Pedro Araújo

Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis

Lignin Biosynthesis Complications Lignin is a polymer that lends its sturdy properties to wood and makes plant cell walls tougher, which creates problems for chemists converting cellulosic plant biomass into biofuels. Vanholme et al. (p. 1103, published online 15 August; see the cover) have identified a new step in the biosynthetic pathway of lignin in Arabidopsis in which caffeoyl shikimate esterase catalyzes synthesis of caffeate. Cellulose from mutant plants, which had reduced amounts of lignin, was more efficiently processed into glucose. A key enzyme involved in lignin biosynthesis is identified and characterized in the model plant Arabidopsis. Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development

Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant

Lignin is a complex phenolic heteropolymer deposited in the secondarily thickened walls of specialized plant cells to provide strength for plants to stand upright and hydrophobicity to conducting cells for long-distance water transport. Although essential for plant growth and development, lignin is the major plant cell-wall component responsible for biomass recalcitrance to industrial processing. Peroxidases and laccases are generally thought to be responsible for lignin polymerization, but, given their broad substrate specificities and large gene families, specific isoforms involved in lignification are difficult to identify. This study used a combination of co-expression analysis, tissue/cell-type-specific expression analysis, and genetic complementation to correlate a sugarcane laccase gene, SofLAC, to the lignification process. A co-expression network constructed from 37 cDNA libraries showed that SofLAC was coordinately expressed with several phenylpropanoid biosynthesis genes. Tissue-specific expression analysis by quantitative RT-PCR showed that SofLAC was expressed preferentially in young internodes and that expression levels decrease with stem maturity. Cell-type-specific expression analysis by in situ hybridization demonstrated the localization of SofLAC mRNA in lignifying cell types, mainly in inner and outer portions of sclerenchymatic bundle sheaths. To investigate whether SofLAC is able to oxidize monolignols during lignification, the Arabidopsis lac17 mutant, which has reduced lignin levels, was complemented by expressing SofLAC under the control of the Arabidopsis AtLAC17 promoter. The expression of SofLAC restored the lignin content but not the lignin composition in complemented lac17 mutant lines. Taken together, these results suggest that SofLAC participates in lignification in sugarcane.

Mass spectrometry imaging: an expeditious and powerful technique for fast in situ lignin assessment in Eucalyptus.

Plant biomass has been suggested as an alternative to produce bioethanol. The recalcitrance of plant biomass to convert cellulose into simpler carbohydrates used in the fermentation process is partially due to lignin, but the standard methods used to analyze lignin composition frequently use toxic solvents and are laborious and time-consuming. MS imaging was used to study lignin in Eucalyptus, since this genus is the main source of cellulose in the world. Hand-cut sections of stems of two Eucalyptus species were covered with silica and directly analyzed by matrix-assisted laser sesorption ionization (MALDI)-imaging mass spectrometry (MS). Information available in the literature about soluble lignin subunits and structures were used to trace their distribution in the sections and using a software image a relative quantification could be made. Matrixes routinely used in MALDI-imaging analysis are not satisfactory to analyze plant material and were efficiently substituted by thin layer chromatography (TLC) grade silica. A total of 22 compounds were detected and relatively quantified. It was also possible to establish a proportion between syringyl and guaiacyl monolignols, characteristic for each species. Because of the simple way that samples are prepared, the MALDI-imaging approach presented here can replace, in routine analysis, complex and laborious MS methods in the study of lignin composition.

Validation of reference genes from Eucalyptus spp. under different stress conditions

BackgroundThe genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR) require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus.ResultsEucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden) and E. uroglobulus (hybrid from E. urograndis X E. globulus) were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase), SAND (SAND protein), ACT (actin), and A-Tub (α-tubulin) genes were the most stable. IDH was the most stable gene in all of the treatments.ConclusionComparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin). It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified genes in this study should be used as reference genes for a wide range of conditions in eucalyptus.

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.

cis-Cinnamic Acid Is a Novel, Natural Auxin Efflux Inhibitor That Promotes Lateral Root Formation

The phenylpropanoid cis-cinnamic acid is a natural auxin efflux inhibitor that promotes lateral root formation. Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

Sugarcane Water Stress Tolerance Mechanisms and Its Implications on Developing Biotechnology Solutions

Sugarcane is a unique crop with the ability to accumulate high levels of sugar and is a commercially viable source of biomass for bioelectricity and second-generation bioethanol. Water deficit is the single largest abiotic stress affecting sugarcane productivity and the development of water use efficient and drought tolerant cultivars is an imperative for all major sugarcane producing countries. This review summarizes the physiological and molecular studies on water deficit stress in sugarcane, with the aim to help formulate more effective research strategies for advancing our knowledge on genes and mechanisms underpinning plant response to water stress. We also overview transgenic studies in sugarcane, with an emphasis on the potential strategies to develop superior sugarcane varieties that improve crop productivity in drought-prone environments.

A model system to study the lignification process in Eucalyptus globulus

Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass-to-biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus.

Citrus sinensis L. Osbeck orthologs of FRUITFULL and SHATTERPROOF are differentially expressed during fruit development

Several regulatory steps and genes involved in fruit development were identified and characterized in Arabidopsis thaliana. FRUITFULL (FUL) and SHATTERPROOF (SHP), which belong to the MADS-box family of transcription factors, act together to promote the differentiation of the dehiscence zone and thus control the process of pod shattering in Arabidopsis. Homologs to these genes have been described in fleshy fruit species, but the specific nature of the regulatory hierarchy and interactions among these key regulators remains elusive in most plant species. Here, Citrus sinensis putative orthologs to FUL and SHP, named CsFUL and CsSHP respectively, were characterized. Phylogenetic comparisons indicated that CsFUL belongs to FRUITFUL sub-clade within the AP1/SQUA major clade while CsSHP falls into PLENA sub-clade from the AG/PLE clade. CsFUL and CsSHP protein sequences possess all of the characteristic conserved domains commonly found in A- and C-lineages of MIKC MADS-box proteins, respectively. Semi-quantitative RT-PCR showed preferential expression of both genes in developing fruits. In situ hybridization and a detailed analysis of Citrus fruit development using scanning electron microscopy allowed further characterization of these genes during C. sinensis fruit development. CsFUL and CsSHP are differentially expressed in exocarp, mesocarp and endocarp tissues in early stages of fruit development but their expression diminishes with fruit maturation. Moreover, the co-localization of CsFUL and CsSHP mRNA during oil glands and juice vesicle formation suggests a potential role in the development of such structures. Altogether, there results might contribute to a better understanding of the molecular mechanisms involved in Citrus fruit development.