Potential of Cell-Free Supernatant from Lactobacillus plantarum NIBR97, Including Novel Bacteriocins, as a Natural Alternative to Chemical Disinfectants.

The recent pandemic of coronavirus disease 2019 (COVID-19) has increased demand for chemical disinfectants, which can be potentially hazardous to users. Here, we suggest that the cell-free supernatant from Lactobacillus plantarum NIBR97, including novel bacteriocins, has potential as a natural alternative to chemical disinfectants. It exhibits significant antibacterial activities against a broad range of pathogens, and was observed by scanning electron microscopy (SEM) to cause cellular lysis through pore formation in bacterial membranes, implying that its antibacterial activity may be mediated by peptides or proteins and supported by proteinase K treatment. It also showed significant antiviral activities against HIV-based lentivirus and influenza A/H3N2, causing lentiviral lysis through envelope collapse. Furthermore, whole-genome sequencing revealed that NIBR97 has diverse antimicrobial peptides, and among them are five novel bacteriocins, designated as plantaricin 1 to 5. Plantaricin 3 and 5 in particular showed both antibacterial and antiviral activities. SEM revealed that plantaricin 3 causes direct damage to both bacterial membranes and viral envelopes, while plantaricin 5 damaged only bacterial membranes, implying different antiviral mechanisms. Our data suggest that the cell-free supernatant from L. plantarum NIBR97, including novel bacteriocins, is potentially useful as a natural alternative to chemical disinfectants.

HGF and IL-10 expressing ALB::GFP reporter cells generated from iPSCs show robust anti-fibrotic property in acute fibrotic liver model.

BACKGROUND: Cell therapy using hepatocytes derived from stem cells has been regarded as a promising alternate to liver transplantation. However, the heterogeneity of these hepatocytes makes them unsuitable for therapeutic use. To overcome this limitation, we generated homogenous hepatocyte like induced hepatocyte-like (iHep) cells. METHODS: iHep cells were generated from induced pluripotent stem cells (iPSCs) integrated with the albumin (ALB) reporter gene. The therapeutic properties of these iHep cells were investigated after transplantation in fibrotic liver tissues of a mouse model. RESULTS: The iHep cells expressed hepatocyte specific genes and proteins, and exhibited high levels of hepatocyte growth factor (HGF) and interleukin (IL)-10 expressions. Transplantation of iHep cells significantly decreased thioacetamide (TAA)-induced liver fibrosis, apoptotic cells in the liver, and ameliorated abnormal liver function. Liver tissues engrafted with iHep cells exhibited decreased expression of pro-inflammatory factors such as transforming growth factor (TGF)-ß, IL-6, and monocyte chemo attractant protein (MCP)-1. Furthermore, an increased number of proliferating hepatocytes and human albumin-expressing iHep cells were detected in mice liver. CONCLUSIONS: This study has investigated and proven the liver regeneration potential of genome-edited iHep cells and promises to be a strong foundation for further studies exploring cell therapy as an alternative therapeutic option for the treatment of liver fibrosis.

Minicircle-based GCP-2 ex vivo gene therapy enhanced the reepithelialization and angiogenic capacity.

Recently, minicircle (MC)-based cell therapy has been emerging as a novel technology for nonviral genetic modification. In this study, we investigated the characteristics of granulocyte chemotactic protein-2 (GCP-2)-overexpressing fibroblasts (GCP-2/MC) using MC microporation technology, as well as its therapeutic mechanism in wound healing. GCP-2 parent plasmid and MC containing GCP-2 were generated. Human dermal fibroblasts (HDF) were transfected with MC containing GCP-2. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), scratch wound assay, and in vivo wound healing assay were performed. Gene and protein expression analysis revealed that GCP-2/MC highly expressed epithelialization growth factor, epidermal growth factor (EGF), chemokines, GCP-2, interleukin (IL)-8, as well as wound healing-associated genes such as insulin growth factor (IGF)-1 and hepatocyte growth factor (HGF). An in vitro scratch wound closure and matrigel tube formation assays demonstrated that the culture medium derived from GCP-2/MC substantially accelerated the wound closure and matrigel network formation. Wounds in nude mice were created by skin excisions followed by injections of GCP-2/MC. Results showed high cell survival potential and that GCP-2/MC transplantation highly accelerated skin wound closure by increasing reepithelialization, capillary density, and enhancing angiogenic factors, suggesting direct benefits for cutaneous closure. Taken together, these data suggest that MC-based GCP-2 overexpression could be a promising alternative strategy for promoting wound healing.

