Malate Accumulation Research Articles

Transcription factors PpWRKY50 and PpWRKY75 play positive roles in malate accumulation in pear

Transcription factors PpWRKY50 and PpWRKY75 play positive roles in malate accumulation in pear

Senescence metabolomics of <i>Nicotiana tabacum</i> L. VBI-0 heterotrophic suspension cultures

BACKGROUND: Heterotrophic cell cultures are widely used as a model in plant biology. During a culture cycle the composition of the medium changes: the sucrose and other substrates are depleted, metabolism products are accumulated and the density increases. Finally, arrest of a growth is followed by cell death in a short time. These processes are accompanied with physiological alterations, corresponding to senescence. AIM: To resolve metabolic features of tobacco cells in growing and stationary senescent suspension cultures VBI-0. MATERIALS AND METHODS: Nicotiana tabacum VBI-0 cells were cultured in suspension MS medium supplied with 3% sucrose. Cells were sampled at 7th day, during intensive growth, and at 28th day, when the culture was in the stationary phase. The GC-MS method was used to profile the metabolites. RESULTS: Sucrose depletion in media caused starvation of heterotrophic tobacco cell culture and was associated with a decrease in the accumulation of free amino acids. At the same time, the level of pentoses and complex sugars, including sucrose, increased, while the levels of glucose and fructose were not changed significantly and levels of hexose phosphates decreased. During culture senescence cells showed higher levels of accumulation of malate, pyruvate and some other carboxylates. CONCLUSIONS: The metabolomic data indicate that culture senescence was associated with a drop in amino acids metabolism, a decrease in the activity of the upper part of glycolysis, and the accumulation of complex sugars, pentoses and carboxylates.

A natural mutation in the promoter of the aconitase gene ZjACO3 influences fruit citric acid content in jujube.

Jujube (Ziziphus jujuba Mill.) is the most economically important fruit tree of the Rhamnaceae and was domesticated from wild or sour jujube (Z. jujuba Mill. var. spinosa Hu). During the process of domestication, there was a substantial reduction in the content of organic acids, particularly malate and citrate, which greatly influence the taste and nutritional value of the fruit. We previously demonstrated that ZjALMT4 is crucial for malate accumulation. However, the mechanism of citrate degradation in jujube remains poorly understood. In the present study, aconitase ZjACO3 was shown to participate in citric acid degradation in the cytoplasm through the GABA pathway. Interestingly, we discovered an E-box mutation in the ZjACO3 promoter (-484A > G; CAAGTG in sour jujube mutated to CAGGTG in cultivated jujube) that was strongly correlated with fruit citrate content; 'A' represented a high-citrate genotype and 'G' represented a low-citrate genotype. We developed and validated an ACO-based Kompetitive allele-specific PCR (KASP) marker for determining citric acid content. Yeast one-hybrid screening, transient dual-luciferase assays, and overexpression analyses showed that the transcription factor ZjbHLH113 protein directly binds to CAGGTG in the promoter of ZjACO3 in cultivated jujube plants, transcriptionally activating ZjACO3 expression, and enhancing citric acid degradation. Conversely, binding ability of the ZjbHLH113 protein to CAAGTG was weakened in sour jujube, thereby promoting citrate accumulation in the fruit. These findings will assist in elucidating the mechanism by which ZjACO3 modulates citrate accumulation in sour jujube and its cultivars.

Accumulation and regulation of malate in fruit cells

Accumulation and regulation of malate in fruit cells

Waterlogging restricted cotton fiber elongation by reducing osmolyte accumulation and cell wall biosynthesis

Waterlogging severely threatens the formation of cotton fiber quality, especially the length. However, the underlying mechanism is still incompletely understood. Here, the experiment (control: soil relative water content at (75 ± 5)%; treatment: waterlogging for 6 days) was conducted during the flowering and boll forming stage using two upland cotton cultivars (Dexiamian 1 and Yuzaomian 9110) to investigate the effects of waterlogging on fiber development from both transcriptomic and physiological levels. The transcriptome analysis of 10 days post anthesis cotton fiber showed that waterlogging significantly regulated the expression of genes associated with fiber elongation events including osmotic solutes accumulation, fiber cell wall biosynthesis, and cell wall remodeling and loosening. Waterlogging decreased the sucrose input into cotton fiber, resulting in reduced sucrose and hexose contents. Under waterlogging, the accumulation of malate and K+ in both cultivars was reduced by decreasing phosphoenolpyruvate carboxylase (PEPC) activity and potassium transporter genes (GhPOT) expression, respectively. Thus, the total concentration of osmolytes was reduced, which dampened cotton fiber elongation. Due to reduced sucrose synthase activity and uridine diphosphate glucose content, the cellulose biosynthesis was limited, leading to decreased fiber cell wall accumulation, which was adverse to fiber elongation. Besides, genes responsible for cell wall remodeling and loosening, including xyloglucan endotransglucosylase/hydrolase genes (GhXTH), α-expansin genes (GhEXPA), and pectin methylesterase genes (GhPME) were also responsive to waterlogging, but in different ways between cultivars. Overall, the shortened cotton fiber could be attributed to reduced osmolytes accumulation and cell wall biosynthesis, whilst the effects of waterlogging on cotton fiber cell wall loosening require further exploration.

Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification

As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH.

PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars (‘Cuiguan’, ‘Hongsucui’) and two P. ussuriensis cultivars (‘Qiuxiang’, ‘Hanhong’), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.

Bacterial Lipoproteins Shift Cellular Metabolism to Glycolysis in Macrophages Causing Bone Erosion.

Belonging to a group of membrane proteins, bacterial lipoproteins (LPPs) are defined by a unique lipid structure at their N-terminus providing the anchor in the bacterial cell membrane. In Gram-positive bacteria, LPPs play a key role in host immune activation triggered through a Toll-like receptor 2 (TLR2)-mediated action resulting in macrophage stimulation and subsequent tissue damage demonstrated in in vivo experimental models. Yet the physiologic links between LPP activation, cytokine release, and any underlying switches in cellular metabolism remain unclear. In this study, we demonstrate that Staphylococcus aureus Lpl1 not only triggers cytokine production but also confers a shift toward fermentative metabolism in bone marrow-derived macrophages (BMDMs). Lpl1 consists of di- and tri-acylated LPP variants; hence, the synthetic P2C and P3C, mimicking di-and tri-acylated LPPs, were employed to reveal their effect on BMDMs. Compared to P3C, P2C was found to shift the metabolism of BMDMs and the human mature monocytic MonoMac 6 (MM6) cells more profoundly toward the fermentative pathway, as indicated by lactate accumulation, glucose consumption, pH reduction, and oxygen consumption. In vivo, P2C caused more severe joint inflammation, bone erosion, and lactate and malate accumulation than P3C. These observed P2C effects were completely abrogated in monocyte/macrophage-depleted mice. Taken together, these findings now solidly confirm the hypothesized link between LPP exposure, a macrophage metabolic shift toward fermentation, and ensuing bone destruction. IMPORTANCE Osteomyelitis caused by S. aureus is a severe infection of the bone, typically associated with severe bone function impairment, therapeutic failure, high morbidity, invalidity, and occasionally even death. The hallmark of staphylococcal osteomyelitis is the destruction of the cortical bone structures, yet the mechanisms contributing to this pathology are hitherto poorly understood. One bacterial membrane constituent found in all bacteria is bacterial lipoproteins (LPPs). Previously, we have shown that injection of purified S. aureus LPPs into wild-type mouse knee joints caused a TLR2-dependent chronic destructive arthritis but failed to elicit such effect in monocyte/macrophage-depleted mice. This observation stirred our interest in investigating the interaction of LPPs and macrophages and analyzing the underlying physiological mechanisms. This ascertainment of LPP-induced changes in the physiology of macrophages provides an important clue in the understanding of the mechanisms of bone disintegration, opening novel avenues to manage the course of S. aureus disease.

Drought shortens cotton fiber length by inhibiting biosynthesis, remodeling and loosening of the primary cell wall

Drought could severely impair the formation of cotton fiber length, yet the underlying mechanism is not fully understood. Here, the effects of drought on cotton fiber elongation processes including primary cell wall biosynthesis, remodeling and loosening and osmolyte accumulation were investigated from transcriptomic and physiological levels by using two upland cotton cultivars (Dexiamian 1 and Yuzaomian 9110). The results showed that the cotton fiber elongation was significantly stunted under drought, and the biggest reduction in fiber length appeared at 10 days post anthesis. The total osmolytes concentration of cotton fiber sap was increased by drought via promoting the accumulation of malate and K+, which was favorable to fiber elongation. Whilst the biosynthesis of fiber primary cell wall components including pectin, hemicellulose and cellulose was inhibited under drought by suppressing the expression of galacturonosyltransferase (GhGAUT) and galacturonosyltransferase-like (GhGATL), xylosyltransferase (GhXXT), and cellulose synthase (GhCesA) respectively, leading to reduced fiber cell wall biomass. Meanwhile, drought restricted the remodeling and loosening of fiber primary cell wall by suppressing the expression of genes related to primary cell wall decomposition including pectin methylesterase (GhPME), xyloglucan endotransglucosylase/hydrolase (GhXTH), and endoglucanase (GhEG), and α-expansin (GhEXPA). Taken together, it is concluded that the shortened fiber length induced by drought was caused by restricted primary fiber cell wall biosynthesis, remodeling, and loosening but not the lowered osmotic potential. Overall, our work provides new insights into understanding the mechanisms by which drought shortens cotton fiber length.

Exogenous γ-aminobutyric acid (GABA) alleviates nitrogen deficiency by mediating nitrate uptake and assimilation in Andrographis paniculata seedlings

γ-Aminobutyric acid (GABA) plays significant metabolic and signaling roles in plant stress responses. Recent studies have proposed that GABA alleviates plant nitrogen (N) deficient stress; however, the mechanism by which GABA mediates plant N deficiency adaptation remains not yet well understood. Herein we found in a medicinal plant Andrographis paniculata that 5 mmol L−1 exogenous GABA promoted plant growth under N deficient (1 mmol L−1 NO3−) condition, with remarkably increments in total N and NO3− concentrations in plants. GABA increased N assimilation and protein synthesis by up-regulating the activities and expression of N metabolic enzymes. GABA also increased the accumulation of α-ketoglutarate and malate, which could facilitate the assimilation of NO3−. Inhibition of NR by Na2WO4 counteracted the promoting effects of GABA on plant growth, and the effects of GABA were not affected by L-DABA and 3-MP, the inhibitors of GABA transaminase (GABA-T) and glutamate decarboxylase (GAD), respectively. These results suggested that the nutritional role of GABA was excluded in promoting plant growth under low N condition. The results of 15N isotopic tracing and NRTs transcription indicated that exogenous GABA could up-regulate NRT2.4 and NRT3.2 to increase plant NO3− uptake under N deficient condition. Interestingly, primidone, an inhibitor of GABA receptor, impeded the effects of GABA on plant growth and N accumulation. Thus, our results revealed that exogenous GABA acted as a signal to up-regulate NRTs via its receptor to increase NO3− uptake, and subsequently promoted NO3− assimilation to alleviate N deficiency in A. paniculata.

ABRE-BINDING FACTOR3-WRKY DNA-BINDING PROTEIN44 module promotes salinity-induced malate accumulation in pear.

Malate impacts fruit acidity and plays a vital role in stress tolerance. Malate accumulation is induced by salinity in various plants as a metabolite in coping with this stress. However, the exact molecular mechanism responsible for salinity-induced malate accumulation remains unclear. Here, we determined that salinity treatment induces malate accumulation in pear (Pyrus spp.) fruit, calli, and plantlets compared to the control. Genetic and biochemical analyses established the key roles of PpWRKY44 and ABRE-BINDING FACTOR3 (PpABF3) transcription factors in promoting malate accumulation in response to salinity. We found that PpWRKY44 is involved in salinity-induced malate accumulation by directly binding to a W-box on the promoter of the malate-associated gene aluminum-activated malate transporter 9 (PpALMT9) to activate its expression. A series of in-vivo and in-vitro assays revealed that the G-box cis-element in the promoter of PpWRKY44 was targeted by PpABF3, which further enhanced salinity-induced malate accumulation. Taken together, these findings suggest that PpWRKY44 and PpABF3 play positive roles in salinity-induced malate accumulation in pears. This research provides insights into the molecular mechanism by which salinity affects malate accumulation and fruit quality.

Ethylene inhibits malate accumulation in apple by transcriptional repression of aluminum-activated malate transporter 9 via the WRKY31-ERF72 network.

Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.

Genome-Wide Characterization and Gene Expression Analyses of Malate Dehydrogenase (MDH) Genes in Low-Phosphorus Stress Tolerance of Chinese Fir (Cunninghamia lanceolata).

