BackgroundRust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions.FindingsWe analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses.ConclusionsFor tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments.

To identify genes that are up and down-regulated by water deficit in sugarcane we used the macroarray methodology and the expression level of 3 575 independent sugarcane cDNAs was measured by hybridization with RNA extracted from plants submitted to mild, moderate and severe water deficit. We identified approximately 1 670 differentially expressed genes from which 62 % were up-regulated by different stress-conditions, whereas many repressed genes were exclusive for each time-point. Analysis of similarity showed that approximately 24 % of the differentially expressed genes shared homology with proteins involved in different processes such as signal transduction, hormone metabolism, photosynthesis, transcription and stress response. Transcripts with no known function accounted for approximately 39 % and those without similarity represented 36 % of the sequences. Five genes analyzed by RT-PCR confirmed the macroarray results.

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The physiological response of four commercial sugarcane genotypes to water stress was evaluated by measuring the photochemical efficiency of the photosystem II (chlorophyll a fluorescence ratio, F v /F m ), estimated chlorophyll content (SPAD unit), leaf temperature (LT) and leaf relative water content (RWC). A field trial was established in the subtropical area with well-watered and water-stressed genotypes, in completely randomized blocks with four replicates in a 4 9 2 9 3 factorial design (genotype 9 irrigation 9 evaluation date). Physiological measurements were done during a 90 day-period of formative stage of plants. The analysis of variance showed that the interaction of genotype 9 irrigation 9 evaluation date had a significant effect for three physiological markers tested, F v /F m , SPAD unit and RWC. Under non-stressed conditions, all genotypes showed similar responses for the four markers. Under water deficiency stress, two droughttolerant genotypes, HOCP01-523 and TCP89-3505 displayed higher values for F v /F m , SPAD unit and RWC, and lower values for LT, and could be classified as tolerant. It is therefore possible to use these physiological water stress associated traits as scorable marker traits for selecting drought-tolerant sugarcane genotypes in future breeding programs.

M. 2001. Heat or cold chronic stress affects organ weights and Hsp70 levels in chicken embryos. Can. J. Anim. Sci. 81: 83-87. This study was carried out with the objective of evaluating the effect of heat (38.8°C) or cold (35.8°C) stress on chicken embryo development and tissues Hsp70 levels, after the 13th day of incubation. Embryo weight (percent egg weight), organ weight (percent embryo weight) and Hsp70 levels (ng Hsp70 µg -1 total protein) in different tissues (liver, breast muscle, heart, lungs, brain and kidney) were studied at the end of incubation. Cold stress induced a lower embryo weight and lower kidney and lungs weights, whereas heart and liver were lighter in heat-stressed embryos. An interaction between temperature and age was obtained only for Hsp70 levels in kidney and heart. Cold-stressed embryos showed higher Hsp70 levels in the brain, lungs and liver; a decrease in brain and breast muscle Hsp70 levels was seen from the 19th to 20th days in control embryos. Hsp70 levels increased with age in kidneys of control embryos and in heart of heat-and cold-stressed embryos. In conclusion, this study showed that chicken embryo organ weights are affected by incubation temperature, and that Hsp70 expression is tissue dependent (higher levels being seen in the brain) being cold-stress more effective in increasing Hsp70 levels in most studied tissues.Key words: Chicken embryo, development, Hsp70, incubation temperature Givisiez, P. E. N., da Silva, M. M., Mazzi, C. M., Ferro, M. I. T., Ferro, J. A., Gonzales, E. et Macari, M. 2001. Le stress chronique causé par la chaleur ou le froid modifie le poids des organes et la concentration de Hsp70 chez l'embryon du poulet. Can. J. Anim. Sci. 81: 83-87. L'étude devait servir à évaluer les effets de la chaleur (38,8°C) ou du froid (35,8°C) sur l'embryogenèse du poulet et sur la concentration de Hsp70 dans les tissus après 13 jours d'incubation. On a mesuré le poids de l'embryon (% du poids de l'oeuf), le poids des organes (% du poids de l'embryon) et la concentration de Hsp70 (ng Hsp70 µg -1 protéines totales) dans divers tissus (foie, muscles de la poitrine, coeur, poumons, encéphale et reins) à la fin de l'Incubation. Le stress causé par le froid diminue le poids de l'embryon et celui des reins et des poumons, tandis que c'est le coeur et le foie qui sont plus petits chez les embryons soumis au stress causé par la chaleur. Une interaction entre la température et l'âge n'a été observée qu'au niveau de concentration de Hsp70 dans les reins et le coeur. Les embryons soumis au stress causé par le froid présentaient plus de Hsp70 dans l'encéphale, les poumons et le foie; chez les embryons témoins, la concentration de Hsp70 dans le cerveau et les muscles de la poitrine diminue entre le 19 e et le 20 e jour d'incubation. La concentration de Hsp70 s'accroît avec l'âge dans les reins des embryons témoins et dans le coeur des embryons soumis à l'un ou l'autre stress thermique. Les auteurs en concluent que chez l'embryon de poulet, le poids des organes est affecté par la te...

