Existing research supports a strictly defined conclusion: under the condition of keeping the daily average temperature basically constant, a moderate positive diurnal temperature range (daytime temperature higher than nighttime temperature) can increase the soluble sugar, sugar-acid ratio and dry matter distribution to the fruit in some tomato fruits, and reduce organic acids; however, the effect exists in terms of temperature mean, diurnal duration and variety threshold. Yuan Xiaokang and Yang Zaiqiang treated Golden Crown No. 5 tomatoes after fruit setting at a daily average of 25℃ and found that the soluble sugar and sugar-acid ratio at +12℃ DIF (31/19℃) were better than those at +18℃ DIF (34/16℃), while at a daily average of 18℃, 12℃ DIF was better than 6℃ DIF, indicating that "the larger the temperature difference, the better" is not true [1][2]. Nighttime cooling can affect the sugar-acid ratio by reducing nighttime respiration carbon loss, changing source-sink relationships, endogenous hormones and fruit development timing; however, these are inferred by multiple mechanisms, and the simple "more synthesis during the day and less consumption at night" cannot be regarded as a complete explanation that has been directly measured.

Regarding volatiles, the characteristic aroma of tomatoes is not determined by a single substance, but by the combined effect of sugars, acids and hundreds of volatiles. C6/C5 aldehydes and alcohols come from linoleic acid and linolenic acid via the lipoxygenation pathway of LOX-HPL-ADH; branched volatiles are related to Leu/Ile/Val amino transfer, and aromatic volatiles are related to Phe; β-ionone, β-damasone, geraniol acetone, 6-methyl-5-hepten-2-one, etc., come from carotenoid cleavage [3][4]. Cold and heat treatments will change C6 volatiles through gene expression and enzyme activity, respectively [5], but the existing evidence mainly comes from postharvest constant temperature, chilling injury or heat shock, and cannot prove that the diurnal temperature difference during cultivation regulates the aroma of field fruits in the same direction or magnitude. Therefore, the most credible direct evidence for this topic is concentrated on sugar and acid metabolism; the evidence for aroma pathways is indirect evidence, and causal inference needs to be verified by adding a joint experiment of diurnal temperature control-metabolomics-transcriptomics-sensory evaluation.

Direct evidence for the sugar-acid ratio supports "moderate positive DIF" but not "maximizing diurnal temperature range".The most direct evidence is the experiment in the artificial climate chamber with daily average temperature as the main factor and DIF as the secondary treatment. Yuan Xiaokang and Yang Zaiqiang set five DIFs at a daily average of 25℃ for Golden Crown No. 5 tomatoes during the fruit enlargement-ripening period after fruit set: 16/34, 19/31, 25/25, 31/19 and 34/16℃; positive DIF increases soluble sugar, sugar-acid ratio and soluble protein, while negative DIF decreases these indicators and increases organic acids, and +12℃ is better than +18℃[1]. In the earlier six-treatment experiment, the fruit sugar and sucrose DIF were highest at 12℃ at a daily average of 18℃, while the DIF was highest at 6℃ at a daily average of 25℃; the distribution of dry matter to the fruit was largest at 6℃ and smallest at 12℃ at both temperatures[2]. Both results show that the temperature mean determines the basal metabolic window, and DIF determines the direction of resource allocation within the window, and the two cannot replace each other.

The volatile evidence currently does not prove that "a large temperature difference produces a stronger tomato aroma".Currently, no studies have been found that use real diurnal temperature cycles throughout the entire process from flowering or fruit set to maturity, under uniform daily average temperature, photoperiod, and humidity, and that quantify the original synthesis of fatty acids, amino acids, and carotenoids using HS-SPME-GC-MS. Seasonal and facility microclimate observations often confuse temperature difference with radiation, mean temperature, humidity, and harvest period; while postharvest low-temperature or heating studies can explain enzyme thermosensitivity, they involve damage, recovery, and substrate recombination, and cannot replace in-situ accumulation in intact fruit. Therefore, this report labels sugar-acid ratio-related materials as "direct evidence," volatile candidate pathways as "indirect evidence," and descriptions of reduced nighttime respiration, net increase in photosynthetic carbon, diurnal hormones, and gene oscillations as "inferences."

