Vacuolar Invertase Research Articles

Functional Characterization of a Cucumber (Cucumis sativus L.) Vacuolar Invertase, CsVI1, Involved in Hexose Accumulation and Response to Low Temperature Stress.

Cucumber (Cucumis sativus L.), an important vegetable plant species, is susceptible to low temperature stress especially during the seedling stage. Vacuolar invertase (VI) plays important roles in plant responses to abiotic stress. However, the molecular and biochemical mechanisms of VI function in cucumber, have not yet been completely understood and VI responses to low temperature stress and it functions in cold tolerance in cucumber seedlings are also in need of exploration. The present study found that hexose accumulation in the roots of cucumber seedlings under low temperature stress is closely related to the observed enhancement of invertase activity. Our genome-wide search for the vacuolar invertase (VI) genes in cucumber identified the candidate VI-encoding gene CsVI1. Expression profiling of CsVI1 showed that it was mainly expressed in the young roots of cucumber seedlings. In addition, transcriptional analysis indicated that CsVI1 expression could respond to low temperature stress. Recombinant CsVI1 proteins purified from Pichia pastoris and Nicotiana benthamiana leaves could hydrolyze sucrose into hexoses. Further, overexpression of CsVI1 in cucumber plants could increase their hexose contents and improve their low temperature tolerance. Lastly, a putative cucumber invertase inhibitor was found could form a complex with CsVI1. In summary, these results confirmed that CsVI1 functions as an acid invertase involved in hexose accumulation and responds to low temperature stress in cucumber seedlings.

Transcriptomics‐based identification and characterization of genes related to sugar metabolism in ‘Hongshuijing’ pitaya

Sugar composition not only affects fruit flavor but is also an important determinant of fruit taste and consumer preference. In this study, changes in the sugar content and sugar-metabolizing enzymes were investigated from different sections of various fruit development phases of ‘Hongshuijing’ pitaya (Hylocereus monacanthus). Genes related to sugar metabolism were also screened by transcriptome analyses. The results indicated that glucose was the major sugar in mature pitaya fruit, and was mainly regulated by vacuolar acid invertase (VAI) and sucrose synthase (SS) (degradative direction). Sugar accumulation varied in pulp between different sections of the pitaya fruit. VAI, neutral invertase (NI) and SS (degradative direction) are crucial enzymes for sugar accumulation in pitaya. The expression of 17 genes related to sucrose metabolism obtained from seven databases [NCBI non-redundant protein database (Nr), NCBI non-redundant nucleotide sequence database (Nt), EuKaryotic Orthologous Groups (KOG), The Protein Families (Pfam), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, and Gene Ontology (GO)] were analyzed in different pitaya pulp sections. HpVAI1 had the highest relative expression level on the 29th day after pollination (DAP). Positive correlations were found between HpVAI1 expression and VAI activity; HpNI4 and NI activity; HpSS2, HpSS5, and SS activity (synthetic direction), indicating that HpVAI1, HpNI4, and HpSS2 and HpSS5 were involved in the regulation of VAI, NI, and SS (synthetic direction), respectively. HpVAI1 and HpNI4 regulated sucrose degradation and the accumulation of glucose and fructose, while HpSS2 and HpSS5 regulated sucrose synthesis. These results suggest that HpVAI1 plays a key role in sugar metabolism during fruit development of ‘Hongshuijing’ pitaya. The results of this study provide new information about sugar metabolism in pitaya fruit that could help improve fruit quality and the breeding of new cultivars.

Responsiveness of Early Response to Dehydration Six-Like Transporter Genes to Water Deficit in Arabidopsis thaliana Leaves.

