NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

Genome-Wide Analysis of Multiple Organellar RNA Editing Factor (MORF) Family in Kiwifruit (Actinidia chinensis) Reveals Its Roles in Chloroplast RNA Editing and Pathogens Stress

Kiwifruit (Actinidia chinensis) is well known for its high vitamin C content and good taste. Various diseases, especially bacterial canker, are a serious threat to the yield of kiwifruit. Multiple organellar RNA editing factor (MORF) genes are pivotal factors in the RNA editosome that mediates Cytosine-to-Uracil RNA editing, and they are also indispensable for the regulation of chloroplast development, plant growth, and response to stresses. Although the kiwifruit genome has been released, little is known about MORF genes in kiwifruit at the genome-wide level, especially those involved in the response to pathogens stress. In this study, we identified ten MORF genes in the kiwifruit genome. The genomic structures and chromosomal locations analysis indicated that all the MORF genes consisted of three conserved motifs, and they were distributed widely across the seven linkage groups and one contig of the kiwifruit genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the kiwifruit MORF gene family members were classified into six groups (Groups A–F). A synteny analysis indicated that two pairs of MORF genes were tandemly duplicated and five pairs of MORF genes were segmentally duplicated. Moreover, based on analysis of RNA-seq data from five tissues of kiwifruit, we found that both expressions of MORF genes and chloroplast RNA editing exhibited tissue-specific patterns. MORF2 and MORF9 were highly expressed in leaf and shoot, and may be responsible for chloroplast RNA editing, especially the ndhB genes. We also observed different MORF expression and chloroplast RNA editing profiles between resistant and susceptible kiwifruits after pathogen infection, indicating the roles of MORF genes in stress response by modulating the editing extend of mRNA. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes, in particular, for clarifying the resistance mechanisms in kiwifruits and breeding new cultivars with high resistance.

Chlorosis seedling lethality 1 encoding a MAP3K protein is essential for chloroplast development in rice

Background Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in eukaryotic organisms and play essential roles in immunity and stress responses. However, the role of MAPKs in chloroplast development remains to be evidently established. Results In this study, a rice chlorosis seedling lethality 1 (csl1) mutant with a Zhonghua11 (ZH11, japonica) background was isolated. Seedlings of the mutant were characterized by chlorotic leaves and death after the trefoil stage, and chloroplasts were observed to contain accumulated starch granules. Molecular cloning revealed that OsCSL1 encoded a MAPK kinase kinase22 (MKKK22) targeted to the endoplasmic reticulum (ER), and functional complementation of OsCSL1 was found to restore the normal phenotype in csl1 plants. The CRISPR/Cas9 technology was used for targeted disruption of OsCSL1, and the OsCSL1-Cas9 lines obtained therein exhibited yellow seedlings which phenocopied the csl1 mutant. CSL1/MKKK22 was observed to establish direct interaction with MKK4, and altered expression of MKK1 and MKK4 was detected in the csl1 mutant. Additionally, disruption of OsCSL1 led to reduced expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded RNA polymerases, nuclear-encoded RNA polymerase, and nuclear-encoded chloroplast genes. Conclusions The findings of this study revealed that OsCSL1 played roles in regulating the expression of multiple chloroplast synthesis-related genes, thereby affecting their functions, and leading to wide-ranging defects, including chlorotic seedlings and severely disrupted chloroplasts containing accumulated starch granules.

The Component of the TAC Complex, TCD7, Controls Rice Chloroplast Development at the Early Seedling Stage under Cold Stress

Transcriptionally active chromosome (TAC) is a component of protein-DNA complexes with RNA polymerase activity found in chloroplasts. Although TAC in Arabidopsis thaliana has been extensively investigated, how the rice (Oryza sativa L.) TAC complex functions remains largely unknown. We report the characterization of the mutant thermosensitive chlorophyll-deficient7 (tcd7) and the cloning of TCD7. tcd7 mutant seedlings displayed an albino phenotype specifically at low temperatures and before the four-leaf stage. We identified TCD7 by map-based cloning followed by transgenic rescue and genome editing tests, showing that TCD7 encodes the putative TAC component FRUCTOKINASE-LIKE 2 (OsFLN2). TCD7 transcripts were highly abundant in green tissues, and the protein localized to chloroplasts. In agreement with the albino phenotype, transcript levels of genes controlling chloroplast development and the establishment of photosynthetic capacity were severely reduced in tcd7 seedlings at low temperatures, but were expressed as in the wild type at high temperatures, implying that TCD7 regulates the PEP pathway and chloroplast development. Moreover, TCD7 interacted with the thioredoxin OsTRXz to form an OsTRXz-TCD7 regulatory module, which might regulate plastid transcription under cold stress. Our results demonstrate that the nucleus-encoded TAC protein TCD7 protects chloroplast development from cold stress via a TRXz-FLN regulatory module.

Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit β and Chloroplast Development in Arabidopsis

Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz. RT-quantitative real-time PCR, northern blot, and run-on analyses indicated that the PEP-dependent transcription in the pd1 mutant was dramatically impaired, whereas the nuclear-encoded RNA polymerase-dependent transcription was up-regulated. The yeast two-hybrid assays and coimmunoprecipitation experiments showed that the PD1 protein interacts with PEP core subunit β (PEP-β), which has been verified to be essential for chloroplast development. The immunoblot analysis indicated that the accumulation of PEP-β was barely detected in the pd1 mutant, whereas the accumulation of the other essential components of the PEP complex, such as core subunits α and β′, were not affected in the pd1 mutant. These observations suggested that the PD1 protein is essential for the accumulation of PEP-β and chloroplast development in Arabidopsis, potentially by direct interaction with PEP-β.

A Novel Chloroplast Protein RNA Processing 8 Is Required for the Expression of Chloroplast Genes and Chloroplast Development in Arabidopsis thaliana

Chloroplast development involves the coordinated expression of both plastids- and nuclear-encoded genes in higher plants. However, the underlying mechanism still remains largely unknown. In this study, we isolated and characterized an Arabidopsis mutant with an albino lethality phenotype named RNA processing 8 (rp8). Genetic complementation analysis demonstrated that the gene AT4G37920 (RP8) was responsible for the mutated phenotype. The RP8 gene was strongly expressed in photosynthetic tissues at both transcription and translation protein levels. The RP8 protein is localized in the chloroplast and associated with the thylakoid. Disruption of the RP8 gene led to a defect in the accumulation of the rpoA mature transcript, which reduced the level of the RpoA protein, and affected the transcription of PEP-dependent genes. The abundance of the chloroplast rRNA, including 23S, 16S, 4.5S, and 5S rRNA, were reduced in the rp8 mutant, respectively, and the amounts of chloroplast ribosome proteins, such as, PRPS1(uS1c), PRPS5(uS5c), PRPL2 (uL2c), and PRPL4 (uL4c), were substantially decreased in the rp8 mutant, which indicated that knockout of RP8 seriously affected chloroplast translational machinery. Accordingly, the accumulation of photosynthetic proteins was seriously reduced. Taken together, these results indicate that the RP8 protein plays an important regulatory role in the rpoA transcript processing, which is required for the expression of chloroplast genes and chloroplast development in Arabidopsis.