Activation of phospholipase PLA2 actvity in Ricinus communis leaves in response to mechanical wounding

Abstract

In order to investigate the defense response in castor bean (Ricinus communis) against predators, we analyzed the effect of mechanical wounding upon the phospholipase A 2 (PLA 2 ) activity of leaf extracts. Time course experiments revealed that the highest levels of increased PLA 2 activity (ca. two fold) occurred 15 min and 60 min after injury. The induced activities demonstrated high sensitivity towards aristolochic acid (10 mM), a PLA 2 inhibitor. Based on SDS-PAGE analysis, the PLA 2 activity induced 15 min after wounding migrated with a molecular mass of 40 kDa and was denoted RcPLA 2 I. The protein activity induced 60 min after wounding, RcPLA 2 II, migrated with a molecular weight of 14 kDa. Furthermore its N-terminal sequence shared homology with PLA 2 from elm and rice. The PLA 2 enzymes were purified to near homogeneity by a combination of gel filtration and electro-elution of protein bands after native PAGE. Key words: cellular defense, castor bean, plant signaling, phospholipase A 2 , Ricinus communis Com o objetivo de avaliar a resposta de defesa da mamona (Ricinus communis) contra predadores, analisaram-se as alterações nas atividades de fosfolipases A 2 (PLA 2 ) em extratos de folhas de plantas, submetidas à injúria mecânica após diferentes intervalos de tempo. Observou-se que os níveis dessa enzima aumentaram de forma significativa (cerca de duas vezes) 15 e 60 min após o ferimento. As atividades que foram induzidas foram sensíveis ao ácido aristolóquico (10 mM), um inibidor específico para PLA 2 . A enzima induzida após 15 min foi denominada Rc-PLA 2 I e apresentou uma massa molecular aparente, determinada por SDS-PAGE, de 40 kDa. A proteína induzida após 60 min, RcPLA 2 II, apresentou uma massa molecular de 14 kDa. A seqüência N-terminal parcial da Rc-PLAII mostrou homologia com PLA 2 de arroz e olmo. As fosfolipases foram parcialmente purificadas por associação da cromatografia de filtração em gel, seguida por eluição das bandas que apresentaram atividade enzimática após separação por PAGE não desnaturante.

Lipid deacylating enzymes in plants: Old activities, new genes

“…The biphasic nature of PLA activation, peaking at 15 and 60 min, observed upon wounding of tomato [78] and castor bean [21] leaves, is in agreement with this hypothesis. Furthermore, in wounded castor bean leaves, two PLA (of 40 and 14 kDa) were identified [21].…”

“…The biphasic nature of PLA activation, peaking at 15 and 60 min, observed upon wounding of tomato [78] and castor bean [21] leaves, is in agreement with this hypothesis. Furthermore, in wounded castor bean leaves, two PLA (of 40 and 14 kDa) were identified [21]. Thus, besides providing FAs for oxylipin biosynthesis, deacylating enzymes are thought to promote intracellular acidification, as an early response to pathogen attack, leading to different responses including expression of antimicrobial genes and development of the HR.…”

“…This group includes the enzyme purified from elm (Ulmus glabra) seeds and from rice (Oryza sativa) shoots [112,113] and more recently from castor bean (Ricinus communis) leaves [21]. sPLA 2 encoding cDNAs were also cloned from rice shoots [112] and from carnation flowers [49].…”

Biology and Biochemistry of Plant Phospholipases

“…sPLA 2 have also been cloned from carnation flowers (Kim et al, 1999), tomato and rice shoots (Stahl et al, 1999). In addition, several sPLA 2 proteins have been identified from elm (Ulmus glabra) seeds, rice (Oryza sativa) shoots and castor bean (Ricinus communis) leaves (Domingues et al, 2007;Stahl et al, 1998;Stahl et al, 1999).…”

Secretory Phospholipases A2 in Durum Wheat (Triticum durum Desf.): Gene Expression, Enzymatic Activity, and Relation to Drought Stress Adaptation

“…As far as the specific sPLA 2 s are concerned, these were first purified from elm seeds [5] and rice shoots [6], and more recently from castor bean leaves [7]. The cDNA for putative sPLA 2 s from rice shoots and carnation flowers were the first ones to be cloned [6,8], followed by the four sPLA 2 s of A. thaliana [9] and, very recently, by the five sPLA 2 s of G. max [10].…”