Development In Arabidopsis Research Articles

Custom methods to identify conserved genetic modules applied to novel transcriptomic data from Amborella trichopoda.

We have devised a procedure for the inter-species comparison of transcriptomic data and used this procedure to reconstruct the expression dynamics of major genetic modules that were present at least 149 million years ago in the most recent common ancestor of living angiosperms. We began by using laser-assisted microdissection to generate novel transcriptomic data from female flower tissues of Amborella trichopoda, the likely sister to all other living angiosperms. We then employed a gene-expression clustering method, followed by a custom procedure to compare genetic modules on the basis of gene orthology between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this protocol, we succeeded in identifying nine major genetic modules that appear to have conserved their expression dynamics from an early stage in angiosperm evolution. The genes of these modules, representing over 5000 orthogroups, include around one third of those known to control female reproductive development in Arabidopsis. Our study constitutes a proof of concept for the comparison of transcriptomic data between widely diverged plant species and represents a first step in the large-scale analysis of gene expression dynamics in a macro-evolutionary context.

SPL9 mediates freezing tolerance by directly regulating the expression of CBF2 in Arabidopsis thaliana

BackgroundFreezing stress inhibits plant development and causes significant damage to plants. Plants therefore have evolved a large amount of sophisticated mechanisms to counteract freezing stress by adjusting their growth and development correspondingly. Plant ontogenetic defense against drought, high salt, and heat stresses, has been extensively studied. However, whether the freezing tolerance is associated with ontogenetic development and how the freezing signals are delivered remain unclear.ResultsIn this study, we found that the freezing tolerance was increased with plant age at the vegetative stage. The expressions of microRNA156 (miR156) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), playing roles in regulation of ontogenetic development, were induced by cold stress. Overexpression of SPL9 (rSPL9) promoted the expression of C-REPEAT BINDING FACTOR 2 (CBF2) and hereafter enhanced the freezing tolerance. Genetic analysis indicated that the effect of rSPL9 on freezing tolerance is partially restored by cbf2 mutant. Further analysis confirmed that SPL9 directly binds to the promoter of CBF2 to activate the expression of CBF2, and thereafter increased the freezing tolerance.ConclusionsTherefore, our study uncovers a new role of SPL9 in fine-tuning CBF2 expression and thus mediating freezing tolerance in plants, and implies a role of miR156-SPL pathway in balancing the vegetative development and freezing response in Arabidopsis.

Genome-Wide Analysis of BpYABs and Function Identification Involving in the Leaf and Silique Development in Transgenic Arabidopsis.

YABs play an important role in the leaf development of the paper mulberry (Broussonetia papyrifera) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYABs were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYABs were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial–abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2, BpYAB3, BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5. These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Non-TZF Transcriptional Activator AtC3H12 Negatively Affects Seed Germination and Seedling Development in Arabidopsis.

CCCH zinc finger proteins are a large protein family and are classified as either tandem CCCH zinc finger (TZF) or non-TZF proteins. The roles of TZF genes in several plants have been well determined, whereas the functions of many non-TZF genes in plants remain uncharacterized. Herein, we describe biological and molecular functions of AtC3H12, an Arabidopsis non-TZF protein containing three CCCH zinc finger motifs. AtC3H12 has orthologs in several plant species but has no paralog in Arabidopsis. AtC3H12-overexpressing transgenic plants (OXs) germinated slower than wild-type (WT) plants, whereas atc3h12 mutants germinated faster than WT plants. The fresh weight (FW) and primary root lengths of AtC3H12 OX seedlings were lighter and shorter than those of WT seedlings, respectively. In contrast, FW and primary root lengths of atc3h12 seedlings were heavier and longer than those of WT seedlings, respectively. AtC3H12 was localized in the nucleus and displayed transactivation activity in both yeast and Arabidopsis. We found that the 97–197 aa region of AtC3H12 is an important part for its transactivation activity. Detection of expression levels and analysis of Arabidopsis transgenic plants harboring a PAtC3H12::GUS construct showed that AtC3H12 expression increases as the Arabidopsis seedlings develop. Taken together, our results demonstrate that AtC3H12 negatively affects seed germination and seedling development as a nuclear transcriptional activator in Arabidopsis. To our knowledge, this is the first report to show that non-TZF proteins negatively affect plant development as nuclear transcriptional activators.

