ShF5H1 overexpression increases syringyl lignin and improves saccharification in sugarcane leaves.

The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing ShF5H1 under the control of the C4H (cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that ShF5H1 overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the ShF5H1 overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.

Remote sensing reveals unprecedented sublethal impacts of a 40-year-old oil spill on mangroves.

Oil spills cause long-lasting mangrove loss, threatening their conservation and ecosystem services worldwide. Oil spills impact mangrove forests at various spatial and temporal scales. Yet, their long-term sublethal effects on trees remain poorly documented. Here, we explore these effects based on one of the largest oil spills ever recorded, the Baixada Santista pipeline leak, which hit the mangroves of the Brazilian southeastern coast in 1983. Historical, Landsat-derived normalized difference vegetation index (NDVI) maps over the spilled mangrove reveal a large dieback of trees within a year following the oil spill, followed by a eight-year recolonization period and a stabilization of the canopy cover, however 20-30% lower than initially observed. We explain this permanent loss by an unexpected persistence of oil pollution in the sediments based on visual and geochemical evidence. Using field spectroscopy and cutting-edge drone hyperspectral imaging, we demonstrate how the continuous exposure of mangrove trees to high levels of pollution affects their health and productivity in the long term, by imposing permanent stressful conditions. Our study also reveals that tree species differ in their sensitivity to oil, giving the most tolerant ones a competitive advantage to recolonize spilled mangroves. By leveraging drone laser scanning, we estimate the loss of forest biomass caused by the oil spill to be 9.8-91.2 t ha-1, corresponding to 4.3-40.1 t C ha-1. Based on our findings, we encourage environmental agencies and lawmakers to consider the sublethal effects of oil spills on mangroves in the environmental cost of these accidents. We also encourage petroleum companies to use drone remote sensing in monitoring routines and oil spill response planning to improve mangrove preservation and impact assessment.

Low phosphorus induces differential metabolic responses in eucalyptus species improving nutrient use efficiency.

Phosphorus (P) is a vital nutrient for plant growth. P availability is generally low in soils, and plant responses to low P availability need to be better understood. In a previous study, we studied the growth and physiological responses of 24 species to low P availability in the soil and verified of eucalypts, five (Eucalyptus acmenoides, E. grandis, E. globulus, E. tereticornis, and Corymbia maculata) contrasted regarding their efficiency and responsiveness to soil P availability. Here, we obtained the metabolomic and lipidomic profile of leaves, stems, and roots from these species growing under low (4.5 mg dm-3) and sufficient (10.8 mg dm-3) P in the soil. Disregarding the level of P in the soils, P allocation was always higher in the stems. However, when grown in the P-sufficient soil, the stems steadily were the largest compartment of the total plant P. Under low P, the relative contents of primary metabolites, such as amino acids, TCA cycle intermediates, organic acids and carbohydrates, changed differently depending on the species. Additionally, phosphorylated metabolites showed enhanced turnover or reductions. While photosynthetic efficiencies were not related to higher biomass production, A/Ci curves showed that reduced P availability increased the eucalypt species' Vcmax, Jmax and photosynthetic P-use efficiency. Plants of E. acmenoides increased galactolipids and sulfolipids in leaves more than other eucalypt species, suggesting that lipid remodelling can be a strategy to cope with the P shortage in this species. Our findings offer insights to understand genotypic efficiency among eucalypt species to accommodate primary metabolism under low soil P availability and eventually be used as biochemical markers for breeding programs.

Nuclei Enrichment from Sugarcane Stems for Proteomics Analyses.

Nuclei enrichment procedures enable a large variety of investigations. These studies include structural characterization of nuclear proteins, identification of posttranslational modifications, and regulation of stress or development-related gene expression. Successful enrichment of nuclei samples from plant tissues is crucial for a comprehensive analysis of the plant nuclear proteome. Here, we describe a method for nuclei enrichment from sugarcane stems and its assessment by western blot.

Visualization of Suberization and Lignification in Sugarcane.

Cell wall biopolymers are major factors responsible for the high recalcitrance of sugarcane biomass. The study of suberization and lignification mechanisms in sugarcane and of the networks that control biosynthesis of these polymers will contribute to the biotechnological improvement of this crop. Here, we describe experiments that allow the visualization of the suberization and lignification mechanism in response to mechanical injury in sugarcane.

Isolation of Promoters and Transcription Factors Involved in the Regulation of Lignin Biosynthesis in Saccharum Species.

