Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases in the sugarcane industry. Using a molecular biological technique to mine sugarcane resistance genes can provide gene resources for further genetic engineering of sugarcane disease-resistant breeding. Jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins, which involved in the responses to plant pathogens and abiotic stresses, are important signaling molecules of the jasmonic acid (JA) pathway. Seven differentially expressed sugarcane JAZ genes, ScJAZ1–ScJAZ7, were mined from the transcriptome of sugarcane after inoculation with S. scitamineum. Bioinformatic analyses revealed that these seven ScJAZ genes encoded basic proteins that contain the TIFY and CCT_2 domains. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that the ScJAZ1–ScJAZ7 genes were tissue specific and differentially expressed under adverse stress. During S. scitamineum infection, the transcripts of ScJAZ4 and ScJAZ5 were both upregulated in the susceptible genotype ROC22 and the resistant genotype Yacheng05–179; ScJAZ1, ScJAZ2, ScJAZ3, and ScJAZ7 were downregulated in Yacheng05–179 and upregulated in ROC22; and the expression of ScJAZ6 did not change in ROC22, but was upregulated in Yacheng05–179. The transcripts of the seven ScJAZ genes were increased by the stimuli of salicylic acid and abscisic acid, particularly methyl jasmonate. The expression of the genes ScJAZ1–ScJAZ7 was immediately upregulated by the stressors hydrogen peroxide, sodium chloride, and copper chloride, whereas slightly induced after treatment with calcium chloride and polyethylene glycol. In addition, the expression of ScJAZ6, as well as seven tobacco immunity-associated marker genes were upregulated, and antimicrobial activity against Pseudomonas solanacearum and Fusarium solani var. coeruleum was observed during the transient overexpression of ScJAZ6 in Nicotiana benthamiana, suggesting that the ScJAZ6 gene is associated with plant immunity. The different expression profiles of the ScJAZ1–ScJAZ7 genes during S. scitamineum infection, the positive response of ScJAZ1–ScJAZ7 to hormones and abiotic treatments, and the function analysis of the ScJAZ6 gene revealed their involvement in the defense against biotic and abiotic stresses. The findings of the present study facilitate further research on the ScJAZ gene family especially their regulatory mechanism in sugarcane.

Sugarcane (Saccharum spp. hybrids) is considered the most globally important sugar-producing crop and raw material for biofuel. Insect attack is a major issue in sugarcane cultivation, resulting in yield losses and sucrose content reductions. Stem borer (Diatraea saccharalis F.) causes serious yield losses in sugarcane worldwide. However, insect-resistant germplasms for sugarcane are not available in any collections all over the world, and the molecular mechanism of insect resistance has not been elucidated. In this study, cry1Ac transgenic sugarcane lines were obtained and the biological characteristics and transgene dosage effect were investigated and a global exploration of gene expression by transcriptome analysis was performed. The transgene copies of foreign cry1Ac were variable and random. The correlation between the cry1Ac protein and cry1Ac gene copies differed between the transgenic lines from FN15 and ROC22. The medium copy lines from FN15 showed a significant linear relationship, while ROC22 showed no definite dosage effect. The transgenic lines with medium copies of cry1Ac showed an elite phenotype. Transcriptome analysis by RNA sequencing indicated that up/down regulated differentially expressed genes were abundant among the cry1Ac sugarcane lines and the receptor variety. Foreign cry1Ac gene and endogenous borer stress-related genes may have a synergistic effect. Three lines, namely, A1, A5, and A6, were selected for their excellent stem borer resistance and phenotypic traits and are expected to be used directly as cultivars or crossing parents for sugarcane borer resistance breeding. Cry1Ac gene integration dramatically improved sugarcane insect resistance. The elite transgenic offspring contained medium transgene copies. Foreign cry1Ac gene integration and endogenous borer stress-related genes may have a synergistic effect on sugarcane insect resistance improvement.

