Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3 (refs. 6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

A mitochondrial ADXR-ADX-P450 electron transport chain is essential for maternal gametophytic control of embryogenesis in Arabidopsis.

Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

Integration of Light and Brassinosteroid Signaling during Seedling Establishment.

Light and brassinosteroid (BR) are external stimuli and internal cue respectively, that both play critical roles in a wide range of developmental and physiological process. Seedlings grown in the light exhibit photomorphogenesis, while BR promotes seedling etiolation. Light and BR oppositely control the development switch from shotomorphogenesis in the dark to photomorphogenesis in the light. Recent progress report that substantial components have been identified as hubs to integrate light and BR signals. Photomorphogenic repressors including COP1, PIFs, and AGB1 have been reported to elevate BR response, while photomorphogenesis-promoting factors such as HY5, BZS1, and NF-YCs have been proven to repress BR signal. In addition, BR components also modulate light signal. Here, we review the current research on signaling network associated with light and brassinosteroids, with a focus on the integration of light and BR signals enabling plants to thrive in the changeable environment.

High-throughput single-cell transcriptomics reveals the female germline differentiation trajectory in Arabidopsis thaliana.

Female germline cells in flowering plants differentiate from somatic cells to produce specialized reproductive organs, called ovules, embedded deep inside the flowers. We investigated the molecular basis of this distinctive developmental program by performing single-cell RNA sequencing (scRNA-seq) of 16,872 single cells of Arabidopsis thaliana ovule primordia at three developmental time points during female germline differentiation. This allowed us to identify the characteristic expression patterns of the main cell types, including the female germline and its surrounding nucellus. We then reconstructed the continuous trajectory of female germline differentiation and observed dynamic waves of gene expression along the developmental trajectory. A focused analysis revealed transcriptional cascades and identified key transcriptional factors that showed distinct expression patterns along the germline differentiation trajectory. Our study provides a valuable reference dataset of the transcriptional process during female germline differentiation at single-cell resolution, shedding light on the mechanisms underlying germline cell fate determination.

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis.

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo.

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

Determinism and variability of the morphogenesis pathways.

Along with a strict determinism of early embryogenesis in most living organisms, some of them exhibit variability of cell fates and developmental pathways. Here we discuss the phenomena of determinism and variability of developmental pathways, defining its dependence upon cell potency, cell sensitivity to the external signals and cell signaling. We propose a set of conjectures on the phenomenon of variability of developmental pathways, and denote a difference between a normal (local) variability, leading to an invariant final structure (e.g., embryo shape), and fundamental one, which is a switching between different developmental pathways, leading to different possible structures. For illustrating our conjectures, we analyzed early developmental stages of plant embryos with different levels of variability of morphogenesis pathways, and provide a set of computational experiments by Morphogenesis Software.

Gene expression variation in Arabidopsis embryos at single-nucleus resolution.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated 'The people behind the papers' interview.

Arabidopsis vascular complexity and connectivity controls PIN-FORMED1 dynamics and lateral vein patterning during embryogenesis.

Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Here, we show that the expression and localization of the auxin efflux carrier PIN-FORMED1 (PIN1) is altered in vcc developing cotyledons and that overexpression of PIN1-GFP partially rescues vascular defects of vcc in a dosage-dependent manner. Genetic analyses suggest that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons.

Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block in Arabidopsis.

BACKGROUND: Hybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. In Arabidopsis, loss of function of NRPD1, encoding the largest subunit of the plant-specific RNA polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. Recent work suggests that suppression of the triploid block by mutants in RdDM components differs, depending on whether the diploid pollen is derived from tetraploid plants or from the omission in second division 1 (osd1) mutant. This study aims to understand this difference. RESULTS: In this study, we find that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While first homozygous generation mutants in RdDM components NRPD1, RDR2, NRPE1, and DRM2 have weak or no ability to rescue the triploid block, they are able to suppress the triploid block with successive generations of inbreeding. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. CONCLUSIONS: Our data reveal that loss of RdDM function differs in its effect in early and late generations, which has important implications when interpreting the effect of RdDM mutants.

