Sugarcane ScOPR1 gene enhances plant disease resistance through the modulation of hormonal signaling pathways.

KEY MESSAGE: Transgenic plants stably overexpressing ScOPR1 gene enhanced disease resistance by increasing the accumulation of JA, SA, and GST, as well as up-regulating the expression of genes related to signaling pathways. 12-Oxo-phytodienoate reductase (OPR) is an oxidoreductase that depends on flavin mononucleotide (FMN) and catalyzes the conversion of 12-oxophytodienoate (12-OPDA) into jasmonic acid (JA). It plays a key role in plant growth and development, and resistance to adverse stresses. In our previous study, we have obtained an OPR gene (ScOPR1, GenBank Accession Number: MG755745) from sugarcane. This gene showed positive responses to methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), and Sporisorium scitamineum, suggesting its potential for pathogen resistance. Here, in our study, we observed that Nicotiana benthamiana leaves transiently overexpressing ScOPR1 exhibited weaker disease symptoms, darker 3,3-diaminobenzidine (DAB) staining, higher accumulation of reactive oxygen species (ROS), and higher expression of hypersensitive response (HR) and SA pathway-related genes after inoculation with Ralstonia solanacearum and Fusarium solanacearum var. coeruleum. Furthermore, the transgenic N. benthamiana plants stably overexpressing the ScOPR1 gene showed enhanced resistance to pathogen infection by increasing the accumulation of JA, SA, and glutathione S-transferase (GST), as well as up-regulating genes related to HR, JA, SA, and ROS signaling pathways. Transcriptome analysis revealed that the specific differentially expressed genes (DEGs) in ScOPR1-OE were significantly enriched in hormone transduction signaling and plant-pathogen interaction pathways. Finally, a functional mechanism model of the ScOPR1 gene in response to pathogen infection was depicted. This study provides insights into the molecular mechanism of ScOPR1 and presents compelling evidence supporting its positive involvement in enhancing plant disease resistance.

Sugarcane ScCAX4 is a Negative Regulator of Resistance to Pathogen Infection.

Calcium (Ca2+) is a second messenger in various physiological processes within plants. The significance of the Ca2+/H+ exchanger (CAX) has been established in facilitating Ca2+ transport in plants; however, disease resistance functions of the CAX gene remain elusive. In this study, we conducted sequence characterization and expression analysis for a sugarcane CAX gene, ScCAX4 (GenBank Accession Number: MW206380). In order to further investigate the disease resistance functions, this gene was then transiently overexpressed in Nicotiana benthamiana leaves, which were subsequently inoculated with Fusarium solani var. coeruleum. Results showed that ScCAX4 overexpression increased the susceptibility of N. benthamiana to pathogen infection by regulating the expression of genes related to salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways, suggesting its negative role in disease resistance. Furthermore, we genetically transformed the ScCAX4 gene into N. benthamiana and obtained three positive T2 generation lines. Interestingly, the symptomatology of transgenic plants was consistent with that of transient overexpression after pathogen inoculation. Notably, the JA content in transgenic overexpression lines was significantly higher than that in the wild-type. RNA-seq revealed that ScCAX4 could mediate multiple signaling pathways, and the JA signaling pathway played a key role in modulating disease resistance. Finally, a regulatory model was depicted for the increased susceptibility to pathogen infection conferred by the ScCAX4 gene. This study provides genetic resources for sugarcane molecular breeding and the research direction for plant CAX genes.

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.

Molecular identification and functional characterization of a transcription factor GeRAV1 from Gelsemium elegans.

