Cristina Ruberti

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line.

The onset of grapevine berry ripening is characterized by ROS accumulation and lipoxygenase-mediated membrane peroxidation in the skin

BackgroundThe ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H2O2) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken.ResultsROS-specific staining allowed to visualize not only H2O2 but also singlet oxygen (1O2) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H2O2 peak in sample skins at véraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at véraison. Mono- and digalactosyl diacylglycerols were found peroxidized on one or both α-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at véraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves.ConclusionsThe present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a véraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.

An AM‐induced, MYB‐family gene of Lotus japonicus (LjMAMI) affects root growth in an AM‐independent manner

The interaction between legumes and arbuscular mycorrhizal (AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like (LjMAMI) transcription factor (TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate (Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of LjMAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive LjMAMI in non-symbiotic functions: i.e. root growth.

Unfolded protein response in plants: one master, many questions.

To overcome endoplasmic reticulum (ER) stress, ER-localized stress sensors actuate distinct downstream organelle-nucleus signaling pathways to invoke a cytoprotective response, known as the unfolded protein response (UPR). Compared to yeast and metazoans, plant UPR studies are more recent but nevertheless fascinating. Here we discuss recent discoveries in plant UPR, highlight conserved and unique features of the plant UPR as well as critical yet-open questions whose answers will likely make significant contributions to the understanding plant ER stress management.

Conserved and plant-unique strategies for overcoming endoplasmic reticulum stress

Stress caused by environmental conditions or physiological growth can lead to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) causing ER stress, which in turn triggers a cytoprotective mechanism termed the unfolded protein response (UPR). Under mild-short stress conditions the UPR can restore ER functioning and cell growth, such as reducing the load of unfolded proteins through the upregulation of genes involved in protein folding and in degrading mis-folded proteins, and through autophagy activation, but it can also lead to cell death under prolonged and severe stress conditions. A diversified suite of sensors has been evolved in the eukaryotic lineages to orchestrate the UPR most likely to suit the cell’s necessity to respond to the different kinds of stress in a conserved as well as species-specific manner. In plants three UPR sensors cooperate with non-identical signaling pathways: the protein kinase inositol-requiring enzyme (IRE1), the ER-membrane-associated transcription factor bZIP28, and the GTP-binding protein β1 (AGB1). In this mini-review, we show how plants differ from the better characterized metazoans and fungi, providing an overview of the signaling pathways of the UPR, and highlighting the overlapping and the peculiar roles of the different UPR branches in light of evolutionary divergences in eukaryotic kingdoms.

FISSION1A, an Arabidopsis Tail-Anchored Protein, Is Localized to Three Subcellular Compartments

