This study examined the effects of red, yellow, green, and blue 40-60% shade nets on carotenoid accumulation in the peel of 'Newhall' navel oranges over two seasons. The results indicated that the green 60% shade net significantly enhanced carotenoid accumulation, whereas the red 60% shade net reduced it compared with the control during both shaded seasons. HPLC analysis further revealed that the green 60% shade net significantly elevated the levels of essential carotenoids, including α-carotene, β-carotene, phytoene, violaxanthin, and lutein. In contrast, the red 60% shade net showed lower levels of these compounds than the open field. Both the green and red 60% shade nets effectively reduced temperature and light intensity while increasing relative humidity (RH). However, photosystem II (PSII) efficiency was superior under green 60% compared to red 60%, indicating optimal photosynthetic performance. The results suggest that variations in the color spectrum directly affect photochemical efficiency in citrus. Furthermore, green 60% increased carotenoid biosynthesis genes (CitPSY, CitLCYB1, and CitLCYBE) while downregulating degradation-related genes (CitCCD4 and CitNCED3), whereas red 60% exhibited the inverse effect. Moreover, the differential expression patterns were particularly evident in the second season, with CitPSY exhibiting maximal induction under green 60% and CitCCD4 reaching its peak under red 60% throughout all shading stages. These results underscore the potential of green 60% as an innovative, environmentally sustainable approach for citrus orchards, as it enhances the quality and coloration of citrus fruits by managing environmental and light conditions, thereby regulating the fundamental mechanisms of fruit color and quality.

Nitrogen (N) is the primary nutrient that initiates the summer-flush (SF) in citrus trees. However, the integrated "soil-root-shoot" feedback mechanism regulating SF intensity remains poorly understood in fertigation-based systems. To quantify dose-dependent effects on SF morphogenesis, nutrient allocation, root-tissue, and soil N concentrations, we conducted a greenhouse pot experiment with two-year-old 'Newhall' navel orange grafted onto Poncirus trifoliata rootstocks. The plants were subjected to six summer N rates (pure N 1, 2, 3, 4, 5, and 7 g plant(-1)). The results showed that: (1) Reducing N supply progressively delayed SF emergence and advanced maturation, thereby shortening the flush period; (2) Compared to high N treatment (5-7 g plant(-1)), moderate N (3-4 g plant(-1)) application treatment significantly suppressed SF growth, reducing shoot number, length, basal diameter, leaf number, emergence rate and biomass by 42.86 %, 8.99 %, 9.76 %, 16.74 %, 29.37 %, and 47.59 %, respectively. Conversely, this optimal N range maximized the net increments in plant height (1.96 cm) and main stem diameter (1.52 cm); (3) Within this range, the summer shoots maintained medium-to-high levels of nutrient content (N, phosphorus, potassium) and physiologically active substances (abscisic acid, chlorophyll, proline), while root growth was moderated to a medium-low level. Meanwhile, the nutrient status of the root zone soil and roots remained in the medium-to-high range; (4) Root zone soil ammonium nitrogen and nitrate nitrogen concentrations and root length density were strongly correlated with SF architectural traits. Multiple regression models incorporating these variables explained >50 % of the variance in SF phenotype. This regulation was attributed to an N-driven "soil-root-shoot" feedback mechanism, where the root zone inorganic N pool, root morphology, and the balance of key shoot physiologically active substances collectively defined summer-flush growth patterns. Therefore, it is advisable to apply 3-4 g N plant(-1) as the ideal summer rate for potted citrus seedlings in controlled environments. This effectively maintains a balance between vegetative growth and nutrient homeostasis through the identified "soil-root-shoot" feedback loop. However, these conclusions need to be verified in orchard-grown, fruit-bearing trees to substantiate their relevance in commercial cultivation systems before broader implementation.