BACKGROUND: Gelsemium elegans is a traditional Chinese medicinal plant and temperature is one of the key factors affecting its growth. RAV (related to ABI3/VP1) transcription factor plays multiple roles in higher plants, including the regulation of plant growth, development, and stress response. However, RAV transcription factor in G. elegans has not been reported. RESULTS: In this study, three novel GeRAV genes (GeRAV1-GeRAV3) were identified from the transcriptome of G. elegans under low temperature stress. Phylogenetic analysis showed that GeRAV1-GeRAV3 proteins were clustered into groups II, IV, and V, respectively. RNA-sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) analyses indicated that the expression of GeRAV1 and GeRAV2 was increased in response to cold stress. Furthermore, the GeRAV1 gene was successfully cloned from G. elegans leaf. It encoded a hydrophilic, unstable, and non-secretory protein that contained both AP2 and B3 domains. The amino acid sequence of GeRAV1 protein shared a high similarity of 81.97% with Camptotheca acuminata CaRAV. Subcellular localization and transcriptional self-activation experiments demonstrated that GeRAV1 was a nucleoprotein without self-activating activity. The GeRAV1 gene was constitutively expressed in the leaves, stems, and roots of the G. elegans, with the highest expression levels in roots. In addition, the expression of the GeRAV1 gene was rapidly up-regulated under abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) stresses, suggesting that it may be involved in hormonal signaling pathways. Moreover, GeRAV1 conferred improved cold and sodium chloride tolerance in Escherichia coli Rosetta cells. CONCLUSIONS: These findings provided a foundation for further understanding on the function and regulatory mechanism of the GeRAV1 gene in response to low-temperature stress in G. elegans.

Deciphering the Atlas of Post-Translational Modification in Sugarcane.

In plants, lysine acetylation (Kac), 2-hydroxyisobutyrylation (Khib), and lysine lactylation (Kla), the three new types of post-translational modification (PTM), play very important roles in growth, development, and resistance to adverse environmental stresses. Herein, we report the first global acetylome, 2-hydroxyisobutyrylome, and lactylome in sugarcane. A total of 8573 Kac, 4637 Khib, and 215 Kla sites across 3903, 1507, and 139 modified proteins were identified. Besides, homology analyses revealed the Kac, Khib, and Kla sites on histones were conserved between sugarcane and rice or poplar. Functional annotations demonstrated that the Kac, Khib, and Kla proteins were mainly involved in energy metabolism. In addition, a number of modified transcription factors and stress-related proteins, which were constitutively expressed in different tissues of sugarcane and induced by drought, cold or Sporisorium scitamineum stress, were identified. Finally, a proposed working mode on how PTM functions in sugarcane was depicted. We thus concluded that PTM should play a role in sugarcane growth, development, and response to biotic and abiotic stresses, but the mechanisms require further investigation. The present study provided the all-new comprehensive profile of proteins Kac, Khib, and Kla and a new perspective to understand the molecular mechanisms of protein PTMs in sugarcane.

Dissecting the features of TGA gene family in Saccharum and the functions of ScTGA1 under biotic stresses.

Sugarcane is an important sugar and energy crop and smut disease caused by Sporisorium scitamineum is a major fungal disease which can seriously reduce the yield and quality of sugarcane. In plants, TGACG motif binding (TGA) transcription factors are involved in the regulation of salicylic acid (SA) and methyl jasmonate (MeJA) signaling pathways, as well as in response to various biotic and abiotic stresses. However, no TGA-related transcription factor has been reported in Saccharum. In the present study, 44 SsTGA genes were identified from Saccharum spontaneum, and were assorted into three clades (I, II, III). Cis-regulatory elements (CREs) analysis revealed that SsTGA genes may be involved in hormone and stress response. RNA-seq data and RT-qPCR analysis indicated that SsTGAs were constitutively expressed in different tissues and induced by S. scitamineum stress. In addition, a ScTGA1 gene (GenBank accession number ON416997) was cloned from the sugarcane cultivar ROC22, which was homologous to SsTGA1e in S. spontaneum and encoded a nucleus protein. It was constitutively expressed in sugarcane tissues and up-regulated by SA, MeJA and S. scitamineum stresses. Furthermore, transient overexpression of ScTGA1 in Nicotiana benthamiana could enhance its resistance to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum, by regulating the expression of immune genes related to hypersensitive response (HR), ethylene (ET), SA and jasmonic acid (JA) pathways. This study should contribute to our understanding on the evolution and function of the SsTGA gene family in Saccharum, and provide a basis for the functional identification of ScTGA1 under biotic stresses.