Drought is one of the main abiotic stresses, which affects plant growth, development, and crop yield. Plant response to drought implies carbon allocation to sink organs and sugar partitioning between different cell compartments, and thereby requires the involvement of sugar transporters (SUTs). Among them, the early response to dehydration six-like (ESL), with 19 members in Arabidopsis thaliana, form the largest subfamily of monosaccharide transporters (MSTs) still poorly characterized. A common feature of these genes is their involvement in plant response to abiotic stresses, including water deficit. In this context, we carried out morphological and physiological phenotyping of A. thaliana plants grown under well-watered (WW) and water-deprived (WD) conditions, together with the expression profiling of 17 AtESL genes in rosette leaves. The drought responsiveness of 12 ESL genes, 4 upregulated and 8 downregulated, was correlated to different water statuses of rosette leaves. The differential expression of each of the tandem duplicated AtESL genes in response to water stress is in favor of their plausible functional diversity. Furthermore, transfer DNA (T-DNA) insertional mutants for each of the four upregulated ESLs in response to water deprivation were identified and characterized under WW and WD conditions. To gain insights into global sugar exchanges between vacuole and cytosol under water deficit, the gene expression of other vacuolar SUTs and invertases (AtTMT, AtSUC, AtSWEET, and AtβFRUCT) was analyzed and discussed.

PpCBF6 is a low-temperature-sensitive transcription factor that binds the PpVIN2 promoter in peach fruit and regulates sucrose metabolism and chilling injury

PpVIN2 is the only vacuolar invertase (VIN) gene in peaches that is sensitive to cold. PpVIN2 functions in sucrose decomposition and chilling injury (CI), but it is unknown how low temperatures induce PpVIN2. Here, we confirmed low-temperature activation of PpVIN2 transcription from a PpVIN2 promoter using a transgenic tobacco model. C-repeat binding factor (CBF) is a conserved cold-responsive gene in plants, and PpCBF6 expression increases rapidly when peaches suffer from cold stress. We studied the interaction between PpCBF6 and PpVIN2 both in vivo and in vitro. Using yeast one-hybrid assay and electrophoretic mobility shift assay, we confirmed that PpCBF6 binds to the promoter of PpVIN2, and results from a dual luciferase assay indicate that PpCBF6 inhibits the promoter activity of PpVIN2. In vivo, transient overexpression of PpCBF6 decreases PpVIN2 expression and VIN activity, and increases sucrose levels. In addition, when PpCBF6 is targeted using virus-induced gene silencing (VIGS), PpVIN2 expression and activity increase, accompanied by a decrease in sucrose content. Compared to the control group, exogenous methyl jasmonate (MeJA) treatment caused a higher expression of PpCBF6, and reduced the rapid rise of PpVIN2 expression level. This resulted in the lower VIN activity and higher sucrose content in MeJA-treated peach fruit, thereby reducing the CI. In general, we proved that PpCBF6 retarded the degradation of sucrose by inhibiting the increased expression of PpVIN2, which improves the chilling resistance of peach fruit.

Key Regulators of Sucrose Metabolism Identified through Comprehensive Comparative Transcriptome Analysis in Peanuts.

Sucrose content is a crucial indicator of quality and flavor in peanut seed, and there is a lack of clarity on the molecular basis of sucrose metabolism in peanut seed. In this context, we performed a comprehensive comparative transcriptome study on the samples collected at seven seed development stages between a high-sucrose content variety (ICG 12625) and a low-sucrose content variety (Zhonghua 10). The transcriptome analysis identified a total of 8334 genes exhibiting significantly different abundances between the high- and low-sucrose varieties. We identified 28 differentially expressed genes (DEGs) involved in sucrose metabolism in peanut and 12 of these encoded sugars will eventually be exported transporters (SWEETs). The remaining 16 genes encoded enzymes, such as cell wall invertase (CWIN), vacuolar invertase (VIN), cytoplasmic invertase (CIN), cytosolic fructose-bisphosphate aldolase (FBA), cytosolic fructose-1,6-bisphosphate phosphatase (FBP), sucrose synthase (SUS), cytosolic phosphoglucose isomerase (PGI), hexokinase (HK), and sucrose-phosphate phosphatase (SPP). The weighted gene co-expression network analysis (WGCNA) identified seven genes encoding key enzymes (CIN, FBA, FBP, HK, and SPP), three SWEET genes, and 90 transcription factors (TFs) showing a high correlation with sucrose content. Furthermore, upon validation, six of these genes were successfully verified as exhibiting higher expression in high-sucrose recombinant inbred lines (RILs). Our study suggested the key roles of the high expression of SWEETs and enzymes in sucrose synthesis making the genotype ICG 12625 sucrose-rich. This study also provided insights into the molecular basis of sucrose metabolism during seed development and facilitated exploring key candidate genes and molecular breeding for sucrose content in peanuts.

