Herbicides in the initial growth and chlorophyll indices of sugarcane in pre-sprouted seedlings.

The aim of the present study was to assess the selectivity of herbicides applied in the pre and post-planting of pre-sprouted seedlings (PSS). The experiment was conducted in a greenhouse, using a completely randomized design, with nine treatments and four repetitions. The IACSP95-5000 cultivar was used. The treatments consisted of herbicides applied in five doses as follows: in pre-planting: sulfentrazone, diclosulam, imazapic, and imazapyr, in post-planting: ethoxysulfuron, halosulfuron, 2,4-D and MSMA, and a control treatment without herbicide. Injury symptoms were assessed at 7, 15, 30, 45, 60, 75, and 90 days after application (DAA), height, diameter, number of tillers, chlorophyll A, B, and total chlorophyll at 30, 60, and 90 DAA, leaf area, shoot and root dry mass at 90 DAA. The post-emergent herbicides were considered selective. Pre-planting herbicides reduced the variables assessed at 90 DAA, observed by linear regression for diclosulam and imazapic, and exponential regression for imazapyr. Treatments with sulfentrazone caused few injury symptoms, with subsequent recovery. In addition, all the variables analyzed were equal to control, with the sulfentrazone considered selective for the IACSP95-5000 cultivar.

Transcriptome analysis of sugarcane reveals rapid defense response of SES208 to Xanthomonas albilineans in early infection.

BACKGROUND: Diseases are the major factor affecting the quality and yield of sugarcane during its growth and development. However, our knowledge about the factors regulating disease responses remain limited. The present study focuses on identifying genes regulating transcriptional mechanisms responsible for resistance to leaf scald caused by Xanthomonas albilineans in S. spontaneum and S. officinarum. RESULTS: After inoculation of the two sugarcane varieties SES208 (S. spontaneum) and LA Purple (S. officinarum) with Xanthomonas albilineans, SES208 exhibited significantly greater resistance to leaf scald caused by X. albilineans than did LA Purple. Using transcriptome analysis, we identified a total of 4323 and 1755 differentially expressed genes (DEGs) in inoculated samples of SES208 and LA Purple, respectively. Significantly, 262 DEGs were specifically identified in SES208 that were enriched for KEGG pathway terms such as plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction. Furthermore, we built a transcriptional regulatory co-expression network that specifically identified 16 and 25 hub genes in SES208 that were enriched for putative functions in plant-pathogen interactions, MAPK signaling, and plant hormone signal transduction. All of these essential genes might be significantly involved in resistance-regulating responses in SES208 after X. albilineans inoculation. In addition, we found allele-specific expression in SES208 that was associated with the resistance phenotype of SES208 when infected by X. albilineans. After infection with X. albilineans, a great number of DEGs associated with the KEGG pathways 'phenylpropanoid biosynthesis' and 'flavonoid biosynthesis' exhibited significant expression changes in SES208 compared to LA Purple that might contribute to superior leaf scald resistance in SES208. CONCLUSIONS: We provided the first systematical transcriptome map that the higher resistance of SES208 is associated with and elicited by the rapid activation of multiple clusters of defense response genes after infection by X. albilineans and not merely due to changes in the expression of genes generically associated with stress resistance. These results will serve as the foundation for further understanding of the molecular mechanisms of resistance against X. albilineans in S. spontaneum.

Redox homeostasis at SAM: a new role of HINT protein.

MAIN CONCLUSION: ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Integrated Analysis of Transcriptome and Metabolome Reveals the Regulation of Chitooligosaccharide on Drought Tolerance in Sugarcane (Saccharum spp. Hybrid) under Drought Stress.

