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Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA-DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis.

Epstein Barr Virus (EBV), is associated with an increasing number of lymphoid and epithelial malignancies. Among the genes expressed by EBV during latency, LMP1 plays a key role for growth transformation and immortalization of B lymphocytes. We have previously shown that antisense oligonucleotides (ONs) directed to LMP1 mRNA, effectively suppressed LMP1 gene expression and substantially reduced proliferation of the infected cells. The use of antisense phosphodiester oligonucleotides as therapeutic agents is limited by inefficient cellular uptake and intracellular transport to the target mRNA. We tested the ability of three cationic carriers internalized by different pathways, to increase the delivery of anti-LMP1-ON to their site of action in EBV-infected B lymphocytes. We report here that liposomes, dendrimers or transferrin-polylysine-conjugated ON were internalized by the cells at an extent several fold higher than that of the naked oligomers. However, only the delivery system exploiting the transferrin receptor pathway of internalization, was able to vectorize biologically active antisense LMP1-ON. © 2006.

It is generally accepted that Epstein-Barr virus (EBV) latent genes EBNA-2, EBNA-3A,-3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A,-3B and-3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors.