The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.Starch is the major carbohydrate reserve in cereal grains, where it is located in the nonliving cells of the starchy endosperm and constitutes up to 60% of total grain dry weight (Aman et al., 1985). Starch consists of the essentially linear (134)-␣-glucan amylose, together with the branched (134,136)-␣-glucan amylopectin. The two polysaccharides are organized in semicrystalline starch granules, which in barley (Hordeum vulgare) grain contain 70% to 75% amylopectin and 25% to 30% amylose (for review, see MacGregor and Fincher, 1993).Following germination, the glucosyl residues of amylose and amylopectin are released to support seedling growth by the concerted action of ␣-amylases, ␤-amylases, debranching enzymes, and ␣-glucosidases. The debranching enzymes catalyze the hydrolysis of (136)-␣-glucosidic linkages in amylopectin or in (134,136)-␣-oligoglucosides released by ␣-amylases. Because the (136)-␣-glucosyl linkages in these oligoglucosides, which are also referred to as limit dextrins, are not hydrolyzed by ␣-or ␤-amylases, and because the action of ␣-glucosidase on branched oligoglucosides is relatively slow, debranching enzymes are considered to play a central role in the complete depolymerization of starch to Glc. The debranched oligosaccharides are susceptible to further hydrolysis by amylases and ␣-glucosidases (Lee et al., 1971).Starch-debranching enzymes have been divided into two groups based on differences in their substate specificities and action patterns (Lee et al., 1971). The first group includes the pullulanases (pullulan 6-glucanohydrolase; EC 3.2.1.41), endohydrolases capable of hydrolyzing (136)-␣-linkages in pullulan, a polysaccharide consisting of maltotriosyl residues linked by (136)-␣-linkages. The second group includes isoamylases (glycogen...

Background: Myxospermy is a process by which the external surfaces of seeds of many plant species produce mucilage-a polysaccharide-rich gel with numerous fundamental research and industrial applications. Due to its functional properties the mucilage can be difficult to remove from the seed and established methods for mucilage extraction are often incomplete, time-consuming and unnecessarily wasteful of precious seed stocks. Results: Here we tested the efficacy of several established protocols for seed mucilage extraction and then downsized and adapted the most effective elements into a rapid, small-scale extraction and analysis pipeline. Within 4 h, three chemically-and functionally-distinct mucilage fractions were obtained from myxospermous seeds. These fractions were used to study natural variation and demonstrate structure-function links, to screen for known mucilage quality markers in a field trial, and to identify research and industry-relevant lines from a large mutant population. Conclusion: The use of this pipeline allows rapid analysis of mucilage characteristics from diverse myxospermous germplasm which can contribute to fundamental research into mucilage production and properties, quality testing for industrial manufacturing, and progressing breeding efforts in myxospermous crops.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanism(s) underpinning whether and how plant inflorescence architecture has responded to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a novel mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognised role of plant MADS-box proteins in floral development.

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.

Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases Toll-like receptor-4 (TLR4)-mediated responses to subsequent stimulation with LPS. To explore the pulmonary innate immune response to ozone exposure further, we investigated the effects of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Whole-lung lavage (WLL) and lung tissue were harvested from C57BL/6 mice after exposure to ozone or filtered air, followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the WLL, the surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. We demonstrated an increased WLL cell influx, increased IL-6 and chemokine KC (Cxcl1), and decreased macrophage inflammatory protein (MIP)-1a and TNF-a in response to Pam3CYS as a result of ozone pre-exposure. We also observed the increased cell surface expression of TLR4, TLR2, and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in the expression of genes related to injury repair and the cell cycle as a result of ozone alone or in combination with Pam3CYS. Our results extend previous findings with ozone/LPS to other TLR ligands, and suggest that the ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level.

I. Introduction to seed mucilage II. Plantago seed mucilage composition and properties III. Same but different: mucilage-related features of key model species and beyond IV. Where next? V. Conclusion and outlook Acknowledgements References Summary 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.

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When wetted, Plantago seeds become covered with a polysaccharide-rich gel called mucilage that has value as a food additive and bulking dietary fibre. Industrially, the dry husk layer that becomes mucilage, called psyllium, is milled off Plantago ovata seeds, the only commercial-relevant Plantago species, while the residual inner seed tissues are either used for low value animal feed or discarded. We suggest that this practice is potentially wasting a highly nutritious resource and here describe the use of histological, physicochemical, and chromatographic analyses to compare whole seed composition/characteristics of P. ovata with 11 relatives already adapted to harsh Australian conditions that may represent novel commercial crop options. We show that substantial interspecific differences in mucilage yield and macromolecular properties are mainly a consequence of differences in heteroxylan and pectin composition and probably represent wide differences in hydrocolloid functionality that can be exploited in industry. We also show that non-mucilage producing inner seed tissues contain a substantial mannan-rich endosperm, high in fermentable sugars, protein, and fats. Whole seed Plantago flour, particularly from some species obtained from harsh Australian environments, may provide improved economic and health benefits compared to purified P. ovata psyllium husk, by retaining the functionality of the seed mucilage and providing additional essential nutrients.