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Meiotic recombination starts with the programmed formation of double-strand breaks (DSB) in DNA, which are catalyzed by SPO11, a type II topoisomerase that is evolutionarily conserved, and several other accessary proteins. Homologs of MEIOSIS INHIBITOR 4 (MEI4/REC24/PRD2) are proteins that are also essential for the generation of meiotic DSBs in budding yeast, mice and Arabidopsis thaliana. In Arabidopsis, the protein ARABIDOPSIS THALIANA PUTATIVE RECOMBINATION INITIATION DEFECTS 2/MULTIPOLAR SPINDLE 1 (AtPRD2/MPS1) has been shown to have additional roles in spindle assembly, indicating a functional diversification. Here we characterize the role of the rice MEI4/PRD2 homolog in meiosis. The osprd2 mutant was completely male and female sterile. In male meiocytes of osprd2, no γH2AX foci were detected and twenty-four univalents were produced at diakinesis, suggesting that OsPRD2 is essential for DSB generation. OsPRD2 showed a dynamic localization during meiosis. For instance, OsPRD2 foci first appeared as discrete signals across chromosome at leptotene, and then became confined to the centromeres during zygotene, suggesting that they might be involved in assembly of the spindle. However we did not observe any obvious aberrant morphologies in neither the organization of the bipolar spindle nor in the orientation of the kinetochore in the mutant. These findings suggest that in rice PRD2 might not be required for spindle assembly and organization, as it does in Arabidopsis. Taken together our results indicate that plant MEI4/PRD2 homologs do play a conserved role in the formation of meiotic DSBs in DNA, but that their involvement in bipolar spindle assembly is rather species-specific. [ABSTRACT FROM AUTHOR]

Soil compaction represents a major agronomic challenge, inhibiting root elongation and impacting crop yields. Roots use ethylene to sense soil compaction as the restricted air space causes this gaseous signal to accumulate around root tips. Ethylene inhibits root elongation and promotes radial expansion in compacted soil, but its mechanistic basis remains unclear. Here, we report that ethylene promotes abscisic acid (ABA) biosynthesis and cortical cell radial expansion. Rice mutants of ABA biosynthetic genes had attenuated cortical cell radial expansion in compacted soil, leading to better penetration. Soil compaction-induced ethylene also up-regulates the auxin biosynthesis gene OsYUC8. Mutants lacking OsYUC8 are better able to penetrate compacted soil. The auxin influx transporter OsAUX1 is also required to mobilize auxin from the root tip to the elongation zone during a root compaction response. Moreover, osaux1 mutants penetrate compacted soil better than the wild-type roots and do not exhibit cortical cell radial expansion. We conclude that ethylene uses auxin and ABA as downstream signals to modify rice root cell elongation and radial expansion, causing root tips to swell and reducing their ability to penetrate compacted soil. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa) is one of our main food crops, feeding ~3.5 billion people worldwide. An increasing number of studies note the importance of the cytoskeleton, including actin filaments and microtubules, in rice development and environmental responses. Yet, reliable in vivo cytoskeleton markers are lacking in rice, which limits our knowledge of cytoskeletal functions in living cells. Therefore, we generated bright fluorescent marker lines of the actin and microtubule cytoskeletons in rice, suitable for live-cell imaging in a wide variety of rice tissues. Using these lines, we show that actin bundles and microtubules engage and co-function during pollen grain development, how the cytoskeletal components are coordinated during root cell development, and that the actin cytoskeleton is robust and facilitates microtubule responses during salt stress. Hence, we conclude that our cytoskeletal marker lines, highlighted by our findings of cytoskeletal associations and dynamics, will substantially further future investigations in rice biology. [ABSTRACT FROM AUTHOR]

Root angle has a major impact on acquisition of nutrients like phosphate that accumulate in topsoil and in many species; low phosphate induces shallower root growth as an adaptive response. Identifying genes and mechanisms controlling root angle is therefore of paramount importance to plant breeding. Here we show that the actin-binding protein Rice Morphology Determinant (RMD) controls root growth angle by linking actin filaments and gravity-sensing organelles termed statoliths. RMD is upregulated in response to low external phosphate and mutants lacking of RMD have steeper crown root growth angles that are unresponsive to phosphate levels. RMD protein localizes to the surface of statoliths, and rmd mutants exhibit faster gravitropic response owing to more rapid statoliths movement. We conclude that adaptive changes to root angle in response to external phosphate availability are RMD dependent, providing a potential target for breeders. [ABSTRACT FROM AUTHOR]

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice. [ABSTRACT FROM AUTHOR]

Plants employ dynamic molecular networks to control development in response to environmental changes, yet the underlying mechanisms are largely unknown. Here we report the identification of two rice leucine-rich repeat receptor-like kinases, Thermo-Sensitive Genic Male Sterile 10 (TMS10) and its close homolog TMS10-Like (TMS10L), which redundantly function in the maintenance of the tapetal cell layer and microspore/pollen viability under normal temperature conditions with TMS10 playing an essential role in higher temperatures (namely, 28 °C). tms10 displays male sterility under high temperatures but male fertility under low temperatures, and the tms10 tms10l double mutant shows complete male sterility under both high and low temperatures. Biochemical and genetic assays indicate that the kinase activity conferred by the intracellular domain of TMS10 is essential for tapetal degeneration and male fertility under high temperatures. Furthermore, indica or japonica rice varieties that contain mutations in TMS10, created by genetic crosses or genome editing, also exhibit thermo-sensitive genic male sterility. These findings demonstrate that TMS10 and TMS10L act as a key switch in postmeiotic tapetal development and pollen development by buffering environmental temperature changes, providing insights into the molecular mechanisms by which plants develop phenotypic plasticity via genotype-environment temperature interaction. TMS10 may be used as a genetic resource for the development of hybrid seed production systems in crops. [ABSTRACT FROM AUTHOR]

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signaling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture. [ABSTRACT FROM AUTHOR]

The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice. [ABSTRACT FROM AUTHOR]

Lipid molecules are key structural components of plant male reproductive organs, such as the anther and pollen. Although advances have been made in the understanding of acyl lipids in plant reproduction, the metabolic pathways of other lipid compounds, particularly glycerolipids, are not fully understood. Here we report that an endoplasmic reticulum-localized enzyme, Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3), plays an indispensable role in anther development and pollen formation in rice. OsGPAT3 is preferentially expressed in the tapetum and microspores of the anther. Compared with wild-type plants, the osgpat3 mutant displays smaller, pale yellow anthers with defective anther cuticle, degenerated pollen with defective exine, and abnormal tapetum development and degeneration. Anthers of the osgpat3 mutant have dramatic reductions of all aliphatic lipid contents. The defective cuticle and pollen phenotype coincide well with the down-regulation of sets of genes involved in lipid metabolism and regulation of anther development. Taking these findings together, this work reveals the indispensable role of a monocot-specific glycerol-3-phosphate acyltransferase in male reproduction in rice. [ABSTRACT FROM AUTHOR]

Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations. [ABSTRACT FROM AUTHOR]

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity. [ABSTRACT FROM AUTHOR]

Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development. [ABSTRACT FROM AUTHOR]

The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPINIb) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis. [ABSTRACT FROM AUTHOR]

Rice is a major staple food worldwide. Making hybrid rice has proved to be an effective strategy to significantly increase grain yield. Current hybrid rice technologies rely on male sterile lines and have been used predominantly in indica cultivars. However, intrinsic problems exist in the implementation of these technologies, such as limited germplasms and unpredictable conversions from sterility to fertility in the field. Here, we describe a photoperiod-controlled male sterile line, carbon starved anther (csa), which contains a mutation in an R2R3 MYB transcription regulator of pollen development. This mutation was introduced into indica and japonica rice, and it rendered male sterility under short-day conditions and male fertility under long-day conditions in both lines. Furthermore, F1 plants of csa and a restorer line JP69 exhibited heterosis (hybrid vigor), suggesting the feasibility of using this mutation to create hybrid rice. The csa-based photoperiod-sensitive male sterile line allows the establishment of a stable two-line hybrid system, which promises to have a significant impact on agriculture. [ABSTRACT FROM AUTHOR]

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The MIR168-mediated feedback regulatory loop regulates ARGONAUTEI (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant agol-27 display ABA hypersensitivity and drought tolerance, while the MIR168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of MIR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of MIR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants. [ABSTRACT FROM AUTHOR]

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELLI (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development. [ABSTRACT FROM AUTHOR]

