Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes.

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Concurrent overexpression of amino acid permease AAP1(3a) and SUT1 sucrose transporter in pea resulted in increased seed number and changed cytokinin and protein levels.

Using pea as our model crop, we sought to understand the regulatory control over the import of sugars and amino acids into the developing seeds and its importance for seed yield and quality. Transgenic peas simultaneously overexpressing a sucrose transporter and an amino acid transporter were developed. Pod walls, seed coats, and cotyledons were analysed separately, as well as leaves subtending developing pods. Sucrose, starch, protein, free amino acids, and endogenous cytokinins were measured during development. Temporal gene expression analyses (RT-qPCR) of amino acid (AAP), sucrose (SUT), and SWEET transporter family members, and those from cell wall invertase, cytokinin biosynthetic (IPT) and degradation (CKX) gene families indicated a strong effect of the transgenes on gene expression. In seed coats of the double transgenics, increased content and prolonged presence of cytokinin was particularly noticeable. The transgenes effectively promoted transition of young sink leaves into source leaves. We suggest the increased flux of sucrose and amino acids from source to sink, along with increased interaction between cytokinin and cell wall invertase in developing seed coats led to enhanced sink activity, resulting in higher cotyledon sucrose at process pea harvest, and increased seed number and protein content at maturity.

Manipulation of sucrose phloem and embryo loading affects pea leaf metabolism, carbon and nitrogen partitioning to sinks as well as seed storage pools.

Seed development largely depends on the long-distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source-to-sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this 'Push-and-Pull' approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source-to-sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1-overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild-type plants. Together, the results demonstrate that the SUT1-overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push-and-Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.

Mesenchymal Cells Support the Oncogenicity and Therapeutic Response of the Hedgehog Pathway in Triple-Negative Breast Cancer.

The paracrine interaction between tumor cells and adjacent stroma has been associated with the oncogenic activity of the Hedgehog (Hh) pathway in triple-negative breast tumors. The present study developed a model of paracrine Hh signaling and examined the impact of mesenchymal cell sources and culture modalities in the oncogenicity of the Hh pathway in breast tumor cells. Studies consisted of tumor cell monocultures and co-cultures with cancer-associated and normal fibroblasts, tumor cells that undergo epithelial-mesenchymal transition (EMT), or adipose-derived mesenchymal stem cells (ADMSCs). Hh ligand and pathway inhibitors, GANT61 and NVP-LDE225 (NVP), were evaluated in both cell cultures and a mouse xenograft model. Results in monocultures show that tumor cell viability and Hh transcriptional activity were not affected by Hh inhibitors. In co-cultures, down-regulation of GLI1, SMO, and PTCH1 in the stroma correlated with reduced tumor growth rates in xenografted tumors and cell cultures, confirming a paracrine interaction. Fibroblasts and EMT cells supported Hh transcriptional activity and enhanced tumor cell growth. Mixed and adjacent culture modalities indicate that tumor growth is supported via fibroblast-secreted soluble factors, whereas enriched tumor stemness requires close proximity between tumor and fibroblasts. Overall this study provides a tumor-mesenchymal model of Hh signaling and highlights the therapeutic value of mesenchymal cells in the oncogenic activity of the Hh pathway.

Expression of Genes Related to Sugar and Amino Acid Transport and Cytokinin Metabolism during Leaf Development and Senescence in Pisum sativum L.

Gene editing is becoming the plant breeding tool of choice, but prior to targeting a gene for editing, a knowledge of the gene family members (GFMs) controlling yield is required in the specific crop plant. Critical to yield are components from senescing leaves. We targeted genes controlling senescence in Pisum sativum and the release and transport of carbohydrates and amino acids from the source leaves to the pods and seeds. The expression of GFMs for cytokinin biosynthesis (IPT) and destruction (CKX), sucrose transporters (SUT), Sugar Will Eventually be Exported Transporters (SWEET), amino acid permeases (AAP), and cell wall invertases, was determined using RT-qPCR. GFMs were differentially expressed in leaves of different ages. The expression of many gene family members was lower in the expanding sink leaves compared with the senescing leaves, with the exception of two PsAAP GFMs and PsCKX5, which were highly expressed. Expression of specific PsSWEETs, SUTs, and AAPs increased in the mature and/or senescing leaves. Expression of PsIPTs was least in the mature source leaves, but as strong in the senescing leaves as in the young source leaves. PsCKX7 was expressed in source and senescing leaves. We discuss the potential impact of the targeted reduction of specific PsCKX GFMs on source-sink relationships.

