Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)-ß-glucan exopolysaccharide in the bacterium Sarcina ventriculi.

Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

CLAW: An automated Snakemake workflow for the assembly of chloroplast genomes from long-read data.

Chloroplasts are photosynthetic organelles in algal and plant cells that contain their own genome. Chloroplast genomes are commonly used in evolutionary studies and taxonomic identification and are increasingly becoming a target for crop improvement studies. As DNA sequencing becomes more affordable, researchers are collecting vast swathes of high-quality whole-genome sequence data from laboratory and field settings alike. Whole tissue read libraries sequenced with the primary goal of understanding the nuclear genome will inadvertently contain many reads derived from the chloroplast genome. These whole-genome, whole-tissue read libraries can additionally be used to assemble chloroplast genomes with little to no extra cost. While several tools exist that make use of short-read second generation and third-generation long-read sequencing data for chloroplast genome assembly, these tools may have complex installation steps, inadequate error reporting, poor expandability, and/or lack scalability. Here, we present CLAW (Chloroplast Long-read Assembly Workflow), an easy to install, customise, and use Snakemake tool to assemble chloroplast genomes from chloroplast long-reads found in whole-genome read libraries (https://github.com/aaronphillips7493/CLAW). Using 19 publicly available reference chloroplast genome assemblies and long-read libraries from algal, monocot and eudicot species, we show that CLAW can rapidly produce chloroplast genome assemblies with high similarity to the reference assemblies. CLAW was designed such that users have complete control over parameterisation, allowing individuals to optimise CLAW to their specific use cases. We expect that CLAW will provide researchers (with varying levels of bioinformatics expertise) with an additional resource useful for contributing to the growing number of publicly available chloroplast genome assemblies.

The cellulose synthase-like F3 (CslF3) gene mediates cell wall polysaccharide synthesis and affects root growth and differentiation in barley.

The barley cellulose synthase-like F (CslF) genes encode putative cell wall polysaccharide synthases. They are related to the cellulose synthase (CesA) genes involved in cellulose biosynthesis, and the CslD genes that influence root hair development. Although CslD genes are implicated in callose, mannan and cellulose biosynthesis, and are found in both monocots and eudicots, CslF genes are specific to the Poaceae. Recently the barley CslF3 (HvCslF3) gene was shown to be involved in the synthesis of a novel (1,4)-ß-linked glucoxylan, but it remains unclear whether this gene contributes to plant growth and development. Here, expression profiling using qRT-PCR and mRNA in situ hybridization revealed that HvCslF3 accumulates in the root elongation zone. Silencing HvCslF3 by RNAi was accompanied by slower root growth, linked with a shorter elongation zone and a significant reduction in root system size. Polymer profiling of the RNAi lines revealed a significant reduction in (1,4)-ß-linked glucoxylan levels. Remarkably, the heterologous expression of HvCslF3 in wild-type (Col-0) and root hair-deficient Arabidopsis mutants (csld3 and csld5) complemented the csld5 mutant phenotype, in addition to altering epidermal cell fate. Our results reveal a key role for HvCslF3 during barley root development and suggest that members of the CslD and CslF gene families have similar functions during root growth regulation.

Targeted mutation of barley (1,3;1,4)-ß-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content.

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-ß-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-ß-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-ß-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-ß-glucan content. (1,3;1,4)-ß-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-ß-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-ß-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-ß-glucan in mature grain.

The goo-d stuff: Plantago as a myxospermous model with modern utility.

Mucilage, a gel-like layer formed around wetted seeds in a process called myxospermy, has importance as a proxy for studying cell wall polysaccharide biosynthesis and interactions and as a source of valuable health supplements and hydrocolloids. Arabidopsis thaliana has provided unrivalled insight into mucilage/cell wall synthesis, but its lack of commercial utility presents an opportunity to develop an alternative myxospermous model linking genetics, chemistry and functionality. Here, we discuss recent advances in the understanding of mucilage production, composition and properties of Plantago, a promising candidate as an alternative model with economic relevance. We outline how genomic/transcriptomic and chemical analysis advances could be made to strengthen Plantago's use as a model system, through challenging but achievable approaches.

Genome-wide association study reveals the genetic complexity of fructan accumulation patterns in barley grain.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan.

Mixed-linkage (1,3;1,4)-ß-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-ß-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-ß-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-ß-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain.

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain.

Patient-Centered Medical Home Activities Associated With Low Medicare Spending and Utilization.