Interleukin 10-Secreting MSCs via TALEN-Mediated Gene Editing Attenuates Left Ventricular Remodeling after Myocardial Infarction.

BACKGROUND/AIMS: Stem cells or progenitor cells have been demonstrated as a novel alternative for cell therapy; however, their sustained efficacy is still debated. This study aimed to evaluate whether interleukin 10 (IL-10) gene-edited amniotic mesenchymal stem cells (AMM/I) contribute to left ventricular (LV) function and remodeling after acute myocardial infarction (AMI). METHODS: The IL-10 gene was integrated into the genomic locus of AMM via transcription activator-like effector nucleases (TALEN) and AMM/I were intramyocardially transplanted into AMI mice models. Cardiac function, quantitative polymerase chain reaction, histology, capillary density, and apoptosis assays were performed. RESULTS: AMM/I transplantation significantly suppressed infiltrated CD68 positive or F4/80 positive inflammatory cells and reduced the expression of pro-inflammatory factors in the infarcted myocardium. In addition, significantly improved LV function and reduced infarct size was noted in mice model with AMM/I transplantation than in those given AMM. Moreover, AMM/I highly inhibited cell apoptosis and increased capillary density in the infarcted myocardium. CONCLUSION: Our study demonstrated that AMM/I recruitment played favorable roles in the early restoration of LV function and remodeling by suppressing inflammation and enhancing cardiac protection and capillary density.

IL-10-secreting human MSCs generated by TALEN gene editing ameliorate liver fibrosis through enhanced anti-fibrotic activity.

Mesenchymal stem cells (MSCs) are known for their ability to repair liver damage. However, their therapeutic potential still needs to be enhanced. In the present study, we produced genome-edited MSCs that secrete interleukin 10 (IL-10) and evaluated their therapeutic potential in a liver fibrosis model. Multiple copies of the IL-10 gene were inserted into a safe harbor genomic locus in amniotic mesenchymal stem cells (AMMs) using transcription activator-like effector nucleases (TALENs). The IL-10 gene-edited AMMs (AMM/I) were characterized by reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), and microarray. The effects of AMM/I-conditioned cell medium (CM) on the activation of hepatic stellate cells (HSC) were analyzed in vitro and in vivo therapeutic assays were performed on a mouse liver fibrosis model. The engineered AMM/I expressed high levels of IL-10. AMM/I-CM inhibited the activation of HSC (in vitro) and TNF-α expression of T cells/macrophage derived from fibrotic liver. In addition, human IL-10 was detected in the serum of the mice transplanted with AMM/I and transplantation of AMM/I significantly inhibited thioacetamide (TAA)-induced liver fibrosis and ameliorated abnormal liver function. Furthermore, a high number of human albumin-expressing AMM/I were successfully engrafted into the liver of recipient mice. Overall, genome-edited AMMs overexpressing anti-fibrotic IL-10 might be a promising alternative therapeutic option for the treatment of liver cirrhosis.

Generation of directly reprogrammed human endothelial cells derived from fibroblast using ultrasound.