Advances about the Roles of Membranes in Cotton Fiber Development.

Cotton fiber is an extremely elongated single cell derived from the ovule epidermis and is an ideal model for studying cell development. The plasma membrane is tremendously expanded and accompanied by the coordination of various physiological and biochemical activities on the membrane, one of the three major systems of a eukaryotic cell. This review compiles the recent progress and advances for the roles of the membrane in cotton fiber development: the functions of membrane lipids, especially the fatty acids, sphingolipids, and phytosterols; membrane channels, including aquaporins, the ATP-binding cassette (ABC) transporters, vacuolar invertase, and plasmodesmata; and the regulation mechanism of membrane proteins, such as membrane binding enzymes, annexins, and receptor-like kinases.

Highest Defoliation Tolerance in Amaranthus cruentus Plants at Panicle Development Is Associated With Sugar Starvation Responses.

Defoliation tolerance (DT) in Amaranthus cruentus is known to reach its apex at the panicle emergence (PE) phase and to decline to minimal levels at flowering (FL). In this study, defoliation-induced changes were recorded in the content of non-structural carbohydrates and raffinose family oligosaccharides (RFOs), and in the expression and/or activity of sugar starvation response-associated genes in plants defoliated at different vegetative and reproductive stages. This strategy identified sugar-starvation-related factors that explained the opposite DT observed at these key developmental stages. Peak DT at PE was associated with increased cytosolic invertase (CI) activity in all organs and with the extensive induction of various class II trehalose-phosphate synthase (TPS) genes. Contrariwise, least DT at FL coincided with a sharp depletion of starch reserves and with sucrose (Suc) accumulation, in leaves and stems, the latter of which was consistent with very low levels of CI and vacuolar invertase activities that were not further modified by defoliation. Increased Suc suggested growth-inhibiting conditions associated with altered cytosolic Suc-to-hexose ratios in plants defoliated at FL. Augmented cell wall invertase activity in leaves and roots, probably acting in a regulatory rather than hydrolytic role, was also associated with minimal DT observed at FL. The widespread contrast in gene expression patterns in panicles also matched the opposite DT observed at PE and FL. These results reinforce the concept that a localized sugar starvation response caused by C partitioning is crucial for DT in grain amaranth.

Differential transcription pathways associated with rootstock-induced dwarfing in breadfruit (Artocarpus altilis) scions

