Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus.…

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism

Abstract Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot b…

The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.

This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular devel…

Supplementary Fig. 4 A Respiration and photosynthesis in P. tricornutum cells from Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

Direct assessment of oxygen consumption by a polarographic approach in both phototrophy (black bar) and mix-otrophy (red bar). B. Fluorescent based-assay to monitoring the changes in respiration using the Redox Dye A in presence of the selected compounds (see methods).

Supplementary Fig. 1 Quantitative analysis of P. tricornutum glycerolipids from Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

TAG profile in a total lipid extract from cells grown in replete conditions (A) and deplete conditions (B) in both mixotrophic and phototrophic mode. Glycerolipids are expressed in nmol / mg of dry cells. Each result is the average of two biological replicates ± SD. PHOT: light in N-replete condition; PHOTO-N: light in N-deplete condi-tion; MIX: light+glycerol in N-replete condition; MIX-N: light+glycerol in N-deplete condition.

Phosphoglycerate Kinases Are Co-Regulated to Adjust Metabolism and to Optimize Growth

[EN] In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis (Arabidopsis thaliana) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the phot…

Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast i…

Deficiency in the Phosphorylated Pathway of Serine Biosynthesis Perturbs Sulfur Assimilation.

Although the plant Phosphorylated Pathway of l-Ser Biosynthesis (PPSB) is essential for embryo and pollen development, and for root growth, its metabolic implications have not been fully investigated. A transcriptomics analysis of Arabidopsis (Arabidopsis thaliana) PPSB-deficient mutants at night, when PPSB activity is thought to be more important, suggested interaction with the sulfate assimilation process. Because sulfate assimilation occurs mainly in the light, we also investigated it in PPSB-deficient lines in the day. Key genes in the sulfate starvation response, such as the adenosine 5′phosphosulfate reductase genes, along with sulfate transporters, especially those involved in sulfat…

Supplementary Fig. 2 Membrane lipid composition in P. tricornutum from Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

Lipid analysis of cells grow in N-replete conditions and N-deplete conditions in both mixotrophic and phototrophic mode. Each result is the average of two biological replicates ± SD. SQDG, sulfoquinovosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PHOT: light in N-replete condition; PHOTO-N: light in N-deplete condition; MIX: light+glycerol in N-replete condition; MIX-N: light+glycerol in N-deplete condition.

Phosphoglycerate dehydrogenase genes differentially affect Arabidopsis metabolism and development.

[EN] Unlike animals, plants possess diverse L-serine (Ser) biosynthetic pathways. One of them, the Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been recently described as essential for embryo, pollen and root development, and required for ammonium and sulfur assimilation. The first and rate limiting step of PPSB is the reaction catalyzed by the enzyme phosphoglycerate dehydrogenase (PGDH). In Arabidopsis, the PGDH family consists of three genes, PGDH1, PGDH2 and PGDH3. PGDH1 is characterized as being the essential gene of the family. However, the biological significance of PGDH2 and PGDH3 remains unknown. In this manuscript, we have functionally characterized PGDH2 and PGDH3. Ph…

Functional characterization of the plastidial 3-phosphoglycerate dehydrogenase family in Arabidopsis.

This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted emb…

Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum , using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affect…

Author response: Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Supplementary Fig. 3 Quantification of intracellular pyruvate by a fluorescence-based method from Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum

A. Pyruvate standard curve. B. Quantification of intracellular pyruvate in cells grown in phototrophy (PHOT) and mixotrophy (MIX).

Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis

This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of later…

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase mutants.

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp)…

The essential role of the phosphorylated pathway of serine biosynthesis inArabidopsis

[EN] In plants, 3 different pathways of serine biosynthesis have been described: the Glycolate pathway, which is associated with photorespiration, and 2 non-photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been known since the 1950s, but has been studied relatively little, probably because it was considered of minor significance as compared with the Glycolate pathway. In the associated study1 , we described for the first time in plants the in vivo functional characterization of the PPSB, by targeting the phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Following a gain—and loss-of-function…

RCD1 Coordinates Chloroplastic and Mitochondrial Electron Transfer through Interaction with ANAC Transcription Factors in Arabidopsis

AbstractSignaling from chloroplasts and mitochondria, both dependent on reactive oxygen species (ROS), merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). ROS produced in the chloroplasts affect the abundance, thiol redox state and oligomerization of RCD1. RCD1 directly interactsin vivowith ANAC013 and ANAC017 transcription factors, which are the mediators of the ROS-related mitochondrial complex III retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation ofRCD1leads to increased expression of ANAC013 and ANAC017-regulated genes belonging to the mitochondrial dysfunction stimulon (MDS), including genes for mitochondrial alternative oxidases(AOXs).Accumulat…

Supplementary Fig. 5 Screening of mixotrophic efficiency by biolog and redox dye assay in P. tricornutum from Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

A. OD750 nm changes (relative to phototrophic growth) of P. tricornutum cells grown for 6 days in BiologTM plates P1 and PM2A that contains 190 carbon compounds (see methods). Each data point represents a different com-pound. B. Growth profile of P. tricornutum on few selected compounds (at 20 mM) and a phototrophic control in 100 mL flasks. C. Areas under the growth curves of Supplementary Fig. 5B normalized to the area of the curve of phototrophic growth.

Autophagy

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide…

The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Viable Pollen Development in Arabidopsis

Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was d…