Physical microenvironment plays an important role in determining cellular reprogramming. In this study, we first generated directly reprogrammed human dermal fibroblasts (HDFs) into endothelial cells (ECs) mediated by environmental transition-guided cellular reprogramming (e/Entr) using ultrasound and characterized e/Entr. Ultrasound stimulus was introduced to ECs culture media and HDFs and induced into ECs-like cells. We performed microarray, RT-PCR, protein analysis, matrigel plug assay and e/Entr were transplanted into ischemic hindlimb mice model. Here we show that the activation of MAPK signaling pathways and the modulation of histone proteins such as Hp1-α, H3K27me3 and H3K4me3 in e/Entr contribute to the changes in chromatin configuration and reprogramming. Microarray data demonstrated that e/Entr highly expressed genes associated with ECs transcription factors and angiogenesis. In addition, the transplantation of e/Entr into hindlimb ischemia showed a high recovery of blood perfusion, limb salvage and e/Entr contributed to the formation of new vessels. In conclusion, the present study provided the first evidence that ultrasound reprogramming can induce postnatal cells to functional ECs. Therefore, our data suggest that physical stimulus-mediated reprogramming is a highly effective and safe strategy for the novel therapeutic alternatives.

Dual chemotactic factors-secreting human amniotic mesenchymal stem cells via TALEN-mediated gene editing enhanced angiogenesis.

BACKGROUND: Even though mesenchymal stem cells (MSCs) have angiogenic property, their cytokine secretory capacity is limited to treat ischemic vascular disorders. In present study, we produced genome-edited MSCs that secreted dual chemokine granulocyte chemotactic protein-2 (GCP-2) and stromal-derived factor-1α (SDF-1α) and determined their therapeutic potential in the context of experimental ischemia. METHODS: GCP-2 and SDF-1α genes were integrated into safe harbor site at the safe harbor genomic locus of amniotic mesenchymal stem cells (AMM) via transcription activator-like effector nucleases (TALEN). GCP-2 and SDF-1α gene-edited AMM (AMM/GS) were used for quantitative (q)-PCR, Matrigel tube formation, cell migration, Matrigel plug assays and in vivo therapeutic assays using hindlimb ischemia mouse model. RESULTS: AMM/GS-derived culture media (CM) induced significantly higher tube lengths and branching points as compared to AMM/S CM and AMM CM. Interestingly, Matrigel plug assays revealed that significantly higher levels of red blood cells were found in AMM/GS than AMM/S and AMM Matigel plugs and exhibited micro-vascular like formation. Cells was transplanted into ischemic mouse hindlimbs and compared with control groups. AMM/GS injection prevented limb loss and augmented blood perfusion, suggesting that enhances neovascularization in hindlimb ischemia. In addition, transplanted AMM/GS revealed high vasculogenic potential in vivo compared with transplanted AMM/S. CONCLUSION: Taken together, genome-edited MSCs that express dual chemokine GCP-2 and SDF-1α might be alternative therapeutic options for the treatment of ischemic vascular disease.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification, localization, and function in cellulose biosynthesis and osmotic stress responses.

Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation of KOR1 at tonoplasts. The degree of successful TGN localization of KOR1 agreed well with in vivo-complementation efficacy of the rsw2-1 mutant, suggesting non-catalytic functions in the TGN. A dynamic interaction network involving microtubules, CSCs, KOR1, and currently unidentified glycoprotein component(s) likely determines stress-triggered re-organization of cellulose biosynthesis and resumption of cell-wall growth under stress.

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-ß1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

Arabidopsis C-terminal domain phosphatase-like 1 functions in miRNA accumulation and DNA methylation.

Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that cpl1 was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of ros1 suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, cpl1 reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.

Loss of function of Arabidopsis C-terminal domain phosphatase-like1 activates iron deficiency responses at the transcriptional level.