Organic acid composition has a major influence on fruit taste and quality. In general, the fruits of common citrus varieties accumulate more citrate than malate. Here, we report that a citrus variety, ‘Haruka’ (Citrus tamurana × natsudaidai), accumulates more malate than citrate at the ripening stage. However, the underlying mechanism is unclear. Organic acid profiles were compared between ‘Haruka’ fruits and a common Ponkan cultivar, ‘Huagan 2’ (Citrus reticulata), during fruit development and ripening. We found that ‘Haruka’ fruit accumulated 75% less citrate than ‘Huagan 2’ fruit and that the malate content was nearly 3-fold greater in ‘Haruka’ fruit than in ‘Huagan 2’ fruit. In addition, 12 aluminum-activated malate transporter (ALMT) genes were identified in the citrus genome, with CsALMT9-like being predominantly expressed in ‘Haruka’ fruit juice sacs. An analysis of genes associated with the accumulation of citrate/malate revealed that the expression levels of genes encoding a P-type proton pump (CsPH8), a citrate/H+ symporter (CsCit), and CsALMT9-like were significantly greater in ‘Haruka’ fruit than in ‘Huagan 2’ fruit. Moreover, CsCit overexpression significantly decreased the citrate content, whereas overexpressing and silencing CsALMT9-like significantly increased and decreased the malate content. In addition, shading significantly increased the expression of CsPH8 but reduced the expression of CsCit and CsALMT9-like and significantly increased the citrate content but decreased the malate content, further confirming that CsCit and CsALMT9-like regulate the accumulation of citrate and malate in ‘Haruka’ fruit, respectively. Taken together, these data indicate that the relatively low citrate content of ‘Haruka’ is mainly due to increases in CsCit expression and that the relatively high malate content of ‘Haruka’ is mainly due to increase in CsALMT9-like expression. This study provides insight into the mechanisms that influence malate content in citrus fruit.

Autumn shoot maturation is important for citrus canopy management and subsequent reproductive development. We compared five foliar treatments but interpret their effects as treatment-specific because the salts were not equimolar and KH₂PO₄ supplied both K and P. Water (CK), 0.3

Leaf morphology is an important agronomic trait that affects photosynthetic efficiency and plant architecture. Some KNOXI (Class I KNOTTED1-LIKE HOMEOBOX) or CIN-TCP (CINCINNATA-LIKE TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTORS) transcription factors control leaf development by influencing GA (gibberellin) content. However, the knowledge about their interaction in regulating leaf morphogenesis is still scarce, especially in woody fruit plants such as citrus. In this study, we found that the expression of CsTCP13 (a citrus CIN-TCP gene) dramatically decreased with a decreasing leaf length-width ratio in citrus. Transiently silencing CsTCP13 in citrus produced wider leaves and significantly decreased CsGA20ox1 (a key citrus GA20-oxidase gene for GA biosynthesis) expression and GAs (GA3, GA4, and GA7) contents. Conversely, stable overexpression of CsTCP13 in tobacco generated narrower leaves and significantly increased GA20ox1 expression and GA1 content; it also significantly shortened the distance between veins and reduced the number of epidermal cells per unit area of leaf. Moreover, transactivation assays showed that CsTCP13 had transcriptional activation, and yeast two-hybrid, split-luciferase complementation, and bimolecular fluorescence complementation assays confirmed that CsTCP13 interacted with CsKNAT1 (a citrus KNOXI protein). Furthermore, yeast one-hybrid and dual-luciferase assays validated that CsTCP13 promoted while CsKNAT1 and its interacting protein CsKNAT6 inhibited CsGA20ox1 expression by binding to its promoter. Interestingly, CsKNAT1 or the CsKNAT1-CsKNAT6 complex interacted with CsTCP13 to attenuate its promotion effect on CsGA20ox1 expression. Taken together, our findings revealed a novel regulatory mechanism that CsTCP13 regulates citrus leaf width through directly influencing CsGA20ox1 expression and then GA content, which can be negatively affected by the interaction with CsKNAT1 or the CsKNAT1-CsKNAT6 complex.

