Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response.

BACKGROUND: Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gß subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling's survival under high salinity remains unclear. RESULTS: We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. CONCLUSIONS: Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression.

A lipid viewpoint on the plant endoplasmic reticulum stress response.

Organisms, including humans, seem to be constantly exposed to various changes, which often have undesirable effects, referred to as stress. To keep up with these changes, eukaryotic cells may have evolved a number of relevant cellular processes, such as the endoplasmic reticulum (ER) stress response. Owing to presumably intimate links between human diseases and the ER function, the ER stress response has been extensively investigated in various organisms for a few decades. Based on these studies, we now have a picture of the molecular mechanisms of the ER stress response, one of which, the unfolded protein response (UPR), is highly conserved among yeasts, mammals, higher plants, and green algae. In this review, we attempt to highlight the plant UPR from the perspective of lipids, especially membrane phospholipids. Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) are the most abundant membrane phospholipids in eukaryotic cells. The ratio of PtdCho to PtdEtn and the unsaturation of fatty acyl tails in both phospholipids may be critical factors for the UPR, but the pathways responsible for PtdCho and PtdEtn biosynthesis are distinct in animals and plants. We discuss the plant UPR in comparison with the system in yeasts and animals in the context of membrane phospholipids.

What's unique? The unfolded protein response in plants.

The investigation of a phenomenon called the unfolded protein response (UPR) started approximately three decades ago, and we now know that the UPR is involved in a number of cellular events among metazoans, higher plants, and algae. The relevance of the UPR in human diseases featuring protein folding defects, such as Alzheimer's and Huntington's diseases, has drawn much attention to the response in medical research to date. While metazoans and plants share similar molecular mechanisms of the UPR, recent studies shed light on the uniqueness of the plant UPR, with plant-specific protein families appearing to play pivotal roles. Given the considerable emphasis on the original discoveries of key factors in metazoans, this review highlights the uniqueness of the plant UPR based on current knowledge.

Smad-Independent BMP Signaling in Somatic Cells Limits the Size of the Germline Stem Cell Pool.

In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. Here, we demonstrate that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. Our results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.

A Targeted RNAi Screen Reveals Drosophila Female-Sterile Genes That Control the Size of Germline Stem Cell Niche During Development.

Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance.

Peeking at a plant through the holes in the wall - exploring the roles of plasmodesmata.

Plasmodesmata (PD) are membrane-lined pores that connect neighbouring plant cells and allow molecular exchange via the symplast. Past studies have revealed the basic structure of PD, some of the transport mechanisms for molecules through PD, and a variety of physiological processes in which they function. Recently, with the help of newly developed technologies, several exciting new features of PD have been revealed. New PD structures were observed during early formation of PD and between phloem sieve elements and phloem pole pericycle cells in roots. Both observations challenge our current understanding of PD structure and function. Research into novel physiological responses, which are regulated by PD, indicates that we have not yet fully explored the potential contribution of PD to overall plant function. In this Viewpoint article, we summarize some of the recent advances in understanding the structure and function of PD and propose the challenges ahead for the community.

Arabidopsis dolichol kinase AtDOK1 is involved in flowering time control.

Dolichols are a class of isoprenoids that consist of highly polymerized and unsaturated long-chain isoprenes. They play crucial roles in protein glycosylation including N-glycosylation, because the oligosaccharide is assembled on a lipid carrier, dolichyl diphosphate. Arabidopsis DOLICHOL KINASE 1, AtDOK1 (At3g45040), encodes a functional dolichol kinase that is involved in plant reproductive processes. The expression of AtDOK1 is limited to highly pluripotent cells although protein glycosylation is thought to be required ubiquitously in the entire plant body. In this study, we further explored AtDOK1 functions by creating leaky knockdown mutants of DOK1. We used a microRNA-mediated gene suppression technique because knockout of DOK1 causes lethality. The DOK1 knockdown mutants showed an early flowering phenotype without any remarkable growth defect in vegetative tissues. Indeed, AtDOK1 was highly expressed in emerging shoot apical meristems as well as inflorescence and floral meristems. A subcellular localization study of DOK1 revealed that DOK1 was localized at the endoplasmic reticulum. Our findings suggest that the endoplasmic reticulum-localized catalytically active DOK1 is highly expressed in the meristems and is involved in the control of flowering time, possibly by post-transcriptional regulation including protein glycosylation.

Endoplasmic Reticulum Stress Response in Arabidopsis Roots.

Roots are the frontier of plant body to perceive underground environmental change. Endoplasmic reticulum (ER) stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on the ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of immunoglobulin-binding protein 3 (BiP3), a representative marker for the response. Roots promptly responded to the TM-induced ER stress through the induction of similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity.

Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

Heterotrimeric G protein subunits differentially respond to endoplasmic reticulum stress in Arabidopsis.

Canonical heterotrimeric G proteins in eukaryotes are major components that localize at plasma membrane and transmit extracellular stimuli into the cell. Genome of a seed plant Arabidopsis thaliana encodes at least one Gα (GPA1), one Gß (AGB1), and 3 Gγ (AGG1, AGG2 and AGG3) subunits. The loss-of-function mutations of G protein subunit(s) cause multiple defects in development as well as biotic and abiotic stress responses. However, it remains elusive how these subunits differentially express these defects. Here, we report that Arabidopsis heterotrimeric G protein subunits differentially respond to the endoplasmic reticulum (ER) stress. An isolated homozygous mutant of AGB1, agb1-3, was more sensitive to the tunicamycin-induced ER stress compared to the wild type and the other loss-of-function mutants of G protein subunits. Moreover, ER stress responsive genes were highly expressed in the agb1-3 plant. Our results indicate that AGB1 positively contributes to ER stress tolerance in Arabidopsis.

Arabidopsis DOK1 encodes a functional dolichol kinase involved in reproduction.

Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation. Dol-P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome-wide search for cytidylyltransferase motif-containing proteins in Arabidopsis, and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S. cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T-DNA-tagged AtDOK1 mutants, dok1-1 and dok1-2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis.

Notch signaling mediates the age-associated decrease in adhesion of germline stem cells to the niche.

Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.

Countercurrent flow of supercritical anti-solvent in the production of pure xanthophylls from Nannochloropsis oculata.

This study examined pilot scaled elution chromatography coupled with supercritical anti-solvent precipitation (using countercurrent flow) in generating zeaxanthin-rich particulates from a micro-algal species. Ultrasonic agitated acetone extract subjected to column fractionation successfully yielded a fraction containing 349.4 mg/g of zeaxanthin with a recovery of 85%. Subsequently, supercritical anti-solvent (SAS) precipitation of the column fraction at 150 bar and 343 K produced submicron-sized particulates with a concentration of 845.5mg/g of zeaxanthin with a recovery of 90%. Experimental results from a two-factor response surface method SAS precipitation indicated that purity, mean size and morphology of the precipitates were significantly affected by the flow type configuration, feed flow rate and injection time.