A device for single leaf labelling with CO2 isotopes to study carbon allocation and partitioning in Arabidopsis thaliana

BACKGROUND: Plant biomass consists primarily of carbohydrates derived from photosynthesis. Monitoring the assimilation of carbon via the Calvin-Benson cycle and its subsequent utilisation is fundamental to understanding plant growth. The use of stable and radioactive carbon isotopes, supplied to plants as CO(2), allows the measurement of fluxes through the intermediates of primary photosynthetic metabolism, long-distance transport of sugars in the vasculature, and the synthesis of structural and storage components. RESULTS: Here we describe the design of a system for supplying isotopically labelled CO(2) to single leaves of Arabidopsis thaliana. We demonstrate that the system works well using short pulses of (14)CO(2) and that it can be used to produce robust qualitative and quantitative data about carbon export from source leaves to the sink tissues, such as the developing leaves and the roots. Time course experiments show the dynamics of carbon partitioning between storage as starch, local production of biomass, and export of carbon to sink tissues. CONCLUSION: This isotope labelling method is relatively simple to establish and inexpensive to perform. Our use of (14)CO(2) helps establish the temporal and spatial allocation of assimilated carbon during plant growth, delivering data complementary to those obtained in recent studies using (13)CO(2) and MS-based metabolomics techniques. However, we emphasise that this labelling device could also be used effectively in combination with (13)CO(2) and MS-based techniques

Starch mobilization in leaves

Starch mobilization is well understood in cereal endosperms, but both the pathway and the regulation of the process are poorly characterized in other types of plant organs. Arabidopsis leaves offer the opportunity for rapid progress in this area, because of the genomic resources available in this species and the ease with which starch synthesis and degradation can be monitored and manipulated. Progress in understanding three aspects of starch degradation is described: the role of disproportionating enzyme, the importance of phosphorolytic degradation, and new evidence about the involvement of a starch‐phosphorylating enzyme in the degradative process. Major areas requiring further research are outline

Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity

Background: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Results: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Conclusions: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process. Keywords: Amylopectin structure; Arabidopsis thaliana; Heterologous expression in yeast; Parallel reaction monitoring, Proteomics; Starch biosynthesis; YFP reporter

The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2

The evolution of functional complexity within the β-amylase gene family in land plants

Background β-Amylases (BAMs) are a multigene family of glucan hydrolytic enzymes playing a key role not only for plant biology but also for many industrial applications, such as the malting process in the brewing and distilling industries. BAMs have been extensively studied in Arabidopsis thaliana where they show a surprising level of complexity in terms of specialization within the different isoforms as well as regulatory functions played by at least three catalytically inactive members. Despite the importance of BAMs and the fact that multiple BAM proteins are also present in other angiosperms, little is known about their phylogenetic history or functional relationship. Results Here, we examined 961 β-amylase sequences from 136 different algae and land plant species, including 66 sequenced genomes and many transcriptomes. The extraordinary number and the diversity of organisms examined allowed us to reconstruct the main patterns of β-amylase evolution in land plants. We identified eight distinct clades in angiosperms, which results from extensive gene duplications and sub- or neo-functionalization. We discovered a novel clade of BAM, absent in Arabidopsis, which we called BAM10. BAM10 emerged before the radiation of seed plants and has the feature of an inactive enzyme. Furthermore, we report that BAM4 – an important protein regulating Arabidopsis starch metabolism – is absent in many relevant starch-accumulating crop species, suggesting that starch degradation may be differently regulated between species. Conclusions BAM proteins originated sometime more than 400 million years ago and expanded together with the differentiation of plants into organisms of increasing complexity. Our phylogenetic analyses provide essential insights for future functional studies of this important class of storage glucan hydrolases and regulatory proteins

The Leadership Processes of Pacific Public Servants in Aotearoa, New Zealand

This dissertation presents research focused on leadership processes among Pacific public servants at multiple levels in the New Zealand Public Service. The current study was guided by this research question: What are the leadership processes currently employed by Pacific public servants in the New Zealand Public Service? This study also explored participants' views on the effect of Pacific cultural backgrounds and organisational contexts on their current experience of leadership processes. The exploration of the topic was developed within a post-positivist research paradigm, using phenomenological methodology to examine the leadership processes of Pacific public servants. It employs qualitative case studies of two New Zealand Public Service organisations in the Wellington region. I employed two data collection tools in these case studies. The first was the use of in-depth interviews, and the second was an analysis of relevant organisational documents. A total of sixteen Pacific public servants participated in my study, eight from each case organisation. The findings indicated that the Pacific participants understood leadership as a social process of collective influence within a context. Participants perceived participating, networking and relationship building, learning about leadership from cultural contexts, and practising the Pacific value of va as important leadership processes for their performance in the organisations in which they were working. This study also found that the organisations' key roles and leadership values, which are embedded in Pacific cultures, shaped participants' experiences of the leadership processes. The findings also highlight some factors that contribute to and constrain the Pacific public servants' leadership processes. This emphasises the need for diverse policies to encompass leadership development. This study also highlights the need for leadership support for Pacific public servants at all levels in their New Zealand organisations. Practical and future research recommendations gained from the findings are discussed. The study contributes to the field of leadership research on Pacific public servants in New Zealand, and provides a different perspective on leadership processes in general leadership theory

The Arabidopsis Framework Model version 2 predicts the organism-level effects of circadian clock gene mis-regulation

Predicting a multicellular organism’s phenotype quantitatively from its genotype is challenging, as genetic effects must propagate across scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour. Here we explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used diverse metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for the vegetative growth of Arabidopsis thaliana, sharing the model and data files in a structured, public resource. The calibrated model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants under standard laboratory conditions. Altered night-time metabolism of stored starch accounted for most of the decrease in whole-plant biomass, as previously proposed. Mobilisation of a secondary store of malate and fumarate was also mis-regulated, accounting for any remaining biomass defect. The three candidate mechanisms tested did not explain this organic acid accumulation. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits