Recent advances in genome editing strategies for balancing growth and defence in sugarcane (Saccharum officinarum).

Sugarcane (Saccharum officinarum ) has gained more attention worldwide in recent decades because of its importance as a bioenergy resource and in producing table sugar. However, the production capabilities of conventional varieties are being challenged by the changing climates, which struggle to meet the escalating demands of the growing global population. Genome editing has emerged as a pivotal field that offers groundbreaking solutions in agriculture and beyond. It includes inserting, removing or replacing DNA in an organism's genome. Various approaches are employed to enhance crop yields and resilience in harsh climates. These techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas). Among these, CRISPR/Cas is one of the most promising and rapidly advancing fields. With the help of these techniques, several crops like rice (Oryza sativa ), tomato (Solanum lycopersicum ), maize (Zea mays ), barley (Hordeum vulgare ) and sugarcane have been improved to be resistant to viral diseases. This review describes recent advances in genome editing with a particular focus on sugarcane and focuses on the advantages and limitations of these approaches while also considering the regulatory and ethical implications across different countries. It also offers insights into future prospects and the application of these approaches in agriculture.

Transcriptional Regulation of SugarCane Response to Sporisorium scitamineum: Insights from Time-Course Gene Coexpression and Ca2+ Signaling.

Sugarcane response to Sporisorium scitamineum is determined by multiple major genes and numerous microeffector genes. Here, time-ordered gene coexpression networks were applied to explore the interaction between sugarcane and S. scitamineum. Totally, 2459 differentially expressed genes were identified and divided into 10 levels, and several stress-related subnetworks were established. Interestingly, the Ca2+ signaling pathway was activated to establish the response to sugarcane smut disease. Accordingly, two CAX genes (ScCAX2 and ScCAX3) were cloned and characterized from sugarcane. They were significantly upregulated under ABA stress but inhibited by MeJA treatment. Furthermore, overexpression of ScCAX2 and ScCAX3 enhanced the susceptibility of transgenic plants to the pathogen infection, suggesting its negative role in disease resistance. A regulatory model for ScCAX genes in disease response was thus depicted. This work helps to clarify the transcriptional regulation of sugarcane response to S. scitamineum stress and the function of the CAX gene in disease response.

Functional characterization of sugarcane ScFTIP1 reveals its role in Arabidopsis flowering.

The timing of floral transition is essential for reproductive success in flowering plants. In sugarcane, flowering time affects the production of sugar and biomass. Although the function of the crucial floral pathway integrators, FLOWERING LOCUS T (FT), in sugarcane, has been uncovered, the proteins responsible for FT export and the underlying mechanism remain unexplored. In this study, we identified a member of the multiple C2 domain and transmembrane region proteins (MCTPs) family in sugarcane, FT-interacting protein 1 (ScFTIP1), which was localized to the endoplasmic reticulum. Ectopic expression of ScFTIP1 in the Arabidopsis mutant ftip1-1 rescued the late-flowering phenotype. ScFTIP1 interacted with AtFT in vitro and in vivo assays. Additionally, ScFTIP1 interacted with ScFT1 and the floral inducer ScFT3. Furthermore, we found that the NAC member, ScNAC23, could directly bind to the ScFTIP1 promoter and negatively regulate its transcription. Overall, our findings revealed the function of ScFTIP1 and proposed a potential mechanism underlying flowering regulation in sugarcane.

Genome-wide identification of JAZ gene family in sugarcane and function analysis of ScJAZ1/2 in drought stress response and flowering regulation.

