RESUMEN
As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of ROS under photoinhibitory light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.
RESUMEN
In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.
Asunto(s)
Complejo de Proteína del Fotosistema II , Tilacoides , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Ribosomas/metabolismo , Tilacoides/metabolismoRESUMEN
Epidermal cells are the main avenue for signal and material exchange between plants and the environment. Leaf epidermal cells primarily include pavement cells, guard cells, and trichome cells. The development and distribution of different epidermal cells are tightly regulated by a complex transcriptional regulatory network mediated by phytohormones, including jasmonic acid, and transcription factors. How the fate of leaf epidermal cells is determined, however, is still largely unknown due to the diversity of cell types and the complexity of their regulation. Here, we characterized the transcriptional profiles of epidermal cells in 3-day-old true leaves of Arabidopsis thaliana using single-cell RNA sequencing. We identified two genes encoding BASIC LEUCINE-ZIPPER (bZIP) transcription factors, namely bZIP25 and bZIP53, which are highly expressed in pavement cells and early-stage meristemoid cells. Densities of pavement cells and trichome cells were found to increase and decrease, respectively, in bzip25 and bzip53 mutants, compared with wild-type plants. This trend was more pronounced in the presence of jasmonic acid, suggesting that these transcription factors regulate the development of trichome cells and pavement cells in response to jasmonic acid.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ciclopentanos , Oxilipinas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células Epidérmicas , Factores de Transcripción , Hojas de la Planta , Tricomas , Análisis de Secuencia de ARN , Regulación de la Expresión Génica de las PlantasRESUMEN
R-loops, three-stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single-stranded DNA, play critical roles in gene expression and genome stability. How R-loop homeostasis is integrated into chloroplast gene expression remains largely unknown. We found an unexpected function of FtsHi1, an inner envelope membrane-bound AAA-ATPase in chloroplast R-loop homeostasis of Arabidopsis thaliana. Previously, this protein was shown to function as a component of the import motor complex for nuclear-encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP-dependent helicase that efficiently unwinds both DNA-DNA and DNA-RNA duplexes, thereby preventing R-loop accumulation. Over-accumulation of R-loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear- and chloroplast-encoded subunits of multi-protein photosynthetic complexes. This study suggests a mechanical link between protein import and R-loop homeostasis in chloroplasts of higher plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Adenosina Trifosfato/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Transporte de Proteínas , Estructuras R-Loop , ARN/metabolismo , ARN Helicasas/genéticaRESUMEN
Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1-4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5-7, the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC-TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.
Asunto(s)
Chlamydomonas reinhardtii , Cloroplastos , Microscopía por Crioelectrón , Complejos Multiproteicos , Transporte de Proteínas , Cloroplastos/química , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Ácido Fítico/metabolismo , Estabilidad Proteica , Especificidad por SustratoRESUMEN
Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.
Asunto(s)
Carotenoides , Nicotiana , Farnesiltransferasa/genética , Nicotiana/genética , Carotenoides/metabolismo , Fotosíntesis , Alineación de SecuenciaRESUMEN
The chloroplast is a complex cellular organelle that not only performs photosynthesis but also synthesizes amino acids, lipids, and phytohormones. Nuclear and chloroplast genetic activity are closely coordinated through signaling chains from the nucleus to chloroplast, referred to as anterograde signaling, and from chloroplast to the nucleus, named retrograde signaling. The chloroplast can act as an environmental sensor and communicates with other cell compartments during its biogenesis and in response to stress, notably with the nucleus through retrograde signaling to regulate nuclear gene expression in response to developmental cues and stresses that affect photosynthesis and growth. Although several components involved in the generation and transmission of plastid-derived retrograde signals and in the regulation of the responsive nuclear genes have been identified, the plastid retrograde signaling network is still poorly understood. Here, we review the current knowledge on multiple plastid retrograde signaling pathways, and on potential plastid signaling molecules. We also discuss the retrograde signaling-dependent regulation of nuclear gene expression within the frame of a multilayered network of transcription factors.
RESUMEN
I present my personal reminiscence of Paul Levine-a highly innovative scientist who did seminal work in photosynthesis. He was among the first to initiate and use a genetic approach toward photosynthesis. He greatly helped in establishing the green unicellular alga Chlamydomonas reinhardtii as a powerful model system not only for understanding the function of the photosynthetic apparatus but also for studying its biogenesis and regulation. During the period he spent at Harvard, he made several ground-breaking contributions such as identifying and establishing the order of some components of the photosynthetic electron transport chain as well as determining their genetic origin. He trained many students and post-doctoral fellows several of whom later became prominent in this field and in other areas of plant science.
Asunto(s)
Chlamydomonas reinhardtii , Humanos , Chlamydomonas reinhardtii/fisiología , Fotosíntesis/fisiologíaRESUMEN
Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.
Asunto(s)
Cloroplastos , Proteínas de Plantas , Núcleo Celular , Proteínas de Cloroplastos , Chaperonas Moleculares , Transporte de ProteínasRESUMEN
Most chloroplast proteins are nucleus-encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP-powered Ycf2 complex consisting of several FtsH-like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co-chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin-proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process.
Asunto(s)
Cloroplastos , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Cloroplastos/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Transporte de Proteínas , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Precursores de Proteínas/metabolismoRESUMEN
The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.
