Project description:A simulated human-tended flight experiment was conducted by growing Arabidopsis thaliana plants on a phytagel matrix-encased coupon which was inserted into a Kennedy Fixation Tube (KFT) preloaded with RNAlater. RNAseq analysis conducted on these plants showed the ability to capture gravitational responses with high reproducibility. To mimick a suborbital flight profile, plants in KFTs were subjected to clinorotation and its transcriptome was compared to various quiescent controls.
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis.
Project description:Little is known of the transcriptome of in vivo-grown pollen tubes, due to the difficulty of collection of pollen tubes elongating within the maternal gynoecium.We obtained the mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana(Col-0).
Project description:Of all the essential nutrients, nitrogen is the one most often limiting for plant growth. Nitrogen can be taken up by plants in two ways. One possibility is through ammonium and nitrate, which are the predominate inorganic forms of nitrogen in soils. The second possibility is the uptake of air-born nitrogen through plant-associated mircoorganisms in root nodules. The majority of plants able to form such nitrogen-fixing root nodules are in the legume family Fabaceae. Here we present a third possibility – a new pathway, termed as nitric oxide (NO)-fixation pathway, which allows plants to fix atmospheric NO and to use it for better growth and development. We identified non-symbiotic hemoglobin class 1 (AtGLB1) and class 2 (AtGLB2) as key proteins of the NO-fixation pathway. In an NO enriched environment NO-fixation is enhanced considerably in plants overexpressing AtGLB1 or AtGLB2 genes. NO uptake resulted in four-fold higher nitrate levels in these plants compared to NO-treated wild-type plants. Correspondingly, the growth parameters like rosettes size and weight, vegetative shoot thickness and also seed yield were 25%, 40%, 30%, and 20% higher, respectively, in the overexpression lines in comparison to wild-type plants. Our results highlight the existence of a NO-fixing pathway in plants. We demonstrated that plant non-symbiotic hemoglobin proteins can fix atmospheric NO and convert it to nitrate, which is further introduced into the N-metabolism. We assume that our results might provide new insights into the field of crop science research and that the NO-fixation capability might serve as a new economically important breeding trait for enhancing biomass, fruit, and seed production. Modifying this pathway might be a promising approach for better and more environment-friendly supply of nitrogen. For example crop plant hemoglobin proteins could be improved for their NO-fixing capability and their expression levels could be increased.
Project description:Along with lipidomic and metabolomic analyses, we analysed the effect of short-term heat stress on Nicotiana tabacum pollen tubes. Tubes were either grown for 3 hours at room temperature, for 6 hours at room temperature or for 3 hours at room temperature and then 37 °C for another 3 hours.
Project description:Little is known of the transcriptome of in vivo-grown pollen tubes, due to the difficulty of collection of pollen tubes elongating within the maternal gynoecium.We obtained the mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana(Col-0). Transgenic Arabidopsis plants (LAT52-HF-RPL18) harboring an epitope tagged ribosomal protein L18 driven by the pollen specific promoter (ProLAT52) were used for mRNA-ribosome complex isolation. After collection of polyribosomal (polysomal) complexes from self-pollinated (in vivo), unpollinated styles (buds), and in vitro-cultured pollen tubes, the actively translated mRNAs (the translatome) were purified, amplified to antisense RNA (aRNA). These aRNAs were hybridized to microarrays.Three independent biological replicates samples of aRNA from Bud, in vivo, and in vitro polysomal mRNA (translatomes) were hybridized to GeneChips to produce CEL files.
Project description:Polyadenylation of mRNAs is critical for their export from the nucleus, stability and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, their distinct mutant phenotypes and transcriptome studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the two very similar PAPS2 and PAPS4 proteins. Such functional specialization raises the possibility of modulating the balance of activities between the isoforms to alter poly(A) lengths of sets of transcripts, providing an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression more strongly than in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes germinate and grow at the same rates as wild-type pollen tubes, they are compromised in locating the micropyles of ovules. Transcriptomic analyses indicate that previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A)-tail lengths of transcripts in mutant and wild-type pollen tubes suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 upregulation during pollen-tube growth through the style plays a key role in the acquisition of competence and support the notion that plants can modulate the balance of activity between PAPS isoforms to regulate gene expression.