Sugarcane (Saccharum spp. hybrid) is an important crop for sugar and biofuels, and often suffers from water shortages during growth. Currently, there is limited knowledge concerning the molecular mechanism involved in sugarcane response to drought stress (DS) and whether chitooligosaccharide could alleviate DS. Here, we carried out a combined transcriptome and metabolome of sugarcane in three different treatment groups: control group (CG), DS group, and DS + chitooligosaccharide group (COS). A total of 12,275 (6404 up-regulated and 5871 down-regulated) differentially expressed genes (DEGs) were identified when comparing the CG and DS transcriptomes (T_CG/DS), and 2525 (1261 up-regulated and 1264 down-regulated) DEGs were identified in comparing the DS and COS transcriptomes (T_DS/COS). GO and KEGG analysis showed that DEGs associated with photosynthesis were significantly enriched and had down-regulated expression. For T_DS/COS, photosynthesis DEGs were also significantly enriched but had up-regulated expression. Together, these results indicate that DS of sugarcane has a significantly negative influence on photosynthesis, and that COS can alleviate these negative effects. In metabolome analysis, lipids, others, amino acids and derivatives and alkaloids were the main significantly different metabolites (SDMs) observed in sugarcane response to DS, and COS treatment reduced the content of these metabolites. KEGG analysis of the metabolome showed that 2-oxocarboxylic acid metabolism, ABC transporters, biosynthesis of amino acids, glucosinolate biosynthesis and valine, leucine and isoleucine biosynthesis were the top-5 KEGG enriched pathways when comparing the CG and DS metabolome (M_CG/DS). Comparing DS with COS (M_DS/COS) showed that purine metabolism and phenylalanine metabolism were enriched. Combined transcriptome and metabolome analysis revealed that pyruvate and phenylalanine metabolism were KEGG-enriched pathways for CG/DS and DS/COS, respectively. For pyruvate metabolism, 87 DEGs (47 up-regulated and 40 down-regulated) and five SDMs (1 up-regulated and 4 down-regulated) were enriched. Pyruvate was closely related with 14 DEGs (|r| > 0.99) after Pearson's correlation analysis, and only 1 DEG (Sspon.02G0043670-1B) was positively correlated. For phenylalanine metabolism, 13 DEGs (7 up-regulated and 6 down-regulated) and 6 SDMs (1 up-regulated and 5 down-regulated) were identified. Five PAL genes were closely related with 6 SDMs through Pearson's correlation analysis, and the novel.31257 gene had significantly up-regulated expression. Collectively, our results showed that DS has significant adverse effects on the physiology, transcriptome, and metabolome of sugarcane, particularly genes involved in photosynthesis. We further show that COS treatment can alleviate these negative effects.

Cell wall formation pathways are differentially regulated in sugarcane contrasting genotypes associated with endophytic diazotrophic bacteria.

MAIN CONCLUSION: Differences in cell wall components between two BNF-contrasting sugarcane genotypes might result from genetic variations particular to the genotype and from the efficiency in diazotrophic bacteria association. Sugarcane is a plant of the grass family (Poaceae) that is highly cultivated in Brazil, as an important energy resource. Commercial sugarcane genotypes may be successfully associated with beneficial endophytic nitrogen-fixing bacteria, which can influence several plant metabolic pathways, such as cell division and growth, synthesis of hormones, and defense compounds. In this study, we investigated how diazotrophic bacteria associated with sugarcane plants could be involved in the regulation of cell wall formation pathways. A molecular and structural characterization of the cell wall was compared between two genotypes of sugarcane with contrasting rates of Biological Nitrogen Fixation (BNF): SP70-1143 (high BNF) and Chunee (low BNF). Differentially expressed transcripts were identified in transcriptomes generated from SP70-1143 and Chunee. Expression profiles of cellulose and lignin genes, which were more expressed in SP70-1134, and callose genes, which were more expressed in Chunee, were validated by RT-qPCR and microscopic analysis of cell wall components in tissue sections. A similar expression profile in both BNF-contrasting genotypes was observed in naturally colonized plants and in plants inoculated with G. diazotrophicus. Cell walls of the high BNF genotype have a greater cellulose content, which might contribute to increase biomass. In parallel, callose was concentrated in the vascular tissues of the low BNF genotype and could possibly represent a barrier for an efficient bacterial colonization and dissemination in sugarcane tissues. Our data show a correlation between the gene profiles identified in the BNF-contrasting genotypes and a successful association with endophytic diazotrophic bacteria.

Assessing the performance of two gridded weather data for sugarcane crop simulations with a process-based model in Center-South Brazil.

