Expansion and improvement of ChinaMu by MuT-seq and chromosome-level assembly of the Mu-starter genome.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

Decoding the gene regulatory network of endosperm differentiation in maize.

The persistent cereal endosperm constitutes the majority of the grain volume. Dissecting the gene regulatory network underlying cereal endosperm development will facilitate yield and quality improvement of cereal crops. Here, we use single-cell transcriptomics to analyze the developing maize (Zea mays) endosperm during cell differentiation. After obtaining transcriptomic data from 17,022 single cells, we identify 12 cell clusters corresponding to five endosperm cell types and revealing complex transcriptional heterogeneity. We delineate the temporal gene-expression pattern from 6 to 7 days after pollination. We profile the genomic DNA-binding sites of 161 transcription factors differentially expressed between cell clusters and constructed a gene regulatory network by combining the single-cell transcriptomic data with the direct DNA-binding profiles, identifying 181 regulons containing genes encoding transcription factors along with their high-confidence targets, Furthermore, we map the regulons to endosperm cell clusters, identify cell-cluster-specific essential regulators, and experimentally validated three predicted key regulators. This study provides a framework for understanding cereal endosperm development and function at single-cell resolution.

Accumulation of 22 kDa α-zein-mediated nonzein protein in protein body of maize endosperm.

Protein bodies (PBs), the major protein storage organelle in maize (Zea mays) endosperm, comprise zeins and numerous nonzein proteins (NZPs). Unlike zeins, how NZPs accumulate in PBs remains unclear. We characterized a maize miniature kernel mutant, mn*, that produces small kernels and is embryo-lethal. After cloning the Mn* locus, we determined that it encodes the mitochondrial 50S ribosomal protein L10 (mRPL10). MN* localized to mitochondria and PBs as an NZP; therefore, we renamed MN* Non-zein Protein 1 (NZP1). Like other mutations affecting mitochondrial proteins, mn* impaired mitochondrial function and morphology. To investigate its accumulation mechanism to PBs, we performed protein interaction assays between major zein proteins and NZP1, and found that NZP1 interacts with 22 kDa α-zein. Levels of NZP1 and 22 kDa α-zein in various opaque mutants were correlated. Furthermore, NZP1 accumulation in induced PBs depended on its interaction with 22 kDa α-zein. Comparative proteomic analysis of PBs between wild-type and opaque2 revealed additional NZPs. A new NZP with plastidial localization was also found to accumulate in induced PBs via interaction with 22 kDa α-zein. This study thus reveals a mechanism for accumulation of NZPs in PBs and suggests a potential application for the accumulation of foreign proteins in maize PBs.

ENB1 encodes a cellulose synthase 5 that directs synthesis of cell wall ingrowths in maize basal endosperm transfer cells.

Development of the endosperm is strikingly different in monocots and dicots: it often manifests as a persistent tissue in the former and transient tissue in the latter. Little is known about the controlling mechanisms responsible for these different outcomes. Here we characterized a maize (Zea mays) mutant, endosperm breakdown1 (enb1), in which the typically persistent endosperm (PE) was drastically degraded during kernel development. ENB1 encodes a cellulose synthase 5 that is predominantly expressed in the basal endosperm transfer layer (BETL) of endosperm cells. Loss of ENB1 function caused a drastic reduction in formation of flange cell wall ingrowths (ingrowths) in BETL cells. Defective ingrowths impair nutrient uptake, leading to premature utilization of endosperm starch to nourish the embryo. Similarly, developing wild-type kernels cultured in vitro with a low level of sucrose manifested early endosperm breakdown. ENB1 expression is induced by sucrose via the BETL-specific Myb-Related Protein1 transcription factor. Overexpression of ENB1 enhanced development of flange ingrowths, facilitating sucrose transport into BETL cells and increasing kernel weight. The results demonstrated that ENB1 enhances sucrose supply to the endosperm and contributes to a PE in the kernel.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis.

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

Arabidopsis retains vertebrate-type telomerase accessory proteins via a plant-specific assembly.