The sugar-acid ratio is not an isolated ratio of "total sugar ÷ total acid", but rather the result of the combined effects of carbon supply, inventory demand, and acid turnover.Tomato pulp sugars are usually mainly fructose and glucose, and some materials also have high sucrose; the sugar-acid ratio is affected by soluble solids or reducing sugars, citric acid and malic acid as well as fruit water content. Yuan Xiaokang and Yang Zaiqiang's unified daily average experiment found that under positive DIF, the soluble sugar and sugar-acid ratio increased and the organic acid decreased, while the negative DIF had the opposite effect [1]. This result can be explained by the fact that the suitable daytime temperature promotes photosynthesis and assimilation loading and input into the fruit, while the lower nighttime temperature reduces carbon consumption caused by non-photorespiration and dark respiration; however, the same evidence can also be used to explain that "nighttime temperature affects fruit sink activity and organic acid turnover", and the existing treatments do not separate the daytime and nighttime carbon flux item by item.

The advantage of nighttime cooling has a threshold; excessively high positive DIF can sacrifice dry matter distribution and total yield.At a daily average of 25℃, the soluble sugar, protein, and vitamin C content of Golden Crown No. 5 at +12℃ DIF was better than that at +18℃ DIF[1]. In another experiment, the distribution of dry matter to fruit was highest at 6℃ DIF and lowest at 12℃ DIF at both daily averages[2]. This indicates that although low night temperatures may reduce respiration per unit time, they may also prolong development, weaken sink strength, or affect transport. If actual management only pursues sugar content, it may result in smaller fruit, lower total dry matter, or altered ripening synchronicity. "Optimal taste" must be measured simultaneously for single fructose content, juice sugar concentration, organic acids, water content, and yield, and cannot be represented by °Brix alone.

Organic acids do not necessarily respond to temperature differences in sync with sugars, and total titratable acids cannot be used to replace metabolic profiles.The improvement in the sugar-acid ratio under positive DIF can come from an increase in sugar and a decrease in acid, or from a change in both simultaneously; under negative DIF, a decrease in sugar and an increase in acid will suppress the ratio [1]. The sour taste of tomatoes is mainly contributed by citric acid and malic acid, both of which are affected by the tricarboxylic acid cycle, malate valve, citrate synthase, aconitase, NAD/NADP malic enzyme, PEPC, vacuole compartmentalization and diurnal proton transport. Existing DIF studies have not systematically reported the citric acid/malic acid ratio, enzyme activity or vacuole pH, so they can only confirm the "direction of total organic acid concentration" and cannot further assert which acid metabolism pathway is changed by temperature difference.

Source-sink, hormones, and phenology are key mediating factors that influence sugar and acid levels due to temperature differences.Yuan and Yang set five DIFs (16/34, 19/31, 25/25, 31/19, 34/16℃) during the fruiting period, maintaining a daily average of 25℃. Positive DIF increased shoot tip GA3, IAA, and ZT, while ABA showed no significant change. It also promoted plant height, stem diameter, fruit diameter, and leaf area, accompanied by increases in soluble sugars, vitamin C, and soluble proteins [6]. This supports the candidate mechanism of "positive DIF—hormone—growth and sink organ expansion—assimilate allocation". However, this evidence is still correlational: hormone changes may come from temperature itself, sugar signals, light signals, or phenological changes; to prove causality, it is necessary to measure phloem sugar flow, sugar signals, hormone synthesis/signal mutants, and exogenous hormone feedback after temperature treatment.

The temperature difference effect should first be broken down into temperature level and temperature amplitude.Temperature levels determine enzyme optima, photorespiration, stomatal conductance, photosynthetic duration, and respiration rate; DIF (diurnal variation factor) alters the day/night distribution while the average level remains constant. Therefore, reporting only a "diurnal temperature variation of 10°C" without specifying the daily average, day length, transition time, and absolute night temperature makes it impossible to determine the mechanism. For example, while both 31/19°C and 21/11°C represent a +12°C DIF, the former is closer to the experimental window of the Kingcrown 5 enzyme, while the latter may have reached the low-temperature metabolic limitation. DIF cannot be interpreted in isolation from absolute temperature.