Genome-wide characterization of sugarcane catalase gene family identifies a ScCAT1 gene associated disease resistance.

In plants, catalase (CAT) mainly scavenges H2O2 from reactive oxygen species (ROS) and regulates the growth and development. So far, genome-wide identification of CAT gene family in Saccharum has not yet been reported. Here, 16 SsCAT genes were identified based on a Saccharum spontaneum genome. They were clustered into three subfamilies, with closer genes sharing similar structures. Most SsCAT proteins contained three conserved amino acids, one active catalytic site, one heme-ligand signature, and three peroxisomal targeting signal 1 (PTS1) sequences. The cis-regulatory element prediction revealed that SsCAT genes were involved in growth and development, and in response to various hormones and stresses. RNA-Seq databases showed that SsCAT genes were differentially expressed in Saccharum tissues and under cold, drought, and Sporisorium scitamineum stresses. The ScCAT1 gene transcript (GenBank accession number KF664183) and relevant CAT activity were up-regulated under S. scitamineum stress. Overexpression of ScCAT1 gene in Nicotiana benthamiana could enhance its resistance to pathogen infection through scavenging of excessive toxic ROS and up-regulated expressions of genes related to hypersensitive response (HR), ROS and salicylic acid (SA) pathways. This study provides comprehensive information for the CAT gene family and sets up a basis for its function identification in sugarcane.

Ectopic Expression of Sugarcane ScAMT1.1 Has the Potential to Improve Ammonium Assimilation and Grain Yield in Transgenic Rice under Low Nitrogen Stress.

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.

Screening of Candidate Genes Associated with Brown Stripe Resistance in Sugarcane via BSR-seq Analysis.

Sugarcane brown stripe (SBS), caused by the fungal pathogen Helminthosporium stenospilum, is one of the most serious threats to sugarcane production. However, its outbreaks and epidemics require suitable climatic conditions, resulting in the inefficient improvement of the SBS resistance by phenotype selection. The sugarcane F1 population of SBS-resistant YT93-159 × SBS-susceptible ROC22 was used for constructing the bulks. Bulked segregant RNA-seq (BSR-seq) was then performed on the parents YT93-159 (T01) and ROC22 (T02), and the opposite bulks of 30 SBS-susceptible individuals mixed bulk (T03) and 30 SBS-resistant individuals mixed bulk (T04) collected from 287 F1 individuals. A total of 170.00 Gb of clean data containing 297,921 SNPs and 70,426 genes were obtained. Differentially expressed genes (DEGs) analysis suggested that 7787 and 5911 DEGs were identified in the parents (T01 vs. T02) and two mixed bulks (T03 vs. T04), respectively. In addition, 25,363 high-quality and credible SNPs were obtained using the genome analysis toolkit GATK for SNP calling. Subsequently, six candidate regions with a total length of 8.72 Mb, which were located in the chromosomes 4B and 7C of sugarcane wild species Saccharum spontaneum, were identified, and 279 genes associated with SBS-resistance were annotated by ED algorithm and ΔSNP-index. Furthermore, the expression profiles of candidate genes were verified by quantitative real-time PCR (qRT-PCR) analysis, and the results showed that eight genes (LRR-RLK, DHAR1, WRKY7, RLK1, BLH4, AK3, CRK34, and NDA2) and seven genes (WRKY31, CIPK2, CKA1, CDPK6, PFK4, CBL2, and PR2) of the 20 tested genes were significantly up-regulated in YT93-159 and ROC22, respectively. Finally, a potential molecular mechanism of sugarcane response to H. stenospilum infection is illustrate that the activations of ROS signaling, MAPK cascade signaling, Ca2+ signaling, ABA signaling, and the ASA-GSH cycle jointly promote the SBS resistance in sugarcane. This study provides abundant gene resources for the SBS resistance breeding in sugarcane.

