Maria Cristina Neves de Oliveira

Are Chemical Compounds Important for Soybean Resistance to Anticarsia gemmatalis

The identification and quantification of flavonoids (rutin and genistin) present in extracts of soybean genotypes, and their effects on the biology and physiology of Anticarsia gemmatalis Hübner (Lep.: Noctuidae) were studied. Analysis of covariance and bicoordinate utilization plots were used to remove the effect of feeding time from pupal weight and consumption as well as to separate pre- and postingestive effects of treatment on A. gemmatalis growth. Genotypes PI 274454, PI 227687, and “IAC-100” extracts in general, caused higher mortality, negatively influenced initial larval and pupal weight, and elongated larval cycle. Larvae fed on the “IAC-100” extract diet ingested larger amounts of food per unit of time, but were less efficient in its conversion to biomass. Leaf extracts of PI 227687 had the largest concentration of rutin (quercitin 3-O-rhamnosylglucoside), followed by PI 274454, and “IAC-100”; PI 74454 also had the highest genistin (genistein 7-O-glucoside) content. The susceptible cultivar “BR-16” showed only a kaempferol-based flavonoid in its chemical profile, indicating that after successive crosses, secondary compounds responsible for plant defenses were eliminated. Genotypes PI 274454, PI 227687, and “IAC-100” showed accentuated resistance characteristics and were considered inadequate sources for the development of A. gemmatalis. Considering rutin and genistin concentration in these genotypes, it is suggested that flavonoids are important factors conferring resistance to A. gemmatalis.

Diurnal oscillations of soybean circadian clock and drought responsive genes.

Rhythms produced by the endogenous circadian clock play a critical role in allowing plants to respond and adapt to the environment. While there is a well-established regulatory link between the circadian clock and responses to abiotic stress in model plants, little is known of the circadian system in crop species like soybean. This study examines how drought impacts diurnal oscillation of both drought responsive and circadian clock genes in soybean. Drought stress induced marked changes in gene expression of several circadian clock-like components, such as LCL1-, GmELF4- and PRR-like genes, which had reduced expression in stressed plants. The same conditions produced a phase advance of expression for the GmTOC1-like, GmLUX-like and GmPRR7-like genes. Similarly, the rhythmic expression pattern of the soybean drought-responsive genes DREB-, bZIP-, GOLS-, RAB18- and Remorin-like changed significantly after plant exposure to drought. In silico analysis of promoter regions of these genes revealed the presence of cis-elements associated both with stress and circadian clock regulation. Furthermore, some soybean genes with upstream ABRE elements were responsive to abscisic acid treatment. Our results indicate that some connection between the drought response and the circadian clock may exist in soybean since (i) drought stress affects gene expression of circadian clock components and (ii) several stress responsive genes display diurnal oscillation in soybeans.

Detrimental effect of rutin on Anticarsia gemmatalis

O efeito de dieta contendo rutina (quercetina 3-O-rutinosideo) no comportamento e na nutricao do principal desfolhador da soja, Anticarsia gemmatalis Hubner (Lep.: Noctuidae) foi avaliado do terceiro instar ate a formacao da pupa. Rutina e um dos flavonois glicosidicos identificados em folhas da soja selvagem PI 227687. O peso das lagartas e o consumo decresceram com o aumento na concentracao de rutina na dieta. Houve interacao entre o tempo de alimentacao e a dieta no crescimento do inseto; lagartas alimentadas com dieta pura, cujo tempo de alimentacao prolongou-se, originaram pupas mais pesadas. O peso das pupas alimentadas com dieta contendo rutina permaneceu estavel, apesar do maior tempo de alimentacao. Rutina afetou negativamente o crescimento do inseto, nao somente pela inibicao alimentar, mas tambem em consequencia de eventos pos-ingestivos, ja que depois do ajuste do peso de pupa pelo consumo (covariavel), o efeito do tratamento permaneceu significativo. Assim, o efeito negativo de rutina em A. gemmatalis resulta de efeitos pre-ingestivos, indicados pela reducao no consumo, e pos-ingestivos, indicados por baixas conversoes do alimento ingerido em biomassa e assimilacao de alimentos.

Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR), genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress), and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity). The raw cycle threshold (Ct) data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M), and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

Characterization of genes Rpp2, Rpp4, and Rpp5 for resistance to soybean rust

Asian rust, caused by the fungus Phakopsora pachyrhizi, is the most severe disease currently threatening soybean crops in Brazil. The development of resistant cultivars is a top priority. Genetic characterization of resistance genes is important for estimating the improvement when these genes are introduced into soybean plants and for planning breeding strategies against this disease. Here, we infected an F2 population of 140 plants derived from a cross between ‘An-76’, a line carrying two resistance genes (Rpp2 and Rpp4), and ‘Kinoshita’, a cultivar carrying Rpp5, with a Brazilian rust population. We scored six characters of rust resistance (lesion color [LC], frequency of lesions having uredinia [%LU], number of uredinia per lesion [NoU], frequency of open uredinia [%OU], sporulation level [SL], and incubation period [IP]) to identify the genetic contributions of the three genes to these characters. Furthermore, we selected genotypes carrying these three loci in homozygosis by marker-assisted selection and evaluated their genetic effect in comparison with their ancestors, An-76, PI230970, PI459025, Kinoshita and BRS184. All three genes contributed to the phenotypes of these characters in F2 population and when pyramided, they significantly contributed to increase the resistance in comparison to their ancestors. Rpp2, previously reported as being defeated by the same rust population, showed a large contribution to resistance, and its resistance allele seemed to be recessive. Rpp5 had the largest contribution among the three genes, especially to SL and NoU. Only Rpp5 showed a significant contribution to LC. No QTLs for IP were detected in the regions of the three genes. We consider that these genes could contribute differently to resistance to soybean rust, and that genetic background plays an important role in Rpp2 activity. All three loci together worked additively to increase resistance when they were pyramided in a single genotype indicating that the pyramiding strategy is one good breeding strategy to increase soybean rust resistance.