BackgroundBreadfruit (Artocarpus altilis) is a traditional staple tree crop throughout the tropics. Through interspecific grafting, a dwarf phenotype with over 50% reduction in plant height was identified when marang (Artocarpus odoratissimus) rootstocks were used. However, the molecular mechanism underlying the rootstock-induced breadfruit dwarfing is poorly understood.ResultsAn RNA-sequencing study of breadfruit scions at 22 months after grafting identified 5409 differentially expressed genes (DEGs) of which 2069 were upregulated and 3339 were downregulated in scion stems on marang rootstocks compared to those on self-graft. The DEGs were predominantly enriched for biological processes involved in carbon metabolism, cell wall organization, plant hormone signal transduction and redox homeostasis. The down-regulation of genes encoding vacuolar acid invertases and alkaline/neutral invertases, was consistent with the decreased activity of both enzymes, accompanying with a higher sucrose but lower glucose and fructose levels in the tissues. Key genes of biosynthetic pathways for amino acids, lipids and cell wall were down regulated, reflecting reduction of sucrose utilisation for stem growth on dwarfing rootstocks. Genes encoding sugar transporters, amino acid transporters, choline transporters, along with large number of potassium channels and aquaporin family members were down-regulated in scion stems on marang rootstocks. Lower activity of plasma membrane H+-ATPase, together with the predominance of genes encoding expansins, wall-associated receptor kinases and key enzymes for biosynthesis and re-modelling of cellulose, xyloglucans and pectins in down-regulated DGEs suggested impairment of cell expansion. Signalling pathways of auxin and gibberellin, along with strigolacton and brassinosteroid biosynthetic genes dominated the down-regulated DEGs. Phenylpropanoid pathway was enriched, with key lignin biosynthetic genes down-regulated, and flavonoid biosynthetic genes upregulated in scions on marang rootstocks. Signalling pathways of salicylic acid, jasmonic acid, ethylene and MAPK cascade were significantly enriched in the upregulated DEGs.ConclusionsRootstock-induced disruption in pathways regulating nutrient transport, sucrose utilisation, cell wall biosynthesis and networks of hormone transduction are proposed to impair cell expansion and stem elongation, leading to dwarf phenotype in breadfruit scions. The information provides opportunity to develop screening strategy for rootstock breeding and selection for breadfruit dwarfing.

Comparative transcriptome analysis reveals key genes potentially related to organic acid and sugar accumulation in loquat.

Organic acids and sugars are the primary components that determine the quality and flavor of loquat fruits. In the present study, major organic acids, sugar content, enzyme activities, and the expression of related genes were analyzed during fruit development in two loquat cultivars, 'JieFangZhong' (JFZ) and 'BaiLi' (BL). Our results showed that the sugar content increased during fruit development in the two cultivars; however, the organic acid content dramatically decreased in the later stages of fruit development. The differences in organic acid and sugar content between the two cultivars primarily occured in the late stage of fruit development and the related enzymes showed dynamic changes in activies during development. Phosphoenolpyruvate carboxylase (PEPC) and mNAD malic dehydrogenase (mNAD-MDH) showed higher activities in JFZ at 95 days after flowering (DAF) than in BL. However, NADP-dependent malic enzyme (NADP-ME) activity was the lowest at 95 DAF in both JFZ and BL with BL showing higher activity compared with JFZ. At 125 DAF, the activity of fructokinase (FRK) was significantly higher in JFZ than in BL. The activity of sucrose synthase (SUSY) in the sucrose cleavage direction (SS-C) was low at early stages of fruit development and increased at 125 DAF. SS-C activity was higher in JFZ than in BL. vAI and sucrose phosphate synthase (SPS) activities were similar in the two both cultivars and increased with fruit development. RNA-sequencing was performed to determine the candidate genes for organic acid and sugar metabolism. Our results showed that the differentially expressed genes (DEGs) with the greated fold changes in the later stages of fruit development between the two cultivars were phosphoenolpyruvate carboxylase 2 (PEPC2), mNAD-malate dehydrogenase (mNAD-MDH), cytosolic NADP-ME (cyNADP-ME2), aluminum-activated malate transporter (ALMT9), subunit A of vacuolar H+-ATPase (VHA-A), vacuolar H+-PPase (VHP1), NAD-sorbitol dehydrogenase (NAD-SDH), fructokinase (FK), sucrose synthase in sucrose cleavage (SS-C), sucrose-phosphate synthase 1 (SPS1), neutral invertase (NI), and vacuolar acid invertase (vAI). The expression of 12 key DEGs was validated by quantitative reverese transcription PCR (RT-qPCR). Our findings will help understand the molecular mechanism of organic acid and sugar formation in loquat, which will aid in breeding high-quality loquat cultivars.