Dear Editor, The multiple targeting proteins that have been described in yeast, mammals, and plants have generated intriguing areas of inquiry, including the evolutionary relevance of common proteins shared by different organelles, the mechanisms that determine targeting to each organelle, and, finally, the extent of this phenomenon. Several dual-targeted proteins have been identified in eukaryotic cells. In particular, in plant cells, mitochondria and chloroplasts share over 100 proteins, mitochondria and peroxisomes share approximately a dozen, and plastidial and peroxisomal dual-targeted proteins have been also reported (Zhang and Hu, 2010; Carrie and Small, 2013). Tail-anchored FISSION1-like proteins are among those that share dual localization to mitochondria and peroxisomes and are involved in organelle fissions in yeasts, mammals, and plants (Koch et al., 2005; Zhang and Hu, 2008). In the Arabidopsis thaliana genome, two closely related orthologs of yeast and human FIS1 proteins are present, namely FIS1A (At3g57090) and FIS1B (At5g12390). Both proteins are localized to the mitochondrial and peroxisomal membranes and are involved in their fission events (Zhang and Hu, 2008). Moreover, FIS1A, but not FIS1B, is also listed in the chloroplast proteome data set (Zybailov et al., 2008). An in silico analysis of the FIS1A protein structure enabled us to define it as a typical TA-protein (Pedrazzini, 2009). In fact, we identified an N-terminal domain (aa 1–139) predicted to be cytosolic, a single transmembrane domain (TMD, aa 143–164), and a 6-amino-acid-long hydrophilic C-terminal sequence (CTS, aa 165–170) (Supplemental Figure 1). To study the in vivo subcellular localization of the FIS1A protein, we examined the YFP-tagged protein in transgenic Arabidopsis plants. YFP was first fused to the N-terminus of the FIS1A coding sequence under the control of the FIS1A native promoter (pFIS1A::YFP–FIS1A, hereafter termed ‘YFP–FIS1A’, Supplemental Figure 1A), and the fusion protein was stably expressed in Arabidopsis Col-0 plants. Next, YFP–FIS1A plants were crossed with Arabidopsis plants harboring the fluorescent protein RFP targeted to the mitochondrial matrix (COX–RFP) or to the peroxisomal lumen (RFP–KSRM). Using confocal laser scanning microscopy (CLSM), YFP–FIS1A was detected in mitochondria (Figure 1A), peroxisomes (Figure 1B), and chloroplasts (Figure 1C) as well as in the tubular protrusions (~0.5 μm in diameter) extending from these different organelles, which have been termed matrixules, peroxules, and stromules, respectively (Köhler and Hanson, 2000; Mathur et al., 2012). By Western blot analysis using an antibody raised against a synthetic peptide corresponding to amino acids 33–47 of the protein, we observed that FIS1A (Mr ~18.7 kDa) exclusively associates with the membrane fraction of wild-type Arabidopsis leaf extracts (Figure 1D). To acquire information on the nature of the FIS1A-containing membranes and to evaluate the FIS1A distribution in different organelles, we carried out subcellular fractionation by differential centrifugation. FIS1A was present in the different protein fractions enriched in chloroplast, mitochondrial, and microsome (Figure 1E), with relative distributions of 36%, 21%, and 43%, respectively (Figure 1E, columns P1, P2, P3), confirming its promiscuous localization. Given the presence of FIS1A in the microsomal-enriched fraction, we further separated this fraction by isopycnic centrifugation on a linear sucrose density-gradient. Fractions from the gradient were analyzed by immunoblot for the presence of FIS1A and for markers specific for different subcellular membranes. Quantification of the immunoblots revealed that FIS1A localizes to the outer membranes of both chloroplasts and mitochondria, to the peroxisomal membrane, and to a lighter fraction of membranes (Figure 1F, red line) that could represent the organellar protrusions observed by CLSM. Organelle protrusions have already been reported in Arabidopsis cells (Köhler and Hanson, 2000; Mathur et al., 2012). Interestingly, we observed that YFP–FIS1A is localized on a high number of protrusions extending from the different organelles (Figure 1G), but never on the ER (Supplemental Figure 2) or in the cytosol. We observed that the number of these protrusions marked by YFP– was significantly different in different organs, being three times higher in root cells than in cotyledons and the hypocotyl cells of 7-day-old seedlings (Supplemental Figure 3). CLSM time-lapse analyses performed on YFP–FIS1A-tagged plants revealed that the protrusions extending from the organelles are highly dynamic. Specifically, they undulated, extended, and retracted within the cytoplasm (Figure 1H). To investigate whether actin filaments play a role in the motility of the organelle protrusions, the 7-day-old

