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.

The L-ascorbate peroxidase 6 gene (APX6) is one of the most important genes for scavenging H₂O₂ and plays a vital role in plant resistance to environmental stresses. In this study, a novel ScAPX6 gene (GenBank Accession No. KT907352) was obtained from a sugarcane variety (ROC22). Bioinformatics analysis showed that ScAPX6 has a cDNA length of 1,086 bp and encoded 333 amino acid residues. Subcellular localization confirmed that ScAPX6 was located in the chloroplast. Enhanced growth of Escherichia coli BL21 cells that expressed ScAPX6 showed high tolerance under copper (Cu) stress. Real-time quantitative PCR analysis revealed that ScAPX6 was constitutively expressed wherein with the highest expression levels in sugarcane pith and leaf and the lowest in the root. ScAPX6 was down-regulated by salicylic acid (SA), hydrogen peroxide (H₂O₂), polyethylene glycol (PEG) and sodium chloride (NaCl) stimuli. Interestingly, it was significantly up-regulated under the stresses of abscisic acid (ABA) and methyl jasmonate (MeJA) wherein with the highest inducible expression levels at 6 h at 6.0- and 70.0-times higher, respectively than that of control. Overexpression of ScAPX6 in Nicotiana benthamiana leaves enhanced the resistance to the infection of tobacco pathogens Pseudomonas solanacearum and Fusarium solani var. coeruleum. These results implied that ScAPX6 might positively respond to ABA, MeJA, and Cu, but might negatively respond to the stresses of SA, H₂O₂, PEG, and NaCl. Keeping in view the current investigation, ScAPX6 could be associated with the hypersensitive response (HR) or immunity of sugarcane, which will provide a baseline for the function identification of sugarcane ScAPX6.

Free calcium ions are common second messengers in plant cells. The calcineurin B-like protein (CBL) is a special calcium sensor that plays an important role in plant growth and stress response. In this study, we obtained three CBL genes (GenBank accession nos. KX013374, KX013375, and KX013376) from sugarcane variety ROC22. The open reading frames of ScCBL genes ranged from 642 to 678 base pairs in length and encoded polypeptides from 213 to 225 amino acids in length. ScCBL2-1, ScCBL3-1, and ScCBL4 were all located in the plasma membrane and cytoplasm. ScCBL2-1 and ScCBL3-1 expression was up-regulated by treatment with salicylic acid (SA), methyl jasmonate (MeJA), hydrogen peroxide (H₂O₂), polyethylene glycol (PEG), sodium chloride (NaCl), or copper chloride (CuCl₂). ScCBL4 expression was down-regulated in response to all of these stresses (abscisic acid (ABA), SA, MeJA, and NaCl) except for H₂O₂, calcium chloride (CaCl₂), PEG, and CuCl₂. Expression in Escherichia coli BL21 cells showed that ScCBLs can enhance tolerance to NaCl or copper stress. Overexpression of ScCBLs in Nicotiana benthamiana leaves promoted their resistance to infection with the tobacco pathogen Ralstonia solanacearum. The results from the present study facilitate further research regarding ScCBL genes, and in particular, their roles in the response to various stresses in sugarcane.

The contamination of aquatic systems with heavy metals is affecting the fish population and hence results in a decline of productivity rate. River Kabul is a transcountry river originating at Paghman province in Afghanistan and inters in Khyber Pakhtunkhwa province of Pakistan and it is the major source of irrigation and more than 54 fish species have been reported in the river. Present study aimed at the estimation of heavy metals load in the fish living in River Kabul. Heavy metals including chromium, nickel, copper, zinc, cadmium, and lead were determined through atomic absorption spectrophotometer after tissue digestion by adopting standard procedures. Concentrations of these metals were recorded in muscles and liver of five native fish species, namely,Wallago attu,Aorichthys seenghala,Cyprinus carpio,Labeo dyocheilus, andOmpok bimaculatus. The concentrations of chromium, nickel, copper, zinc, and lead were higher in both of the tissues, whereas the concentration of cadmium was comparatively low. However, the concentration of metals was exceeding the RDA (Recommended Dietary Allowance of USA) limits. Hence, continuous fish consumption may create health problems for the consumers. The results of the present study are alarming and suggest implementing environmental laws and initiation of a biomonitoring program of the river.

Sugarcane smut is a fungal disease caused by Sporisorium scitamineum. Cultivation of smut-resistant sugarcane varieties is the most effective way to control this disease. The interaction between sugarcane and S. scitamineum is a complex network system. However, to date, there is no report on the identification of microRNA (miRNA) target genes of sugarcane in response to smut pathogen infection by degradome technology. TaqMan qRT-PCR detection and enzyme activity determination showed that S. scitamineum rapidly proliferated and incurred significant enzyme activity changes in the reactive oxygen species metabolic pathway and phenylpropanoid metabolic pathway at 2 d and 5 d after inoculation, which was the best time points to study target gene degradation during sugarcane and S. scitamineum interaction. A total of 122.33 Mb of raw data was obtained from degradome sequencing analysis of YC05–179 (smut-resistant) and ROC22 (smut-susceptible) after inoculation. The Q30 of each sample was > 93%, and the sequence used for degradation site analysis exactly matched the sugarcane reference sequence. A total of 309 target genes were predicted in sugarcane, corresponding to 97 known miRNAs and 112 novel miRNAs, and 337 degradation sites, suggesting that miRNAs can efficiently direct cleavage at multiple sites in the predicted target mRNAs. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the predicted target genes were involved in various regulatory processes, such as signal transduction mechanisms, inorganic ion transport and metabolism, defense mechanisms, translation, posttranslational modifications, energy production and conversion, and glycerolipid metabolism. qRT-PCR analysis of the expression level of 13 predicted target genes and their corresponding miRNAs revealed that there was no obvious negative regulatory relationship between miRNAs and their target genes. In addition, a number of putative resistance-related target genes regulated by miRNA-mediated cleavage were accumulated in sugarcane during S. scitamineum infection, suggesting that feedback regulation of miRNAs may be involved in the response of sugarcane to S. scitamineum infection. This study elucidates the underlying response of sugarcane to S. scitamineum infection, and also provides a resource for miRNAs and their predicted target genes for smut resistance improvement in sugarcane.

Red rod is an economically important disease of sugarcane caused by the fungus Colletotrichum falcatum. We used a simple sequence repeat (SSR)-based marker system to identify and analyze genetic relationships of red rot resistant and susceptible sugarcane cultivars grown in Pakistan. Twenty-one highly polymorphic SSR markers were used for DNA fingerprinting and genetic diversity analysis of 20 sugarcane cultivars. These SSR markers were found to be highly robust; we identified 144 alleles, with 3-11 alleles per marker and a mean of 6.8. Three SSR markers were able to identify all 20 cultivars. DNAMAN(®)-generated homology tree was used to analyze genetic diversity among these cultivars; all cultivars shared 58% or more similarity. We correlated polymorphism information content and resolving power values with marker effectiveness in the process of sugarcane cultivar identification. We concluded that a small number of SSR-derived DNA markers will allow breeders to identify red rot resistant and susceptible cultivars.