ATHB2 is a negative regulator of germination in Arabidopsis thaliana seeds.

The germination timing of seeds is of the utmost adaptive importance for plant populations. Light is one of the best characterized factors promoting seed germination in several species. The germination is also finely regulated by changes in hormones levels, mainly those of gibberellin (GA) and abscisic acid (ABA). Here, we performed physiological, pharmacological, and molecular analyses to uncover the role of ATHB2, an HD-ZIP II transcription factor, in germination of Arabidopsis seeds. Our study demonstrated that ATHB2 is a negative regulator and sustains the expression of transcription factors to block germination promoted by light. Besides, we found that ATHB2 increases ABA sensitivity. Moreover, ABA and auxin content in athb2-2 mutant is higher than wild-type in dry seeds, but the differences disappeared during the imbibition in darkness and the first hours of exposition to light, respectively. Some ABA and light transcription factors are up-regulated by ATHB2, such as ABI5, ABI3, XERICO, SOMNUS and PIL5/PIF1. In opposition, PIN7, an auxin transport, is down-regulated. The role of ATHB2 as a repressor of germination induced by light affecting the gemination timing, could have differential effects on the establishment of seedlings altering the competitiveness between crops and weeds in the field.

An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication.

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

RUS6, a DUF647-containing protein, is essential for early embryonic development in Arabidopsis thaliana.

BACKGROUND: The Arabidopsis RUS (ROOT UV-B SENSITIVE) gene family contains six members, each of which encodes a protein containing a DUF647 (domain of unknown function 647) that is commonly found in eukaryotes. Previous studies have demonstrated that RUS1 and RUS2 play critical roles in early seedling development. All six RUS genes are expressed throughout the plant, but little is known about the functional roles of RUS3, RUS4, RUS5 and RUS6. RESULTS: We used a reverse-genetic approach to identify knockout mutants for RUS3, RUS4, RUS5 and RUS6. Each mutant was confirmed by direct DNA sequencing and genetic segregation analysis. No visible phenotypic differences were observed in rus3, rus4, or rus5 knockout mutants under standard growth conditions, but rus6 knockout mutants displayed a strong embryo-lethal phenotype. Two independent knockout lines for RUS6 were characterized. The rus6 mutations could only be maintained through a heterozygote, because rus6 homozygous mutants did not survive. Closer examinations of homozygous rus6 embryos from rus6/ + parent plants revealed that RUS6 is required for early embryo development. Loss of RUS6 resulted in embryo lethality, specifically at the mid-globular stage. The embryo-lethality phenotype was complemented by a RUS6::RUS6-GFP transgene, and GFP signal was detected throughout the embryo. Histological analyses with the ß-glucuronidase reporter gene driven by the RUS6 promoter showed tissue- and development-specific expression of RUS6, which was highest in floral tissues. CONCLUSION: Our data revealed that RUS6 is essential for early embryo development in Arabidopsis, and that the RUS gene family functions in multiple stages of plant development.

Mutation of an Arabidopsis Golgi membrane protein ELMO1 reduces cell adhesion.

Plant growth, morphogenesis and development involve cellular adhesion, a process dependent on the composition and structure of the extracellular matrix or cell wall. Pectin in the cell wall is thought to play an essential role in adhesion, and its modification and cleavage are suggested to be highly regulated so as to change adhesive properties. To increase our understanding of plant cell adhesion, a population of ethyl methanesulfonate-mutagenized Arabidopsis were screened for hypocotyl adhesion defects using the pectin binding dye Ruthenium Red that penetrates defective but not wild-type (WT) hypocotyl cell walls. Genomic sequencing was used to identify a mutant allele of ELMO1 which encodes a 20 kDa Golgi membrane protein that has no predicted enzymatic domains. ELMO1 colocalizes with several Golgi markers and elmo1-/- plants can be rescued by an ELMO1-GFP fusion. elmo1-/- exhibits reduced mannose content relative to WT but no other cell wall changes and can be rescued to WT phenotype by mutants in ESMERALDA1, which also suppresses other adhesion mutants. elmo1 describes a previously unidentified role for the ELMO1 protein in plant cell adhesion.

Antagonistic activities of cotranscriptional regulators within an early developmental window set FLC expression level.

Quantitative variation in expression of the Arabidopsis floral repressor FLC influences whether plants overwinter before flowering, or have a rapid cycling habit enabling multiple generations a year. Genetic analysis has identified activators and repressors of FLC expression but how they interact to set expression level is poorly understood. Here, we show that antagonistic functions of the FLC activator FRIGIDA (FRI) and the repressor FCA, at a specific stage of embryo development, determine FLC expression and flowering. FRI antagonizes an FCA-induced proximal polyadenylation to increase FLC expression and delay flowering. Sector analysis shows that FRI activity during the early heart stage of embryo development maximally delays flowering. Opposing functions of cotranscriptional regulators during an early embryonic developmental window thus set FLC expression levels and determine flowering time.

A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene.

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.

TOR coordinates nucleotide availability with ribosome biogenesis in plants.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.

Accumulation of the Auxin Precursor Indole-3-Acetamide Curtails Growth through the Repression of Ribosome-Biogenesis and Development-Related Transcriptional Networks.

The major auxin, indole-3-acetic acid (IAA), is associated with a plethora of growth and developmental processes including embryo development, expansion growth, cambial activity, and the induction of lateral root growth. Accumulation of the auxin precursor indole-3-acetamide (IAM) induces stress related processes by stimulating abscisic acid (ABA) biosynthesis. How IAM signaling is controlled is, at present, unclear. Here, we characterize the ami1rooty double mutant, that we initially generated to study the metabolic and phenotypic consequences of a simultaneous genetic blockade of the indole glucosinolate and IAM pathways in Arabidopsisthaliana. Our mass spectrometric analyses of the mutant revealed that the combination of the two mutations is not sufficient to fully prevent the conversion of IAM to IAA. The detected strong accumulation of IAM was, however, recognized to substantially impair seed development. We further show by genome-wide expression studies that the double mutant is broadly affected in its translational capacity, and that a small number of plant growth regulating transcriptional circuits are repressed by the high IAM content in the seed. In accordance with the previously described growth reduction in response to elevated IAM levels, our data support the hypothesis that IAM is a growth repressing counterpart to IAA.

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development.

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

nMAT3 is an essential maturase splicing factor required for holo-complex I biogenesis and embryo development in Arabidopsis thaliana plants.

Group-II introns are self-splicing mobile genetic elements consisting of catalytic intron-RNA and its related intron-encoded splicing maturase protein cofactor. Group-II sequences are particularly plentiful within the mitochondria of land plants, where they reside within many critical gene loci. During evolution, the plant organellar introns have degenerated, such as they lack regions that are are required for splicing, and also lost their evolutionary related maturase proteins. Instead, for their splicing the organellar introns in plants rely on different host-acting protein cofactors, which may also provide a means to link cellular signals with respiratory functions. The nuclear genome of Arabidopsis thaliana encodes four maturase-related factors. Previously, we showed that three of the maturases, nMAT1, nMAT2 and nMAT4, function in the excision of different group-II introns in Arabidopsis mitochondria. The function of nMAT3 (encoded by the At5g04050 gene locus) was found to be essential during early embryogenesis. Using a modified embryo-rescue method, we show that nMAT3-knockout plants are strongly affected in the splicing of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria, resulting in complex-I biogenesis defects and altered respiratory activities. Functional complementation of nMAT3 restored the organellar defects and embryo-arrested phenotypes associated with the nmat3 mutant line. Notably, nMAT3 and nMA4 were found to act on the same RNA targets but have no redundant functions in the splicing of nad1 transcripts. The two maturases, nMAT3 and nMAT4 are likely to cooperate together in the maturation of nad1 pre-RNAs. Our results provide important insights into the roles of maturases in mitochondria gene expression and the biogenesis of the respiratory system during early plant life.