Identification of QTL Combinations that Cause Spikelet Sterility in Rice Derived from Interspecific Crosses.

BACKGROUND: The exploitation of useful genes through interspecific and intersubspecific crosses has been an important strategy for the genetic improvement of rice. Postzygotic reproductive isolation routinely occurs to hinder the growth of pollen or embryo sacs during the reproductive development of the wide crosses. RESULT: In this study, we investigated the genetic relationship between the hybrid breakdown of the population and transferred resistance genes derived from wide crosses using a near-isogenic population composed of 225 lines. Five loci (qSS12, qSS8, qSS11, ePS6-1, and ePS6-2) associated with spikelet fertility (SF) were identified by QTL and epistatic analysis, and two out of five epistasis interactions were found between the three QTLs (qSS12, qSS8 and qSS11) and background marker loci (ePS6-1 and ePS6-2) on chromosome 6. The results of the QTL combinations suggested a genetic model that explains most of the interactions between spikelet fertility and the detected loci with positive or negative effects. Moreover, the major-effect QTLs, qSS12 and qSS8, which exhibited additive gene effects, were narrowed down to 82- and 200-kb regions on chromosomes 12 and 8, respectively. Of the 13 ORFs present in the target regions, Os12g0589400 and Os12g0589898 for qSS12 and OS8g0298700 for qSS8 induced significantly different expression levels of the candidate genes in rice at the young panicle stage. CONCLUSION: The results will be useful for obtaining a further understanding of the mechanism causing the hybrid breakdown of a wide cross and will provide new information for developing rice cultivars with wide compatibility.

Upstream regulatory architecture of rice genes: summarizing the baseline towards genus-wide comparative analysis of regulatory networks and allele mining.

Dissecting the upstream regulatory architecture of rice genes and their cognate regulator proteins is at the core of network biology and its applications to comparative functional genomics. With the rapidly advancing comparative genomics resources in the genus Oryza, a reference genome annotation that defines the various cis-elements and trans-acting factors that interface each gene locus with various intrinsic and extrinsic signals for growth, development, reproduction and adaptation must be established to facilitate the understanding of phenotypic variation in the context of regulatory networks. Such information is also important to establish the foundation for mining non-coding sequence variation that defines novel alleles and epialleles across the enormous phenotypic diversity represented in rice germplasm. This review presents a synthesis of the state of knowledge and consensus trends regarding the various cis-acting and trans-acting components that define spatio-temporal regulation of rice genes based on representative examples from both foundational studies in other model and non-model plants, and more recent studies in rice. The goal is to summarize the baseline for systematic upstream sequence annotation of the rapidly advancing genome sequence resources in Oryza in preparation for genus-wide functional genomics. Perspectives on the potential applications of such information for gene discovery, network engineering and genomics-enabled rice breeding are also discussed.

Phenotypic, physiological, and molecular evaluation of rice chilling stress response at the vegetative stage.

Rice is a chilling-sensitive plant that is particularly prone to injury during the early stages of seedling development and during flowering. Significant variation exists between subspecies with japonica cultivars generally being less sensitive than most indica cultivars. In most temperate and subtropical countries where rice is grown, crop damage often occurs during the early stages of seedling development due to occasional cold snaps coinciding with the first few weeks after direct seeding in late spring to early summer. Irreversible injuries often result in seedling mortality or if the crop survives a stress episode, plant vigor and resistance to pests and diseases are severely compromised. Recent physiological and molecular studies have shown that oxidative stress is the primary cause of early chilling injuries in rice and the differential responses of indica and japonica cultivars are defined to a large extent by gene expression related to oxidative signaling and defenses. In this chapter, we summarize basic phenotypic, physiological, and molecular procedures that can be adopted for routine evaluation of differential responses between cultivars as well as for functional genomics studies.

Cis-regulatory signatures of orthologous stress-associated bZIP transcription factors from rice, sorghum and Arabidopsis based on phylogenetic footprints.

BACKGROUND: The potential contribution of upstream sequence variation to the unique features of orthologous genes is just beginning to be unraveled. A core subset of stress-associated bZIP transcription factors from rice (Oryza sativa) formed ten clusters of orthologous groups (COG) with genes from the monocot sorghum (Sorghum bicolor) and dicot Arabidopsis (Arabidopsis thaliana). The total cis-regulatory information content of each stress-associated COG was examined by phylogenetic footprinting to reveal ortholog-specific, lineage-specific and species-specific conservation patterns. RESULTS: The most apparent pattern observed was the occurrence of spatially conserved 'core modules' among the COGs but not among paralogs. These core modules are comprised of various combinations of two to four putative transcription factor binding site (TFBS) classes associated with either developmental or stress-related functions. Outside the core modules are specific stress (ABA, oxidative, abiotic, biotic) or organ-associated signals, which may be functioning as 'regulatory fine-tuners' and further define lineage-specific and species-specific cis-regulatory signatures. Orthologous monocot and dicot promoters have distinct TFBS classes involved in disease and oxidative-regulated expression, while the orthologous rice and sorghum promoters have distinct combinations of root-specific signals, a pattern that is not particularly conserved in Arabidopsis. CONCLUSIONS: Patterns of cis-regulatory conservation imply that each ortholog has distinct signatures, further suggesting that they are potentially unique in a regulatory context despite the presumed conservation of broad biological function during speciation. Based on the observed patterns of conservation, we postulate that core modules are likely primary determinants of basal developmental programming, which may be integrated with and further elaborated by additional intrinsic or extrinsic signals in conjunction with lineage-specific or species-specific regulatory fine-tuners. This synergy may be critical for finer-scale spatio-temporal regulation, hence unique expression profiles of homologous transcription factors from different species with distinct zones of ecological adaptation such as rice, sorghum and Arabidopsis. The patterns revealed from these comparisons set the stage for further empirical validation by functional genomics.