Project description:Of all the essential nutrients, nitrogen is the one most often limiting for plant growth. Nitrogen can be taken up by plants in two ways. One possibility is through ammonium and nitrate, which are the predominate inorganic forms of nitrogen in soils. The second possibility is the uptake of air-born nitrogen through plant-associated mircoorganisms in root nodules. The majority of plants able to form such nitrogen-fixing root nodules are in the legume family Fabaceae. Here we present a third possibility M-bM-^@M-^S a new pathway, termed as nitric oxide (NO)-fixation pathway, which allows plants to fix atmospheric NO and to use it for better growth and development. We identified non-symbiotic hemoglobin class 1 (AtGLB1) and class 2 (AtGLB2) as key proteins of the NO-fixation pathway. In an NO enriched environment NO-fixation is enhanced considerably in plants overexpressing AtGLB1 or AtGLB2 genes. NO uptake resulted in four-fold higher nitrate levels in these plants compared to NO-treated wild-type plants. Correspondingly, the growth parameters like rosettes size and weight, vegetative shoot thickness and also seed yield were 25%, 40%, 30%, and 20% higher, respectively, in the overexpression lines in comparison to wild-type plants. Our results highlight the existence of a NO-fixing pathway in plants. We demonstrated that plant non-symbiotic hemoglobin proteins can fix atmospheric NO and convert it to nitrate, which is further introduced into the N-metabolism. We assume that our results might provide new insights into the field of crop science research and that the NO-fixation capability might serve as a new economically important breeding trait for enhancing biomass, fruit, and seed production. Modifying this pathway might be a promising approach for better and more environment-friendly supply of nitrogen. For example crop plant hemoglobin proteins could be improved for their NO-fixing capability and their expression levels could be increased. WT-Arabidopsis thaliana plants were fumigated with Ambient NO and 3 ppm NO air in three completely independent biological experiments. Total RNA was isolated from four-week old rosette leaves of these plants to determine the gene expression signature of each samples using Agilent one-color microarray. Differences in the gene expression signatures between Ambient NO and 3 ppm NO treated samples were analyzed to see the effect of NO fumigation on the WT Arabidopsis plants at the transcript level.
Project description:The triose-phosphate/phosphate translocator (TPT) of the chloroplast inner envelope membrane mediates the counter-exchange of stromal triose phosphates derived from CO2 fixation with cytosolic phosphate, thus providing the cytosol with precursors for sucrose synthesis. We have isolated an Arabidopsis mutant (tpt-1) in which the gene encoding TPT is disrupted by a T-DNA insertion. During growth in low light tpt-1 plants are phenotypically normal, but in high light photosynthesis is inhibited and growth is retarded relative to wildtype. This mutant compensates for the absence of TPT by diverting photosynthate into starch which is hydrolysed and exported from the chloroplast as glucose that is subsequently phosphorylated by hexokinase. In low light the capacity of the pathway of starch synthesis is sufficient to accommodate the normal rate of CO2 fixation, but in high light it is unable to match the potential rate of CO2 fixation. Consequently, in high light-grown plants there are measurable effects on the redoxstate of several components. Thus, the tpt-1 mutation influences the carbohydrate status within the cell, alters the form in which carbon is received by the cytosol, and changes the redox signals that are important in photosynthetic acclimation. Method: Plants will be grown in a 8h light:16h dark regime at both 400 and 100 µmol PAR m-2 s-1. Total RNA will be extracted from pools of individual illuminated leaves from at least eight plants at growth stage 3.70. Leaves will be harvested 2 h into the photoperiod to maximise differences between plant lines in the expression of genes of photosynthesis and carbohydrate metabolism. We anticipate that expression of many genes will be affected by the absence of TPT and by changes in light intensity. However, by comparing differences in transcript levels between wildtype and TPT mutant grown in high light with the differences that occur in plants grown in low light we will discriminate between genes whose expression is affected by changes in the pattern of carbohydrate metabolism and those influenced by redox poise of the thylakoid photochemical components. These comparisons will also highlight genes affected by recently identified regulatory interactions between sugar-sensing and redox-sensing. A genome-wide expression study will establish the extent to which gene expression is altered by the absence of TPT in leaves, and will provide the basis for more detailed analysis of a selected range of transcripts whose levels of expression differ between plant lines.
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis. Experiment Overall Design: Pollen and pollen tubes were collected as described in the protocols section for RNA extraction and hybridization on Affymetrix ATH1 Genechip microarrays.