Genome-Wide Identification of Auxin-Responsive GH3 Gene Family in Saccharum and the Expression of ScGH3-1 in Stress Response.

Gretchen Hagen3 (GH3), one of the three major auxin-responsive gene families, is involved in hormone homeostasis in vivo by amino acid splicing with the free forms of salicylic acid (SA), jasmonic acid (JA) or indole-3-acetic acid (IAA). Until now, the functions of sugarcane GH3 (SsGH3) family genes in response to biotic stresses have been largely unknown. In this study, we performed a systematic identification of the SsGH3 gene family at the genome level and identified 41 members on 19 chromosomes in the wild sugarcane species, Saccharum spontaneum. Many of these genes were segmentally duplicated and polyploidization was the main contributor to the increased number of SsGH3 members. SsGH3 proteins can be divided into three major categories (SsGH3-I, SsGH3-II, and SsGH3-III) and most SsGH3 genes have relatively conserved exon-intron arrangements and motif compositions. Diverse cis-elements in the promoters of SsGH3 genes were predicted to be essential players in regulating SsGH3 expression patterns. Multiple transcriptome datasets demonstrated that many SsGH3 genes were responsive to biotic and abiotic stresses and possibly had important functions in the stress response. RNA sequencing and RT-qPCR analysis revealed that SsGH3 genes were differentially expressed in sugarcane tissues and under Sporisorium scitamineum stress. In addition, the SsGH3 homolog ScGH3-1 gene (GenBank accession number: OP429459) was cloned from the sugarcane cultivar (Saccharum hybrid) ROC22 and verified to encode a nuclear- and membrane-localization protein. ScGH3-1 was constitutively expressed in all tissues of sugarcane and the highest amount was observed in the stem pith. Interestingly, it was down-regulated after smut pathogen infection but up-regulated after MeJA and SA treatments. Furthermore, transiently overexpressed Nicotiana benthamiana, transduced with the ScGH3-1 gene, showed negative regulation in response to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum. Finally, a potential model for ScGH3-1-mediated regulation of resistance to pathogen infection in transgenic N. benthamiana plants was proposed. This study lays the foundation for a comprehensive understanding of the sequence characteristics, structural properties, evolutionary relationships, and expression of the GH3 gene family and thus provides a potential genetic resource for sugarcane disease-resistance breeding.

Identification of Low-Nitrogen-Related miRNAs and Their Target Genes in Sugarcane and the Role of miR156 in Nitrogen Assimilation.

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.

Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq.

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca2+ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.

Sugarcane ScDREB2B-1 Confers Drought Stress Tolerance in Transgenic Nicotiana benthamiana by Regulating the ABA Signal, ROS Level and Stress-Related Gene Expression.

The dehydration-responsive element-binding protein (DREB) is a subgroup member of the AP2/ERF family and actively participates in the response of plants to abiotic stress. Although DREB genes have been studied in a variety of plant species, there are few reports of DREB genes in sugarcane (Saccharum spp.). In this study, a novel full-length cDNA sequence of the ScDREB2B-1 gene was cloned from the Saccharum hybrid ROC22, whose encoding protein contained only one AP2-conserved domain and was clustered into the DREB (A-2) subgroup. The diverse promoter elements in the ScDREB2B-1 gene and the accumulated transcripts of its homologous gene (SsAP2/ERF-107) in S. spontaneum under drought stress suggest that the ScDREB2B-1 gene may play a role in drought response. In addition, reverse transcription quantitative PCR analysis showed that the expression level of the ScDREB2B-1 gene was upregulated in the root and leaf of ROC22 under polyethylene glycol, sodium chloride and abscisic acid (ABA) treatments. The yeast two-hybrid experiment demonstrated that ScDREB2B-1 had transcriptional self-activation activity. Compared with wild-type plants, the overexpression of the ScDREB2B-1 gene improved the drought tolerance of the transgenic Nicotiana benthamiana by activating the ABA pathway to enhance the expression of the ABA-responsive gene (NbNCED) and ABA content, regulate the intracellular reactive oxygen species (ROS) level (enhance the transcripts of ROS synthase-related gene NbRbohB and the activities of catalase, peroxidase and superoxide dismutase) and increase the relative water content, proline content and expression level of osmotic stress-related genes (NbERD and NbLEA). Collectively, our data indicate that ScDREB2B-1 is a stress-inducible and ABA-responsive transcription factor gene that responds to drought stress by regulating ABA signaling, ROS levels and stress-related gene expression. This study contributes to a better understanding of the biological function of ScDREB2B-1, which could serve as a foundation for future resistance breeding in sugarcane.