The JASMONATE ZIM DOMAIN (JAZ) proteins are a key inhibitors of the jasmonic acid (JA) signaling pathway that play an important role in the regulation of plant growth and development and environmental stress responses. However, there is no systematic identification and functional analysis of JAZ gene family members in sugarcane. In this study, a total of 49 SsJAZ genes were identified from the wild sugarcane species Saccharum spontaneum genome that were unevenly distributed on 13 chromosomes. Phylogenetic analysis showed that all SsJAZ members can be divided into six groups, and most of the SsJAZ genes contained photoreactive and ABA-responsive elements. RNA-seq analysis revealed that SsJAZ1-1/2/3/4 and SsJAZ7-1 were significantly upregulated under drought stress. The transcript level of ScJAZ1 which is the homologous gene of SsJAZ1 in modern sugarcane cultivars was upregulated by JA, PEG, and abscisic acid (ABA). Moreover, ScJAZ1 can interact with three other JAZ proteins to form heterodimers. The spatial and temporal expression analysis showed that SsJAZ2-1/2/3/4 were highly expressed in different tissues and growth stages and during the day-night rhythm between 10:00 and 18:00. Overexpression of ScJAZ2 in Arabidopsis accelerated flowering through activating the expression of AtSOC1, AtFT, and AtLFY. Moreover, the transcription level of ScJAZ2 was about 30-fold in the early-flowering sugarcane variety than that of the non-flowering variety, indicating ScJAZ2 positively regulated flowering. This first systematic analysis of the JAZ gene family and function analysis of ScJAZ1/2 in sugarcane provide key candidate genes and lay the foundation for sugarcane breeding.

Silicon induces ROS scavengers, hormone signalling, antifungal metabolites, and silicon deposition against brown stripe disease in sugarcane.

Bipolaris setariae is known to cause brown stripe disease in sugarcane, resulting in significant yield losses. Silicon (Si) has the potential to enhance plant growth and biotic resistance. In this study, the impact of Si on brown stripe disease was investigated across susceptible and resistant sugarcane varieties, utilizing four Si concentrations (0, 15, 30, and 45 g per barrel of Na2SiO3·5H2O). Si significantly reduced the incidence of brown stripe disease (7.41-59.23%) and alleviated damage to sugarcane growth parameters, photosynthetic parameters, and photosynthetic pigments. Submicroscopic observations revealed that Si induced the accumulation of silicified cells in leaves, reduced spore accumulation, decreased stomatal size, and protected organelles from B. setariae damage. In addition, Si increased the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), reduced reactive oxygen species production (malondialdehyde and hydrogen peroxide) and modulated the expression of genes associated with hormone signalling (PR1, TGA, AOS, AOC, LOX, PYL8, and SnRK2), leading to the accumulation of abscisic acid and jasmonic acid and inhibiting SA synthesis. Si also activated the activity of metabolism-related enzymes (polyphenol oxidase and phenylalanine ammonia lyase) and the gene expression of PAL-dependent genes (PAL, C4H, and 4CL), regulating the accumulation of metabolites, such as chlorogenic acid and lignin. The antifungal test showed that chlorogenic acid (15ug µL-1) had a significant inhibitory effect on the growth of B. setariae. This study is the first to demonstrate the inhibitory effect of Si on B. setariae in sugarcane, highlighting Si as a promising and environmentally friendly strategy for managing brown stripe disease.

Transcriptome and small RNA analysis unveils novel insights into the C4 gene regulation in sugarcane.

MAIN CONCLUSION: This study reveals miRNA indirect regulation of C4 genes in sugarcane through transcription factors, highlighting potential key regulators like SsHAM3a. C4 photosynthesis is crucial for the high productivity and biomass of sugarcane, however, the miRNA regulation of C4 genes in sugarcane remains elusive. We have identified 384 miRNAs along the leaf gradients, including 293 known miRNAs and 91 novel miRNAs. Among these, 86 unique miRNAs exhibited differential expression patterns, and we identified 3511 potential expressed targets of these differentially expressed miRNAs (DEmiRNAs). Analyses using Pearson correlation coefficient (PCC) and Gene Ontology (GO) enrichment revealed that targets of miRNAs with positive correlations are integral to chlorophyll-related photosynthetic processes. In contrast, negatively correlated pairs are primarily associated with metabolic functions. It is worth noting that no C4 genes were predicted as targets of DEmiRNAs. Our application of weighted gene co-expression network analysis (WGCNA) led to a gene regulatory network (GRN) suggesting miRNAs might indirectly regulate C4 genes via transcription factors (TFs). The GRAS TF SsHAM3a emerged as a potential regulator of C4 genes, targeted by miR171y and miR171am, and exhibiting a negative correlation with miRNA expression along the leaf gradient. This study sheds light on the complex involvement of miRNAs in regulating C4 genes, offering a foundation for future research into enhancing sugarcane's photosynthetic efficiency.

