LncRNA HOTAIRM1 functions in DNA double-strand break repair via its association with DNA repair and mRNA surveillance factors.

The eukaryotic exon junction complex component Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. Using immunoprecipitation-RNA-seq, we identified a set of Y14-associated long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1 serves as a strong candidate that mediates the interaction between Y14 and the NHEJ complex. HOTAIRM1 localized to near ultraviolet laser-induced DNA damage sites. Depletion of HOTAIRM1 delayed the recruitment of DNA damage response and repair factors to DNA lesions and compromised the efficiency of NHEJ-mediated DSB repair. Identification of the HOTAIRM1 interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in a HOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude that HOTAIRM1 serves as an assembly scaffold for both DNA repair and mRNA surveillance factors that act in concert to repair DSBs.

Upregulation of PD-L1 by SARS-CoV-2 promotes immune evasion.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T-cell sequestration, cytokine storm, and mortality. However, it remains largely unknown how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated nuclear factor-κB (NF-κB) and interferon regulatory factor 1 (IRF1) pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 chromatin immunoprecipitation sequencing. Cross-referencing our transcriptomic and epigenomic data sets revealed that coupling NF-κB and IRF1 pathways mediate programmed death ligand-1 (PD-L1) immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity.

Maize Golden2-like transcription factors boost rice chloroplast development, photosynthesis, and grain yield.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

The Impact of Dysregulated microRNA Biogenesis Machinery and microRNA Sorting on Neurodegenerative Diseases.

MicroRNAs (miRNAs) are 22-nucleotide noncoding RNAs involved in the differentiation, development, and function of cells in the body by targeting the 3'- untranslated regions (UTR) of mRNAs for degradation or translational inhibition. miRNAs not only affect gene expression inside the cells but also, when sorted into exosomes, systemically mediate the communication between different types of cells. Neurodegenerative diseases (NDs) are age-associated, chronic neurological diseases characterized by the aggregation of misfolded proteins, which results in the progressive degeneration of selected neuronal population(s). The dysregulation of biogenesis and/or sorting of miRNAs into exosomes was reported in several NDs, including Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD). Many studies support the possible roles of dysregulated miRNAs in NDs as biomarkers and therapeutic treatments. Understanding the molecular mechanisms underlying the dysregulated miRNAs in NDs is therefore timely and important for the development of diagnostic and therapeutic interventions. In this review, we focus on the dysregulated miRNA machinery and the role of RNA-binding proteins (RBPs) in NDs. The tools that are available to identify the target miRNA-mRNA axes in NDs in an unbiased manner are also discussed.

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes.

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Decoding the differentiation of mesenchymal stem cells into mesangial cells at the transcriptomic level.

BACKGROUND: Mesangial cells play an important role in the glomerulus to provide mechanical support and maintaine efficient ultrafiltration of renal plasma. Loss of mesangial cells due to pathologic conditions may lead to impaired renal function. Mesenchymal stem cells (MSC) can differentiate into many cell types, including mesangial cells. However transcriptomic profiling during MSC differentiation into mesangial cells had not been studied yet. The aim of this study is to examine the pattern of transcriptomic changes during MSC differentiation into mesangial cells, to understand the involvement of transcription factor (TF) along the differentiation process, and finally to elucidate the relationship among TF-TF and TF-key gene or biomarkers during the differentiation of MSC into mesangial cells. RESULTS: Several ascending and descending monotonic key genes were identified by Monotonic Feature Selector. The identified descending monotonic key genes are related to stemness or regulation of cell cycle while ascending monotonic key genes are associated with the functions of mesangial cells. The TFs were arranged in a co-expression network in order of time by Time-Ordered Gene Co-expression Network (TO-GCN) analysis. TO-GCN analysis can classify the differentiation process into three stages: differentiation preparation, differentiation initiation and maturation. Furthermore, it can also explore TF-TF-key genes regulatory relationships in the muscle contraction process. CONCLUSIONS: A systematic analysis for transcriptomic profiling of MSC differentiation into mesangial cells has been established. Key genes or biomarkers, TFs and pathways involved in differentiation of MSC-mesangial cells have been identified and the related biological implications have been discussed. Finally, we further elucidated for the first time the three main stages of mesangial cell differentiation, and the regulatory relationships between TF-TF-key genes involved in the muscle contraction process. Through this study, we have increased fundamental understanding of the gene transcripts during the differentiation of MSC into mesangial cells.

Hyperbaric Oxygen Treatment Ameliorates Hearing Loss and Auditory Cortex Injury in Noise Exposed Mice by Repressing Local Ceramide Accumulation.

