Large-scale Transcriptome Analysis Of Two Sugarcane Genotypes Contrasting For Lignin Content

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.108Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Brazilian National Council for Scientific and Technological DevelopmentFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [08/58035-6, 08/58031-0

Physiological and molecular characterization of drought tolerance and its relation with the root system in Coffea species

Orientador: Paulo MazzaferaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Plantas das cultivares Catuaí Vermelho e Mundo Novo de Coffea arabica L. e da cultivar Apoatã de C. canephora Pierre ex Froehner, e plantas de Mundo Novo enxertadas sobre Apoatã foram avaliadas quanto a possíveis estratégias utilizadas para suportar a baixa disponibilidade de água no solo. Em paralelo, foi conduzido um ensaio com suspensões celulares de Catuaí Vermelho sob estresse osmótico induzido por PEG. Assim, este estudo teve por objetivo examinar alguns mecanismos de tolerância à seca em café através da avaliação de aspectos morfológicos (sistema radicular), fisiológicos (ajuste osmótico) e moleculares (expressão de deidrinas e aquaporinas) em plantas jovens e cultura de células. A análise de expressão do gene CaDh1 (deidrina) em suspensão celular de café tratada com PEG (15% m/v), por 8, 24 e 72 h, mostrou aumento na atividade sob estresse osmótico, o que poderia estar associado a um possível mecanismo de proteção de membranas e macromoléculas contra a dessecação. Não foi verificada alteração na resposta dos genes PIP1, PIP2 e TIP1(aquaporinas) sob condição de estresse nas células de café. Plantas de café cultivadas em vasos (4,0 L) foram submetidas a estresse hídrico moderado por suspensão da irrigação, sob condições de casa de vegetação, até que o potencial hídrico da folha no "predawn" atingisse cerca de -2,0 MPa. Verificou-se que as taxas de crescimento nos genótipos de café arábica, avaliada pelo acúmulo de biomassa total (folhas e raízes), foram intensamente afetadas pela seca, com redução em torno de 26,5% em Catuaí e 29,0% em Mundo Novo. Por outro lado, as plantas enxertadas não apresentaram redução significativa de crescimento em relação às plantas irrigadas, sugerindo uma contribuição positiva da enxertia para melhorar a eficiência na captação de água pelo porta-enxerto Apoatã, sob condição de baixa disponibilidade de água no solo. O conteúdo de açúcares solúveis totais nas folhas das plantas irrigadas foi maior que o observado para as plantas sob déficit hídrico. A maior redução foi constatada entre os genótipos de café arábica (23,0% em Catuaí e 43,8% em Mundo Novo), enquanto as reduções em Apoatã e MN/Apoatã não foram significativas. Verificou-se maior acúmulo de aminoácidos totais nas folhas e raízes de plantas de Catuaí sob estresse hídrico comparado a Mundo Novo. Apoatã e MN/Apoatã não mostraram aumentos significativos em relação às respectivas plantas irrigadas. Em todos os genótipos analisados, os teores de prolina (Pro) nas folhas e raízes de plantas sob estresse foram aumentados e, nas raízes, esse acúmulo foi bastante acentuado, sendo 6 vezes maior nos genótipos de café arábica, 4 vezes em Apoatã e 16 vezes na enxertia MN/Apoatã em relação às plantas irrigadas. Entretanto, tais aumentos não parecem estar diretamente associados a maiores graus de tolerância à seca em café. Os conteúdos dos aminoácidos asparagina (Asn), ácido ?- aminobutírico (Gaba), glutamato (Glu) e alanina (Ala) também foram alterados, observandose, de modo geral, aumento nos teores de Asn e Gaba e redução de Glu e Ala nas folhas e raízes das plantas sob estresse. Em conclusão observou-se que os genótipos com sistema radicular mais vigoroso como Apoatã e Catuaí foram menos afetados pela seca, provavelmente porque foram mais hábeis em explorar o perfil do solo para a captação de água.Abstract: Seedlings of the cultivars Catuaí Vermelho and Mundo Novo of Coffea arabica L., of the cultivar Apoatã of C. canephora Pierre ex Froehner, and plants of Mundo Novo grafted on Apoatã were evaluated for possible strategies to withstand low water availability in the soil. In parallel, an assay with cellular suspension culture (Catuaí Vermelho) was carried out under osmotic stress induced by PEG. Thus, this study aimed to examine morphological (root systems), physiological (osmotic adjustment) and molecular (expression of dehydrins and aquaporins) traits in coffee plants and coffee cell suspension cultures as possible mechanisms of drought tolerance. The expression analysis of CaDh1 (dehydrin) in coffee cell suspensions grown with PEG (15% w/v) for 8, 24 and 72 h, showed an increase in activity under osmotic stress indicating a possible protection mechanism against desiccation. No changes were observed in the response of PIP1, PIP2 and TIP1 genes (aquaporins) under stress condition in coffee cells. Under greenhouse conditions, coffee seedlings were cultivated in 4.0 L pots submitted to moderate drought stress by suspending irrigation until leaf water potential at predawn reach about -2.0 MPa. Among the genotypes of arabica coffee subjected to water stress there was reduction in total dry weight, around 26.5% in Catuaí and 29.0% in Mundo Novo when compared with irrigated control plants. On the other hand, the grafted plants were not affected, suggesting a positive contribution of the grafting due to bigger efficiency in the water acquisition by the rootstock Apoatã under low water availability in the soil. The content of total soluble sugars was greater in leaves of irrigated plants than plants under water stress. The greatest reduction was verified among the genotypes of arabica coffee (23.0% in Catuaí and 43.8% in Mundo Novo) while the reduction in Apoatã and MN/Apoatã was not statistically significant. The accumulation of total amino acids was higher in leaves and roots of water stressed Catuaí than Novo Mundo. Apoatã and MN/Apoatã did not show significant increase in the content of amino acids in relation to irrigated control plants. In all genotypes, water stress induced an increase in the levels of proline (Pro) in leaves and roots. In the roots this increased was about 6 times in the genotypes of arabica coffee, 4 times in Apoatã and 16 times in the grafting MN/Apoatã in relation to irrigated control plants. However this increase in the levels of Pro does not seem to be directly associated to higher degrees of drought tolerance in coffee. The content of amino acids asparagine (Asn), ?-aminobutiric acid (Gaba), glutamate (Glu) and alanine (Ala) was also changed in the leaves and roots of stressed plants. In general, there was increase in levels of Asn and Gaba while was observed reduction in content of Glu and Ala. In conclusion it was found that the genotypes with more vigorous root system as Apoatã and Catuaí were less affected by drought, probably because they were more efficient in exploring the soil profile for the water absorption.MestradoBiologia VegetalMestre em Biologia Vegeta