PURPOSE: To identify components of the patient-centered medical home (PCMH) model of care that are associated with lower spending and utilization among Medicare beneficiaries. METHODS: Regression analyses of changes in outcomes for Medicare beneficiaries in practices that engaged in particular PCMH activities compared with beneficiaries in practices that did not. We analyzed claims for 302,719 Medicare fee-for-service beneficiaries linked to PCMH surveys completed by 394 practices in the Centers for Medicare & Medicaid Services' 8-state Multi-Payer Advanced Primary Care Practice demonstration. RESULTS: Six activities were associated with lower spending or utilization. Use of a registry to identify and remind patients due for preventive services was associated with all 4 of our outcome measures: total spending was $69.77 less per beneficiary per month (PBPM) (P = 0.00); acute-care hospital spending was $36.62 less PBPM (P = 0.00); there were 6.78 fewer hospital admissions per 1,000 beneficiaries per quarter (P1KBPQ) (P = 0.003); and 11.05 fewer emergency department (ED) visits P1KBPQ (P = 0.05). Using a patient registry for pre-visit planning and clinician reminders was associated with $29.31 lower total spending PBPM (P = 0.05). Engaging patients with chronic conditions in goal setting and action planning was associated with 4.62 fewer hospital admissions P1KBPQ (P = 0.01) and 11.53 fewer ED visits P1KBPQ (P = 0.00). Monitoring patients during hospital stays was associated with $22.06 lower hospital spending PBPM (P = 0.03). Developing referral protocols with commonly referred-to clinicians was associated with 11.62 fewer ED visits P1KBPQ (P = 0.00). Using quality improvement approaches was associated with 13.47 fewer ED visits P1KBPQ (P =0.00). CONCLUSIONS: Practices seeking to deliver more efficient care may benefit from implementing these 6 activities.

Accumulation of volatile phenol glycoconjugates in grapes following grapevine exposure to smoke and potential mitigation of smoke taint by foliar application of kaolin.

MAIN CONCLUSION: The accumulation of volatile phenol glycoconjugates in smoke-exposed grapes was monitored following grapevine exposure to smoke, with different glycoconjugate profiles observed for fruit sampled 1 and 7 days after smoke exposure, and at maturity. Foliar application of kaolin reduced the concentration of volatile phenol glycoconjugates in smoke-exposed fruit, but efficacy depended on the rate of application and extent of coverage. Smoke taint can be found in wines made from grapes exposed to smoke from bushfires or prescribed burns. It is characterized by objectionable smoky and ashy aromas and flavors, which have been attributed to the presence of smoke-derived volatile phenols, in free and glycoconjugate forms. This study investigated: (1) the accumulation of volatile phenol glycoconjugates in grapes following the application of smoke to Sauvignon Blanc, Chardonnay and Merlot grapevines at approximately 10 days post-veraison; and (2) the potential mitigation of smoke taint as a consequence of foliar applications of kaolin (a clay-based protective film) prior to grapevine smoke exposure. Varietal differences were observed in the glycoconjugate profiles of smoke-exposed grapes; the highest glycoconjugate levels were found in Merlot grapes, being pentose-glucosides of guaiacol, cresols, and phenol, and gentiobiosides of guaiacol and syringol. Changes in volatile phenol glycoconjugate profiles were also observed with time, i.e., between fruit sampled 1 day after smoke exposure and at maturity. The application of kaolin did not significantly affect the glycoconjugate profiles of Sauvignon Blanc and Chardonnay grapes, but significantly lower volatile phenol glycoconjugate levels were observed in Merlot fruit that was treated with kaolin prior to smoke exposure. The potential for control and smoke-exposed grapes to be differentiated by measurement of spectral reflectance was also demonstrated.

Loss of LOFSEP Transcription Factor Function Converts Spikelet to Leaf-Like Structures in Rice.

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.

Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution.

Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase (CesA) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM, that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-ß-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae.

Isolation of tissues and preservation of RNA from intact, germinated barley grain.

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19 611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24 h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination.

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants.

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis.

Genetic and environmental factors contribute to variation in cell wall composition in mature desi chickpea (Cicer arietinum L.) cotyledons.