Transcriptome scale analysis to decode the differential sucrose accumulation mechanisms in sugarcane under the effect of gibberellin.

In the present study, we analyzed GA3 (gibberellin)-treated sugarcane samples at the transcriptomic level to elucidate the differential expression of genes that influence sucrose accumulation. Previous research has suggested that GA3 application can potentially delay sink saturation by enhancing sink strength and demand, enabling the accommodation of more sucrose. To investigate the potential role of GA-induced modification of sink capacity in promoting higher sucrose accumulation, we sought to unravel the differential expression of transcripts and analyze their functional annotation. Several genes homologous to the sugar-phosphate/phosphate translocator, UTP-glucose-1-phosphate uridylyltransferase, and V-ATPases (vacuolar-type H+ ATPase) were identified as potentially associated with the increased sucrose content observed. A differentially expressed transcript was found to be identical to the mRNA of an unknown protein. Homology-based bioinformatics analysis suggested it to be a hydrolase enzyme, which could potentially act as a stimulator of sucrose buildup. The database of differentially expressed transcripts obtained in this study under the influence of GA3 represents a valuable addition to the sugarcane transcriptomics and functional genomics knowledge base.

A chromosomal-scale genome assembly of modern cultivated hybrid sugarcane provides insights into origination and evolution.

Sugarcane is a vital crop with significant economic and industrial value. However, the cultivated sugarcane's ultra-complex genome still needs to be resolved due to its high ploidy and extensive recombination between the two subgenomes. Here, we generate a chromosomal-scale, haplotype-resolved genome assembly for a hybrid sugarcane cultivar ZZ1. This assembly contains 10.4 Gb genomic sequences and 68,509 annotated genes with defined alleles in two sub-genomes distributed in 99 original and 15 recombined chromosomes. RNA-seq data analysis shows that sugar accumulation-associated gene families have been primarily expanded from the ZZSO subgenome. However, genes responding to pokkah boeng disease susceptibility have been derived dominantly from the ZZSS subgenome. The region harboring the possible smut resistance genes has expanded significantly. Among them, the expansion of WAK and FLS2 families is proposed to have occurred during the breeding of ZZ1. Our findings provide insights into the complex genome of hybrid sugarcane cultivars and pave the way for future genomics and molecular breeding studies in sugarcane.

Identification and characterization of PAL genes involved in the regulation of stem development in Saccharum spontaneum L.

BACKGROUND: Saccharum spontaneum L. is a closely related species of sugarcane and has become an important genetic component of modern sugarcane cultivars. Stem development is one of the important factors for affecting the yield, while the molecular mechanism of stem development remains poorly understanding in S. spontaneum. Phenylalanine ammonia-lyase (PAL) is a vital component of both primary and secondary metabolism, contributing significantly to plant growth, development and stress defense. However, the current knowledge about PAL genes in S. spontaneum is still limited. Thus, identification and characterization of the PAL genes by transcriptome analysis will provide a theoretical basis for further investigation of the function of PAL gene in sugarcane. RESULTS: In this study, 42 of PAL genes were identified, including 26 SsPAL genes from S. spontaneum, 8 ShPAL genes from sugarcane cultivar R570, and 8 SbPAL genes from sorghum. Phylogenetic analysis showed that SsPAL genes were divided into three groups, potentially influenced by long-term natural selection. Notably, 20 SsPAL genes were existed on chromosomes 4 and 5, indicating that they are highly conserved in S. spontaneum. This conservation is likely a result of the prevalence of whole-genome replications within this gene family. The upstream sequence of PAL genes were found to contain conserved cis-acting elements such as G-box and SP1, GT1-motif and CAT-box, which collectively regulate the growth and development of S. spontaneum. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that SsPAL genes of stem had a significantly upregulated than that of leaves, suggesting that they may promote the stem growth and development, particularly in the + 6 stem (The sixth cane stalk from the top to down) during the growth stage. CONCLUSIONS: The results of this study revealed the molecular characteristics of SsPAL genes and indicated that they may play a vital role in stem growth and development of S. spontaneum. Altogether, our findings will promote the understanding of the molecular mechanism of S. spontaneum stem development, and also contribute to the sugarcane genetic improving.

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.

Unveiling the predominance of Saccharum spontaneum alleles for resistance to orange rust in sugarcane using genome-wide association.

KEY MESSAGE: Six QTLs of resistance to sugarcane orange rust were identified in modern interspecific hybrids by GWAS. For five of them, the resistance alleles originated from S. spontaneum. Altogether, they efficiently predict disease resistance. Sugarcane orange rust (SOR) is a threatening emerging disease in many sugarcane industries worldwide. Improving the genetic resistance of commercial cultivars remains the most promising solution to control this disease. In this study, an association panel of 568 modern interspecific sugarcane hybrids (Saccharum officinarum x S. spontaneum) from Réunion's breeding program was evaluated for its resistance to SOR under natural conditions of infection. Two genome-wide association studies (GWAS) were conducted between disease reactions and 183,842 single nucleotide polymorphism (SNP) markers obtained by targeted genotyping-by-sequencing. Five resistance quantitative trait loci (QTLs), named Oru1, Oru2, Oru3, Oru4 and Oru5, were identified using a single-locus GWAS (SL-GWAS). These five QTLs all originated from the species S. spontaneum. A multi-locus GWAS (ML-GWAS) uncovered an additional but less significant resistance QTL named Oru6, which originated from S. officinarum. All six QTLs had a moderate to major phenotypic effect on disease resistance. Prediction accuracy estimated with linear regression models based on each of the five QTLs identified by SL-GWAS was between 0.16-0.41. Altogether, these five QTLs provided a relatively high prediction accuracy of 0.60. In comparison, accuracies obtained with six genome-wide prediction models (i.e., GBLUP, Bayes-A, Bayes-B, Bayes-C, Bayesian Lasso and RKHS) reached only 0.65. The good prediction accuracy of disease resistance provided by the QTLs and the predominant S. spontaneum origin of their resistance alleles pave the way for effective marker-assisted breeding strategies.

The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

A Comprehensive Analysis of Transcriptomics and Metabolomics Revealed Key Pathways Involved in Saccharum spontaneum Defense against Sporisorium scitamineum.

Sugarcane smut, caused by Sporisorium scitamineum, poses a severe threat to sugarcane production. The genetic basis of sugarcane resistance to S. scitamineum remains elusive. A comparative transcriptomic and metabolomic study was conducted on two wild Saccharum species of S. spontaneum with contrast smut resistance. Following infection, the resistant line exhibited greater down-regulation of genes and metabolites compared to the susceptible line, indicating distinct biological processes. Lignan and lignin biosynthesis and SA signal transduction were activated in the resistant line, while flavonoid biosynthesis and auxin signal transduction were enhanced in the susceptible line. TGA2.2 and ARF14 were identified as playing positive and negative roles, respectively, in plant defense. Exogenous auxin application significantly increased the susceptibility of S. spontaneum to S. scitaminum. This study established the significant switching of defense signaling pathways in contrast-resistant S. spontaneum following S. scitamineum infection, offering a hypothetical model and candidate genes for further research into sugarcane smut disease.

Amino acid metabolism and MAP kinase signaling pathway play opposite roles in the regulation of ethanol production during fermentation of sugarcane molasses in budding yeast.

Sugarcane molasses is one of the main raw materials for bioethanol production, and Saccharomyces cerevisiae is the major biofuel-producing organism. In this study, a batch fermentation model has been used to examine ethanol titers of deletion mutants for all yeast nonessential genes in this yeast genome. A total of 42 genes are identified to be involved in ethanol production during fermentation of sugarcane molasses. Deletion mutants of seventeen genes show increased ethanol titers, while deletion mutants for twenty-five genes exhibit reduced ethanol titers. Two MAP kinases Hog1 and Kss1 controlling the high osmolarity and glycerol (HOG) signaling and the filamentous growth, respectively, are negatively involved in the regulation of ethanol production. In addition, twelve genes involved in amino acid metabolism are crucial for ethanol production during fermentation. Our findings provide novel targets and strategies for genetically engineering industrial yeast strains to improve ethanol titer during fermentation of sugarcane molasses.