The Phloem Intercalated With Xylem-Correlated 3 Receptor-Like Kinase Constitutively Interacts With Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 and Is Involved in Vascular Development in Arabidopsis.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) play fundamental roles in cell-to-cell and plant-environment communication. LRR-RLKs can function as receptors perceiving endogenous or external ligands, or as coreceptors, which stabilize the complex, and enhance transduction of the intracellular signal. The LRR-RLK BAK1 is a coreceptor for different developmental and immunity pathways. In this article, we identified PXY-CORRELATED 3 (PXC3) as a BAK1-interacting LRR-RLK, which was previously reported to be transcribed in vascular tissues co-expressed with PHLOEM INTERCALATED WITH XYLEM (PXY), the receptor of the TDIF/CLE41 peptide. Characterization of pxc3 loss-of-function mutants revealed reduced hypocotyl stele width and vascular cells compared to wild type, indicating that PXC3 plays a role in the vascular development in Arabidopsis. Furthermore, our data suggest that PXC3 might function as a positive regulator of the CLE41/TDIF–TDR/PXY signaling pathway.

The N-terminal acetyltransferase Naa50 regulates tapetum degradation and pollen development in Arabidopsis

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9–11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

CFI 25 Subunit of Cleavage Factor I is Important for Maintaining the Diversity of 3' UTR Lengths in Arabidopsis thaliana (L.) Heynh.

Cleavage and polyadenylation at the 3' end of the pre-mRNA is essential for mRNA function, by regulating its translatability, stability and translocation to the cytoplasm. Cleavage factor I (CFI) is a multi-subunit component of the pre-mRNA 3' end processing machinery in eukaryotes. Here, we report that plant CFI 25 subunit of CFI plays an important role in maintaining the diversity of the 3' ends of mRNA. The genome of Arabidopsis thaliana (L.) Heynh. contained four genes encoding three putative CFI subunits (AtCFI 25, AtCFI 59 and AtCFI 68), orthologous to the mammalian CFI subunits. There were two CFI 25 paralogs (AtCFI 25a and AtCFI 25b) that shared homology with human CFI 25. Two null alleles of AtCFI 25a displayed smaller rosette leaves, longer stigmatic papilla, smaller anther, earlier flowering and lower fertility compared to wild-type plants. Null alleles of AtCFI 25b, as well as, plants ectopically expressing full-length cDNA of AtCFI 25a, displayed no obvious morphological defects. AtCFI 25a was shown to interact with AtCFI 25b, AtCFI 68 and itself, suggesting various forms of CFI in plants. Furthermore, we show that AtCFI 25a function was essential for maintaining proper diversity of the 3' end lengths of transcripts coding for CFI subunits, suggesting a self-regulation of the CFI machinery in plants. AtCFI 25a was also important to maintain 3' ends for other genes to different extent. Collectively, AtCFI 25a, but not AtCFI 25b, seemed to play important roles during Arabidopsis development by maintaining proper diversity of the 3' UTR lengths.

Potent inhibition of TCP transcription factors by miR319 ensures proper root growth in Arabidopsis.

Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

Loss of Cap1 Leads to the Abnormality of Arabidopsis Growth and Development

Loss of Cap1 Leads to the Abnormality of Arabidopsis Growth and Development

Dynamic roles and intricate mechanisms of ethylene in epidermal hair development in Arabidopsis and cotton.