Malate dehydrogenase (MDH) genes play vital roles in developmental control and environmental stress tolerance in sessile plants by modulating the organic acid-malic acid level. However, MDH genes have not yet been characterized in gymnosperm, and their roles in nutrient deficiency are largely unexplored. In this study, 12 MDH genes were identified in Chinese fir (Cunninghamia lanceolata), namely, ClMDH-1, -2, -3, …, and -12. Chinese fir is one of the most abundant commercial timber trees in China, and low phosphorus has limited its growth and production due to the acidic soil of southern China. According to the phylogenetic analysis, MDH genes were classified into five groups, and Group 2 genes (ClMDH-7, -8, -9, and 10) were only found to be present in Chinese fir but not in Arabidopsis thaliana and Populus trichocarpa. In particular, the Group 2 MDHs also had specific functional domains-Ldh_1_N (malidase NAD-binding functional domain) and Ldh_1_C (malate enzyme C-terminal functional domain)-indicating a specific function of ClMDHs in the accumulation of malate. All ClMDH genes contained the conserved MDH gene characteristic functional domains Ldh_1_N and Ldh_1_C, and all ClMDH proteins exhibited similar structures. Twelve ClMDH genes were identified from eight chromosomes, involving fifteen ClMDH homologous gene pairs, each with a Ka/Ks ratio of <1. The analysis of cis-elements, protein interactions, and transcription factor interactions of MDHs showed that the ClMDH gene might play a role in plant growth and development, and in response to stress mechanisms. The results of transcriptome data and qRT-PCR validation based on low-phosphorus stress showed that ClMDH1, ClMDH6, ClMDH7, ClMDH2, ClMDH4, ClMDH5, ClMDH10 and ClMDH11 were upregulated under low-phosphorus stress and played a role in the response of fir to low-phosphorus stress. In conclusion, these findings lay a foundation for further improving the genetic mechanism of the ClMDH gene family in response to low-phosphorus stress, exploring the potential function of this gene, promoting the improvement of fir genetics and breeding, and improving production efficiency.

The SLC25A47 locus controls gluconeogenesis and energy expenditure

Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations betweenSLC25A47and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion ofSLC25A47impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.

Photosynthesis and leaf structure of F1 hybrids between Cymbidium ensifolium (C3) and C. bicolor subsp. pubescens (CAM).

The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. Seven F1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical and physiological traits involved in CAM in their leaves were investigated. Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F1s. The δ13C values of the F1s were intermediate between those of the parents. Leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium, and those of the F1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium, and that of the F1s was intermediate between them. Interestingly, one F1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F1s. Nevertheless, all F1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate, Pi dikinase as C. bicolor subsp. pubescens. CAM traits were intricately inherited in the F1 hybrids, the level of CAM expression varied widely among F1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F1s.

Photon costs of shoot and root NO3-, and root NH4+, assimilation in terrestrial vascular plants considering associated pH regulation, osmotic and ontogenetic effects.

The photon costs of photoreduction/assimilation of nitrate (NO3-) into organic nitrogen in shoots and respiratory driven NO3- and NH4+ assimilation in roots are compared for terrestrial vascular plants, considering associated pH regulation, osmotic and ontogenetic effects. Different mechanisms of neutralisation of the hydroxyl (OH-) ion necessarily generated in shoot NO3- assimilation are considered. Photoreduction/assimilation of NO3- in shoots with malic acid synthesis and either accumulation of malate in leaf vacuoles or transport of malate to roots and catabolism there have a similar cost which is around 35% less than that for root NO3- assimilation and around 20% less than that for photoreduction/assimilation of NO3-, oxalate production and storage of Ca oxalate in leaf vacuoles. The photon cost of root NH4+ assimilation with H+ efflux to the root medium is around 70% less than that of root NO3- assimilation. These differences in photon cost must be considered in the context of the use of a combination of locations of NO3- assimilation and mechanisms of acid-base regulation, and a maximum of 4.9-9.1% of total photon absorption needed for growth and maintenance that is devoted to NO3- assimilation and acid-base regulation.

Fructose Treatment Targets Myeloid Leukemogenesis in IDH2 Mutants By Exhausting Alpha-Ketoglutarate Pools