Genomic investigation of duplication, functional conservation, and divergence in the LRR-RLK Family of Saccharum.

BACKGROUND: Sugarcane (Saccharum spp.) holds exceptional global significance as a vital crop, serving as a primary source of sucrose, bioenergy, and various by-products. The optimization of sugarcane breeding by fine-tuning essential traits has become crucial for enhancing crop productivity and stress resilience. Leucine-rich repeat receptor-like kinases (LRR-RLK) genes present promising targets for this purpose, as they are involved in various aspects of plant development and defense processes. RESULTS: Here, we present a detailed overview of phylogeny and expression of 288 (495 alleles) and 312 (1365 alleles) LRR-RLK genes from two founding Saccharum species, respectively. Phylogenetic analysis categorized these genes into 15 subfamilies, revealing considerable expansion or reduction in certain LRR-type subfamilies. Compared to other plant species, both Saccharum species had more significant LRR-RLK genes. Examination of cis-acting elements demonstrated that SsLRR-RLK and SoLRR-RLK genes exhibited no significant difference in the types of elements included, primarily involved in four physiological processes. This suggests a broad conservation of LRR-RLK gene function during Saccharum evolution. Synteny analysis indicated that all LRR-RLK genes in both Saccharum species underwent gene duplication, primarily through whole-genome duplication (WGD) or segmental duplication. We identified 28 LRR-RLK genes exhibiting novel expression patterns in response to different tissues, gradient development leaves, and circadian rhythm in the two Saccharum species. Additionally, SoLRR-RLK104, SoLRR-RLK7, SoLRR-RLK113, and SsLRR-RLK134 were identified as candidate genes for sugarcane disease defense response regulators through transcriptome data analysis of two disease stresses. This suggests LRR-RLK genes of sugarcane involvement in regulating various biological processes, including leaf development, plant morphology, photosynthesis, maintenance of circadian rhythm stability, and defense against sugarcane diseases. CONCLUSIONS: This investigation into gene duplication, functional conservation, and divergence of LRR-RLK genes in two founding Saccharum species lays the groundwork for a comprehensive genomic analysis of the entire LRR-RLK gene family in Saccharum. The results reveal LRR-RLK gene played a critical role in Saccharum adaptation to diverse conditions, offering valuable insights for targeted breeding and precise phenotypic adjustments.

Assessment of physio-biochemical assessment and gene expression analysis of sugarcane genotypes under water stress.

BACKGROUND: Sugarcane, an economically important crop cultivated for its unique character of accumulating sucrose into its stalk and the world's major crop according to production quantity. Sugarcane production is negatively influenced by abiotic stresses because it faces all types of environments due to its long-life cycle period. Among the various abiotic stresses, drought is one of the major limiting factors creates obstacle in sugarcane production. Thus, an attempt was made to assess the molecular insights into sugarcane genotypes under water stress. A preliminary screening was done in ten sugarcane genotypes grown under semi-arid region of India through physiological, biochemical and antioxidant responses of these genotypes under two water deficit levels. METHODS: In the current study, drought was imposed on ten sugarcane genotypes during their formative stage (110 DAP) by depriving them of irrigation. A pot experiment was carried out to see how several commercial sugarcane genotypes responded to water scarcity. Sugarcane received two treatments, the first after 125 days and the second after 140 days. The physio-biochemical and antioxidant responses recorded were RWC, MSI, SCMR, Proline accumulation, SOD, Catalase, Peroxidase and Lipid peroxidation. The significant variations were recorded in responses of all genotypes. On the basis of physio-biochemical, three genotypes Cos 98,014, Cos 13,235 and Colk 14,201 were selected for differential gene expression pattern analysis. The total RNA was isolated and reverse transcribe to cDNA and real time PCR was performed for expression analysis under 10 genes. RESULTS: Under drought conditions, all sugarcane genotypes showed significantly decreased RWC, chlorophyll content, and MSI. However, when water was scarce, proline buildup, malondialdehyde (MDA) contents, enzymatic antioxidant activity (CAT, POD, and SOD), and contents all increased dramatically. Finally, in all physiological and biochemical parameters, Co 98,014 genotype displayed superior adaptation responses to drought stress, followed by Co 018, Cos 13,235, and Colk 14,201. For gene expression analysis out of 21 genes, 10 genes were expressed in sugarcane genotypes, in which 7 genes (Shbbx2, Shbbx3, Shbbx4, Shbbx5, Shbbx8, Shbbx15 and Shbbx20) were upregulated and 3 genes (Shbbx1, Shbbx16 and Shbbx17) were downregulated. CONCLUSION: The statistical analysis conducted in this study demonstrated that drought stress had a negative impact on physiological responses, including RWC, SPAD, and MSI, in sugarcane crops. However, it was found that the crops were able to survive in these stress conditions by increasing their biochemical parameters, all while maintaining their growth and function.

