Pathway Of Breakdown Research Articles

The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies.

As the kynurenine pathway's links to inflammation, the immune system, and neurological disorders became more apparent, it attracted more and more attention. It is the main pathway through which the liver breaks down Tryptophan and the initial step in the creation of nicotinamide adenine dinucleotide (NAD+) in mammals. Immune system activation and the buildup of potentially neurotoxic substances can result from the dysregulation or overactivation of this pathway. Therefore, it is not shocking that kynurenines have been linked to neurological conditions (Depression, Parkinson's, Alzheimer's, Huntington's Disease, Schizophrenia, and cognitive deficits) in relation to inflammation. Nevertheless, preclinical research has demonstrated that kynurenines are essential components of the behavioral analogs of depression and schizophrenia-like cognitive deficits in addition to mediators associated with neurological pathologies due to their neuromodulatory qualities. Neurodegenerative diseases have been extensively associated with neuroactive metabolites of the kynurenine pathway (KP) of tryptophan breakdown. In addition to being a necessary amino acid for protein synthesis, Tryptophan is also transformed into the important neurotransmitters tryptamine and serotonin in higher eukaryotes. In this article, a summary of the KP, its function in neurodegeneration, and the approaches being used currently to target the route therapeutically are discussed.

TMIC-38. CD39 EXPRESSION IN GLIOBLASTOMA TUMOR MICROENVIRONMENT DOES NOT AFFECT SURVIVAL OR T-CELL EXHAUSTION

Abstract Glioblastoma (GBM) is the most common malignant brain tumor with a median survival of 15 months. GBM is very difficult to treat due to numerous factors, including the paucity of immune infiltration of the tumor itself. Efforts to overcome this have led to use of immune checkpoint inhibitors such as PD-1 inhibitor, which have revolutionized treatment of many other solid tumors, and yet have not been successful in the treatment of GBM. This has led to many studies investigating various other similar markers. One such marker is CD39, which is highly upregulated in GBM and functions as the first enzyme in the metabolic pathway of ATP breakdown to adenosine. Additionally, CD39 is thought to contribute to T-cell exhaustion. Like PD-1, CD39 inhibition has had preliminary success in the treatment of other solid tumors. However, when tested on GBM, blocking the ATP breakdown pathway did not improve survival in mice, even in conjunction with PD-1 inhibition. Our team sought to determine if the expression of CD39 on the cell surface functioned as an exhaustion marker by injecting two different cell lines of GBM into CD39 global null mice. There was no survival benefit seen in either cell line when compared to wild type mice. Next, expression of known exhaustion markers such as PD-1, TIM3, and LAG3 on tumor infiltrating lymphocytes were analyzed. Expression of TIM3 and 2B4 were found to be increased in CD39KO mice in comparison to wild type (p < 0.01). Additionally, numerous exhaustion markers such as LAG3, TIGIT, and CTLA4 were decreased on tumor in comparison to peripheral blood (p < 0.01). These results demonstrate the complex nature of immune signaling in GBM tumor microenvironment that our team continues to investigate.

Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background

The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.

Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac β Myosin.

Human cardiac β myosin undergoes the cross-bridge cycle as part of the force-generating mechanism of cardiac muscle. The recovery stroke is considered one of the key steps of the kinetic cycle as it is the conformational rearrangement required to position the active site residues for hydrolysis of ATP and interaction with actin. We explored the free-energy surface of the transition and investigated the effect of the genetic cardiomyopathy causing mutations R453C, I457T, and I467T on this step using metadynamics. This work extends previous studies on Dictyostelium myosin II with engineered mutations. Here, like previously, we generated an unbiased thermodynamic ensemble of reactive trajectories for the chemical step using transition path sampling. Our methodologies were able to predict the changes to the dynamics of the recovery stroke as well as predict the pathway of breakdown of ATP to ADP and HPO42- with the stabilization of the metaphosphate intermediate. We also observed clear differences between the Dictyostelium myosin II and human cardiac β myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.

Proteolysis and Process-Induced Modifications in Synbiotic Yogurt Investigated by Peptidomics and Phosphopeptidomics.