Acute Myeloid Leukemia (AML) is a heterogeneous group of hematological malignancies and represents the most frequent cause of leukemia-related deaths. The uncontrolled growth of myeloblasts in the Bone Marrow (BM) leads to accelerated metabolic processes and high consumption of nutrients, especially glucose. Isocitrate dehydrogenase (IDH) 1 and 2 are frequently mutated in human cancer, affecting up to 15% of AML patients. The neo-morphic activity conferred by IDH 1 or 2 mutations (mIDH1/2) converts α-ketoglutarate (α-KG) to the R-enantiomer of 2-hydroxyglutarate (2-HG), which competitively inhibits α-KG-dependent dioxygenases (Figueroa et al., 2010, Su et al.,2018, Li et al.,2017) and contributes to a block in myeloid differentiation and leukemic transformation. mIDH1/2 are clinically validated therapeutic targets as specific inhibitors have been approved by the FDA for the treatment of AML(DiNardo et al., 2018; Rohle et al., 2013; Stein et al., 2019; Wang et al., 2013) However, only 40% or less of mIDH AML patients respond to IDH inhibitor therapy (DiNardo et al., 2018; Stein et al., 2017) and little is known of how IDH mutations can be exploited for new metabolic vulnerabilities. We recently reported a novel metabolic dependency in AML cells that express low levels of the fructose transporter GLUT5. In fructose conditions these AMLs rewired their metabolism away from glycolysis and toward the serine synthesis pathway (SSP), (Jeong and Savino, Cell Metabolism 2021). Inhibiting the SSP by blocking PHGDH under these metabolic conditions resulted in reduced tumor burden. Additionally, patients with FLT-ITD alterations were sensitive to SSP blockade (Bjelosevic et. al. 2021). Although we previously found that myeloid leukemia cells grow normally in fructose, AML cell lines engineered to express IDH2 R140Q (TF1 knock in, and transduced MOLM13, U937 and OCIAML2) had a 50% reduction in cellular growth and 1.6-fold increase in differentiation (CD15 marker). Remarkably, a similar effect was observed in mIDH1 or 2 chondrosarcoma cell lines, suggesting that this metabolic program is more generally applicable. Excitingly, this new fructose induced therapeutic vulnerability could be recapitulated in vivo. We found that the fructose treated mice could delay myeloid leukemogenesis by 9-10 days (MOLM13 IDH2 R140Q) or in a primary patient sample successfully engrafted with IDH2 R140Q (n=6 mice/group p=0.0012; 3 weeks, I.P. 4 g/kg). To understand the basic mechanism for this new metabolic vulnerability, we first focused on the flux though the SSP. We found an increase in the end product of the SSP, glycine, as measured by LC-MS in the fructose condition compared to glucose. This SSP addiction under fructose was further demonstrated by reduced growth upon PHGDH inhibition (mIDH2: 75% reduction vs 50% WT IDH2 p=.0004; NCT503 5uM). Utilization of the SSP results in the generation of cytosolic α-KG, the substrate of IDH mutant enzymes. Thus, we hypothesized that mIDH AML cells might display enhanced dependency due to the requirement of α-KG for SSP, mIDH2 activity and to sustain the otherwise defective TCA cycle. Consistent with α-KG's central role in fructose driven metabolic addiction, we rescued the cell growth reduction by treating with cell permeable a-KG. Furthermore, in fructose conditions, we observed significant accumulation of the TCA cycle intermediate malate as well as aspartate, suggesting an engagement of the malate-aspartate shuttle in maintaining the redox balance. Previously, we found that AML cell lines (i.e THP1 cells) with moderate to high abundance of surface GLUT5, were not dependent on SSP in fructose. Surprisingly, by introducing IDH2 R140Q or by treating the cells with exogenous D-2HG, we found a 2-fold reduction of surface GLUT5 protein. This reduction of GLUT5 rewired the metabolism toward the SSP-addiction and resulted in a 50% reduction in cell growth in fructose. Interestingly, GLUT5 was controlled through transcriptional regulation of the locus as early as 8hrs post D-2HG treatment. Importantly, this relationship was found to be associated in patients, where PHGDH expression was significantly increased in mIDH2 AML patients with low vs high GLUT5 (n=15, n=16 p<.038) compared to other mutational subtypes (n=155, n=155 ns). In summary, we find a surprising new metabolic dependency based on sugar conditions in mIDH2 AML that could pave the way for a new therapeutic strategy.

Low-acidity ALUMINUM-DEPENDENT MALATE TRANSPORTER4 genotype determines malate content in cultivated jujube.