WGCNA Identifies a Comprehensive and Dynamic Gene Co-Expression Network That Associates with Smut Resistance in Sugarcane.

Sugarcane smut is a major fungal disease caused by Sporisorium scitamineum, which seriously reduces the yield and quality of sugarcane. In this study, 36 transcriptome data were collected from two sugarcane genotypes, YT93-159 (resistant) and ROC22 (susceptible) upon S. scitamineum infection. Data analysis revealed 20,273 (12,659 up-regulated and 7614 down-regulated) and 11,897 (7806 up-regulated and 4091 down-regulated) differentially expressed genes (DEGs) in YT93-159 and ROC22, respectively. A co-expression network was then constructed by weighted gene co-expression network analysis (WGCNA), which identified 5010 DEGs in 15 co-expressed gene modules. Four of the 15 modules, namely, Skyblue, Salmon, Darkorange, and Grey60, were significantly associated with smut resistance. The GO and KEGG enrichment analyses indicated that the DEGs involving in these four modules could be enriched in stress-related metabolic pathways, such as MAPK and hormone signal transduction, plant-pathogen interaction, amino acid metabolism, glutathione metabolism, and flavonoid, and phenylpropanoid biosynthesis. In total, 38 hub genes, including six from the Skyblue module, four from the Salmon module, 12 from the Darkorange module, and 16 from the Grey60 module, were screened as candidate hub genes by calculating gene connectivity in the corresponding network. Only 30 hub genes were amplifiable with RT-qPCR, of which 27 were up-regulated upon S. scitamineum infection. The results were consistent with the trend of gene expression in RNA-Seq, suggesting their positive roles in smut resistance. Interestingly, the expression levels of AOX, Cyb5, and LAC were higher in ROC22 than in YT93-159, indicating these three genes may act as negative regulators in response to S. scitamineum infection. This study revealed the transcriptome dynamics in sugarcane challenged by S. scitamineum infection and provided gene targets for smut resistance breeding in sugarcane.

Genome-Wide Identification, Characterization, and Expression Analysis of Glutamate Receptor-like Gene (GLR) Family in Sugarcane.

The plant glutamate receptor-like gene (GLR) plays a vital role in development, signaling pathways, and in its response to environmental stress. However, the GLR gene family has not been comprehensively and systematically studied in sugarcane. In this work, 43 GLR genes, including 34 in Saccharum spontaneum and 9 in the Saccharum hybrid cultivar R570, were identified and characterized, which could be divided into three clades (clade I, II, and III). They had different evolutionary mechanisms, the former was mainly on the WGD/segmental duplication, while the latter mainly on the proximal duplication. Those sugarcane GLR proteins in the same clade had a similar gene structure and motif distribution. For example, 79% of the sugarcane GLR proteins contained all the motifs, which proved the evolutionary stability of the sugarcane GLR gene family. The diverse cis-acting regulatory elements indicated that the sugarcane GLRs may play a role in the growth and development, or under the phytohormonal, biotic, and abiotic stresses. In addition, GO and KEGG analyses predicted their transmembrane transport function. Based on the transcriptome data, the expression of the clade III genes was significantly higher than that of the clade I and clade II. Furthermore, qRT-PCR analysis demonstrated that the expression of the SsGLRs was induced by salicylic acid (SA) treatment, methyl jasmonic acid (MeJA) treatment, and abscisic acid (ABA) treatment, suggesting their involvement in the hormone synthesis and signaling pathway. Taken together, the present study should provide useful information on comparative genomics to improve our understanding of the GLR genes and facilitate further research on their functions.

