Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.     

Chaperones of the endoplasmic reticulum are required for Ve1‐mediated resistance to Verticillium

The tomato receptor‐like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1‐enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)‐resident chaperones HSP70 binding proteins (BiPs) and a lectin‐type calreticulin (CRT). Knock‐down of the different BiPs and CRTs in tomato resulted in compromised Ve1‐mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP‐type Cladosporium fulvum resistance protein Cf‐4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf‐4 protein. In contrast, knock‐down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf‐4‐ and Ve1‐mediated immunity.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

An endoplasmic reticulum-derived structure that is induced under stress conditions in Arabidopsis

The endoplasmic reticulum (ER) body is a characteristic structure derived from ER and is referred to as a proteinase-sorting system that assists the plant cell under various stress conditions. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing green fluorescent protein fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate (MeJA), a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies was suppressed by ethylene. An electron microscopic analysis showed that typical ER bodies were induced in the non-transgenic rosette leaves treated with MeJA. An experiment using coi1 and etr1-4 mutant plants showed that the induction of ER bodies was strictly coupled with the signal transduction of MeJA and ethylene. These results suggested that the formation of ER bodies is a novel and unique type of endomembrane system in the response of plant cells to environmental stresses. It is possible that the biological function of ER bodies is related to defense systems in higher plants.     

Glucosylation of the phytoalexin N‐feruloyl tyramine modulates the levels of pathogen‐responsive metabolites in Nicotiana benthamiana

Enzyme promiscuity, a common property of many uridine diphosphate sugar‐dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC‐MS‐guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen‐induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N‐feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen‐induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.     

Peroxisome Formation Requires the Endoplasmic Reticulum Channel Protein Sec61

In peroxisome formation, models of near‐autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec 61 complex, the protein‐conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec 61 in yeast disables peroxisome formation. The ER entry of the early peroxisomal membrane protein Pex 3 engineered with a glycosylation tag is reduced in sec61 mutant cells. Moreover, we were able to reconstitute Pex 3 import into ER membranes in vitro, and we identified a variant of a signal anchor sequence for ER translocation at the Pex 3 N‐terminus. Our findings are consistent with a Sec 61 requirement for peroxisome formation and a fundamental role of the ER in peroxisome biogenesis.     

TGD5 is required for normal morphogenesis of non-mesophyll plastids,but not mesophyll chloroplasts,in Arabidopsis

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.     

Functional characterization of Nicotiana benthamiana chromomethylase 3 in developmental programs by virus‐induced gene silencing

DNA methylation is essential for normal developmental processes and genome stability. DNA methyltransferases are key enzymes catalyzing DNA methylation. Chromomethylase (CMT) genes are specific to the plant kingdom and encode chromodomain‐containing methyltransferases. However, the function of CMT genes in plants remains elusive. In this study, we isolated and characterized a CMT gene from Nicotiana benthamiana, designated NbCMT3. Alignment of the NbCMT3 amino acid sequence with other plant CMT3s showed conservation of bromo‐adjacent‐homology and methyltransferase catalytic domains. We investigated the expression patterns of NbCMT3 and its function in developmental programs. NbCMT3 was expressed predominately in proliferating tissues such as apical shoots and young leaves. NbCMT3 protein showed a nuclear location, which could be related to its putative cellular functions. Knocking down NbCMT3 expression by virus‐induced gene silencing revealed its vital role(s) in leaf morphogenesis. The formation of palisade cells was defective in NbCMT3‐silenced plants as compared with controls. NbCMT3 has a role in developmental programs.     

Elicitin‐like proteins Oli‐D1 and Oli‐D2 from Pythium oligandrum trigger hypersensitive response in Nicotiana benthamiana and induce resistance against Botrytis cinerea in tomato

The biocontrol agent Pythium oligandrum and its elicitin‐like proteins oligandrins have been shown to induce disease resistance in a range of plants. In the present study, the ability of two oligandrins, Oli‐D1 and Oli‐D2, to induce an immune response and the possible molecular mechanism regulating the defence responses in Nicotiana benthamiana and tomato were investigated. Infiltration of recombinant Oli‐D1 and Oli‐D2 proteins induced a typical immune response in N. benthamiana including the induction of a hypersensitive response (HR), accumulation of reactive oxygen species and production of autofluorescence. Agrobacterium‐mediated transient expression assays revealed that full‐length Oli‐D1 and Oli‐D2 were required for full HR‐inducing activity in N. benthamiana, and virus‐induced gene silencing‐mediated knockdown of some of the signalling regulatory genes demonstrated that NbSGT1 and NbNPR1 were required for Oli‐D1 and Oli‐D2 to induce HR in N. benthamiana. Subcellular localization analyses indicated that both Oli‐D1 and Oli‐D2 were targeted to the plasma membrane of N. benthamiana. When infiltrated or transiently expressed in leaves, Oli‐D1 and Oli‐D2 induced resistance against Botrytis cinerea in tomato and activated the expression of a set of genes involved in the jasmonic acid/ethylene (JA/ET)‐mediated signalling pathway. Our results demonstrate that Oli‐D1 and Oli‐D2 are effective elicitors capable of inducing immune responses in plants, probably through the JA/ET‐mediated signalling pathway, and that both Oli‐D1 and Oli‐D2 have potential for the development of bioactive formulae for crop disease control in practice.     

Spider mites (Tetranychus urticae) perform poorly on and disperse from plants exposed to methyl jasmonate

Jasmonates are plant hormones involved in wound and defense responses against herbivorous arthropods. Methyl jasmonate (MeJA) is used experimentally to induce defense responses in plants. In experiments outlined here we utilized a novel preference assay with unwounded plants that allowed us to study the impact of a MeJA spray on subsequent Tetranychus urticae Koch (Acari: Tetranychidae) proliferation and preference. Spraying plants with 100 μm MeJA 1 day before infestation caused mites to disperse within 2 days from treated impatiens [Impatiens wallerana Hook f., ‘Super Elfin Pink’ (Balsaminaceae)], pansy [Viola × wittrockiana Gams, ‘Imperial Beaconsfield’ (Violaceae)], and tomato [Solanum lycopersicum L., ‘Big Boy’ (Solanaceae)] plants. In addition, MeJA application reduced mite proliferation rate on impatiens and pansy by 60% (measured 22–34 days after infestation). Proteinase inhibitor (PI) assays suggested that MeJA‐induced PIs alone were not responsible for the observed results in pansy and impatiens but may have been a factor in tomato. Implications of these results in the context of MeJA‐induced resistance responses and possible directions for future research and application are discussed.