Performance of Hevea brasiliensis under drought conditions on osmoregulation and antioxidant activity through evaluation of vacuolar invertase and reducing sugars

Rubber tree cultivation is limited in many regions by abiotic factors such as drought. We investigated the biochemical mechanisms responsible for responses to, and recovery from, drought conditions during the establishment phase of four high latex producing rubber tree clones (RRIM600, IAC40, PR255 and GT1). Five-month-old plants were exposed to 32 days of water restriction, followed by 15 days of soil rehydration. Leaf area, as well as their osmolyte accumulations, saccharolytic enzyme activity, and oxidative stress markers, were accompanied. Although clones IAC40 and PR255 responded more precociously to drought conditions, halting leaf expansion before clones GT1 and RRIM600, they demonstrated slow recuperation after reestablishing irrigation. The greater tolerances of clones RRIM600 and GT1 to drought conditions were related to greater vacuolar invertase (VINV) activity in their leaves, which guaranteed more significant accumulations of vacuolar reducing sugars (RS). Similar to RS, glycine betaine accumulations were related to osmoprotection and to reducing oxidative damage (lipidic peroxidation) caused by water deficit conditions. The observed decreases in cytosol neutral invertase (AINV) and cell wall insoluble invertase (CWINV) activities, which resulted in cytosol hexose decreases, may be related to increases in antioxidant enzyme (superoxide dismutase and ascorbate peroxidase) activities in the leaves in response to water deficit conditions. As such, the introduction of specific sugars (RS) and the modulation of key carbon metabolism enzymes, such as VINV, are promising strategies for promoting drought tolerance in rubber tree clones.

StRAP2.3, an ERF\u2010VII transcription factor, directly activates StInvInh2 to enhance cold-induced sweetening resistance in potato

Potato invertase inhibitor (StInvInh2) positively regulates cold-induced sweetening (CIS) resistance by inhibiting the activity of vacuolar invertase. The distinct expression patterns of StInvInh2 have been thoroughly characterized in different potato genotypes, but the related CIS ability has not been characterized. The understanding of the regulatory mechanisms that control StInvInh2 transcription is unclear. In this study, we identified an ERF‐VII transcription factor, StRAP2.3, that directly regulates StInvInh2 to positively modulate CIS resistance. Acting as a nuclear-localized transcriptional activator, StRAP2.3 directly binds the ACCGAC cis-element in the promoter region of StInvInh2, enabling promoter activity. Overexpression of StRAP2.3 in CIS-sensitive potato tubers induced StInvInh2 mRNA abundance and increased CIS resistance. In contrast, silencing StRAP2.3 in CIS-resistant potato tubers repressed the expression of StInvInh2 and decreased CIS resistance. We conclude that cold-responsive StInvInh2 is due to the binding of StRAP2.3 to the ACCGAC cis-element in the promoter region of StInvInh2. Overall, these findings indicate that StRAP2.3 directly regulates StInvInh2 to positively modulate CIS resistance, which may provide a strategy to improve the processing quality of potatoes.

Comprehensive analysis of sucrolytic enzyme gene families in carnation (Dianthus caryophyllus L.)