[Identification of GeERF transcription factors in Gelsmium elegans and their expression under low temperature stress].

With prominent medicinal value, Gelsemium elegans has been overexploited, resulting in the reduction of the wild resource. As a result, artificial cultivation turns out to be a solution. However, this medicinal species is intolerant to low temperature, and thus genes responding to the low temperature are important for the cultivation of this species. Based on the transcriptome database of G. elegans at 4 ℃, 29 differentially expressed GeERF genes were identified. Bioinformatics analysis of 21 GeERF gene sequences with intact open reading frames showed that 12 and 9 of the GeERF proteins respectively clustered in DREB subgroup and ERF subgroup. GeDREB1 A-1-GeERF6 B-1, with molecular weight of 23.78-50.96 kDa and length of 212-459 aa, were all predicted to be hydrophilic and in nucleus. Furthermore, the full-length cDNA sequence of GeERF2B-1 was cloned from the leaves of G. elegans. Subcellular localization suggested that GeERF2B-1 was located in the nucleus. According to the quantitative reverse-transcription PCR(qRT-PCR), GeERF2B-1 showed constitutive expression in roots, stems, and leaves of G. elegans, and the expression was the highest in roots. In terms of the response to 4 ℃ treatment, the expression of GeERF2B-1 was significantly higher than that in the control and peaked at 12 h, suggesting a positive response to low temperature. This study lays a scientific basis for the functional study of GeERF transcription factors and provides gene resources for the improvement of stress resistance of G. elegans.

Characterization and Functional Implications of the Nonexpressor of Pathogenesis-Related Genes 1 (NPR1) in Saccharum.

Sugarcane (Saccharum spp.) is an important sugar and energy crop worldwide. As a core regulator of the salicylic acid (SA) signaling pathway, nonexpressor of pathogenesis-related genes 1 (NPR1) plays a significant role in the response of the plant to biotic and abiotic stresses. However, there is currently no report on the NPR1-like gene family in sugarcane. In this study, a total of 18 NPR1-like genes were identified in Saccharum spontaneum and classified into three clades (clade I, II, and III). The cis-elements predicted in the promotors revealed that the sugarcane NPR1-like genes may be involved in various phytohormones and stress responses. RNA sequencing and quantitative real-time PCR analysis demonstrated that NPR1-like genes were differentially expressed in sugarcane tissues and under Sporisorium scitamineum stress. In addition, a novel ShNPR1 gene from Saccharum spp. hybrid ROC22 was isolated by homologous cloning and validated to be a nuclear-localized clade II member. The ShNPR1 gene was constitutively expressed in all the sugarcane tissues, with the highest expression level in the leaf and the lowest in the bud. The expression level of ShNPR1 was decreased by the plant hormones salicylic acid (SA) and abscisic acid (ABA). Additionally, the transient expression showed that the ShNPR1 gene plays a positive role in Nicotiana benthamiana plants' defense response to Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provided comprehensive information for the NPR1-like family in sugarcane, which should be helpful for functional characterization of sugarcane NPR1-like genes in the future.

Systematic identification of miRNA-regulatory networks unveils their potential roles in sugarcane response to Sorghum mosaic virus infection.

