Sequencing and de novo assembly of a near complete indica rice genome.

A high-quality reference genome is critical for understanding genome structure, genetic variation and evolution of an organism. Here we report the de novo assembly of an indica rice genome Shuhui498 (R498) through the integration of single-molecule sequencing and mapping data, genetic map and fosmid sequence tags. The 390.3 Mb assembly is estimated to cover more than 99% of the R498 genome and is more continuous than the current reference genomes of japonica rice Nipponbare (MSU7) and Arabidopsis thaliana (TAIR10). We annotate high-quality protein-coding genes in R498 and identify genetic variations between R498 and Nipponbare and presence/absence variations by comparing them to 17 draft genomes in cultivated rice and its closest wild relatives. Our results demonstrate how to de novo assemble a highly contiguous and near-complete plant genome through an integrative strategy. The R498 genome will serve as a reference for the discovery of genes and structural variations in rice.

Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.).

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli-tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.

Expression patterns of purple acid phosphatase genes in Arabidopsis organs and functional analysis of AtPAP23 predominantly transcribed in flower.

Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin.

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1(t) and 1Dx2(t)) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.

Inducing rye 1R chromosome structural changes in common wheat cv. Chinese spring by the gametocidal chromosome 2C of Aegilops cylindrica.

To generate 1 R deletion and translocation lines, we introduced a 2C chromosome,which was derived from Aegilops cylindrica and was known to have a gametocidal function when added monosomically into common wheat cv. Chinese Spring (CS) and its derivative, into a wheat-rye 1R chromosome disomic addition line (CS-1R"). When the individuals with chromosome constitution 21" + 1R" + 2C' (2n = 45) were selfed, the 1R chromosome structural changes were found to be induced with high frequency (24.1%) among the progenies. By using C-banding and GISH analysis, we analyzed 1R structural changes in 46 F3 individuals, which came from 23 F2 plants. The rearranged 1R chromosomes could be characterized in about 85% of the F3 individuals. This included telosome 1RL (39.1%), iso-chromosome 1 RL (2.2%), whole arm translocation involving 1RL (32.6%), telosome 1RS (4.3%), iso-chromosome 1RS (4.3%), and 1R deletion mutant with break point in the long arm (2.2%). The mutant 1R lines obtained in this study will potentially be useful in mapping the chromosome locations of agronomically important genes located in 1R. This study also demonstrated that molecular markers might be used to identify wheat chromosome arm involved in translocation with 1R.

Interaction between the movement protein of barley yellow dwarf virus and the cell nuclear envelope: role of a putative amphiphilic alpha-helix at the N-terminus of the movement protein.

The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment.

Equilibrative nucleoside transporters of Arabidopsis thaliana. cDNA cloning, expression pattern, and analysis of transport activities.

Equilibrative nucleoside transporters (ENTs) occur in diverse organisms. In the model plant Arabidopsis thaliana, eight potential ENTs (AtENTs) have been predicted by genome sequencing. We here report the cloning of the cDNAs for AtENTs 2, 3, 4, 6, 7, and 8. Conceptual translation of the cDNAs of AtENTs 2, 3, 4, 6, 7, and 8 yielded polypeptides possessing strong similarities to ENTs characterized previously. Eleven putative transmembrane domains were identified in each of the six AtENTs. In suspension cells, the transcription of AtENTs 1, 3, 4, 6, and 8 was increased by two treatments (nitrogen deprivation, application of 5-fluorouracil and methotrexate) that inhibited the de novo pathway of nucleotide synthesis, indicating that multiple members of the Arabidopsis ENT family may function in the salvage pathway of nucleotide synthesis. Except for AtENT1, the transcription of the remaining six AtENTs showed varying degrees of organ specificity. However, all seven AtENTs were expressed in the leaf and flower. In plant, insect, and yeast cells, ectopically expressed AtENT3 was targeted to the plasma membrane. AtENT3 expressed in yeast cells transported adenosine and uridine with high affinity. Furthermore, the activities of AtENT3 appear not to require a transmembrane proton gradient because protonophores did not abolish adenosine or uridine transport. In competition experiments, the transport of [3H]adenosine by AtENT3 was most significantly inhibited by a number of different purine and pyrimidine nucleosides and 2'-deoxynucleosides, although certain nucleobases and nucleotides were also found to have some inhibitory effect. This indicates that AtENT3 may possess broad substrate specificity. Adenosine and uridine transport by AtENT3, although partly sensitive to the vasodilator drugs dilazep and dipyridamole, was resistant to the nucleoside analogue nitrobenzylmercaptopurine ribonucleoside. We conclude that AtENT3 represents the first ei type ENT characterized from higher plants. The potential functions of ENTs in the biology of A. thaliana are discussed.

Purple acid phosphatases of Arabidopsis thaliana. Comparative analysis and differential regulation by phosphate deprivation.

Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family. They are characterized by the presence of seven conserved amino acid residues involved in coordinating the dimetal nuclear center in their reactive site. We compared the 29 PAPs predicted for Arabidopsis thaliana in their varieties of potential metal-ligating residues. Although 24 members possessed sets of metal-ligating residues typical of known PAPs, 1 member lacked four of the seven residues. For the remaining four members, potential metal-ligating residues were generally more similar to those in metal-dependent exonucleases and related proteins. Evidence was obtained for the expression of the majority of the 29 PAPs. To facilitate future investigations, a scheme for naming Arabidopsis PAPs and a system for classifying the 29 PAPs are proposed. The cDNA sequences and the responses to phosphate deprivation of seven Arabidopsis PAPs (AtPAP7-AtPAP13) were characterized. For some AtPAPs analyzed, there were fully processed transcripts as well as splice variants. The splice variants of AtPAP10 were found to associate with polyribosomes and may be translated into a NH(2)-terminal truncated protein. Phylogenetic investigations showed that AtPAPs 7 and 8, together with similar enzymes from other plant species, formed the low molecular weight plant PAP group. Members of this group were more closely related to PAPs from mammalian cells. AtPAPs 9-13, together with kidney bean PAP, formed the high molecular weight PAP group. In phosphate deprivation experiments, gene transcription of AtPAP11 and AtPAP12 was induced and increased, respectively, whereas that of the remaining five AtPAPs was not affected by phosphate deprivation. The present work demonstrates that structure variation and expression regulation of plant PAPs are more complex than previously described and provides a framework for comprehensive molecular genetic and biochemical studies of all Arabidopsis PAPs in the future.