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Genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds
Background:
Tocopherols are natural antioxidants with both in vivo (vitamin E) and in vitro activity. Sunflower seeds contain predominantly alpha-tocopherol (>90% of total tocopherols), with maximum vitamin E effect but lower in vitro antioxidant action than other tocopherol forms such as gamma-tocopherol. Sunflower germplasm with stable high levels of gamma-tocopherol (>85%) has been developed. The trait is controlled by recessive alleles at a single locus Tph2 underlying a gamma-tocopherol methyltransferase (gamma-TMT). Additionally, unstable expression of increased gamma-tocopherol content in the range from 5 to 85% has been reported. The objective of this research was to determine the genetic basis of unstable expression of high gamma-tocopherol content in sunflower seeds.
Results:
Male sterile plants of nuclear male sterile line nmsT2100, with stable high gamma-tocopherol content, were crossed with plants of line IAST-1, with stable high gamma-tocopherol content but derived from a population that exhibited unstable expression of the trait. F2 seeds showed continuous segregation for gamma-tocopherol content from 1.0 to 99.7%. Gamma-tocopherol content in F2 plants (average of 24 individual F3 seeds) segregated from 59.4 to 99.4%. A genetic linkage map comprising 17 linkage groups (LGs) was constructed from this population using 109 SSR and 20 INDEL marker loci, including INDEL markers for tocopherol biosynthesis genes. QTL analysis revealed a major QTL on LG 8 that corresponded to the gamma-TMT Tph2 locus, which suggested that high gamma-tocopherol lines nmsT2100 and IAST-1 possess different alleles at this locus. Modifying genes were identified at LGs 1, 9, 14 and 16, corresponding in most cases with gamma-TMT duplicated loci.
Conclusions:
Unstable expression of high gamma-tocopherol content is produced by the effect of modifying genes on tph2a allele at the gamma-TMT Tph2 gene. This allele is present in line IAST-1 and is different to allele tph2 present in line nmsT2100, which is not affected by modifying genes. No sequence differences at the gamma-TMT gene were found associated to allelic unstability. Our results suggested that modifying genes are mostly epistatically interacting gamma-TMT duplicated loci.
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Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells
Background:
In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells.
Results:
We used fluorescent protein fusions to visualize and track Golgi stacks, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that all four organelles are gradually distributed in protonemata from the tip of the apical cell to the base of the sub-apical cell. For example, the number of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonema-ta and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi stacks and less chlo-roplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protone-mata move with low persistency and instantaneous velocities ranging from 29 to 75 nm / sec, which are at least three orders of magnitude slower than those of pollen tube or root hair orga-nelles.
Conclusions:
To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and by comparing the distribution and dynamics of organelles from dif-ferent tip growing plant cells, may help better understand the mechanisms of plant polarized cell growth.
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Development of high amylose wheat through TILLING
Background:
Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes.
Results:
Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels of up to 12% compared to levels of less than 1% in wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry.
Conclusions:
We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.
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miRFANs: an integrated database for Arabidopsis thaliana microRNA function annotations
Background:
Plant microRNAs (miRNAs) have been revealed to play important roles in developmental control, hormone secretion, cell differentiation and proliferation, and response to environmental stresses. However, our knowledge about the regulatory mechanisms and functions of miRNAs remains very limited. The main difficulties lie in two aspects. On one hand, the number of experimentally validated miRNA targets is very limited and the predicted targets often include many false positives, which constrains us to reveal the functions of miRNAs. On the other hand, the regulation of miRNAs is known to be spatio-temporally specific, which increases the difficulty for us to understand the regulatory mechanisms of miRNAs.Description: In this paper we present miRFANs, an online database for Arabidopsis thaliana miRNA function annotations. We integrated various type of datasets, including miRNA-target interactions, transcription factor (TF) and their targets, expression profiles, genomic annotations and pathways, into a comprehensive database, and developed various statistical and mining tools, together with a user-friendly web interface. For each miRNA target predicted by psRNATarget, TargetAlign and UEA target-finder, or recorded in TarBase and miRTarBase, the effect of its up-regulated or down-regulated miRNA on the expression level of the target gene is evaluated by carrying out differential expression analysis of both miRNA and targets expression profiles acquired under the same (or similar) experimental condition and in the same tissue. Moreover, each miRNA target is associated with gene ontology and pathway terms, together with the target site information and regulating miRNAs predicted by different computational methods. These associated terms may provide valuable insight for the functions of each miRNA.
Conclusion:
First, a comprehensive collection of miRNA targets for Arabidopsis thaliana provides valuable information about the functions of plant miRNAs. Second, a highly informative miRNA-mediated genetic regulatory network is extracted from our integrative database. Third, a set of statistical and mining tools is equipped for analyzing and mining the database. And fourth, a user-friendly web interface is developed to facilitate the browsing and analysis of the collected data.
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A generalized deceptive pollination system of Doritis pulcherrima (Aeridinae: Orchidaceae) with non-reconfigured pollinaria
Background:
As one of largest angiosperm families, orchids have long fascinated evolutionary biologists with their staggering diversity in floral design and display to promote outcrossing. Two of the most intriguing aspects of orchid pollination that promote cross-pollination are pollinarium reconfiguration (PR) and deceptive pollination. PR and generalized food deception employ virtually antagonistic methods of promoting cross-pollination: PR occurs through delayed pollination, involving the relatively long visitation periods that are typically observed for the pollinators of one flower or inflorescence; conversely, generalized food deception leads to reductions in the visitation periods of pollinators to one flower or inflorescence. Thus, it is logical to hypothesize that PR is unnecessary or PR happens soon in generalized food-deceptive orchids in the promotion of cross-pollination. Using Doritis pulcherrima as a model, the aim of this study was to understand the following: (1) the pollination and breeding system of D. pulcherrima; (2) the morphological interactions between orchids and their pollinators; and (3) whether PR is necessary in the promotion of cross-pollination in D. pulcherrima.
Results:
Our observations indicated that Doritis pulcherrima is pollinated almost exclusively by Amegilla nigritar (Hymenoptera: Apidae) and possesses pollinia that are deposited on the "occiputs" (cervical membranes) of these insects. All of evidences are indicated that D. pulcherrima is a generalized food-deceptive orchid. Our morphometric measurements of the flowers and pollinators show that the heights of the "occiputs" with un-oriented pollinaria were equal to the distances between stigmas and surfaces of the middle lobes, suggesting that pollinarium reconfiguration is not necessary in Doritis pulcherrima.
Conclusions:
Our observation and analyses supported the hypothesis that pollinarium reconfiguration is unnecessary in generalized food-deceptive orchids, such as Doritis pulcherrima, for the promotion of cross-pollination. This conclusion was indirectly supported by the abundance of deceptive orchids that do not exhibit pollinarium reconfiguration. There are two mechanisms (i.e. clone-growing characteristics and a long flowering season) that promote fruit sets in the epiphytic food-deceptive orchids in tropical regions.
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Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H+-ATPase activity and abundance
Background:
The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level.
Results:
The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process.
Conclusions:
Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.
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Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses
Background:
Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition proteins (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we use comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand B. distachyon's potential as a model species for agriculturally important temperate grasses
Results:
Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species.
Conclusions:
We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.
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Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat
Background:
Bread wheat, one of the world's staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat.
Results:
The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2 % of t3AS with an average DNA sequence read every 19 kb. Overall, 79 % of the sequence consisted of repetitive elements, 1.38 % as coding regions (estimated 2,850 genes) and another 19 % of unknown origin. Comparative sequence analysis suggested that 70-77 % of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, Gypsy/Sabrina (12.4 %) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29 %) were 3A-specific and compared to 17 (18 %) for 96 SSRs.
Conclusion:
This work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping.
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A novel mesh processing based technique for 3D plant analysis
Background:
In recent years, imaging based, automated, non-invasive, and non-destructive high-throughput plant phenotyping platforms have become popular tools for plant biology, underpinning the field of plant phenomics. Such platforms acquire and record large amounts of raw data that must be accurately and robustly calibrated, reconstructed, and analysed, requiring the development of sophisticated image understanding and quantification algorithms. The raw data can be processed in different ways, and the past few years have seen the emergence of two main approaches: 2D image processing and 3D mesh processing algorithms. Direct image quantification methods (usually 2D) dominate the current literature due to comparative simplicity. However, 3D mesh analysis provides the tremendous potential to accurately estimate specific morphological features cross-sectionally and monitor them over-time.Result:In this paper, we present a novel 3D mesh based technique developed for temporal high-throughput plant phenomics and perform initial tests for the analysis of Gossypium hirsutum vegetative growth. Based on plant meshes previously reconstructed from multi-view images, the methodology involves several stages, including morphological mesh segmentation, phenotypic parameters estimation, and plant organs tracking over time. The initial study focuses on presenting and validating the accuracy of the methodology on dicotyledons such as cotton but we believe the approach will be more broadly applicable. This study involved applying our technique to a set of six Gossypium hirsutum (cotton) plants studied over four time-points. Manual measurements, performed for each plant at every time-point, were used to assess the accuracy of our pipeline and quantify the error on the morphological parameters estimated.
Conclusion:
By directly comparing our automated mesh based quantitative data with manual measurements of individual stem height, leaf width and leaf length, we obtained the mean absolute errors of 9.34%, 5.75%, 8.78%, and correlation coefficients 0.88, 0.96, and 0.95 respectively. The temporal matching of leaves was accurate in 95% of the cases and the average execution time required to analyse a plant over four time-points was 4.9 minutes. The mesh processing based methodology is thus considered suitable for quantitative 4D monitoring of plant phenotypic features.
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The response and recovery of the Arabidopsis thaliana transcriptome to phosphate starvation
Background:
Over application of phosphate fertilizers in modern agriculture contaminates waterways and disrupts natural ecosystems. Nevertheless, this is a common practice among farmers, especially in developing countries as abundant fertilizers are believed to boost crop yields. The study of plant phosphate metabolism and its underlying genetic pathways is key to discovering methods of efficient fertilizer usage. The work presented here describes a genome-wide resource on the molecular dynamics underpinning the response and recovery in roots and shoots of Arabidopsis thaliana to phosphate-starvation.
Results:
Genome-wide profiling by micro- and tiling-arrays (accessible from GEO: GSE34004) revealed minimal overlap between root and shoot transcriptomes suggesting two independent phosphate-starvation regulons. Novel gene expression patterns were detected for over 1000 candidates and were classified as either initial, persistent, or latent responders. Comparative analysis to AtGenExpress identified cohorts of genes co-regulated across multiple stimuli. The hormone ABA displayed a dominant role in regulating many phosphate-responsive candidates. Analysis of co-regulation enabled the determination of specific versus generic members of closely related gene families with respect to phosphate-starvation. Thus, among others, we showed that PHR1-regulated members of closely related phosphate-responsive families (PHT1;1, PHT1;7-9, SPX1-3, and PHO1;H1) display greater specificity to phosphate-starvation than their more generic counterparts.
Conclusion:
Our results uncover a much larger, staged responses to phosphate-starvation than previously described. To our knowledge, this work describes the most complete genome-wide data on plant nutrient stress to-date.
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