Excessive and random outgrowth of axillary buds (AxBs) produce a mass of summer shoots, resulting in overly tall and dense canopies that complicate disease and pest control. Heavy fruit load reduces the number of summer shoots. However, the underlying molecular mechanism for this suppression remains unclear. In this study, we found that transcript levels of two well-characterized dormancy marker genes (CsBRC1 and CsDRM1) were significantly higher in AxBs from fruit-bearing branchlet (FB) than from vegetative branchlet (VB). However, de-fruiting significantly decreased them within 9 days. Transcriptome analysis identified 5638 differentially expressed genes (DEGs) after de-fruiting, many of which are associated with sugar and phytohormone homeostasis. Interestingly, transcript levels of sink strength-related genes (CsCwINV6 and CsSUS6) and auxin-efflux carrier gene CsPIN1a were significantly increased within 2 h after de-fruiting. Furthermore, their expression was markedly lower in FB AxBs than in VB AxBs and was significantly induced by sucrose supplementation. Collectively, these results suggested that a heavy fruit load suppresses the expression of sink strength-associated genes and CsPIN1a, thereby reducing sugar availability and impairing auxin export from axillary buds, ultimately inhibiting the outgrowth of AxBs. These findings provide valuable insights into the molecular basis of poor vegetative growth under heavy fruit load and contribute to simplifying canopy management through rationalizing fruit load. Heavy fruit load weakens the sink strength-associated genes and CsPIN1a, which ultimately inhibits the outgrowth of AxBs through reducing sugar availability and hindering auxin depletion in AxBs.

The excessive and random production of summer shoots poses significant challenges to pest and disease management and the improvement of fruit quality in citrus orchards. Although heavy fruit load has been observed to reduce summer shoot numbers, the mechanism is not well understood. This study combined a field investigation with a de-fruiting experiment to demonstrate that significant negative correlation exists between fruit load and summer shoot numbers in citrus orchard. Metabolomic analysis further indicated that fruits at the cell expansion stage function as dominant carbohydrate sinks, attracting more soluble sugars. De-fruiting significantly elevated sugar content and upregulated the transcript levels of sink strength-related genes (Sucrose synthase, CsSUS4/5/6) by more than 3.0-fold in the axillary buds. Additionally, exogenous application of sugar-related DAMs (differentially accumulated metabolites), such as sucrose, significantly promoted axillary bud outgrowth. Taken together, our findings confirm that heavy fruit load suppresses shoot branching, primarily through competing for soluble sugars. This provides a physiological basis for managing summer shoots by regulating fruit load, offering a practical strategy to enhance citrus orchard management and the effectiveness of pest and disease control programs.

Shade nets are frequently used to protect fruit-bearing trees from unfavorable weather conditions. However, the impact of colored shade nets on plant growth and fruit quality of horticultural crops remains poorly understood. This experiment was designed to investigate the effect of colored shade nets on photosystem II efficiency (Fv/ Fm), vegetative growth, and fruit quality of tangerine cultivar known as Murcott (Citrus reticulata, 'Murcott'). The experimental design consisted of a randomized complete block with six replications and five different shade net treatments: red 60 %, red 40 %, green 60 %, green 40 %, and an open field as the control. The nets were placed on steel rectangular structures, which had a height of 4.5 m, for a period of three months during the summer season. The results indicated that all shade net treatments led to an increase in relative humidity and a decrease in temperature, as compared to the control group. The green 60 % had the highest Fv/Fm value, even though it had low rates of photosynthetic active radiation (PAR) and light intensity (LUX). On the other hand, the open field had the lowest rate, despite having a high rate of PAR and LUX. Trees under green 60 % had the highest leaf chlorophyll content, while those under red 40 % had the lowest. The open field and green 60 % had the highest shoot growth rate, whereas the lowest rates were observed in trees under red 60 % and green 40 %. There was no significant impact of any of the shade net treatments on leaf area and total yield compared to the control. Compared to the open field, all shade net treatments resulted in a decrease in sunburn incidence. The highest levels of total soluble solids (TSS) were observed under green shade nets and the control group, whereas the lowest levels were detected under the red nets. Furthermore, it was observed that the green 60 % resulted in a significant increase in several parameters including titratable acidity (TA), antioxidant capacity, total phenols, total carotenoids, and color index (Lab*). Given these positive attributes, the green 60 % option is considered to be a viable choice for covering citrus trees.

