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.

Targeting MELK improves PD-1 blockade efficiency in cervical cancer via enhancing antitumor immunity.

The balance between T helper 1 (Th1) and T helper 2 (Th2) has a critical function in determining intratumoral immune response and anti-PD-1 immunotherapy. The level of maternal embryonic leucine zipper kinase (MELK) is reported to correlate with infiltration of immune cells in cancers, but the underlying molecular mechanism is not clarified. In the present study, we aimed to elucidate the potential function of MELK in cervical cancer. We found that MELK was upregulated and played an oncogenic role in cervical cancer. MELK overexpression shifted Th1/Th2 balance toward Th2 predisposition in mouse cervical tumors in vivo and naive T cells from human PBMCs in vitro, whereas MELK knockdown exhibited opposite effects. MELK overexpression activated NF-κB signaling and promoted IL-6 secretion by cervical cancer cells. Depletion of IL-6 by neutralization antibodies abrogated the influence of MELK on Th1/Th2 balance. In addition, MELK modulated the antitumor activity of cytotoxic CD8+ T cells in cervical tumors, but depletion of Th2 cells by IL-4 neutralization abrogated this effect. Finally, MELK overexpression conferred tolerance to PD-1 blockade in cervical tumors, whereas targeting MELK by OTSSP167 significantly enhanced PD-1 blockade efficiency. Our data elucidated a novel role of MELK in regulating Th1/Th2 balance and anti-PD-1 immunotherapy in cervical cancer.

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.

A Mott-Schottky Heterojunction with Strong Chemisorption and Fast Conversion Effects for Room-Temperature Na-S Batteries.

The practical application of the room-temperature sodium-sulfur (RT Na-S) batteries is currently limited by low reversible capacity and serious capacity decay due to the sluggish reaction kinetics and shuttle effect. It is necessary to design a suitable sulfur host integrated with electrocatalysts to realize effective chemisorption and catalysis of sodium polysulfides (NaPSs). Herein, under the guidance of theoretical calculation, the Mott-Schottky heterojunction with a built-in electric field composed of iron (Fe) and iron disulfide (FeS2 ) components anchored on a porous carbon matrix (Fe/FeS2 -PC) is designed and prepared. The enhanced chemisorption effect of Fe, the fast electrocatalytic effect of FeS2 , and the fast transfer effect of the built-in electric field within the Fe/FeS2 heterojunction in the cathode of RT Na-S batteries work together to effectively improve the electrochemical performance. As a result, the Fe/FeS2 -PC@S cathode exhibits high reversible capacity (815 mAh g-1 after 150 cycles at 0.2 A g-1 ) and excellent stability (516 mAh g-1 after 600 cycles at 5 A g-1 , with only 0.07% decay per cycle). The design of the Fe/FeS2 heterojunction electrocatalyst provides a new strategy for the development of highly stable RT Na-S batteries.

Identification of monotonically expressed long non-coding RNA signatures for breast cancer using variational autoencoders.

As breast cancer is a multistage progression disease resulting from a genetic sequence of mutations, understanding the genes whose expression values increase or decrease monotonically across pathologic stages can provide insightful clues about how breast cancer initiates and advances. Utilizing variational autoencoder (VAE) networks in conjunction with traditional statistical testing, we successfully ascertain long non-coding RNAs (lncRNAs) that exhibit monotonically differential expression values in breast cancer. Subsequently, we validate that the identified lncRNAs really present monotonically changed patterns. The proposed procedure identified 248 monotonically decreasing expressed and 115 increasing expressed lncRNAs. They correspond to a total of 65 and 33 genes respectively, which possess unique known gene symbols. Some of them are associated with breast cancer, as suggested by previous studies. Furthermore, enriched pathways by the target mRNAs of these identified lncRNAs include the Wnt signaling pathway, human papillomavirus (HPV) infection, and Rap 1 signaling pathway, which have been shown to play crucial roles in the initiation and development of breast cancer. Additionally, we trained a VAE model using the entire dataset. To assess the effectiveness of the identified lncRNAs, a microarray dataset was employed as the test set. The results obtained from this evaluation were deemed satisfactory. In conclusion, further experimental validation of these lncRNAs with a large-sized study is warranted, and the proposed procedure is highly recommended.

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.

Identification and characterization of WAK gene family in Saccharum and the negative roles of ScWAK1 under the pathogen stress.

Wall-associated kinase (WAK) is widely involved in signal transduction, reproductive growth, responses to pathogen infection and metal ion stress in plants. In this study, 19, 12, and 37 SsWAK genes were identified in Saccharum spontaneum, Saccharum hybrid and Sorghum bicolor, respectively. Phylogenetic tree showed that they could be divided into three groups. These WAK genes contained multiple cis-acting elements related to stress, growth and hormone response. RNA-seq analysis demonstrated that SsWAK genes were constitutively expressed in different sugarcane tissues and involved in response to smut pathogen (Sporisorium scitamineum) stress. Additionally, ScWAK1 (GenBank Accession No. OP479864), was then isolated from sugarcane cultivar ROC22. It was highly expressed in leaves and roots and its expression could be induced under SA and MeJA stress. Besides, ScWAK1 was significantly downregulated in both smut-resistant and susceptible sugarcane cultivars in response to S. scitamineum infection. ScWAK1 was a membrane protein without self-activating activity. Furthermore, transient expression of ScWAK1 in Nicotiana benthamiana enhanced the susceptibility of tobacco to the inoculation of Ralstonia solanacearum and Fusarium solani var. coeruleum, suggesting its negative role in disease resistance. The present study reveals the origin, distribution and evolution of WAK gene family and provides potential gene resources for sugarcane molecular breeding.

Evaluation of the Mayo Additive Staging System in patients with newly diagnosed multiple myeloma: A real-world analysis.

OBJECTIVES: Recently, the Mayo Clinic introduced a new staging system (the Mayo Additive Staging System [MASS]) for patients with newly diagnosed multiple myeloma (NDMM) based on the number of high-risk (HR) abnormalities, including HR IgH translocations, 1q gain/amplification, chromosome 17 abnormalities, International Staging System (ISS)-III, and elevated lactate dehydrogenase. Patients with 0, 1, or ≥2 HR abnormalities were defined as stage I, II, or III, respectively. We aimed to validate the real-world prognostic value of the MASS. METHODS: We retrospectively analyzed the cytogenetic and laboratory results of 544 patients with NDMM at a single center. RESULTS: Ninety (16.5%) patients had no HR factors (MASS I), 193 (35.5%) had 1 HR factor (MASS II), and 261 (48%) had ≥2 HR factors (MASS III). The median progression-free survival (PFS) and overall survival (OS) times were 48, 28, and 20 months and 137, 73, and 39 months in the three groups, respectively (p < .001). In the subgroup analysis, patients had different OS outcomes based on the MASS when grouped by age, renal function, or therapeutic regimens. The MASS identified patients with the worst outcomes among those rated revised ISS II. CONCLUSION: The MASS system is a reliable risk stratification tool for patients with NDMM in real-world clinical practice.

The applicability of the Second Revision of the International Staging System for patients with multiple myeloma receiving immunomodulatory drugs or proteasome inhibitor-based regimens as induction treatment: A real-world analysis.

The Second Revision of the International Staging System (R2-ISS) was recently introduced to improve risk stratification over that provided by the extensively applied standard revised International Staging System (R-ISS). In addition to the variables included in the R-ISS, the R2-ISS incorporates chromosome 1q gain/amplification and divides the patients into 4 groups with different survival outcomes, better stratifying patients within the R-ISS intermediate-risk. The new model was developed based on a great quantity of data from patients participating in uniform clinical trials and has not been validated in real-world clinical practice. Therefore, we retrospectively analyzed the prognostic value of the R2-ISS in 474 consecutive patients with multiple myeloma receiving immunomodulatory drugs or proteasome inhibitor-based regimens as their first-line treatment. According to the R2-ISS, 41 (8.6%), 76 (16%), 275 (58%), and 82 (17.3%) patients were identified as R2-ISS I, R2-ISS II, R2-ISS III, and R2-ISS IV, respectively. The median progression-free survival (PFS) was 48 (95% CI: 38-58), 35 (95% CI: 23-47), 24 (95% CI: 21-27), and 12 (95% CI: 7-17) months, and the estimated median overall survival (OS) was 110 (95% CI: 42-178), 88 (95% CI: 75-101), 50 (95% CI: 43-57), and 26 (95% CI: 19-33) months (p < 0.001) in the 4 groups, respectively. The R2-ISS could also classify groups with distinct survival among patients with renal impairment or classified as R-ISS II. Adjusted by age, sex, treatment approaches and transplantation status, the R2-ISS was an independent prognostic factor associated with OS with a hazard ratio of 7.055 (95% CI: 3.626-13.726) (p < 0.001) for R2-ISS IV versus R2-ISS I and 2.707 (95% CI: 1.436-5.103) (p = 0.002) for R2-ISS III versus R2-ISS I. In conclusion, our results suggest that the R2-ISS is a simple and robust risk stratification tool for patients with multiple myeloma treated with novel drugs and could be used in everyday clinical practice.