The recent discovery of the bona-fide telomerase RNA (TR) from plants reveals conserved and unique secondary structure elements and the opportunity for new insight into the telomerase RNP. Here we examine how two highly conserved proteins previously implicated in Arabidopsis telomere maintenance, AtPOT1a and AtNAP57 (dyskerin), engage plant telomerase. We report that AtPOT1a associates with Arabidopsis telomerase via interaction with TERT. While loss of AtPOT1a does not impact AtTR stability, the templating domain is more accessible in pot1a mutants, supporting the conclusion that AtPOT1a stimulates telomerase activity but does not facilitate telomerase RNP assembly. We also show, that despite the absence of a canonical H/ACA binding motif within AtTR, dyskerin binds AtTR with high affinity and specificity in vitro via a plant specific three-way junction (TWJ). A core element of the TWJ is the P1a stem, which unites the 5' and 3' ends of AtTR. P1a is required for dyskerin-mediated stimulation of telomerase repeat addition processivity in vitro, and for AtTR accumulation and telomerase activity in vivo. The deployment of vertebrate-like accessory proteins and unique RNA structural elements by Arabidopsis telomerase provides a new platform for exploring telomerase biogenesis and evolution.

shrunken4 is a mutant allele of ZmYSL2 that affects aleurone development and starch synthesis in maize.

Minerals are stored in the aleurone layer and embryo during maize seed development, but how they affect endosperm development and activity is unclear. Here, we cloned the gene underlying the classic maize kernel mutant shrunken4 (sh4) and found that it encodes the YELLOW STRIPE-LIKE oligopeptide metal transporter ZmYSL2. sh4 kernels had a shrunken phenotype with developmental defects in the aleurone layer and starchy endosperm cells. ZmYSL2 showed iron and zinc transporter activity in Xenopus laevis oocytes. Analysis using a specific antibody indicated that ZmYSL2 predominately accumulated in the aleurone and sub-aleurone layers in endosperm and the scutellum in embryos. Specific iron deposition was observed in the aleurone layer in wild-type kernels. In sh4, however, the outermost monolayer of endosperm cells failed to accumulate iron and lost aleurone cell characteristics, indicating that proper functioning of ZmYSL2 and iron accumulation are essential for aleurone cell development. Transcriptome analysis of sh4 endosperm revealed that loss of ZmYSL2 function affects the expression of genes involved in starch synthesis and degradation processes, which is consistent with the delayed development and premature degradation of starch grains in sh4 kernels. Therefore, ZmYSL2 is critical for aleurone cell development and starchy endosperm cell activity during maize seed development.

Maize endosperm development.

Recent breakthroughs in transcriptome analysis and gene characterization have provided valuable resources and information about the maize endosperm developmental program. The high temporal-resolution transcriptome analysis has yielded unprecedented access to information about the genetic control of seed development. Detailed spatial transcriptome analysis using laser-capture microdissection has revealed the expression patterns of specific populations of genes in the four major endosperm compartments: the basal endosperm transfer layer (BETL), aleurone layer (AL), starchy endosperm (SE), and embryo-surrounding region (ESR). Although the overall picture of the transcriptional regulatory network of endosperm development remains fragmentary, there have been some exciting advances, such as the identification of OPAQUE11 (O11) as a central hub of the maize endosperm regulatory network connecting endosperm development, nutrient metabolism, and stress responses, and the discovery that the endosperm adjacent to scutellum (EAS) serves as a dynamic interface for endosperm-embryo crosstalk. In addition, several genes that function in BETL development, AL differentiation, and the endosperm cell cycle have been identified, such as ZmSWEET4c, Thk1, and Dek15, respectively. Here, we focus on current advances in understanding the molecular factors involved in BETL, AL, SE, ESR, and EAS development, including the specific transcriptional regulatory networks that function in each compartment during endosperm development.

Maize kernel development.

Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endosperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics, maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development, functional gene cloning of kernel mutants, and genetic engineering of kernel traits.

Identification and Quantification of Small RNAs.

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

OsWUS promotes tiller bud growth by establishing weak apical dominance in rice.