Sugar is a key factor, significantly affecting fruit flavor quality through sucrose metabolism and accumulation. This study investigated the effect of short-day shading on the accumulation of soluble sugars in citrus fruits by using 'Nanfeng' tangerine (Citrus reticulata cv. Nanfeng) as materials. Results revealed that using a black net with 12 % light transmittance for 18-day shading remarkably increased soluble sugars and decreased starch content in fruits. Moreover, expresion levels of two cell wall invertase genes (CwINV6/7) and one sucrose synthase genes (CsSUS6) in the segment membrane (SM) were increased significantly at 18 DAS. On the other hand, in juice sacs (JS), expression levels of vacuolar invertase (VINV), five sucrose synthase genes (CsSUS1-5), two sucrose phosphate synthases (CsSPS1/3), some sugar transporter genes including STP7, SUT1/2/3, two SWEET genes (SWEET5/10), two vacuolar glucose transporter genes (VGT1/2), two tonoplast monosaccharide transporter genes (TMT1/2), and two citrus type I V-PPase genes (CsVPP1/2) were increased significantly at 18DAS as compared to control. Furthermore, starch degradation related gene ISA3, BAM3, GWD and PWD were also significantly induced by 18-day shading. Overall, this study suggested that short-day shading (12 %) enhances soluble sugar accumulation in citrus fruit, primarily by promoting starch degradation and soluble sugar storage in the JS, except for the increase of sucrose distribution to the fruits.

Organic acid composition has a major influence on fruit taste and quality. In general, the fruits of common citrus varieties accumulate more citrate than malate. Here, we report that a citrus variety, u2018Harukau2019 (Citrus tamurana u00D7 natsudaidai), accumulates more malate than citrate at the ripening stage. However, the underlying mechanism is unclear. Organic acid profiles were compared between u2018Harukau2019 fruits and a common Ponkan cultivar, u2018Huagan 2u2019 (Citrus reticulata), during fruit development and ripening. We found that u2018Harukau2019 fruit accumulated 75% less citrate than u2018Huagan 2u2019 fruit and that the malate content was nearly 3-fold greater in u2018Harukau2019 fruit than in u2018Huagan 2u2019 fruit. In addition, 12 aluminum-activated malate transporter (ALMT) genes were identified in the citrus genome, with CsALMT9-like being predominantly expressed in u2018Harukau2019 fruit juice sacs. An analysis of genes associated with the accumulation of citrate/malate revealed that the expression levels of genes encoding a P-type proton pump (CsPH8), a citrate/H+ symporter (CsCit), and CsALMT9-like were significantly greater in u2018Harukau2019 fruit than in u2018Huagan 2u2019 fruit. Moreover, CsCit overexpression significantly decreased the citrate content, whereas overexpressing and silencing CsALMT9-like significantly increased and decreased the malate content. In addition, shading significantly increased the expression of CsPH8 but reduced the expression of CsCit and CsALMT9-like and significantly increased the citrate content but decreased the malate content, further confirming that CsCit and CsALMT9-like regulate the accumulation of citrate and malate in u2018Harukau2019 fruit, respectively. Taken together, these data indicate that the relatively low citrate content of u2018Harukau2019 is mainly due to increases in CsCit expression and that the relatively high malate content of u2018Harukau2019 is mainly due to increase in CsALMT9-like expression. This study provides insight into the mechanisms that influence malate content in citrus fruit.

Citric acid accumulation is an essential process in citrus fruits that determines fruit flavor and marketability. The MBW complex transcription factor genes, CsAN11, CsAN1, and CsPH4 play key roles in regulating citric acid accumulation. Although how to regulate CsAN1 or CsPH4 was widely investigated, studies on the regulation of CsAN11 are scarce. In this study, we characterized the AP2/ERF (APETALA2/ethylene response factor) transcription factor gene CsAIL6, which is lowly expressed in high-acid citrus varieties and highly expressed in low-acid citrus varieties. Overexpressing CsAIL6 obviously decreased the citric acid content in citrus fruits, calli, or tomatoes, whereas silencing CsAIL6 significantly increased the fruit citric acid content. Additionally, transcript levels of CsAN11, CsAN1, and CsPH4 were significantly increased by silencing CsAIL6; only the CsAN11 transcript level was significantly decreased by overexpressing CsAIL6. Similarly, only the tomato AN11 (SIAN11) transcript level in CsAIL6 stably overexpressing fruits was markedly lower than that in wild-type (WT) fruits. Further experiments revealed that overexpressing CsAN11 significantly increased the organic acid content but had no obvious influence on the CsAIL6 transcript level; in addition, CsAIL6 only interacts with CsAN11, rather than with CsAN1 and CsPH4 of the MBW complex. Taken together, our findings verified that CsAIL6 negatively regulates citric acid accumulation through directly interacting with the WD40 protein CsAN11, which provides a new mechanism for citric acid accumulation in fruits through the regulation of the MBW complex.