Sugarcane bagasse has received attention as a raw material for the production of second-generation ethanol (E2G). However, its use is limited because of the cell wall recalcitrance, mostly conferred by lignin. Recently our knowledge of the genes coding for the enzymes of the lignin biosynthesis pathway has increased; however, still little is known about the transcription factors controlling the expression of these genes in sugarcane. Here we describe protocols to optimize the isolation of the promoters of the lignin biosynthetic genes ShCAD8, ShCOMT and ShF5H and the transcription factors (TFs) ShMYB85 and ShMYB58/63 in Saccharum species. To confirm whether these TFs are able to activate the target promoters, a transactivation assay in BY2 protoplasts of Nicotiana tabacum is also detailed.

Metallothionein production is a common tolerance mechanism in four species growing in polluted Cu mining areas in Peru.

Cu pollution is a problem in mining areas in Peru. Here we evaluate the phytoextraction capacity, physiological and proteomic responses of four species growing in copper-contaminated areas in Arequipa, Peru. The plants used in the experiments were obtained by collecting seedlings (Tessaria integrifolia, Bacharis salicifolia), rhizomes (Eleocharis montevidensis) and seeds (Chenopodium murale) along a polluted river. They were exposed to solutions containing 2, 4, 8, 16 and 32 mg Cu L-1 during 20 days. Growth was affected in a concentration-dependent way. According to the tolerance index, B. salicifolia and C. murale were the most sensitive species, but with greater Cu phytoextraction capacity and accumulation in the biomass. The content and ratio of photosynthetic pigments changed differently for each specie and carotenoids level were less affected than chlorophyll. Cu also induced changes in the protein and sugar contents. Antioxidant enzyme activities (catalase and superoxide dismutase) increased with a decrease in the malondialdehyde. There were marked changes in the protein 2D-PAGE profiles with an increase in the abundance of metallothioneins (MT) of class II type I and II. Our results suggest that these species can grow in Cu polluted areas because they developed multiple tolerance mechanisms, such as and MTs production seems a important one.

Enhancing potassium content in leaves and stems improves drought tolerance of eucalyptus clones.

Eucalyptus are widely planted in regions with low rainfall, occasioning frequent drought stresses. To alleviate the stress-induced effects on plants growing in these environments, soil fertilization with potassium (K) may affect drought-adaptive plant mechanisms, notably on tropical soils with low K availability. This work aimed to evaluate the K dynamic nutrition in eucalyptus in response to soil-K and -water availabilities, correlating the K-nutritional status with the physiological responses of contrasting eucalyptus clones to drought tolerance. A complete randomized design was used to investigate the effects of three water regimes (well-watered, moderate water deficit, and severe water deficit) and two K soil supplies (sufficient and low K) on growth and physiological responses of two elite eucalyptus clones: "VM01" (Eucalyptus urophylla × camaldulensis) and "AEC 0144" (E. urophylla). Results depicted that the K-well-nourished E. urophylla × camaldulensis clone under severe water deficit maintained shoot biomass accumulation by upregulating the K-content in leaves and stems, gas exchange, water-use efficiency (WUEI ), leaf water potential (Ψw), and chlorophyll a fluorescence parameters, compared to E. urophylla clone. Meanwhile, E. urophylla with a severe water deficit showed a decreased of K content in leaves and stem, as well as a reduction in the accumulation of dry mass. Therefore, the K-use efficiency and the apparent electron transport rate through photosystem II were positively correlated in plants grown in low K, indicating the importance of K in maintaining leaf photochemical processes. In conclusion, management strategy should seek to enhance K-nutrition to optimize water-use efficiencies and photosynthesis.

Revisiting the stem proteome of Eucalyptus grandis and Eucalyptus globulus: Identification of temperature-induced changes.

Eucalyptus grandis and Eucalyptus globulus are important species for the Brazilian forestry industry. E. grandis plantations are mainly found in tropical regions, yet E. globulus plants are usually cultivated under moderate to low temperature conditions. As temperature seems to be a key factor for the planting of these species, we revisited our previously generated shotgun proteomics dataset to identify the main patterns of proteome regulation induced by thermal stimulus and to pinpoint specific proteins involved in the environmental response. Large-scale analysis has pointed out the different proteomic responses of E. grandis and E. globulus under temperature stimulus, with 296 proteins considered to be differentially regulated in the stems of Eucalyptus spp. grown at different temperatures. A stringent filtering approach was used to identify the most differentially regulated proteins. Through the stringent criteria, 66 proteins were found to be enriched in the plant species. Cultivation of E. globulus plants in low-temperature conditions induced the highest number of differentially regulated proteins. Additionally, metabolic proteins were mostly down-regulated, while stress-related proteins were majorly up-regulated in both species. Finally, the subset of the most differentially regulated proteins comprised new candidates of protein markers of temperature stress.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

KEY MESSAGE: A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes ß-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography.

