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Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis , endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets., Competing Interests: Competing interests statement:The authors declare no competing interest.

Sphingolipids are pivotal for plant development and stress responses. Growing interest has been directed toward fully comprehending the regulatory mechanisms of the sphingolipid pathway. We explore its de novo biosynthesis and homeostasis in Arabidopsis thaliana cell cultures, shedding light on fundamental metabolic mechanisms. Employing 15 N isotope labeling and quantitative dynamic modeling approach, we obtained data with notable variations and developed a regularized and constraint-based dynamic metabolic flux analysis (r-DMFA) framework to predict metabolic shifts due to enzymatic changes. Our analysis revealed key enzymes such as sphingoid-base hydroxylase (SBH) and long-chain-base kinase (LCBK) to be critical for maintaining sphingolipid homeostasis. Disruptions in these enzymes were found to affect cellular viability and increase the potential for programmed cell death (PCD). Despite challenges posed by data variability, this work enhances our understanding of sphingolipid metabolism and demonstrates the utility of dynamic modeling in analyzing complex metabolic pathways., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy., Competing Interests: HL, JC, AG, KC, JB, SS, AM, RV, KE, MO No competing interests declared, (© 2023, Lee et al.)

The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB- MPK6- ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.

Endocytosis, secretion, and endosomal trafficking are key cellular processes that control the composition of the plasma membrane. Through the coordination of these trafficking pathways, cells can adjust the composition, localization, and turnover of proteins and lipids in response to developmental or environmental cues. Upon being incorporated into vesicles and internalized through endocytosis, plant plasma membrane proteins are delivered to the trans-Golgi network (TGN). At the TGN, plasma membrane proteins are recycled back to the plasma membrane or transferred to multivesicular endosomes (MVEs), where they are further sorted into intralumenal vesicles for degradation in the vacuole. Both types of plant endosomes, TGN and MVEs, act as sorting organelles for multiple endocytic, recycling, and secretory pathways. Molecular assemblies such as retromer, ESCRT (endosomal sorting complex required for transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with specific domains of endosomal membranes to mediate different sorting and membrane-budding events. In this review, we discuss the mechanisms underlying the recognition and sorting of proteins at endosomes, membrane remodeling and budding, and their implications for cellular trafficking and physiological responses in plants., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Barley malting depends on hydrolytic enzymes that degrade storage macromolecules. Identifying barley cultivars with proteolytic activity that guarantees appropriate foaming, flavor, and aroma in the beer is of great importance. In this work, the proteolytic activity and profiles of brewing malt from Mexican barley cultivars were analyzed. Data showed that Cys- (at 50°C) and Ser-proteases (at 70°C) are the major contributors to proteolytic activity during mashing. Essential amino acids, necessary for fermentation and production of good flavor and aroma in beer, were detected at the end of mashing. According to our results, Mexican cultivar HV2005-19 exhibits similar proteolytic activities as those from cultivar Metcalfe, which is one of the most utilized for the brewing industry. Moreover, we propose Cys- and Ser-proteases as biochemical markers during mashing at 50 and 70°C, respectively, to select barley cultivars for beer production. PRACTICAL APPLICATIONS: Proteolytic activity, which depends on activation and de novo synthesis of proteases in the aleurone layer of barley seeds, is crucial in beer production. Identifying new barley varieties that have optimal proteolytic activities is of great interest for genetic improvement programs. In this study, we propose the variety HV2005-19 as a genotype with Cys- and Ser-proteases activity similar to that from Metcalfe, which is a top variety in the brewing industry., (© 2022 Wiley Periodicals LLC.)

Lipid structures affect membrane biophysical properties such as thickness, stability, permeability, curvature, fluidity, asymmetry, and interdigitation, contributing to membrane function. Sphingolipids are abundant in plant endomembranes and plasma membranes (PMs) and comprise four classes: ceramides, hydroxyceramides, glucosylceramides, and glycosylinositolphosphoceramides (GIPCs). They constitute an array of chemical structures whose distribution in plant membranes is unknown. With the aim of describing the hydrophobic portion of sphingolipids, 18 preparations from microsomal (MIC), vacuolar (VM), PM, and detergent-resistant membranes (DRM) were isolated from Arabidopsis (Arabidopsis thaliana) leaves. Sphingolipid species, encompassing pairing of long-chain bases and fatty acids, were identified and quantified in these membranes. Sphingolipid concentrations were compared using univariate and multivariate analysis to assess sphingolipid diversity, abundance, and predominance across membranes. The four sphingolipid classes were present at different levels in each membrane: VM was enriched in glucosylceramides, hydroxyceramides, and GIPCs; PM in GIPCs, in agreement with their key role in signal recognition and sensing; and DRM in GIPCs, as reported by their function in nanodomain formation. While a total of 84 sphingolipid species was identified in MIC, VM, PM, and DRM, only 34 were selectively distributed in the four membrane types. Conversely, every membrane contained a different number of predominant species (11 in VM, 6 in PM, and 17 in DRM). This study reveals that MIC, VM, PM, and DRM contain the same set of sphingolipid species but every membrane source contains its own specific assortment based on the proportion of sphingolipid classes and on the predominance of individual species., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In plants, pathogen triggered programmed cell death (PCD) is frequently mediated by polar lipid molecules referred as long chain bases (LCBs) or ceramides. PCD interceded by LCBs is a well-organized process where several cell organelles play important roles. In fact, light-dependent reactions in the chloroplast have been proposed as major players during PCD, however, the functional aspects of the chloroplast during PCD are largely unknown. For this reason, we investigated events that lead to disassembly of the chloroplast during PCD mediated by LCBs. To do so, LCB elevation was induced with Pseudomonas syringae pv. tomato (a non-host pathogen) or Fumonisin B1 in Phaseolus vulgaris. Then, we performed biochemical tests to detect PCD triggering events (phytosphingosine rises, MPK activation and H 2 O 2 generation) followed by chloroplast structural and functional tests. Observations of the chloroplast, via optical phenotyping methods combined with microscopy, indicated that the loss of photosynthetic linear electron transport coincides with the organized ultrastructure disassembly. In addition, structural changes occurred in parallel with accumulation of H 2 O 2 inside the chloroplast. These features revealed the collapse of chloroplast integrity and function as a mechanism leading to the irreversible execution of the PCD promoted by LCBs.

Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plasmodesmata-intercellular channels that communicate adjacent cells-possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures.

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations, and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)