Drought can promote soluble sugar accumulation in fruits by increasing the fruit sink strength. Cell wall invertase (CwINV) plays a pivotal role in determining sink strength by regulating sucrose partitioning into the extracellular matrix. Research has demonstrated that drought stress significantly increases the transcript level of citrus CwINV6, but the transcriptional mechanisms governing its regulation under drought conditions remain elusive. In this study, we characterised the MYB transcription factor gene CsMYB1 from the citrus genome. CsMYB1 is localised in the cell nucleus, and CwINV6 is localised in the cell wall. Furthermore, the transcript levels of both CsMYB1 and CwINV6 significantly increased in 'Nanfeng' tangerine fruits (Citrus reticulata) in response to drought or ABA treatment. Transient overexpression of CsMYB1 or CwINV6 promoted the accumulation of glucose and fructose in 'Nanfeng' fruits. Conversely, transient VIGS of CsMYB1 or CwINV6 resulted in the opposite trend. Additionally, stable overexpression of CsMYB1 or CwINV6 significantly increased the soluble sugar content in the fruits of the 'Micro-Tom' tomato lines. Y1H and luciferase assays confirmed that CsMYB1 can bind to the CwINV6 promoter and positively regulate its expression. Taken together, our findings reveal that drought promotes soluble sugar distribution in citrus fruits by increasing sink strength via the CsMYB1-CwINV6 module.

Excessively and randomly producing summer shoots will lead to difficulty in citrus orchard management, specially in pest and disease control. Heavy fruit load can reduce the summer shoot number. However, the mechanism is still unclear. In this study, field investigation and de-fruiting treatment confirmed that heavy fruit load reduces the number of citrus summer shoots, which is zero when the yield surpasses 3.3 kg per 125 dm3 of tree canopy. Metabolite analysis indicated that fruits at the cell expansion stage attract more soluble sugars and de-fruiting significantly increase the content of sugars and the transcript levels of sink strength-related genes, CsSUS4/5/6 to over 3.0 fold in the axillary buds. Moreover, exogenous application of some sugar-related DAMs (differently accumulated metabolites) such as sucrose obviously promoted axillary bud outgrowth. Taken together, these results confirmed that heavy fruit load plays a role in inhibiting axillary bud outgrowth or shoot branching primarily through competing for soluble sugars, which provides the basis for the inhibition of summer shoots by increasing the fruit load in citrus orchard and for the improvement of pest and disease management effectiveness.

BACKGROUND:The trifoliate orange is used as rootstock in the Citrus genus; nevertheless, propagation from cuttings is challenging due to low rooting ability. The underlying mechanism for low rooting ability is still unclear. This study aims to investigate hormonal biosynthesis and signaling cascades during Adventitious Root (AR) formation. METHODS AND RESULTS:We systematically compared trifoliate orange and Micro-Tom tomato cuttings grown in water. The results indicated Micro-Tom tomato cuttings produced ARs within 3 days, whereas trifoliate orange developed ARs at 21 days after cuttings (DAC). The key endogenous hormones contents: indole-3-acetic acid (IAA), Jasmonic acid (JA), and salicylic acid (SA), were significantly lower in trifoliate orange than Micro-Tom tomato cuttings at earlier stages 1, 2, and 3 DAC. Moreover, IAA-related and ethylene biosynthesis genes demonstrated differential expression patterns during different developmental stages between trifoliate orange and Micro-Tom tomato. Specially, the expression levels of auxin biosynthesis genes (YUCCA6/4, GH3.3/3.6) and response factor (ARF8) were significantly elevated in Micro-Tom tomato cuttings, while lower in trifoliate orange cuttings at the earliest stages. Additionally, transcripts related to auxin signaling and transport (PIN2, LAX2, AUX1, TR2a, IAA11/12) and ethylene-related transcripts (ACS3/7, ASA1, ERF003/109) were lower in trifoliate orange than Micro-Tom tomato cuttings during the earliest stages. CONCLUSIONS:Our results confirmed AR formation in trifoliate orange was delayed compared to Micro-tom tomato. The low level IAA hormones, significantly lower expression IAA and ethylene-related genes at the earliest stages could be attributed to the delay of AR formation in trifoliate orange cuttings.

