Plant Reproduction最新文献

Key message: ClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds. Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.

Key message: The first record of gene expression during seed development within the Nymphaeales provides evidence for a variety of biological processes, including dynamic epigenetic patterning during sexual reproduction in the water lily Nymphaea thermarum. Studies of gene expression during seed development have been performed for a growing collection of species from a phylogenetically broad sampling of flowering plants (angiosperms). However, angiosperm lineages whose origins predate the divergence of monocots and eudicots have been largely overlooked. In order to provide a new resource for understanding the early evolution of seed development in flowering plants, we sequenced transcriptomes of whole ovules and seeds from three key stages of reproductive development in the waterlily Nymphaea thermarum, an experimentally tractable member of the Nymphaeales. We first explore patterns of gene expression, beginning with mature ovules and continuing through fertilization into early- and mid-stages of seed development. We find patterns of gene expression that corroborate histological/morphological observations of seed development in this species, such as expression of genes involved in starch synthesis and transcription factors that have been associated with embryo and endosperm development in other species. We also find evidence for processes that were previously not known to be occurring during seed development in this species, such as epigenetic modification. We then examine the expression of genes associated with patterning DNA and histone methylation-processes that are essential for seed development in distantly related and structurally diverse monocots and eudicots. Around 89% of transcripts putatively homologous to DNA and histone methylation modifiers are expressed during seed development in N. thermarum, including homologs of genes known to pattern imprinting-related epigenetic modifications. Our results suggest that dynamic epigenetic patterning is a deeply conserved aspect of angiosperm seed development.

It is widely known that an optimal nucleotide sequence context immediately upstream of the AUG start codon greatly improves the efficiency of translation initiation of mRNA in mammalian and plant somatic cells, which in turn increases protein levels. However, it is still unclear whether a similar regulatory mechanism is also present in highly differentiated cells. Here, we surveyed this issue in Arabidopsis thaliana sperm cells and found that the sequence context-mediated regulation of translation initiation in sperm cells is generally similar to that in somatic cells. A simple motif of four adenine nucleotides at positions - 1 to - 4 greatly improved the efficiency of translation initiation, and when the motif was present there, translation was even initiated at some non-AUG codons in sperm cells. However, unlike that in mammalian cells, a mainly effective nucleotide site to regulate the efficiency of translation initiation was not present at positions - 1 to - 4 in sperm cells. Meanwhile, different from somatic cells, sperm cells did not use eukaryotic translation initiation factor 1 to regulate the efficiency in a poor context consisting of the lowest frequency nucleotides. All these results contribute to our understanding of the cytoplasmic event of translation initiation in highly differentiated sperm cells.

Key message: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.

Although the seed remains small in size during the initial stage of seed development (the lag phase), several studies indicate that environment and assimilate supply level manipulations during the lag phase affect the final seed size. However, the manipulations were not only at the lag phase, making it difficult to understand the specific role of the lag phase in final seed size determination. It also remained unclear whether environmental cues are sensed by plants and regulate seed development or if it is simply the assimilate supply level, changed by the environment, that affects the subsequent seed development. We investigated soybean (Glycine max L. Merr.) seed phenotypes grown in a greenhouse using different source-sink manipulations (shading and removal of flowers and pods) during the lag phase. We show that assimilate supply is the key factor controlling flower and pod abortion and that the assimilate supply during the lag phase affects the subsequent potential seed growth rate during the seed filling phase. In response to low assimilate supply, plants adjust flower/pod abortion and lag phase duration to supply the minimum assimilate per pod/seed. Our results provide insight into the mechanisms whereby the lag phase is crucial for seed development and final seed size potential, essential parameters that determine yield.

Plastid ribosomal proteins (PRPs) can play essential roles in plastid ribosome functioning that affect plant function and development. However, the roles of many PRPs remain unknown, including elucidation of which PRPs are essential or display redundancy. Here, we report that the nuclear-encoded PLASTID RIBOSOMAL PROTEIN L5 (PRPL5) is essential for early embryo development in A. thaliana, as homozygous loss-of-function mutations in the PRPL5 gene impairs chloroplast development and leads to embryo failure to develop past the globular stage. We confirmed the prpl5 embryo-lethal phenotype by generating a mutant CRISPR/Cas9 line and by genetic complementation. As PRPL5 underwent transfer to the nuclear genome early in the evolution of Embryophyta, PRPL5 can be expected to have acquired a chloroplast transit peptide. We identify and validate the presence of an N-terminal chloroplast transit peptide, but unexpectedly also confirm the presence of a conserved and functional Nuclear Localization Signal on the protein C-terminal end. This study highlights the fundamental role of the plastid translation machinery during the early stages of embryo development in plants and raises the possibility of additional roles of plastid ribosomal proteins in the nucleus.

Key message: The pollen tube is being subjected to control by a complex network of communication that regulates its shape and the misfunction of a single component causes specific deformations. In flowering plants, the pollen tube is a tubular extension of the pollen grain required for successful sexual reproduction. Indeed, maintaining the unique shape of the pollen tube is essential for the pollen tube to approach the embryo sac. Many processes and molecules (such as GTPase activity, phosphoinositides, Ca²⁺ gradient, distribution of reactive oxygen species and nitric oxide, nonuniform pH values, organization of the cytoskeleton, balance between exocytosis and endocytosis, and cell wall structure) play key and coordinated roles in maintaining the cylindrical shape of pollen tubes. In addition, the above factors must also interact with each other so that the cell shape is maintained while the pollen tube follows chemical signals in the pistil that guide it to the embryo sac. Any intrinsic changes (such as erroneous signals) or extrinsic changes (such as environmental stresses) can affect the above factors and thus fertilization by altering the tube morphology. In this review, the processes and molecules that enable the development and maintenance of the unique shape of pollen tubes in angiosperms are presented emphasizing their interaction with specific tube shape. Thus, the purpose of the review is to investigate whether specific deformations in pollen tubes can help us to better understand the mechanism underlying pollen tube shape.

Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2, and 5) in the cytoplasm and the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.

Complex epigenetic changes occur during plant reproduction. These regulations ensure the proper transmission of epigenetic information as well as allowing for zygotic totipotency. In Arabidopsis, the main DNA methyltransferase is called MET1 and is responsible for methylating cytosine in the CG context. The Arabidopsis genome encodes for three additional reproduction-specific homologs of MET1, namely MET2a, MET2b and MET3. In this paper, we show that the DNA methyltransferase MET3 is expressed in the seed endosperm and its expression is later restricted to the chalazal endosperm. MET3 is biallelically expressed in the endosperm but displays a paternal expression bias. We found that MET3 expression is regulated by the Polycomb complex proteins FIE and MSI1. Seed development is not impaired in met3 mutant, and we could not observe significant transcriptional changes in met3 mutant. MET3 might regulates gene expression in a Polycomb mutant background suggesting a further complexification of the interplay between H3K27me3 and DNA methylation in the seed endosperm. KEY MESSAGE: The DNA METHYLTRANSFERASE MET3 is controlled by Polycomb group complex during endosperm development.