The Novel Chloroplast Outer Membrane Kinase KOC1 Is a Required Component of the Plastid Protein Import Machinery

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis

Plasma membrane H⁺ -ATPase regulation is required for auxin gradient formation preceding phototropic growth.

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺ -ATPases that are required to control apoplastic pH. Our results show that H⁺ -ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺ -ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development through Its Interaction with an FtsH12-FtsHi Protease Complex

Malate dehydrogenases (MDHs) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. mutants lacking plastidial NAD-dependent MDH () are embryo-lethal, and constitutive silencing (1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid, and protochlorophyllide levels in 1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of , while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles 1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function

Ontologie de l'appareil transcriptionnel plastidial au cours de la germination et du développement de la jeune plantule

La graine au cours de la germination utilise ses réserves puis acquiert des capacités d'autotrophie. Cette transition de mode de nutrition dépend de la différenciation des proplastes, présents dans les graines matures, en chloroplastes. Ces derniers organites possèdent un génome d'environ 150 kpb chez les végétaux supérieurs. Malgré cette taille réduite, la transcription du génome plastidial fait intervenir au moins 2 types d'ARN polymérases : les NEP, ARN polymérase de type phagique codées dans le noyau, et la PEP, d'origine procaryote et dont les sous unités catalytiques sont codées dans le plastome. Nous nous sommes intéressés au fonctionnement de ces ARN polymérases lors de la germination et des premières phases de développement de la jeune plantule, afm de savoir si, d'une part, ces polymérases sont exprimées et actives lors des stades précédant la mise en place de la photosynthèse et, d'autre part, d'évaluer l'importance de leurs activités dans le processus germinatif. Après avoir démontré que ces deux types d' ARN polymérases sont présentes dans les graines de deux plantes, chez Arabidopsis et l'épinard, nous avons cherché à caractériser le rôle de chacune d'entre elles. Nous avons utilisé pour cela plusieurs approches, combinant l'utilisation d'un inhibiteur spécifique de la PEP et de mutants d'expression des NEP, à des méthodes d'analyse à petite échelle (Northem blot, extension d'amorces) et à grande échelle (transcriptomique, protéomique).Nous montrons pour la première fois que les deux types d'ARN polymérases plastidiales sont actives simultanément et dès le début de la germination (imbibition) contredisant l'idée courante d'une activité séquentielle des polymérases.Nous montrons également pour la première fois l'expression antisens d'un groupe de gènes plastidiaux dont certains sont présents dans les graines.Nous montrons enfm que les toutes premières étapes de la transcription plastidiale concernent l'opéron ribosomique et que l'expression de cet opéron pendant la germination fait intervenir à la fois la PEP et une des NEP. L'absence d'activité de l'une ou l'autre provoque essentiellement une déficience de transcription des ARN ribosomiques plastidiaux et induit un retard de germination.En conclusion notre travail souligne l'importance et la nécessité de la transcription plastidiale au cours de la germination.During the germination process the seed uses all the reserves that it contains and only afterwards becomes autotrophic. This transition in the nutrition mode is dependent on the differentiation of proplastids (that are present in the mature seed) into chloroplasts.ln higher plants, chloroplasts contain a genome of around 150 kb. However, despite the small size, the genome is transcribed by two different types of RNA polymerases: the NEPs enzymes, which are phage-type polymerases and are encoded by the nucleus Œucleus-gncoded RNA rolymerase), and the PEP enzyme, which is a eubacterial-type polymerase and contains plastid-encoded subunits. Aim of this study is to investigate the role of the plastid RNA polymerases during germination and early plant development. First of all, we have analysed if all the RNA polymerases are expressed and active in the stages before the building up of the photosynthetic apparatus and if they are required by the germination process. We have indeed shown that both the NEP and the PEP polymerases are aIready present in the seeds oftwo plant species, Arabidopsis and Spinach. ln order to determine the role of each enzyme, we have analysed the transcriptome and the proteome of plants where the plastid polymerases were inactive. For this reason, we have either analysed plants that contain mutations in the genes coding for the NEP enzymes or that were treated with a specifie inhibitor of the PEP polymerases. Both large scale and small scale approaches were used.For the first time, we have shown that both types ofplastid RNA polymerases are active at the same time and since the beginning ofthe germination process (imbibition). Our data are in opposition to the CUITent opinion that suggests that NEP and PEP polymerases transcribe the plastid genome in a sequential fashion.For the first time, we have also shown the anti-sense expression of a group of plastid genes, in same cases even in the seeds.Finally, we have shown that during germination one of the fust gene-unit to be transcribed in the plastid is the ribosomal operon. Interestingly, the expression of the ribosomal operon needs both NEP and PEP polymerases. Therefore, the main consequence of the absence of either one of the two polymerases types is the reduction of the ribosomal RNAs synthesis that leads to a germination delay. ln conclusion, our study shows the importance and the requirement of the transcription of the plastid genome during the germination process.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Perception and Signaling of Ultraviolet-B Radiation in Plants

Ultraviolet-B (UV-B) radiation is an intrinsic fraction of sunlight that plants perceive through the UVR8 photoreceptor. UVR8 is a homodimer in its ground state that monomerizes upon UV-B photon absorption via distinct tryptophan residues. Monomeric UVR8 competitively binds to the substrate binding site of COP1, thus inhibiting its E3 ubiquitin ligase activity against target proteins, which include transcriptional regulators such as HY5. The UVR8-COP1 interaction also leads to the destabilization of PIF bHLH factor family members. Additionally, UVR8 directly interacts with and inhibits the DNA binding of a different set of transcription factors. Each of these UVR8 signaling mechanisms initiates nuclear gene expression changes leading to UV-B-induced photomorphogenesis and acclimation. The two WD40-repeat proteins RUP1 and RUP2 provide negative feedback regulation and inactivate UVR8 by facilitating redimerization. Here, we review the molecular mechanisms of the UVR8 pathway from UV-B perception and signal transduction to gene expression changes and physiological UV-B responses

Coping with 'Dark Sides of the Sun' through Photoreceptor Signaling

Plants grow in constantly changing environments, including highly variable light intensities. Sunlight provides the energy that drives photosynthesis and is thus of the utmost importance for plant growth and the generation of oxygen, which the majority of life on Earth depends on. However, exposure to either insufficient or excess levels of light can have detrimental effects and cause light stress. Whereas exposure to insufficient light limits photosynthetic activity, resulting in 'energy starvation', exposure to excess light can damage the photosynthetic apparatus. Furthermore, strong sunlight is associated with high levels of potentially damaging UV-B radiation. Different classes of photoreceptors play important roles in coping with the negative aspects of sunlight, for which specific mechanisms are emerging that are reviewed here