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids.

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.

Changes in the relative abundance and movement of insect pollinators during the flowering cycle of Brassica rapa crops: implications for gene flow.

The potential movement of transgenes from genetically modified crops to non-genetically modified crops via insect-mediated pollen dispersal has been highlighted as one of the areas of greatest concern in regards to genetically modified crops. Pollen movement depends sensitively on spatial and temporal variation in the movement of insect pollinators between crop fields. This study tested the degree of variation in the diversity and relative abundance of flower-visiting insects entering versus leaving pak choi, Brassica rapa var. chinensis L. (Brassicales: Brassicaceae), crops throughout different stages of the flowering cycle. The relative abundance of flower-visiting insects varied significantly with Brassica crop phenology. Greater numbers of flower-visiting insects were captured inside rather than outside the crop fields, with the highest capture rates of flower-visitors coinciding with the peak of flowering in both spring-flowering and summer-flowering crops. Moreover, the ratio of flower-visiting insects entering versus leaving crop fields also varied considerably with changing crop phenology. Despite high variation in relative capture rates, the data strongly indicate non-random patterns of variation in insect movement in relation to crop phenology, with early-season aggregation of flower-visiting insects entering and remaining in the crop, and then mass emigration of flower-visiting insects leaving the crop late in the flowering season. Although pollen movement late in the flowering cycle might contribute relatively little to total seed set (and hence crop production), the findings here suggest that extensive late-season pollinator redistribution in the landscape could contribute disproportionately to long-distance gene movement between crops.

Developing psychotherapists' competence through clinical supervision: protocol for a qualitative study of supervisory dyads.

BACKGROUND: Mental health professionals face unique demands and stressors in their work, resulting in high rates of burnout and distress. Clinical supervision is a widely adopted and valued mechanism of professional support, development, and accountability, despite the very limited evidence of specific impacts on therapist or client outcomes. The current study aims to address this by exploring how psychotherapists develop competence through clinical supervision and what impact this has on the supervisees' practice and their clients' outcomes. This paper provides a rationale for the study and describes the protocol for an in-depth qualitative study of supervisory dyads, highlighting how it addresses gaps in the literature. METHODS/DESIGN: The study of 16-20 supervisor-supervisee dyads uses a qualitative mixed method design, with two phases. In phase one, supervisors who are nominated as expert by their peers are interviewed about their supervision practice. In phase two, supervisors record a supervision session with a consenting supervisee; interpersonal process recall interviews are conducted separately with supervisor and supervisee to reflect in depth on the teaching and learning processes occurring. All interviews will be transcribed, coded and analysed to identify the processes that build competence, using a modified form of Consensual Qualitative Research (CQR) strategies. Using a theory-building case study method, data from both phases of the study will be integrated to develop a model describing the processes that build competence and support wellbeing in practising psychotherapists, reflecting the accumulated wisdom of the expert supervisors. DISCUSSION: The study addresses past study limitations by examining expert supervisors and their supervisory interactions, by reflecting on actual supervision sessions, and by using dyadic analysis of the supervisory pairs. The study findings will inform the development of future supervision training and practice and identify fruitful avenues for future research.

Managing difficulties in supervision: supervisors' perspectives.

