A short review on sugarcane: its domestication, molecular manipulations and future perspectives.

Sugarcane (Saccharum spp.) is a special crop plant that underwent anthropogenic evolution from a wild grass species to an important food, fodder, and energy crop. Unlike any other grass species which were selected for their kernels, sugarcane was selected for its high stem sucrose accumulation. Flowering in sugarcane is not favored since flowering diverts the stored sugar resources for the reproductive and developmental energy needs. Cultivars are vegetatively propagated and sugarcane breeding is still essentially focused on conventional methods, since the knowledge of sugarcane genetics has lagged that of other major crops. Cultivar improvement has been extremely challenging due to its polyploidy and aneuploidy nature derived from a few interspecific hybridizations between Saccharum officinarum and Saccharum spontaneum, revealing the coexistence of two distinct genome organization modes in the modern variety. Alongside implementation of modern agricultural techniques, generation of hybrid clones, transgenics and genome edited events will help to meet the ever-growing bioenergy needs. Additionally, there are two common biotechnological approaches to improve plant stress tolerance, which includes marker-assisted selection (MAS) and genetic transformation. During the past two decades, the use of molecular approaches has contributed greatly to a better understanding of the genetic and biochemical basis of plant stress-tolerance and in some cases, it led to the development of plants with enhanced tolerance to abiotic stress. Hence, this review mainly intends on the events that shaped the sugarcane as what it is now and what challenges ahead and measures taken to further improve its yield, production and maximize utilization to beat the growing demands.

TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response.

Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network.

Deep sequencing of suppression subtractive library identifies differentially expressed transcripts of Saccharum spontaneum exposed to salinity stress.

Saccharum spontaneum, a wild relative of sugarcane, is highly tolerant to drought and salinity. The exploitation of germplasm resources for salinity tolerance is a major thrust area in India. In this study, we utilized suppression subtractive hybridization (SSH) followed by sequencing for the identification of upregulated transcripts during salinity stress in S. spontaneum clones coming from different geographical regions of India. Our sequencing of the SSH library revealed that 95% of the transformants contained inserts of size 200-1500 bp. We have identified 314 differentially expressed transcripts in the salinity-treated samples after subtraction, which were subsequently validated by quantitative real-time polymerase chain reaction. Functional annotation and pathway analysis revealed that the upregulated transcripts were a result of protein modifications, stress, and hormone signaling along with cell wall development and lignification. The prominently upregulated transcripts included UDP glucose dehydrogenase, cellulose synthase, ribulose, cellulose synthase COBRA, leucine-rich protein, NAC domain protein, pectin esterase, ABA-responsive element binding factor 1, and heat stress protein. Our results is a step forward the understanding of the molecular response of S. spontaneum under salinity stress, which will lead to the identification of genes and transcription factors as novel targets for salinity tolerance in sugarcane.

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

Gene expression studies of Saccharum spontaneum, a wild relative of sugarcane in response to salinity stress.

The study is aimed to assess the morphological, physiological, and molecular responses of seven Saccharum spontaneum clones for salinity stress. These clones (IND-07-1462, IND-07-1465, IND-07-1470, IND-07-1471, IND 16-1761, IND 16-1762, and IND 16-1763) were subjected to salinity stress at two different concentrations of electrical conductivity 6 and 8 ds/m after 60 days of planting. All seven genotypes showed a decrease in relative water content and nitrate reductase activity with an increase in severity of salt stress. The effect was more pronounced in IND-07-1471, while IND-16-1762 exhibited only a minimum drop. Similarly we observed an increase in proline content and lipid peroxidation activity for the genotype IND-07-1471, while IND-16-1762 showed minimum increase. Molecular profiling of genes/transcription factors like salt overly sensitive, responsive to abscissic acid, dirigent, myeloblastosis, ethylene responsive factor associated with salinity stress tolerance showed 19-, 18-, 17-, 10-, and 9-fold increased expression at 8 ds/m of salinity stress, respectively, in IND-16-1762 showed. Based on the evidences obtained from expression profiling, we have cloned the conserved regions of RAB and SOS1 genes. The domain of SOS and RAB was identified as a regulatory subunit of cAMP-dependent protein kinases which is involved in a signaling pathway.

Halophilic archaea and their potential to generate renewable fuels and chemicals.

Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value-added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5-5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt-based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value-added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered "too salty" for most microbial processes including wastes from the olive-mill, shell fish, and biodiesel industries.

High-throughput sequencing reveals genes associated with high-temperature stress tolerance in sugarcane.

Sugarcane (Saccharum spp.) is a major sugar crop grown in tropical and sub-tropical areas throughout the world which is vulnerable to high temperature stress due to climate change. In this present study, we have generated a transcriptome profile of sugarcane variety Co 99004 exposed to high-temperature stress (47 °C). The Illumina Nexseq2500 platform yielded a total of 39.28 and 13.44 million reads, corresponding to 3.9 and 1.3 gigabase pair (Gb) of the processed reads for control and high-temperature-stressed samples, respectively. Initially, the reads were de novo assembled into 118,017 unigenes with an average length of 780 bp. The longest sequence in the assembly was 21 kb. Further, these transcripts were BLASTed against GO, KEGG and COG databases to identify the novel genes/transcripts expressed due to elevated temperature conditions. The different expression analysis showed 1137 transcripts which were up-regulated during heat temperature stress when compared to control conditions. Analysis of relative gene expression showed phytepsin, ferredoxin-dependent glutamate synthase, and stress protein DDR-48 threefold increased expression during heat stress. These findings reveal novel targets for subsequent research on the genomics genetic manipulation and molecular mechanism of elevated stress tolerance in sugarcane.

