Abstract Ascorbate peroxidases (APXs) are heme peroxidases involved in the control of hydrogen peroxide levels and signal transduction pathways related to development and stress responses. Here, a total of 238 APX, 30 APX-related (APX-R), and 34 APX-like (APX-L) genes were identified from 24 species from the Poaceae family. Phylogenetic analysis of APX indicated five distinct clades, equivalent to cytosolic (cAPX), peroxisomal (pAPX), mitochondrial (mitAPX), stromal (sAPX), and thylakoidal (tAPX) isoforms. Duplication events contributed to the expansion of this family and the divergence times. Different from other APX isoforms, the emergence of Poaceae mitAPXs occurred independently after eudicot and monocot divergence. Our results showed that the constitutive silencing of mitAPX genes is not viable in rice plants, suggesting that these isoforms are essential for rice regeneration or development. We also obtained rice plants silenced individually to sAPX isoforms, demonstrating that, different to plants double silenced to both sAPX and tAPX or single silenced to tAPX previously obtained, these plants do not show changes in the total APX activity and hydrogen peroxide content in the shoot. Among rice plants silenced to different isoforms, plants silenced to cAPX showed a higher decrease in total APX activity and an increase in hydrogen peroxide levels. These results suggest that the cAPXs are the main isoforms responsible for regulating hydrogen peroxide levels in the cell, whereas in the chloroplast, this role is provided mainly by the tAPX isoform. In addition to broadening our understanding of the core components of the antioxidant defense in Poaceae species, the present study also provides a platform for their functional characterization.

Abstract Drought limits wheat production in the Brazilian Cerrado biome. In order to search for candidate genes associated to the response to water deficit, we analyzed the gene expression profiles, under severe drought stress, in roots and leaves of the cultivar MGS1 Aliança, a well-adapted cultivar to the Cerrado. A set of 4,422 candidate genes was found in roots and leaves. The number of down-regulated transcripts in roots was higher than the up-regulated transcripts, while the opposite occurred in leaves. The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis revealed a 0.78 correlation with the expression data. The candidate genes were distributed across all chromosomes and component genomes, but a greater number was mapped on the B genome, particularly on chromosomes 3B, 5B and 2B. When considering both tissues, 116 different pathways were induced. One common pathway, among the top three activated pathways in both tissues, was starch and sucrose metabolism. These results pave the way for future marker development and selection of important genes and are useful for understanding the metabolic pathways involved in wheat drought response.

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.

Environmental stresses caused by either abiotic or biotic factors greatly affect agriculture. As for soybean [Glycine max (L.) Merril], one of the most important crop species in the world, the situation is not different. In order to deal with these stresses, plants have evolved a variety of sophisticated molecular mechanisms, to which the transcriptional regulation of target-genes by transcription factors is crucial. Even though the involvement of several transcription factor families has been widely reported in stress response, there still is a lot to be uncovered, especially in soybean. Therefore, the objective of this study was to investigate the role of bHLH and trihelix-GT transcription factors in soybean responses to environmental stresses. Gene annotation, data mining for stress response, and phylogenetic analysis of members from both families are presented herein. At least 45 bHLH (from subgroup 25) and 63 trihelix-GT putative genes reside in the soybean genome. Among them, at least 14 bHLH and 11 trihelix-GT seem to be involved in responses to abiotic/biotic stresses. Phylogenetic analysis successfully clustered these with members from other plant species. Nevertheless, bHLH and trihelix-GT genes encompass almost three times more members in soybean than in Arabidopsis or rice, with many of these grouping into new clades with no apparent near orthologs in the other analyzed species. Our results represent an important step towards unraveling the functional roles of plant bHLH and trihelix-GT transcription factors in response to environmental cues.

When plants are exposed to stressful environmental conditions, the production of Reactive Oxygen Species (ROS) increases and can cause significant damage to the cells. Antioxidant defenses, which can detoxify ROS, are present in plants. A major hydrogen peroxide detoxifying system in plant cells is the ascorbate-glutathione cycle, in which, ascorbate peroxidase (APX) enzymes play a key role catalyzing the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Different APX isoforms are present in distinct subcellular compartments, such as chloroplasts, mitochondria, peroxisome, and cytosol. The expression of APX genes is regulated in response to biotic and abiotic stresses as well as during plant development. The APX responses are directly involved in the protection of plant cells against adverse environmental conditions. Furthermore, mutant plants APX genes showed alterations in growth, physiology and antioxidant metabolism revealing those enzymes involvement in the normal plant development.

Antioxidant metabolism protects cells from oxidative damage caused by reactive oxygen species (ROS). In plants, several enzymes act jointly to maintain redox homeostasis. Moreover, isoform diversity contributes to the fine tuning necessary for plant responses to both exogenous and endogenous signals influencing antioxidant metabolism. This study aimed to provide a comprehensive view of the major classes of antioxidant enzymes in the woody species Eucalyptus grandis. A careful survey of the FORESTs data bank revealed 36 clusters as encoding antioxidant enzymes: six clusters encoding ascorbate peroxidase (APx) isozymes, three catalase (CAT) proteins, three dehydroascorbate reductase (DHAR), two glutathione reductase (GR) isozymes, four monodehydroascorbate reductase (MDHAR), six phospholipid hydroperoxide glutathione peroxidases (PhGPx), and 12 encoding superoxide dismutases (SOD) isozymes. Phylogenetic analysis demonstrated that all clusters (identified herein) grouped with previously characterized antioxidant enzymes, corroborating the analysis performed. With respect to enzymes involved in the ascorbate-glutathione cycle, both cytosolic and chloroplastic isoforms were putatively identified. These sequences were widely distributed among the different ESTs libraries indicating a broad gene expression pattern. Overall, the data indicate the importance of antioxidant metabolism in eucalyptus.

A embriogênese somática em berinjela foi induzida a partir de explantes cotiledonares cultivados em meio MS suplementado com 54 µM de ANA. A análise anatômica do início de formação e desenvolvimento de embriões somáticos foi realizada durante as primeiras quatro semanas. A formação de pré-embriões foi observada após o segundo dia de cultura, diretamente a partir de células perivasculares ou a partir de massas pré-embriogênicas derivadas de massas meristemáticas indeterminadas (IMMs) formadas a partir do tecido vascular. As IMMs também deram origem a primórdios radiculares após 10 dias de cultura. A origem dos embriões é discutida, assim como as similaridades entre a formação de embriões somáticos e raízes adventícias.

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

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.

As plantas respondem ao estresse provocado por temperaturas elevadas através da síntese de um grupo de proteínas denominadas proteínas de choque térmico (Heat Shock Proteins-HSPs) que estão associadas com a obtenção de termotolerância. Nesse estudo, foi avaliada a indução de proteínas de choque térmico de baixo peso molecular (sHSPs) em oito cultivares de feijoeiro através de "Northern blot" utilizando o cDNA da W HSP 16,9 como uma sonda heteróloga. Caupi foi utilizado como controle, uma vez que, ao contrário do feijoeiro, é uma espécie que se apresenta bem adaptada às condições de temperaturas elevadas como as observadas no Semi-árido brasileiro. Após um período de crescimento, as plantas foram submetidas a um período de duas horas de choque térmico a 40 ºC. Todas as variedades avaliadas foram capazes de induzir mRNAs para sHSP que hibridizou com a sonda W HSP 16,9. Entretanto, foi observada uma diferença significativa no padrão de indução entre as diferentes variedades. Os níveis de mRNA e sHSPs induzidos em caupi após o choque térmico foram bem maiores que os observados para as variedades de feijoeiro. Além disso, a expressão de sHSPs foi também avaliada em relação ao acúmulo de proteínas através da análise de "Western-blot" para caupi e as variedades de feijoeiro IPA 7 e Negro Argel. O padrão de proteínas observado confirma que as sHSPs são diferencialmente expressas em diferentes variedades de feijoeiro de acordo com a tolerância ao estresse térmico.

Plants respond to temperature stress by synthesizing a set of heat shock proteins (HSPs), which may be responsible for the acquisition of thermotolerance. In this study, the induction of small HSPs (sHSPs) in eight common bean varieties was evaluated by Northern blot analysis using the W HSP 16.9 cDNA as heterologous probe. Cowpea was used, as a positive control since this plant, as opposed to common bean, is known to grow well under high temperature regimes such as that found in the Brazilian semi-arid region. After the growth period, the plants were submitted to two h of heat shock at 40 ºC. All varieties tested were able to induce sHSP mRNAs that hybridized with W HSP 16.9 probe. However, significant kinetic differences were found when comparing different varieties. SHSP mRNA levels induced after heat shock in cowpea was higher than the levels observed on the bean varieties displaying the highest expression of these proteins. Besides, the sHSP expression was also assessed at the protein accumulation level by Western-blot analysis for cowpea and both IPA 7 and Negro Argel varieties of bean plants. The revealed protein pattern confirmed that sHSPs are differentially expressed in distinct varieties of common bean according their heat stress tolerance.

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.