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.

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.

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.

Identification of a novel autophagy-related prognostic signature and small molecule drugs for glioblastoma by bioinformatics.

OBJECTIVE: To explore the autophagy-related prognostic signature (ARPs) via data mining in gene expression profiles for glioblastoma (GBM). METHODS: Using the Cancer Genome Atlas (TCGA) database, we obtained 156 GBM samples and 5 adjacent normal samples, and denoted them as discovery cohort. Univariate Cox regression analysis was used to screen autophagy genes that related to GBM prognosis. Then, the least absolute shrinkage and selection operator Cox regression model was used to construct an autophagy-based ARPs, which was validated in an external cohort containing 80 GBM samples. The patients in the above-mentioned cohorts were divided into low-risk group and high-risk group according to the median prognostic risk score, and the diagnostic performance of the model was assessed by receiver operating characteristic curve analyses. The gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analyses were performed between the high-risk and low-risk patients. Additionally, the genetic features of ARPs, such as genetic variation profiles, correlations with tumor-infiltrating lymphocytes (TILs), and potential drug sensitivity, were further assessed in the TCGA-GBM data set. RESULTS: A signature of ARPs including NDUFB9, BAK1, SUPT3H, GAPDH, CDKN1B, CHMP6, and EGFR were detected and validated. We identified a autophagy-related prognosis 7-gene signature correlated survival prognosis, immune infiltration, level of cytokines, and cytokine receptor in tumor microenvironment. Furthermore, the signature was tested in several pathways related to disorders of tumor microenvironment, as well as cancer-related pathways. Additionally, a range of small molecular drugs, shown to have a potential therapeutic effect on GBM. CONCLUSIONS: We constructed an autophagy-based 7-gene signature, which could serve as an independent prognostic indicator for cases of GBM and sheds light on the role of autophagy as a potential therapeutic target in GBM.

Circular RNA Protein Tyrosine Kinase 2 Promotes Cell Proliferation, Migration and Suppresses Apoptosis via Activating MicroRNA-638 Mediated MEK/ERK, WNT/ß-Catenin Signaling Pathways in Multiple Myeloma.

Our previous study observed that circular RNA protein tyrosine kinase 2 (circ-PTK2) was upregulated and correlated with worse clinical features and unfavorable prognosis in multiple myeloma (MM) patients. Thus, this study aimed to further characterize the regulatory function of circ-PTK2 on cell malignant activities and its target microRNA-638 (miR-638) as well as downstream MEK/ERK, WNT/ß-catenin signaling pathways in MM. The effect of circ-PTK2 on MM cell proliferation, apoptosis, migration, invasion and its potential target miRNAs was assessed by transfecting circ-PTK2 overexpression plasmids into U226 cells and circ-PTK2 knock-down plasmids into LP-1 cells. Furthermore, the interaction between circ-PTK2 and miR-638 mediated MEK/ERK and WNT/ß-catenin signaling pathways was validated by rescue experiments. Circ-PTK2 was overexpressed in most MM cell lines compared to normal plasma cells. Overexpressing circ-PTK2 promoted proliferation and migration, inhibited apoptosis in U266 cells, but did not affect cell invasion; knocking down circ-PTK2 achieved opposite effect in LP-1 cells. Besides, circ-PTK2 reversely regulated miR-638 expression but not miR-4690, miR-6724, miR-6749 or miR-6775. The following luciferase reporter assay illustrated the direct bind of circ-PTK2 towards miR-638. In rescue experiments, overexpressing miR-638 suppressed proliferation, migration, while promoted apoptosis in both wild U266 cells and circ-PTK2-overexpressed U266 cells; meanwhile, overexpressing miR-638 also suppressed MEK/ERK and WNT/ß-catenin pathways in both wild U266 cells and circ-PTK2-overexpressed U266 cells. Knocking down miR-638 achieved opposite effect in both wild LP-1 cells and circ-PTK2-knocked-down LP-1 cells. In conclusion, circ-PTK2 promotes cell proliferation, migration, suppresses cell apoptosis via miR-638 mediated MEK&ERK and WNT&ß-catenin signaling pathways in MM.

The CaCA superfamily genes in Saccharum: comparative analysis and their functional implications in response to biotic and abiotic stress.

