Copper-containing amine oxidases contribute to terminal polyamine oxidation in peroxisomes and apoplast of Arabidopsis thaliana

Polyamines (PAs) are oxidatively deaminated at their primary or secondary amino-groups by copper-containing amine oxidases (CuAOs) or FAD-dependent amine oxidases (PAOs), respectively. Both enzymes have long been considered to be apoplastic proteins. However, three out of five PAO isoforms in Arabidopsis thaliana are localized in peroxisomes, while the other two PAOs are predicted to be cytosolic. Interestingly, most of these PAOs do not contribute to terminal PA oxidation, but instead are involved in the back-conversion pathway, producing spermidine from spermine and putrescine from spermidine, which in turn is inhibited by putrescine. This opens the question as to whether PAs are catabolized in the apoplast of Arabidopsis and if the terminal oxidation occurs in the peroxisomes. The main objective of this study was to know if these catabolic processes are mediated by CuAOs. Results A. thaliana contains ten genes annotated as CuAOs, but only one (ATAO1) has been characterized at the protein level. Reported herein is the characterization of three genes encoding putative Arabidopsis CuAOs (AtCuAO1, AtCuAO2 and AtCuAO3). These genes encode functional CuAOs that use putrescine and spermidine as substrates. AtCuAO1, like ATAO1, is an extracellular protein, while AtCuAO2 and AtCuAO3 are localized in peroxisomes. The three genes present a different expression profile in response to exogenous treatments, such as application of abcisic acid, methyl jasmonate, salycilic acid, flagellin 22 and wounding. Conclusions PA catabolism in the Arabidopsis apoplast is mediated predominantly by CuAOs, while in peroxisomes the co-localization of CuAO-dependent terminal catabolism with PAO-back-conversion machineries might contribute to modulating putrescine-mediated inhibition of the back-conversion, suggesting the occurrence of a tight coordination between both catabolic pathways. The expression profile of AtCuAO1-3 in response to different exogenous treatments, together with the different localization of the corresponding proteins, provides evidence for the functional diversification of Arabidopsis CuAO proteins

Polyamines under abiotic stress: Metabolic crossroads and hormonal crosstalks in plants

metabolite

Polyamines under Abiotic Stress: Metabolic Crossroads and Hormonal Crosstalks in Plants

Polyamines are essential compounds for cell survival and have key roles in plant stress protection. Current evidence points to the occurrence of intricate cross-talks between polyamines, stress hormones and other metabolic pathways required for their function. In this review we integrate the polyamine metabolic pathway in the context of its immediate metabolic network which is required to understand the multiple ways by which polyamines can maintain their homeostasis and participate in plant stress responses

Complex interplays between phytosterols and plastid development

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs. Despite their variety of structures, all isoprenoids derive from the common C₅ precursor isopentenyl diphosphate (IPP). Plants synthesize IPP through two different metabolic pathways, the mevalonic acid (MVA) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways that operate in the cytosol-RE and plastids, respectively. MEP-derived isoprenoids include important compounds for chloroplast function and as such, knock-out mutant plants affected in different steps of this pathway display important alterations in plastid structure. These alterations often lead to albino phenotypes and lethality at seedling stage. MVA knock-out mutant plants show, on the contrary, lethal phenotypes already exhibited at the gametophyte or embryo developmental stage. However, the recent characterization of conditional knock-down mutant plants of farnesyl diphosphate synthase (FPS), a central enzyme in cytosolic and mitochondrial isoprenoid biosynthesis, revealed an unexpected role of this pathway in chloroplast development and plastidial isoprenoid metabolism in post-embryonic stages. Upon FPS silencing, chloroplast structure is severely altered, together with a strong reduction in the levels of MEP pathway-derived major end products. This phenotype is associated to misregulation of genes involved in stress responses predominantly belonging to JA and Fe homeostasis pathways. Transcriptomic experiments and analysis of recent literature indicate that sterols are the cause of the observed alterations through an as yet undiscovered mechanism

Structural and functional analysis of tomato sterol C22 desaturase

Background: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function. Results: In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding. Conclusions: Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism

Effects of impaired steryl ester biosynthesis on tomato growth and developmental processes

Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS. Depending on the donor substrate, these enzymes are called acyl-CoA:sterol acyltransferases (ASAT), when the substrate is a long-chain acyl-CoA, and phospholipid:sterol acyltransferases (PSAT), which use a phospholipid as a donor substrate. We have recently identified and preliminary characterized the tomato (Solanum lycopersicum cv. Micro-Tom) SlASAT1 and SlPSAT1 enzymes. To gain further insight into the biological role of these enzymes and SE biosynthesis in tomato, we generated and characterized CRISPR/Cas9 single knock-out mutants lacking SlPSAT1 (slpsat1) and SlASAT1 (slasat1), as well as the double mutant slpsat1 x slasat1. Analysis of FS and SE profiles in seeds and leaves of the single and double mutants revealed a strong depletion of SE in slpsat1, that was even more pronounced in the slpsat1 x slasat1 mutant, while an increase of SE levels was observed in slasat1. Moreover, SlPSAT1 and SlASAT1 inactivation affected in different ways several important cellular and physiological processes, like leaf lipid bo1dies formation, seed germination speed, leaf senescence, and the plant size. Altogether, our results indicate that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. It is also worth to mention that some of the metabolic and physiological responses in the tomato mutants lacking functional SlPSAT1 or SlASAT1 are different from those previously reported in Arabidopsis, being remarkable the synergistic effect of SlASAT1 inactivation in the absence of a functional SlPSAT1 on the early germination and premature senescence phenotypes

Downregulation of Tomato STEROL GLYCOSYLTRANSFERASE 1 Perturbs Plant Development and Facilitates Viroid Infection

[EN] Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but rather hypersusceptible to the viroid. A combination of phenotypic, molecular and metabolic analyses of amiRNAexpressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus confirming that downregulation of SlSGT1 was responsible for the viroid hypersusceptibility phenotype. Our results highlight the role of SGTs in proper plant development and indicate that the unbalance of sterol glycosylation levels favors viroid infection most likely by facilitating viroid movement.Cisneros, AE.; Lisón, P.; Campos, L.; López-Tubau, JM.; Altabella, T.; Ferrer, A.; Daròs, J.... (2022). Downregulation of Tomato STEROL GLYCOSYLTRANSFERASE 1 Perturbs Plant Development and Facilitates Viroid Infection. Journal of Experimental Botany. 1-37. https://doi.org/10.1093/jxb/erac36113

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Pseudomonas germanica sp. nov., isolated from Iris germanica rhizomes

Through bacterial plant-endophyte extraction from rhizomes of Iris germanica plant, a Gram-stain-negative, aerobic, catalase- and oxidase-positive gammaproteobacterial strain, referred to as FIT28T, was isolated. FIT28T shows vigorous growth on nutrient rich media within the temperature range of 4-35 °C, with optimal growth at 28 °C, a wide pH tolerance from pH 5 to 11, and salt tolerance up to 6 % (w/v) NaCl. Colonies are white-yellow and quickly become mucoid. The results of analysis of the 16S rRNA gene sequence placed the strain within the genus Pseudomonas, and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, gyrB and rpoD concatenated sequences revealed that the closest relatives of FIT28T are Pseudomonas zeae OE48.2T, 'Pseudomonas crudilactis' UCMA 17988, Pseudomonas tensinigenes ZA5.3T, Pseudomonas helmanticensis OHA11T, Pseudomonas baetica a390T, Pseudomonas iridis P42T, Pseudomonas atagonensis PS14T and Pseudomonas koreensis Ps 9-14T, within the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens lineage. The genome size of FIT28T is about 6.7 Mb with 59.09 mol% DNA G+C content. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values calculated from the genomic sequences of FIT28T, and the closely related P. zeae OE48.2T are 95.23 and 63.4 %, respectively. Biochemical, metabolic and chemotaxonomic studies further support our proposal that Pseudomonas germanica sp. nov., should be considered a novel species of the genus Pseudomonas. Hence, the type strain FIT28T (=LMG 32353T=DSM 112698T) has been deposited in public cell-type culture centres