Rice GA3ox1 modulates pollen starch granule accumulation and pollen wall development.

The rice GA biosynthetic gene OsGA3ox1 has been proposed to regulate pollen development through the gametophytic manner, but cellular characterization of its mutant pollen is lacking. In this study, three heterozygotic biallelic variants, "-3/-19", "-3/-2" and "-3/-10", each containing one null and one 3bp-deletion allele, were obtained by the CRISPR/Cas9 technique for the functional study of OsGA3ox1. The three homozygotes, "-19/-19", "-2/-2" and "-10/-10", derived from heterozygotic variants, did not affect the development of most vegetative and floral organs but showed a significant reduction in seed-setting rate and in pollen viability. Anatomic characterizations of these mutated osga3ox1 pollens revealed defects in starch granule accumulation and pollen wall development. Additional molecular characterization suggests that abnormal pollen development in the osga3ox1 mutants might be linked to the regulation of transcription factors OsGAMYB, OsTDR and OsbHLH142 during late pollen development. In brief, the rice GA3ox1 is a crucial gene that modulates pollen starch granule accumulation and pollen wall development at the gametophytic phase.

The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene.

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line 'TNG71-Bph45' was developed from the Oryza sativa japonica variety 'Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.

Two genomic regions of a sodium azide induced rice mutant confer broad-spectrum and durable resistance to blast disease.

Rice blast, one of the most destructive epidemic diseases, annually causes severe losses in grain yield worldwide. To manage blast disease, breeding resistant varieties is considered a more economic and environment-friendly strategy than chemical control. For breeding new resistant varieties, natural germplasms with broad-spectrum resistance are valuable resistant donors, but the number is limited. Therefore, artificially induced mutants are an important resource for identifying new broad-spectrum resistant (R) genes/loci. To pursue this approach, we focused on a broad-spectrum blast resistant rice mutant line SA0169, which was previously selected from a sodium azide induced mutation pool of TNG67, an elite japonica variety. We found that SA0169 was completely resistant against the 187 recently collected blast isolates and displayed durable resistance for almost 20 years. Linkage mapping and QTL-seq analysis indicated that a 1.16-Mb region on chromosome 6 (Pi169-6(t)) and a 2.37-Mb region on chromosome 11 (Pi169-11(t)) conferred the blast resistance in SA0169. Sequence analysis and genomic editing study revealed 2 and 7 candidate R genes in Pi169-6(t) and Pi169-11(t), respectively. With the assistance of mapping results, six blast and bacterial blight double resistant lines, which carried Pi169-6(t) and/or Pi169-11(t), were established. The complementation of Pi169-6(t) and Pi169-11(t), like SA0169, showed complete resistance to all tested isolates, suggesting that the combined effects of these two genomic regions largely confer the broad-spectrum resistance of SA0169. The sodium azide induced mutant SA0169 showed broad-spectrum and durable blast resistance. The broad resistance spectrum of SA0169 is contributed by the combined effects of two R regions, Pi169-6(t) and Pi169-11(t). Our study increases the understanding of the genetic basis of the broad-spectrum blast resistance induced by sodium azide mutagenesis, and lays a foundation for breeding new rice varieties with durable resistance against the blast pathogen.

Comparisons within the Rice GA 2-Oxidase Gene Family Revealed Three Dominant Paralogs and a Functional Attenuated Gene that Led to the Identification of Four Amino Acid Variants Associated with GA Deactivation Capability.

