Allelic mutant series reveal distinct functions for Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis

Sterols have multiple functions in all eukaryotes. In plants, sterol biosynthesis is initiated by the enzymatic conversion of 2,3-oxidosqualene to cycloartenol. This reaction is catalyzed by cycloartenol synthase 1 (CAS1), which belongs to a family of 13 2,3-oxidosqualene cyclases in Arabidopsis thaliana. To understand the full scope of sterol biological functions in plants, we characterized allelic series of cas1 mutations. Plants carrying the weak mutant allele cas1–1 were viable but developed albino inflorescence shoots because of photooxidation of plastids in stems that contained low amounts of carotenoids and chlorophylls. Consistent with the CAS1 catalyzed reaction, mutant tissues accumulated 2,3-oxidosqualene. This triterpenoid precursor did not increase at the expense of the pathway end products. Two strong mutations, cas1–2 and cas1–3, were not transmissible through the male gametes, suggesting a role for CAS1 in male gametophyte function. To validate these findings, we analyzed a conditional CRE/loxP recombination-dependent cas1–2 mutant allele. The albino phenotype of growing leaf tissues was a typical defect observed shortly after the CRE/loxP-induced onset of CAS1 loss of function. In the induced cas1–2 seedlings, terminal phenotypes included arrest of meristematic activity, followed by necrotic death. Mutant tissues accumulated 2,3-oxidosqualene and contained low amounts of sterols. The vital role of sterols in membrane functioning most probably explains the requirement of CAS1 for plant cell viability. The observed impact of cas1 mutations on a chloroplastic function implies a previously unrecognized role of sterols or triterpenoid metabolites in plastid biogenesis

Biosynthesis of secondary metabolites in sugarcane

A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST) database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC); chalcone synthase (CHS), the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS), involved in plant defense and root nodulation; isoflavone reductase (IFR), a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.<br>Este trabalho foi realizado com os objetivos de gerar uma coleção de genes relacionados ao metabolismo secundário da cana de açúcar e investigar o padrão de expressão gênica de enzimas chaves reguladoras das principais vias biossintéticas ativas nos diferentes tipos de tecidos e situações de estresse físico-químico e biológico a que estão submetidas plantas cultivadas em casas de vegetação, campo ou in vitro. A estratégia de mineração dos dados da database de sequências expressas de cana de açúcar, SUCEST, usando ferramentas de bioinformática, focalizou classes de compostos como isoprenóides e fenilpropanóides que comprovadamente desempenham um papel na resposta de plantas a variações ambientais. Foram identificados e caracterizados genes que codificam enzimas chaves para a síntese de terpenóides, como a sesquiterpeno ciclase (SC); (CHS) para síntese de flavonóides; isoflavona sintase (IFS) envolvida na biossíntese de isoflavonóides que desempenharm importante papel na defesa de plantas e nodulação de raízes; isoflavona redutases (IFR) enzimas chaves para a síntese de fenilpropanóide fitoalexinas, bem como enzimas relacionadas à síntese de precursores de lignina, como a enzima ácido caféico- O- metiltransferase. O efeito do estresse causado por bactérias como Herbaspirillum rubri e Gluconacetobacter diazotroficans também foi avaliado tendo sido constatada a indução da expressão de chalcona sintase (CHS) em plântulas infectadas com esses agentes, sugerindo a ativação da via de flavonóides em resposta a este estresse biológico. Esses resultados apontam para o fato de que as vias do metabolismo de isopropanóides e de fenilpropanóides em cana de açúcar são ativadas de acordo com o estágio de desenvolvimento, especificidade de tecidos e em resposta a situações de estresse. Essas observações deverão ser confirmadas por meio de experimentação genética e bioquímica