Probiotic and synbiotic yogurt preparations were manufactured at the semi-industrial pilot scale with Lactobacillus acidophilus and Bifidobacteria strains without inulin or fortified with 1 and 3% (w/w) inulin. The pathway of casein breakdown was determined in probiotic, synbiotic, conventional yogurt, and nonstarted milk base using HPLC-ESI-MS/MS-based peptidomics and phosphopeptidomics; in the latter case, casein phosphorylated peptides (CPPs) were previously enriched by hydroxyapatite chromatography. Compared with traditional yogurt, casein proteolysis increased in probiotic and even more in synbiotic yogurt with 1% inulin. Fortification with 3% inulin greatly modified the proteolytic pattern, indicating a characteristic contribution of probiotics to proteolysis. The enhanced proteolysis in synbiotic yogurt exposed the neo-formed peptides to progressively increase enzymatic or chemical modifications, such as dephosphorylation of CPPs, methionine oxidation, and formation of N-terminal pyroglutamic acids. These modifications might constitute molecular signature descriptors of metabolic processes mediated by complex bacterial communities, with technological, nutritional, and sensorial significance.

Cryptic chlorophyll breakdown in non-senescent green Arabidopsis thaliana leaves.

Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables photosynthesis, it appears to have no role in photoactive green leaves. Here, colorless PBs were detected in green, non-senescent leaves of Arabidopsis thaliana. The PBs from the green leaves had structures entirely consistent with the PAO/phyllobilin pathway and the mutation of a single Chl catabolic enzyme completely abolished PBs with the particular modification. Hence, the PAO/phyllobilin pathway was active in the absence of visible senescence and expression of genes encoding Chl catabolic enzymes was observed in green Arabidopsis leaves. PBs accumulated to only sub-% amounts compared to the Chls present in the green leaves, excluding a substantial contribution of Chl breakdown from rapid Chl turnover associated with photosystem II repair. Indeed, Chl turnover was shown to involve a Chl a dephytylation and Chl a reconstitution cycle. However, non-recyclable pheophytin a is also liberated in the course of photosystem II repair, and is proposed here to be scavenged and degraded to the observed PBs. Hence, a cryptic form of the established pathway of Chl breakdown is indicated to play a constitutive role in photoactive leaves.

Oleic acid-induced defective autolysosome shows impaired lipid degradation

Recent studies suggest an alternative pathway of lipid breakdown called lipophagy, which delivers lipid droplets (LDs) to lysosomes for degradation of LDs. However, molecular mechanisms regulating lipophagy are still largely unknown. In this study, we evaluated the effect of oleic acid (OA) on lipophagy in cells. We found that OA treatment results in accumulation of p62 and LC3-II proteins and reduces red fluorescence in cells stably expressing mCherry-GFP-LC3. In addition, OA inhibits the co-localization of LC3 with LAMP1 under serum-deprived condition, suggesting that OA blocks autophagosome-lysosome fusion. In the cells with ATG5 or ULK1 gene deletion, LDs did not increase upon OA treatment more than in wild type cells. However, cell starvation following OA removal resulted in reduced lipid accumulation by lipophagy and recovery of autophagy flux, suggesting that the specific condition of OA treatment and cell starvation are important for lipophagy flux activity.

Characterization of the pheophorbide a oxygenase/phyllobilin pathway of chlorophyll breakdown in grasses.

Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

Identification of a mutarotase gene involved in D-galactose utilization in Aspergillus nidulans

Aldose 1-epimerases or mutarotases (EC 5.1.3.3) are catalyzing the interconversion of α- and β-anomers of hemiacetals of aldose sugars such as D-glucose and D-galactose, and are presumed to play an auxiliary role in carbohydrate metabolism as mutarotation occurs spontaneously in watery solutions. The first step in the Leloir pathway of D-galactose breakdown is preceded by accelerated conversion of β-D-galactopyranose into the α-anomer, the substrate of the anomer-specific D-galactose 1-kinase. Here, we identified two putative aldose-1-epimerase genes (galmA and galmB) in the model organism Aspergillus nidulans, and characterized them upon generation of single- and double deletion mutant strains, as well as overexpressing mutants carrying multiple copies of either. Assaying cell-free extracts from the galmB single- and galm double mutants, we observed that the mutarotation hardly exceeded spontaneous anomer conversion, while galmB multicopy strains displayed higher activities than the wild type, increasing with the copy number. When grown on D-galactose in submerged cultures, biomass formation and D-galactose uptake rates in mutants lacking galmB were considerably reduced. None such effects were observed studying galmA deletion mutants, which consistently behave like the wild type. We conclude that GalmB is the physiologically relevant mutarotase for the utilization of D-galactose in A. nidulans.

Exposure of articular chondrocytes to wear particles induces phagocytosis, differential inflammatory gene expression, and reduced proliferation.