BACKGROUND: GA 2-oxidases (GA2oxs) are involved in regulating GA homeostasis in plants by inactivating bioactive GAs through 2ß-hydroxylation. Rice GA2oxs are encoded by a family of 10 genes; some of them have been characterized, but no comprehensive comparisons for all these genes have been conducted. RESULTS: Rice plants with nine functional GA2oxs were demonstrated in the present study, and these genes not only were differentially expressed but also revealed various capabilities for GA deactivation based on their height-reducing effects in transgenic plants. Compared to that of wild-type plants, the relative plant height (RPH) of transgenic plants was scored to estimate their reducing effects, and 8.3% to 59.5% RPH was observed. Phylogenetic analysis of class I GA2ox genes revealed two functionally distinct clades in the Poaceae. The OsGA2ox3, 4, and 8 genes belonging to clade A showed the most severe effect (8.3% to 8.7% RPH) on plant height reduction, whereas the OsGA2ox7 gene belonging to clade B showed the least severe effect (59.5% RPH). The clade A OsGA2ox3 gene contained two conserved C186/C194 amino acids that were crucial for enzymatic activity. In the present study, these amino acids were replaced with OsGA2ox7-conserved arginine (C186R) and proline (C194P), respectively, or simultaneously (C186R/C194P) to demonstrate their importance in planta. Another two amino acids, Q220 and Y274, conserved in OsGA2ox3 were substituted with glutamic acid (E) and phenylalanine (F), respectively, or simultaneously to show their significance in planta. In addition, through sequence divergence, RNA expression profile and GA deactivation capability analyses, we proposed that OsGA2ox1, OsGA2ox3 and OsGA2ox6 function as the predominant paralogs in each of their respective classes. CONCLUSIONS: This study demonstrates rice has nine functional GA2oxs and the class I GA2ox genes are divided into two functionally distinct clades. Among them, the OsGA2ox7 of clade B is a functional attenuated gene and the OsGA2ox1, OsGA2ox3 and OsGA2ox6 are the three predominant paralogs in the family.

Using Machine Learning Approaches to Predict Target Gene Expression in Rice T-DNA Insertional Mutants.

To change the expression of the flanking genes by inserting T-DNA into the genome is commonly used in rice functional gene research. However, whether the expression of a gene of interest is enhanced must be validated experimentally. Consequently, to improve the efficiency of screening activated genes, we established a model to predict gene expression in T-DNA mutants through machine learning methods. We gathered experimental datasets consisting of gene expression data in T-DNA mutants and captured the PROMOTER and MIDDLE sequences for encoding. In first-layer models, support vector machine (SVM) models were constructed with nine features consisting of information about biological function and local and global sequences. Feature encoding based on the PROMOTER sequence was weighted by logistic regression. The second-layer models integrated 16 first-layer models with minimum redundancy maximum relevance (mRMR) feature selection and the LADTree algorithm, which were selected from nine feature selection methods and 65 classified methods, respectively. The accuracy of the final two-layer machine learning model, referred to as TIMgo, was 99.3% based on fivefold cross-validation, and 85.6% based on independent testing. We discovered that the information within the local sequence had a greater contribution than the global sequence with respect to classification. TIMgo had a good predictive ability for target genes within 20 kb from the 35S enhancer. Based on the analysis of significant sequences, the G-box regulatory sequence may also play an important role in the activation mechanism of the 35S enhancer.

Phalaenopsis orchid miniaturization by overexpression of OsGA2ox6, a rice GA2-oxidase gene.

BACKGROUND: Phalaenopsis orchids are one of the most common potted orchids sold worldwide. Most Phalaenopsis cultivars have long inflorescences that cause shipping problems and increase handling costs. Miniaturization of Phalaenopsis orchids not only reduces overall production costs but also can expand the appeal of the orchids to a different group of consumers who prefer to keep flowers on desks or tabletops. Although some miniature Phalaenopsis plants can be obtained via hybridization or mutation, they are unpredictable and limited in variety. We therefore used the transgenic approach of overexpressing gibberellin 2-oxidase 6 (OsGA2ox6), a rice GA deactivation gene, to investigate its functional effect in miniaturizing Phalaenopsis and to create a stable miniaturization platform to facilitate a supply for the potential demands of the miniature flower market. RESULTS: A commercial moth orchid, Phalaenopsis Sogo Yukidian 'SPM313', was transformed with the plasmid vector Ubi:OsGA2ox6 and successfully overexpressed the OsGA2ox6 gene in planta. The transgenic lines displayed darker-green, shorter, and wider leaves, thicker roots and much shorter flower spikes (10 cm vs 33 cm) than the nontransgenic line with a normal flower size and blooming ability and are therefore an ideal miniaturized form of Phalaenopsis orchids. CONCLUSIONS: We demonstrated that the ectopic expression of OsGA2ox6 can miniaturize Phalaenopsis Sogo Yukidian 'SPM313' while preserving its blooming ability, providing an alternative, useful method for miniaturizing Phalaenopsis species. This miniaturization by a transgenic approach can be further expanded by using GA2ox genes from different plant species or different gene variants, thereby expanding the technical platform for miniaturizing Phalaenopsis species to meet the potential demands of the miniature Phalaenopsis flower market.

