in citric acid content between the two varieties mainly
   stemmed from differences in the regulation of the citric acid
   degradation pathway, which relies mainly on the γ-aminobutyric acid
   (GABA) branch rather than the isocitric acid lyase (ICL) pathway.
   Citric acid accumulation in pomegranate fruit was driven by metabolic
   fluxes rather than vesicular storage capacity. Weighted gene
   co-expression network analysis (WGCNA) uncovered a significant citric
   acid content associated module (r = −0.72) and predicted six core
   transcriptional regulators (bHLH42, ERF4, ERF062, WRKY6, WRKY23, and
   WRKY28) within this network. Notably, bHLH42, ERF4, and WRKY28 showed
   significant positive correlations with citric acid content, whereas
   ERF062, WRKY6, and WRKY23 demonstrated significant negative
   correlations. Our findings provide comprehensive insights into the
   genetic architecture governing soluble sugars and organic acids
   homeostasis in pomegranate, offering both a novel mechanistic
   understanding of fruit acidity regulation and valuable molecular
   targets for precision breeding of fruit quality traits.

   Keywords: pomegranate, soluble sugars, organic acids, transcriptomic
   profile, differentially expressed genes, transcription factor

1. Introduction

   Pomegranate (Punica granatum L.), a deciduous shrub or small tree
   belonging to the Lythraceae family (genus Punica), is a horticulturally
   important crop originating from Central Asia that is subsequently
   domesticated in Mediterranean and Middle Eastern regions [[30]1]. As
   one of the earliest cultivated fruit species in human history [[31]2],
   pomegranate has gained global agricultural significance due to its
   multifunctional value, encompassing economic, nutritional, medicinal,
   and ornamental applications [[32]3]. The fruit is particularly renowned
   for its high anthocyanin content [[33]4], which confers potent
   antioxidant properties [[34]5] and demonstrates therapeutic potential
   in mitigating inflammation and preventing carcinogenesis [[35]6,[36]7].
   The global pomegranate market has witnessed sustained growth, fueled by
   increasing consumer demand for functional foods. Given its commercial
   importance, substantial research efforts have been devoted to
   elucidating its bioactive compounds and optimizing cultivation
   practices.

   Soluble sugars and organic acids are the primary determinants of fruit
   flavor, playing pivotal roles not only in regulating the balance
   between sweetness and acidity but also in influencing texture,
   coloration [[37]8,[38]9], processing characteristics, marketability,
   and economic value. Fruit flavor profiles are primarily shaped by
   complex interactions involving the ratio of soluble sugars to organic
   acids, their specific molecular composition, and synergistic effects
   [[39]10,[40]11]. The predominant soluble sugars in fruit tissues
   include sucrose, glucose, and fructose [[41]12,[42]13]. In most fruit
   crops, these metabolites serve dual physiological roles—acting both as
   energy substrates for respiration and as osmoregulators critical for
   maintaining cellular homeostasis during fruit development and
   postharvest storage. Sucrose is the dominant photoassimilate
   transported through the phloem [[43]14], where it undergoes cytosolic
   metabolism before being sequestered in the vacuole [[44]15]. Among
   organic acids, malic acid and citric acid are the most abundant
   [[45]16,[46]17], functioning as both respiratory substrates [[47]18]
   and pH regulators [[48]19].

   The metabolic regulation of soluble sugars and organic acid
   accumulation in fruits involves a sophisticated, multilayered control
   network governed by coordinated processes including biosynthesis,
   catabolism, transmembrane transport, vacuolar sequestration,
   transcriptional regulation [[49]20,[50]21,[51]22], and environmental
   modulation [[52]23,[53]24,[54]25]. Currently, the development of
   various molecular biology tools including transcriptomics has greatly
   helped us to gain a deeper understanding of the regulatory mechanisms
   of soluble sugars and organic acids metabolism. Transcriptomics, which
   analyzes the composition and dynamic changes of the complete set of
   transcripts under specific physiological states, reveals gene
   expression patterns and regulatory networks, playing a pivotal role in
   functional gene discovery, expression divergence analysis, and
   molecular mechanism inference. For instance, transcriptomic studies in
   loquat [[55]26], litchi [[56]27] and sweet cherry [[57]28] have
   identified multiple gene families that regulate soluble sugar and
   organic acid biosynthesis through differential expression. Comparative
   analyses reveal substantial interspecific variation in the key genetic
   determinants of these metabolic pathways, suggesting that the molecular
   control of soluble sugars and organic acid balance is influenced by
   complex interactions among species-specific genetic backgrounds,
   variety characteristics, and agronomic management practices [[58]27].

   Current applications of transcriptomics in pomegranate research have
   predominantly focused on abiotic stress tolerance [[59]28], anthocyanin
   biosynthesis [[60]29], fruit cracking mechanisms [[61]30], and floral
   pigmentation [[62]31], leaving a significant knowledge gap regarding
   the molecular basis of flavor compound accumulation in this species.
   This gap impedes targeted breeding efforts to meet consumer preferences