Asunto(s)
Cotiledón , Regulación de la Expresión Génica de las Plantas , Regulación de la Expresión Génica de las Plantas/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , ARN/metabolismo , PlantonesRESUMEN
Although numerous studies have been carried out on chloroplast development and biogenesis, the underlying regulatory mechanisms are still largely elusive. Here, we characterized a chloroplast stromal protein Chloroplast Development and Biogenesis1 (CDB1). The knockout cdb1 mutant exhibits a seedling-lethal and ivory leaf phenotype. Immunoblot and RNA blot analyses show that accumulation of chloroplast ribosomes is compromised in cdb1, resulting in an almost complete loss of plastid-encoded proteins including the core subunits of the plastid-encoded RNA polymerase (PEP) RpoB and RpoC2, and therefore in impaired PEP activity. Orthologs of CDB1 are found in green algae and land plants. Moreover, a protein shows high similarity with CDB1, designated as CDB1-Like (CDB1L), is present in angiosperms. Absence of CDB1L results in impaired embryo development. While CDB1 is specifically located in the chloroplast stroma, CDB1L is localized in both chloroplasts and mitochondria in Arabidopsis. Thus, our results demonstrate that CDB1 is indispensable for chloroplast development and biogenesis through its involvement in chloroplast ribosome assembly whereas CDB1L may fulfill a similar function in both mitochondria and chloroplasts.
RESUMEN
There are numerous exchanges of signals and materials between leaves and roots, including nitrogen, which is one of the essential nutrients for plant growth and development. In this study we identified and characterized the Chlorophyll A/B-Binding Protein (CAB) (named coe2 for CAB overexpression 2) mutant, which is defective in the development of chloroplasts and roots under normal growth conditions. The phenotype of coe2 is caused by a mutation in the Nitric Oxide Associated (NOA1) gene that is implicated in a wide range of chloroplast functions including the regulation of metabolism and signaling of nitric oxide (NO). A transcriptome analysis reveals that expression of genes involved in metabolism and lateral root development are strongly altered in coe2 seedlings compared with WT. COE2 is expressed in hypocotyls, roots, root hairs, and root caps. Both the accumulation of NO and the growth of lateral roots are enhanced in WT but not in coe2 under nitrogen limitation. These new findings suggest that COE2-dependent signaling not only coordinates gene expression but also promotes chloroplast development and function by modulating root development and absorption of nitrogen compounds.
Asunto(s)
Nitrógeno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Clorofila A/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Mutación , Fenotipo , Desarrollo de la Planta/genética , Fenómenos Fisiológicos de las Plantas , Unión Proteica , Transducción de SeñalRESUMEN
The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes del Cloroplasto , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Transformación Genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Cloroplastos/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Regiones Promotoras Genéticas , RiboswitchRESUMEN
Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Azufre/metabolismo , Fotosíntesis/fisiologíaRESUMEN
In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Vitamina K Epóxido Reductasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Oxidación-Reducción , Fosforilación , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Compuestos de Sulfhidrilo/metabolismo , Vitamina K Epóxido Reductasas/genéticaRESUMEN
In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Complejos Multiproteicos/genética , Proteínas de Plantas/genética , Compartimento Celular , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Complejos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Atp11p and Atp12p are members of two chaperone families essential for assembly of the mitochondrial ATP synthase in Saccharomyces cerevisiae and Homo sapiens. However, the role of their homologs in higher plants is unclear with regard to the assembly of both chloroplast ATP synthase (cpATPase) and mitochondrial ATP synthase (mtATPase). Here, we show that loss of either Atp11 or Atp12 is lethal in Arabidopsis. While Atp12 is only localized in mitochondria, Atp11 is present both in chloroplasts and mitochondria. Yeast two-hybrid analyses showed that, as their homologs in yeast, Atp11 specifically interacts with the ß subunit of the mtATPase and cpATPase, and Atp12 interacts with the α subunit of the mtATPase, implying that Atp11 and Atp12 fulfill a conserved task during assembly of ATP synthase. However, the binding sites for Atp11 in the ß subunit of mtATPase and cpATPase are slightly different, suggesting that the mechanisms of action may have evolved in different ways. Although Atp11 interacts with cpATPase ß subunit as the two assembly factors BFA3 and BFA1, they bind to different sites of the ß subunit. These results indicate that Atp11 is involved in the assembly of both cpATPase and mtATPase but Atp12 is specifically required for the assembly of mtATPase in higher plants.
RESUMEN
The regulation of stomatal lineage cell development has been extensively investigated. However, a comprehensive characterization of this biological process based on single-cell transcriptome analysis has not yet been reported. In this study, we performed RNA sequencing on 12 844 individual cells from the cotyledons of 5-day-old Arabidopsis seedlings. We identified 11 cell clusters corresponding mostly to cells at specific stomatal developmental stages using a series of marker genes. Comparative analysis of genes with the highest variable expression among these cell clusters revealed transcriptional networks that regulate development from meristemoid mother cells to guard mother cells. Examination of the developmental dynamics of marker genes via pseudo-time analysis revealed potential interactions between these genes. Collectively, our study opens the door for understanding how the identified novel marker genes participate in the regulation of stomatal lineage cell development.