Proteomic analysis of the medicinal plant Artemisia annua: data from leaf and trichome extracts

This article contains raw and processed data related to research published by Bryant et al. [1]. Data was obtained by MS-based proteomics, analysing trichome-enriched, trichome-depleted and whole leaf samples taken from the medicinal plant Artemisia annua and searching the acquired MS/MS data against a recently published contig database [2] and other genomic and proteomic sequence databases for comparison. The processed data shows that an order-of-magnitude more proteins have been identified from trichome-enriched Artemisia annua samples in comparison to previously published data. Proteins known to have a role in the biosynthesis of artemisinin and other highly abundant proteins were found which imply additional enzymatically driven processes occurring within the trichomes that are significant for the biosynthesis of artemisinin.

Trichome structure and evolution in Neotropical lianas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glandular trichome density and essential oil composition in leaves and inflorescences of Lippia origanoides Kunth (Verbenaceae) in the Brazilian Cerrado

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glandular trichome diversity on leaves of Lippia origanoides and Lippia stachyoides (Verbenaceae): morphology, histochemistry, and ultrastructure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH]cyt is reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.

Gibberellins Promote Trichome Formation by Up-Regulating GLABROUS1 in Arabidopsis1

Trichome development is dependent on gibberellin (GA) signaling in Arabidopsis thaliana. Using the GA-deficient mutant ga1–3, the GA-response mutant spy-5, and uniconazol (a GA-biosynthesis inhibitor), we show that the GA level response correlates positively with both trichome number and trichome branch number. Two genes, GL1 and TTG, are required for trichome initiation. In ga1–3, coexpression of GL1 and R, the maize TTG functional homolog, under control of the constitutive 35S promoter, restored trichome development, whereas overexpression of neither GL1 nor R alone was sufficient to significantly suppress the glabrous phenotype. We next focused on GL1 regulation by GAs. In the double mutant the gl1–1 glabrous phenotype is epistatic to the spy-5 phenotype, suggesting that GL1 acts downstream of the GA signal transduction pathway. The activity of a β-glucuronidase reporter gene driven by the GL1 promoter was decreased in the wild type grown on uniconazol and showed a clear GA-dependent activation in ga1–3. Finally, quantification of GL1 transcript levels by reverse transcriptase-polymerase chain reaction demonstrated that relative to wild type, ga1–3 plants contained less transcript. These data support the hypothesis that GAs induce trichome development through up-regulation of GL1 and possibly TTG genes.

Phyllosphere of Cotton as a Habitat for Diazotrophic Microorganisms

Positive nitrogenase activities ranging from 0.18 to 0.78 nmol of C2H4 cm−2 h−1 were detected on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants. Beijerinckia sp. was observed to be the predominant nitrogen-fixing microorganism in the phyllosphere of these varieties. A higher level of phyllosphere nitrogen-fixing activity was recorded in the variety Varalaxmi despite a low C/N ratio in the leaf leachates. Leaf surfaces of the above variety possessed the largest number of hairy outgrowths (trichomes) which entrapped a majority of microbes. Immersion of plant roots in nutrient medium containing 32Pi led to the accumulation of label in the trichome-borne microorganisms, thereby indicating a possible transfer of nutrients from leaf to microbes via trichomes. Extrapolation of acetylene reduction values suggested that 1.6 to 3.2 kg of N ha−1 might be contributed by diazotrophs in the phyllosphere of the variety Varalaxmi during the entire growth period.

Species and Hybrids: Chemical and Physical Foliar Attributes and Implications for Herbivory.

Hybridization is an important biological phenomenon that can be used to understand the evolutionary process of speciation of plants and their associated pests and diseases. Interactions between hybrid plants and the herbivores of the parental taxa may be used to elucidate the various cues being used by the pests for host location or other processes. The chemical composition of plants, and their physical foliar attributes, including leaf thickness, trichome density, moisture content and specific leaf weight were compared between allopatric pure and commercial hybrid species of Corymbia, an important subtropical hardwood taxon. The leaf-eating beetle Paropsis atomaria, to which the pure taxa represented host (C. citriodora subsp. variegata) and non-host (C. torelliana) plants, was used to examine patterns of herbivory in relation to these traits. Hybrid physical foliar traits, chemical profiles, and field and laboratory beetle feeding preference, while showing some variability, were generally intermediate to those exhibited by parent taxa, thus suggesting an additive inheritance pattern. The hybrid susceptibility hypothesis was not supported by our field or laboratory studies, and there was no strong relationship between adult preference and larval performance. The most-preferred adult host was the sympatric taxon, although this species supported the lowest larval survival, while the hybrid produced significantly smaller pupae than the pure species. The results are discussed in relation to plant chemistry and physical characteristics. The findings suggest a chemical basis for host selection behavior and indicate that it may be possible to select for resistance to this insect pest in these commercially important hardwood trees.