High-quality measured weather data (MWD) are essential for long-term and in-season crop model applications. When MWD is not available, one alternative for crop simulations is to employ gridded weather data (GWD), which needs to be evaluated a priori. Therefore, this study aimed to evaluate the impact of weather data from two GWD sources (NASA and XAVIER), in the way that they are available for end users, on simulating sugarcane crop performance within the APSIM-Sugar model at traditional sites where sugarcane is grown in Center-South Brazil, compared to simulations with MWD. Besides, this study also evaluated the impact of replacing GWD rainfall by the site-specific measured data on such simulations. A common sugarcane cropping system was repeatedly simulated between 1997 and 2015 for different combinations of climate input. Both NASA and XAVIER appear to be interesting for applications that only require temperature and solar radiation for predictions, such as crop phenology and potential yield. Nonetheless, GWD should be used with caution for crop model applications that rely on accurate estimation of crop water balance, canopy development, and biomass accumulation, at least with crop models that run at a daily time-step. The replacement of gridded rainfall with measured rainfall was pivotal for improving sugarcane simulations, as observed for cane yield, by increasing both agreement (NASA d index from 0.67 to 0.90; XAVIER d from 0.73 to 0.93) and R2 (NASA from 0.35 to 0.76; XAVIER from 0.43 to 0.79) and reducing root mean square errors (RMSE) from 32.8 to 16.3 t/ha when simulated with other variables of NASA data and from 27.9 to 12.7 t/ha when having XAVIER data as input. Therefore, while using both GWD sets without any correction, it is recommended to replace gridded rainfall by measured values, whenever possible, to improve sugarcane simulations in Center-South Brazil.

Molecular Detection and Quantification of Xanthomonas albilineans in Juice from Symptomless Sugarcane Stalks Using a Real-Time Quantitative PCR Assay.

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A sensitive and robust quantitative PCR (qPCR) assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis groups. The detection thresholds were 100 copies/µl of plasmid DNA, 100 fg/µl of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) of symptomless stalk samples, whereas only 28.4% (155/546) of samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi Province) and Zhanjiang (Guangdong Province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Metabolic Regulation and Development of Energy Cane Setts upon Auxin Stimulus.

Energy cane is a bioenergy crop with an outstanding ability to bud sprouting and increasing yield in ratoon cycles even in marginal lands. Bud fate control is key to biomass production and crop profits due to vegetative propagation and tiller dependency, as well as phenotype plasticity to withstand harsh environmental conditions. During the establishment stage (plant cane cycle), energy cane has a tendency for low root:shoot ratio, which might hamper the ability to cope with stress. Auxin is known to modulate bud sprouting and stimulate rooting in sugarcane. Hence, we treated a slow and a fast bud sprouting energy cane cultivars with auxin or controls (with and without water soaking) for 6 h prior to planting and evaluate plant growth parameters and metabolic profiling using two techniques (gas chromatography with time-of-flight mass spectrometer and nuclear magnetic resonance) to characterize the effect and identify metabolite markers associated with bud inhibition and outgrowth. Auxin inhibited bud burst and promote rooting in setts changing the root:shoot ratio of plantlets. Metabolome allowed the identification of lactate, succinate and aspartate family amino acids as involved in bud fate control through the potential modulation of oxygen and energy status. Investigating environmental and biochemical factors that regulate bud fate can be incremental to other monocot species. Our study provides new insights into bud quiescence and outgrowth in cane hybrids, with the potential to leverage our understanding of yield-related traits, crop establishment and adaptation to global climate change.

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.

Characterization of an AP endonuclease from sugarcane - ScARP1.