Lignin is a heterogeneous aromatic polymer responsible for cell wall stiffness and protection from pathogen attack. However, lignin represents a bottleneck to biomass degradation due to its recalcitrance related to the natural cell wall resistance to release sugars for fermentation or further processing. A biological approach involving genetics and molecular biology was used to disrupt lignin pathway synthesis and decrease lignin deposition. Here, we imaged three-dimensional fragments of the petioles of wild type and C4H lignin mutant Arabidopsis thaliana plants by synchrotron cryo-ptychography. The three-dimensional images revealed the heterogeneity of vessels, parenchyma, and fibre cell wall morphologies, highlighting the relation between disturbed lignin deposition and vessel implosion (cell collapsing and obstruction of water flow). We introduce a new parameter to accurately define cell implosion conditions in plants, and we demonstrate how cryo-ptychographic X-ray computed tomography (cryo-PXCT) provides new insights for plant imaging in three dimensions to understand physiological processes.

Should I stay or should I go: are chlorogenic acids mobilized towards lignin biosynthesis?

Chlorogenic acids (CGAs) and the biopolymer lignin are both products of the phenylpropanoid pathway. Whereas CGAs have been reported to play a role during stress responses, lignin is a major component of secondary cell walls, providing physical strength and hydrophobicity to supportive and water-conducting tissues. Because the chemical structure of CGAs largely resembles those of some lignin intermediates and because CGAs can be converted back to hydroxycinnamoyl-CoAs in vitro, CGAs have been considered authentic intermediates of the lignin biosynthetic pathway. However, it is still unclear whether and how the CGA pool can be channeled towards the production of lignin monomers in response to developmental or environmental signals. Comprehensive studies on the catalytic activity of recombinant enzymes together with functional characterizations in planta have been very useful in understanding the potential interdependence between these two metabolic routes. Here we present the current understanding on CGA metabolism and discuss the biochemical and molecular evidence of the metabolic re-routing of CGAs towards lignin.

Label-Free Quantitative Proteomics of Enriched Nuclei from Sugarcane (Saccharum ssp) Stems in Response to Drought Stress.

Drought is considered the major abiotic stress limiting crop productivity. This study seeks to identify proteins involved in the drought response in sugarcane stems submitted to drought stress. The integration of nuclei enrichment sample preparation with the shotgun proteomic approach results in great coverage of the sugarcane stem proteome with 5381 protein groups identified. A total of 1204 differentially accumulated proteins are detected in response to drought, among which 586 and 618 are increased and reduced in abundance, respectively. A total of 115 exclusive proteins are detected, being 41 exclusives of drought-stressed plants and 74 exclusives of control plants. In the control plants, most of these proteins are related to cell wall metabolism, indicating that drought affects negatively the cell wall metabolism. Also, 37 transcription factors (TFs) are identified, which are low abundant nuclear proteins and are differentially accumulated in response to drought stress. These TFs are associated to protein domains such as leucine-rich (bZIP), C2H2, NAC, C3H, LIM, Myb-related, heat shock factor (HSF) and auxin response factor (ARF). Increased abundance of chromatin remodeling and RNA processing proteins are also observed. It is suggested that these variations result from an imbalance of protein synthesis and degradation processes induced by drought.

Diel oscillations in cell wall components and soluble sugars as a response to short-day in sugarcane (Saccharum sp.).

