Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

The first two whole mitochondrial genomes for the genus Dactylis species: assembly and comparative genomics analysis.

BACKGROUND: Orchardgrass (Dactylis glomerata L.), a perennial forage, has the advantages of rich leaves, high yield, and good quality and is one of the most significant forage for grassland animal husbandry and ecological management in southwest China. Mitochondrial (mt) genome is one of the major genetic systems in plants. Studying the mt genome of the genus Dactylis could provide more genetic information in addition to the nuclear genome project of the genus. RESULTS: In this study, we sequenced and assembled two mitochondrial genomes of Dactylis species of D. glomerata (597, 281 bp) and D. aschersoniana (613, 769 bp), based on a combination of PacBio and Illumina. The gene content in the mitochondrial genome of D. aschersoniana is almost identical to the mitochondrial genome of D. glomerata, which contains 22-23 protein-coding genes (PCGs), 8 ribosomal RNAs (rRNAs) and 30 transfer RNAs (tRNAs), while D. glomerata lacks the gene encoding the Ribosomal protein (rps1) and D. aschersoniana contains one pseudo gene (atp8). Twenty-three introns were found among eight of the 30 protein-coding genes, and introns of three genes (nad 1, nad2, and nad5) were trans-spliced in Dactylis aschersoniana. Further, our mitochondrial genome characteristics investigation of the genus Dactylis included codon usage, sequences repeats, RNA editing and selective pressure. The results showed that a large number of short repetitive sequences existed in the mitochondrial genome of D. aschersoniana, the size variation of two mitochondrial genomes is due largely to the presence of a large number of short repetitive sequences. We also identified 52-53 large fragments that were transferred from the chloroplast genome to the mitochondrial genome, and found that the similarity was more than 70%. ML and BI methods used in phylogenetic analysis revealed that the evolutionary status of the genus Dactylis. CONCLUSIONS: Thus, this study reveals the significant rearrangements in the mt genomes of Pooideae species. The sequenced Dactylis mt genome can provide more genetic information and improve our evolutionary understanding of the mt genomes of gramineous plants.

Complete Chloroplast Genomes of 14 Subspecies of D. glomerata: Phylogenetic and Comparative Genomic Analyses.

Orchardgrass (Dactylis glomerata L.) is a species in the Gramineae family that is highly important economically and valued for its role in ecology. However, the phylogeny and taxonomy of D. glomerata are still controversial based on current morphological and molecular evidence. The study of chloroplast (cp) genomes has developed into a powerful tool to develop molecular markers for related species and reveal the relationships between plant evolution and phylogenetics. In this study, we conducted comparative genomic analyses and phylogenetic inferences on 14 cp genomes of D. glomerata originating from the Mediterranean and Eurasia. The genome size ranged from 134,375 bp to 134,993 bp and exhibited synteny of gene organization and order. A total of 129-131 genes were identified, including 85-87 protein coding genes, 38 tRNA genes and 8 rRNA genes. The cp sequences were highly conserved, and key sequence variations were detected at the junctions of inverted repeats (IRs)/small single-copy (SSC) regions. Moreover, nine highly variable regions were identified among the subspecies based on a sequence divergence analysis. A total of 285 RNA editing sites were detected that were relevant to 52 genes, where rpoB exhibited the most abundant RNA editing sites. The phylogenetic analysis revealed that all Dactylis subspecies clustered into a monophyletic group and most branches provided a high support bootstrap. The main divergence time of D. glomerata was dated to the Miocene era, and this could have been due to changes in the climate. These findings will provide useful insights for further studies on phylogeny, the identification of subspecies and the development of hypotheses for the evolutionary history of the genus Dactylis and of the Gramineae family.

Palladium-Catalyzed Decarbonylation of Amino Acid Derivatives via C-C Bond and C-N Bond Dual Activations.

A unique decarbonylation of an amino acid derivative catalytic system has been established via palladium-catalyzed C-C bond and C-N bond dual activations. By employing 8-aminoquinoline as the directing group, this transformation has been found to facilitate the high chemoselectivity to decarbonylation of amino acid derivatives rather than intramolecular deamination or cross-dehydrogenative coupling reactions. This method provides a straightforward avenue for constructing diverse functionalized amide compounds in good to excellent yields. We proposed a possible reaction pathway that may go through the C-C bond and C-N bond dual activations on the basis of the mechanistic studies.