Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Molecular evolution of Bowman-Birk type proteinase inhibitors in flowering plants.

The Bowman-Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.

Bauhinia forficata link shoot regeneration: histological analysis of organogenesis pathway

Plant regeneration was achieved from cells of callus induced from hypocotyl segments of Bauhinia forficata on half strength Murashige and Skoog culture medium supplemented with several concentrations of BAP. Within 40 days of culture shoot buds formation was observed on callus surface. Calli were then transferred to a same composition culture medium without plant growth regulator in order to induce shoot elongation. Histological studies indicated that in vitro plant regeneration in B. forficata occurred through indirect organogenesis. Meristemoids consisting of small cells with dense cytoplasm and prominent nuclei were randomly distributed throughout the callus surface indicating early stages of shoot bud differentiation. Shoots developed de novo from superficial layers of cells and the pattern of shoot origin and development were very similar to those previously described for other leguminous species.

A regeneração de plantas de Bauhinia forficata a partir de calos induzidos em segmentos de hipocótilo foi obtida em meio de cultura contendo metade da formulação de sais de Murashige & Skoog, suplementado com várias concentrações de BAP. Após 40 dias de cultura, ocorreu a formação de gemas adventícias na superfície do calo. Os calos que apresentavam tais formações, foram então transferidos para o mesmo meio de cultura mas sem a adição de fitorreguladores, visando o alongamento das gemas. Estudos histológicos indicaram que a regeneração in vitro de plantas de B. forficata ocorre pela via organogenética. Meristemóides caracterizados por células pequenas, de citoplasma denso e núcleo e nucléolo proeminentes se encontravam distribuídos pela superfície do calo, caracterizando os estágios iniciais da diferenciação de gemas caulinares. Partes aéreas se desenvolveram de novo a partir de camadas superficiais de células e seu padrão de origem e desenvolvimento foi muito similar aos previamente descritos na literatura para outras espécies de leguminosas.