The expression of genes that control iron (Fe) uptake and distribution (i.e. Fe utilization-related genes) is tightly regulated. Fe deficiency strongly induces Fe utilization-related gene expression; however, little is known about the mechanisms that regulate this response in plants. Transcriptome analysis of an Arabidopsis (Arabidopsis thaliana) mutant defective in RNA polymerase II C-terminal domain-phosphatase-like1 (CPL1) revealed significant up-regulation of Fe utilization-related genes (e.g. IRON-REGULATED TRANSPORTER1), suggesting the importance of RNA metabolism in Fe signaling. An analysis using multiple cpl1 alleles established that cpl1 mutations enhanced specific transcriptional responses to low Fe availability. Changes in protein level were less prominent than those in transcript level, indicating that cpl1-2 mainly affects the Fe deficiency response at the transcriptional level. However, Fe content was significantly increased in the roots and decreased in the shoots of cpl1-2 plants, indicating that the cpl1 mutations do indeed affect Fe homeostasis. Furthermore, root growth of cpl1-2 showed improved tolerance to Fe deficiency and cadmium (Cd) toxicity. cpl1-2 plants accumulated more Cd in the shoots, suggesting that Cd toxicity in the roots of this mutant is averted by the transport of excess Cd to the shoots. Genetic data indicate that cpl1-2 likely activates Fe deficiency responses upstream of both FE-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent and -independent signaling pathways. Interestingly, various osmotic stress/abscisic acid (ABA)-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. We propose that the cpl1 mutations enhance Fe deficiency signaling and promote cross talk with a branch of the osmotic stress/ABA signaling pathway.

Functional characterization of ObgC in ribosome biogenesis during chloroplast development.

The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT.

AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis.

Obg is a ribosome-associated GTPase essential for bacterial viability and is conserved in most organisms, from bacteria to eukaryotes. Obg is also expressed in plants, which predicts an important role for this molecule in plant viability; however, the functions of the plant Obg homologs have not been reported. Here, we first identified Arabidopsis AtObgC as a plant chloroplast-targeting Obg and elucidated its molecular biological and physiological properties. AtObgC encodes a plant-specific Obg GTPase that contains an N-terminal region for chloroplast targeting and has intrinsic GTP hydrolysis activity. A targeting assay using a few AtObgC N-terminal truncation mutants revealed that AtObgC localizes to chloroplasts and its transit peptide consists of more than 50 amino acid residues. Interestingly, GFP-fused full-length AtObgC exhibited a punctate staining pattern in chloroplasts of Arabidopsis protoplasts, which suggests a dimerization or multimerization of AtObgC. Moreover, its Obg fold was indispensable for the generation of the punctate staining pattern, and thus, was supposed to be important for such oligomerization of AtObgC by mediating the protein-protein interaction. In addition, the T-DNA insertion AtObgC null mutant exhibited an embryonic lethal phenotype that disturbed the early stage of embryogenesis. Altogether, our results provide a significant implication that AtObgC as a chloroplast targeting GTPase plays an important role at the early embryogenesis by exerting its function in chloroplast protein synthesis.

Arabidopsis thaliana PRP40s are RNA polymerase II C-terminal domain-associating proteins.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions as a scaffold for RNA processing machineries that recognize differentially phosphorylated conserved (YSPTSPS)(n) repeats. Evidence indicates that proteins that regulate the phosphorylation status of the CTD are determinants of growth, development, and stress responses of plants; however, little is known about the mechanisms that translate the CTD phosphoarray into physiological outputs. We report the bioinformatic identification of a family of three phospho-CTD-associated proteins (PCAPs) in Arabidopsis and the characterization of the AtPRP40 (Arabidopsis thaliana PRE-mRNA-PROCESSING PROTEIN 40) family as PCAPs. AtPRP40s-CTD/CTD-PO(4) interactions were confirmed using the yeast two-hybrid assay and far-Western blotting. WW domains at the N-terminus of AtPRP40b mediate the AtPRP40b-CTD/CTD-PO(4) interaction. Although AtPRP40s interact with both phosphorylated and unphosphorylated CTD in vitro, there is a strong preference for the phosphorylated form in Arabidopsis cell extract. AtPRP40s are ubiquitously expressed and localize to the nucleus. These results establish that AtPRP40s are specific PCAPs, which is consistent with the predicted function of the AtPRP40 family in pre-mRNA splicing.

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling.

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.