EAT-Rice: A predictive model for flanking gene expression of T-DNA insertion activation-tagged rice mutants by machine learning approaches.

T-DNA activation-tagging technology is widely used to study rice gene functions. When T-DNA inserts into genome, the flanking gene expression may be altered using CaMV 35S enhancer, but the affected genes still need to be validated by biological experiment. We have developed the EAT-Rice platform to predict the flanking gene expression of T-DNA insertion site in rice mutants. The three kinds of DNA sequences including UPS1K, DISTANCE, and MIDDLE were retrieved to encode and build a forecast model of two-layer machine learning. In the first-layer models, the features nucleotide context (N-gram), cis-regulatory elements (Motif), nucleotide physicochemical properties (NPC), and CG-island (CGI) were used to build SVM models by analysing the concealed information embedded within the three kinds of sequences. Logistic regression was used to estimate the probability of gene activation which as feature-encoding weighting within first-layer model. In the second-layer models, the NaiveBayesUpdateable algorithm was used to integrate these first layer-models, and the system performance was 88.33% on 5-fold cross-validation, and 79.17% on independent-testing finally. In the three kinds of sequences, the model constructed by Middle had the best contribution to the system for identifying the activated genes. The EAT-Rice system provided better performance and gene expression prediction at further distances when compared to the TRIM database. An online server based on EAT-rice is available at http://predictor.nchu.edu.tw/EAT-Rice.

Large-scale phenomics analysis of a T-DNA tagged mutant population.

Rice, Oryza sativa L., is one of the most important crops in the world. With the rising world population, feeding people in a more sustainable and environmentally friendly way becomes increasingly important. Therefore, the rice research community needs to share resources to better understand the functions of rice genes that are the foundation for future agricultural biotechnology development, and one way to achieve this goal is via the extensive study of insertional mutants. We have constructed a large rice insertional mutant population in a japonica rice variety, Tainung 67. The collection contains about 93 000 mutant lines, among them 85% with phenomics data and 65% with flanking sequence data. We screened the phenotypes of 12 individual plants for each line grown under field conditions according to 68 subcategories and 3 quantitative traits. Both phenotypes and integration sites are searchable in the Taiwan Rice Insertional Mutants Database. Detailed analyses of phenomics data, T-DNA flanking sequences, and whole-genome sequencing data for rice insertional mutants can lead to the discovery of novel genes. In addition, studies of mutant phenotypes can reveal relationships among varieties, cultivation locations, and cropping seasons.

Tumor Hypoxia Regulates Forkhead Box C1 to Promote Lung Cancer Progression.

Forkhead box C1 (FOXC1) is a member of the forkhead family of transcription factors that are characterized by a DNA-binding forkhead domain. Increasing evidence indicates that FOXC1 is involved in tumor progression. However, the role of tumor hypoxia in FOXC1 regulation and its impact on lung cancer progression are unclear. Here, we report that FOXC1 was upregulated in hypoxic areas of lung cancer tissues from rodents or humans. Hypoxic stresses significantly induced FOXC1 expression. Moreover, hypoxia activated FOXC1 transcription via direct binding of hypoxia-inducible factor-1α (HIF-1α) to the hypoxia-responsive element (HRE) in the FOXC1 promoter. FOXC1 gain-of-function in lung cancer cells promoted cell proliferation, migration, invasion, angiogenesis, and epithelial-mesenchymal transition in vitro. However, a knockdown of FOXC1 in lung cancer cells inhibited these effects. Notably, knockdown of tumor hypoxia-induced FOXC1 expression via HIF-1-mediated FOXC1 shRNAs in lung cancer xenograft models suppressed tumor growth and angiogenesis. Finally, systemic delivery of FOXC1 siRNA encapsulated in lipid nanoparticles inhibited tumor growth and increased survival time in lung cancer-bearing mice. Taken together, these data indicate that FOXC1 is a novel hypoxia-induced transcription factor and plays a critical role in tumor microenvironment-promoted lung cancer progression. Systemic FOXC1 blockade therapy may be an effective therapeutic strategy for lung cancer.

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice.

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops.

Genetic resources offer efficient tools for rice functional genomics research.

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.

The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos.

KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.