Sucrose plays crucial roles in growth and responses of plants to the environment, including those in ornamental species. During post-harvest handling of cut flowers, sucrose degradation is an essential process of inter- and intra-cellular carbon partitioning affecting flower opening and senescence and, subsequently, flower quality. However, complete information about the molecular basis of sucrose degradation in ornamental flowers, which can be catalyzed by two kinds of sucrolytic enzymes, invertase (INV), and sucrose synthase (SUS), is not available from past reports. The present study shows that sucrose treatment of carnation (Dianthus caryophyllus L.) florets increased starch content in petals, accompanied by decreased vacuolar INV (VIN) activity and increased SUS activity. However, hypoxic treatment of carnation florets decreased sucrose content and cell-wall INV (CWIN) activity in petals. In silico analysis using the carnation genome database identified six CWIN, three VIN, eight cytoplasmic INV (CIN), and five SUS genes. Real-time RT-PCR analysis confirmed that these genes are differentially expressed in carnation petals in response to sucrose and hypoxic treatments, partially corresponding to the changes in enzyme activities. In contrast to DcSUS1 (Dca4507.1), a SUS gene already reported in carnation, which showed preferential expression under aerated conditions, the expression of DcSUS2 (Dca22218.1), an undescribed carnation SUS gene, was enhanced under hypoxia similarly to an alcohol dehydrogenase gene DcADH1 (Dca18671.1). These results suggest that sugar metabolism in carnation petals is regulated in response to environmental cues, accompanied by modulated activities and gene expression of a set of sucrolytic enzymes.

Regulation of Maize Kernel Carbohydrate Metabolism by Abscisic Acid Applied at the Grain-Filling Stage at Low Soil Water Potential

A water deficit during the grain-filling stage increases the frequency of yield loss in maize (Zea mays L.). Abscisic acid (ABA) plays a regulatory role in many stages of plant growth; however, its effects on sucrose-metabolizing enzyme activities under stress are poorly understood. The activities of cell-wall-bound acid invertase, vacuolar invertase, cytoplasmic invertase, and sucrose synthase decreased continuously under drought stress, whereas ABA treatment partially restored these activities. In addition, the increase and development of sucrose content under drought stress were related to invertase activity. Up-regulation of the activities and gene expression of cell-wall-bound acid invertase and vacuolar invertase with ABA treatment contributed to the increase in the number of rows and number of grains per row. Furthermore, ABA inhibited the increase in the length of the bald tip. Compared with the control group, water stress significantly reduced the yield index, with the lowest yield index on the 10th day of stress. These results suggest that the increase in ABA-induced sucrose-metabolizing enzyme activity might be an effective mechanism to improve maize drought resistance at the grain-filling stage.

Polystyrene microplastics disturb the redox homeostasis, carbohydrate metabolism and phytohormone regulatory network in barley

As emerging contaminants, microplastics (mPS, <5 mm) have been reported to adversely affect the plant growth; however, the mechanisms of mPS-induced growth limitation are rarely known. Here, it was found that the plastic particles were absorbed and accumulated in barley plants, which limited the development of rootlets. The mPS-treated plants had significantly higher concentrations of H2O2 and O2- in roots than the control. The mPS significantly increased the activities of dehydroascorbate reductase, glutathione reductase, ADP-Glucose pyrophosphorylase, fructokinase and phosphofructokinase, while decreased the activities of cell wall peroxidase, vacuolar invertase, sucrose synthase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and phosphoglucoisomerase in roots. The changes in activities of carbohydrate and ROS metabolism enzymes in leaves showed a different trend from that in roots. The mPS plants possessed a higher trans-zeatin concentration while lower concentrations of indole-3-acetic acid, indole-3-butyric acid and dihydrozeatin than the control plants in leaves. However, the phytohormone changes in roots were distinct from those in leaves under mPS. In addition, significant correlations between enzyme activities and phytohormone concentrations were found. It was suggested that the phytohormone regulatory network plays key roles in regulating the activities of key enzymes involved in carbohydrate and ROS metabolisms in response to mPS in barley.

