Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

Genome-wide identification and abiotic stress responses of <Emphasis Type="Italic">DGK</Emphasis> gene family in maize

Diacylglycerol kinase (DGK) is a kind of phosphokinase that catalyzes the formation of signaling molecule phosphatidic acid. In this study, seven maize (Zea mays) DGK gene family members were identified by an exploration of maize genome via multiple online databases, and designated as ZmDGK1-7, respectively. The proteins encoded by ZmDGKs ranged from 487 to 716 amino acids, and had a molecular weight (MWs) between 54.6 and 80.2 kDa. Phylogenetic analysis revealed that ZmDGKs grouped into three clusters as described for known plant DGK families: Cluster I was composed of three maize DGKs, ZmDGK1, ZmDGK4 and ZmDGK5, cluster II contained ZmDGK6, and the isoforms ZmDGK2, ZmDGK3 and ZmDGK7 fell into cluster III. ZmDGK proteins featured the typical functional domains, while all seven ZmDGKs have a conserved catalytic domain DGKc, only the cluster I ZmDGKs have the DAG/PE binding domain. Most ZmDGK genes showed ubiquitous expression profiles at various developmental stages, while a high relative expression was observed at the tasseling stage. ZmDGK genes exhibited differential expression patterns in response to abiotic stresses including cold, salinity and drought, and all ZmDGK genes were found obviously up-regulated by cold. The distinct roles of ZmDGKs in cold response was also supported by the finding that an accumulation of DGK products–PA under low temperature. This study will help to better understand the roles of DGKs in the development and abiotic stress responses in major crops.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

A new <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant <Emphasis Type="Italic">apetala1-20</Emphasis>

A new deletion allele of the APETALA1 (AP1) gene encoding a type II MADS-box protein with the key role in the initiation of flowering and development of perianth organs has been identified in A. thaliana. The deletion of seven amino acids in the conserved region of the K domain in the ap1-20 mutant considerably delayed flowering and led to a less pronounced abnormality in the corolla development compared to the weak ap1-3 and intermediate ap1-6 alleles. At the same time, a considerable stamen reduction has been revealed in ap1-20 as distinct from ap1-3 and ap1-6 alleles. These data indicate that the K domain of AP1 can be crucial for the initiation of flowering and expression regulation of B-class genes controlling stamen development.     

Characterization of the inner membrane protein BB0173 from <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background

The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3β1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made.

Results

Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions.

Conclusions

Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.

     

Comparative pan genome analysis of oral <Emphasis Type="Italic">Prevotella</Emphasis> species implicated in periodontitis

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a “Por_secre_tail” domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.     

Isolation and characterization of two distinct Class II <Emphasis Type="Italic">PR4</Emphasis> genes from the oriental lily hybrid Sorbonne

Pathogenesis-related (PR) proteins are generally involved in the defense of plants and are important contributors in the disease resistance of plants. Among the 17 PRs that are currently recognized, the PR4 family of proteins is divided into two classes and features a conserved barwin domain. In this study, we isolated two Class II PR4s from the oriental hybrid lily cultivar Sorbonne using the rapid amplification of the cDNA ends (RACE) method, and designated these two PR4s LhSorPR4a and LhSorPR4b. LhSorPR4a and LhSorPR4b were 627 and 617 bp in length, respectively, and encoded two corresponding PR4s of 141 and 143 amino acids. These deciphered LhSorPR4a and LhSorPR4b protein sequences shared a sequence similarity of 90.7%, but their theoretical isoelectric points were distinctively different (7.74 and 4.08, respectively). The three-dimensional structures of LhSorPR4a and LhSorPR4b predicted by homology modeling showed high similarity to their corresponding papaya barwin-like protein template. Analysis of expression by qPCR revealed that both LhSorPR4a and LhSorPR4b were responsive to methyl jasmonate and ethephon treatments. The LhSorPR4b expression was also significantly induced by sodium salicylate (SS); however, LhSorPR4a was unresponsive to the SS treatment. Both LhSorPR4a and LhSorPR4b were expressed in Escherichia coli (E. coli) and successfully purified. The PR4s characterized in this study (LhSorPR4a and LhSorPR4b) are the first two PR4 family genes isolated from the Lilium genus, and they could therefore play an important role in lily disease resistance.     

Contribution of <Emphasis Type="Italic">Eutrema salsugineum</Emphasis> Cold Shock Domain Structure to the Interaction with RNA

Plant cold shock domain proteins (CSDPs) are DNA/RNA-binding proteins. CSDPs contain the conserved cold shock domain (CSD) in the N-terminal part and a varying number of the CCHC-type zinc finger (ZnF) motifs alternating with glycine-rich regions in the C-terminus. CSDPs exhibit RNA chaperone and RNA-melting activities due to their non-specific interaction with RNA. At the same time, there are reasons to believe that CSDPs also interact with specific RNA targets. In the present study, we used three recombinant CSDPs from the saltwater cress plant (Eutrema salsugineum)-EsCSDP1, EsCSDP2, EsCSDP3 with 6, 2, and 7 ZnF motifs, respectively, and showed that their nonspecific interaction with RNA is determined by their C-terminal fragments. All three proteins exhibited high affinity to the single-stranded regions over four nucleotides long within RNA oligonucleotides. The presence of guanine in the single-or double-stranded regions was crucial for the interaction with CSDPs. Complementation test using E. coli BX04 cells lacking four cold shock protein genes (ΔcspA, ΔcspB, ΔcspE, ΔcspG) revealed that the specific binding of plant CSDPs with RNA is determined by CSD.     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.     

Genome-wide survey and expression analysis of the stress-associated protein gene family in desert poplar, <Emphasis Type="Italic">Populus euphratica</Emphasis>

Stress-associated proteins (SAPs) are a novel class of zinc finger proteins that extensively participate in abiotic stress responses. To date, no overall analysis and expression profiling of SAP genes in woody plants have been reported. Populus euphratica is distributed in desert regions and is extraordinarily adaptable to abiotic stresses. Thus, it is regarded as a promising candidate for studying abiotic stress resistance mechanisms of woody plants. In this study, 18 non-redundant SAP genes were identified from the genome of P. euphratica using basic local alignment search tool algorithms and functional domain verification. Among these 18 PeuSAP genes, 15 were intronless. To investigate the evolutionary relationships of SAP genes in P. euphratica and other Salicaceae plants, phylogenetic analyses were performed. Subsequently, the expression profiles of the 18 PeuSAP genes were analyzed in different tissues and under various stresses (drought, salt, heat, cold, and abscisic acid (ABA) treatment) using quantitative real-time PCR. Tissue expression analysis indicated that PeuSAPs showed no tissue specificity. PeuSAPs were induced by multiple abiotic stresses, especially drought, salt, and heat stresses, perhaps because of abundant cis-acting heat shock elements and drought-inducible elements in the promoter regions of the PeuSAPs. Moreover, single nucleotide polymorphisms (SNPs) variant analysis revealed many synonymous and non-synonymous SNPs in PeuSAP genes, but the zinc finger structure was conserved during evolution. These results provide an overview of the SAP gene family in P. euphratica and a reference for further functional research on PeuSAP genes.