Sugarcane borer is the most common and harmful pest in Chinese sugarcane fields, and can cause damage to the whole plant during the entire growing season. To improve borer resistance in sugarcane, we constructed a plant expression vector pGcry2A0229 with the bar gene as the marker and the cry2A gene as the target, and introduced it into embryogenic calli of most widely cultivated sugarcane cultivar ROC22 by particle bombardment. After screening with phosphinothricin in vitro and Basta spray, 21 resistance-regenerated plants were obtained, and 10 positive transgenic lines harboring the cry2A gene were further confirmed by conventional PCR detection. Real-time quantitative PCR (RT-qPCR) analysis showed that the copy number of the cry2A gene varied among different transgenic lines but did not exceed four copies. Quantitative ELISA analysis showed that there was no linear relationship with copy number but negatively correlated with the percentage of borer-infested plants. The analysis of industrial and agronomic traits showed that the theoretical sugar yields of transgenic lines TR-4 and TR-10 were slightly lower than that of the control in both plant cane and ratoon cane; nevertheless, TR-4 and TR-10 lines exhibited markedly lower in frequency of borer-infested plants in plant cane and in the ratoon cane compared to the control. Our results indicate that the introduction of the cry2A gene via bombardment produces transgenic lines with obviously increased stem borer resistance and comparable sugar yield, providing a practical value in direct commercial cultivation and crossbreeding for ROC22 has been used as the most popular elite genitor in various breeding programs in China.

Polyploid Saccharum with complex genomes hindered the progress of sugarcane improvement, while their chloroplast genomes are much smaller and simpler. Chloroplast (cp), the vital organelle, is the site of plant photosynthesis, which also evolves other functions, such as tolerance to environmental stresses. In this study, the cp genome of two sugarcane ancestors Saccharum officinarum and S. spontaneum were sequenced, and genome comparative analysis between these two species was carried out, together with the photosynthetic ability. The length is 141,187 bp for S. officinarum and that is 7 bp longer than S. spontaneum, with the same GC content (38.44%) and annotated gene number (134), 13 with introns among them. There is a typical tetrad structure, including LSC, SSC, IRb and IRa. Of them, LSC and IRa/IRb are 18 bp longer and 6 bp shorter than those in S. spontaneum (83,047 bp and 22,795 bp), respectively, while the size of SSC is same (12,544 bp). Five genes exhibit contraction and expansion at the IR junctions, but only one gene ndhF with 29 bp expansion at the border of IRb/SSC. Nucleotide diversity (Pi) based on sliding window analysis showed that the single copy and noncoding regions were more divergent than IR- and coding regions, and the variant hotspots trnG-trnM, psbM-petN, trnR-rps14, ndhC-trnV and petA-psbJ in the LSC and trnL-ccsA in the SSC regions were detected, and petA-psbJ with the highest divergent value of 0.01500. Genetic distances of 65 protein genes vary from 0.00000 to 0.00288 between two species, and the selective pressure on them indicated that only petB was subjected to positive selection, while more genes including rpoC2, rps3, ccsA, ndhA, ndhA, psbI, atpH and psaC were subjected to purifying or very strong purifying selection. There are larger number of codons in S. spontaneum than that in S. officinarum, while both species have obvious codon preference and the codons with highest-(AUG) and lowest frequency (AUA) are same. Whilst, the most abundant amino acid is leucine in both S. officinarum and S. spontaneum, with number of 2175 (10.88% of total) and 2228 (10.90% of total) codons, respectively, and the lowest number is cysteine, with only 221 (1.105%) and 224 (1.096%), respectively. Protein collinearity analysis showed the high collinearity though several divergences were present in cp genomes, and identification of simple sequence repeats (SSRs) were included in this study. In addition, in order to compare cold tolerance and explore the expanding function of this environmental stress, the chlorophyll relative content (SPAD) and chlorophyll fluorescence Fv/Fm were measured. The significantly higher SPAD were observed in S. spontaneum than those in S. officinarum, no matter what the control conditions, exposure to low temperature or during recovery, and so was for Fv/Fm under exposure to low temperature, together with higher level of SPAD in S. spontaneum in each measurement. Aforementioned results suggest much stronger photosynthetic ability and cold tolerance in S. spontaneum. Our findings build a foundation to investigate the biological mechanism of two sugarcane ancestor chloroplasts and retrieve reliable molecular resources for phylogenetic and evolutionary studies, and will be conducive to genetic improvement of photosynthetic ability and cold resistance in modern sugarcane.

