Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development.

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, ß-, σ-, and µ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The µ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2-dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.

Ectopic expression of an esterase, which is a candidate for the unidentified plant cutinase, causes cuticular defects in Arabidopsis thaliana.

Cutinase is an esterase that degrades the polyester cutin, a major component of the plant cuticle. Although cutinase activity has been detected in pollen, the genes encoding this enzyme have not been identified. Here, we report the identification and characterization of Arabidopsis CDEF1 (cuticle destructing factor 1), a novel candidate gene encoding cutinase. CDEF1 encodes a member of the GDSL lipase/esterase family of proteins, although fungal and bacterial cutinases belong to the alpha/beta hydrolase superfamily which is different from the GDSL lipase/esterase family. According to the AtGenExpress microarray data, CDEF1 is predominantly expressed in pollen. The ectopic expression of CDEF1 driven by the 35S promoter caused fusion of organs, including leaves, stems and flowers, and increased surface permeability. Ultrastructural analysis revealed that the cuticle of the transgenic plants was often disrupted and became discontinuous. Subcellular analysis with green fluorescent protein (GFP)-tagged CDEF1 showed that the protein is secreted to the extracellular space in leaves. The recombinant CDEF1 protein has esterase activity. These results are consistent with cutinase being secreted from cells and directly degrading the polyester in the cuticle. CDEF1 promoter activity was detected in mature pollen and pollen tubes, suggesting that CDEF1 is involved in the penetration of the stigma by pollen tubes. Additionally, we found CDEF1 expression at the zone of lateral root emergence, which suggests that CDEF1 degrades cell wall components to facilitate the emergence of the lateral roots. Our findings suggest that CDEF1 is a candidate gene for the unidentified plant cutinase.

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana.

SUMMARY: Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana, and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants (ole1, ole2, ole3 and ole4) and generated three double mutants (ole1 ole2, ole1 ole3 and ole2 ole3). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2, which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4 degrees C abolished seed germination of single mutants (ole1, ole2 and ole3), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22 degrees C instead of 4 degrees C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.