Transcriptome profiling characterizes phosphate deficiency effects on carbohydrate metabolism in rice leaves.

Phosphorus (P) is a structural component of nucleic acids and phospholipids and plays important roles in plant growth and development. P accumulation was significantly reduced (about 35%) in rice leaves from plants grown under low (32 µM) P compared to 320 µM P grown plants. Genome response to low P was examined using the rice 60K oligonucleotide DNA microarrays. At the threshold significance of |log2| fold>2.0, 21,033 genes (about 33.7% of all genes on the microarray) were affected by P deficiency. Among all genes on the microarray, 4271 genes were sorted into 51 metabolic pathways. Low P affected 1494 (35.0%) genes and the largest category of genes was related to sucrose degradation to ethanol and lactate pathway. To survey the role of P in rice, 25 pathways were selected based on number of affected genes. Among these pathways, cytosolic glycolysis contained the least number of upregulated but most down-regulated genes. Low P decreased glucose, pyruvate and chlorophyll, and genes related to carbon metabolism and chlorophyllide a biosynthesis. However, sucrose and starch levels increased. These results indicate that P nutrition affects diverse metabolic pathways mostly related to glucose, pyruvate, sucrose, starch, and chlorophyll a.

Supra-optimal expression of the cold-regulated OsMyb4 transcription factor in transgenic rice changes the complexity of transcriptional network with major effects on stress tolerance and panicle development.

The R2R3-type OsMyb4 transcription factor of rice has been shown to play a role in the regulation of osmotic adjustment in heterologous overexpression studies. However, the exact composition and organization of its underlying transcriptional network has not been established to be a robust tool for stress tolerance enhancement by regulon engineering. OsMyb4 network was dissected based on commonalities between the global chilling stress transcriptome and the transcriptome configured by OsMyb4 overexpression. OsMyb4 controls a hierarchical network comprised of several regulatory sub-clusters associated with cellular defense and rescue, metabolism and development. It regulates target genes either directly or indirectly through intermediary MYB, ERF, bZIP, NAC, ARF and CCAAT-HAP transcription factors. Regulatory sub-clusters have different combinations of MYB-like, GCC-box-like, ERD1-box-like, ABRE-like, G-box-like, as1/ocs/TGA-like, AuxRE-like, gibberellic acid response element (GARE)-like and JAre-like cis-elements. Cold-dependent network activity enhanced cellular antioxidant capacity through radical scavenging mechanisms and increased activities of phenylpropanoid and isoprenoid metabolic processes involving various abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), ethylene and reactive oxygen species (ROS) responsive genes. OsMyb4 network is independent of drought response element binding protein/C-repeat binding factor (DREB/CBF) and its sub-regulons operate with possible co-regulators including nuclear factor-Y. Because of its upstream position in the network hierarchy, OsMyb4 functions quantitatively and pleiotrophically. Supra-optimal expression causes misexpression of alternative targets with costly trade-offs to panicle development.

An early response regulatory cluster induced by low temperature and hydrogen peroxide in seedlings of chilling-tolerant japonica rice.

BACKGROUND: Plants respond to low temperature through an intricately coordinated transcriptional network. The CBF/DREB-regulated network of genes has been shown to play a prominent role in freeze-tolerance of Arabidopsis through the process of cold acclimation (CA). Recent evidence also showed that the CBF/DREB regulon is not unique to CA but evolutionarily conserved between chilling-insensitive (temperate) and chilling-sensitive (warm-season) plants. In this study, the wide contrast in chilling sensitivity between indica and japonica rice was used as model to identify other regulatory clusters by integrative analysis of promoter architecture (ab initio) and gene expression profiles. RESULTS: Transcriptome analysis in chilling tolerant japonica rice identified a subset of 121 'early response' genes that were upregulated during the initial 24 hours at 10 degrees C. Among this group were four transcription factors including ROS-bZIP1 and another larger sub-group with a common feature of having as1/ocs-like elements in their promoters. Cold-induction of ROS-bZIP1 preceded the induction of as1/ocs-like element-containing genes and they were also induced by exogenous H2O2 at ambient temperature. Coordinated expression patterns and similar promoter architectures among the 'early response' genes suggest that they belong to a potential regulon (ROS-bZIP-as1/ocs regulatory module) that responds to elevated levels of ROS during chilling stress. Cultivar-specific expression signatures of the candidate genes indicate a positive correlation between the activity of the putative regulon and genotypic variation in chilling tolerance. CONCLUSION: A hypothetical model of an ROS-mediated regulon (ROS-bZIP-as1/ocs) triggered by chilling stress was assembled in rice. Based on the current results, it appears that this regulon is independent of ABA and CBF/DREB, and that its activation has an important contribution in configuring the rapid responses of rice seedlings to chilling stress.