Isolation, cloning and expression analysis of cinnamyl alcohol dehydrogenase (CAD) involved in phenylpropanoid pathway of Erianthus arundinaceus, a wild relative of sugarcane.

In this study we have cloned and characterized cinnamyl alcohol dehydrogenase (CAD) involved in phenylpropanoid pathway which can be utilized for biomass modification for improved saccharification efficiency. The full length gene CAD is of 4 kb containing four exons and three introns, among which the exon 1 and 2 of 88 and 116 bp were conserved with sorghum and Miscanthus CADs. The coding region of CAD was identified with 1098 bp open reading frame (ORF), for 365 amino acids. In the PROSITE analysis, a zinc-containing alcohol dehydrogenase signature (GHEVVGEVVEVGPEV) and an NADP-binding domain motif (GLGGLG) was identified, while the motif analysis showed unique signature sequence of "LEPYLA" at 258-264 aa which was absent in the CAD sequences of other crops. This sequence information on CAD from Erianthus a bioenergy crop might be useful for subsequent research on lignin engineering for improved biomass conversion and for unravelling the impact of lignin on cell wall mechanics.

Transcriptome analysis highlights key differentially expressed genes involved in cellulose and lignin biosynthesis of sugarcane genotypes varying in fiber content.

Sugarcane (Saccharum spp. hybrids) is a potential lignocellulosic feedstock for biofuel production due to its exceptional biomass accumulation ability, high convertible carbohydrate content and a favorable energy input/output ratio. Genetic modification of biofuel traits to improve biomass conversion requires an understanding of the regulation of carbohydrate and lignin biosynthesis. RNA-Seq was used to investigate the transcripts differentially expressed between the immature and mature tissues of the sugarcane genotypes varying in fiber content. Most of the differentially expressed transcripts were found to be down-regulated during stem maturation, highlighting their roles in active secondary cell-wall development in the younger tissues of both high and low fiber genotypes. Several cellulose synthase genes (including CesA2, CesA4, CesA7 and COBRA-like protein), lignin biosynthesis-related genes (ρ-coumarate 3-hydroxylase, ferulate 5-hydroxylase, cinnamyl alcohol dehydrogenase and gentiobiase) and transcription regulators for the secondary cell-wall synthesis (including LIM, MYB, PLATZ, IAA24, C2H2 and C2C2 DOF zinc finger gene families) were exclusively differentially expressed between immature and mature tissues of high fiber genotypes. These findings reveal target genes for subsequent research on the regulation of cellulose and lignin metabolism.

A survey of the complex transcriptome from the highly polyploid sugarcane genome using full-length isoform sequencing and de novo assembly from short read sequencing.

BACKGROUND: Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms. RESULTS: The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes. CONCLUSIONS: The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.

Molecular Cloning, Characterization, and Expression Analysis of Lignin Genes from Sugarcane Genotypes Varying in Lignin Content.

Sugarcane (Saccharum spp.) is one of the highest biomass-producing plant and the best lignocellulosic feedstock for ethanol production. To achieve more efficient conversion of biomass to ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Therefore, with this objective, here, we report a systematic study on lignin content, deposition, identification, and cloning of genes involved in lignin biosynthesis and their differential expression in five sugarcane clones, EC11003, EC11010, IK 76-91, IK 76-99, and Co 86032. Lignin content among the clones varied from 26.87 to 23.19 % with the highest in the clone EC11010 and the lowest in high sugar Co86032. Lignin deposition studied through phloroglucinol staining of the cell walls implied that the sclerenchyma cells of the energy canes (EC11010 and EC11003) have more lignin deposition followed by the Erianthus (IK 76-91 and IK 76-99) clones whereas Co86032 has the minimum amount of lignin deposition. We cloned partial coding regions of important genes of lignification COMT (650 bp), CCR (332 bp), and PAL (650 bp) from Erianthus, wild relative of sugarcane followed by the expression analysis through real-time PCR. Differential expression analysis showed high level of expression for the three genes in the energy cane EC11010.

Mature embryo-derived wheat transformation for tolerance to moisture stress.

We have developed marker-free transgenic wheat using a transcription factor, AtDREB1A cloned from Arabidopsis. Southern hybridization confirmed a transgenic event with a single copy insertion. PCR analysis of the T1 plants showed four were positive only for AtDREB1A. A T1 plant (HRCB3#17-37) was marker-free and had good expression of drought tolerance in comparison with untransformed plants. The leaf relative water content of this T1 transgenic plant was 12-15% higher than that of the wild type during stress with an 8% higher yield under water deficit conditions compared with wild type plants.