Quantitative differential expression of alpha and beta ryanodine receptor genes in PSE (Pale, Soft, Exudative) meat from two chicken lines: broiler and layer

ABSTRACT Total RNA isolated from Pectoralis major muscle from PSE (L* 24h >53.0, pH 0.05) in α-ryr gene expression regardless of line studied. The β-ryr RQ results suggested that in PSE samples an alteration might occur in the regular ratio (1:1) of α-RyR/β-RyR normally found in avian muscles. These results provided the first evidence of PSE meat occurrence as a result of the differential expression of ryanodine receptor genes which might lead to an increased in Ca 2+ availability at the cell milieu. Key words: malignant hyperthermia, poultry, gene expression, real time PCR * Author for correspondence: mshimo@uel.br

Expression patterns of GmAP2/EREB-like transcription factors involved in soybean responses to water deficit.

Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a GmDRIP-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 GmAP2/EREB-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the GmDREB1F-like gene, for example, was four times higher in roots than in leaves). The GmDRIP-like gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several GmDREB-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.

Do different casein concentrations increase the adverse effect of rutin on the biology of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)?

The flavonoid rutin is recognized as playing an important role in the protection of plants against lepidopterans. Bioassays with this compound are generally carried out using artificial diets. Proteins of high energy value, such as casein, are important ingredients of insect artificial diets as a source of essential amino acids. However, such proteins can generally increase the allelochemical activity. Our objective was to evaluate the effects of rutin on larvae of the velvetbean caterpillar Anticarsia gemmatalis Hübner by incorporating this allelochemical into diets with different concentrations of casein. Three casein concentrations (0, 7 g, or 14 g) combined with none, 0.65%, or 1.30% of rutin were added to the rearing diet and offered to the larvae from hatching to pupation. Rutin negatively affected larval development, the amount of food consumed, and pupal weight of A. gemmatalis. These negative effects were clearly seen in insects fed on diets with 7 g of casein to which any concentration of rutin was added. The effects of rutin when added to the diets without casein were stronger than in diets containing a suitable amount of casein (14 g). The greater negative effects of rutin in diets containing suboptimal concentrations of casein indicate that casein can increase the effects of rutin only when the diets are nutritionally unsuitable for insect development.

Escala diagramática para avaliação da severidade da mancha alvo da soja

A mancha alvo da soja, causada pelo fungo Corynespora cassiicola, e encontrada em praticamente todas as regioes de cultivo de soja do Brasil e pode causar perdas economicas. Esse trabalho teve como objetivo elaborar e validar uma escala diagramatica para avaliacao da severidade da mancha alvo da soja. Para tal, foram coletados foliolos de soja com sintomas da doenca, onde se mediu a severidade, determinando-se os limites minimos e maximos, e os niveis intermediarios da escala de acordo com a lei do estimulo visual de Weber-Fechner. A escala elaborada apresentou sete niveis de severidade: 1, 2, 5, 9, 19, 33 e 52%. A validacao foi realizada por oito avaliadores, sem experiencia em quantificar essa doenca, que estimaram a severidade de 40 foliolos com diferentes niveis de severidade da mancha alvo, sem e com o auxilio da escala. A utilizacao da escala diagramatica resultou em melhor acuracia e precisao das estimativas realizadas, sendo uma ferramenta de facil e rapida utilizacao, que pode ser adotada para auxiliar na avaliacao da severidade da mancha alvo da soja.

Environmental and genetic variation of isoflavone content of soybean seeds grown in Brazil

Abstract – The objective of this work was to evaluate isoflavoneconcentrations in seeds of different Brazilian soybean cultivars grown in a range of locations and environmental conditions in Brazil. Seeds of 233 cultivars grown in Ponta Grossa, PR, Brazil, during the 2001/2002 soybean season, and of 22 cultivars sown in different locations of Brazilian Northeast, Southeast on South regions were analyzed for total isoflavones,including daidzin, glycitin, ‑1genistin and acetylgenistin. The total isoflavonesranged from 12 mg 100 g (cv. Embrapa 48) to 461 mg 100 g ‑1 (cv. CS 305) among the 233 cultivars grown in Ponta Grossa, and the differences among them are due to genetic effects since all cultivars were grown and collected at the same locatation and year. This is an indication of the possibility of breeding for isoflavonecontent. Differences in isoflavonecontent observed in the cultivars grown in different locations permit the selection of locations for optimum isoflavone content (low or high), depending on the uses of soybean. In the Northeast region (5–8°S), higher concentrations of ‑1total isoflavoneswere observed at Sao Raimundo das Mangabeiras (232 mg 100 g ) and Tasso Fragoso (284 mg 100 g