Abstract The diversity of diacylglycerol acyltransferases (DGATs) indicates alternative roles for these enzymes in plant metabolism besides triacylglycerol (TAG) biosynthesis. In this work, we functionally characterized castor bean (Ricinus communis L.) DGATs assessing their subcellular localization, expression in seeds, capacity to restore triacylglycerol (TAG) biosynthesis in mutant yeast and evaluating whether they provide tolerance over free fatty acids (FFA) in sensitive yeast. RcDGAT3 displayed a distinct subcellular localization, located in vesicles outside the endoplasmic reticulum (ER) in most leaf epidermal cells. This enzyme was unable to restore TAG biosynthesis in mutant yeast; however, it was able to outperform other DGATs providing higher tolerance over FFA. RcDAcTA subcellular localization was associated with the ER membranes, resembling RcDGAT1 and RcDGAT2, but it failed to rescue the long-chain TAG biosynthesis in mutant yeast, even with fatty acid supplementation. Besides TAG biosynthesis, our results suggest that RcDGAT3 might have alternative functions and roles in lipid metabolism.

Abstract Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

As microRNAs (miRNAs) are important regulators of many biological processes, a series of small RNAomes from plants have been produced in the last decade. However, miRNA data from several groups of plants are still lacking, including some economically important crops. Here microRNAs from Coffea canephora leaves were profiled and 58 unique sequences belonging to 33 families were found, including two novel microRNAs that have never been described before in plants. Some of the microRNA sequences were also identified in Coffea arabica that, together with C. canephora, correspond to the two major sources of coffee production in the world. The targets of almost all miRNAs were also predicted on coffee expressed sequences. This is the first report of novel miRNAs in the genus Coffea, and also the first in the plant order Gentianales. The data obtained establishes the basis for the understanding of the complex miRNA-target network on those two important crops.

Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.

Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five "genetic clusters" (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three "genetic clusters". These results are in agreement with the pattern of geographic distribution of the studied populations.

The occurrence of quasi-repetitive glycine-rich peptides has been reported in different organisms. Glycine-rich regions are proposed to be involved in protein-protein interactions in some mammalian protein families. In plants, a set of glycine-rich proteins (GRPs) was characterized several years ago, and since then a wealth of new GRPs have been identified. GRPs may have very diverse sub-cellular localization and functions. The only common feature among all different GRPs is the presence of glycine-rich repeat domains. The expression of genes encoding GRPs is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. In addition to the highly modulated expression, several GRPs also show tissue-specific localization. GRPs specifically expressed in xylem, phloem, epidermis, anther tapetum and roots have been described. In this paper, the structural and functional features of these proteins in Eucalyptus are summarized. Since this is the first description of GRPs in this species, particular emphasis has been given to the expression pattern of these genes by analyzing their abundance and prevalence in the different cDNA-libraries of the Eucalyptus Genome Sequencing Project Consortium (ForEST). The comparison of GRPs from Eucalyptus and other species is also discussed.

MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants ranging from root to flower and fruit development. Members of the MADS-box gene family share a highly conserved sequence of approximately 180 nucleotides that encodes a DNA-binding domain. We used bioinformatics tools to investigate the information generated by the Eucalyptus Expressed Sequence Tag (FORESTs) genome project in order to identify and annotate MADS-box genes. The comparative phylogenetic analysis of the Eucalyptus MADS-box genes with Arabidopsis homologues allowed us to group them into one of the well-known subfamilies. Trends in gene expression of these putative Eucalyptus MADS-box genes were investigated by hierarchical clustering analysis. Among 24 MADS-box genes identified by our analysis, 12 are expressed in vegetative organs. Out of these, five are expressed predominately in wood. Understanding of the molecular mechanisms performed by MADS-box proteins underlying Eucalyptus growth, development and stress reactions would provide important insights into tree development and could reveal means by which tree characteristics could be modified for the improvement of industrial properties.

The expression of AtchitIV gene was analysed in Arabidopsis plants submitted to abiotic stresses. Transcript accumulation was detected in leaves in response to UV light exposure, exogenous salicylic acid administration and wounding. Transgenic Arabidopsis plants carrying AtchitIV promoter::gus fusion also showed differential expression of the reporter gene in response to these treatments. The AtchitIV expression was also analysed during Arabidopsis embryo development. GUS assay demonstrated AtchitIV promoter activation in zygotic embryos from torpedo stage up to full maturation. Promoter deletion analysis indicated that all the 5' cis-acting elements responsible for the specific tissue expression are located in a region of 1083 bp, adjacent to the start of transcription. A negative regulatory region located between portions -1083 and -600 was also observed.

Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB) como pertencentes à sub-sub-classe E.C.3.4.22, foram usadas na busca de clusters no banco de dados do SUCEST (SUgarCane EST project), utilizando-s o programa T-BLAST-n. Homologia de seqüências foram encontradas com 76 clusters que correspondem a prováveis proteinases cisteínicas. O alinhamento destas seqüências com a de outras proteases cisteínicas, de diversas origens, forneceu informação quanto à classificação e possível função das proteinases de cana-de-açúcar. Além disso, o padrão de expressão de cada gene foi postulado a partir da correlação direta com as bibliotecas de cDNA do SUCEST dos quais os clusters foram derivados. Uma vez que nenhum gene de protease cisteínica foi anteriormente evidenciado em cana-de-açúcar, este estudo representa uma etapa inicial para o estudo de novos aspectos bioquímicos, fisiológicos e biotecnológicos destas enzimas.

Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST) data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.