Plants are sessile organisms that need to cope with different conditions. The Base Excision Repair (BER) pathway is an important mechanism protecting the genome from DNA lesions. Apurinic/apyrimidinic (AP) endonucleases are key BER enzymes that process AP sites arising either spontaneously or as BER intermediates. In Arabidopsis there are three AP endonucleases: AtARP1, AtAPE1L, and AtAPE2, and in sugarcane two AtARP1 homologues have been identified: ScARP1 and ScARP3. ScARP1 shares 59% sequence identity with Arabidopsis AtARP. Protein modeling of ScARP1 and AtARP1 revealed conserved active sites and metal binding sites. For biochemical characterisation, recombinant ScARP1 protein displayed AP endonuclease activity both in the presence of MnCl2 or MgCl2 and the optimal temperature for its activity was 37 °C. Under these conditions, 3'-exonuclease, 3'-phosphatase, and 3'-phosphodiesteterase activities were not detectable. We also show that ScARP1 protein is able to complement mutant atarp-/- cell extracts deficient in AP endonuclease activity. These results suggest that AP endonucleases from different plant species preserve AP endonuclease activity. The biochemical characterisation of ScARP1 extends our knowledge of the BER pathway to a monocot crop plant group.

Decarboxylation mechanisms of C4 photosynthesis in Saccharum spp.: increased PEPCK activity under water-limiting conditions.

BACKGROUND: C4 plants have been classified into three subtypes based on the enzymes used to decarboxylate C4 acids in the bundle sheath cells (NADP-ME, NAD-ME and PEPCK pathways). Evidences indicate that, depending on environmental factors, C4 plants may exhibit a certain degree of flexibility in the use of the decarboxylation mechanisms. In this context, the objective was to extend the knowledge on the degree of flexibility between the pathways of decarboxylation in sugarcane, a NADP-ME species, at different levels of water deficit. RESULTS: An experiment was carried out with two cultivars - RB92579 (tolerant to water deficit) and SP80-3280 (susceptible to water deficit) subjected to moderate level (- 1.5 to - 1.8 MPa), severe level (below - 2.0 MPa) and recovery (48 h after rehydration) and changes in the activities of the enzymes involved in the three C4 mechanisms and in gene expression were investigated. Our results showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both cultivars. CONCLUSIONS: The results obtained here, show that sugarcane increases the use of the PEPCK pathway as a decarboxylation mechanism, in addition to the NADP-ME pathway, under conditions of water deficit, particularly in the tolerant cultivar.

Target enrichment sequencing of 307 germplasm accessions identified ancestry of ancient and modern hybrids and signatures of adaptation and selection in sugarcane (Saccharum spp.), a 'sweet' crop with 'bitter' genomes.

Sugarcane (Saccharum spp.) is a highly energy-efficient crop primarily for sugar and bio-ethanol production. Sugarcane genetics and cultivar improvement have been extremely challenging largely due to its complex genomes with high polyploidy levels. In this study, we deeply sequenced the coding regions of 307 sugarcane germplasm accessions. Nearly five million sequence variations were catalogued. The average of 98× sequence depth enabled different allele dosages of sequence variation to be differentiated in this polyploid collection. With selected high-quality genome-wide SNPs, we performed population genomic studies and environmental association analysis. Results illustrated that the ancient sugarcane hybrids, S. barberi and S. sinense, and modern sugarcane hybrids are significantly different in terms of genomic compositions, hybridization processes and their potential ancestry contributors. Linkage disequilibrium (LD) analysis showed a large extent of LD in sugarcane, with 962.4 Kbp, 2739.2 Kbp and 3573.6 Kbp for S. spontaneum, S. officinarum and modern S. hybrids respectively. Candidate selective sweep regions and genes were identified during domestication and historical selection processes of sugarcane in addition to genes associated with environmental variables at the original locations of the collection. This research provided an extensive amount of genomic resources for sugarcane community and the in-depth population genomic analyses shed light on the breeding and evolution history of sugarcane, a highly polyploid species.

Comparative Transcriptome Profiling of Resistant and Susceptible Sugarcane Cultivars in Response to Infection by Xanthomonas albilineans.