Grass plants develop unique floral patterns that determine grain production. However, the molecular mechanism underlying the specification of floral organ identities and meristem determinacy, including the interaction among floral homeotic genes, remains largely unknown in grasses. Here, we report the interactions of rice (Oryza sativa) floral homeotic genes, OsMADS3 (a C-class gene), OsMADS13 (a D-class gene), and DROOPING LEAF (DL), in specifying floral organ identities and floral meristem determinacy. The interaction among these genes was revealed through the analysis of double mutants, osmads13-3 osmads3-4 displayed a loss of floral meristem determinacy and generated abundant carpelloid structures containing severe defective ovules in the flower center, which were not detectable in the single mutant. In addition, in situ hybridization and yeast two-hybrid analyses revealed that OsMADS13 and OsMADS3 did not regulate each other's transcription or interact at the protein level. This indicates that OsMADS3 plays a synergistic role with OsMADS13 in both ovule development and floral meristem termination. Strikingly, osmads3-4 dl-sup6 displayed a severe loss of floral meristem determinacy and produced supernumerary whorls of lodicule-like organs at the forth whorl, suggesting that OsMADS3 and DL synergistically terminate the floral meristem. Furthermore, the defects of osmads13-3 dl-sup6 flowers appeared identical to those of dl-sup6, and the OsMADS13 expression was undetectable in dl-sup6 flowers. These observations suggest that DL and OsMADS13 may function in the same pathway specifying the identity of carpel/ovule and floral meristem. Collectively, we propose a model to illustrate the role of OsMADS3, DL, and OsMADS13 in the specification of flower organ identity and meristem determinacy in rice. [ABSTRACT FROM AUTHOR]

Synthesis of lipidic components in anthers, including of the pollen exine, is essential for plant male reproductive development. Plant lipid transfer proteins (LTPs) are small, abundant lipid-binding proteins that have the ability to exchange lipids between membranes in vitro. However, their biological role in male reproductive development remains less understood. Here, we report the crucial role of OsC6 in regulating postmeiotic anther development in rice (Oryza sativa). Found in monocots, OsC6 belongs to a distinct clade from previously identified LTP1 and LTP2 family members found in both dicots and monocots. OsC6 expression is mainly detectable in tapetal cells and weakly in microspores from stage 9 to stage 11 of anther development. Immunological assays indicated that OsC6 is widely distributed in anther tissues such as the tapetal cytoplasm, the extracellular space between the tapetum and middle layer, and the anther locule and anther cuticle. Biochemical assays indicated that recombinant OsC6 has lipid binding activity. Moreover, plants in which OsC6 was silenced had defective development of orbicules (i.e. Ubisch bodies) and pollen exine and had reduced pollen fertility. Furthermore, additional evidence is provided that the expression of OsC6 is positively regulated by a basic helix-loop-helix transcription factor, Tapetum Degeneration Retardation (TDR). Extra granule-like structures were observed on the inner surface of the tdr tapetal layer when the expression of OsC6 was driven by the TDR promoter compared with the tdr mutant. These data suggest that OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice. [ABSTRACT FROM AUTHOR]

Grass plants develop distinct inflorescences and spikelets that determine grain yields. However, the mechanisms underlying the specification of inflorescences and spikelets in grasses remain largely unknown. Here, we report the biological role of one SEPALLATA (SEP)-like gene, OsMADS34, in controlling the development of inflorescences and spikelets in rice (Oryza sativa). OsMADS34 encodes a MADS box protein containing a short carboxyl terminus without transcriptional activation activity in yeast cells. We demonstrate the ubiquitous expression of OsMADS34 in roots, leaves, and primordia of inflorescence and spikelet organs. Compared with the wild type, osmads34 mutants developed altered inflorescence morphology, with an increased number of primary branches and a decreased number of secondary branches. In addition, osmads34 mutants displayed a decreased spikelet number and altered spikelet morphology, with lemma/leaf-like elongated sterile lemmas. Moreover, analysis of the double mutant osmads34 osmads1 suggests that OsMADS34 specifies the identities of floral organs, including the lemma/palea, lodicules, stamens, and carpel, in combination with another rice SEP-like gene, OsMADS1. Collectively, our study suggests that the origin and diversification of OsMADS34 and OsMADS1 contribute to the origin of distinct grass inflorescences and spikelets. [ABSTRACT FROM AUTHOR]

Estimation of the homoplasmy of transgene in chloroplast is a necessary step in chloroplast transformation. This task is usually achieved by Southern blot analysis, which is laborious, time-consuming, requires large amounts of plant materials and needs hazardous radioisotopes in some cases. To develop a fast, sensitive, stable and effective technique for determining the homoplasmy of transgene in chloroplast, one real-time PCR system based on TaqMan probe technique was developed for evaluating the homoplasmy degree. In the real-time PCR system, one assay targets the exogenous faeG gene, and another one the tobacco chloroplast reference gene, maturase gene within the trnK intron ( matK). The homoplasmy of the transgene was determined by the comparison of copy number of faeG and matK. The analyzed results of 17 transplastomic tobacco lines using the developed real-time PCR system were consistent with those from Southern blot analysis, indicating that the real-time PCR method is suitable for estimating the homoplasmy degree of transplastomic plant with the advantages of high efficiency and throughput, low cost and saving time. [ABSTRACT FROM AUTHOR]

