BiP-overexpressing soybean plants display accelerated hypersensitivity response (HR) affecting the SA-dependent sphingolipid and flavonoid pathways.

Biotic and abiotic environmental stresses have limited the increase in soybean productivity. Overexpression of the molecular chaperone BiP in transgenic plants has been associated with the response to osmotic stress and drought tolerance by maintaining cellular homeostasis and delaying hypersensitive cell death. Here, we evaluated the metabolic changes in response to the hypersensitivity response (HR) caused by the non-compatible bacteria Pseudomonas syringae pv. tomato in BiP-overexpressing plants. The HR-modified metabolic profiles in BiP-overexpressing plants were significantly distinct from the wild-type untransformed. The transgenic plants displayed a lower abundance of HR-responsive metabolites as amino acids, sugars, carboxylic acids and signal molecules, including p-aminobenzoic acid (PABA) and dihydrosphingosine (DHS), when compared to infected wild-type plants. In contrast, salicylic acid (SA) biosynthetic and signaling pathways were more stimulated in transgenic plants, and both pathogenesis-related genes (PRs) and transcriptional factors controlling the SA pathway were more induced in the BiP-overexpressing lines. Furthermore, the long-chain bases (LCBs) and ceramide biosynthetic pathways showed alterations in gene expression and metabolite abundance. Thus, as a protective pathway against pathogens, HR regulation by sphingolipids and SA may account at least in part by the enhanced resistance of transgenic plants. GmNAC32 transcriptional factor was more induced in the transgenic plants and it has also been reported to regulate flavonoid synthesis in response to SA. In fact, the BiP-overexpressing plants showed an increase in flavonoids, mainly prenylated isoflavones, as precursors for phytoalexins. Our results indicate that the BiP-mediated acceleration in the hypersensitive response may be a target for metabolic engineering of plant resistance against pathogens.

Remodeling of the cell wall as a drought-tolerance mechanism of a soybean genotype revealed by global gene expression analysis.

Drought stress is major abiotic stress that affects soybean production. Therefore, it is widely desirable that soybean becomes more tolerant to stress. To provide insights into regulatory mechanisms of the stress response, we compared the global gene expression profiles from leaves of two soybean genotypes that display different responses to water-deficit (BR 16 and Embrapa 48, drought-sensitive and drought-tolerant, respectively). After the RNA-seq analysis, a total of 5335 down-regulated and 3170 up-regulated genes were identified in the BR16. On the other hand, the number of genes differentially expressed was markedly lower in the Embrapa 48, 355 up-regulated and 471 down-regulated genes. However, induction and expression of protein kinases and transcription factors indicated signaling cascades involved in the drought tolerance. Overall, the results suggest that the metabolism of pectin is differently modulated in response to drought stress and may play a role in the soybean defense mechanism against drought. This occurs via an increase of the cell wall plasticity and crosslink, which contributed to a higher hydraulic conductance (K f) and relative water content (RWC%). The drought-tolerance mechanism of the Embrapa 48 genotype involves remodeling of the cell wall and increase of the hydraulic conductance to the maintenance of cell turgor and metabolic processes, resulting in the highest leaf RWC, photosynthetic rate (A), transpiration (E) and carboxylation (A/C i). Thus, we concluded that the cell wall adjustment under drought is important for a more efficient water use which promoted a more active photosynthetic metabolism, maintaining higher plant growth under drought stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00043-4.

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) / Purificação de uma enzima "tipo tripsina" não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner)

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima "tipo-tripsina" ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) e submetida à uma cromatografia de afinidade em uma coluna de p-aminobenzamidina, seguida por uma cromatografia de troca-aniônica. O rendimento da enzima purificada foi de 11% com fator de purificação de 143 e uma atividade específica final de 18.6 µM min.-1 mg-1 de proteína usando N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) como substrato. Após a separação da amostra purificada por SDS-PAGE e incubação subsequente com caseína, uma única banda ativa com massa molecular aparente de 25 kDa foi observada. A massa molecular determinada por espectrometria de massa (MALDI-TOF) foi de 28,632 ± 26 Da. O baixo rendimento e as dificuldades em purificar grandes quantidades da enzima limitaram caracterização complementar. A observação desta enzima, no entanto, é mais uma etapa no processo de conhecer as endoproteases presentes no intestino de A. gemmatalis, alvos potenciais de inibidores de proteases naturais ou especificamente projetados.

A herbivore that manipulates plant defence.

Phytopathogens and herbivores induce plant defences. Whereas there is evidence that some pathogens suppress these defences by interfering with signalling pathways involved in the defence, such evidence is scarce for herbivores. We found that the invasive spider mite Tetranychus evansi suppresses the induction of the salicylic acid and jasmonic acid signalling routes involved in induced plant defences in tomato. This was reflected in the levels of inducible defence compounds, such as proteinase inhibitors, which in mite-infested plants were reduced to even lower levels than the constitutive levels in herbivore-free plants. Additionally, the spider mite suppressed the release of inducible volatiles, which are implicated in plant defence. Consequently, the mites performed much better on previously attacked plants than on non-attacked plants. These findings provide a new perspective on plant-herbivore interactions, plant protection and plant resistance to invasive species.

Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus

Two α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ºC were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65ºC, respectively. The K M app for ρ -nitrophenyl-α -D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the K M app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50ºC and invertase maintained about 90 percent after 6 h at 55 ºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.

Duas α-galactosidases (P1 e P2) e uma invertase produzidas no sobrenadante da cultura do fungo Aspergillus terreus quando crescido por 168 h a 28ºC com farelo de trigo como fonte de carbono foram parcialmente purificadas por cromatografias de gel filtração e interação hidrofóbica. O pH e temperatura ótimos para as preparações P1, P2 e invertase foram entre 4,5-5,0, 5,5 e 4,0 e 60, 55 e 65ºC, respectivamente. O K M app para ρ-nitrofenil-α-D-galactopiranosideo foi 1.32 mM e 0.72 mM para P1 e P2, respectivamente. O valor de K M app para invertase usando sacarose como substrato foi de 15,66 mM. As preparações enzimáticas P1 e P2 mantiveram suas atividades após 3 h de pré-incubação a 50 ºC e a invertase manteve cerca de 90 por cento após 6 h a 55 ºC. P1 e P2 foram diferentemente sensíveis à inibição por Ag+, D-galactose e SDS. As preparações enzimáticas hidrolisaram os galactooligossacarídeos presentes em melaço de soja.

Effect of flavonoids morin: quercetin and nicotinic acid on lipid metabolism of rats experimentally fed with triton

Aterosclerose é uma doença coronária onde a deposiçäo de lipídeos nas artérias leva a problemas na circulaçäo sanguínea. O presente trabalho avalia a açäo dos flavonóides morina, quercetina e ácido nicotínico isoladamente e em associaçäo no metabolismo lipídico em ratos hiperlipidêmicos. Foram analisados os níveis de colesterol, colesterol HDL e tricilfliceróis no soro sanguíneo após administraçäo via intraperitoneal, dos compostos flavononoídicos dissolvidos em propileno glicol na dose de 5mg/kg de peso corporal. Quercetina mostrou o maior percentual de reduçäo do colesterol. para colesterol HDL os melhores resultados foram obtidos com ácido nicotínico isoladamente, enquanto a associaçäo morina-ácido nicotínico mostrou os melhores resultados para triacilgliceróis. Os resultados mostraram que os flavonóides podem ser benéficos no tratamento de doença coronariana