Water Stress Alters Lignin Content And Related Gene Expression In Two Sugarcane Genotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The lignin deposition in the stem of two sugarcane genotypes was assessed on exposure to water stress. The lignin content and the morphoanatomical characterization of the stem indicated that IACSP94-2094 plants are more lignified than those of IACSP95-5000 genotype, under normal water supply conditions, which was especially associated with higher lignin contents in the rind of mature internodes. Water deficit had negative impact on the biomass production, mostly with IACSP94-2094 plants, possibly due to stress severity or higher susceptibility of that genotype during the stem-lengthening phase. Water deficit led to significant alterations in the expression levels of lignin biosynthesis genes and led to an approximate 60% increase of lignin content in the rind of young internodes in both genotypes. It is concluded that the young rind region was more directly affected by water stress and, depending on the genotype, a higher lignin accumulation may occur in the stem, thus implying lower quality biomass for bioethanol production.631947084720Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58035-6

Lignin biosynthesis in sugarcane is affected by low temperature

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Sugarcane is an abundant and promising source of plant biomass for biofuel production. The use of biomass for conversion to ethanol is limited by the recalcitrance of lignocellulosic material mainly due to the presence of lignin. This study characterised some aspects of lignification in the stem of two sugarcane genotypes grown under low (Cl') and warmer (HT - reference) temperatures. Stems were separated in young and mature culms and the culms were separated in cortex (rind) and medulla (pith). Plants of the genotype IACSP04-627, which has more lignin in the stem, grew better (fresh weight of the stem) than IACSP04-065 under HT. Cold negatively affected plant growth but apparently IACSPO4-065 was more sensitive than the other genotype. Lignin content was significantly increased in the young rind of IACSP04-627 plants at CT, what could not be directly correlated with the expression profile of genes of the monolignols biosynthesis. Lignin content in mature rind was reduced in IACSP04-065 plants exposed to CT, what could be correlated with the low expression level of the genes ShCAD2, ShCOMT1 and ShCCoAOMT1. The high expression of ShF5H occurred preferentially in mature pith of both sugarcane genotypes, what is possibly related with the early formation of the secondary cell wall induced by low temperature. In conclusion, lignin deposition in sugarcane under low temperature seems to be differentially regulated in rind and pith tissues and it is genotype-dependent. (C) 2015 Elsevier B.V. All rights reserved.Sugarcane is an abundant and promising source of plant biomass for biofuel production. The use of biomass for conversion to ethanol is limited by the recalcitrance of lignocellulosic material mainly due to the presence of lignin. This study characterised1203142FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58035-6]2008/58035-6SEM INFORMAÇÃOSEM INFORMAÇÃOThis study was funded by Fundac ̧ão de Amparo à Pesquisa doEstado de São Paulo (FAPESP – grant 2008/58035-6). The authors thank Coordenac ̧ão de Aperfeic ̧oamento de Pessoal de Nível Supe-rior (Capes – Brazil) and Conselho Nacional de DesenvolvimentoCien

Enzyme Characterisation, Isolation And Cdna Cloning Of Polyphenol Oxidase In The Hearts Of Palm Of Three Commercially Important Species.

Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid.49970-

Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content

<div><p>Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and <i>de novo</i> assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.</p></div

Transcript levels of lignin genes in the contrasting sugarcane genotypes.

<p>(*) indicates transcripts that are significantly (p-value ≤ 1e^-4) different in abundance between the two sugarcane genotypes.</p

Transcripts of the cluster enriched for flavonoid metabolism (C16), as derived from the K-means analysis.

<p>Transcripts of the cluster enriched for flavonoid metabolism (C16), as derived from the K-means analysis.</p