An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and Single-Cell RNA Sequencing

The recent advent of single-cell RNA sequencing (scRNA-seq) has enabled access to the developmental landscape of a complex organ by monitoring the differentiation trajectory of every specialized cell type at the single-cell level. A main challenge in this endeavor is dissociating plant cells from the rigid cell walls and some species are recalcitrant to such cellular isolation. Here, we describe the establishment of a simple and efficient protocol for protoplast preparation in Chirita pumila, which includes two consecutive digestion processes with different enzymatic buffers. Using this protocol, we generated viable cell suspensions suitable for an array of expression analyses, including scRNA-seq. The universal application of this protocol was further tested by successfully isolating high-quality protoplasts from multiple organs (petals, fruits, tuberous roots, and gynophores) from representative species on the key branches of the angiosperm lineage. This work provides a robust method in plant science, overcoming barriers to isolating protoplasts in diverse plant species and opens a new avenue to study cell type specification, tissue function, and organ diversification in plants

Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats

Klotho (KL) is a glycosyl hydrolase and aging-suppressor gene. Stress is a risk factor for depression and anxiety, which are highly comorbid with each other. The aim of this study is to determine whether KL is regulated by estrogen and plays an important role in sex differences in stress resilience. Our results showed that KL is regulated by estrogen in rat hippocampal neurons in vivo and in vitro and is essential for the estrogen-mediated increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1)-positive clusters on the dendrites of hippocampal neurons. The role of KL in sex differences in stress response was examined in rats using 3-week chronic unpredictable mild stress (CUMS). CUMS produced a deficit in spatial learning and memory, anhedonic-like behaviors, and anxiety-like behaviors in male but not female rats, which was accompanied by a reduction in KL protein levels in the hippocampus of male but not female rats. This demonstrated the resilience of female rats to CUMS. Interestingly, the knockdown of KL protein levels in the rat hippocampus of both sexes caused a decrease in stress resilience in both sexes, especially in female rats. These results suggest that the regulation of KL by estrogen plays an important role in estrogen-mediated synapse formation and that KL plays a critical role in the sex differences in cognitive deficit, anhedonic-like behaviors, and anxiety-like behaviors induced by chronic stress in rats, highlighting an important role of KL in sex differences in stress resilience

Table_7_Vertical transfer and functional characterization of cotton seed core microbiome.xlsx

IntroductionMicrobiome within plant tissues is pivotal for co-evolution with host plants. This microbiome can colonize the plant, with potential transmission via seeds between parents and offspring, affecting seedling growth and host plant adaptability to the environment.MethodsWe employed 16S rRNA gene amplicon analysis to investigate the vertical distribution of core microbiome in cotton seeds across ecological niches [rhizosphere, root, stem, leaf, seed and seed-P (parental seed)] of the three cotton genotypes.ResultsThe findings demonstrated a significant decrease in microbiome diversity and network complexity from roots, stems, and leaves to seeds. The microenvironment exerted a more substantial influence on the microbiome structure of cotton than the genotypes. The core endophytic microorganisms in cotton seeds comprised 29 amplicon sequence variants (ASVs) affiliated with Acidimicrobiia, Alphaproteobacteria, Bacilli, Bacteroidia, Clostridia, Gammaproteobacteria, and unclassified_Proteobacteria. These vertically transmitted taxa are widely distributed in cotton plants. Through 16S rRNA gene-based function prediction analysis of the cotton microbiome, we preliminarily understood that there are potential differences in metabolic capabilities and phenotypic traits among microbiomes in different microhabitats.DiscussionIn conclusion, this study demonstrated the crucial role of the microenvironment in influencing the cotton microbiome and offered insights into the structures and functions of the cotton seed microbiome, facilitating future crop yield enhancement through core seed microbiome regulation.</p

Image_1_Vertical transfer and functional characterization of cotton seed core microbiome.pdf

IntroductionMicrobiome within plant tissues is pivotal for co-evolution with host plants. This microbiome can colonize the plant, with potential transmission via seeds between parents and offspring, affecting seedling growth and host plant adaptability to the environment.MethodsWe employed 16S rRNA gene amplicon analysis to investigate the vertical distribution of core microbiome in cotton seeds across ecological niches [rhizosphere, root, stem, leaf, seed and seed-P (parental seed)] of the three cotton genotypes.ResultsThe findings demonstrated a significant decrease in microbiome diversity and network complexity from roots, stems, and leaves to seeds. The microenvironment exerted a more substantial influence on the microbiome structure of cotton than the genotypes. The core endophytic microorganisms in cotton seeds comprised 29 amplicon sequence variants (ASVs) affiliated with Acidimicrobiia, Alphaproteobacteria, Bacilli, Bacteroidia, Clostridia, Gammaproteobacteria, and unclassified_Proteobacteria. These vertically transmitted taxa are widely distributed in cotton plants. Through 16S rRNA gene-based function prediction analysis of the cotton microbiome, we preliminarily understood that there are potential differences in metabolic capabilities and phenotypic traits among microbiomes in different microhabitats.DiscussionIn conclusion, this study demonstrated the crucial role of the microenvironment in influencing the cotton microbiome and offered insights into the structures and functions of the cotton seed microbiome, facilitating future crop yield enhancement through core seed microbiome regulation.</p

Table_2_Vertical transfer and functional characterization of cotton seed core microbiome.xlsx

IntroductionMicrobiome within plant tissues is pivotal for co-evolution with host plants. This microbiome can colonize the plant, with potential transmission via seeds between parents and offspring, affecting seedling growth and host plant adaptability to the environment.MethodsWe employed 16S rRNA gene amplicon analysis to investigate the vertical distribution of core microbiome in cotton seeds across ecological niches [rhizosphere, root, stem, leaf, seed and seed-P (parental seed)] of the three cotton genotypes.ResultsThe findings demonstrated a significant decrease in microbiome diversity and network complexity from roots, stems, and leaves to seeds. The microenvironment exerted a more substantial influence on the microbiome structure of cotton than the genotypes. The core endophytic microorganisms in cotton seeds comprised 29 amplicon sequence variants (ASVs) affiliated with Acidimicrobiia, Alphaproteobacteria, Bacilli, Bacteroidia, Clostridia, Gammaproteobacteria, and unclassified_Proteobacteria. These vertically transmitted taxa are widely distributed in cotton plants. Through 16S rRNA gene-based function prediction analysis of the cotton microbiome, we preliminarily understood that there are potential differences in metabolic capabilities and phenotypic traits among microbiomes in different microhabitats.DiscussionIn conclusion, this study demonstrated the crucial role of the microenvironment in influencing the cotton microbiome and offered insights into the structures and functions of the cotton seed microbiome, facilitating future crop yield enhancement through core seed microbiome regulation.</p