Nighttime cooling may improve net saccharide accumulation, but current direct evidence does not measure net carbon gain.The verifiable inference chain is that under moderate daytime temperatures, photosynthetic carbon fixation, sucrose synthesis, and phloem loading are relatively strong; after nighttime temperatures decrease, dark respiration, glycolysis, and growth-related carbon consumption in leaves and fruits decrease; more carbon is retained as sucrose, fructose, and glucose or temporarily enters starch, which is hydrolyzed to supply sugar in the later stages of ripening. Robinson et al.'s work on carbon metabolism in tomato fruit development has demonstrated that there is a brief accumulation of starch in the early stage and an increase in soluble hexose in the later stage, which is related to the activity of enzymes such as ADP-glucose pyrophosphorylase and sucrose synthase [7]; however, this study is not a DIF experiment and can only be used to establish pathway candidate genes, and cannot prove that their expression is controlled by diurnal temperature variation.

Temperature may alter the “maturation time” through phenological compression, rather than changing the synthesis rate per unit time.High temperatures typically accelerate flowering, fruit setting, color change, and ripening; rapid ripening may shorten the effective time for carbon input, acid degradation, and aroma formation. Delgado-Vargas et al. increased the average daily temperature from about 27.0°C to 29.9°C and found that the effects of high temperatures varied depending on the variety and the time of occurrence; when heat was applied during the rapid growth period, the sugar and acid content of Money Maker and Campeche 40 were more significantly affected, while Chapingo F1 had higher sugar content, lower acid content, and larger fruit [8]. This indicates that sugar and acid content is not a simple linear function of diurnal temperature, and the fruit stage is an important modifier of the effect.

Water, nutrients, and CO2 can alter the direction and magnitude of the DIF effect.High light, adequate water, potassium supply and balanced nitrogen can enhance source strength and sink transport; water stress can "concentrate" sugar and acid when the fruit water content decreases, but it is not equivalent to an absolute increase in total sugar. There is an interaction between CO2 and temperature: higher CO2 at 27/22℃ can reduce the adverse effects of high temperature on flowering and fruit setting, but warm temperature treatment reduces the soluble sugar in the fruit and aggravates the negative response of some mineral nutrients, carotenoids and vitamin C under high CO2[9]. Therefore, if only the temperature difference is adjusted in the facility experiment and CO2, vapor pressure deficit and irrigation fluctuation are allowed, the sugar and acid changes obtained may be mistakenly attributed to the temperature difference.

The composition of sugar and the sugar-acid ratio together determine sweetness; sugar content alone is insufficient to predict flavor.The main sweetness of mature tomatoes comes from fructose and glucose, and some materials also contain measurable sucrose. After being synthesized in the source leaves, sucrose is transported through the phloem and can be converted into hexose in the fruit by sucrose synthase (SUS/Sucrose synthase) and invertase (INV); fructose kinase, hexokinase, UDP-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase (AGPase), starch phosphorylase and other enzymes are involved in its distribution. Among different varieties, the configuration of INV, SUS, SPS, SUT and SWEET transport systems in sucrose-accumulating and hexose-accumulating fruits is different [10]. For comparison of “taste tomatoes”, at least fructose, glucose, sucrose, starch and °Brix should be measured, and the sugar-acid ratio should be calculated in molar concentration, rather than just reporting the mass percentage.

Organic acid metabolism is the other half of the sugar-acid ratio; citric acid and malic acid must be measured separately.The diurnal and developmental variations of citric and malic acid in fruits are influenced by mitochondrial TCA, cytoplasmic PEPC, malate shuttle, citrate and malate transport, and vacuole compartmentalization. A decrease in acidity does not necessarily indicate that respiration "consumes more acid"; it is more likely due to vacuole instability, degradation, transamination, or redistribution as part of the carbon skeleton. Conversely, a decrease in acidity can enhance the perception of sweetness; therefore, "sweetness" in sensory evaluation is a combined result of sugar concentration, acid concentration, volatile matter, and the interaction of sweetness measurements, and cannot be equated with total sugar.