Sugarcane (Saccharum spp. hybrids) is a major source of sugar and renewable bioenergy crop worldwide and suffers serious yield losses due to many pathogen infections. Leaf scald caused by Xanthomonas albilineans is a major bacterial disease of sugarcane in most sugarcane-planting countries. The molecular mechanisms of resistance to leaf scald in this plant are, however, still unclear. We performed a comparative transcriptome analysis between resistant (LCP 85-384) and susceptible (ROC20) sugarcane cultivars infected by X. albilineans using the RNA-seq platform. 24 cDNA libraries were generated with RNA isolated at four time points (0, 24, 48, and 72 h post inoculation) from the two cultivars with three biological replicates. A total of 105,783 differentially expressed genes (DEGs) were identified in both cultivars and the most upregulated and downregulated DEGs were annotated for the processes of the metabolic and single-organism categories, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the 7612 DEGs showed that plant-pathogen interaction, spliceosome, glutathione metabolism, protein processing in endoplasmic reticulum, and plant hormone signal transduction contributed to sugarcane's response to X. albilineans infection. Subsequently, relative expression levels of ten DEGs determined by quantitative reverse transcription-PCR (qRT-PCR), in addition to RNA-Seq data, indicated that different plant hormone (auxin and ethylene) signal transduction pathways play essential roles in sugarcane infected by X. albilineans. In conclusion, our results provide, for the first time, valuable information regarding the transcriptome changes in sugarcane in response to infection by X. albilineans, which contribute to the understanding of the molecular mechanisms underlying the interactions between sugarcane and this pathogen and provide important clues for further characterization of leaf scald resistance in sugarcane.

Plant Cell Wall Proteomics: A Focus on Monocot Species, Brachypodium distachyon, Saccharum spp. and Oryza sativa.

Plant cell walls mostly comprise polysaccharides and proteins. The composition of monocots' primary cell walls differs from that of dicots walls with respect to the type of hemicelluloses, the reduction of pectin abundance and the presence of aromatic molecules. Cell wall proteins (CWPs) differ among plant species, and their distribution within functional classes varies according to cell types, organs, developmental stages and/or environmental conditions. In this review, we go deeper into the findings of cell wall proteomics in monocot species and make a comparative analysis of the CWPs identified, considering their predicted functions, the organs analyzed, the plant developmental stage and their possible use as targets for biofuel production. Arabidopsis thaliana CWPs were considered as a reference to allow comparisons among different monocots, i.e., Brachypodium distachyon, Saccharum spp. and Oryza sativa. Altogether, 1159 CWPs have been acknowledged, and specificities and similarities are discussed. In particular, a search for A. thaliana homologs of CWPs identified so far in monocots allows the definition of monocot CWPs characteristics. Finally, the analysis of monocot CWPs appears to be a powerful tool for identifying candidate proteins of interest for tailoring cell walls to increase biomass yield of transformation for second-generation biofuels production.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

miRNA alteration is an important mechanism in sugarcane response to low-temperature environment.

BACKGROUND: Cold is a major abiotic stress limiting the production of tropical and subtropical crops in new production areas. Sugarcane (Saccharum spp.) originates from the tropics but is cultivated primarily in the sub-tropics where it frequently encounters cold stress. Besides regulating plant growth, miRNAs play an important role in environmental adaption. RESULTS: In this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. Among them, 62 exhibited significant differential expression under cold stress, with 34 being upregulated and 28 being downregulated. The expression of 13 miRNAs and 12 corresponding targets was validated by RT-qPCR, with the majority being consistent with the sequencing data. GO and KEGG analysis indicated that these miRNAs were involved in stress-related biological pathways. To further investigate the involvement of these miRNAs in tolerance to abiotic stresses, sugarcane miR156 was selected for functional analysis. RT-qPCR revealed that miR156 levels increased in sugarcane during cold, salt and drought stress treatments. Nicotiana benthamiana plants transiently overexpressing miR156 exhibited better growth status, lower ROS levels, higher anthocyanin contents as well as the induction of some cold-responsive genes, suggesting its positive role in the plant cold stress response. CONCLUSIONS: This study provides a global view of the association of miRNA expression with the sugarcane response to cold stress. The findings have enriched the present miRNA resource and have made an attempt to verify the involvement of miR156 in plant response to cold stress.

GBS-based single dosage markers for linkage and QTL mapping allow gene mining for yield-related traits in sugarcane.