Two branching strategies are exhibited in crops: enhanced apical dominance, as in maize; or weak apical dominance, as in rice. However, the underlying mechanism of weak apical dominance remains elusive. OsWUS, an ortholog of Arabidopsis WUSCHEL (WUS) in rice, is required for tiller development. In this study, we identified and functionally characterized a low-tillering mutant decreased culm number 1 (dc1) that resulted from loss-of-function of OsWUS. The dc1 tiller buds are viable but repressed by the main culm apex, leading to stronger apical dominance than that of the wild-type (WT). Auxin response is enhanced in the dc1 mutant, and knocking out the auxin action-associated gene ABERRANT SPIKELET AND PANICLE 1 (ASP1) de-repressed growth of the tiller buds in the dc1 mutant, suggesting that OsWUS and ASP1 are both involved in outgrowth of the rice tiller bud. Decapitation triggers higher contents of cytokinins in the shoot base of the dc1 mutant compared with those in the WT, and exogenous application of cytokinin is not sufficient for sustained growth of the dc1 tiller bud. Transcriptome analysis indicated that expression levels of transcription factors putatively bound by ORYZA SATIVA HOMEOBOX 1 (OSH1) are changed in response to decapitation and display a greater fold change in the dc1 mutant than that in the WT. Collectively, these findings reveal an important role of OsWUS in tiller bud growth by influencing apical dominance, and provide the basis for an improved understanding of tiller bud development in rice.

A SnRK1-ZmRFWD3-Opaque2 Signaling Axis Regulates Diurnal Nitrogen Accumulation in Maize Seeds.

Zeins are the predominant storage proteins in maize (Zea mays) seeds, while Opaque2 (O2) is a master transcription factor for zein-encoding genes. How the activity of O2 is regulated and responds to external signals is yet largely unknown. Here, we show that the E3 ubiquitin ligase ZmRFWD3 interacts with O2 and positively regulates its activity by enhancing its nuclear localization. Ubiquitination of O2 enhances its interaction with maize importin1, the α-subunit of Importin-1 in maize, thus enhancing its nuclear localization ability. We further show that ZmRFWD3 can be phosphorylated by a Suc-responsive protein kinase, ZmSnRK1, which leads to its degradation. We demonstrated that the activity of O2 responds to Suc levels through the ZmSnRK1-ZmRFWD3-O2 signaling axis. Intriguingly, we found that Suc levels, as well as ZmRFWD3 levels and the cytonuclear distribution of O2, exhibit diurnal patterns in developing endosperm, leading to the diurnal transcription of O2-regulated zein genes. Loss of function in ZmRFWD3 disrupts the diurnal patterns of O2 cytonuclear distribution and zein biosynthesis, and consequently changes the C/N ratio in mature seeds. We therefore identify a SnRK1-ZmRFWD3-O2 signaling axis that transduces source-to-sink signals and coordinates C and N assimilation in developing maize seeds.

Degradation of SERRATE via ubiquitin-independent 20S proteasome to survey RNA metabolism.

SERRATE (SE) is a key factor in RNA metabolism. Here, we report that SE binds 20S core proteasome α subunit G1 (PAG1) among other components and is accumulated in their mutants. Purified PAG1-containing 20S proteasome degrades recombinant SE via an ATP- and ubiquitin-independent manner in vitro. Nevertheless, PAG1 is a positive regulator for SE in vivo, as pag1 shows comparable molecular and/or developmental defects relative to se. Furthermore, SE is poorly assembled into macromolecular complexes, exemplified by the microprocessor in pag1 compared with Col-0. SE overexpression triggered the destruction of both transgenic and endogenous protein, leading to similar phenotypes of se and SE overexpression lines. We therefore propose that PAG1 degrades the intrinsically disordered portion of SE to secure the functionality of folded SE that is assembled and protected in macromolecular complexes. This study provides insight into how the 20S proteasome regulates RNA metabolism through controlling its key factor in eukaryotes.

Maize pentatricopeptide repeat protein DEK53 is required for mitochondrial RNA editing at multiple sites and seed development.

Pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing in plant mitochondria and plastids. In this study, we characterized maize (Zea mays) kernel mutant defective kernel 53 (dek53), which has an embryo lethal and collapsed endosperm phenotype. Dek53 encodes an E-subgroup PPR protein, which possesses a short PLS repeat region of only seven repeats. Subcellular localization analysis indicated that DEK53 is localized in the mitochondrion. Strand- and transcript-specific RNA-seq analysis showed that the dek53 mutation affected C-to-U RNA editing at more than 60 mitochondrial C targets. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the assembly of mitochondrial complex III in dek53. Transmission electron microscopic examination showed severe morphological defects of mitochondria in dek53 endosperm cells. In addition, yeast two-hybrid and luciferase complementation imaging assays indicated that DEK53 can interact with the mitochondrion-targeted non-PPR RNA editing factor ZmMORF1, suggesting that DEK53 might be a functional component of the organellar RNA editosome.

Lack of endoplasmic reticulum quality control (ERQC) promotes tonoplast (TP) targeting of KORRIGAN 1 (KOR1).

Cellular dynamics of KORRIGAN 1 (KOR1) is closely linked with cellulose biosynthesis and plant osmotic stress tolerance. Cycling of KOR1 between the plasma membrane (PM) and trans-Golgi Network (TGN) is maintained by sequence motifs and protein structures that are recognized by cellular transport and quality control mechanisms. Several mutations in KOR1, as well as in the host genetic background, promote the mistargeting of KOR1 and induce KOR1 accumulation in the tonoplast (TP). Yet, little is known about how retention and sorting of KOR1 are regulated in the PM-TGN cycle. Forward genetic screening for GFP-KOR1 mislocalizing phenotype resulted in several mutant lines with different localization patterns or signal intensity of GFP-KOR1. One of the identified mutants were disrupted at UDP-glucose:glycoprotein glucosyltransferase (UGGT) locus, which is essential for the protein quality control in the ER. Our finding suggests the mis/unfolded structure of KOR1 triggers the TP targeting.

Multiple Quality Control Mechanisms in the ER and TGN Determine Subcellular Dynamics and Salt-Stress Tolerance Function of KORRIGAN1.

Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-ß-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1's sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L48L49→A48A49 variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans.

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

Arabidopsis Serrate Coordinates Histone Methyltransferases ATXR5/6 and RNA Processing Factor RDR6 to Regulate Transposon Expression.

Serrate (SE) is a key component in RNA metabolism. Little is known about whether and how it can regulate epigenetic silencing. Here, we report histone methyltransferases ATXR5 and ATXR6 (ATXR5/6) as novel partners of SE. ATXR5/6 deposit histone 3 lysine 27 monomethylation (H3K27me1) to promote heterochromatin formation, repress transposable elements (TEs), and control genome stability in Arabidopsis. SE binds to ATXR5/6-regulated TE loci and promotes H3K27me1 accumulation in these regions. Furthermore, SE directly enhances ATXR5 enzymatic activity in vitro. Unexpectedly, se mutation suppresses the TE reactivation and DNA re-replication phenotypes in the atxr5 atxr6 mutant. The suppression of TE expression results from triggering RNA-dependent RNA polymerase 6 (RDR6)-dependent RNA silencing in the se atxr5 atxr6 mutant. We propose that SE facilitates ATXR5/6-mediated deposition of the H3K27me1 mark while inhibiting RDR6-mediated RNA silencing to protect TE transcripts. Hence, SE coordinates epigenetic silencing and RNA processing machineries to fine-tune the TE expression.

Actions of plant Argonautes: predictable or unpredictable?

Argonaute (AGO) proteins are the key effector of RNA-induced silencing complex (RISC). Land plants typically encode numerous AGO proteins, and they can be typically divided into two major functional groups based on the species of their housed small RNAs (sRNAs). One group of AGOs, guided by 24-nucleotide (nt) sRNAs, canonically function in nuclei to implement transcriptional gene silencing (TGS), whereas the other group of AGOs, guided by 21-nt sRNAs, act in the cytoplasm to fulfill posttranscriptional gene silencing (PTGS). Many new discoveries have been recently made on functions and mechanisms of AGO proteins in plants, and some of the findings change our views on the conventional classification and roles of AGO proteins. In this review, we summarize our current knowledge of AGO proteins in plants.