Chemical nitrogen (N) fertilizer is widely used in sugarcane production, especially in China and India. Understanding the molecular mechanisms and mining miRNAs and their target genes associated with nitrogen use efficiency (NUE) in sugarcane can aid in developing the N-efficient varieties, and thus is beneficial to reduce N fertilizer application. In this study, the root miRNA database of N-efficient sugarcane variety ROC22 under low N stress (0.3 mM NH₄NO₃) for 3 h was constructed, along with their transcriptome-rearranged data. KEGG analysis indicated that those candidate target genes, corresponding to differentially expressed miRNAs, were mainly enriched in N metabolism, amino acid metabolism, carbohydrate metabolism, photosynthesis, and hormone signal transduction pathways. It was found that under low N stress for 0–24 h, there was a negative correlation between miR168 and SPX, along with miR396 and acnA. Furthermore, the expression of miR156 in the roots of ROC22 was significantly up-regulated under low N treatment. Compared with the wild-type, the Arabidopsis plants overexpressing sugarcane miR156 exhibited significantly improved length and surface area of roots, while the expression of one NO₃⁻ transporter gene NRT1.1, three N assimilation key genes (NR1, NIR1, and GS), and the activity of two N assimilation key enzymes (NR and GS) were up-regulated under low N treatment. It can be reasonably deduced that sugarcane miR156 can enhance the nitrogen assimilation ability of the overexpressed Arabidopsis plants under low N application, and thus has a potential ability for improving sugarcane NUE. The present study should be helpful for understanding the molecular regulatory network in the N-efficient sugarcane genotype responding to low N stress and could provide the candidate miRNAs with a potential function in improving sugarcane NUE.

In China, nitrogen (N) fertilizer is excessively used in sugarcane planting areas, while the nitrogen use efficiency (NUE) of sugarcane is relatively low. Mining and identifying the key genes in response to low N stress in sugarcane can provide useful gene elements and a theoretical basis for developing sugarcane varieties with high NUE. In our study, RNA-Seq combined with qRT-PCR analysis revealed that the ScAMT1.1 gene responded positively to low N stress, resulting in the stronger low N tolerance and high NUE ability of sugarcane cultivar ROC22. Then, ScAMT1.1 was cloned from sugarcane. The full-length cDNA of the ScAMT1.1 gene is 1868 bp, containing a 1491 bp open reading frame (ORF), and encoding 496 amino acids. ScAMT1.1 belongs to the AMT superfamily and shares 91.57% homologies with AMT1.1 from Oryza sativa. Furthermore, it was stably overexpressed in rice (O. sativa). Under low N treatment, the plant height and the fresh weight of the ScAMT1.1-overexpressed transgenic rice were 36.48% and 51.55% higher than that of the wild-type, respectively. Both the activity of ammonium assimilation key enzymes GS and GDH, and the expression level of ammonium assimilation key genes, including GS1.1, GS1.2, GDH, Fd-GOGAT, and NADH-GOGAT2 in the transgenic plants, were significantly higher than that of the wild-type. The grain number and grain yield per plant in the transgenic rice were 6.44% and 9.52% higher than that of the wild-type in the pot experiments, respectively. Taken together, the sugarcane ScAMT1.1 gene has the potential to improve ammonium assimilation ability and the yield of transgenic rice under low N fertilizer conditions. This study provided an important functional gene for improving sugarcane varieties with high NUE.