Noise-induced hearing loss (NIHL) relates closely to auditory cortex (AC) injury, so countermeasures aiming at the AC recovery would be of benefit. In this work, the effect of hyperbaric oxygen treatment on NIHL was elucidated, which was imposed on mice before (HBOP), during (HBOD) or after (HBOA) noise exposure. Morphology of neurons was assayed by hematoxylin-eosin or Nissl staining. Ceramide (Cer) level was measured through immunohistochemistry analysis. Apoptotic neurons were counted using transferase-mediated dUTP nick end labeling (TUNEL) staining. We demonstrated that the intense, broad band noise raised the threshold of auditory brainstem response, evoked neuronal degeneration or apoptosis and triggered the Cer accumulation in AC, all of which were restored significantly by HBOP, but not HBOD or HBOA. Cer over-generation reversed the advantages of HBOP significantly, while its curtailment recapitulated the effect. Next, noise exposure raised the superoxide or malondialdehyde (MDA) production which was blocked by HBOP or Cer repression. Oxidative control not only attenuated the hearing loss or neurodegeneration but, in turn, reduced the Cer formation significantly. In summary, mutual regulation between Cer and oxidative stress underlies the HBOP's curative effect on hearing loss and neuronal damage in noise-exposed mice.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats.

Structural adaptation of arteries to weightlessness might lower the working ability or even threaten the physical health of astronauts, but the underlying mechanism is unclear. Acid sphingomyelinase (ASM) catalyzes ceramide (Cer) generation controlling arterial remodeling through multiple signaling pathways. In the present study, we aimed to investigate the contribution of ASM/Cer to the changes of common carotid artery intima-media thickness (CIMT) induced by simulated weightlessness. Hindlimb-unloaded tail-suspended (HU) rats were used to simulate the effect of weightlessness. Morphology of the carotid artery (CA) was examined by hematoxylin-eosin staining. Protein content of ASM or proliferating cell nuclear antigen (PCNA) was detected by Western blot. Cer level was measured by immunohistochemistry analysis. Apoptosis events were observed by transferase-mediated dUTP nick end labeling (TUNEL) staining. During 4 weeks of tail suspension, CIMT was increased gradually in HU but not in their synchronous control rats (P < 0.05). Correspondingly, the CA of HU rats had a lower apoptosis and higher proliferation of vascular smooth muscle cells (VSMCs). As compared to the control, both ASM protein expression and Cer content were reduced significantly in CA of HU rats (P < 0.05), incubation of which with permeable Cer reversed the changes in apoptosis and proliferation substantially. Furthermore, when the ASM protein content as well as Cer level in CA of control rats was diminished by using an ASM inhibitor, an increase of CIMT along with reduced apoptosis and enhanced proliferation of VSMCs was found. Our results suggest that by controlling the balance between apoptosis and proliferation, ASM/Cer plays an important role in the regulation of CIMT during simulated weightlessness.

Effects of hyperbaric oxygen on hippocampal neuronal apoptosis in rats with acute carbon monoxide poisoning.

INTRODUCTION: Acute carbon monoxide (CO) poisoning causes serious health problems such as neuropsychological sequelae. This study aimed to investigate neuronal apoptosis and the effects of hyperbaric oxygen (HBO2) on different regions of the rat hippocampus after CO poisoning. METHODS: 90 mature male Sprague Dawley rats were randomly divided into three groups: the normal control group (NC group), the acute carbon monoxide-poisoned group (CO group) and the hyperbaric oxygen treatment group (HBO2 group). CO exposure included 0, 1, 3, 7, 14 and 21 treatment days, one exposure on the first day, and sacrifice on each of the following days. HBO2 exposure included treatment for 0, 1, 3, 7, 14 and 21 days, daily treatment after CO exposure, and sacrifice after the last HBO2 treatment on each of those days. Hematoxylin-eosin staining, immunohistochemical staining, immunofluorescence staining, and western blot analysis were performed to detect apoptosis in brain tissue samples. RESULTS: MMP-9 and caspase-3 were prominently increased by CO exposure and inhibited by HBO2 in the CA3 region in the hippocampus at one, three and seven days (immunohistochemical staining [IHC]: P ⟨ 0.05). Neu N and the ratio of Bcl-2/ BAX were prominently decreased by CO exposure and rescued by HBO2 in the CA3 region after seven days of treatment (IHC: P ⟨ 0.05). CONCLUSION: These findings indicated that neuronal apoptosis in the rat hippocampus could be induced by acute CO exposure, especially in the CA3 region. HBO2 could effectively inhibit neuronal apoptosis, especially in the CA3 region after seven days of treatment. The application of HBO2 to inhibit MMP-9 and apoptosis may contribute to brain recovery after acute CO poisoning.

Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination.

Our anatomical analysis revealed that a dry maize seed contains four to five embryonic leaves at different developmental stages. Rudimentary kranz structure (KS) is apparent in the first leaf with a substantial density, but its density decreases toward younger leaves. Upon imbibition, leaf expansion occurs rapidly with new KSs initiated from the palisade-like ground meristem cells in the middle of the leaf. In parallel to the anatomical analysis, we obtained the time course transcriptomes for the embryonic leaves in dry and imbibed seeds every 6 h up to hour 72. Over this time course, the embryonic leaves exhibit transcripts of 30,255 genes at a level that can be regarded as "expressed." In dry seeds, ∼25,500 genes are expressed, showing functional enrichment in transcription, RNA processing, protein synthesis, primary metabolic pathways, and calcium transport. During the 72-h time course, ∼13,900 genes, including 590 transcription factor genes, are differentially expressed. Indeed, by 30 h postimbibition, ∼2,200 genes expressed in dry seeds are already down-regulated, and ∼2,000 are up-regulated. Moreover, the top 1% expressed genes at 54 h or later are very different from those before 30 h, reflecting important developmental and physiological transitions. Interestingly, clusters of genes involved in hormone metabolism, signaling, and responses are differentially expressed at various time points and TF gene expression is also modular and stage specific. Our dataset provides an opportunity for hypothesizing the timing of regulatory actions, particularly in the context of KS development.

Functional evolution of cardiac microRNAs in heart development and functions.

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression either by degrading target mRNAs or by suppressing protein translation. miRNAs have been found to be involved in many biological processes, such as development, differentiation, and growth. However, the evolution of miRNA regulatory functions and networks has not been well studied. In this study, we conducted a cross-species analysis to study the evolution of cardiac miRNAs and their regulatory functions and networks. We found that conserved cardiac miRNA target genes have maintained highly conserved cardiac functions. Additionally, most of cardiac miRNA target genes in human with annotations of cardiac functions evolved from the corresponding homologous targets, which are also involved in heart development-related functions. On the basis of these results, we investigated the functional evolution of cardiac miRNAs and presented a functional evolutionary map. From this map, we identified the evolutionary time at which the cardiac miRNAs became involved in heart development or function and found that the biological processes of heart development evolved earlier than those of heart functions, for example, heart contraction/relaxation or cardiac hypertrophy. Our study of the evolution of the cardiac miRNA regulatory networks revealed the emergence of new regulatory functional branches during evolution. Furthermore, we discovered that early evolved cardiac miRNA target genes tend to participate in the early stages of heart development. This study sheds light on the evolution of developmental features of genes regulated by cardiac miRNAs.

Maize and millet transcription factors annotated using comparative genomic and transcriptomic data.

BACKGROUND: Transcription factors (TFs) contain DNA-binding domains (DBDs) and regulate gene expression by binding to specific DNA sequences. In addition, there are proteins, called transcription coregulators (TCs), which lack DBDs but can alter gene expression through interaction with TFs or RNA Polymerase II. Therefore, it is interesting to identify and classify the TFs and TCs in a genome. In this study, maize (Zea mays) and foxtail millet (Setaria italica), two important species for the study of C4 photosynthesis and kranz anatomy, were selected. RESULT: We conducted a comprehensive genome-wide annotation of TFs and TCs in maize B73 and in two strains of foxtail millet, Zhang gu and Yugu1, and classified them into families. To gain additional support for our predictions, we searched for their homologous genes in Arabidopsis or rice and studied their gene expression level using RNA-seq and microarray data. We identified many new TF and TC families in these two species, and described some evolutionary and functional aspects of the 9 new maize TF families. Moreover, we detected many pseudogenes and transposable elements in current databases. In addition, we examined tissue expression preferences of TF and TC families and identified tissue/condition-specific TFs and TCs in maize and millet. Finally, we identified potential C4-related TF and TC genes in maize and millet. CONCLUSIONS: Our results significantly expand current TF and TC annotations in maize and millet. We provided supporting evidence for our annotation from genomic and gene expression data and identified TF and TC genes with tissue preference in expression. Our study may facilitate the study of regulation of gene expression, tissue morphogenesis, and C4 photosynthesis in maize and millet. The data we generated in this study are available at http://sites.google.com/site/jjlmmtf.

Genetic insights into carbohydrate sulfotransferase 8 and its impact on the immunotherapy efficacy of cancer.

