Silencing ScGUX2 reduces xylan glucuronidation and improves biomass saccharification in sugarcane.

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

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.

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.

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.

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.