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.

Sugarcane calcineurin B-like (CBL) genes play important but versatile roles in regulation of responses to biotic and abiotic stresses.

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.

Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix.

Human pluripotent stem cell-derived neural crest stem cells (NCSCs) provide a promising cell source for generating Schwann cells in the treatment of neurodegenerative diseases and traumatic injuries in the peripheral nervous system. Influencing cell behavior through a synthetic matrix topography has been shown to be an effective approach to directing stem cell proliferation and differentiation. Here we have investigated the effect of nanofiber topography on the differentiation of human embryonic stem cell-derived NCSCs towards the Schwann cell lineage. Using electrospun fibers of different diameters and alignments we demonstrated that aligned fiber matrices effectively induced cell alignment, and that fiber matrices with average diameters of 600nm and 1.6µm most effectively promoted NCSC differentiation towards the Schwann cell lineage compared with random fibers and two-dimensional tissue culture plates. More importantly, human NCSCs that were predifferentiated in Schwann cell medium for 2weeks exhibited higher sensitivity to the aligned fiber topography than undifferentiated NCSCs. This study provides an efficient protocol for Schwann cell derivation by combining an aligned nanofiber matrix and an optimized differentiation medium, and highlights the importance of matching extrinsic matrix signaling with cell intrinsic programming in a temporally specific manner.

Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold.

The traditional notion that injured neurons are unable to regenerate in the adult mammalian brain and spinal cord has long been a concern. This view has led to methodology designed to overcome this problem, most recently by advancements in tissue engineering. Here, neural precursor cells (NPCs) and the Nogo receptor antibody (NgR-Ab) or poly-L-lysine (PLL) were tested in concert with hyaluronic acid hydrogel scaffolds (HA). In particular, we wished to optimize viability and differentiation of NPCs within HA hydrogel scaffolds. Our results show that HA hydrogels can be modified physically or chemically to improve NPCs attachment on the scaffolding doped with NgR-Ab or PLL. Both the HA hydrogels and their modifications support the viability of NPCs. NPCs were also able to differentiate into neurons and glial cells on HA hydrogels, although this was affected by the different modifications. Immunofluorescence showed that fewer beta-III-tubulin antibody and antineurofilament antibody-positive cells were found on HA-PLL hydrogel compared with HA or HA NgR-Ab hydrogels. This indicates that the PLL-modified HA hydrogels may inhibit differentiation of NPCs, whereas modification by NgR-Ab had no such effect. Finally, the NgR-Ab-modified HA scaffold can be used as not only a NPC delivery system but also a bioactive factor transportation system for CNS repair.

Performance improvement of injectable poly(ethylene glycol) dimethacrylate-based hydrogels with finely dispersed hydroxyapatite.

Injectable hydrogels are attractive materials for biomedical application. In this work, a chemical mixing technique was developed to promote the dispersion of hydroxyapatite (HA) in injectable poly(ethylene glycol) dimethacrylate (PEGDMA)-based hydrogels. Nano-sized HA particles were distributed homogenously within the organic network, whereby HA crystals were formed in the presence of PEGDMA macromers. In addition, hydrogels were also prepared by physical mixing of dry HA particles with PEGDMA, as a comparison. Transmission electron microscopy was used to evaluate the morphology and crystal structure of HA formed in the PEGDMA aqueous solution before polymerization. According to Fourier transform infrared spectra and x-ray diffraction results, hydrogels prepared by different methods have similar components and crystal structures. Scanning electron microscopy was used to observe the hydrogels' morphology, which showed that HA in hydrogels made by chemical mixing was well dispersed and nano sized. Mechanical evaluation indicated that the mean value of the compressive strength and modulus of hydrogels prepared by physical mixing were 0.137 MPa and 0.518 MPa, respectively, while those of hydrogels prepared by chemical mixing were 0.290 MPa and 0.696 MPa, respectively. Furthermore, temperature measurement showed that the mean value of the maximum temperature in the crosslinking process of hydrogels made by chemical mixing was 38.0 degrees C, which was significantly lower than that of for hydrogels made by physical mixing (38.6 degrees C). The results indicated that the performance of composite hydrogels could be promoted by chemical mixing of the inorganic network into a polymer network.

Preparation and characterization of fibroin/hyaluronic acid composite scaffold.

Hyaluronic acid (HA) was added into fibroin solution to prepare fibroin-based porous composite scaffolds. HA exhibited important effects on pore formation and hydrophilicity of fibroin-based scaffold. The aqueous-fibroin/HA scaffolds had highly homogeneous and interconnected pores with porosity of above 90% and controllable pore size ranging from 123 to 253 microm. The water take-up of fibroin/HA scaffolds increased significantly with the increase of HA content. Containing HA at a defined content range, such as 3-6%, fibroin-based scaffolds' affinity to primary neural cells was improved. In 6%HA/fibroin scaffolds, neurosphere-forming cell migrated from their original aggregate and adhered tightly to the surface of scaffolds.

In vitro behavior of neural stem cells in response to different chemical functional groups.

Neural stem cells (NSCs) cultured on glass surfaces modified by different chemical groups, including hydroxyl (-OH), sulfonic (-SO3H), amino (-NH2), carboxyl (-COOH), mercapto (-SH) and methyl (-CH3) groups, are shown here to commit to phonotypes with extreme sensitivity to surface chemical groups. The adhering NSCs at the level of single cells exhibited morphological changes in response to different chemical groups. NSCs on -SO(3)H surfaces had the largest contact area and the most flattened morphology, while those on -CH(3) surfaces exhibited the smallest contact area and the most rounded morphology. After 5 days of culture, the migration of NSCs from their original aggregates onto these test surfaces followed the trend: -NH2>-COOH=-SH>>-SO3H>-CH3>-OH. The expression of specific markers, including nestin, beta-Tubulin-III, glial fibrillary acidic protein and O4, were used to examine NSCs lineage specification. The -SO3H surfaces favored NSCs differentiation into oligodendrocytes, while NSCs in contact with -COOH, -NH2, -SH and -CH3 had the ability to differentiate into neurons, astrocytes and oligodendrocytes. Compared to -COOH surfaces, -NH2 seemed to promote neuronal differentiation. These chemically modified surfaces exhibited regulation of NSCs on adhesion, migration and differentiation potential, providing chemical means for the design of biomaterials to direct NSCs lineage specification for neural tissue engineering.