BACKGROUND: In plants, Calcium (Ca2+) acts as a universal messenger in various signal transduction pathways, including responses to biotic and abiotic stresses and regulation of cellular and developmental processes. The Ca2+/cation antiporter (CaCA) superfamily proteins play vital roles in the transport of Ca2+ and/or other cations. However, the characteristics of these superfamily members in Saccharum and their evolutionary and functional implications have remained unclear. RESULTS: A total of 34 CaCA genes in Saccharum spontaneum, 5 CaCA genes in Saccharum spp. R570, and 14 CaCA genes in Sorghum bicolor were identified and characterized. These genes consisted of the H+/cation exchanger (CAX), cation/Ca2+ exchanger (CCX), EF-hand / CAX (EFCAX), and Mg2+/H+ exchanger (MHX) families, among which the CCX and EFCAX could be classified into three groups while the CAX could be divided into two groups. The exon/intron structures and motif compositions suggested that the members in the same group were highly conserved. Synteny analysis of CaCAs established their orthologous and paralogous relationships among the superfamily in S. spontaneum, R570, and S. bicolor. The results of protein-protein interactions indicated that these CaCA proteins had direct or indirect interactions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that most members of Saccharum CaCA genes exhibited a similar expression pattern in response to hormonal (abscisic acid, ABA) treatment but played various roles in response to biotic (Sporisorium scitamineum) and abiotic (cold) stresses. Furthermore, ScCAX4, a gene encoding a cytoplasm, plasma membrane and nucleus positioning protein, was isolated from sugarcane. This gene was constitutively expressed in different sugarcane tissues and its expression was only induced at 3 and 6 h time points after ABA treatment, however was inhibited and indued in the whole process under cold and S. scitamineum stresses, respectively. CONCLUSIONS: This study systematically conducted comparative analyses of CaCA superfamily genes among S. spontaneum, R570, and S. bicolor, delineating their sequence and structure characteristics, classification, evolutionary history, and putative functions. These results not only provided rich gene resources for exploring the molecular mechanism of the CaCA superfamily genes but also offered guidance and reference for research on other gene families in Saccharum.

Genome-wide identification, characterization and expression analysis of the carotenoid cleavage oxygenase (CCO) gene family in Saccharum.

Carotenoid cleavage oxygenases (CCOs) play crucial roles in plant growth and development, as well as in the response to phytohormonal, biotic and abiotic stresses. However, comprehensive and systematic research on the CCO gene family has not yet been conducted in Saccharum. In this study, 47 SsCCO and 14 ShCCO genes were identified and characterized in Saccharum spontaneum and Saccharum spp. R570 cultivar, respectively. The SsCCOs consisted of 38 SsCCDs and 9 SsNCEDs, while ShCCOs contained 11 ShCCDs and 3 ShNCEDs. The SsCCO family could be divided into 7 groups, while ShCCO family into 5 groups. The genes/proteins contained similar compositions within the same group, and the evolutionary mechanisms differed between S. spontaneum and R570. Gene Ontology annotation implied that CCOs were involved in many physiological and biochemical processes. Additionally, 41 SsCCOs were regulated by 19 miRNA families, and 8 ShCCOs by 9 miRNA families. Cis-regulatory elements analysis suggested that CCO genes functioned in the process of growth and development or under the phytohormonal, biotic and abiotic stresses. qRT-PCR analysis indicated that nine CCO genes from different groups exhibited similar expression patterns under abscisic acid treatment, while more divergent profiles were observed in response to Sporisorium scitamineum and cold stresses. Herein, comparative genomics analysis of the CCO gene family between S. spontaneum and R570 was conducted to investigate its evolution and functions. This is the first report on the CCO gene family in S. spontaneum and R570, thus providing valuable information and facilitating further investigation into its function in the future.

A class III WRKY transcription factor in sugarcane was involved in biotic and abiotic stress responses.

WRKY transcription factors play significant roles in plant stress responses. In this study, a class III WRKY gene ScWRKY5, was successfully isolated from sugarcane variety ROC22. The ScWRKY5 was a nucleus protein with transcriptional activation activity. The ScWRKY5 gene was constitutively expressed in all the sugarcane tissues, with the highest expression level in the stem epidermis and the lowest in the root. After inoculation with Sporisorium scitamineum for 1 d, the expression level of ScWRKY5 was significantly increased in two smut-resistant varieties (YZ01-1413 and LC05-136), while it was decreased in three smut-susceptible varieties (ROC22, YZ03-103, and FN40). Besides, the expression level of ScWRKY5 was increased by the plant hormones salicylic acid (SA) and abscisic acid (ABA), as well as the abiotic factors polyethylene glycol (PEG) and sodium chloride (NaCl). Transient overexpression of the ScWRKY5 gene enhanced the resistance of Nicotiana benthamiana to the tobacco bacterial pathogen Ralstonia solanacearum, however the transiently overexpressed N. benthamiana was more sensitive to the tobacco fungal pathogen Fusarium solani var. coeruleum. These results provide a reference for further research on the resistance function of sugarcane WRKY genes.

