Stéphanie Pascal

Autophagy is a conserved intracellular catabolic process, critical for plant stress tolerance. Upon their delivery in the vacuole, how autophagic bodies containing cargo are hydrolyzed to warrant autophagy degradation remains unclear in multicellular organisms. Here, we found that two Arabidopsis phospholipases, LCAT4 and LCAT3, traffic to the vacuolar lumen and converge on autophagic bodies through fundamentally different routes. While LCAT4 directly binds ATG8 and uses autophagy as a transport system to reach the vacuole prepackaged within autophagosomes, LCAT3 traffics to the lytic compartment independently of autophagosome formation. Knocking out both genes causes an accumulation of autophagic bodies accompanied with a reduction in autophagy degradation. In vivo reconstitution demonstrated that LCAT3 can hydrolyse the membrane of autophagic bodies, enabling the activity of LCAT4 to enhance this process. Together, this work sheds light on the vacuolar stages of autophagy, showing that plants have evolved a multi-component pathway for the efficient disruption of autophagosomal membranes as a critical step for the completion of the autophagy pathway.

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

The permeability of roots to water and nutrients is controlled through a variety of mechanisms and one of the most conspicuous is the presence of the Casparian strips and suberin lamellae. Roots actively regulate the creation of these structures developmentally, along the length of the root, and in response to the environment, including drought. In the current study, we characterized the suberin composition along the length of grapevine fine roots during development and in response to water deficit, and in the same root systems we quantified changes in expression of suberin biosynthesis‐ and deposition‐related gene families (via RNAseq) allowing the identification of drought‐responsive suberin‐related genes. Grapevine suberin composition did not differ between primary and lateral roots, and was similar to that of other species. Under water deficit there was a global upregulation of suberin biosynthesis which resulted in an increase of suberin specific monomers, but without changes in their relative abundances, and this upregulation took place across all the developmental stages of fine roots. These changes corresponded to the upregulation of numerous suberin biosynthesis‐ and export‐related genes which included orthologs of the previously characterized AtMYB41 transcriptional factor. Functional validation of two grapevine MYB41 orthologs, VriMYB41 and VriMYB41‐like, confirmed their ability to globally upregulate suberin biosynthesis, export, and deposition. This study provides a detailed characterization of the developmental and water deficit induced suberization of grapevine fine roots and identifies important orthologs responsible for suberin biosynthesis, export, and its regulation in grape.

The permeability of roots to water and nutrients is controlled through a variety of mechanisms and one of the most conspicuous is the presence of structures such as the Casparian strips and suberin lamellae. Roots actively regulate the creation of these structures developmentally, along the length of the root, and in response to the environment, including abiotic stresses such as drought. In the current study, we characterized the suberin composition along the length of grapevine fine roots during development and in response to water deficit. In parallel samples we quantified changes in expression of suberin biosynthesis- and deposition-related gene families (via RNAseq) allowing the identification of drought-responsive suberin-related genes. Grapevine suberin composition did not differ between primary and lateral roots, and was similar to that of other species. Under water deficit there was a global upregulation of suberin biosynthesis which resulted in an increase of suberin specific monomers, but without changes in their relative abundances, and this upregulation took place across all the developmental stages of fine roots. These changes corresponded to the upregulation of numerous suberin biosynthesis- and deposition-related genes which included orthologs of the previously characterized AtMYB41 transcriptional factor. Functional validation of two grapevine MYB41 orthologs, VviMYB41 and VviMYB41-like, confirmed their ability to globally upregulate suberin biosynthesis and deposition. This study provides a detailed characterization of the developmental and water deficit induced suberization of grapevine fine roots and identifies important orthologs responsible for suberin biosynthesis, deposition, and its regulation in grape. One sentence summary: Our study details the biochemical changes and molecular regulation of how grapevines decrease their root permeability during drought.

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an “auto-immune” type interaction. Plant embryogenesis occurs deep within the tissues of the developing seed, and leads to the production of the mature embryo. In Arabidopsis and many other plant species embryo-derive structure (such as the cotyledons) are suddenly exposed to environmental stresses such as low humidity. In these species the embryonic cuticle provides a primary defence against environmental stress, and particularly dehydration, at germination. The formation of an intact and functional cuticle during embryogenesis is thus of key importance for seedling survival. Our work shows that a signalling pathway involving receptor-kinases expressed in the embryo epidermis, and a protease expressed in the endosperm tissue surrounding the embryo, is critical for ensuring the production of an intact cuticle. Furthermore, we show that a component of stress-related MAP-Kinase signalling in plants acts downstream in this pathway, possibly to mediate transcriptional responses characteristic of responses to stress. We propose that plants have redeployed a signalling pathway associated with stress resistance to ensure the formation of an intact embryonic cuticle prior to germination, and thus ensure seedling survival at germination.

Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A’s to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, lipid staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an “auto-immune” type interaction.

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of ageing Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in ageing leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in ageing leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition, suggesting a role for LDs in the recycling of plastid lipids. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the level of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data show that leaf LDs are structurally and functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER.