Profiling Gene Expression in Germinating Brassica Roots.

Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

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.

Transcriptional regulatory network triggered by oxidative signals configures the early response mechanisms of japonica rice to chilling stress.

BACKGROUND: The transcriptional regulatory network involved in low temperature response leading to acclimation has been established in Arabidopsis. In japonica rice, which can only withstand transient exposure to milder cold stress (10 degrees C), an oxidative-mediated network has been proposed to play a key role in configuring early responses and short-term defenses. The components, hierarchical organization and physiological consequences of this network were further dissected by a systems-level approach. RESULTS: Regulatory clusters responding directly to oxidative signals were prominent during the initial 6 to 12 hours at 10 degrees C. Early events mirrored a typical oxidative response based on striking similarities of the transcriptome to disease, elicitor and wounding induced processes. Targets of oxidative-mediated mechanisms are likely regulated by several classes of bZIP factors acting on as1/ocs/TGA-like element enriched clusters, ERF factors acting on GCC-box/JAre-like element enriched clusters and R2R3-MYB factors acting on MYB2-like element enriched clusters.Temporal induction of several H2O2-induced bZIP, ERF and MYB genes coincided with the transient H2O2 spikes within the initial 6 to 12 hours. Oxidative-independent responses involve DREB/CBF, RAP2 and RAV1 factors acting on DRE/CRT/rav1-like enriched clusters and bZIP factors acting on ABRE-like enriched clusters. Oxidative-mediated clusters were activated earlier than ABA-mediated clusters. CONCLUSION: Genome-wide, physiological and whole-plant level analyses established a holistic view of chilling stress response mechanism of japonica rice. Early response regulatory network triggered by oxidative signals is critical for prolonged survival under sub-optimal temperature. Integration of stress and developmental responses leads to modulated growth and vigor maintenance contributing to a delay of plastic injuries.

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.

Oxygen dependency of germinating Brassica seeds.

Establishing plants in space, Moon or Mars requires adaptation to altered conditions, including reduced pressure and composition of atmospheres. To determine the oxygen requirements for seed germination, we imbibed Brassica rapa seeds under varying oxygen concentrations and profiled the transcription patterns of genes related to early metabolism such as starch degradation, glycolysis, and fermentation. We also analyzed the activity of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH), and measured starch degradation. Partial oxygen pressure (pO2) greater than 10% resulted in normal germination (i.e., protrusion of radicle about 18 hours after imbibition) but lower pO2 delayed and reduced germination. Imbibition in an oxygen-free atmosphere for three days resulted in no germination but subsequent transfer to air initiated germination in 75% of the seeds and the root growth rate was transiently greater than in roots germinated under ambient pO2. In hypoxic seeds soluble sugars degraded faster but the content of starch after 24 h was higher than at ambient oxygen. Transcription of genes related to starch degradation, α-amylase (AMY) and Sucrose Synthase (SUS), was higher under ambient O2 than under hypoxia. Glycolysis and fermentation pathway-related genes, glucose phosphate isomerase (GPI), 6-phosphofructokinase (PFK), fructose 1,6-bisphosphate aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate decarboxylase (PDC), LDH, and ADH, were induced by low pO2. The activity of LDH and ADH was the highest in anoxic seeds. Germination under low O2 conditions initiated ethanolic fermentation. Therefore, sufficient oxygen availability is important for germination before photosynthesis provides necessary oxygen and the determination of an oxygen carrying capacity is important for uniform growth in space 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.

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.

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.

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.

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.

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.