棉纤维发育相关基因的克隆和鉴定

棉纤维是棉花子房内的胚珠外珠被上的一种单细胞表皮毛.为了解胚珠表皮细胞发育启动为棉纤维的分子控制机制,我们用同源克隆的方法,从棉纤维发育早期的幼嫩子房和胚珠中克隆到了72个棉花MYB基因.序列分析的结果表明它们属于MYB转录因子家族的55个成员,其中有一个MYB蛋白-GhMYB9的氨基酸序列与控制拟南芥单细胞表皮毛的发育启动基因GL1和WER高度同源.它们之间的序列一致性,不仅表现在保守区-DNA结合区,而且也表现在5’和3’的非保守区内.根据序列相似功能相似的原理,我们推测GhMYB9很可能是控制棉纤维发育启动的一个MYB基因.对GhMYB9的Southern杂交结果表明,该基因在棉花基因组中是单拷贝基因;对它的Northern表达分析可知,该基因在花前5天的子房、花前3天的胚珠、花期及花后3天的胚珠中都有表达,但在花前3天的棉纤维发育启动期（我们的扫描电镜结果）高表达.此外,还构建了一个陆地棉可转化人工染色体TAC文库,并通过PCR的方法从中筛选到了含有GhMYB9的TAC克隆.这些结果为进一步对GhMYB9做功能分析、揭示棉纤维发育启动的分子机制奠定了了事实上的基础.

Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

Desenvolvimento e validação de método analítico para quantificação da boldina em peumus boldus mol. (monimiaceae) e avaliação preliminar de sua estabilidade

Peumus boldus Molina (Monimiaceae) é uma árvore comum e abundante no Chile, e suas folhas são amplamente empregadas pela medicina tradicional para o tratamento de uma variedade de afecções do sistema digestivo e hepatobiliar. Seus principais constituintes químicos são alcalóides do tipo aporfínicos, sendo a boldina seu maior e mais característico representante, cujas propriedades antioxidantes são extensivamente relatadas na literatura, servindo desta forma como marcador químico para a espécie. Com o objetivo de contribuir com parâmetros de controle de qualidade, um estudo botânico macro e microscópico das folhas e do pó da espécie foi realizado. Características morfológicas, identificadas pela análise macroscópica, como pilosidade, tipologia dos tricomas, proeminência ao redor da base dos tricomas e aparência do bordo, e características microscópicas como estômatos anomocíticos, folha hipoestomática e ocorrência de cristais de oxalato de cálcio denotam valor taxonômico. Parâmetros farmacopéicos como cinzas totais e perda por dessecação foram determinados, e um método de quantificação da boldina foi desenvolvido e validado utilizando a CLAE. O limite mínimo de boldina observado na análise de 10 amostras adquiridas foi de aproximadamente 0,016%. A fim de avaliarse o comportamento de seus constituintes químicos frente a variações de temperatura e umidade, realizou-se estudo de estabilidade acelerada (40ºC ± 2ºC / 75% ± 5% U.R.). A degradação máxima de boldina foi observada após 90 dias em câmara climática chegando a aproximadamente 52%. A reação de degradação sugerida foi de segunda ordem, e o tempo de vida útil e meia-vida calculados foram de aproximadamente 18 dias e 165 dias, respectivamente. Por fim, realizou-se a quantificação de alguns metais pesados nas folhas da espécie, que decresceram na ordem de Fe> Mn> Cu> Ni, sendo que Pb, Cr e Co tiveram níveis abaixo do limite de detecção de 5 μg/g.

Nonpreference of whitefly for oviposition in tomato genotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resistência de genótipos de soja à mosca branca Bemisia tabaci (Genn.) biótipo B (Hemiptera: Aleyrodidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aspectos biológicos do ácaro Brevipalpus phoenicis vetor da leprose dos citros em plantas de buva (Conyza canadensis)

O ácaro Brevipalpus phoenicis, vetor da leprose dos citros, é uma espécie polífaga que tem ampla gama de hospedeiros alternativos nos pomares cítricos, nos quais se pode manter e/ou incrementar suas populações. O objetivo deste trabalho foi estudar a sobrevivência, a oviposição e o modo de alimentação do ácaro B. phoenicis em plantas de buva (Conyza canadensis) com diferentes tamanhos. Para avaliação da sobrevivência e oviposição do ácaro B. phoenicis, foi utilizado o delineamento estatístico inteiramente casualizado, com cinco tratamentos e doze repetições. Os tratamentos estudados foram: (1) secções de caules de plantas menores que 20 cm, (2) entre 21 e 50 cm, (3) entre 51 e 100 cm e (4) maiores que 101 cm de altura; e (5) secções de ramos de laranja. Dez fêmeas adultas de B. phoenicis, procedentes de uma criação-estoque, foram transferidas para cada secção de caule de buva e ramos de citros como testemunha. Avaliou-se a sobrevivência e a oviposição dos ácaros até 120 horas após a transferência. Alguns aspectos comportamentais do ácaro B. phoenicis foram fotomicrografados, utilizando-se microscópico eletrônico de varredura. Verificou-se que as plantas de maior altura de buva foram mais favoráveis à sobrevivência do ácaro B. phoenicis do que as de menor tamanho. Contudo, a quantidade de ovos postos pelo ácaro não diferiu em relação à altura das plantas de buva. Com base nas fotomicrografias, constatou-se a preferência do ácaro B. phoenicis por se alimentar na base dos tricomas presentes na superfície do caule da planta de buva, justificado, possivelmente, pela maior turgescência das células encontradas nessa região da planta. Esses resultados reforçam a importância do manejo de buva nos pomares cítricos, pois a sua presença no campo pode favorecer a sobrevivência e o desenvolvimento do ácaro B. phoenicis, servindo, portanto, como hospedeira alternativa, o que pode contribuir para a disseminação do ácaro e, consequentemente, da leprose nos pomares cítricos.