Labeling Lysosomes and Tracking Lysosome-Dependent Apoptosis with a Cell-Permeable Activity-Based Probe

In this study, we describe a new strategy for labeling and tracking lysosomes with a cell-permeable fluorescent activity-based probe (CpFABP) that is covalently bound to select lysosomal proteins. Colocalization studies that utilized LysoTracker probes as standard lysosomal markers demonstrated that our novel probe is effective in specifically labeling lysosomes in various kinds of live cells. Furthermore, our studies revealed that this probe has the ability to label fixed cells, permeabilized cells, and NH₄Cl-treated cells, unlike LysoTracker probes, which show ineffective labeling under the same conditions. Remarkably, when applied to monitor the process of lysosome-dependent apoptosis, our probe not only displayed the expected release of lysosomal cathepsins from lysosomes into the cytosol but also revealed additional information about the location of the cathepsins during apoptosis, which is undetectable by other chemical lysosome markers. These results suggest a wide array of promising applications for our probe and provide useful guidelines for its use as a lysosome marker in lysosome-related studies

Comparative Proteomics of Contrasting Maize Genotypes Provides Insights into Salt-Stress Tolerance Mechanisms

Salt stress is a major abiotic factor limiting maize yield. To characterize the mechanism underlying maize salt tolerance, we compared the seedling root proteomes of salt-tolerant Jing724 and salt-sensitive D9H. The germination rate and growth parameter values (weight and length) were higher for Jing724 than for D9H under saline conditions. Using an iTRAQ-based method, we identified 513 differentially regulated proteins (DRPs), with 83 and 386 DRPs specific to Jing724 and D9H, respectively. In salt-stressed Jing724, the DRPs were primarily associated with the pentose phosphate pathway, glutathione metabolism, and nitrogen metabolism. Key DRPs, such as glucose-6-phosphate 1-dehydrogenase, NADPH-producing dehydrogenase, glutamate synthase, and glutamine synthetase, were identified based on pathway enrichment and protein–protein interaction analyses. Moreover, salt-responsive proteins in Jing724 seedlings were implicated in energy management, maintenance of redox homeostasis, detoxification of ammonia, regulation of osmotic homeostasis, stress defense and adaptation, biotic cross-tolerance, and regulation of gene expression. Quantitative analyses of superoxide dismutase activity, malondialdehyde content, relative electrolyte leakage, and proline content were consistent with the predicted changes based on DRP functions. Furthermore, changes in the abundance of eight representative DRPs were correlated with the corresponding mRNA levels. Our results may be useful for elucidating the molecular networks mediating salt tolerance

Identification of Genes Potentially Associated with the Fertility Instability of S-Type Cytoplasmic Male Sterility in Maize via Bulked Segregant RNA-Seq

<div><p>S-type cytoplasmic male sterility (CMS-S) is the largest group among the three major types of CMS in maize. CMS-S exhibits fertility instability as a partial fertility restoration in a specific nuclear genetic background, which impedes its commercial application in hybrid breeding programs. The fertility instability phenomenon of CMS-S is controlled by several minor quantitative trait locus (QTLs), but not the major nuclear fertility restorer (<i>Rf3</i>). However, the gene mapping of these minor QTLs and the molecular mechanism of the genetic modifications are still unclear. Using completely sterile and partially rescued plants of fertility instable line (FIL)-B, we performed bulk segregant RNA-Seq and identified six potential associated genes in minor effect QTLs contributing to fertility instability. Analyses demonstrate that these potential associated genes may be involved in biological processes, such as floral organ differentiation and development regulation, energy metabolism and carbohydrates biosynthesis, which results in a partial anther exsertion and pollen fertility restoration in the partially rescued plants. The single nucleotide polymorphisms (SNPs) identified in two potential associated genes were validated to be related to the fertility restoration phenotype by KASP marker assays. This novel knowledge contributes to the understanding of the molecular mechanism of the partial fertility restoration of CMS-S in maize and thus helps to guide the breeding programs.</p></div

Pollen staining with I₂-KI revealed the normal development of a small portion of pollen from the partially rescued FIL-B.

<p>Representative images of the stained pollen of Jing724 (<b>A</b>) and the partially rescued (<b>B</b>) and sterile (<b>C</b>) individuals of FIL-B. Pollen was collected when the anthers exserted in the partially rescued plants. Round pollen with black staining was recorded as normal. The scale bars represent 200 μm.</p

The physical position and number of genes in the five identified genomic regions on chromosome 2.

<p>The physical position and number of genes in the five identified genomic regions on chromosome 2.</p

Identification of genomic regions contributing to the fertility instability in FIL-B by BSR-Seq using the Euclidean distance (ED) algorithm.

<p><b>A.</b> The ED scores raised to the fifth power across the genome. Each dot represents each SNP identified from the RNA-Seq, and the different colors designate the different chromosomes as indicated on the X-axis. For all of the panels, the gray vertical dotted lines delineate the chromosome edges, and the width of the chromosome represents the relative numbers of SNPs identified. The pink horizontal dotted lines represent the significant threshold of the 99% percentile of the ED⁵. <b>B.</b> The ED⁵ scores of a close-up of chromosome 2. <b>C.</b> The Loess fit curve calculated from A. <b>D.</b> The Loess fit curve of a close-up of chromosome 2 with the physical position indicated on X-axis. Each peak represents a possible associated genomic region.</p

Twelve genes with differential expression from the 5 genomic regions identified on chromosome 2.

<p>Twelve genes with differential expression from the 5 genomic regions identified on chromosome 2.</p

Partially restored anther exsertion in the FIL-B population.

<p><b>A.</b> Representative image of the tassels of Jing724 and FIL-B individuals with partial anther exsertion and complete sterility at six days after tasseling. The scale bars represent 3 cm. <b>B.</b> Representative image of the anthers harvested from A. The scale bars represent 3 cm.</p

Paraffin slides show normal microspore development in the partially rescued FIL-B.

<p>Microscopic images of anther transverse sections of Jing724 (<b>A</b>) and the partially rescued (<b>B</b>) and sterile (<b>C</b>) individuals of FIL-B. The black round microspores were considered as normal. The scale bars represent 100 μm.</p

Comparison of the transcript levels of the six potential associated genes from the partially rescued (F) and sterile (S) plants of FIL-B, as detected by QPCR.

<p>Each bar represents the mean±SE of the biological replicates. The values are calculated using Actin as an internal control. The asterisks show the statistically significant difference compared to the partially rescued plants, as determined by the analysis of variance: * (P<0.05), **(P<0.01), ***(P<0.001).</p