A transposable element insertion within ZmGE2 gene is associated with increase in embryo to endosperm ratio in maize

Most of the maize kernel oil is located in the embryo while the majority of starch is located in the endosperm. Maize kernel composition and value are affected significantly by the ratio of the embryo size to the endosperm size; however, the genetic regulation of embryo to endosperm ratio (EER) in maize is unknown. Here we identified ZmGE2 gene, which encodes a cytochrome p450 protein, as a gene associated with EER variation in maize. We first expressed rice Giant Embryo (GE) gene driven by oleosin promoter in maize and detected a 23.2?% reduction in EER in transgenic seeds, demonstrating the existence of evolutionarily conserved mechanisms for EER determination in rice and maize. We next identified maize GE2, a homolog of rice GE sharing 70?% identity in amino sequence, as a candidate based on the similar expression pattern and co-localization with a previously detected QTL for EER. Followed by linkage and association mapping, a 247-bp transposable element (TE) insertion in 3′-untranslated region of ZmGE2 gene was identified to be associated with increase in EER and kernel oil content. Expression level of the favorable ZmGE2 allele containing the 247-bp TE insertion was strongly reduced. In addition, the 247-bp TE insertion site was a selection target during the artificial long-term selection for the high EER trait in a high oil population. This is the first report that demonstrates an association of ZmGE2 with EER variation in maize and identifies ZmGE2 gene as a promising target for manipulation of EER and grain composition by either transgenic approach or molecular breeding in maize.     

Molecular characterization of rice OsBIANK1, encoding a plasma membrane-anchored ankyrin repeat protein, and its inducible expression in defense responses

A rice gene, OsBIANK1, encoding a protein containing a typical ankyrin repeat domain, was cloned and identified. The OsBIANK1 protein, consisting of 329 amino acids, contains a conserved ankyrin repeat domain with two ankyrin repeats organized in tandem and was showed to be localized on cytoplasmic membrane during transient expression in onion epidermal cells. Expression of OsBIANK1 was induced by treatment with benzothiadiazole (BTH), a chemical inducer capable of inducing disease resistance response in rice. In BTH-treated rice seedlings, expression of OsBIANK1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, as compared with those in water-treated seedlings. Our preliminary results confirm previous evidences that OsBIANK1 may be involved in regulation of disease resistance response in rice.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

Occurrence and characterization of PEND proteins in angiosperms

The PEND protein is a DNA-binding protein in the inner envelope membrane of the developing chloroplast. It consists of a short pre-sequence, an N-terminal DNA-binding domain (cbZIP), a central repeat domain, and a C-terminal transmembrane domain. PEND homologs have been detected in various angiosperms, including Arabidopsis thaliana, Brassica napus, Medicago truncatula, cucumber and cherry. Monocot homologs have also been detected in barley and rice, but sequence conservation was low in monocots. PEND-related sequences have not been detected in non-flowering plants and algae. Green fluorescent protein fusions consisting of the N-terminal as well as full-length PEND homologs in A. thaliana and B. napus were targeted to chloroplasts, and localized to nucleoids and chloroplast periphery, respectively. Immunoblot analysis suggested that crucifer homologs were present in chloroplasts probably as a dimer, as in the case of pea. These results suggest that PEND protein is present in angiosperms, and the homologs in crucifers are functionally analogous to the PEND protein in pea.     

The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.     

Genome-wide identification and phylogenetic analysis of rice FTIP gene family

FT-INTERACTING PROTEIN (FTIP) gene family in rice are the members of multiple C2 domain and transmembrane region proteins (MCTPs). There are many homologs of OsFTIPs in plants; however, the bioinformatics of them remains unclear. In the studies, 13 OsFTIP genes are identified in rice. OsFTIPs are unevenly located in 12 chromosomes. The OsFTIPs are phylogenetically divided into three clades. Cis-elements respond to abiotic stress, light, and hormones are found in the promoter region of OsFTIPs which are induced by the stimuli. All OsFTIPs are expressed with different profiles. Syntenic analysis of 128 OsFTIPs and FTIP-like homologs reveals that various number of gene pairs are identified between rice and other species. The 128 FTIP-like homologs are divided into six groups which fall into three classes. Ten motifs are shared by most OsFTIPs and their homologs. The studies provide a theoretical basis for further elucidating the functions of OsFTIP gene family.     

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na⁺/H⁺ antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4 % identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na⁺ and accumulate more K⁺. Expression of HtSOS1 decreased Na⁺ content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.     

Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd²⁺ and Cu²⁺ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu²⁺ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu²⁺ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu²⁺ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.     

A CRISPR/Cas9 deletion into the phosphate transporter SlPHO1;1 reveals its role in phosphate nutrition of tomato seedlings

In vascular (Arabidopsis thaliana) and non‐vascular (Physcomitrella patens) plants, PHOSPHATE 1 (PHO1) homologs play important roles in the acquisition and transfer of phosphate. The tomato genome contains six genes (SlPHO1;1‐SlPHO1;6) homologous to AtPHO1. The six proteins have typical characteristics of the plant PHO1 family, such as the three Syg1/Pho81/XPRI (SPX) subdomains in the N‐terminal portion and one ERD1/XPR1/SYG1 (EXS) domain in the C‐terminal portion. Phylogenetic analysis revealed that the SlPHO1 family is subdivided into three clusters. A pairwise comparison indicated that SlPHO1;1 showed the highest level of sequence identity/similarity (67.39/76.21%) to AtPHO1. SlPHO1;1 deletion mutants induced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 displayed typical phenotypes of Pi starvation, such as decreased shoot fresh weight and increased root fresh weight, therefore having a greater root‐to‐shoot ratio. Mutants also accumulated more anthocyanin and had more soluble Pi content in the root and less in the shoot. These results indicate that SlPHO1;1 plays an important role in Pi transport in the tomato at seedling stage.     

Physcomitrella patens Has Kinase-LRR R Gene Homologs and Interacting Proteins

Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein–protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis.     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.     

Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors

The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors.