Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic Acid, metabolism, and transitory starch accumulation

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (NADP-ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects in fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformats, which suggest that is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positional labelled glucoses of fruits lacking in plastidic NADP-malic enzyme and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate which suggests that excess of oxaloacetate OAA is converted to aspartate and reintroduced in the TCA via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-malic enzyme antisense lines were characterized by no changes in respiration rates and TCA cycle flux and together with an increase of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicates that pyruvate is supply through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening

Transcriptomics of C4 photosynthesis in rice paddy

This is the author accepted manuscript. The final version is available from the American Society of Plant Biologists via http://dx.doi.org/​10.​1104/​pp.​15.​00889The C₄ pathway is a highly complex trait that increases photosynthetic efficiency in over sixty plant lineages. Although the majority of C₄ plants occupy disturbed, arid and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C₄ E. glabrescens and C₃ rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C₄ photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C₃ species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C₄) and Brachypodium distachyon (C₃) were also found to accumulate chloroplastic PEPC. We identified a molecular signature associated with C₄ photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C₄ plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture

Purification of the pancreatic cholecystokinin receptor

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28032/1/0000471.pd

Changes in the concentrations and transcripts for gibberellins and other hormones in a growing leaf and roots of wheat seedlings in response to water restriction

Background Bread wheat (Triticum aestivum) is a major source of nutrition globally, but yields can be seriously compromised by water limitation. Redistribution of growth between shoots and roots is a common response to drought, promoting plant survival, but reducing yield. Gibberellins (GAs) are necessary for shoot and root elongation, but roots maintain growth at lower GA concentrations compared with shoots, making GA a suitable hormone for mediating this growth redistribution. In this study, the effect of progressive drought on GA content was determined in the base of the 4th leaf and root tips of wheat seedlings, containing the growing regions, as well as in the remaining leaf and root tissues. In addition, the contents of other selected hormones known to be involved in stress responses were determined. Transcriptome analysis was performed on equivalent tissues and drought-associated differential expression was determined for hormone-related genes. Results After 5 days of applying progressive drought to 10-day old seedlings, the length of leaf 4 was reduced by 31% compared with watered seedlings and this was associated with significant decreases in the concentrations of bioactive GA(1) and GA(4) in the leaf base, as well as of their catabolites and precursors. Root length was unaffected by drought, while GA concentrations were slightly, but significantly higher in the tips of droughted roots compared with watered plants. Transcripts for the GA-inactivating gene TaGA2ox4 were elevated in the droughted leaf, while those for several GA-biosynthesis genes were reduced by drought, but mainly in the non-growing region. In response to drought the concentrations of abscisic acid, cis-zeatin and its riboside increased in all tissues, indole-acetic acid was unchanged, while trans-zeatin and riboside, jasmonate and salicylic acid concentrations were reduced. Conclusions Reduced leaf elongation and maintained root growth in wheat seedlings subjected to progressive drought were associated with attenuated and increased GA content, respectively, in the growing regions. Despite increased TaGA2ox4 expression, lower GA levels in the leaf base of droughted plants were due to reduced biosynthesis rather than increased catabolism. In contrast to GA, the other hormones analysed responded to drought similarly in the leaf and roots, indicating organ-specific differential regulation of GA metabolism in response to drought

Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering

Chemical intervention in plant sugar signalling increases yield and resilience

The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a ‘signalling-precursor’ concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)