Background: Total internal reflection fluorescence microscopy (TIRFM) is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM) was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results: Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein) and clathrin light chain (a vesicle coat protein) support our theoretical analysis. Conclusions: These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

Background: In sorghum (Sorghum bicolor), one paramount breeding objective is to increase grain quality. The nutritional quality and end use value of sorghum grains are primarily influenced by the proportions of tannins, starch and proteins, but the genetic basis of these grain quality traits remains largely unknown. This study aimed to dissect the natural variation of sorghum grain quality traits and identify the underpinning genetic loci by genome-wide association study.Results: Levels of starch, tannins and 17 amino acids were quantified in 196 diverse sorghum inbred lines, and 44 traits based on known metabolic pathways and biochemical interactions amongst the 17 amino acids calculated. A Genome-wide association study (GWAS) with 3,512,517 SNPs from re-sequencing data identified 14, 15 and 711 significant SNPs which represented 14, 14, 492 genetic loci associated with levels of tannins, starch and amino acids in sorghum grains, respectively. Amongst these significant SNPs, two SNPs were associated with tannin content on chromosome 4 and colocalized with three previously identified loci forTannin1, and orthologs ofZm1andTT16genes. One SNP associated with starch content colocalized with sucrose phosphate synthase gene. Furthermore, homologues ofopaque1andopaque2genes associated with amino acid content were identified. Using the KEGG pathway database, six and three candidate genes of tannins and starch were mapped into 12 and 3 metabolism pathways, respectively. Thirty-four candidate genes were mapped into 16 biosynthetic and catabolic pathways of amino acids. We finally reconstructed the biosynthetic pathways for aspartate and branched-chain amino acids based on 15 candidate genes identified in this study.Conclusion: Promising candidate genes associated with grain quality traits have been identified in the present study. Some of them colocalized with previously identified genetic regions, but novel candidate genes involved in various metabolic pathways which influence grain quality traits have been dissected. Our study acts as an entry point for further validation studies to elucidate the complex mechanisms controlling grain quality traits such as tannins, starch and amino acids in sorghum.

Cellulose is an important component of cell wall, yet its location and function in pollen tubes remain speculative. In this paper, we studied the role of cellulose synthesis in pollen tube elongation in Pinus bungeana Zucc. by using the specific inhibitor, 2, 6-dichlorobenzonitrile (DCB). In the presence of DCB, the growth rate and morphology of pollen tubes were distinctly changed. The organization of cytoskeleton and vesicle trafficking were also disturbed. Ultrastructure of pollen tubes treated with DCB was characterized by the loose tube wall and damaged organelles. DCB treatment induced distinct changes in tube wall components. Fluorescence labeling results showed that callose, and acidic pectin accumulated in the tip regions, whereas there was less cellulose when treated with DCB. These results were confirmed by FTIR microspectroscopic analysis. In summary, our findings showed that inhibition of cellulose synthesis by DCB affected the organization of cytoskeleton and vesicle trafficking in pollen tubes, and induced changes in the tube wall chemical composition in a dose-dependent manner. These results confirm that cellulose is involved in the establishment of growth direction of pollen tubes, and plays important role in the cell wall construction during pollen tube development despite its lower quantity.