Identification and characterization of invertase family genes reveal their roles in vacuolar sucrose metabolism during Pyrus bretschneideri Rehd. fruit development

23 invertase (PbrInvs) genes, including eight vacuolar invertases (PbrvacInvs), five cell wall invertases (PbrcwInvs) and 10 alkaline/neutral invertases (PbrA/N-Invs), were identified from P. bretschneideri Rehd. genome, with diverse chromosome locations, cis-acting elements, gene structures and motifs. Their expression profiles were tissue-specific, and postharvest light or temperature treatment would alter their expression profiles. During ‘Dangshansuli’ pear development, in association with visual/inner quality change was the alternations of invertase activity and the expression profiles of PbrInvs. In combination with results of subcellular sugar distribution as well as correlation analysis among sugar content, invertase activity and PbrInv mRNA abundance, PbrvacInv1 might be involved in sucrose decomposition during pear development. PbrvacInv1-GFP fusion protein mainly accumulated on the tonoplast (vacuolar membrane); meanwhile, transient overexpression of PbrvacInv1 in pear fruit would upregulate vacInv activity, causing higher fructose and lower sucrose when compared with that of the control. Furthermore, invertase inhibitor 5 (PbrInvInh5) could interact with PbrvacInv1.

Role of sucrose phosphate synthase and vacuolar invertase in postharvest sweetening of immature sweetpotato tuberous roots (Ipomoea batatas (L.) Lam cv ‘Xinxiang’)

Postharvest sweetening can improve the eating quality of sweetpotato, particularly of immature sweetpotato. In this study, soluble sugar content, activities of sucrose phosphate synthase (SPS) and vacuolar invertase (VIN) enzymes and expression of three IbSPSs, five IbVINs and two vacuolar invertase inhibitory factor (IbVIFs) genes were examined in immature sweetpotato cv ‘Xinxiang’ during 30 days storage at 20, 13 and 4 °C. Results showed that sucrose content increased at all temperatures, while glucose and fructose mainly increased at 4 °C. However, chilling injury developed at 4 °C and postharvest sweetening at 20 °C is potentially a safer option. The sucrose level was correlated with SPS activity and SPS activity is not manipulated at transcriptional stage. Fructose/glucose content was correlated with VIN activity. Transcript level of IbVIN1 was correlated with VIN activity and sweetness index in sweetpotato roots. Therefore, IbVIN1 could be a candidate gene in cultivating variety high in sweetness.

Coupling kinetic models and advection–diffusion equations. 2. Sensitivity analysis of an advection–diffusion–reaction model

Abstract The accompanying paper (Uys et al., in silico Plants, 2021: diab013) presented a core model of sucrose accumulation within the advection–diffusion–reaction framework, which is able to capture the spatio-temporal evolution of the system from a set of initial conditions. This paper presents a sensitivity analysis of this model. Because this is a non-steady-state model based on partial differential equations, we performed the sensitivity analysis using two approaches from engineering. The Morris method is based on a one-at-a-time design, perturbing parameters individually and calculating the influence on model output in terms of elementary effects. Fourier amplitude sensitivity test (FAST) is a global sensitivity analysis method, where all parameters are perturbed simultaneously, oscillating at different frequencies, enabling the calculation of the contribution of each parameter through Fourier analysis. Overall, both methods gave similar results. Perturbations in reactions tended to have a large influence on their own rate, as well as on directly connected metabolites. Sensitivities varied both with the time of the simulation and the position along the sugarcane stalk. Our results suggest that vacuolar sucrose concentrations are most sensitive to vacuolar invertase in the centre of the stalk, but that phloem unloading and vacuolar sucrose uptake also contribute, especially towards the stalk edges. Sucrose in the phloem was most sensitive to phloem loading at the nodes, but most sensitive to phloem unloading in the middle of the internodes. Sink concentrations of sucrose in the symplast were most sensitive to phloem unloading in the middle of the internodes, but at the nodes cytosolic invertase had the greatest effect.

