Long-Term Imaging of Endoplasmic Reticulum Morphology in Embryos During Seed Germination.

Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool, which enables subtle perturbations in the ER network to be measured.

Quantitation of ER Morphology and Dynamics.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

A tell tail sign: a conserved C-terminal tail-anchor domain targets a subset of pathogen effectors to the plant endoplasmic reticulum.

The endoplasmic reticulum (ER) is the entry point to the secretory pathway and, as such, is critical for adaptive responses to biotic stress, when the demand for de novo synthesis of immunity-related proteins and signalling components increases significantly. Successful phytopathogens have evolved an arsenal of small effector proteins which collectively reconfigure multiple host components and signalling pathways to promote virulence; a small, but important, subset of which are targeted to the endomembrane system including the ER. We identified and validated a conserved C-terminal tail-anchor motif in a set of pathogen effectors known to localize to the ER from the oomycetes Hyaloperonospora arabidopsidis and Plasmopara halstedii (downy mildew of Arabidopsis and sunflower, respectively) and used this protein topology to develop a bioinformatic pipeline to identify putative ER-localized effectors within the effectorome of the related oomycete, Phytophthora infestans, the causal agent of potato late blight. Many of the identified P. infestans tail-anchor effectors converged on ER-localized NAC transcription factors, indicating that this family is a critical host target for multiple pathogens.

Chloroplasts play a central role in facilitating MAMP-triggered immunity, pathogen suppression of immunity and crosstalk with abiotic stress.

Microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) research has traditionally centred around signal transduction pathways originating from activated membrane-localized pattern recognition receptors (PRRs), culminating in nuclear transcription and posttranslational modifications. More recently, chloroplasts have emerged as key immune signalling hubs, playing a central role in integrating environmental signals. Notably, MAMP recognition induces chloroplastic reactive oxygen species (cROS) that is suppressed by pathogen effectors, which also modify the balance of chloroplast-synthesized precursors of the defence hormones, jasmonic acid, salicylic acid (SA) and abscisic acid. This study focuses on how well-characterized PRRs and coreceptors modulate chloroplast physiology, examining whether diverse signalling pathways converge to similarly modulate chloroplast function. Pretreatment of receptor mutant plants with MAMP and D(Damage)AMP peptides usually protect against effector modulation of chlorophyll fluorescence and prevent Pseudomonas syringae effector-mediated quenching of cROS and suppression of maximum dark-adapted quantum efficiency (the ratio of variable/maximum fluorescence [Fv /Fm ]). The MTI coreceptor double mutant, bak1-5/bkk1-1, exhibits a remarkable decrease in Fv /Fm compared to control plants during infection, underlining the importance of MTI-mediated signalling in chloroplast immunity. Further probing the role of the chloroplast in immunity, we unexpectedly found that even moderate changes in light intensity can uncouple plant immune signalling.

The Passage of H2O2 from Chloroplasts to Their Associated Nucleus during Retrograde Signalling: Reflections on the Role of the Nuclear Envelope.

The response of chloroplasts to adverse environmental cues, principally increases in light intensity, stimulates chloroplast-to-nucleus retrograde signalling, which leads to the induction of immediate protective responses and longer-term acclimation. Hydrogen peroxide (H2O2), generated during photosynthesis, is proposed to both initiate and transduce a retrograde signal in response to photoinhibitory light intensities. Signalling specificity achieved by chloroplast-sourced H2O2 for signal transduction may be dependent upon the oft-observed close association of a proportion of these organelles with the nucleus. In this review, we consider more precisely the nature of the close association between a chloroplast appressed to the nucleus and the requirement for H2O2 to cross both the double membranes of the chloroplast and nuclear envelopes. Of particular relevance is that the endoplasmic reticulum (ER) has close physical contact with chloroplasts and is contiguous with the nuclear envelope. Therefore, the perinuclear space, which transducing H2O2 molecules would have to cross, may have an oxidising environment the same as the ER lumen. Based on studies in animal cells, the ER lumen may be a significant source of H2O2 in plant cells arising from the oxidative folding of proteins. If this is the case, then there is potential for the ER lumen/perinuclear space to be an important location to modify chloroplast-to-nucleus H2O2 signal transduction and thereby introduce modulation of it by additional different environmental cues. These would include for example, heat stress and pathogen infection, which induce the unfolded protein response characterised by an increased H2O2 level in the ER lumen.

Analysis of the impact of indole-3-acetic acid (IAA) on gene expression during leaf senescence in Arabidopsis thaliana.

Leaf senescence is an important developmental process for the plant life cycle. It is controlled by endogenous and environmental factors and can be positively or negatively affected by plant growth regulators. It is characterised by major and significant changes in the patterns of gene expression. Auxin, especially indole-3-acetic acid (IAA), is a plant growth hormone that affects plant growth and development. The effect of IAA on leaf senescence is still unclear. In this study, we performed microarray analysis to investigate the role of IAA on gene expression during senescence in Arabidopsis thaliana. We sprayed IAA on plants at 3 different time points (27, 31 or 35 days after sowing). Following spraying, PSII activity of the eighth leaf was evaluated daily by measurement of chlorophyll fluorescence parameters. Our results show that PSII activity decreased following IAA application and the IAA treatment triggered different gene expression responses in leaves of different ages.

Arabidopsis Lunapark proteins are involved in ER cisternae formation.

The plant endoplasmic reticulum (ER) is crucial to the maintenance of cellular homeostasis. The ER consists of a dynamic and continuously remodelling network of tubules and cisternae. Several conserved membrane proteins have been implicated in formation and maintenance of the ER network in plants, such as RHD3 and the reticulon proteins. Despite the recent work in mammalian and yeast cells, the detailed molecular mechanisms of ER network organization in plants remain largely unknown. Recently, novel ER network-shaping proteins called Lunapark (LNP) have been identified in yeast and mammalian cells. Here we identify two Arabidopsis LNP homologues and investigate their subcellular localization via confocal microscopy and potential function in shaping the ER network using protein-protein interaction assays and mutant analysis. We show that AtLNP1 overexpression in tobacco leaf epidermal cells mainly labels cisternae in the ER network, whereas AtLNP2 labels the whole ER. Overexpression of LNP proteins results in an increased abundance of ER cisternae and lnp1 and lnp1lnp2 amiRNA lines display a reduction in cisternae and larger polygonal areas. Thus, we hypothesize that AtLNP1 and AtLNP2 are involved in determining the network morphology of the plant ER, possibly by regulating the formation of ER cisternae.

Long-Term Imaging of Endoplasmic Reticulum Morphology in Embryos During Seed Germination.

Imaging plant embryos at the cellular level over time is technically challenging, since the embryo, once its protective seed coat is removed, must be kept viable and unstressed on a microscope slide for the duration of the experiment. Here we describe a procedure and suitable apparatus for the visualization, over several days, of changes in endoplasmic reticulum (ER) morphology associated with the process of germination in Arabidopsis thaliana seeds. Moreover, we also present a user-friendly image analysis tool which enables subtle perturbations in the ER network to be measured.

A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

A local regulatory network around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves.

A model is presented describing the gene regulatory network surrounding three similar NAC transcription factors that have roles in Arabidopsis leaf senescence and stress responses. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1-hybrid analysis and modelling using gene expression time course data has been applied to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modelling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions. Gene expression analysis in mutants of ANAC019 and ANAC055 at different times during leaf senescence has revealed a distinctly different role for each of these genes. Yeast 1-hybrid analysis is shown to be a valuable tool that can distinguish clades of binding proteins and be used to test and quantify protein binding to predicted promoter motifs.