Plain Language Summary of the PAISLEY study: deucravacitinib as a treatment for people with systemic lupus erythematosus.

What is this summary about?This plain language summary describes the results of the phase 2 study called PAISLEY which tested deucravacitinib, a new medicine under investigation before approval, in people living with lupus. In this trial, researchers wanted to find out if deucravacitinib would be safe and reduce the symptoms and disease activity in people living with lupus. PAISLEY looked at the type of lupus known as systemic lupus erythematosus, shortened to SLE.What happened in the study?The study included 363 people from 17 countries who had SLE and were between 18 and 75 years of age. The participants were divided into 4 groups at random. One group was given placebo (a fake or dummy pill that contains no medicine) and the other 3 groups took deucravacitinib, a pill taken by mouth. Each of the groups taking deucravacitinib took a different dose, either 3 milligrams (mg) twice daily, 6 mg twice daily, or 12 mg once daily. After 32 and 48 weeks, researchers measured the number of people in each group who had improvements in their SLE symptoms and disease activity, as measured by different tests. They also looked at any side effects people experienced, which may or may not have been caused by the medicine.What do the results mean?After 32 weeks of treatment, SLE symptoms and disease activity improved in more people in each of the deucravacitinib dose groups compared with the people taking placebo (the dummy pill). After 48 weeks of treatment, SLE symptoms and disease activity were still improved in more people taking deucravacitinib compared with people taking placebo, and this was measured in several different ways. The best results were seen in people taking deucravacitinib 3 mg twice daily. The number of serious side effects was similar for people taking deucravacitinib and those taking placebo. The most common side effects that were seen in people taking deucravacitinib were infections such as sore throat, cough, or bronchitis (upper respiratory tract), infltion in the nose (nasopharyngitis), headaches, and urinary tract infections. More people taking deucravacitinib than placebo had acne, rash, and cold sores (oral herpes). These were not serious and did not have any long-term effects on patient health or lead to patients stopping treatment.How to say (double click sound icon to play sound)… Systemic lupus erythematosus: SIS-teh-MIC LOO-puhs Eh-RE-the-ma-TOE-susDeucravacitinib: doo-KRAV-a-sih-ti-nibEnzyme: EN-zimeInterferon: in-tur-FER-onPlacebo: pluh-SEE-bohTyrosine kinase: TY-ruh-seen KY-naysTYK2: TIK-tu.

Deucravacitinib, a Tyrosine Kinase 2 Inhibitor, in Systemic Lupus Erythematosus: A Phase II, Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: To assess the efficacy and safety of deucravacitinib, an oral, selective, allosteric inhibitor of TYK2, in a phase II trial in adult patients with active systemic lupus erythematosus (SLE). METHODS: Adults with active SLE were enrolled from 162 sites in 17 countries. Patients (n = 363) were randomized 1:1:1:1 to receive deucravacitinib 3 mg twice daily, 6 mg twice daily, 12 mg once daily, or placebo. The primary end point was SLE Responder Index 4 (SRI-4) response at week 32. Secondary outcomes assessed at week 48 included SRI-4, British Isles Lupus Assessment Group-based Composite Lupus Assessment (BICLA) response, Cutaneous Lupus Erythematosus Disease Area and Severity Index 50 (CLASI-50), Lupus Low Disease Activity State (LLDAS), and improvements in active (swollen plus tender), swollen, and tender joint counts. RESULTS: At week 32, the percentage of patients achieving SRI-4 response was 34% with placebo compared to 58% with deucravacitinib 3 mg twice daily (odds ratio [OR] 2.8 [95% confidence interval (95% CI) 1.5, 5.1]; P < 0.001 versus placebo), 50% with 6 mg twice daily (OR 1.9 [95% CI 1.0, 3.4]; P = 0.02 versus placebo), and 45% with 12 mg once daily (OR 1.6 [95% CI 0.8, 2.9]; nominal P = 0.08 versus placebo). Response rates were higher with deucravacitinib treatment for BICLA, CLASI-50, LLDAS, and joint counts compared to placebo. Rates of adverse events were similar across groups, except higher rates of infections and cutaneous events, including rash and acne, with deucravacitinib treatment. Rates of serious adverse events were comparable, with no deaths, opportunistic infections, tuberculosis infections, major adverse cardiovascular events, or thrombotic events reported. CONCLUSION: Deucravacitinib treatment elicited higher response rates for SRI-4 and other end points compared with placebo, with an acceptable safety profile, in adult patients with active SLE.

A dominant mutation in ß-AMYLASE1 disrupts nighttime control of starch degradation in Arabidopsis leaves.

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

Restriction of cytosolic sucrose hydrolysis profoundly alters development, metabolism, and gene expression in Arabidopsis roots.

Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth, and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

Efficacy and Safety of Epratuzumab in Moderately to Severely Active Systemic Lupus Erythematosus: Results From Two Phase III Randomized, Double-Blind, Placebo-Controlled Trials.

OBJECTIVE: Epratuzumab, a monoclonal antibody that targets CD22, modulates B cell signaling without substantial reductions in the number of B cells. The aim of this study was to report the results of 2 phase III multicenter randomized, double-blind, placebo-controlled trials, the EMBODY 1 and EMBODY 2 trials, assessing the efficacy and safety of epratuzumab in patients with moderately to severely active systemic lupus erythematosus (SLE). METHODS: Patients met ≥4 of the American College of Rheumatology revised classification criteria for SLE, were positive for antinuclear antibodies and/or anti-double-stranded DNA antibodies, had an SLE Disease Activity Index 2000 (SLEDAI-2K) score of ≥6 (increased disease activity), had British Isles Lupus Assessment Group 2004 index (BILAG-2004) scores of grade A (severe disease activity) in ≥1 body system or grade B (moderate disease activity) in ≥2 body systems (in the mucocutaneous, musculoskeletal, or cardiorespiratory domains), and were receiving standard therapy, including mandatory treatment with corticosteroids (5-60 mg/day). BILAG-2004 grade A scores in the renal and central nervous system domains were excluded. Patients were randomized 1:1:1 to receive either placebo, epratuzumab 600 mg every week, or epratuzumab 1,200 mg every other week, with infusions delivered for the first 4 weeks of each 12-week dosing cycle, for 4 cycles. Patients across all 3 treatment groups also continued with their standard therapy. The primary end point was the response rate at week 48 according to the BILAG-based Combined Lupus Assessment (BICLA) definition, requiring improvement in the BILAG-2004 score, no worsening in the BILAG-2004 score, SLEDAI-2K score, or physician's global assessment of disease activity, and no disallowed changes in concomitant medications. Patients who discontinued the study medication were classified as nonresponders. RESULTS: In the EMBODY 1 and EMBODY 2 trials of epratuzumab, 793 patients and 791 patients, respectively, were randomized, 786 (99.1%) and 788 (99.6%), respectively, received study medication, and 528 (66.6%) and 533 (67.4%), respectively, completed the study. There was no statistically significant difference in the primary end point between the groups, with the week 48 BICLA response rates being similar between the epratuzumab groups and the placebo group (response rates ranging from 33.5% to 39.8%). No new safety signals were identified. CONCLUSION: In patients with moderate or severely active SLE, treatment with epratuzumab + standard therapy did not result in improvements in response rates over that observed in the placebo + standard therapy group.

Efficacy and safety of epratuzumab in patients with moderate/severe active systemic lupus erythematosus: results from EMBLEM, a phase IIb, randomised, double-blind, placebo-controlled, multicentre study.

OBJECTIVE: To identify a suitable dosing regimen of the CD22-targeted monoclonal antibody epratuzumab in adults with moderately to severely active systemic lupus erythematosus (SLE). METHODS: A phase IIb, multicentre, randomised controlled study (NCT00624351) was conducted with 227 patients (37-39 per arm) receiving either: placebo, epratuzumab 200 mg cumulative dose (cd) (100 mg every other week (EOW)), 800 mg cd (400 mg EOW), 2400 mg cd (600 mg weekly), 2400 mg cd (1200 mg EOW), or 3600 mg cd (1800 mg EOW). The primary endpoint (not powered for significance) was the week 12 responder rate measured using a novel composite endpoint, the British Isles Lupus Assessment Group (BILAG)-based Combined Lupus Assessment (BICLA). RESULTS: Proportion of responders was higher in all epratuzumab groups than with placebo (overall treatment effect test p=0.148). Exploratory pairwise analysis demonstrated clinical improvement in patients receiving a cd of 2400 mg epratuzumab (OR for 600 mg weekly vs placebo: 3.2 (95% CI 1.1 to 8.8), nominal p=0.03; OR for 1200 mg EOW vs placebo: 2.6 (0.9 to 7.1), nominal p=0.07). Post-hoc comparison of all 2400 mg cd patients versus placebo found an overall treatment effect (OR=2.9 (1.2 to 7.1), nominal p=0.02). Incidence of adverse events (AEs), serious AEs and infusion reactions was similar between epratuzumab and placebo groups, without decreases in immunoglobulin levels and only partial reduction in B-cell levels. CONCLUSIONS: Treatment with epratuzumab 2400 mg cd was well tolerated in patients with moderately to severely active SLE, and associated with improvements in disease activity. Phase III studies are ongoing.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion.

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night.

Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a "glucosyl buffer" to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.

Altered starch turnover in the maternal plant has major effects on Arabidopsis fruit growth and seed composition.

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

Data quality challenges in systemic lupus erythematosus trials: how can this be optimized?

Major scientific advances in basic science, pharmacology, and translational medicine have allowed the discovery of new molecular targets whose manipulation by new chemical entities has led to treatments for inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. Development of new agents for systemic lupus erythematosus (SLE) has lagged, however, because the protean manifestations of SLE present challenges for measuring therapeutic effects in a consistent manner. Composite end points combining several Disease Activity Indices (DAIs) are being used in ongoing global studies, but the uniform application of these complex DAIs across large numbers of clinical sites has proven difficult. We describe herein approaches that are being utilized to facilitate collection, review, and analysis of the clinical measures utilizing independent central adjudication committees.

Callose synthase GSL7 is necessary for normal phloem transport and inflorescence growth in Arabidopsis.

One isoform of callose synthase, Glucan Synthase-Like7 (GSL7), is tightly coexpressed with two isoforms of sucrose synthase (SUS5 and SUS6) known to be confined to phloem sieve elements in Arabidopsis (Arabidopsis thaliana). Investigation of the phenotype of gsl7 mutants of Arabidopsis revealed that the sieve plate pores of stems and roots lack the callose lining seen in wild-type plants. Callose synthesis in other tissues of the plant appears to be unaffected. Although gsl7 plants show only minor phenotypic alterations during vegetative growth, flowering stems are reduced in height and all floral parts are smaller than those of wild-type plants. Several lines of evidence suggest that the reduced growth of the inflorescence is a result of carbohydrate starvation. Levels of sucrose, hexoses, and starch are lower in the terminal bud clusters of gsl7 than in those of wild-type plants. Transcript levels of "starvation" genes expressed in response to low sugars are elevated in the terminal bud clusters of gsl7 plants, at the end of the night, and during an extended night. Pulse-chase experiments with (14)CO(2) show that transport of assimilate in the flowering stem is much slower in gsl7 mutants than in wild-type plants. We suggest that the callose lining of sieve plate pores is essential for normal phloem transport because it confers favorable flow characteristics on the pores.

Phase 1 safety and tolerability study of BMP-7 in symptomatic knee osteoarthritis.

BACKGROUND: There are no proven therapies that modify the structural changes associated with osteoarthritis (OA). Preclinical data suggests that intra-articular recombinant human BMP-7 (bone morphogenetic protein-7) has reparative effects on cartilage, as well as on symptoms of joint pain. The objective of this study was to determine the safety and tolerability as well as dose-limiting toxicity and maximal tolerated dose of intra-articular BMP-7. The secondary objectives were to determine the effect on symptomatic responses through 24 weeks. METHODS: This was a Phase 1, double-blind, randomized, multi-center, placebo-controlled, single-dose escalation safety study consisting of 4 dosing cohorts in participants with knee OA. Each cohort was to consist of 8 treated participants, with treatment allocation in a 3:1 active (intra-articular BMP-7) to placebo ratio. Eligible participants were persons with symptomatic radiographic knee OA over the age of 40. The primary objective of this study was to determine the safety and tolerability of BMP-7 including laboratory assessments, immunogenicity data and radiographic assessments. Secondary objectives were to determine the proportion of participants with a 20%, 50%, and 70% improvement in the WOMAC pain and function subscales at 4, 8, 12, and 24 weeks. Other secondary outcomes included the change from baseline to 4, 8, 12, and 24 weeks for the OARSI responder criteria. RESULTS: The mean age of participants was 60 years and 73% were female. All 33 participants who were enrolled completed the study and most adverse events were mild or moderate and were similar in placebo and BMP-7 groups. The 1 mg BMP-7 group showed a higher frequency of injection site pain and there was no ectopic bone formation seen on plain x-rays. By week 12, most participants in both the BMP-7 and placebo groups experienced a 20% improvement in pain and overall the BMP-7 group was similar to placebo with regard to this measurement. In the participants who received 0.1 mg and 0.3 mg BMP-7, there was a trend toward greater symptomatic improvement than placebo. The other secondary endpoints showed similar trends including the OARSI responder criteria for which the BMP-7 groups had more responders than placebo. CONCLUSIONS: There was no dose limiting toxicity identified in this study. The suggestion of a symptom response, together with the lack of dose limiting toxicity provide further support for the continued development of this product for the treatment of osteoarthritis.

