From genome to function: the Arabidopsis aquaporins

BACKGROUND: In the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well. RESULTS: The Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo. CONCLUSIONS: Our preliminary data indicate a strongly transcellular component for the flux of water in roots

Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii

HighlightCharacterization of regulatory networks in Chlamydomonas reinhardtii led to the identification of regulatory hubs that control the repatterning of cellular metabolism that leads to triacylglycerol accumulation in microalgae.Microalgae-based biofuels are promising sources of alternative energy, but improvements throughout the production process are required to establish them as economically feasible. One of the most influential improvements would be a significant increase in lipid yields, which could be achieved by altering the regulation of lipid biosynthesis and accumulation. Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N) deprivation. Although a few important regulatory genes have been identified that are involved in controlling this process, a global understanding of the larger regulatory network has not been developed. In order to uncover this network in this species, a combined omics (transcriptomic, proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414 predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated versus down-regulated and early response versus late response relative to two phases of polar lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite profiling generated compound accumulation levels that were integrated with the transcript dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this proposed regulatory network led to the identification of several regulatory hubs that control many aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism, photosynthesis and lipid metabolism

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and 13C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700+ reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic 13CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates

An Osmotic Model of the Growing Pollen Tube

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

Sulfur Transfer through an Arbuscular Mycorrhiza1

Despite the importance of sulfur (S) for plant nutrition, the role of the arbuscular mycorrhizal (AM) symbiosis in S uptake has received little attention. To address this issue, 35S-labeling experiments were performed on mycorrhizas of transformed carrot (Daucus carota) roots and Glomus intraradices grown monoxenically on bicompartmental petri dishes. The uptake and transfer of 35SO42− by the fungus and resulting 35S partitioning into different metabolic pools in the host roots was analyzed when altering the sulfate concentration available to roots and supplying the fungal compartment with cysteine (Cys), methionine (Met), or glutathione. Additionally, the uptake, transfer, and partitioning of 35S from the reduced S sources [35S]Cys and [35S]Met was determined. Sulfate was taken up by the fungus and transferred to mycorrhizal roots, increasing root S contents by 25% in a moderate (not growth-limiting) concentration of sulfate. High sulfate levels in the mycorrhizal root compartment halved the uptake of 35SO42− from the fungal compartment. The addition of 1 mm Met, Cys, or glutathione to the fungal compartment reduced the transfer of sulfate by 26%, 45%, and 80%, respectively, over 1 month. Similar quantities of 35S were transferred to mycorrhizal roots whether 35SO42−, [35S]Cys, or [35S]Met was supplied in the fungal compartment. Fungal transcripts for putative S assimilatory genes were identified, indicating the presence of the trans-sulfuration pathway. The suppression of fungal sulfate transfer in the presence of Cys coincided with a reduction in putative sulfate permease and not sulfate adenylyltransferase transcripts, suggesting a role for fungal transcriptional regulation in S transfer to the host. A testable model is proposed describing root S acquisition through the AM symbiosis

From genome to function: the Arabidopsis aquaporins

BACKGROUND:In the post-genomic era newly sequenced genomes can be used to deduce organismal functions from our knowledge of other systems. Here we apply this approach to analyzing the aquaporin gene family in Arabidopsis thaliana. The aquaporins are intrinsic membrane proteins that have been characterized as facilitators of water flux. Originally termed major intrinsic proteins (MIPs), they are now also known as water channels, glycerol facilitators and aqua-glyceroporins, yet recent data suggest that they facilitate the movement of other low-molecular-weight metabolites as well.RESULTS:The Arabidopsis genome contains 38 sequences with homology to aquaporin in four subfamilies, termed PIP, TIP, NIP and SIP. We have analyzed aquaporin family structure and expression using the A. thaliana genome sequence, and introduce a new NMR approach for the purpose of analyzing water movement in plant roots in vivo.CONCLUSIONS:Our preliminary data indicate a strongly transcellular component for the flux of water in roots.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Triacylglyceride Metabolism by Fusarium graminearum During Colonization and Sexual Development on Wheat

Fusarium graminearum, a devastating pathogen of small grains, overwinters on crop residues and produces ephemeral perithecia. Accumulation of lipids in overwintering hyphae would provide reserves for overwinter survival and perithecium development. Fatty acid composition of cultures during perithecium development indicated a drop in neutral lipid levels during development but little change in fatty acid composition across stages. Microscopic examination of cultures early in sexual development revealed hyphal cells engorged with lipid bodies. In comparison, vegetative hyphae contained few lipid bodies. Microarray analysis was performed on wheat stems at stages of colonization through perithecium development. Gene expression analysis during stages of perithecium development both in planta and in vitro (previously published) supports the view that lipid biosynthesis occurs during early stages of wheat colonization leading to sexual development and that lipid oxidation occurs as perithecia are developing. Analysis of gene expression during the stages of wheat stem colonization also revealed sets of genes unique to these stages. These results support the view that lipids accumulate in hyphae colonizing wheat stalks and are subsequently used in perithecium formation on stalk tissue. These results indicate that extensive colonization of plant tissue prior to harvest is essential for subsequent sporulation on crop residues and, thus, has important implications for inoculum reduction