A Method for Characterizing Embryogenesis in <em>Arabidopsis</em>

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Capital Normal University

Shanxi Normal University

To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive L: -arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of L: -arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.

Refolding of a staphylokinase variant y1-Sak by reverse dilution.

Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis.

SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Paf1 complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Paf1 complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degradation, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.

The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Paf1 Complex in Arabidopsis.

Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants.

Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.

Suspensor development is essential for early embryogenesis. The filamentous suspensor plays vital roles in supporting the embryo proper and in exchanging nutrients and information between the embryo proper and embryo sac. In addition, at the globular stage, the uppermost suspensor cell differentiates into the hypophysis, which generates the progenitors of the quiescent center and columella stem cells. In naa10 and naa15 mutant plants, suspensor cell identity was found to be abnormal and embryo development was disturbed, leading to embryonic lethality. Therefore, the NatA complex is required for proper suspensor development in Arabidopsis.

Jinlin Feng

College of Life Sciences,
Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement,
College of Life Sciences, Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement

Capital Normal University

A Method for Characterizing Embryogenesis in Arabidopsis