Genome resources for the elite bread wheat cultivar Aikang 58 and mining of elite homeologous haplotypes for accelerating wheat improvement.

Despite recent progress in crop genomics studies, the genomic changes brought about by modern breeding selection are still poorly understood, thus hampering genomics-assisted breeding, especially in polyploid crops with compound genomes such as common wheat (Triticum aestivum). In this work, we constructed genome resources for the modern elite common wheat variety Aikang 58 (AK58). Comparative genomics between AK58 and the landrace cultivar Chinese Spring (CS) shed light on genomic changes that occurred through recent varietal improvement. We also explored subgenome diploidization and divergence in common wheat and developed a homoeologous locus-based genome-wide association study (HGWAS) approach, which was more effective than single homoeolog-based GWAS in unraveling agronomic trait-associated loci. A total of 123 major HGWAS loci were detected using a genetic population derived from AK58 and CS. Elite homoeologous haplotypes (HHs), formed by combinations of subgenomic homoeologs of the associated loci, were found in both parents and progeny, and many could substantially improve wheat yield and related traits. We built a website where users can download genome assembly sequence and annotation data for AK58, perform blast analysis, and run JBrowse. Our work enriches genome resources for wheat, provides new insights into genomic changes during modern wheat improvement, and suggests that efficient mining of elite HHs can make a substantial contribution to genomics-assisted breeding in common wheat and other polyploid crops.

A rice chloroplast-localized ABC transporter ARG1 modulates cobalt and nickel homeostasis and contributes to photosynthetic capacity.

Transport and homeostasis of transition metals in chloroplasts, which are accurately regulated to ensure supply and to prevent toxicity induced by these metals, are thus crucial for chloroplast function and photosynthetic performance. However, the mechanisms that maintain the balance of transition metals in chloroplasts remain largely unknown. We have characterized an albino-revertible green 1 (arg1) rice mutant. ARG1 encodes an evolutionarily conserved protein belonging to the ATP-binding cassette (ABC) transporter family. Protoplast transfection and immunogold-labelling assays showed that ARG1 is localized in the envelopes and thylakoid membranes of chloroplasts. Measurements of metal contents, metal transport, physiological and transcriptome changes revealed that ARG1 modulates cobalt (Co) and nickel (Ni) transport and homeostasis in chloroplasts to prevent excessive Co and Ni from competing with essential metal cofactors in chlorophyll and metal-binding proteins acting in photosynthesis. Natural allelic variation in ARG1 between indica and temperate japonica subspecies of rice is coupled with their different capabilities for Co transport and Co content within chloroplasts. This variation underpins the different photosynthetic capabilities in these subspecies. Our findings link the function of the ARG1 transporter to photosynthesis, and potentially facilitate breeding of rice cultivars with improved Co homeostasis and consequently improved photosynthetic performance.

[Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection].

OBJECTIVE: To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection. METHODS: The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 µmol/L EPA) and EPA (0, 25, 50, 100, and 200 µmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant. RESULTS: After EPA treatment (25 and 50 µmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 µmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 µmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 µmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 µmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). CONCLUSIONS: EPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

The F-box protein OsFBK12 targets OsSAMS1 for degradation and affects pleiotropic phenotypes, including leaf senescence, in rice.

Leaf senescence is related to the grain-filling rate and grain weight in cereals. Many components involved in senescence regulation at either the genetic or physiological level are known. However, less is known about molecular regulation mechanisms. Here, we report that OsFBK12 (an F-box protein containing a Kelch repeat motif) interacts with S-ADENOSYL-l-METHIONINE SYNTHETASE1 (SAMS1) to regulate leaf senescence and seed size as well as grain number in rice (Oryza sativa). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays indicate that OsFBK12 interacts with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and with OsSAMS1. Biochemical and physiological data showed that OsFBK12 targets OsSAMS1 for degradation. OsFBK12-RNA interference lines and OsSAMS1 overexpression lines showed increased ethylene levels, while OsFBK12-OX lines and OsSAMS1-RNA interference plants exhibited decreased ethylene. Phenotypically, overexpression of OsFBK12 led to a delay in leaf senescence and germination and increased seed size, whereas knockdown lines of either OsFBK12 or OsSAMS1 promoted the senescence program. Our results suggest that OsFBK12 is involved in the 26S proteasome pathway by interacting with Oryza sativa S-PHASE KINASE-ASSOCIATED PROTEIN1-LIKE PROTEIN and that it targets the substrate OsSAMS1 for degradation, triggering changes in ethylene levels for the regulation of leaf senescence and grain size. These data have potential applications in the molecular breeding of rice.