Few studies have examined the practice wisdom of expert supervisors. This study addresses this gap by exploring how experienced supervisors manage difficulties in supervision in the context of the supervisory relationship. The supervisors were a purposive sample of 16 senior members of the profession with considerable expertise in supervision. In-depth interviews were first conducted with the supervisors. An interpersonal process recall method was then used to explore their reflections on one of their DVD-recorded supervision sessions. Analysis of transcripts was completed using a modified consensual qualitative research method. Major difficulties included the broad domains of supervisee competence and ethical behavior, supervisee characteristics, supervisor countertransference, and problems in the supervisory relationship. Supervisors managed these difficulties using 4 key approaches: relational (naming, validating, attuning, supporting, anticipating, exploring parallel process, acknowledging mistakes, and modeling); reflective (facilitating reflectivity, remaining mindful and monitoring, remaining patient and transparent, processing countertransference, seeking supervision, and case conceptualizing); confrontative (confronting tentatively, confronting directly, refusing/terminating supervision, taking formal action, referring to personal therapy, and becoming directive); and avoidant interventions (struggling on, withholding, and withdrawing). Two brief case studies illustrate the process of applying these strategies sequentially in managing difficulties. The study highlights the importance of relational strategies to maintain an effective supervisory alliance, reflective strategies-particularly when difficulties pertain to clinical material and the supervisory relationship-and confrontative strategies with unhelpful supervisee characteristics and behaviors that impede supervision.

Increased phloem transport of S-methylmethionine positively affects sulfur and nitrogen metabolism and seed development in pea plants.

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.

Metabolic effects of soy supplementation in postmenopausal Caucasian and African American women: a randomized, placebo-controlled trial.

OBJECTIVE: We sought to determine the effect of daily soy supplementation on abdominal fat, glucose metabolism, and circulating inflammatory markers and adipokines in obese, postmenopausal Caucasian and African American women. STUDY DESIGN: In a double-blinded controlled trial, 39 postmenopausal women were randomized to soy supplementation or to a casein placebo without isoflavones. In all, 33 completed the study and were analyzed. At baseline and at 3 months, glucose disposal and insulin secretion were measured using hyperglycemic clamps, body composition and body fat distribution were measured by computed tomographic scan and dual energy x-ray absorptiometry, and serum levels of C-reactive protein, interleukin-6, tumor necrosis factor-alpha, leptin, and adiponectin were measured by immunoassay. RESULTS: Soy supplementation reduced total and subcutaneous abdominal fat and interleukin-6. No difference between groups was noted for glucose metabolism, C-reactive protein, tumor necrosis factor-alpha, leptin, or adiponectin. CONCLUSION: Soy supplementation reduced abdominal fat in obese postmenopausal women. Caucasians primarily lost subcutaneous and total abdominal fat, and African Americans primarily lost total body fat.

A hidden heritage.

THIS YEAR, as we celebrate the bicentenary of the passing of a parliamentary bill to abolish the slave trade in the then British Empire, we also have an ideal opportunity to reflect on the continuing contributions of black and minority ethnic (BME) staff to the NHS.

Pine (Pinus radiata).

This chapter describes the transformation of Pinus radiata using organogenic cotyledon explants rather than the more common somatic embryogenesis methods for conifers. The advantages of our method are the year round availability of seed and that over 80% of genotypes can be easily regenerated from the mature cotyledon explants. The transformation efficiency (i.e., the number of transformed shoots regenerated from excised cotyledons) is 1.7% and, as with other Agrobacterium tumefaciens transformation methods, the majority of transgene integrations are single copy. Critical factors for success are survival of the cotyledons, Agrobacterium strain, and selection pressure after cocultivation.

Peas (Pisum sativum L.).

In this chapter we describe a robust method for transformation of peas that has been successfully used in our laboratory since 1992. Using immature pea seed collected from field- or greenhouse-grown plants, we have produced transgenic lines for over 30 genotypes including named pea cultivars and advanced breeding lines. This method uses immature cotyledons as the explant, and the transformation efficiency is in the range 0.2 to 13.5% of cotyledons producing at least one independently transformed line. Agrobacterium tumefaciens strains AGL1 and KYRT1 are the most successful in our procedure, and kanamycin, phosphinothricin, and hygromycin are reliable selectable markers. Potentially useful genes have been introduced for pest and disease resistance, altering quality traits, and investigating metabolic pathways and are being studied in transgenic pea lines.