Characterization of sugarcane mutants developed through gamma irradiations for their lignin content and caffeic acid-O-methyl transferase (COMT) gene mutations.

PURPOSE: Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. MATERIALS AND METHODS: The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. RESULTS: In comparison to the control (21.5%), the mutant plants' lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. CONCLUSIONS: Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

Small secreted effector protein from Fusarium sacchari suppresses host immune response by inhibiting ScPi21-induced cell death.

Fusarium sacchari is one of the primary pathogens causing pokkah boeng disease, which impairs the yield and quality of sugarcane around the world. Understanding the molecular mechanisms of the F. sacchari effectors that regulate plant immunity is of great importance for the development of novel strategies for the persistent control of pokkah boeng disease. In a previous study, Fs00367 was identified to inhibit BAX-induced cell death. In this study, Fs00367nsp (without signal peptide) was found to suppress BAX-induced cell death, reactive oxygen species bursts and callose accumulation. The amino acid region 113-142 of Fs00367nsp is the functional region. Gene mutagenesis indicated that Fs00367 is important for the full virulence of F. sacchari. A yeast two-hybrid assay revealed an interaction between Fs00367nsp and sugarcane ScPi21 in yeast that was further confirmed using bimolecular fluorescence complementation, pull-down assay and co-immunoprecipitation. ScPi21 can induce plant immunity, but this effect could be blunted by Fs00367nsp. These results suggest that Fs00367 is a core pathogenicity factor that suppresses plant immunity through inhibiting ScPi21-induced cell death. The findings of this study provide new insights into the molecular mechanisms of effectors in regulating plant immunity.

Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor.

Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.

Use of continuous genotypes for genomic prediction in sugarcane.

Genomic selection in sugarcane faces challenges due to limited genomic tools and high genomic complexity, particularly because of its high and variable ploidy. The classification of genotypes for single nucleotide polymorphisms (SNPs) becomes difficult due to the wide range of possible allele dosages. Previous genomic studies in sugarcane used pseudo-diploid genotyping, grouping all heterozygotes into a single class. In this study, we investigate the use of continuous genotypes as a proxy for allele-dosage in genomic prediction models. The hypothesis is that continuous genotypes could better reflect allele dosage at SNPs linked to mutations affecting target traits, resulting in phenotypic variation. The dataset included genotypes of 1318 clones at 58K SNP markers, with about 26K markers filtered using standard quality controls. Predictions for tonnes of cane per hectare (TCH), commercial cane sugar (CCS), and fiber content (Fiber) were made using parametric, non-parametric, and Bayesian methods. Continuous genotypes increased accuracy by 5%-7% for CCS and Fiber. The pseudo-diploid parametrization performed better for TCH. Reproducing kernel Hilbert spaces model with Gaussian kernel and AK4 (arc-cosine kernel with hidden layer 4) kernel outperformed other methods for TCH and CCS, suggesting that non-additive effects might influence these traits. The prevalence of low-dosage markers in the study may have limited the benefits of approximating allele-dosage information with continuous genotypes in genomic prediction models. Continuous genotypes simplify genomic prediction in polyploid crops, allowing additional markers to be used without adhering to pseudo-diploid inheritance. The approach can particularly benefit high ploidy species or emerging crops with unknown ploidy.