Knockdown of SAMS genes encoding S-adenosyl-l-methionine synthetases causes methylation alterations of DNAs and histones and leads to late flowering in rice.

S-Adenosyl-l-methionine synthetase (SAMS) [EC 2.5.1.6] catalyzes to produce SAM (S-adenosyl-l-methionine), a universal methyl group donor in biochemical reactions in cells. However, less is known how SAMS controls plant development. Here, we demonstrate that OsSAMS1, 2 and 3 are essential for histone H3K4me3 and DNA methylation to regulate gene expression related to flowering in Oryza sativa. RNA interference (RNAi) transgenic rice with downregulated transcripts of OsSAMS1, 2 and 3 showed pleiotropic phenotypes, including dwarfism, reduced fertility, delayed germination, as well as late flowering. Delayed germination was largely rescued by application of SAM in the knockdown lines. Knockdown of OsSAMS1, 2 and 3 led to distinguished late flowering and greatly reduced the expression of the flowering key genes, Early heading date 1 (Ehd1), Hd3a and RFT1 (rice FT-like genes). Moreover, the histone H3K4me3 and symmetric DNA methylation at these genes were greatly reduced. Thus, SAM deficiency suppressing DNA and H3K4me3 transmethylations at flowering key genes led to a late-flowering phenotype in rice. This information could help elucidate the mechanism of epigenetic control flowering transition.

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development.

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

AtSOFL1 and AtSOFL2 act redundantly as positive modulators of the endogenous content of specific cytokinins in Arabidopsis.

BACKGROUND: Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited. METHODOLOGY/PRINCIPAL FINDINGS: Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2's overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N(6)-(Delta(2)-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.

Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner.

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.

Overexpression of OsERF1, a novel rice ERF gene, up-regulates ethylene-responsive genes expression besides affects growth and development in Arabidopsis.

Ethylene-responsive factors (ERFs), composing the largest group of AP2/EREBP transcription factors, are involved in diverse functions and some of them have been identified in plants. However, even in model plants Arabidopsis and rice, most of the genes in this group are functionally unknown yet. Especially in rice, ERF genes involved in ethylene response have not been reported previously. Here, we report a novel member of ERF group in rice, OsERF1 (Ethylene Response Factor gene in Oryza sativa). OsERF1 expressed consistently in different organs of rice and could be up-regulated by ethylene, a plant hormone associated with stress response. Overexpression of OsERF1 in Arabidopsis up-regulated the expression of two known ethylene-responsive genes, PDF1.2 and b-chitinase, and also significantly affected the growth and development of transgenic Arabidopsis. These results suggest the involvement of OsERF1 in ethylene response.

Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis.

We used a cDNA microarray approach to monitor the expression profile of rice (Oryza sativa) under cold stress and identified 328 cold-regulated genes. Thirteen such genes encoding MYB, homeodomain, and zinc finger proteins with unknown functions showed a significant change in expression under 72-h cold stress. Among them, OsMYB3R-2 was selected for further study. Unlike most plant R2R3 MYB transcription factors, OsMYB3R-2 has three imperfect repeats in the DNA-binding domain, the same as in animal c-MYB proteins. Expression of OsMYB3R-2 was induced by cold, drought, and salt stress. The Arabidopsis (Arabidopsis thaliana) transgenic plants overexpressing OsMYB3R-2 showed increased tolerance to cold, drought, and salt stress, and the seed germination of transgenic plants was more tolerant to abscisic acid or NaCl than that of wild type. The expression of some clod-related genes, such as dehydration-responsive element-binding protein 2A, COR15a, and RCI2A, was increased to a higher level in OsMYB3R-2-overexpressing plants than in wild type. These results suggest that OsMYB3R-2 acts as a master switch in stress tolerance.