A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism.

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

The plastidial glucose-6-phosphate/phosphate antiporter GPT1 is essential for morphogenesis in Arabidopsis embryos.

The glucose-6-phosphate/phosphate antiporter GPT1 is a major route of entry of carbon into non-photosynthetic plastids. To discover its importance in oilseeds, we used a seed-specific promoter to generate lines of Arabidopsis thaliana with reduced levels of GPT1 in developing embryos. Strong reductions resulted in seed abortion at the end of the globular stage of embryo development, when proplastids in normal embryos differentiate and acquire chlorophyll. Seed abortion was partly dependent on the light level during silique development. Embryos in seeds destined for abortion failed to undergo normal morphogenesis and were 'raspberry-like' in appearance. They had ultrastructural and biochemical defects including proliferation of peroxisomes and starch granules, and altered expression of genes involved in starch turnover and the oxidative pentose phosphate pathway. We propose that GPT1 is necessary for early embryo development because it catalyses import into plastids of glucose-6-phosphate as the substrate for NADPH generation via the oxidative pentose phosphate pathway. We suggest that low NADPH levels during plastid differentiation and chlorophyll synthesis may result in generation of reactive oxygen species and triggering of embryo cell death.

Starch turnover in developing oilseed embryos.

*Starch accumulates early during embryo development in Arabidopsis and oilseed rape, then disappears during oil accumulation. Little is known about the nature and importance of starch metabolism in oilseed embryos. *Histochemical and quantitative measures of starch location and content were made on developing seeds and embryos from wild-type Arabidopsis plants, and from mutants lacking enzymes of starch synthesis and degradation with established roles in leaf starch turnover. Feeding experiments with [(14)C]sucrose were used to measure the rate of starch synthesis in oilseed rape embryos within intact siliques. *The patterns of starch turnover in the developing embryo are spatially and temporally complex. Accumulation is associated with zones of cell division. Study of mutant plants reveals a major role in starch turnover for glucan, water dikinase (absent from the sex1 mutant) and isoforms of beta-amylase (absent from various bam mutants). Starch is synthesized throughout the period of its accumulation and loss in embryos within intact siliques of oilseed rape. *We suggest that starch turnover is functionally linked to cell division and differentiation rather than to developmental or storage functions specific to embryos. The pathways of embryo starch metabolism are similar in several respects to those in Arabidopsis leaves.

Plastidial glycolysis in developing Arabidopsis embryos.

During oilseed embryo development, carbon from sucrose is utilized for fatty acid synthesis in the plastid. The role of plastidial glycolysis in Arabidopsis embryo oil accumulation was investigated. Genes encoding enolases (ENO) and phosphoglyceromutases (PGlyM) were identified, and activities and subcellular locations were established by expression of recombinant and green fluorescent protein (GFP)-fusion proteins. Mutant Arabidopsis plants lacking putative plastidial isoforms were characterized with respect to isoform composition and embryo oil content. In the developing embryo, ENO1 and ENO2 account for most or all of the plastidial and cytosolic ENO activity, respectively, and PGLYM1 accounts for most or all of the plastidial PGlyM activity. The eno1 and pglym1 mutants, in which plastidic ENO and PGlyM activities were undetectable, had wild-type amounts of seed oil at maturity. It is concluded that although plastids of developing Arabidopsis embryos have the capacity to carry out the lower part of the glycolytic pathway, the cytosolic glycolytic pathway alone is sufficient to support the flux from 3-phosphoglycerate to phosphoenolpyruvate required for oil production. The results highlight the importance for oil production of translocators that facilitate interchange of glycolytic intermediates between the cytosol and the plastid stroma.

Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase.

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds.

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [(14)C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [(14)C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [(14)C]Suc converted to hexose falls dramatically. Instead, most of the [(14)C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.

Isolation of amyloplasts.

Two different methods for the preparation of starch-rich plastids are described together with protocols for the determination of plastid yield, purity, and intactness. The preparation of amyloplasts from maize endosperm and oilseed rape embryos are given as examples, but the protocols could be adapted for the isolation of starch-rich plastids from other plant organs. A method for the determination of the quantitative distribution of an enzyme between the plastids and cytosol is given. Typical results and references for marker enzymes for a range of subcellular compartments are listed.

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos.

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.