C6 and C5 volatiles are the fast reaction modules of the lipid oxygenation pathway, but temperature can affect the enzyme, membrane lipid substrate, and compartment contact simultaneously.Linoleic acid (18:2) and α-linolenic acid (18:3) generate corresponding hydroperoxides under the action of lipoxygenase, which are then converted into C6 aldehydes, such as hexanal and cis-3-hexenal, via HPL, and then into hexanol and cis-3-hexenol via ADH; C5 volatiles can also be formed along the branch. TomLoxC is functionally important for fatty acid-derived C6 aldehydes and alcohols, and HPL and ADH2 are also key nodes [3][5]. Bai et al. compared the effects of 5℃ for 5 days and 52℃ for 15 minutes on ripe red Tasti-Lee and Sanibel fruits: chilling injury downregulated TomloxA/B/C (TomloxD was not significant) and inhibited the expression of HPL and ADH genes and subsequent enzyme activity; heating upregulated the expression of TomloxB/C and HPL/ADH, but the activity of HPL and ADH was still temporarily inhibited [5]. This shows that "low temperature slows down enzymes" is not enough to summarize the effect: the same phenotype can be caused by transcriptional downregulation or post-translational inactivation.

Branched and aromatic nitrogenous volatile compounds directly link amino acid metabolism to tomato flavor.Leu, Ile, and Val can be transaminated and decarboxylated by branched-chain aminotransferases (BCAT) to produce 2-/3-methylbutanal, 2-/3-methylbutanol, and 2-methylpropanol acetate, which present malty, fruity, or wine-like aromas; Phe can be converted into 2-phenylethanol and 2-phenylacetaldehyde by aromatic amino acid decarboxylases. Homologous BCATs in Arabidopsis/tomato are related to branched-chain volatiles, but the substrate specificity of specific isomers in tomato fruit for various branched-chain volatiles is still not fully determined [4]. This means that the increase in amino acids does not automatically lead to a better aroma: the final amount is determined by the supply of free amino acids, transamination, decarboxylation, redox, acyl transfer, and volatile release.

Carotenoid cleavage products provide low-concentration, high-odor contribution of floral and fruity aromas, and are the intersection of the temperature difference-light-carotenoid network.LeCCD1A/1B can oxidatively cleave various linear and cyclic carotenoids to form pseudoionone, β-ionone, β-damascone, geraniol acetone, 6-methyl-5-hepten-2-one, etc. [4]. Among them, 6-methyl-5-hepten-2-one is associated with tomato-like and sweet floral aromas. Maturation-related nodes PSY, PDS, ZDS, CRTISO, LCY-E/B, CYCB and CCD1 link transcription factors such as light, temperature, ethylene and RIN/NOR/AP2a to isoprene volatiles [3][4]. If positive DIF increases photosynthesis and carotenoids by prolonging the optimal temperature and light period, it can theoretically increase the precursors of this library; however, there is no direct experimental evidence in this study to prove the diurnal phase change of this pathway in the diurnal cycle.

Esters link sugar metabolism with lipid-derived alcohols and acyl donors, and are the most easily overlooked layer in the "sugar-acid-aroma" network.Fatty acid-derived alcohols can be acetylated by alcohol acetyltransferase AAT1 to form acetates. The absence or low expression of TomloxC/AAT/CCD1 can affect the characteristic aroma of different varieties [3]. Glycolysis and TCA can provide lipid production and energy supply, and can also provide acyl donors via acetyl-CoA; however, these connections have not been quantitatively studied in the diurnal temperature range of tomato fruits. Therefore, when measuring volatiles, precursors (free fatty acids, amino acids, carotenoids), enzyme activity/transcriptions and end products must be included. The GC peak area alone cannot distinguish between "reduced synthesis, reduced release, insufficient precursors or increased degradation".