The production of wear debris particulate remains a concern due to its association with implant failure through complex biologic interactions. In the setting of unicompartmental knee arthroplasty (UKA), damage and wear of the components may introduce debris particulate into the adjacent, otherwise, healthy compartment. The purpose of this study was to investigate the in vitro effect of polymeric and metallic wear debris particles on cell proliferation, extracellular matrix regulation, and phagocytosis index of normal human articular chondrocytes (nHACs). In culture, nHACs were exposed to both cobalt-chromium-molybdenum (CoCrMo) and polymethyl-methacrylate (PMMA) wear debris particulate for 3 and 10 days. At 3 days, no significant difference in cell proliferation was found between control cells and cells exposed to both CoCrMo or PMMA particles. However, cell proliferation was significantly decreased for CoCrMo exposed nHACs at both 6 (P < 0.001) and 10 days (P < 0.001) and PMMA at 10 days (P < 0.001). Target gene expression displayed both a time- and material-dependent response to CoCrMo and PMMA particles. Significant differences in COL10A1, ACAN, VCAN, IL-1β, TNF-α, MMP3, ADAMTS1, CASP3, and CASP9 regulation were found between CoCrMo and PMMA exposed nHACs at day 3 with gene regulation returning to near baseline at 10 days. Results from our study indicate a role of wear debris induced cartilage degeneration after exposure to polymeric and metallic wear debris particulate, suggesting an additional pathway of cartilage breakdown, potentially manifesting in traditional clinical symptoms.

The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.

Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner-Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO2 via the Calvin-Benson cycle.

Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Dangerous Membranes: Viruses That Subvert Autophagosomes

Dangerous Membranes: Viruses That Subvert Autophagosomes

Direct study of fluorescently-labelled barley β-glucan fate in an in vitro human colon digestion model

β-Glucans from cereals are β(1–3)(1–4)-mixed linkage linear homopolysaccharides of d-glucopyranosyl residues, recently recognised as functional components of foods with benefits in maintaining the health of the digestive tract not least through a prebiotic effect. Here we describe the development of methodology to facilitate the study of β-glucans as prebiotics. Relatively short β-glucan fragments (DP 6–50) were produced by partial hydrolysis of β-glucan fibres with Lichenase then functionalised at their reducing end with a tetramethylrhodamine dye. Their enzymatic break down by human colon microbiota in an in vitro fermentation model was examined. Digestion products were isolated by virtue of their fluorescence labels, identified and characterised using capillary electrophoresis and mass spectrometry. Complete digestion of the labelled substrates was indicated, as fluorescently labelled glucose was obtained as the final product. Furthermore, a pathway of enzymatic breakdown was proposed on the basis of a time course experiment; initial fast hydrolysis with an endo-1,3(4)-β-glucanase was followed by slow degradation with an exo-1,4-β-glucanase and finally slow action of an exo-1,3-β-glucanase.

Water deficit induces chlorophyll degradation via the 'PAO/phyllobilin' pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants.

Angiosperm resurrection plants exhibit poikilo- or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit-induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non-enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO-dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit-induced poikilochlorophylly occurs via the well-characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.

The effects of 30 days resveratrol supplementation on adipose tissue morphology and gene expression patterns in obese men

Polyphenolic compounds, such as resveratrol, have recently received widespread interest because of their ability to mimic effects of calorie restriction. The objective of the present study was to gain more insight into the effects of 30 days resveratrol supplementation on adipose tissue morphology and underlying processes. Eleven healthy obese men were supplemented with placebo and resveratrol for 30 days (150 mg per day), separated by a 4-week washout period in a double-blind randomized crossover design. A postprandial abdominal subcutaneous adipose tissue biopsy was collected to assess adipose tissue morphology and gene expression using microarray analysis. Resveratrol significantly decreased adipocyte size, with a shift toward a reduction in the proportion of large and very-large adipocytes and an increase in small adipocytes. Microarray analysis revealed downregulation of Wnt and Notch signaling pathways and upregulation of pathways involved in cell cycle regulation after resveratrol supplementation, suggesting enhanced adipogenesis. Furthermore, lysosomal/phagosomal pathway and transcription factor EB were upregulated reflecting an alternative pathway of lipid breakdown by autophagy. Further research is necessary to investigate whether resveratrol improves adipose tissue function.

A dioxobilane as product of a divergent path of chlorophyll breakdown in Norway maple.