Ethylene affects many aspects of plant growth and development, including root hairs and trichomes growth in Arabidopsis, as well as fiber development in cotton, though the underlying mechanism is unclear. In this article, we update the research progress associated with the main genes in ethylene biosynthesis and signaling pathway, and we propose a clear ethylene pathway based on genome-wide identification of homologues in cotton. Expression pattern analysis using transcriptome data revealed that some candidate genes may contribute to cotton fiber development through the ethylene pathway. Moreover, we systematically summarized the effects of ethylene on the development of epidermal hair and the underlying regulatory mechanisms in Arabidopsis. Based on the knowledge of ethylene-promoted cell differentiation, elongation, and development in different tissues or plants, we advised a possible regulatory network for cotton fiber development with ethylene as the hub. Importantly, we emphasized the roles of ethylene as an important node in regulating cotton vegetative growth, and stress resistance, and suggested utilizing multiple methods to subtly modify ethylene synthesis or signaling in a tissue or spatiotemporal-specific manner to clarify its exact effect on architecture, adaptability of the plant, and fiber development, paving the way for basic research and genetic improvement of the cotton crop.

ADH2/GSNOR1 is a key player in limiting genotoxic damage mediated by formaldehyde and UV-B in Arabidopsis.

Maintenance of genome stability is an essential requirement for all living organisms. Formaldehyde and UV-B irradiation cause DNA damage and affect genome stability, growthand development, but the interplay between these two genotoxic factors is poorly understood in plants. We show that Arabidopsis adh2/gsnor1 mutant, which lacks alcohol dehydrogenase 2/S-nitrosoglutathione reductase 1 (ADH2/GSNOR1), are hypersensitive to low fluence UV-B irradiation or UV-B irradiation-mimetic chemicals. Although the ADH2/GSNOR1 enzyme can act on different substrates, notably on S-hydroxymethylglutathione (HMG) and S-nitrosoglutathione (GSNO), our study provides several lines of evidence that the sensitivity of gsnor1 to UV-B is caused mainly by UV-B-induced formaldehyde accumulation rather than other factors such as alteration of the GSNO concentration. Our results demonstrate an interplay between formaldehyde and UV-B that exacerbates genome instability, leading to severe DNA damage and impaired growth and development in Arabidopsis, and show that ADH2/GSNOR1 is a key player in combating these effects.

AaCycTL Regulates Cuticle and Trichome Development in Arabidopsis and Artemisia annua L.

Artemisinin is an important drug for resistance against malaria. Artemisinin is derived from the glandular trichome of leaves, stems, or buds of the Chinese traditional herb Artemisia annua. Increasing the trichome density may enhance the artemisinin content of A. annua. It has been proven that cyclins are involved in the development of trichomes in tomato, Arabidopsis, and tobacco, but it is unclear whether the cyclins in A. annua influence trichome development. In this study, we showed that AaCycTL may regulate trichome development and affect the content of artemisinin. We cloned AaCycTL and found that it has the same expression files as the artemisinin biosynthesis pathway gene. We overexpressed AaCycTL in Arabidopsis, and the results indicated that AaCycTL changed the wax coverage on the surface of Arabidopsis leaves. The trichome density decreased as well. Using yeast two-hybrid and BiFC assays, we show that AaCycTL can interact with AaTAR1. Moreover, we overexpressed AaCycTL in A. annua and found that the expression of AaCycTL was increased to 82–195%. Changes in wax coverage on the surface of transgenic A. annua leaves or stems were found as well. We identified the expression of the artemisinin biosynthesis pathway genes ADS, CYP71AV1, and ALDH1 has decreased to 88–98%, 76–97%, and 82–97% in the AaCycTL-overexpressing A. annua lines, respectively. Furthermore, we found reduced the content of artemisinin. In agreement, overexpression of AaCycTL in A. annua or Arabidopsis may alter waxy loading, change the initiation of trichomes and downregulate trichome density. Altogether, AaCycTL mediates trichome development in A. annua and thus may serve to regulate trichome density and be used for artemisinin biosynthesis.

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.

B1‐type cyclins control microtubule organization during cell division in Arabidopsis

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.

A novel strategy to uncover specific GO terms/phosphorylation pathways in phosphoproteomic data in Arabidopsis thaliana

BackgroundProteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database.ResultsIn this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings.ConclusionsThis study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.

Phosphorylation of Histone H2A at Serine 95 Is Essential for Flowering Time and Development in Arabidopsis.