Effect of excessive nitrogen on levels of amino acids and sugars, and differential response to post-harvest cold storage in potato (Solanum tuberosum L.) tubers

Nitrogen (N) is an important nutrient for increased potato tuber yield. However, excessive N can decrease tuber quality. Furthermore, the impact of optimal and higher N levels of potato tuber metabolic profile at harvest and cold storage remains unclear. This study aimed to investigate the metabolic profiling of free amino acids and sugars in potato tubers affected by different nitrogen levels (optimal, ON; and excessive, EN) at harvest (AH) and cold storage (CS) (~4 °C, 4 weeks) through untargeted GC–TOF–MS, and targeted UHPLC–QqQ–MS. Carbohydrate content and vacuolar invertase activity (IV) were determined. Principal component analysis of metabolite data indicated a distinct separation between ON and EN treatments at harvest and cold storage. Multivariate data analysis revealed that sucrose, reducing sugars, and free asparagine were the most altered metabolites (VIP > 1 and P < 0.05), which were involved in starch and sucrose metabolism, and alanine, aspartate and glutamate metabolism. At harvest, the absolute contents of various free amino acids including asparagine were higher (by 1.3–1.5 fold) in the EN treatment than ON treatment, and this difference was maintained at 4-week cold storage. Under the EN treatment, tuber maturity was reduced, and sucrose accumulation was increased at harvest, while IV was increased after cold storage, reducing sugar also accumulated. These results highlighted the negative effects of EN on free amino acid and sugars metabolism in the post–harvest tubers and provided useful information for understanding the underpinning physiological mechanisms.

PpINH1, an invertase inhibitor, interacts with vacuolar invertase PpVIN2 in regulating the chilling tolerance of peach fruit

Sucrose metabolism, particularly the decomposition of sucrose by invertase, plays a central role in plant responses to cold stress. Invertase inhibitors (INHs) evolved in higher plants as essential regulators of sucrose metabolism. By limiting invertase activity, INHs keep cellular sugar levels elevated, which provides enhanced protection to plants under stress. Our results showed that the expression of PpVIN2, the only vacuolar invertase (VIN) gene in peach fruit sensitive to chilling temperatures, increases significantly during cold storage, while VIN enzyme activity increases more modestly. We also found that peach fruit transiently overexpressing PpINH1 had decreased VIN activity. Interactions of PpINH1 and PpVIN2 with recombinant proteins were shown by yeast two-hybrid assays and bimolecular fluorescence complementation assays, as well as in vitro. During cold storage, trehalose-treated peach fruit had significantly increased PpINH1 expression, decreased VIN enzyme activity, and significantly higher sucrose content than did untreated fruit. As a result, the treated fruit had enhanced resistance to chilling injury. Collectively, our data show that the post-translational repression of VIN enzyme activity by PpINH1 helps maintain sucrose levels in peach fruit during cold storage, thereby improving resistance to chilling injury.

The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively affect lateral root development by repressing the vacuolar invertase VIN2 in Arabidopsis.

The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively modulate lateral root development by repressing vacuolar invertase VIN2 activity. Lateral root (LR) architecture greatly affects the efficiency of nutrient absorption and the anchorage of plants. Although the internal phytohormone regulatory mechanisms that control LR development are well known, how external nutrients influence lateral root development remains elusive. Here, we characterized the function of two FK506-binding proteins, namely, FKBP15-1 and FKBP15-2, in Arabidopsis. FKBP15-1/15-2 genes were expressed prominently in the vascular bundles of the root basal meristem region, and the FKBP15-1/15-2 proteins were localized to the endoplasmic reticulum of the cells. Using IP-MS, Co-IP, and BiFC assays, we demonstrated that FKBP15-1 and FKBP15-2 interacted with vacuolar invertase 2 (VIN2). Compared to Col-0 and the single mutants, the fkbp15-1fkbp15-2 double mutant had more LRs, and presented higher sucrose catalytic activity. Moreover, genetic analysis showed genetic epistasis of VIN2 over FKBP15-1/FKBP15-2 in controlling LR development. Our results indicate that FKBP15-1 and FKBP15-2 participate in the control of LR number by inhibiting the catalytic activity of VIN2. Owing to the conserved peptidylprolyl cis-trans isomerase activity of FKBP family proteins, our results provide a clue for further analysis of the interplay between lateral root development and protein modification by FKBPs.