Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzymeencoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: "What regulates flux through this pathway in vivo?" Previous proteomic experiments with Arabidopsis discussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway in Arabidopsis: the NADP-TRX reductase a and b double mutant (ntra ntrb) and the mitochondrially located thioredoxin o1 (trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when 13 Cglucose, 13 C-malate, or 13 C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.Arabidopsis | redox regulation | thioredoxin TCA cycle regulation | citric acid cycle regulation | ATP-citrate lyase A s in animals and aerobic microorganisms (1, 2), the tricarboxylic acid (TCA) cycle of plant mitochondria is composed of a set of eight enzymes that oxidize pyruvate and malate formed in the cytosol to CO 2 and NADH (3). The CO 2 is released and the NADH is oxidized by the electron transport chain for the generation of ATP. Recent years have witnessed major advances in our understanding of the cycle in plants, including its different modes of operation and properties of its constituent enzymes (4-6). We also now understand a great deal about the physiological role, kinetic features, and transcriptional and posttranslational regulation of enzymes participating in the cycle.In addition to these studies, experiments have focused on functional interactions taking place between mitochondria and the other organelle that generates energy in plant cells, namely, the chloroplast (7, 8). The results suggest that the two compartments are tightly linked by regulatory mechanisms acting at the levels of the gene and interorganellar metabolite transport (9-13). Further, a long-standing body of evidence indicates that the cycle is regul...

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-highperformance 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.

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.

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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.

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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.