Plant Reproduction最新文献

Female gametophyte (FG) development is essential for plant reproduction since it is necessary for pollen tube guidance, fertilization, and maternal control of seed development. FG development entails dynamic cellular and developmental changes including multiple cell divisions and cell differentiations with biologically distinct roles in fertilization, a critical process for grain yield in cereal crops. Rice (Oryza sativa) is a model monocotyledonous plant with invaluable relevance for food security as it is one of the most important staple food crops. Therefore, to study rice FG development, a robust and precise staging method is required to explore the molecular dynamics of this important organ. Here, we provide a pistil length-based staging framework for defining developmental stages in rice ovules. We systematically analyzed FG developmental progress using several reproductive morphological parameters. Six unique developmental stages that are morphologically distinguishable and encompass the entirety of FG development were identified. Using transcriptional expression of developmental marker genes that are temporally regulated during FG development we validated this staging method. Our method provides a systematic and accurate staging framework that could be used to explore temporally and developmentally dynamic molecular events during FG development, including transcriptional, epigenetic, and metabolic regulation.

In plant cells, lipids serve various roles facilitating membrane bilayer formation, energy storage and signaling molecules. Acyl lipids are the most common in distinct plant cell compartments. Lipids regulated by key genes encoding fatty acid desaturases, diacylglycerol acyltransferase, 3-ketoacyl-CoA synthase and acyl-CoA-binding proteins (ACBPs) are deemed crucial during floral development. ACBPs, along with long-chain acyl-CoA synthase, acetyl-CoA carboxylase, fatty acid synthase, acyl-acyl carrier protein desaturases, acyl-ACP thioesterases and the ATP-binding cassette transporter subfamily A, contribute to fatty acid (FA) production, lipid transport and seed oil accumulation, making them bioengineering targets. To investigate lipid function, it is important to use appropriate analytical strategies because different lipid classes contain distinct FA patterns. These well-developed techniques include advanced lipidomic studies using multi-dimensional liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry imaging, lipid-binding assays and x-ray crystallography. As these techniques continue to evolve, further updates on lipid function are expected to rapidly materialize.

The male gametophyte in flowering plants, pollen, both performs the critical role of fertilization and represents a unique and accessible system for interrogating plant cell mechanics. A key component in this robust mechanical system is MscS-Like 8 (MSL8), a mechanosensitive ion channel. We previously proposed that MSL8 serves as an "osmotic safety valve", regulating pressure in the germinating pollen tube by releasing anions in response to plasma membrane tension. However, we subsequently identified defects in the cell walls of msl8 mutant pollen that suggested a role independent of osmoregulation. Here, we show that pollen tubes lacking MSL8 channel function by genetic knockout or channel-blocking point mutation lose major growth pauses, have altered pectin esterification patterns, and are sensitive to pectin crosslinking. Together, these data suggest a mechanism whereby anion efflux through mechanosensitive channels regulates cell wall composition and growth dynamics.

Key message: Loss of SPL10 and SPL11 increases penetrance of abnormal phenotypes in dcl1 embryos. The first division of the Arabidopsis zygote is asymmetric, resulting in an apical cell lineage that generates most of the embryo proper, and a basal cell lineage that produces the root meristem and the extraembryonic suspensor. Loss of function mutations in the microRNA processing enzyme genes DICER-LIKE 1 (DCL1) and SERRATE (SE) show cell division defects in the embryo proper, hypophyseal cell, and suspensor. Previous transcriptome analyses showed that the microRNA156-targeted transcription factor genes SQUAMOSA PROMOTER BINDING PROTEIN-LIKE2 (SPL2), SPL3 and SPL11 were upregulated in both globular stage dcl1 and se embryos, while SPL10 was upregulated in dcl1. It was previously proposed that upregulation of SPL10 and SPL11 could explain some abnormal phenotypes in dcl1 embryos. In this work, we used T-DNA and CRISPR-Cas9-induced loss of function alleles to further explore the function of SPL2, SPL3, SPL10 and SPL11 in early embryogenesis and their contribution to the dcl1 phenotype. On their own, spl2, spl3, spl10, and spl11 single mutants and an spl10 spl11 double mutant showed no abnormal cell divisions in early embryogenesis. In the dcl1/+ background, loss of function of SPL2 or SPL3 did not change the proportion of cell division defects in hypophyseal cells or suspensors observed in dcl1/+. Loss of spl10 or spl11 in dcl1/+ resulted in a slight decrease or increase (respectively) in the penetrance of abnormal suspensor divisions in heart stage embryos, while the spl10 spl11 double mutant caused a small increase in the penetrance of abnormal hypophyseal divisions in dcl1 embryos. The differences between our results and previous studies are likely due to genetic redundancy of miR156-targeted SPL genes, variable environmental conditions or the effect of genetic background on the penetrance of the dcl1 phenotype. In the future, analysis of higher order mutations in SPL and MIR156 genes will help to better understand the role of these important developmental regulators in early embryo development.

