The Complex Story of Plant Cyclic Nucleotide-Gated Channels

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.     

The Respiratory Burst Oxidase Homolog Protein D (GhRbohD) Positively Regulates the Cotton Resistance to Verticillium dahliae

Verticillium wilt, mainly caused by a soil-inhabiting fungus Verticillium dahliae, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to V. dahliae, and found early differentially expressed protein burst oxidase homolog protein D (GhRbohD). However, the role of GhRbohD-mediated ROS in cotton defense against V. dahliae needs further investigation. In this study, we analyzed the function of GhRbohD-mediated resistance of cotton against V. dahliae in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of GhRbohD was significantly upregulated after V. dahliae inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H₂O₂ and Ca²⁺ contents of GhRbohD-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of GhRbohD-silenced cotton’s defense against V. dahliae. The expression levels of resistance-related genes were downregulated in GhRbohD-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of GhRbohD enhanced resistance of transgenic Arabidopsis to V. dahliae challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that GhRbohD activates ROS production to positively regulate the resistance of plants against V. dahliae.     

Predicted ligand interactions for 3',5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response tothe binding of 3',5'-cyclic nucleotides. Members of the CNGCfamily vary as much as 100-fold in their ability to respondto cAMP and cGMP. Molecular models of the nucleotide bindingdomains of the bovine retina and catfish and rat olfactory CNGCswere built from the crystal structure of cAMP bound to catabolitegene activator protein (CAP) with AMMP, a program for molecularmechanics and dynamics. The nucleotide conformation can be predictedfrom the number of strong and weak interactions between thepurine ring and the binding site. The amino acids predictedto be important for determining the nucleotide affinity andspecificity are residues 61, 83 (mediated through a water molecule),119 and 127 (CAP sequence numbers) which interact with the purinering. These residues also dictate the conformation of the ligandin the binding pocket cGMP is preferentially bound in the synconformation in bovine retina, bovine olfactory and rat olfactoryCNGCs due to Thr83, while either conformation can bind in catfisholfactory CNGC. cAMP is predicted to bind either in syn or anticonformation, depending on the interaction with residue 119:the anti conformation is preferentially bound in olfactory CNGCs.     

GhKWL1 Upregulates GhERF105 but Its Function Is Impaired by Binding with VdISC1, a Pathogenic Effector of Verticillium dahliae

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.     

An Overview of Cotton Gland Development and Its Transcriptional Regulation

Cotton refers to species in the genus Gossypium that bear spinnable seed coat fibers. A total of 50 species in the genus Gossypium have been described to date. Of these, only four species, viz. Gossypium, hirsutum, G. barbadense, G. arboretum, and G. herbaceum are cultivated; the rest are wild. The black dot-like structures on the surfaces of cotton organs or tissues, such as the leaves, stem, calyx, bracts, and boll surface, are called gossypol glands or pigment glands, which store terpenoid aldehydes, including gossypol. The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that stores gossypol and its derivatives. It provides an ideal system for studying cell differentiation and organogenesis. However, only a few genes involved in the process of gland formation have been identified to date, and the molecular mechanisms underlying gland initiation remain unclear. The terpenoid aldehydes in the lysigenous glands of Gossypium species are important secondary phytoalexins (with gossypol being the most important) and one of the main defenses of plants against pests and diseases. Here, we review recent research on the development of gossypol glands in Gossypium species, the regulation of the terpenoid aldehyde biosynthesis pathway, discoveries from genetic engineering studies, and future research directions.     

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.     

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep.     

Using Staphylococcus aureus Cas9 to Expand the Scope of Potential Gene Targets for Genome Editing in Soybean

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a revolutionary genome editing technology that has been used to achieve site-specific gene knock-out, large fragment deletion, or base editing in many plant species including soybean (Glycine max). The Streptococcus pyogenes Cas9 (SpCas9) is widely used in plants at present, although there are some reports describing the application of CRISPR/Cpf1 in soybean. Therefore, the selection range of PAM (protospacer adjacent motif) sequences for soybean is currently limited to 5′-NGG-3′ (SpCas9) or 5′-TTTN-3′ (Cpf1), which in turn limits the number of genes that can be mutated. Another Cas9 enzyme from Staphylococcus aureus (SaCas9) recognizes the PAM sequence 5′-NNGRRT-3′ (where R represents A or G), which can provide a wider range of potential target sequences. In this study, we developed a CRISPR/SaCas9 system and used this tool to specifically induce targeted mutations at five target sites in the GmFT2a (Glyma.16G150700) and GmFT5a (Glyma.16G044100) genes in soybean hairy roots. We demonstrated that this tool can recognize the PAM sequences 5′-AAGGGT-3′, 5′-GGGGAT-3′, 5′-TTGAAT-3′, and 5′-TAGGGT-3′ in soybean, and it achieved mutation rates ranging from 34.5% to 73.3%. Our results show that we have established a highly efficient CRISPR/SaCas9 tool that is as suitable as SpCas9 for genome editing in soybean, and it will be useful for expanding the range of target sequences for genome editing.     

Genomewide Identification and Characterization of the Genes Involved in the Flowering of Cotton

Flowering is a prerequisite for flowering plants to complete reproduction, and flowering time has an important effect on the high and stable yields of crops. However, there are limited reports on flowering-related genes at the genomic level in cotton. In this study, genomewide analysis of the evolutionary relationship of flowering-related genes in different cotton species shows that the numbers of flowering-related genes in the genomes of tetraploid cotton species Gossypium hirsutum and Gossypium barbadense were similar, and that these numbers were approximately twice as much as the number in diploid cotton species Gossypium arboretum. The classification of flowering-related genes shows that most of them belong to the photoperiod and circadian clock flowering pathway. The distribution of flowering-related genes on the chromosomes of the At and Dt subgenomes was similar, with no subgenomic preference detected. In addition, most of the flowering-related core genes in Arabidopsis thaliana had homologs in the cotton genome, but the copy numbers and expression patterns were disparate; moreover, flowering-related genes underwent purifying selection throughout the evolutionary and selection processes. Although the differentiation and reorganization of many key genes of the cotton flowering regulatory network occurred throughout the evolutionary and selection processes, most of them, especially those involved in the important flowering regulatory networks, have been relatively conserved and preferentially selected.