Desde o isolamento da primeira proteína rica em glicina (GRP) em plantas, um grande número de novas GRPs vem sendo identificado. Seu padrão de expressão altamente específico, embora variado, em conjunto com as diferentes localizações sub-celulares de alguns dos tipos de GRPs, claramente indica que estas proteínas encontram-se envolvidas em diversos processos fisiológicos independentes. Embora ainda sem uma clara definição do papel de GRPs na célula vegetal, estudos realizados com estas proteínas têm resultado em novos e interessantes esclarecimentos da biologia celular e molecular de plantas. Promotores com regulação complexa, assim como distintos mecanismos de regulação da expressão gênica tem sido demonstrados. Novas vias de endereçamento de proteínas tais como a exportação de GRPs para diferentes tipos celulares, tem sido observados. Estes dados mostram que as GRPs podem constituir marcadores e/ou modelos interessantes para a compreensão de distintos aspectos da biologia vegetal. Neste trabalho, características estruturais e funcionais deste tipo de proteínas em cana-de-açúcar (Saccharum officinarum L.) foram analisadas. Uma vez que esta é a primeira descrição deste tipo de proteínas em cana-de-açúcar, especial atenção foi dada para o padrão de expressão destes genes, analisando-se a abundância e prevalência de cada um dos genes nas diferentes bibliotecas de cDNA do projeto Sequenciamento de ESTs de cana-de-açúcar (SUCEST). A comparação das GRPs de cana-de-açúcar com as GRPs descritas em outras espécies também será discutida.

Since the isolation of the first glycine-rich proteins (GRPs) in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L.) are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST) project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

As cistatinas são inibidores competitivos e reversíveis de proteinases cisteínicas, compondo uma superfamília de proteínas evolutivamente relacionadas. Esta superfamília está dividida em três famílias: a primeira, das estefinas, é composta de proteínas destituídas de pontes de enxofre; a segunda, das cistatinas, agrupa proteínas que possuem pontes de sulfeto; a terceira família, dos kininogênios, se caracteriza por ser formada por glicoproteínas de alto peso molecular com três domínios repetidos similares aos da família das cistatinas. As fitocistatinas constituem uma família de cistatinas que possui representantes exclusivamente em plantas. Elas são proteínas que não possuem pontes de enxofre e contém uma seqüência conservada L-A-R-[FY]-A-[VI]-X3-N na região N-terminal. Neste trabalho nos usamos seqüências derivadas de sete fitocistatinas, identificadas no projeto genoma de Arabidopsis, para identificar possíveis membros da superfamília das cistatinas presentes no banco de dados de cana-de-açúcar do SUCEST. Foram identificadas vinte e cinco possíveis fitocistatinas de cana-de-açúcar. Uma análise filogenética permitiu agrupar as fitocistatinas em quatro grupos: (i) contendo o consenso N-terminal ; (ii) contendo o consenso N-terminal e mais uma extensão C-terminal; (iii) destituído do consenso N-terminal e (iv) destituído do consenso N-terminal e do motivo conservado QxVxG encontrado em todos os membros da superfamília.

The cystatins are tightly binding, but reversible, inhibitors of cysteine proteinases, which constitute a superfamily of evolutionary related proteins. They have been subdivided into three families: the cystatin family which contain two disulfide bonds, the stefin family which lack disulfide bonds, and the kininogen family composed of large glycoproteins containing three repeats similar to those found in the cystatin family. Members of the cystatin superfamily occurring in plants are currently known as phytocystatins, defined as proteins lacking disulfide bonds but possessing a conserved N-terminal amino acid sequence (L-A-R-[FY]-A-[VI]-X(3)-N). We have used the protein sequence deduced from seven phytocystatins (from the Arabidopsis thaliana genome project) and from the SUgarCane EST project (SUCEST) database to identify 25 possible sugarcane phytocystatins. Phylogenetic analysis has allowed us to cluster these phytocystatins into four distinct groups: (i) those with a characteristic N-terminal consensus, (ii) those with the same consensus plus a long C-terminal extension; (iii) those that lack the consensus but contain the highly conserved QxVxG motif found in all members of the superfamily and (iv) those that lack both the consensus and the QxVxG motif.

O melhoramento vegetal vem sendo praticado pelo homem há milhares de anos. Entretanto, esse processo de domesticação tornou os vegetais mais susceptíveis a pragas e doenças. O melhoramento clássico permitiu, por cruzamento, a manipulação genética dos vegetais com conseqüente aumento na produtividade agrícola. Recentemente, a tecnologia do DNA recombinante ampliou as possibilidades de integração de genes exógenos ao genoma vegetal, resultando na produção das plantas transgênicas. Apesar das grandes discussões em torno do assunto, essas plantas representam hoje um caminho promissor para o melhoramento vegetal. Inúmeros exemplos de estratégias de transferência de genes conferiram, com sucesso, resistência a herbicidas, vírus, fungos, bactérias e insetos ou produziram um aumento na qualidade dos alimentos. Além das aplicações biotecnológicas, as plantas transgênicas têm contribuído significativamente para o estudo do funcionamento dos genes, tais como a análise da regulação da expressão gênica e o estudo das funções das proteínas codificadas pelos diferentes genes da planta.

Plant breeding has been a human practice for some thousands of years. However, this process of domestication has made plants more vulnerable to pests and diseases. Classical plant breeding has allowed the genetic manipulation of plants through crossings with a resulting increase in crop productivity. Recently, the recombinant DNA technology has increased the possibilities of integration of exogenous genes to the plant genome, resulting in the production of transgenic plants. Despite the great debate on this issue, such plants represent to date a promising avenue for plant breeding. There are many examples of gene transference strategies which have been successful in promoting resistance to herbicides, viruses, fungi, bacteria and insects, or in producing an increase in food quality. In addition to biotechnological applications, transgenic plants have made a significant contribution to the study of gene functioning, such as the analysis of genic expression regulation and the study of protein functions codified by distinct plant genes.