Key message: Cultured Arabidopsis flowers inmedium produce diploid microspores due to occurrence of defective meiotic cytokinesis, which can be partially rescued by anexogenous application of sucrose. Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the set up of a healthy ex vivo culture system for inflorescences ensuring that the captured chromosomes dynamics approaches the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of the half-strength Murashige and Skoog basal salt, MES, Myo-inositol, sucrose and agar produce diploid microspores due to occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within the CCM flowers. Immunolocalization of α-tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, the CCM flowers showed a gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, an increased supply of sucrose or its metabolite fructose in culture medium promoted the expression of AMS and partially rescued haploid microspore formation in the CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants.

Key message: Generative cell internalisation establishes distinctive 'cell-within-a-cell' pollen organization in angiosperms. Comparative and transgenic expression analyses provide valuable insights into the cytological and genetic regulation underlying generative cell internalisation in Arabidopsis. For double fertilisation in flowering plants, a three-celled male gametophyte (pollen grain) delivers two non-motile sperm cells to the female gametes through a rapidly elongating pollen tube. This process is facilitated by a distinctive'cell-within-a-cell' pollen organisation, where the smaller generative cell becomes internalised into the larger vegetative cytoplasm through precisely regulated morphogenetic process and callose dynamics. However, the genetic mechanisms underlying generative cell internalisation remain unclear. Therefore, this study aims to comparatively analyse Arabidopsis wild-type with two different mutants that exhibit immobilised generative cells from the loss-of-function of DUF707 STICKY GENERATIVE CELL (SGC) and bHLH transcription factors BONOBO (BNB1) and BNB2, respectively. The results show that wild-type generative cells internalise through four successive steps. In contrast, bnb1;bnb2 mutants fail to progress to step 3 due to incomplete dissolution of the hemispherical callose and sgc-1 mutant proceeds to step 3 but arrests before step 4 with the intine-side callose dissolution disturbed. Moreover, transgenic analyses employing SGC promoter reporters and various BNB expression constructs revealed that SGC expression is positively regulated by BNB transcription factors. These findings enhance the understanding of the cytological and genetic regulation involving BNB transcription factors and downstream components, including SGC, during generative cell internalisation.

Key message: TubeTracker provides a method to partially automate analysis of pollen tube growth using live imaging. Pollen function is critical for successful plant reproduction and crop productivity and it is important to develop accessible methods to quantitatively analyze pollen performance to enhance reproductive resilience. Here we introduce TubeTracker as a method to quantify key parameters of pollen performance such as, time to pollen grain germination, pollen tube tip velocity and maintenance of pollen tube integrity. TubeTracker integrates manual and automatic image processing routines and the graphical interface allows the user to interact with the software to make manual corrections of automated steps. TubeTracker does not depend on training data sets required to implement machine learning approaches and thus can be immediately implemented using readily available imaging systems. Furthermore, TubeTracker is an excellent tool to produce the pollen performance data sets necessary to take advantage of emerging AI-based methods to fully automate analysis. We tested TubeTracker and found it to be accurate in measuring pollen tube germination and pollen tube tip elongation across multiple cultivars of tomato.