ABSTRACT. Drought is one of the most frequent abiotic stresses limiting the productivity and geographical distribution of sugarcane culture. The use of drought-tolerant genotypes is one approach for overcoming the effects of water stress. We conducted a comparative study to identify gene expression profiles under water stress in tolerant sugarcane roots. Two different cultivars, 1 drought tolerant (RB867515) and 1 drought susceptible (SP86-155), were evaluated at 4 sampling time points (1, 3, 5, and 10 days) using the cDNA-amplified fragment length polymorphism technique. A total of 173 fragments were found to be differentially expressed in response to water stress in the tolerant cultivar. Seventy of these were cloned, sequenced, and categorized. Similarity analysis using Candidate genes to drought tolerance in sugarcane BLAST revealed that 64% of the fragments differentially expressed code proteins classified as no hits (23%), hypothetical (21%), or involved in stress response (20%), with others were involved in communication pathways and signal transduction, bioenergetics, secondary metabolism, and growth and development. Four genes were analyzed and validated using real-time quantitative polymerase chain reaction to determine their expression and showed consistency with the cDNA-amplified fragment length polymorphism analyses. Our results contribute insight into the molecular responses to water stress in sugarcane and possibility to the development of cultivars with improved tolerance to drought.

1. The relationship between repeated thermal treatments and hepatic synthesis of Hsp 70 was studied in broiler chickens. 2. Sixty broilers were submitted to 5 different treatments (12 birds each) from day 1 to day 42. Four groups were kept in a thermoneutral environment and subjected to 0, 1, 2 and 3 heat stress episodes at 35 degrees C for 4 h per week (TN-0, TN-1, TN-2 and TN-3, respectively). The last group (HT-35) was reared at a room temperature of 35 degrees C. 3. From 39 to 42 old, the birds experienced acute heat stress at 41 degrees C. Resistance to heat stress was evaluated by the time taken for rectal temperature to increase by 3 degrees C above the pre-treatment value. Livers were collected (before and after heat stress) and Hsp70 was determined using Western Blot analysis with monoclonal anti-Hsp70 antibody. 4. Resistance to heat stress and concentration of Hsp70 were higher in those birds subjected to more heat stress episodes during the experimental period (TN-3) and HT-35. A positive correlation was observed between Hsp70 concentration and the time taken for a 3 degrees C increase in rectal temperature (r = 0.42; P<0.01). 5. Exposing birds to episodes of heat stress (35 degrees C) during rearing may improve their resistance to acute heat stress, but the previous thermal history did not seem to influence the hepatocyte Hsp70 content after exposure to more severe heat stress (41 degrees C).

In the present study we have investigated the effects of heat acclimation on brain and hepatic Hsp70 protein levels and body temperature of broiler chickens in response to gradual heat stress. Two groups of broilers were raised up to 47 days of age under distinct temperature conditions: thermoneutral (TN, according to bird age) or hot environmental (HS, 31-33°C). At 46 days of age, the birds reared at high ambient temperature were transferred to thermoneutrality conditions. After 18 h, these birds and the birds reared at thermoneutral temperature were submitted to gradual heat stress in a climatic chamber so that environment temperature was increased from 28 to 40ºC at a rate of 2ºC/h. Colonic temperature was measured using a thermometer sensor probe at each two hours, and hepatic and brain tissues were collected immediately after slaughter in order to assess Hsp70 protein level by Western blotting analysis. The colonic temperatures of birds reared at high temperature increased steeply during the first 2 h of heat stress (1.06ºC/h) and more slowly thereafter (0.59ºC/h). Broilers reared at thermoneutral temperature showed a small increase in the first 4 h of heat stress (0.18ºC/h) and then colonic temperature increased sharply (0.72ºC/h). Nevertheless, both groups presented similar final colonic temperature by the end of the stress period. Hsp70 levels (ng Hsp70 µg total protein-1) did not change in the liver or brain of the birds reared at high temperature. On the other hand, both liver and brain Hsp70 levels increased significantly during heat stress in the animals reared at thermoneutrality, with a higher expression of this peptide in brain tissue