Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs controlling the nodulation process.

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with the N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where the atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of the cells of the mature soybean nodule have not yet been characterized. Applying single nucleus RNA-seq and Molecular CartographyTM technologies, we precisely characterized the transcriptomic signature of the soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule including those actively involved in nitrogen fixation, and those engaged in senescence. The mining of the single cell-resolution nodule transcriptome atlas and associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes controlling the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein controlling rhizobia infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of 'Zaosu' pear fruit by L-glutamate treatment.

'Zaosu' pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of 'Zaosu' pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and ß-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbß-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of 'Zaosu' pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

The roles of a novel CDKB/KRP/FB3 cell cycle core complex in rice gametes and initiation of embryogenesis.

The cell cycle controls division and proliferation of all eukaryotic cells and is tightly regulated at multiple checkpoints by complexes of core cell cycle proteins. Due to the difficulty in accessing female gametes and zygotes of flowering plants, little is known about the molecular mechanisms underlying embryogenesis initiation despite the crucial importance of this process for seed crops. In this study, we reveal three levels of factors involved in rice zygotic cell cycle control and characterize their functions and regulation. Protein-protein interaction studies, including within zygote cells, and in vitro biochemical analyses delineate a model of the zygotic cell cycle core complex for rice. In this model, CDKB1, a major regulator of plant mitosis, is a cyclin (CYCD5)-dependent kinase; its activity is coordinately inhibited by two cell cycle inhibitors, KRP4 and KRP5; and both KRPs are regulated via F-box protein 3 (FB3)-mediated proteolysis. Supporting their critical roles in controlling the rice zygotic cell cycle, mutations in KRP4, KRP5 and FB3 result in the compromised function of sperm cells and abnormal organization of female germ units, embryo and endosperm, thus significantly reducing seed-set rate. This work helps reveal regulatory mechanisms controlling the zygotic cell cycle toward seed formation in angiosperms.

Acibenzolar-S-methyl activates phenylpropanoid pathway to enhance resistance against Alternaria alternata in pear fruit.

BACKGROUND: Alternaria alternata is a causal agent of black spot rot of pear fruit after harvest. Acibenzolar-S-methyl (ASM) has been shown to be a potential elicitor of tolerance in several horticultural products. This work was performed to research the influence of ASM on black spot rot of Docteur Jules Guyot pears and vital enzyme activity and gene expression in the phenylpropanoid pathway. RESULTS: ASM remarkably decreased the lesion diameter of A. alternata-inoculated pears. ASM also increased phenylalanine ammonialyase, cinnamate 4-hydroxylase, cinnamyl alcohol dehydrogenase, peroxidase, polyphenol oxidase activities and gene expression, and enhanced 4-coumarate/coenzyme A ligase activity in pears. Moreover, ASM improved the content of phenylalanine, total phenolic compounds, caffeic acid, flavonoids, anthocyanin and lignin in pears. CONCLUSION: ASM could modulate vital enzyme activity and gene expression in the phenylpropanoid pathway to accelerate metabolite synthesis, thereby enhancing resistance against A. alternata in pears. © 2022 Society of Chemical Industry.

Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells-the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

Genome-wide redistribution of 24-nt siRNAs in rice gametes.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Step-by-step protocols for rice gamete isolation.

KEY MESSAGE: A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented. Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.

Mechanism of the long range anti-silencing function of targeted histone acetyltransferases in yeast.

Transcriptionally silent chromatin in Saccharomyces cerevisiae is associated with histone hypoacetylation and is formed through the action of the Sir histone deacetylase complex. A histone acetyltransferase (HAT) targeted near silent chromatin can overcome silencing at a distance by increasing histone acetylation in a sizable region. However, how a tethered HAT acetylates distant nucleosomes has not been resolved. We demonstrate here that targeting the histone H3-specific HAT Gcn5p promotes acetylation of not only histone H3 but also histone H4 in a broad region. We also show that long range anti-silencing and histone acetylation by targeted HATs can be blocked by nucleosome-excluding sequences. These results are consistent with the contention that a tethered HAT promotes stepwise propagation of histone acetylation along the chromatin. Because histone hypoacetylation is key to the formation and maintenance of transcriptionally silent chromatin, it is believed that acetylation promoted by a targeted HAT disrupts silent chromatin thereby overcoming silencing. However, we show that the acetylated and transcriptionally active region created by a tethered HAT retains structural hallmarks of Sir-dependent silent chromatin and remains associated with Sir proteins indicating that tethered HATs overcome silencing without completely dismantling silent chromatin.