Chickpea (Cicer arietinum L.) is an important nutritionally rich legume crop that is consumed worldwide. Prior to cooking, desi chickpea seeds are most often dehulled and cleaved to release the split cotyledons, referred to as dhal. Compositional variation between desi genotypes has a significant impact on nutritional quality and downstream processing, and this has been investigated mainly in terms of starch and protein content. Studies in pulses such as bean and lupin have also implicated cell wall polysaccharides in cooking time variation, but the underlying relationship between desi chickpea cotyledon composition and cooking performance remains unclear. Here, we utilized a variety of chemical and immunohistological assays to examine details of polysaccharide composition, structure, abundance, and location within the desi chickpea cotyledon. Pectic polysaccharides were the most abundant cell wall components, and differences in monosaccharide and glycosidic linkage content suggest both environmental and genetic factors contribute to cotyledon composition. Genotype-specific differences were identified in arabinan structure, pectin methylesterification, and calcium-mediated pectin dimerization. These differences were replicated in distinct field sites and suggest a potentially important role for cell wall polysaccharides and their underlying regulatory machinery in the control of cooking time in chickpea.

Characteristics of Patient-Centered Medical Home Initiatives that Generated Savings for Medicare: a Qualitative Multi-Case Analysis.

BACKGROUND: Through the Multi-Payer Advanced Primary Care Practice (MAPCP) Demonstration, Medicare, Medicaid, and private payers offered supplemental payments to 849 primary care practices that became patient-centered medical homes (PCMHs) in eight states; practices also received technical assistance and data reports. Average Medicare payments were capped at $10 per beneficiary per month in each state. OBJECTIVE: Since there was variation in the eight participating states' demonstration designs, experiences, and outcomes, we conducted a qualitative multi-case analysis to identify the key factors that differentiated states that were estimated to have generated net savings for Medicare from states that did not. PARTICIPANTS: States' MAPCP Demonstration initiatives were comprehensively profiled in case studies based on secondary document review, three rounds of annual interviews with state staff, payers, practices, and other stakeholders, and other data sources. APPROACH: Case study findings were summarized in a case-ordered predictor-outcome matrix, which identified the presence or absence of key demonstration design features and experiences and arrayed states based on the amount of net savings or losses they generated for Medicare. We then used this matrix to identify initiative features that were present in at least three of the four states that generated net savings and absent from at least three of the four states that did not generate savings. RESULTS: A majority of the states that generated net savings: required practices to be recognized PCMHs to enter the demonstration, did not allow late entrants into the demonstration, used a consistent demonstration payment model across participating payers, and offered practices opportunities to earn performance bonuses. Practices in states that generated net savings also tended to report receiving the demonstration payments and bonuses they expected to receive, without any issues. CONCLUSIONS: Designers of future PCMH initiatives may increase their likelihood of generating net savings by incorporating the demonstration features we identified.

Perspectives on Implementing Quality Improvement Collaboratives Effectively: Qualitative Findings from the CHIPRA Quality Demonstration Grant Program.

BACKGROUND: The most frequently pursued intervention in the $100 million, 18-state Children's Health Insurance Program Reauthorization Act of 2009 (CHIPRA) quality demonstration (2010-2015) was quality improvement collaboratives, which 12 states offered to more than 300 primary care practices. A study was conducted to identify which aspects of these collaboratives were viewed by organizers and participants as working well and which were not. METHODS: Some 223 interviews were conducted in these states near the end of their collaboratives. Interview notes were coded and analyzed to identify trends. RESULTS: Aspects of collaboratives that interviewees valued were aimed at attracting participation, maintaining engagement, or facilitating learning. To attract participants, interviewees recommended offering maintenance-of-certification credits, aligning content with existing financial incentives, hiring a knowledgeable collaborative organizer of the same medical specialty as participants, and having national experts speak at meetings. Positively viewed approaches for maintaining engagement included meeting one-on-one with practices to articulate participation expectations in advance, tying disbursal of stipends to meeting participation expectations, and soliciting feedback and making mid-course adjustments. To facilitate learning, interviewees liked learning from other practices, interactive exercises, practical handouts, and meeting face-to-face with new referral partners. CONCLUSION: Prior studies have tended to focus on strategies to maintain engagement. The interviewees valued these features but also valued aspects of collaboratives that attracted participants in the first place and facilitated learning after participants were actively engaged. The findings suggest that a wider array of features may be important when developing or evaluating collaboratives. Collaborative organizers may benefit from incorporating the recommended collaborative features into their own collaboratives.

Method for hull-less barley transformation and manipulation of grain mixed-linkage beta-glucan.

Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-ß-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm.

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.