Although AGAMOUS-LIKE6 (AGL6) MADS-box genes are ancient with wide distributions in gymnosperms and angiosperms, their functions remain poorly understood. Here, we show the biological role of the AGL6-like gene, OsMADS6, in specifying floral organ and meristem identities in rice (Oryza sativa L.). OsMADS6 was strongly expressed in the floral meristem at early stages. Subsequently, OsMADS6 transcripts were mainly detectable in paleas, lodicules, carpels and the integument of ovule, as well as in the receptacle. Compared to wild type plants, osmads6 mutants displayed altered palea identity, extra glume-like or mosaic organs, abnormal carpel development and loss of floral meristem determinacy. Strikingly, mutation of a SEPALLATA (SEP)-like gene, OsMADS1 (LHS1), enhanced the defect of osmads6 flowers, and no inner floral organs or glume-like structures were observed in whorls 2 and 3 of osmads1-z osmads6-1 flowers. Furthermore, the osmads1-z osmads6-1 double mutants developed severely indeterminate floral meristems. Our finding, therefore, suggests that the ancient OsMADS6 gene is able to specify “floral state” by determining floral organ and meristem identities in monocot crop rice together with OsMADS1. [ABSTRACT FROM AUTHOR]

Abstract  To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21–47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm−3. Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B. [ABSTRACT FROM AUTHOR]

Background: Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed. Results: GMO Detection method Database (GMDD) has collected almost all the previous developed and reported GMOs detection methods, which have been grouped by different strategies (screen-, gene-, construct-, and event-specific), and also provide a user-friendly search service of the detection methods by GMO event name, exogenous gene, or protein information, etc. In this database, users can obtain the sequences of exogenous integration, which will facilitate PCR primers and probes design. Also the information on endogenous genes, certified reference materials, reference molecules, and the validation status of developed methods is included in this database. Furthermore, registered users can also submit new detection methods and sequences to this database, and the newly submitted information will be released soon after being checked. Conclusion: GMDD contains comprehensive information of GMO detection methods. The database will make the GMOs analysis much easier. [ABSTRACT FROM AUTHOR]

To understand the molecular mechanism regulating meristem development in the monocot rice (Oryza sativa), we describe here the isolation and characterization of three floral organ number4 (fon4) alleles and the cloning of the FON4 gene. The fon4 mutants showed abnormal enlargement of the embryonic and vegetative shoot apical meristems (SAMs) and the inflorescence and floral meristems. Likely due to enlarged SAMs, fon4 mutants produced thick culms (stems) and increased numbers of both primary rachis branches and floral organs. We identified FON4 using a map-based cloning approach and found it encodes a small putatively secreted protein, which is the putative ortholog of the Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) gene. FON4 transcripts mainly accumulated in the small group of cells at the apex of the SAMs, whereas the rice ortholog of CLV1 (FON1) is expressed throughout the SAMs, suggesting that the putative FON4 ligand might be sequestered as a possible mechanism for rice meristem regulation. Exogenous application of the peptides FON4p and CLV3p corresponding to the CLV3/ESR-related (CLE) motifs of FON4 and CLV3, respectively, resulted in termination of SAMs in rice, and treatment with CLV3p caused consumption of both rice and Arabidopsis root meristems, suggesting that the CLV pathway in limiting meristem size is conserved in both rice and Arabidopsis. However, exogenous FON4p did not have an obvious effect on limiting both rice and Arabidopsis root meristems, suggesting that the CLE motifs of Arabidopsis CLV3 and FON4 are potentially functionally divergent. [ABSTRACT FROM AUTHOR]

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning. [ABSTRACT FROM AUTHOR]

The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form well-supported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs. [ABSTRACT FROM AUTHOR]

To understand the expansion of multicopy microRNA (miRNA) families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miR160 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.Cell Research (2006) 16: 507–518. doi:10.1038/sj.cr.7310062; published online 15 May 2006 [ABSTRACT FROM AUTHOR]

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 μg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions. [ABSTRACT FROM AUTHOR]