Alexandra Bottcher

Lignification in Sugarcane: Biochemical Characterization, Gene Discovery, and Expression Analysis in Two Genotypes Contrasting for Lignin Content

Biochemical, histological, and transcriptional characterization of lignification identifies substantial differences in two sugarcane genotypes. Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes.

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

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.

Association with arbuscular mycorrhizal fungi influences alkaloid synthesis and accumulation in Catharanthus roseus and Nicotiana tabacum plants

Frequently disregarded, plant associations with arbuscular mycorrhizal fungi (AMF) can influence plant specialized metabolism with important ecological and/or economic implications. In this study, we report on both the influence of mycorrhization on the content of a wide range of alkaloids and differential gene expression of some enzymes involved in alkaloid biosynthetic pathways in the leaves and roots of Catharanthus roseus and Nicotiana tabacum plants. These plants were divided into several treatments: mycorrhizal, inoculated with AMF; non-AMF inoculated plants; and non-AMF inoculated plants with an extra supply of phosphorus. The contents of vindoline, vinblastine, vincristine, catharanthine, ajmalicine and serpentine in C. roseus and of nicotine, anabasine and nornicotine in N. tabacum tobacco plants were determined. Mycorrhizal inoculation increased ajmalicine and serpentine contents in C. roseus roots suggesting that mycorrhization had a greater influence on the accumulation of alkaloids in roots than it did in shoots. The youngest leaves of mycorrhizal C. roseus plants showed lower transcript levels of the genes analysed; however, in older leaves, the expression levels were higher when compared with the leaves of non-mycorrhizal plants. In the case of tobacco, higher leaf to root ratios for nicotine and anabasine were found in plants with a mycorrhizal association. Our results showed that mycorrhization changed the alkaloid content and expression pattern of the genes analysed in both species; however, differences were found between the roots and shoots. In nature, such changes may have a direct influence on the interactions between plants and insects (herbivory) and pathogens. These interactions must be studied further to reveal the ecological influence mycorrhizae may have on chemical defences in a broader sense.

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.

Expression Profile of Sugarcane Transcription Factor Genes Involved in Lignin Biosynthesis

Cell wall recalcitrance, which is conferred in part by lignin, is the main bottleneck in lignocellulosic ethanol production. Transcription factors (TFs) have been suggested as targets to reduce or modify lignin. Here we analysed the expression profile of nine sugarcane TFs, their relationships with genes of the monolignol biosynthesis pathway, and their effects on lignin content and composition. Our assays compared two sugarcane genotypes with different lignin contents. To identify differences between tissue types and between the top and bottom of the plant, the culm was divided into intermediary and mature internodes, and the internodes were separated into pith and rind. The expression profiles obtained for the nine TFs were rather complex, showing that not only the genotype but also the tissue type and developmental stage influenced the results. Pearson correlation analysis indicated that ShMYB58/63 was positively correlated with the syringyl/guaiacyl ratio. In addition, a Bayesian network showed predicted interactions between the TFs and genes for lignin biosynthesis that were previously reported in the literature, as well as novel interactions such as those between ShMYB58/63 and ShF5H. These findings suggest that in sugarcane culm, the differential lignin deposition between tissue types (rind and pith) and at different developmental stages is under transcriptional regulation.

Water stress alters lignin content and related gene expression in two sugarcane genotypes.

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.

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane

Dirigent (DIR) proteins, encoded by DIR genes, are referred to as “dirigent” because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (−) pinoresinol, the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of ShDIR1-like transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the ShDIR1-like transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.

Elicitation of tobacco alkaloid biosynthesis by disrupted spores and filtrate of germinating spores of the arbuscular mycorrhizal fungi Glomus etunicatum

Molecules released by soil microorganisms, such as arbuscular mycorrhizal fungi (AMF), trigger plant responses prior to any physical contact. Here, it is shown that disrupted spores (DS) homogenates and exudates from germinating spores (GS) of Glomus etunicatum caused marked alterations in the content of the tobacco alkaloids nicotine, anabasine, and nornicotine and the genes involved in their biosynthesis. GS and DS were applied to the base of Nicotiana tabacum seedling stems, and 3 or 10 days later, the leaves and roots were harvested for analyses. The alkaloid contents were influenced by both elicitors and varied depending on the harvest day. In general, such variations agree with the transcript levels of putrescine N-methyltransferase, oxide reductase – A622 and nicotine N-demethylase – CYP82E4. The results are discussed in light of recent insights on chemical signaling processes between plants and AMF able to trigger different elicitation responses and their possible effects on secondary metabolism in plants.

Antioxidative responses of cell suspension cultures of two Coffea arabica varieties to low aluminum levels at pH 5.8

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

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

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 secondgeneration 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.