Cloning and characterization of a rice cDNA encoding glutamate decarboxylase.

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7 % and 61.9 % identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2 % identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both Ca(2+) and calmodulin. The GAD-encoded 56 approximately 58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a Ca(2+)/ calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

Enhanced antioxidant enzymes are associated with reduced hydrogen peroxide in barley roots under saline stress.

Antioxidant enzymes are related to the resistance to various abiotic stresses including salinity. Barley is relatively tolerant to saline stress among crop plants, but little information is available on barley antioxidant enzymes under salinity stress. We investigated temporal and spatial responses of activities and isoform profiles of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), non-specific peroxidase (POX), and glutathione reductase (GR) to saline stress in barley seedlings treated with 200 mM NaCl for 0, 1, 2, 5 days, respectively. In the control plant, hydrogen peroxide content was about 2-fold higher in the root than in the shoot. Under saline stress, hydrogen peroxide content was decreased drastically by 70% at 2 d after NaCl treatment (DAT) in the root. In the leaf, however, the content was remained unchanged by 2 DAT and increased about 14 % at 5 DAT. In general, the activities of antioxidant enzymes were increased in the root and shoot under saline stress. But the increase was more significant and consistent in the root. The activities of SOD, CAT, APX, POX, and GR were increased significantly in the root within 1 DAT, and various elevated levels were maintained by 5 DAT. Among the antioxidant enzymes, CAT activity was increased the most drastically. The significant increase in the activities of SOD, CAT, APX, POX, and GR in the NaCl-stressed barley root was highly correlated with the increased expression of the constitutive isoforms as well as the induced ones. The hydrogen peroxide content in the root.

Expression of gamma-tocopherol methyltransferase transgene improves tocopherol composition in lettuce (Latuca sativa L.).

A cDNA encoding gamma-tocopherol methyltransferase (gamma-TMT) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce (T0) containing the gamma-TMT transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and gamma-TMT activities. The ratio of alpha-/gamma-tocopherol content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the T0 plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 T1 progenies of the T0 transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and gamma-TMT activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in T2 progenies of T1 plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis gamma-TMT transgene in lettuce results in a higher enzyme activity and the conversion of the gamma-tocopherol pool to alpha-tocopherol in transgenic lettuce.

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants.

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.

Differential responses of rice acid phosphatase activities and isoforms to phosphorus deprivation.

Acid phosphatases (APases) play a role in the release of phosphate in organic complexes in soil. We investigated tissue- and isoform-specific responses of APases to phosphorus (P) deficiency in three rice genotypes; Dasan-byeo, Sobi-byeo, and Palawan. The levels of shoot APase activity per protein were similar in the three genotypes. They significantly decreased with P deprivation that was longer than seven days. Root APase activity per protein was two- to three-fold higher in Dasan than in Sobi and Palawan. In all genotypes the APase activity increased in P-deficient plants, but the increase was higher in Sobi and Palawan. After 21 days of P deprivation, secreted APase activity increased more than eight-fold in Dasan and two-fold in Sobi and Palawan. Isoform profiles of shoot and root APases were most diverse in Dasan. The activities of the major isoforms in P-deficient shoots decreased in all three genotypes. Depending on the genotypes, further increases in constitutive isoforms and new induction of one to four isoforms occurred in P-deficient roots. The results indicate that tissue and genotype differences in the response of APase to P deficiency are primarily facilitated by the different responses of the isoforms.

Induction of phenylalanine ammonia-lyase gene expression by paraquat and stress-related hormones in Rehmannia glutinosa.

Rehmannia glutinosa is a medicinal herb that is tolerant to the non-selective herbicide paraquat. Acteoside, a phenolic compound present in the plant, has been shown to inhibit paraquat. To understand regulation of the phenylpropanoid pathway that produces the acteoside moiety, we isolated a phenylalanine ammonia-lyase (PAL) cDNA clone (RgPAL1) and used it to examine PAL expression. The deduced 712 amino acid sequence of the open reading frame contains the conserved active site and potential phosphorylation sites of other plant PALs. RgPAL1 mRNA was detected in the leaves, flowers and roots of healthy plants, and the level of the mRNA was higher in leaves than in flowers and roots. RgPAL1 mRNA was induced in leaves by paraquat, H2O2, UV light, wounding, yeast extract, jasmonic acid and ethephon. The transcript level and enzyme activity increased gradually from 6 to 24 h after exposure to paraquat or jasmonic acid. Induction of RgPAL1 by paraquat and stress-related phytohormones suggests that it is involved in the regulation of the phenylpropanoid pathway under oxidative stress.