BACKGROUND: Sugarcane is a tropical crop that can accumulate high concentration of sucrose in the stem as a storage carbohydrate. For that reason, sugarcane accounts for approximately 75% of all the sugar produced in the world and has become the main sugar source to produce first-generation bioethanol in Brazil. Daily rhythms cause plants to adapt and coordinate their metabolism to achieve maximum photosynthesis and carbohydrate production throughout the day. Circadian rhythms arise from the interaction of an internal oscillator and external stimuli, whereas diel rhythms occur in response to a light-dark cycle. Diel signalling contributes to synchronizing circadian rhythms to photoperiods, and levels of carbohydrates oscillate in a diel fashion. Under regular photoperiods, they are synthesized during the daytime and consumed throughout the night as an energy reserve. However, short days can induce higher rates of synthesis during daytime and lower rates of consumption in the dark. Cell wall carbohydrates are also diurnally regulated, and it has been shown that celluloses, hemicelluloses and pectin are deposited/degraded at different times of the day. To assess the diel carbohydrate profile in young sugarcane plants, we measured soluble sugars and cell wall components along a time course in plants subjected either to a regular day or short day. RESULTS: Short-day influenced sucrose synthesis and cell wall components. In short-day a 44% increase in sucrose concentration was detected in the dark, but was stable during the day. Cellulose, hemicellulose and pectin also fluctuate within a 24 h interval when subjected to a short day. A 38% increase in leaf sheath cellulose was observed from the middle of the day to the first hour of the night. Leaf sheath pectin and hemicellulose also increased from the day to the night, while it decreased in leaves. CONCLUSIONS: The presented data show diurnal patterns of soluble sugar metabolism together with temporal regulation of cell wall metabolism for a short day, suggesting that diel signalling has a role in how sugarcane manages sugar accumulation and partitioning. Understanding cell wall synthesis/degradation dynamics may help to improve the yield of sugarcane.

Evolution of phytochemical diversity in Pilocarpus (Rutaceae).

The evolution of phytochemical diversity and biosynthetic pathways in plants can be evaluated from a phylogenetic and environmental perspective. Pilocarpus Vahl (Rutaceae), an economically important medicinal plant in the family Rutaceae, has a great diversity of imidazole alkaloids and coumarins. In this study, we used phylogenetic comparative methods to determine whether there is a phylogenetic signal for chemical traits across the genus Pilocarpus; this included ancestral reconstructions of continuous and discrete chemical traits. Bioclimatic variables found to be associated with the distribution of this genus were used to perform OLS regressions between chemical traits and bioclimatic variables. Next, these regression models were evaluated to test whether bioclimatic traits could significantly predict compound concentrations. Our study found that in terms of compound concentration, variation is most significantly associated with adaptive environmental convergence rather than phylogenetic relationships. The best predictive model of chemical traits was the OLS regression that modeled the relationship between coumarin and precipitation in the coldest quarter. However, we also found one chemical trait was dependent on phylogenetic history and bioclimatic factors. These findings emphasize that consideration of both environmental and phylogenetic factors is essential to tease out the intricate processes in the evolution of chemical diversity in plants. These methods can benefit fields such as conservation management, ecology, and evolutionary biology.

Deposition of lignin in four species of Saccharum.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

Ecometabolomic Analysis of Wild Populations of Pilocarpus pennatifolius (Rutaceae) Using Unimodal Analyses.

Studies examining the diversity of plant specialized metabolites suggest that biotic and abiotic pressures greatly influence the qualitative and quantitative diversity found in a species. Large geographic distributions expose a species to a great variety of environmental pressures, thus providing an enormous opportunity for expression of environmental plasticity. Pilocarpus, a neotropical genus of Rutaceae, is rich in alkaloids, terpenoids, and coumarins, and is the only commercial source of the alkaloid pilocarpine for the treatment of glaucoma. Overharvesting of species in this genus for pilocarpine, has threatened natural populations of the species. The aim of this research was to understand how adaptation to environmental variation shapes the metabolome in multiple populations of the widespread species Pilocarpus pennatifolius. LCMS data from alkaloid and phenolic extracts of leaf tissue were analyzed with environmental predictors using unimodal unconstrained and constrained ordination methods for an untargeted metabolomics analysis. PLS-DA was used to further confirm the chemoecotypes of each site. The most important variables contributing to the alkaloid variation between the sites: mean temperature of wettest quarter, as well as the soil content of phosphorus, magnesium, and base saturation (V%). The most important contributing to the phenolic variation between the sites: mean temperature of the wettest quarter, temperature seasonality, calcium and soil electrical conductivity. This research will have broad implications in a variety of areas including biocontrol for pests, environmental and ecological plant physiology, and strategies for species conservation maximizing phytochemical diversity.

Biomass Accumulation and Cell Wall Structure of Rice Plants Overexpressing a Dirigent-Jacalin of Sugarcane (ShDJ) Under Varying Conditions of Water Availability.

A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T0 were selected and evaluated in both the T1 and T4 generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.