Phosphorylation of H2A at serine 95 (H2AS95ph) mediated by MLK4 promotes flowering and H2A.Z deposition. However, little is known about MLK1, MLK2, and MLK3 during the flowering time. Here, we systemically analyze the functions of MLK family in flowering time and development. Mutation in MLK3, but not MLK1 and MLK2, displayed late-flowering phenotype. Loss of MLK3 function enhanced the late-flowering phenotype of mlk4 mutant, but not reinforced the late-flowering phenotype of mlk1 mlk2 double mutants. MLK3 displayed the kinase activity to histone H2AS95ph in vitro. The global H2AS95ph levels were reduced in mlk3 mlk4, but not in mlk3 and mlk4 single mutant and mlk1 mlk2 double mutant, and the H2AS95ph levels in mlk1 mlk3 mlk4 and mlk2 mlk3 mlk4 were similar to those in mlk3 mlk4 double mutant. MLK3 interacted with CCA1, which binds to the promoter of GI. Correspondingly, the transcription levels and H2AS95ph levels of GI were reduced in mlk3 and mlk4 single mutant, and greatly decreased in mlk3 mlk4 double mutant, but not further attenuated in mlk1 mlk3 mlk4 and mlk2 mlk3 mlk4 triple mutant. Together, our results suggested that H2AS95ph deposition mediated by MLK3 and MLK4 is essential for flowering time in Arabidopsis.

Overexpression of the Selaginella lepidophylla bHLH transcription factor enhances water-use efficiency, growth, and development in Arabidopsis

Abiotic stresses have the greatest impact on the growth and productivity of crops, especially under current and future extreme weather events due to climate change. Thus, it is vital to explore novel strategies to improve crop plant abiotic stress tolerance to feed an ever-growing world population. Selaginella lepidophylla is a desiccation-tolerant spike moss with specialized adaptations that allow it to tolerate water loss down to 4% relative water content. A candidate basic helix-loop-helix (bHLH) transcription factor was highly expressed at 4% relative water content in S. lepidophylla (SlbHLH). This SlbHLH gene was codon-optimized (SlbHLHopt) and overexpressed in Arabidopsis for functional characterization. Overexpression of the SlbHLHopt gene not only significantly increased plant growth, development, and integrated water-use efficiency, but also significantly increased seed germination and green cotyledon emergence rates under water-deficit stress and salt stress conditions. Under a 150 mM NaCl salt stress condition, SlbHLHopt-overexpressing lines increased primary root length, the number of lateral roots, and fresh and dry biomass at the seedling stage compared to control lines. Interestingly, SlbHLHopt-overexpressing lines also have significantly higher flavonoid content. Altogether, these results suggest that SlbHLH functions as an important regulator of plant growth, development, abiotic stress tolerance, and water-use efficiency.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Deciphering the synergistic and redundant roles of CG and non-CG DNA methylation in plant development and transposable element silencing.

Summary DNA methylation plays key roles in transposable element (TE) silencing and gene expression regulation. DNA methylation occurs at CG, CHG and CHH sequence contexts in plants. However, the synergistic and redundant roles of CG and non‐CG methylation are poorly understood.By introducing CRISPR/Cas9‐induced met1 mutation into the ddcc (drm1 drm2 cmt2 cmt3) mutant, we attempted to knock out all five DNA methyltransferases in Arabidopsis and then investigate the synergistic and redundant roles of CG and non‐CG DNA methylation.We found that the homozygous ddcc met1 quintuple mutants are embryonically lethal, although met1 and ddcc mutants only display some developmental abnormalities. Unexpectedly, the ddcc met1 quintuple mutations only reduce transmission through the male gametophytes. The ddcc met1+/− mutants show apparent size divergence, which is not associated with difference in DNA methylation patterns, but associated with the difference in the levels of DNA damage. Finally, we show that a group of TEs are specifically activated in the ddcc met1+/− mutants.This work reveals that CG and non‐CG DNA methylation synergistically and redundantly regulate plant reproductive development, vegetative development and TE silencing in Arabidopsis. Our findings provide insights into the roles of DNA methylation in plant development.