BACKGROUND: Sugarcane (Saccharum spp.) is predominantly an autopolyploid plant with a variable ploidy level, frequent aneuploidy and a large genome that hampers investigation of its organization. Genetic architecture studies are important for identifying genomic regions associated with traits of interest. However, due to the genetic complexity of sugarcane, the practical applications of genomic tools have been notably delayed in this crop, in contrast to other crops that have already advanced to marker-assisted selection (MAS) and genomic selection. High-throughput next-generation sequencing (NGS) technologies have opened new opportunities for discovering molecular markers, especially single nucleotide polymorphisms (SNPs) and insertion-deletion (indels), at the genome-wide level. The objectives of this study were to (i) establish a pipeline for identifying variants from genotyping-by-sequencing (GBS) data in sugarcane, (ii) construct an integrated genetic map with GBS-based markers plus target region amplification polymorphisms and microsatellites, (iii) detect QTLs related to yield component traits, and (iv) perform annotation of the sequences that originated the associated markers with mapped QTLs to search putative candidate genes. RESULTS: We used four pseudo-references to align the GBS reads. Depending on the reference, from 3,433 to 15,906 high-quality markers were discovered, and half of them segregated as single-dose markers (SDMs) on average. In addition to 7,049 non-redundant SDMs from GBS, 629 gel-based markers were used in a subsequent linkage analysis. Of 7,678 SDMs, 993 were mapped. These markers were distributed throughout 223 linkage groups, which were clustered in 18 homo(eo)logous groups (HGs), with a cumulative map length of 3,682.04 cM and an average marker density of 3.70 cM. We performed QTL mapping of four traits and found seven QTLs. Our results suggest the presence of a stable QTL across locations. Furthermore, QTLs to soluble solid content (BRIX) and fiber content (FIB) traits had markers linked to putative candidate genes. CONCLUSIONS: This study is the first to report the use of GBS for large-scale variant discovery and genotyping of a mapping population in sugarcane, providing several insights regarding the use of NGS data in a polyploid, non-model species. The use of GBS generated a large number of markers and still enabled ploidy and allelic dosage estimation. Moreover, we were able to identify seven QTLs, two of which had great potential for validation and future use for molecular breeding in sugarcane.

Small RNA sequencing reveals a role for sugarcane miRNAs and their targets in response to Sporisorium scitamineum infection.

BACKGROUND: Sugarcane smut caused by Sporisorium scitamineum leads to a significant reduction in cane yield and sucrose content. MicroRNAs (miRNAs) play an important role in regulating plant responses to biotic stress. The present study was the first to use two sugarcane genotypes, YA05-179 (smut-resistant) and ROC22 (smut-susceptible), to identify differentially expressed miRNAs in sugarcane challenged with S. scitamineum by using high-throughput sequencing. RESULTS: The predicted target gene number corresponding to known differentially expressed miRNAs in YA05-179 was less than that in ROC22, however most of them were in common. Expression of differential miRNAs under S. scitamineum challenge was mostly downregulated, with similar trends in the two varieties. Gene ontology (GO) analysis showed that the target gene classification of known miRNAs was similar to that of the newly identified miRNAs. These were mainly associated with cellular processes and metabolic processes in the biological process category, as well as combination and catalytic activity in the molecular function category. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these predicted target genes involved in a series of physiological and biochemical pathways or disease resistance-related physiological metabolism and signal transduction pathways, suggesting that the molecular interaction mechanism between sugarcane and S. scitamineum was a complex network system. These findings also showed certain predicted target genes of miR5671, miR5054, miR5783, miR5221, and miR6478 play roles in the mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, and plant-pathogen interaction. Quantitative real-time PCR (qRT-PCR) analysis showed that majority of the known miRNAs and its predicted target genes followed a negatively regulated mode. Seven out of eight predicted target genes showed identical expression after 12 h treatment and reached the highest degree of matching at 48 h, indicating that the regulatory role of miRNAs on the target genes in sugarcane was maximized at 48 h after S. scitamineum challenge. CONCLUSIONS: Taken together, our findings serve as evidence for the association of miRNA expression with the molecular mechanism underlying the pathogenesis of sugarcane smut, particularly on the significance of miRNA levels in relation to the cultivation of smut-resistant sugarcane varieties.

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane.

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.