The rice OsRad21-4, an orthologue of yeast Rec8 protein, is required for efficient meiosis.

In yeast, Rad21/Scc1 and its meiotic variant Rec8 are key players in the establishment and subsequent dissolution of sister chromatid cohesion for mitosis and meiosis, respectively, which are essential for chromosome segregation. Unlike yeast, our identification revealed that the rice genome has 4 RAD21-like genes that share lower than 21% identity at polypeptide levels, and each is present as a single copy in this genome. Here we describe our analysis of the function of OsRAD21-4 by RNAi. Western blot analyses indicated that the protein was most abundant in young flowers and less in leaves and buds but absent in roots. In flowers, the expression was further defined to premeiotic pollen mother cells (PMCs) and meiotic PMCs of anthers. Meiotic chromosome behaviors were monitored from male meiocytes of OsRAD21-4-deficient lines mediated by RNAi. The male meiocytes showed multiple aberrant events at meiotic prophase I, including over-condensation of chromosomes, precocious segregation of homologues and chromosome fragmentation. Fluorescence in situ hybridization experiments revealed that the deficient lines were defective in homologous pairing and cohesion at sister chromatid arms. These defects resulted in unequal chromosome segregation and aberrant spore generation. These observations suggest that OsRad21-4 is essential for efficient meiosis.

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth.

As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.

Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana.

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.

Studies on the effects of fpf1 gene on Artemisia annua flowering time and on the linkage between flowering and artemisinin biosynthesis.

The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBIfpf1, and induced shoots. Transgenic plants were obtained through the selection with kanamycin. PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A. annua genome. RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants. These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.

Cloning and characteristics of an allene oxide synthase gene (TaAOS) of winter wheat.

Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase pathway which leads to the formation of jasmonic acid (JA). A full length cDNA of TaAOS was cloned in winter wheat (Triticum aestivum L. cv. Jinghua No.3) seedlings. The open reading frame encompassed 1410 bp encoding a polypeptide of 470 amino acids with calculated molecular mass of 51.9 kD. Southern blot analysis suggested there are three copies of the gene in wheat genome. The TaAOS mRNA could be strongly induced by exogenous JA, and the highest level JA was observed after a 10 h induction. In situ RNA hybridization of seedling indicated preferential gene expression in young leaves, especially in the parenchyma cells around the vascular bundles, and the hybridization also showed that exogenous La(3+) could not suppress the expression of TaAOS induced by JA.

Changes in transcript levels of chloroplast psbA and psbD genes during water stress in wheat leaves.

In order to examine whether the decrease in gene expression of chloroplast DNA-encoded polypetides contributes to the inhibition of photosystem II (PSII) function during water stress, changes in transcript and template levels of chloroplast psbA and psbD genes (encoding the D1 and D2 reaction center proteins of PSII, respectively) were investigated in spring wheat leaves (Triticum aestivum L. cv. Longchun No. 10) using northern, Southern and dot blot analyses. The results of northern hybridization indicated that stressing wheat seedlings in polyethylene glycol (PEG) solutions with an osmotic potential of -0.5 MPa for 0, 24, 48 and 72 h, caused marked declines in the steady state levels of the psbA and psbD transcripts but did not alter their transcript processing patterns. RNA dot blot analysis further demonstrated that over the whole range of water stress investigated, the transcript levels of the two genes declined by 2- and 3-fold, respectively, relative to the same amount of total RNA. As total RNA decreased 3-fold during the process of stress, the transcript levels of psbA and psbD genes actually declined by 6- and 9-fold, respectively. These results suggest that water stress affects the expression of the psbA and psbD genes, possibly at the transcriptional level. Southern and DNA dot blot analyses consistently showed that water stress did not affect the template levels of either psbA or psbD genes, suggesting that the decreased abundance of psbA and psbD transcripts under water stress is not due to limited gene templates but likely a result of lowered gene transcriptional activity and/or changed mRNA stability.