New insights into the evolution and functional divergence of the CIPK gene family in Saccharum.

BACKGROUND: Calcineurin B-like protein (CBL)-interacting protein kinases (CIPKs) are the primary components of calcium sensors, and play crucial roles in plant developmental processes, hormone signaling transduction, and in the response to exogenous stresses. RESULTS: In this study, 48 CIPK genes (SsCIPKs) were identified from the genome of Saccharum spontaneum. Phylogenetic reconstruction suggested that the SsCIPK gene family may have undergone six gene duplication events from the last common ancestor (LCA) of SsCIPKs. Whole-genome duplications (WGDs) served as the driving force for the amplification of SsCIPKs. The Nonsynonymous to synonymous substitution ratio (Ka/Ks) analysis showed that the duplicated genes were possibly under strong purifying selection pressure. The divergence time of these duplicated genes had an average duplication time of approximately 35.66 Mya, suggesting that these duplication events occurred after the divergence of the monocots and eudicots (165 Mya). The evolution of gene structure analysis showed that the SsCIPK family genes may involve intron losses. Ten ScCIPK genes were amplified from sugarcane (Saccharum spp. hybrids). The results of real-time quantitative polymerase chain reaction (qRT-PCR) demonstrated that these ten ScCIPK genes had different expression patterns under abscisic acid (ABA), polyethylene glycol (PEG), and sodium chloride (NaCl) stresses. Prokaryotic expression implied that the recombinant proteins of ScCIPK3, - 15 and - 17 could only slightly enhance growth under salinity stress conditions, but the ScCIPK21 did not. Transient N. benthamiana plants overexpressing ScCIPKs demonstrated that the ScCIPK genes were involved in responding to external stressors through the ethylene synthesis pathway as well as to bacterial infections. CONCLUSIONS: In generally, a comprehensive genome-wide analysis of evolutionary relationship, gene structure, motif composition, and gene duplications of SsCIPK family genes were performed in S. spontaneum. The functional study of expression patterns in sugarcane and allogenic expressions in E. coli and N. benthamiana showed that ScCIPKs played various roles in response to different stresses. Thus, these results improve our understanding of the evolution of the CIPK gene family in sugarcane as well as provide a basis for in-depth functional studies of CIPK genes in sugarcane.

The alcohol dehydrogenase gene family in sugarcane and its involvement in cold stress regulation.

BACKGROUND: Alcohol dehydrogenases (ADHs) in plants are encoded by a multigene family. ADHs participate in growth, development, and adaptation in many plant species, but the evolution and function of the ADH gene family in sugarcane is still unclear. RESULTS: In the present study, 151 ADH genes from 17 species including 32 ADH genes in Saccharum spontaneum and 6 ADH genes in modern sugarcane cultivar R570 were identified. Phylogenetic analysis demonstrated two groups of ADH genes and suggested that these genes underwent duplication during angiosperm evolution. Whole-genome duplication (WGD)/segmental and dispersed duplications played critical roles in the expansion of ADH family in S. spontaneum and R570, respectively. ScADH3 was cloned and preferentially expressed in response to cold stress. ScADH3 conferred improved cold tolerance in E. coli cells. Ectopic expression showed that ScADH3 can also enhance cold tolerance in transgenic tobacco. The accumulation of reactive oxygen species (ROS) in leaves of transgenic tobacco was significantly lower than in wild-type tobacco. The transcript levels of ROS-related genes in transgenic tobacco increased significantly. ScADH3 seems to affect cold tolerance by regulating the ROS-related genes to maintain the ROS homeostasis. CONCLUSIONS: This study depicted the size and composition of the ADH gene family in 17 species, and investigated their evolution pattern. Comparative genomics analysis among the ADH gene families of S. bicolor, R570 and S. spontaneum revealed their close evolutionary relationship. Functional analysis suggested that ScADH3, which maintained the steady state of ROS by regulating ROS-related genes, was related to cold tolerance. These findings will facilitate research on evolutionary and functional aspects of the ADH genes in sugarcane, especially for the understanding of ScADH3 under cold stress.