A proteomic study of rice cultivar TNG67 and its high aroma mutant SA0420.

Fragrance is a very important economic trait for rice cultivars. To identify the aroma genes in rice, we performed a proteomics analysis of aroma-related proteins between Tainung 67 (TNG67) and its high aroma mutant SA0420. Seventeen of the differentially identified proteins were close related with the aroma phenotype of SA0420. Among them, 9 were found in leaves and 8 were found in grains. One protein (L3) was identified as the chloroplastic glyceraldehyde-3-phosphate dehydrogenase B (OsGAPDHB) which was less abundant in SA0420 than TNG67. Sequence analysis demonstrated that this protein in SA0420 carries a P425S mutation in the C-terminal extension domain, which might hinder the formation of holoenzyme, thereby changing the profile of aroma compounds. The protein profile of OsGAPDHB showed only a weak correlation to its transcription profile. This result indicated that the reduction of OsGAPDHB in SA0420 is regulated by post-translational processes and can only be analyzed by proteomics approach. Transgenic lines suppressing OsGAPDHB through RNAi harbored more fragrance than TNG67 but less than SA0420. With betaine-aldehyde dehydrogenase as the only fragrance gene identified in rice to date, OsGAPDHB may serve as the second protein known to contribute to the aroma phenotype.

Ectopic expression of OsMADS45 activates the upstream genes Hd3a and RFT1 at an early development stage causing early flowering in rice.

BACKGROUND: The rice gene, OsMADS45, which belongs to the MADS-box E class gene, participates in the regulation of floral development. Previous studies have revealed that ectopic expression of OsMADS45 induces early flowering and influences reduced plant height under short-day (SD) conditions. However, the regulation mechanism of OsMADS45 overexpression remains unknown. We introduce an OsMADS45 overexpression construct Ubi:OsMADS45 into TNG67 plants (an Hd1 (Heading date 1) and Ehd1 (Early heading date 1) defective rice cultivar grown in Taiwan), and we analyzed the expression patterns of various floral regulators to understand the regulation pathways affected by OsMADS45 expression. RESULTS: The transgenic rice exhibit a heading date approximately 40 days earlier than that observed in TNG67 plants, and transgenic rice display small plant size and low grain yield. OsMADS45 overexpression did not alter the oscillating rhythm of the examined floral regulatory genes but advanced (by approximately 20 days) the up-regulate of two florigens, Hd3a (Heading Date 3a) and RFT1 (RICE FLOWERING LOCUS T1) and suppressed the expression of Hd1 at the juvenile stage. The expression levels of OsMADS14 and OsMADS18, which are two well-known reproductive phase transition markers, were also increased at early developmental stages and are believed to be the major regulators responsible for early flowering in OsMADS45-overexpressing transgenic rice. OsMADS45 overexpression did not influence other floral regulator genes upstream of Hd1 and Ehd1, such as OsGI (OsGIGANTEA), Ehd2/Osld1/RID1 and OsMADS50. CONCLUSION: These results indicate that in transgenic rice, OsMADS45 overexpressing ectopically activates the upstream genes Hd3a and RFT1 at early development stage and up-regulates the expression of OsMADS14 and OsMADS18, which induces early flowering.

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

Porcine lactoferrin expression in transgenic rice and its effects as a feed additive on early weaned piglets.

The purpose of this study is to determine the growth performance and immune characteristics of early weaned piglets receiving rice bran expressing porcine lactoferrin as a feed additive. Full-length cDNA encoding porcine lactoferrin (LF) driven by a rice actin promoter was transformed into rice plants, and its integration into the rice genome was verified by Southern blot analysis. The expression of recombinant LF (rLF) in whole grains and rice bran was also confirmed, and the amount of rLF accumulated in rice bran was estimated by immunoblot assay to be approximately 0.1% of rice bran weight. An iron-binding assay showed that the rLF retained iron-binding activity and the binding capacity of 1 mg/mL rLF would be saturated by 100 microM of FeCl(3). Thirty-six early weaned piglets at 21 days old were randomly selected into two groups and fed a diet containing 5% transgenic rice bran containing 50 mg/kg rLF (rLF group) and 5% rice bran (control group) to investigate the piglets' growth performance and immune characteristics. The results showed no significant difference in growth performance between the groups during the feeding period. However, the aerobic bacteria, anaerobic bacteria, and coliform counts in the cecal contents of the rLF-fed group were significantly lower than those of the control group. Additional immune characteristics such as the IgG concentration in the rLF group was higher than the control group at the 28th day, but leukocyte counts and the peripheral lymphocyte ratio remained similar. In summary, porcine LF expressed in rice bran, a byproduct of rice, can be used as a functional additive to improve antimicrobial capabilities and IgG concentration of early weaned piglets.

Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms.

Expression of OLE16 and OLE18, two oleosin isoforms in oil bodies of rice seeds, was suppressed by RNA interference. Electron microscopy revealed a few large, irregular oil clusters in 35S::ole16i transgenic seed cells, whereas accumulated oil bodies in 35S::ole18i transgenic seed cells were comparable to or slightly larger than those in wild-type seed cells. Large and irregular oil clusters were observed in cells of double mutant seeds. These unexpected differences observed in oil bodies of 35S::ole16i and 35S::ole18i transgenic seeds were further analyzed. In comparison to wild-type plants, OLE18 levels were reduced to approximately 40% when OLE16 was completely eliminated in 35S::ole16i transgenic plants. In contrast, OLE16 was reduced to only 80% of wild-type levels when OLE18 was completely eliminated in 35S::ole18i transgenic plants. While the triacylglycerol content of crude seed extracts of 35S::ole16i and 35S::ole18i transgenic seeds was reduced to approximately 60% and 80%, respectively, triacylglycerol in isolated oil bodies was respectively reduced to 45% and 80% in accordance with the reduction of their oleosin contents. Oil bodies isolated from both 35S::ole16i and 35S::ole18i transgenic seeds were found to be of comparable size and stability to those isolated from wild-type rice seeds, although they were merely sheltered by a single oleosin isoform. The drastic difference between the triacylglycerol contents of crude seed extracts and isolated oil bodies from 35S::ole16i transgenic plants could be attributed to the presence of large, unstable oil clusters that were sheltered by insufficient amounts of oleosin and therefore could not be isolated together with stable oil bodies.

Tandem organization of StarkB element (22.8 kb) in the maize B chromosome.

A very large repetitive element (StarkB, 22.8 kb) is present in the maize B chromosome, presumably not organized as tandem arrays. Results of the current study are contrary to this notion. Out of eighteen StarkB-carrying sequences, nine were the expected internal fragment of StarkB, and nine others were fragments spanning two StarkB elements. One of the two StarkB components, GrandeB, was flanked in all clones with identical target sequences, as opposed to other Grandes that are associated with different target sequences. Also observed was a prominent Southern signal associated with a fragment representing the junction of two adjacent StarkB units. A clone possessing a structure inverse to that of the second component of StarkB is proposed to be the initial element into which a GrandeB inserted to derive StarkB. Most, if not all, isolated StarkB arrays were not the original form, being disrupted by the invasion of various mobile elements intertwined with various stages of amplification.

A novel class of gibberellin 2-oxidases control semidwarfism, tillering, and root development in rice.

Gibberellin 2-oxidases (GA2oxs) regulate plant growth by inactivating endogenous bioactive gibberellins (GAs). Two classes of GA2oxs inactivate GAs through 2beta-hydroxylation: a larger class of C(19) GA2oxs and a smaller class of C(20) GA2oxs. In this study, we show that members of the rice (Oryza sativa) GA2ox family are differentially regulated and act in concert or individually to control GA levels during flowering, tillering, and seed germination. Using mutant and transgenic analysis, C(20) GA2oxs were shown to play pleiotropic roles regulating rice growth and architecture. In particular, rice overexpressing these GA2oxs exhibited early and increased tillering and adventitious root growth. GA negatively regulated expression of two transcription factors, O. sativa homeobox 1 and TEOSINTE BRANCHED1, which control meristem initiation and axillary bud outgrowth, respectively, and that in turn inhibited tillering. One of three conserved motifs unique to the C(20) GA2oxs (motif III) was found to be important for activity of these GA2oxs. Moreover, C(20) GA2oxs were found to cause less severe GA-defective phenotypes than C(19) GA2oxs. Our studies demonstrate that improvements in plant architecture, such as semidwarfism, increased root systems and higher tiller numbers, could be induced by overexpression of wild-type or modified C(20) GA2oxs.