Fleshy fruit taste and quality are influenced by their soluble sugar contents. In order to improve the fruit quality, it is imperative to better understand how soluble sugars accumulate in fruit vacuoles. A previous report showed that the type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruits, but its transcriptional regulation still remains elusive. In this study, the NAC transcription factor gene CsNAC47 was functionally characterized and found to localize predominantly within the nucleus, consistent with its predicted role in transcriptional regulation. Moreover, drought stress markedly upregulated the transcript levels of CsNAC47 and CsVPP1/2 in Nanfeng tangerine (Citrus reticulata) fruits, indicating their potential role in sugars accumulation. Transient overexpression of CsNAC47 and CsVPP1/2 led to enhanced accumulation of soluble sugars in citrus fruit juice sacs, while virus-induced gene silencing (VIGS) of CsNAC47 and CsVPP1/2 inhibited sugar accumulation. Stable transformation in Micro-Tom tomato indicated that overexpression of CsNAC47 and CsVPP1/2 significantly elevated the vacuolar storage of soluble sugar contents. We found out through yeast one-hybrid screening assay and dual luciferase assays that CsNAC47 directly binds to the promoters of CsVPP1/2 and regulates their gene expression. This study offers novel insights into the transcriptional regulation of soluble sugar accumulation in citrus fruits under drought stress, specifically through the interaction between CsNAC47 and CsVPP1/2, which will ultimately contribute to improving fruit quality in the future.

Trifoliate orange (Poncirus trifoliata) is the commonly used rootstock for citrus grafting propagation. Accelerating the secondary growth of trifoliate orange stem is important to reduce the grafting propagation time. However, the factors and molecular mechanisms that may accelerate its secondary growth are still largely unknown. In this study, we found that exogenous auxin application dramatically increased the stem diameter, xylem cell layers, and xylem width in trifoliate orange as compared to the control group. Differently, exogenous cytokinin resulted in a marked increase of branches, while gibberellin or 1-aminocyclopropane-1-carboxylic acid (ACC) treatment didn't significantly influence the stem diameter. Subsequently, qRT-PCR analysis showed that exogenous auxin notably upregulated transcript levels of key secondary growth-related genes, including CYCD3, C3H17, SND1, WOX4, and PXY, as well as Auxin Response Factor 7 gene (ARF7). Subcellular localization and transcriptional activity analysis indicated that ARF7 functions as a transcriptional activator. We then conducted yeast one-hybrid assays and revealed that ARF7 can bind to the promoters of CYCD3, C3H17, and WOX4; furthermore, dual-luciferase assays indicated ARF7 can enhance their expression. In conclusion, our results demonstrated that auxin promotes the secondary growth of trifoliate orange stems at least through ARF7 inducing CYCD3, C3H17, and WOX4 expression. These findings provide a novel clue in research for shortening grafting propagation time and improving seedling efficiency in the citrus industry.

Topping, an important tree shaping and pruning technique, can promote the outgrowth of citrus axillary buds. However, the underlying molecular mechanism is still unclear. In this study, spring shoots of Citrus reticulata 'Huagan No.2' were topped and transcriptome was compared between axillary buds of topped and untopped shoots at 6 and 11 days after topping (DAT). 1944 and 2394 differentially expressed genes (DEGs) were found at 6 and 11 DAT, respectively. KEGG analysis revealed that many DEGs were related to starch and sucrose metabolism, signal transduction of auxin, cytokinin and abscisic acid. Specially, transcript levels of auxin synthesis, transport, and signaling-related genes (SAURs and ARF5), cytokinin signal transduction related genes (CRE1, AHP and Type-A ARRs), ABA signal responsive genes (PYL and ABF) were up-regulated by topping; while transcript levels of auxin receptor TIR1, auxin responsive genes AUX/IAAs, ABA signal transduction related gene PP2Cs and synthesis related genes NCED3 were down-regulated. On the other hand, the contents of sucrose and fructose in axillary buds of topped shoots were significantly higher than those in untopped shoots; transcript levels of 16 genes related to sucrose synthase, hexokinase, sucrose phosphate synthase, endoglucanase and glucosidase, were up-regulated in axillary buds after topping. In addition, transcript levels of genes related to trehalose 6-phosphate metabolism and glycolysis/tricarboxylic acid (TCA) cycle, as well to some transcription factors including Pkinase, Pkinase_Tyr, Kinesin, AP2/ERF, P450, MYB, NAC and Cyclin_c, significantly responded to topping. Taken together, the present results suggested that topping promoted citrus axillary bud outgrowth through comprehensively regulating plant hormone and carbohydrate metabolism, as well as signal transduction. These results deepened our understanding of citrus axillary bud outgrowth by topping and laid a foundation for further research on the molecular mechanisms of citrus axillary bud outgrowth.

Sugar accumulation is influenced by various fertilizer treatments, of which potassium spraying is the most effective. However, the effect of different potassium sources as a foliar application at different fruit development stages on citrus fruits is still unclear. In this study, three different potassium fertilizers and one water (F1: 0.65% KNO3, F2: 0.88% KH2PO4, F3: 0.56% K2SO4, and F4: water) were sprayed on Citrus reticulata cv. Nanfeng at cell division, cell expansion, fruit ripening, and throughout fruit developmental stages, respectively. Results showed that the six-time K2SO4 application had the best function in enhancing fruit physiological attributes such as fruit weight and the total carotenoids; also, this treatment significantly decreased TA (titratable acid), increased fruit TSS (total soluble solids), soluble sugar content, and TSS:TA ratio. Furthermore, K2SO4 spraying obviously increased the expression of CsCWINV-2/6 in the segment membrane, and CsSUT-1/2 and CsVPP-1/2 in fruit juices. Taken together, six-time application of K2SO4 throughout fruit developmental stages produced better fruit quality, at least through enhancing sink strength and promoting sugar transportation in citrus fruits. This study might effectively contribute to maximizing fruit quality and its marketability.

Accumulation of soluble sugars promotes the formation of fruit nutritional quality and flavor while proper application of Magnesium (Mg) can increase the sugar content in citrus fruits. However, the underlying mech-anism is still unclear. In the present study by using 'Huagan No.2 ' (Citrus reticulate cv. Huagan No.2) as research material, foliar spraying of MgSO4 with two or three times was performed during the fruit expansion period. Results indicated that foliar spraying of Mg significantly increased the total soluble solids (TSS), as well as the contents of sucrose, fructose and glucose in fruits; moreover, the activities of sucrose metabolizing enzymes including sucrose synthase (SS), sucrose phosphate synthesis (SPS) and acid invertase (AI), as well as the tran-script levels of SS2, SPS1-3 and vacuolar acid invertase gene (VINV) were significantly increased in the Mg-treated fruits. In addition, the transcript levels of the sucrose transport-related gene (SUT3) and the vacuolar pyrophosphate proton pump type I genes (CsVPP-1 and CsVPP-2) in the fruits were also significantly increased by Mg treatment. These results were further verified by soaking the fruit-bearing branches in 0.5% MgSO4 solution. In conclusion, foliar spraying Mg with two or three times promotes soluble sugar accumulation in fruits through enhancing the sink strength by inducing the activity of sucrose synthase via inducing SS2 transcript level, and improving sucrose transport ability by inducing transcript levels of SUT3, CsVPP-1, CsVPP-2 and VINV.

Abstract Soluble sugars primarily contribute to fruit flavouring and are crucial for fruit quality. Carbohydrate accumulation in fruits is a multifaceted process that can be influenced by their biosynthesis capacity, sink strength, homeostasis, and vacuolar storage ability. Moreover, total soluble sugar contents in fruits also vary among different varieties/species and environmental conditions. Numerous studies have elucidated the carbohydrate regulatory mechanism in fruits; however, there is a knowledge gap regarding the most important contributor to soluble sugar accumulation in perennial fruits. Here, we briefly discussed the recent advances, including carbohydrate long-distance transportation, metabolism and vacuolar storage, and transcript factors for soluble sugar accumulation in fruits. Most importantly, we elucidated that the sink strength in fruit is the most crucial factor that can trap more soluble sugars by maintaining continuous unloading to fruit by genes of cell wall invertase; moreover, genes of sucrose transporters/pyrophosphatase play key roles in vacuolar storage of soluble sugars in perennial fruits. This review delivers a comprehensive summary of recent findings for soluble sugar regulation and fruit quality improvement, which may facilitate sugar optimisation in fruit crops for enhanced fruit quality.