Actual DIF tests and post-harvest temperature tests must be compared separately.The former treats the entire plant, canopy, and even roots, and the fruit undergoes source-sink and transport changes; the latter typically treats fruit that has already reached commercial maturity, and low temperatures may simultaneously cause chilling injury, membrane phase transition, and ripening arrest. The “Evidence Type” in the list below is used to emphasize this distinction.

Temperature range and absolute nighttime temperature cannot be summarized by a single DIF value.The same +12℃ can be 31/19℃, where the daytime temperature is moderate and the nighttime temperature is moderate, or it can be 34/16℃, where the daytime temperature is close to the high temperature and the nighttime temperature is close to the low temperature stress. The positive DIF results of Yuan Xiaokang and Yang Zaiqiang have proven that the latter is not better[1]. Therefore, the research plan should report the daytime temperature, nighttime temperature, daily average value, and DIF as four related variables respectively; if DIF is the main treatment, the daily average value must be controlled, otherwise the DIF effect and the average temperature effect will be completely confused.

Photoperiod and light intensity are non-negligible covariates of the temperature difference effect.Light provides photosynthetic carbon, influences photoresponse nodes such as PSY/LCY, and may directly alter carotenoid pyrolysis volatiles. If temperature treatment uses long-day supplemental lighting, the increase in sugar may come from an increase in total light energy rather than DIF; if far-red or white light is supplemented at night, unnatural DIF may be generated, causing confusion with the light signal. Artificial climate chambers should be set with the same photoperiod, PAR, spectrum, air velocity, humidity, or VPD control, and at least two different light intensities or cumulative light doses should be used for nested treatment.

Sugar allocation nodes should be verified using a triplet of enzyme activity, transcript, and transport flux.Candidates related to sucrose loading/unloading and fruit input include SUT/SUC, SWEET, and STP/Hexose transporter; transformation side includes cell wall INV, vacuolar INV, and cytoplasmic neutral INV; sucrose and starch resynthesis side includes SPS, SUS, AGPase, and starch phosphorylase/amylase. Robinson et al. pointed out that early starch accumulation in tomato fruit development is related to the activity time of AGPase and SUS, and starch degradation is characterized by phosphorolytic activity [7]. However, the temperature sensitivity of these genes should be determined by qPCR, protein or enzyme activity, and 13C tracing. Relying solely on transcriptomics will miss enzyme activity and substrate control.

The organic acid node should be upgraded from total titratable acid to metabolic flux.Citric acid-related nodes include CS, aconitase, isocitrate dehydrogenase, PEPC, PEPCK, and mitochondrial electron transport; malic acid-related nodes include NAD/NADP-malic enzyme, malic dehydrogenase, PEPC, tonoplast H+-ATPase/PPi enzyme, and malic transporter. Measuring only total acid cannot determine whether the increase in acid due to temperature difference is caused by increased synthesis, increased vacuolar retention, or differences in maturity at the time of sampling. It is recommended to simultaneously measure pH, citrate, malic acid, key enzyme activities, and vacuolar transport expression at day-night transition points, before dawn, and after dusk.

LOX/HPL/ADH is the most valuable candidate thermometer for C6 volatiles.TomLoxC, TomloxA/B, HPL, and ADH2 constitute the core chain from membrane lipid hydroperoxide to hexanal/hexenal and then to alcohol [3][5]. The experiment should distinguish three mechanisms: first, transcriptional inhibition caused by cold injury; second, protein inactivation caused by high temperature; and third, substrate availability or lipoxygenase isomer compensation. The results of Bai et al. particularly warned that the total LOX activity and the single Tomlox transcript can change inversely, and C6 throughput cannot be judged solely by qPCR.

Amino acid branched chains and aromatic volatiles need to be analyzed according to precursor categories.The changes in BCAT1/2 during ripening support their involvement in Leu/Ile/Val-derived volatiles, but the substrate specificity of the six BCAT members has not been fully elucidated [4]. The experiment should simultaneously quantify the activities of free Leu, Ile, Val, Phe, Glu, Asp, and transaminase; otherwise, the misjudgment of "increased total free amino acids, unchanged key branched volatiles" may occur.