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p < 5 × 10-8) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress T cell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction.

Regulating TKT activity inhibits proliferation of human acute lymphoblastic leukemia cells.

Among pediatric blood cancers, acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy. Within ALL, T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10 to 15% of all pediatric cases, and ~25% of adult cases. For T-ALL, its recurrence and relapse after treatment remain problematic. Therefore, it is necessary to develop new therapies for T-ALL. Recent studies suggested regulating energy metabolism is a novel approach to inhibit tumor growth, likely a promising treatment. Transketolase (TKT) is an important enzyme for modulating glucose metabolize in the pentose phosphate pathway (PPP). In this study, we treated T-ALL cells with different doses of niclosamide and primary T-ALL PBMCs were analyzed by RNA sequencing. T-ALL cells treated with niclosamide were analyzed with the Western blotting and TKT activity assay. Metabolism of T-ALL cells was evaluated by ATP assay and seahorse analyses. Lastly, we used a T-ALL xenograft murine model to determine effects of TKT knockdown on T-ALL tumor growth. Tumor samples were analyzed by H&E and IHC stainings. We found that niclosamide reduced T-ALL cell viability, and reduced expressions of TKT, Transketolase-Like Protein 1/2 (TKTL1/2) and transaldolase. In addition, niclosamide inhibited TKT enzyme activity, aerobic metabolism and glycolysis, finally leading to lower production of ATP. TKT knockdown inhibited tumor growth of xenograft T-ALL mice. Findings showed that niclosamide inhibits T-ALL cell growth by inhibiting TKT and energy metabolism.

Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Galectin-3 aggravates microglial activation and tau transmission in tauopathy.

Alzheimer's disease is characterized by the accumulation of amyloid-ß plaques, aggregation of hyperphosphorylated tau (pTau), and microglia activation. Galectin-3 (Gal3) is a ß-galactoside-binding protein that has been implicated in amyloid pathology. Its role in tauopathy remains enigmatic. Here, we showed that Gal3 was upregulated in the microglia of humans and mice with tauopathy. pTau triggered the release of Gal3 from human induced pluripotent stem cell-derived microglia in both its free and extracellular vesicular-associated (EV-associated) forms. Both forms of Gal3 increased the accumulation of pathogenic tau in recipient cells. Binding of Gal3 to pTau greatly enhanced tau fibrillation. Besides Gal3, pTau was sorted into EVs for transmission. Moreover, pTau markedly enhanced the number of EVs released by iMGL in a Gal3-dependent manner, suggesting a role of Gal3 in biogenesis of EVs. Single-cell RNA-Seq analysis of the hippocampus of a mouse model of tauopathy (THY-Tau22) revealed a group of pathogenic tau-evoked, Gal3-associated microglia with altered cellular machineries implicated in neurodegeneration, including enhanced immune and inflammatory responses. Genetic removal of Gal3 in THY-Tau22 mice suppressed microglia activation, reduced the level of pTau and synaptic loss in neurons, and rescued memory impairment. Collectively, Gal3 is a potential therapeutic target for tauopathy.

Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis.

To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C(4) metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.

Historical profiling of maize duplicate genes sheds light on the evolution of C4 photosynthesis in grasses.

C4 plants evolved from C3 plants through a series of complex evolutionary steps. On the basis of the evolution of key C4 enzyme genes, the evolution of C4 photosynthesis has been considered a story of gene/genome duplications and subsequent modifications of gene function. If whole-genome duplication has contributed to the evolution of C4 photosynthesis, other genes should have been duplicated together with these C4 genes. However, which genes were co-duplicated with C4 genes and whether they have also played a role in C4 evolution are largely unknown. In this study, we developed a simple method to characterize the historical profile of the paralogs of a gene by tracing back to the most recent common ancestor (MRCA) of the gene and its paralog(s) and then counting the number of paralogs at each MRCA. We clustered the genes into clusters with similar duplication profiles and inferred their functional enrichments. Applying our method to maize, a familiar C4 plant, we identified many genes that show similar duplication profiles with those of the key C4 enzyme genes and found that the functional preferences of the C4 gene clusters are not only similar to those identified by an experimental approach in a recent study but also highly consistent with the functions required for the C4 photosynthesis evolutionary model proposed by S.F. Sage. Some of these genes might have co-evolved with the key C4 enzyme genes to increase the strength of C4 photosynthesis. Moreover, our results suggested that most key C4 enzyme genes had different origins and have undergone a long evolutionary process before the emergence of C4 grasses (Andropogoneae), consistent with the conclusion proposed by previous authors.