Chlorophyll breakdown is a hallmark of leaf senescence and a major contributor to the emergence of the fall colors.[1, 2] Strikingly, essential pieces of the puzzle of this biological phenomenon have been solved only within the last two decades.[2–5] A breakthrough was the identification and structure elucidation of a colorless tetrapyrrolic chlorophyll catabolite, thereafter named Hv-NCC-1.[6] The further characterization of tetrapyrrolic chlorophyll catabolites in higher plants has meanwhile provided the structural basis for detailed insight into the pathway of chlorophyll breakdown.[7, 8] Colorless tetrapyrroles, such as Hv-NCC-1, typically accumulate in senescent leaves of higher plants and they have been classified as “nonfluorescent” chlorophyll catabolites (NCCs).[7] Indeed, NCCs have been considered to be the “final” breakdown products of a largely common and well-controlled “linear” catabolic pathway in senescent leaves (see Scheme 1).[7, 9]

The role of transitory starch in C3, CAM, and C4 metabolism and opportunities for engineering leaf starch accumulation

Essentially all plants store starch in their leaves during the day and break it down the following night. This transitory starch accumulation acts as an overflow mechanism when the sucrose synthesis capacity is limiting, and transitory starch also acts as a carbon store to provide sugar at night. Transitory starch breakdown can occur by either of two pathways; significant progress has been made in understanding these pathways in C(3) plants. The hydrolytic (amylolytic) pathway generating maltose appears to be the primary source of sugar for export from C(3) chloroplasts at night, whereas the phosphorolytic pathway supplies carbon for chloroplast reactions, in particular in the light. In crassulacean acid metabolism (CAM) plants, the hydrolytic pathway predominates when plants operate in C(3) mode, but the phosphorolytic pathway predominates when they operate in CAM mode. Information on transitory starch metabolism in C(4) plants has now become available as a result of combined microscopy and proteome studies. Starch accumulates in all cell types in immature maize leaf tissue, but in mature leaf tissues starch accumulation ceases in mesophyll cells except when sugar export from leaves is blocked. Proper regulation of the amount of carbon that goes into starch, the pathway of starch breakdown, and the location of starch accumulation could help ensure that engineering of C(4) metabolism is coordinated with the downstream reactions required for efficient photosynthesis.

Stay-green protein, defective in Mendel's green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway.

Type C stay-green mutants are defined as being defective in the pathway of chlorophyll breakdown, which involves pheophorbide a oxygenase (PAO), required for loss of green color. By analyzing senescence parameters, such as protein degradation, expression of senescence-associated genes and loss of photosynthetic capacity, we demonstrate that JI2775, the green cotyledon (i) pea line used by Gregor Mendel to establish the law of genetics, is a true type C stay-green mutant. STAY-GREEN (SGR) had earlier been shown to map to the I locus. The defect in JI2775 is due to both reduced expression of SGR and loss of SGR protein function. Regulation of PAO through SGR had been proposed. By determining PAO protein abundance and activity, we show that PAO is unaffected in JI2775. Furthermore we show that pheophorbide a accumulation in the mutant is independent of PAO. When silencing SGR expression in Arabidopsis pao1 mutant, both pheophorbide a accumulation and cell death phenotype, typical features of pao1, are lost. These results confirm that SGR function within the chlorophyll catabolic pathway is independent and upstream of PAO.

Starch breakdown: recent discoveries suggest distinct pathways and novel mechanisms.

The aim of this article is to provide an overview of current models of starch breakdown in leaves. We summarise the results of our recent work focusing on Arabidopsis, relating them to other work in the field. Early biochemical studies of starch containing tissues identified numerous enzymes capable of participating in starch degradation. In the non-living endosperms of germinated cereal seeds, starch breakdown proceeds by the combined actions of α-amylase, limit dextrinase (debranching enzyme), β-amylase and α-glucosidase. The activities of these enzymes and the regulation of some of the respective genes on germination have been extensively studied. In living plant cells, additional enzymes are present, such as α-glucan phosphorylase and disproportionating enzyme, and the major pathway of starch breakdown appears to differ from that in the cereal endosperm in some important aspects. For example, reverse-genetic studies of Arabidopsis show that α-amylase and limit-dextrinase play minor roles and are dispensable for starch breakdown in leaves. Current data also casts doubt on the involvement of α-glucosidase. In contrast, several lines of evidence point towards a major role for β-amylase in leaves, which functions together with disproportionating enzyme and isoamylase (debranching enzyme) to produce maltose and glucose. Furthermore, the characterisation of Arabidopsis mutants with elevated leaf starch has contributed to the discovery of previously unknown proteins and metabolic steps in the pathway. In particular, it is now apparent that glucan phosphorylation is required for normal rates of starch mobilisation to occur, although a detailed understanding of this step is still lacking. We use this review to give a background to some of the classical genetic mutants that have contributed to our current knowledge.