RIN, NOR, AP2a, TAGL1, FUL1/FUL2, SlMYB1, etc. constitute the upper-level control of ripening, but how temperature and DIF change them remains a blank.RIN and other MADS transcription factors regulate the ripening module and connect ethylene, pigments and volatiles; Klee and Tieman’s systematic review integrates the volatile precursor pathway with ripening control[3]. SlMYB1 has been reported to bind to promoters such as PSY2 and LCY1 and affect carotenoids and sugars/proteins[12]. However, the complete causal chain of “temperature-RIN/Ethylene-CCD1/LOX-volatiles” has not been verified under diurnal temperature variation conditions.

Input layer: Absolute temperature, temperature difference, and light environment together define the diurnal climate.Daytime temperature, nighttime temperature, daily average, DIF, photoperiod, PAR, spectrum, and diurnal transition slope determine the duration of photosynthesis, photorespiration, canopy temperature, stomatalization, and transpiration. VPD, CO2, soil or substrate temperature, and rhizosphere oxygen also need to be controlled in the facility. Failure to control any of these can lead to confounding sources of sugars, acids, or volatiles.

The first metabolic layer: the source determines carbon supply, and the sink determines distribution.During the day, leaves fix CO2, forming sucrose which is then transported to the fruit via the phloem. The fruit receives sucrose via SUT/SWEET/HXT, and INV, SUS, SPS, and AGPase direct it to hexoses, transient starch, or respiration. Lowering nighttime temperatures can reduce dark respiration without decreasing total photosynthesis, but excessively low temperatures can impair leaf function and delay development. The ultimate change in the sugar-acid ratio is a net result of increased input, decreased consumption, altered fruit sink strength, and a dilution effect.

The second metabolic layer: organic acids contribute not only to sour taste but also to carbon and redox nodes.Citric acid and malic acid are in dynamic equilibrium between TCA, PEPC, the malate shuttle, and the vacuolar compartment. DIF may alter total titratable acid by changing nocturnal respiration, diurnal carbon skeleton flux, and fruit development rate; however, existing data can only confirm the direction of total acid, but cannot identify a unified master controlling enzyme.

The third metabolic layer: amino acids, membrane lipids, and carotenoids provide volatile precursors.Leu/Ile/Val forms malty/fruity branched volatiles via BCAT and subsequent reactions; Phe forms aromatic volatiles via AADC, etc.; 18:2/18:3 forms green C6/C5 compounds via LOX/HPL/ADH; carotenoids form floral and fruity isoprene compounds via CCD1, etc.; alcohols form esters via AAT1. Glycolysis, TCA, and acetyl-CoA link sugar metabolism to acyl donors. Temperature can simultaneously affect substrate accumulation, enzyme kinetics, enzyme stability, protein localization, and membrane state.

The fourth layer: The maturation clock shifts metabolism to the volatile burst window.Ethylene, RIN, NOR, AP2a, etc., control the initiation of ripening and pigment accumulation; if DIF changes the color change time or the duration of ripening, it will change the accumulation window of volatiles. Therefore, GC-MS should sample within the same color level/DAA matrix; otherwise, the differences measured may be differences in ripeness rather than a direct effect of temperature.

Output layer: Sensory perception is not a simple sum of sugars, acids, and volatiles.Sugars reduce sourness, while acid enhances the perception of sweetness. Some low-concentration volatiles can even "enhance sweetness" without changing the sugar concentration. Sensory experiments are recommended to simultaneously evaluate sweetness, sourness, tomato aroma, green aroma, floral and fruity aroma, bitterness/off-flavor, juiciness, and overall preference, and to use OAV (Odor Activity Value) instead of simply peak area; relying solely on chemical concentration will miss sensory interactions.