BACKGROUND: Sugarcane mosaic disease (SMD) is a major viral disease of sugarcane (Saccharum spp.) worldwide. Sorghum mosaic virus (SrMV) is the dominant pathogen of SMD in the sugarcane planting areas of China. There is no report on miRNAs and their regulatory networks in sugarcane response to SrMV infection. RESULTS: In this study, small RNA sequencing (sRNA-seq) of samples from the leaves of SMD-susceptible variety ROC22 and -resistant variety FN39 infected by SrMV was performed. A total of 132 mature miRNAs (55 known miRNAs and 77 novel miRNAs) corresponding to 1,037 target genes were identified. After the SrMV attack, there were 30 differentially expressed miRNAs (17 up-regulated and 13 down-regulated) in FN39 and 19 in ROC22 (16 up-regulated and 3 down-regulated). Besides, there were 18 and 7 variety-specific differentially expressed miRNAs for FN39 and ROC22, respectively. KEGG enrichment analysis showed that the differentially expressed miRNAs targeted genes involved in several disease resistance-related pathways, such as mRNA surveillance, plant pathway interaction, sulfur metabolism, and regulation of autophagy. The reliability of sequencing data, and the expression patterns / regulation relationships between the selected differentially expressed miRNAs and their target genes in ROC22 and FN39 were confirmed by quantitative real-time PCR. A regulatory network diagram of differentially expressed miRNAs and their predicted target genes in sugarcane response to SrMV infection was sketched. In addition, precursor sequences of three candidate differentially expressed novel miRNAs (nov_3741, nov_22650 and nov_40875) were cloned from the ROC22 leaf infected by SrMV. Transient overexpression demonstrated that they could induce the accumulation of hydrogen peroxide and the expression level of hypersensitive response marker genes, salicylic acid-responsive genes and ethylene synthesis-depended genes in Nicotiana benthamiana. It is thus speculated that these three miRNAs may be involved in regulating the early immune response of sugarcane plants following SrMV infection. CONCLUSIONS: This study lays a foundation for revealing the miRNA regulation mechanism in the interaction of sugarcane and SrMV, and also provides a resource for miRNAs and their predicted target genes for SrMV resistance improvement in sugarcane.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Genome-wide identification and expression analysis of the coronatine-insensitive 1 (COI1) gene family in response to biotic and abiotic stresses in Saccharum.

BACKGROUND: The coronatine insensitive 1 (COI1) gene is the core member of jasmonate signaling pathway, which is closely related to plant biotic and abiotic resistance. However, there have been no reports on COI1 in sugarcane (Sacharum spp.). Hence, systematically investigating the characteristics of the COI1 multigene family in sugarcane can provide a means to study and manipulate the jasmonic acid signaling pathway. RESULTS: A total of 156 COI1 proteins were obtained from the genomes of 19 land plants, while none were obtained from five algae species. A phylogenetic tree demonstrated that these COI1 proteins were classified into four groups, while 31 proteins of SsCOI1 from Saccharum spontaneum, SbCOI1 from Sorghum bicolor, and ShCOI1 from Saccharum spp. hybrid cultivar R570 clustered into three groups. Synteny analysis and duplication patterns revealed that COI1 genes expanded through various genome replication events and could have experienced strong purifying selective pressure during evolution in S. spontaneum, S. bicolor, and R570. An investigation of cis-acting elements suggests that COI1 genes may be involved in plant growth and development and response to various stresses. Expression analysis implied that 21 SsCOI1 genes were constitutively expressed, and had positive responses to drought, cold, and Sporisorium scitamineum stresses with different expression patterns. Among them, seven SsCOI1 haplotype genes may play different roles in response to methyl jasmonate. Furthermore, the ShCOI1-4, ShCOI1-5, and ShCOI1-6 genes were cloned from Saccharum spp. hybrid cultivar ROC22. Real-time quantitative PCR (RT-qPCR) analysis demonstrated that these three ShCOI1 genes had divergent expression profiles in response to salicylic acid, abscisic acid, polyethylene glycol, cold, and S. scitamineum. CONCLUSIONS: These results suggest that COI1 genes may act in sugarcane growth, development, and response to various stresses via different regulatory mechanisms, which laying a foundation for the functional identification of the sugarcane COI1 gene.