Nitric oxide induces monosaccharide accumulation through enzyme S-nitrosylation.

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.

COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability.

Seasonal changes in day length are perceived by plant photoreceptors and transmitted to the circadian clock to modulate developmental responses such as flowering time. Blue-light-sensing cryptochromes, the E3 ubiquitin-ligase COP1, and clock-associated proteins ELF3 and GI regulate this process, although the regulatory link between them is unclear. Here we present data showing that COP1 acts with ELF3 to mediate day length signaling from CRY2 to GI within the photoperiod flowering pathway. We found that COP1 and ELF3 interact in vivo and show that ELF3 allows COP1 to interact with GI in vivo, leading to GI degradation in planta. Accordingly, mutation of COP1 or ELF3 disturbs the pattern of GI cyclic accumulation. We propose a model in which ELF3 acts as a substrate adaptor, enabling COP1 to modulate light input signal to the circadian clock through targeted destabilization of GI.

Nitric oxide restrain root growth by DNA damage induced cell cycle arrest in Arabidopsis thaliana.

Nitric oxide (NO) participates in the regulation of diverse functions in plant cells. However, different NO concentrations may trigger different pathways during the plant development. At basal levels of NO, plants utilize the NO signaling transduction pathway to facilitate plant growth and development, whereas higher concentrations trigger programmed cell death (PCD). Our results show that NO lower than the levels causing PCD, but higher than the basal levels induce DNA damage in root cells in Arabidopsis as witnessed by a reduction in root growth, rather than cell death, since cells retain the capacity to differentiate root hairs. The decrease in meristematic cells and increase in DNA damage signals in roots in responses to NO are in a dose dependent manner. The restraint of root growth is due to cell cycle arrest at G1 phase which is caused by NO induced DNA damage, besides a second arrest at G2/M existed in NO supersensitive mutant cue1. The results indicate that NO restrain root growth via DNA damage induced cell cycle arrest.

The specific glycerolipid composition is responsible for maintaining the membrane stability of Physcomitrella patens under dehydration stress.

Land colonization is a major event in plant evolution. Little is known about the evolutionary characteristics of lipids during this process. Here, we proved that Physcomitrella patens, a bryophyte that appeared in the early evolution of terrestrial plants, has short-term desiccation resistance. The maintenance of membrane integrity is related to its specific glycerolipid composition and key genes for lipid metabolism. We analyzed 414 types of lipid molecules, and found that phospholipids accounted for 61.7%, mainly PC and PI; glycolipids accounted for only 26.5%, with a special MGDG molecular map. The most abundant MDGD, that is, MGDG34:6, contained rare 15- and 19-carbon acyl chains; the level of neutral lipids was higher. This was consistent with the results observed by TEM, with fewer lamellae and obvious lipid droplets. Slight dehydration accumulated a large number of TAG molecules, and severe dehydration degraded phospholipids and caused membrane leakage, but PA and MGDG fluctuated less. The key genes of lipid metabolism, DGAT and PAP, were actively transcribed, suggesting that PA was one of the main DAG sources for TAG synthesis. This work proves that Physcomitrella patens adopts high-constitutive PC and PI similar to plant seeds, abundant TAG, and its own specific MGDG to resist extreme dehydration. This result provides a new insight into the lipid evolution of early terrestrial plants against unfavorable terrestrial environments.

Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips.

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.

PIF3 is involved in the primary root growth inhibition of Arabidopsis induced by nitric oxide in the light.

PHYTOCHROME INTERACTING FACTOR3 (PIF3) is an important component in the phytochrome signaling pathway and mediates plant responses to various environmental conditions. We found that PIF3 is involved in the inhibition of root growth of Arabidopsis thaliana seedlings induced by nitric oxide (NO) in light. Overexpression of PIF3 partially alleviated the inhibitory effect of NO on root growth, whereas the pif3-1 mutant displayed enhanced sensitivity to NO in terms of root growth. During phytochrome signaling, the photoreceptor PHYB mediates the degradation of PIF3. We found that the phyB-9 mutant had a similar phenotype to that of PIF3ox in terms of responsiveness to NO. Furthermore, NO treatment promoted the accumulation of PHYB, and thus reduced PIF3 content. Our results further show that the activity of PIF3 is regulated by the DELLA protein RGL3[RGA (repressor of ga1-3) LIKE 3]. Therefore, we speculate that PIF3 lies downstream of PHYB and RGL3, and plays an important role in the inhibitory effect of NO on root growth of Arabidopsis seedlings in light.

Feasible and reliable quantification of mRNA in Arabidopsis thaliana using optical thin-film biosensor chips.

mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly(A) tails was reverse transcribed with oligo(dT)(20) primers and dNTPs mixed with digoxigenin(DIG)-11-dUTP. The transcribed first strand cDNA was hybridized with oligo(dA)(20) nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP (alkaline phosphatase) conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip surface. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.

Detection of six genetically modified maize lines using optical thin-film biosensor chips.

As more and more genetically modified organisms (GMO) are commercialized, efficient and inexpensive assays are required for their quick detection. An event-specific detection strategy based on the unique and specific sequences of integration junctions is useful because of its high specificity. This study developed a system for detecting six GM maize lines (Bt11, Bt176, GA21, MON810, NK603, and T25) using optical silicon thin-film biosensor chips. Aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface. Biotinylated PCR amplicons were then hybridized with the probes. After washing and brief incubation with an anti-biotin IgG horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated PCR amplicons perfectly matched with the probes can be visualized by the color change on the chip surface (gold to blue/purple). This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low through moderate to high throughput.

Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays.

BACKGROUND: Legumes are the third largest family of flowering plants and are unique among crop species in their ability to fix atmospheric nitrogen. As a result of recent genome sequencing efforts, legumes are now one of a few plant families with extensive genomic and transcriptomic data available in multiple species. The unprecedented complexity and impending completeness of these data create opportunities for new approaches to discovery. RESULTS: We report here a transcriptional analysis in six different organ types of syntenic regions totaling approximately 1 Mb between the legume plants barrel medic (Medicago truncatula) and soybean (Glycine max) using oligonucleotide tiling microarrays. This analysis detected transcription of over 80% of the predicted genes in both species. We also identified 499 and 660 transcriptionally active regions from barrel medic and soybean, respectively, over half of which locate outside of the predicted exons. We used the tiling array data to detect differential gene expression in the six examined organ types and found several genes that are preferentially expressed in the nodule. Further investigation revealed that some collinear genes exhibit different expression patterns between the two species. CONCLUSION: These results demonstrate the utility of genome tiling microarrays in generating transcriptomic data to complement computational annotation of the newly available legume genome sequences. The tiling microarray data was further used to quantify gene expression levels in multiple organ types of two related legume species. Further development of this method should provide a new approach to comparative genomics aimed at elucidating genome organization and transcriptional regulation.

A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips.

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.

Distinct reorganization of the genome transcription associates with organogenesis of somatic embryo, shoots, and roots in rice.

Most plant cells retain the capacity to differentiate into all the other cell and organ types that constitute a plant. However, genome-wide transcriptional activities underlying the process of cell differentiation are poorly understood, especially in monocot plants. Here we used a rice (Oryza sativa) cell culture system to generate somatic embryos, which were further induced into shoots and roots. The global transcriptional reorganization during the development of somatic embryos, shoots, and roots from cultured cells was studied using a rice whole genome microarray and verified by RNA blotting analysis of representative genes. Overall, only 1-3% of expressed genes were differentially regulated during each organogenesis process at the examined time point. Also metabolic pathways were minimally regulated. Thus the genes that dictating organ formation should be relatively small in number. Comparison of these three transcriptomes revealed little overlap during these three organogenesis processes. These results indicate that each organogenesis involves specific reorganization of genome expression.

Molecular analysis of early rice stamen development using organ-specific gene expression profiling.

Elucidating the regulatory mechanisms of plant organ formation is an important component of plant developmental biology and will be useful for crop improvement applications. Plant organ formation, or organogenesis, occurs when a group of primordial cells differentiates into an organ, through a well-orchestrated series of events, with a given shape, structure and function. Research over the past two decades has elucidated the molecular mechanisms of organ identity and dorsalventral axis determinations. However, little is known about the molecular mechanisms underlying the successive processes. To develop an effective approach for studying organ formation at the molecular level, we generated organ-specific gene expression profiles (GEPs) reflecting early development in rice stamen. In this study, we demonstrated that the GEPs are highly correlated with early stamen development, suggesting that this analysis is useful for dissecting stamen development regulation. Based on the molecular and morphological correlation, we found that over 26 genes, that were preferentially up-regulated during early stamen development, may participate in stamen development regulation. In addition, we found that differentially expressed genes during early stamen development are clustered into two clades, suggesting that stamen development may comprise of two distinct phases of pattern formation and cellular differentiation. Moreover, the organ-specific quantitative changes in gene expression levels may play a critical role for regulating plant organ formation.

Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (Cucumis sativus L.).

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.

DNA damage in the early primordial anther is closely correlated with stamen arrest in the female flower of cucumber (Cucumis sativus L.).

To investigate the regulatory mechanisms of sex expression in cucumber, morphological observations and biochemical analyses were carried out on inappropriate stamen development of female flowers of cucumber. It was found that developmental arrest of the inappropriate stamen mainly occurs at the anther primordium. This arrest is closely correlated with DNA damage, as detected by TUNEL assay, and might result from anther-specific DNase activation. It was also found that the DNA damage does not lead to cell degeneration, although chromatin condensation is observed in the anther primordia.