It is recommended to adopt a four-factor, phased randomized block design, rather than setting only two seasons or two greenhouse temperature levels.The main plot represents temperature treatments, while the subplots represent varieties and fruit development stages. Temperature factors should simultaneously define daytime temperature, nighttime temperature, daily mean, and DIF (Digital Intensity Factor), avoiding listing only DIF. Recommended baseline windows are 20/14, 24/18, and 28/22℃; 31/19℃ can be added for positive DIF studies. Controls must include treatments with different mean values of 25/25℃ and 24/18℃ to differentiate between "daytime warmth" and "temperature difference" effects. Each treatment should have at least 3-4 independent climate chambers or greenhouse plots, with individual plants representing true biological replicates; fruit replicates should not substitute for plant replicates.

Treatment should cover the entire fruit development process and include stage-specific switching.Treatment was carried out continuously from 7 days before flowering, on the day of flowering, or from fruit set until marketable red ripeness; additional switching groups were set up for "single-stage only" and "color change-maturity only". The flowering date was recorded on a tag for each fruit cluster, and samples were taken using both DAA and color charts. This allows verification of whether the DIF sensitivity window coincides with the sugar input, acid degradation, or volatile matter synthesis period. A linear transition of 1-2 hours is recommended for temperature changes, recording the dawn/dusk slope, as instantaneous switching introduces non-physiological thermal shock.

Light, CO2, water and fertilizer, and root temperature must be treated as controlled covariates.Maintain a fixed photoperiod (e.g., 16 h day/8 h night) and PAR (e.g., 300, 500, 700 μmol m−2 s−1, pre-tested based on material and facility light tolerance) to ensure spectral consistency; CO2 can be set at two levels: ambient background and approximately 800 μmol mol−1. Irrigation should be controlled using a weighing method or capacitive sensors to maintain uniform water treatment; record EC, pH, N, P, K, Ca, Mg, and K/Ca ratio. Root zone temperature should be monitored at least, ideally with independent temperature control; otherwise, aboveground DIF and root temperature changes will become intertwined.

The sugar and acid indicators should simultaneously cover concentration, total amount, growth curve, and metabolic enzymes.Sampling should be conducted at the onset of color change, color breaking, 1/2 red, fully red, and 2 days after fully red for each treatment. Differentiating between peel, pulp, and gelatinous/placental material increases the difficulty of interpretation; after preliminary testing, whole fruit homogenate or designated parts can be collected uniformly. Indicators include fresh weight, dry weight, moisture content, °Brix, fructose, glucose, sucrose, starch, total soluble sugars, titratable acid, pH, citric acid, and malic acid. Enzymatic assays include INV, SUS, SPS, AGPase, CS, NAD/NADP-ME, PEPC, H+-ATPase, and diurnal time curves. The sugar-acid ratio should be clearly defined using a formula; it is recommended to report both the mass concentration ratio and the molar concentration ratio.

Volatile compounds must be measured for in situ synthesis and sensory activity; GC analysis of fragmented tissues alone is insufficient.Dynamic headspace trapping or low-temperature pre-equilibration HS-SPME was employed, combined with GC×GC-TOF-MS or GC-MS/MS; a whole-fruit spatiotemporal release assay was also included to reduce spurious peaks caused by LOX activation due to fruit breakage. Compounds were annotated by fatty acids, branched-chain amino acids, aromatic amino acids, carotenoids, esters, terpenes, and other categories; retention indices, standard or NIST library matching, peak areas, and internal standard quantification were reported. Sensory evaluation should be conducted with training for the descriptive analysis team and consumer testing; OAV of key volatiles was calculated to avoid misidentifying high-value but high-threshold compounds as primary aroma components.

Mechanism validation should be performed simultaneously with metabolomics, transcriptomics, and necessary perturbations.Fruit peel/pulp samples were collected at four time points throughout the day (before dawn, morning, afternoon, and after dusk) to determine amino acids, free fatty acids, carotenoids, and sugars and acids. Transcriptome analysis focused on the LOX family, HPL, ADH, AAT, BCAT, AADC, CCD1/4, PSY, PDS, LCY, SUT/SWEET/HXT, INV/SUS/SPS/AGPase, and RIN/NOR/AP2a/SlMYB1. Candidate modules were validated using VIGS, CRISPR, or sta