Jujube (Ziziphus jujuba Mill.), the most economically important fruit tree in Rhamnaceae, was domesticated from sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). During domestication, fruit sweetness increased and acidity decreased. Reduction in organic acid content is crucial for the increase in sweetness of jujube fruit. In this study, the determination of malate content among 46 sour jujube and 35 cultivated jujube accessions revealed that malate content varied widely in sour jujube (0.90-13.31 mg g-1) but to a lesser extent in cultivated jujube (0.33-2.81 mg g-1). Transcriptome sequencing analysis showed that the expression level of Aluminum-Dependent Malate Transporter 4 (ZjALMT4) was substantially higher in sour jujube than in jujube. Correlation analysis of mRNA abundance and fruit malate content and transient gene overexpression showed that ZjALMT4 participates in malate accumulation. Further sequencing analyses revealed that three genotypes of the W-box in the promoter of ZjALMT4 in sour jujube associated with malate content were detected, and the genotype associated with low malate content was fixed in jujube. Yeast one-hybrid screening showed that ZjWRKY7 binds to the W-box region of the high-acidity genotype in sour jujube, whereas the binding ability was weakened in jujube. Transient dual-luciferase and overexpression analyses showed that ZjWRKY7 directly binds to the promoter of ZjALMT4, activating its transcription, and thereby promoting malate accumulation. These findings provide insights into the mechanism by which ZjALMT4 modulates malate accumulation in sour jujube and jujube. The results are of theoretical and practical importance for the exploitation and domestication of germplasm resources.

Diversity in Acidity between Core and Pulp of Asian Pear Fruit Is Mainly Regulated by the Collaborative Activity of PH8.1 and DIC2 Genes during Fruit Development

The pear (Pyrus pyrifolia) is an important accessory fruit in which the pear core is tarter than the pear pulp. However, the reason for the acidic core and diversity in the taste of the same fruit is not clear. In this study, we observed that the citrate contents were three times higher in the core than in the pulp, while the malate content decreased along with fruit development and was significantly lower in the core than in the pulp at 110 days after flowering. Overall transcript levels for citrate-malate synthesis-related genes increased more in the pear core than the pulp at early fruit development, while degradation-related genes activity was nearly similar or non-significant between the core and pulp during fruit development. The lesser malate accumulation in the pear core compared to the pulp at 110 DAF was possibly due to the reduced activity of tDT2 gene. Regarding citrate accumulation, we identified five important p-type H+-ATPase genes in pear and found that the relative expression level of the PH8.1 gene was four-fold higher in the core than in the pulp during fruit development. Moreover, the expression level of di-carboxylate carrier gene 2 (DIC2) was constantly and significantly higher in the core than in the pulp. In addition, correlation analysis signified that the transcript levels of the two genes PH8.1 and DIC2 positively and significantly correlated with the citrate contents. These results suggested that the increased and collaborative activity of PH8.1 and DIC2 played a key role in the higher citrate accumulation in the core than the pulp, thus, with the help of molecular breeding tools, the citrate contents can be optimized in pear fruit for divers and improved fruit flavoring.

Molecular elucidation for the variance of organic acid profile between citrus top and bottom canopy fruits

Organic acids are key metabolites for diverse fruit flavoring and vital for fruit quality measures. Their contents are significantly varied regarding the canopy position. However, the underlying mechanism is still unclear. In this study by using Citrus raticulata cv. Bendizao as research material, a significant 2-fold increase in citrate content and a 2-fold decrease in malate content were noticed in bottom-canopy fruits subjected to less light and low temperature compared to top-canopy fruits. Moreover, transcript levels of genes like citrate synthase (CS1), aconitate hydratase genes (ACO1 and ACO2), NAD-dependent malic enzyme gene (NAD-ME) and malate dehydrogenase genes (MDH1 and MDH2) were significantly higher, while transcript levels of other genes such as citrate utilization genes (ACL2, ACL3 and ACO3), malate synthase gene (MS) and cytosolic NAD-dependent malate dehydrogenase gene (cytNAD-MDH) were significantly lower in bottom-canopy fruits compared to top-canopy fruits. Additionally, transcript levels of proton pump genes (CsPHs and VHA), two transporter genes (MATE and ALMT9), and four transcription factor genes (MYB73, Noemi, MYC-N, and bHLH) were significantly higher, while mRNA levels of two transcription factor genes (MYB10 and WRKY14) and transporter genes (CsCit1 and ALMT9-like) were significantly lower in bottom-canopy fruits than those in top-canopy fruits. Taken together, the variance of organic acid profiles in the canopy could be attributed to the microclimate change, which comprehensively influences the organic acid accumulation-related genes’ transcript levels; reduced malate accumulation in bottom-canopy fruits is mainly due to the minimal expression of MS and cytNAD-MDH, and high expression of malate utilization genes (MDH and NAD-ME), while the higher citrate content in the bottom-canopy fruits is probably due to the enhanced vacuolar storage through increasing vacuolar proton pump genes and MATE transcript levels, and the minimal citrate degradation and efflux through decreasing ACL2, ACL3, ACO3 and CsCit1 transcript levels.