Mitochondria Change Dynamics and Morphology during Grapevine Leaf Senescence

Leaf senescence is the last stage of development of an organ and is aimed to its ordered disassembly and nutrient reallocation. Whereas chlorophyll gradually degrades during senescence in leaves, mitochondria need to maintain active to sustain the energy demands of senescing cells. Here we analysed the motility and morphology of mitochondria in different stages of senescence in leaves of grapevine (Vitis vinifera), by stably expressing a GFP (green fluorescent protein) reporter targeted to these organelles. Results show that mitochondria were less dynamic and markedly changed morphology during senescence, passing from the elongated, branched structures found in mature leaves to enlarged and sparse organelles in senescent leaves. Progression of senescence in leaves was not synchronous, since changes in mitochondria from stomata were delayed. Mitochondrial morphology was also analysed in grapevine cell cultures. Mitochondria from cells at the end of their growth curve resembled those from senescing leaves, suggesting that cell cultures might represent a useful model system for senescence. Additionally, senescence-associated mitochondrial changes were observed in plants treated with high concentrations of cytokinins. Overall, morphology and dynamics of mitochondria might represent a reliable senescence marker for plant cells.

The D3cpv Cameleon reports Ca2+ dynamics in plant mitochondria with similar kinetics of the YC3.6 Cameleon, but with a lower sensitivity

Mitochondria are key organelles involved in many aspects of plant physiology and, their ability to generate specific Ca2+ signatures in response to abiotic and biotic stimuli has been reported as one of their roles. The recent identification of the mammalian mitochondrial Ca2+ uniporter opens a new research area in plant biology. To study the mitochondrial Ca2+ handling, it is essential to have a reliable probe. Here we have reported the generation of an Arabidopsis transgenic line expressing the genetically encoded probe Cameleon D3cpv targeted to mitochondria, and compared its properties with the already known Cameleon YC3.6.

Maintaining the factory: The roles of the unfolded protein response in cellular homeostasis in plants

Much like a factory, the endoplasmic reticulum (ER) assembles simple cellular building blocks into complex molecular machines known as proteins. In order to protect the delicate protein folding process and ensure the proper cellular delivery of protein products under environmental stresses, eukaryotes have evolved a set of signaling mechanisms known as the unfolded protein response (UPR) to increase the folding capacity of the ER. This process is particularly important in plants, because their sessile nature commands adaptation for survival rather than escape from stress. As such, plants make special use of the UPR, and evidence indicates that the master regulators and downstream effectors of the UPR have distinct roles in mediating cellular processes that affect organism growth and development as well as stress responses. In this review we outline recent developments in this field that support a strong relevance of the UPR to many areas of plant life.

Recovery from temporary endoplasmic reticulum stress in plants relies on the tissue-specific and largely independent roles of bZIP28 and bZIP60, as well as an antagonizing function of BAX-Inhibitor 1 upon the pro-adaptive signaling mediated by bZIP28

The unfolded protein response (UPR) is an ancient signaling pathway that commits to life-or-death outcomes in response to proteotoxic stress in the endoplasmic reticulum (ER). In plants, the membrane-tethered transcription factor bZIP28 and the ribonuclease-kinase IRE1 along with its splicing target, bZIP60, govern the two cytoprotective UPR signaling pathways known to date. The conserved ER membrane-associated BAX inhibitor 1 (BI1) modulates ER stress-induced programmed cell death through yet-unknown mechanisms. Despite the significance of the UPR for cell homeostasis, in plants the regulatory circuitry underlying ER stress resolution is still largely unmapped. To gain insights into the coordination of plant UPR strategies, we analyzed the functional relationship of the UPR modulators through the analysis of single and higher order mutants of IRE1, bZIP60, bZIP28 and BI1 in experimental conditions causing either temporary or chronic ER stress. We established a functional duality of bZIP28 and bZIP60, as they exert partially independent tissue-specific roles in recovery from ER stress, but redundantly actuate survival strategies in chronic ER stress. We also discovered that BI1 attenuates the pro-survival function of bZIP28 in ER stress resolution and, differently to animal cells, it does not temper the ribonuclease activity of inositol-requiring enzyme 1 (IRE1) under temporary ER stress. Together these findings reveal a functional independence of bZIP28 and bZIP60 in plant UPR, and identify an antagonizing role of BI1 in the pro-adaptive signaling mediated by bZIP28, bringing to light the distinctive complexity of the unfolded protein response (UPR) in plants.