Gene expression analysis of flax seed development

<p>Abstract</p> <p>Background</p> <p>Flax, <it>Linum usitatissimum </it>L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.</p> <p>Results</p> <p>We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions <ext-link ext-link-id="LIBEST_026995" ext-link-type="gen">LIBEST_026995</ext-link> to <ext-link ext-link-id="LIBEST_027011" ext-link-type="gen">LIBEST_027011</ext-link>) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for <it>in silico </it>expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.</p> <p>Conclusions</p> <p>We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.</p

Role of cytokinins in arabidopsis flower development

This study was conducted to investigate the possible role of cytokinins (CKs) in the gene-controlled flowering program in Arabidopsis thaliana . Four different experimental approaches and two groups of floral mutants, i.e., floral meristem identity (FMI) and floral organ identity (FOI) mutants were used to analyze the roles of CKs during the development of flowers. In vivo application of both a synthetic and a natural CK induced abnormalities in floral phenotypes of wild type plants. In particular, application of 3 [mu]l of 10-3 M benzylaminopurine (BAP) induced floral phenocopies of FMI mutants in both the wild type and FOI mutants. BAP also the phenotype of the weak allele, apetala2-1 (ap2-6), to resemble the strong allele, ap2-6. BAP application to FMI mutants, ap1-1 and ap1-3 strong and weak alleles of APETALA1 respectively, resulted in the enhanced conversion of flowers to inflorescence-like structures. Similar conversions of flowers to inflorescence-like structures were also observed in BAP treated ap2-1 mutants. In vitro culture of inflorescence explants showed that wild type inflorescences produced flowers in the absence of CKs, and the addition of CKs to the culture medium induced abnormalities similar to those observed in in vivo experiments. These results demonstrate that in vitro growth of Arabidopsis flowers can take place in the absence of exogenous CKs, and suggest that exogenous CKs suppress FMI gene functions as observed in in vivo FMI experiments. This led to a study of mode of CK interaction with FMI genes. Two possible postulates were considered for the roles of CKs in FMI: (1) mutations in FMI genes alter the biosynthesis or metabolism of CKs, and (2) CKs suppress the expression of FMI genes. The first postulate was tested by analysing endogenous CKs in the wild type and FMI mutants, ap1-1 and clavata1-1 (clv1-1). The predominant CK in the wild type and in both FMI mutant tissues was dihydrozeatin (DZ). In the clv1-1 mutant, DZ levels were 10 to 13 fold higher in comparison to the wild type. The ap1-1 mutants did not show major changes in CK levels. This suggested that the CLV1 gene negatively regulates CK levels in the floral meristems of Arabidopsis. The second postulate was studied using altered meristem program1-1 ( amp1-1), a CK-overproducer mutant, to construct double mutant combinations with FMI mutants ap1-1, ap2-1and clv1-1. The double mutants, amp1-1 ap1-1, and amp1-1 ap2-1, showed enhanced conversions of flowers to inflorescence-like structures, supporting the view that high endogenous CKs suppress FMI function in the absence of a functional FMI gene. However, amp1-1 clv1-1 double mutant did not show any such conversions indicating that the role of CLV1 in the establishment of FMI is secondary, and that the role of CKs in FMI becomes obvious only in the absence of the AP1 or AP2 gene. Based on this study a model is proposed which suggests that in wild-type floral meristems, the 'CLV1' gene negatively regulates CK levels which allow the complete activation of FMI genes, and resulting in the formation of a flower

Conserved, divergent and heterochronic gene expression during Brachypodium and Arabidopsis embryo development

Key message: Developmental and transcriptomic analysis of Brachypodium embryogenesis and comparison with Arabidopsis identifies conserved and divergent phases of embryogenesis and reveals widespread heterochrony of developmental gene expression. Abstract: Embryogenesis, transforming the zygote into the mature embryo, represents a fundamental process for all flowering plants. Current knowledge of cell specification and differentiation during plant embryogenesis is largely based on studies of the dicot model plant Arabidopsis thaliana. However, the major crops are monocots and the transcriptional programs associated with the differentiation processes during embryogenesis in this clade were largely unknown. Here, we combined analysis of cell division patterns with development of a temporal transcriptomic resource during embryogenesis of the monocot model plant Brachypodium distachyon. We found that early divisions of the Brachypodium embryo were highly regular, while later stages were marked by less stereotypic patterns. Comparative transcriptomic analysis between Brachypodium and Arabidopsis revealed that early and late embryogenesis shared a common transcriptional program, whereas mid-embryogenesis was divergent between species. Analysis of orthology groups revealed widespread heterochronic expression of potential developmental regulators between the species. Interestingly, Brachypodium genes tend to be expressed at earlier stages than Arabidopsis counterparts, which suggests that embryo patterning may occur early during Brachypodium embryogenesis. Detailed investigation of auxin-related genes shows that the capacity to synthesize, transport and respond to auxin is established early in the embryo. However, while early PIN1 polarity could be confirmed, it is unclear if an active response is mounted. This study presents a resource for studying Brachypodium and grass embryogenesis and shows that divergent angiosperms share a conserved genetic program that is marked by heterochronic gene expression

Dissecting the Roles of Cuticular Wax in Plant Resistance to Shoot Dehydration and Low-Temperature Stress in Arabidopsis

Cuticular waxes are a mixture of hydrophobic very-long-chain fatty acids and their derivatives accumulated in the plant cuticle. Most studies define the role of cuticular wax largely based on reducing nonstomatal water loss. The present study investigated the role of cuticular wax in reducing both low-temperature and dehydration stress in plants using Arabidopsis thaliana mutants and transgenic genotypes altered in the formation of cuticular wax. cer3-6, a known Arabidopsis wax-deficient mutant (with distinct reduction in aldehydes, n-alkanes, secondary n-alcohols, and ketones compared to wild type (WT)), was most sensitive to water loss, while dewax, a known wax overproducer (greater alkanes and ketones compared to WT), was more resistant to dehydration compared to WT. Furthermore, cold-acclimated cer3-6 froze at warmer temperatures, while cold-acclimated dewax displayed freezing exotherms at colder temperatures compared to WT. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis identified a characteristic decrease in the accumulation of certain waxes (e.g., alkanes, alcohols) in Arabidopsis cuticles under cold acclimation, which was additionally reduced in cer3-6. Conversely, the dewax mutant showed a greater ability to accumulate waxes under cold acclimation. Fourier Transform Infrared Spectroscopy (FTIR) also supported observations in cuticular wax deposition under cold acclimation. Our data indicate cuticular alkane waxes along with alcohols and fatty acids can facilitate avoidance of both ice formation and leaf water loss under dehydration stress and are promising genetic targets of interest

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA]

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis

Dissecting the Roles of Cuticular Wax in Plant Resistance to Shoot Dehydration and Low-Temperature Stress in <em>Arabidopsis</em>

Cuticular waxes are a mixture of hydrophobic very-long-chain fatty acids and their derivatives accumulated in the plant cuticle. Most studies define the role of cuticular wax largely based on reducing nonstomatal water loss. The present study investigated the role of cuticular wax in reducing both low-temperature and dehydration stress in plants using Arabidopsis thaliana mutants and transgenic genotypes altered in the formation of cuticular wax. cer3-6, a known Arabidopsis wax-deficient mutant (with distinct reduction in aldehydes, n-alkanes, secondary n-alcohols, and ketones compared to wild type (WT)), was most sensitive to water loss, while dewax, a known wax overproducer (greater alkanes and ketones compared to WT), was more resistant to dehydration compared to WT. Furthermore, cold-acclimated cer3-6 froze at warmer temperatures, while cold-acclimated dewax displayed freezing exotherms at colder temperatures compared to WT. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis identified a characteristic decrease in the accumulation of certain waxes (e.g., alkanes, alcohols) in Arabidopsis cuticles under cold acclimation, which was additionally reduced in cer3-6. Conversely, the dewax mutant showed a greater ability to accumulate waxes under cold acclimation. Fourier Transform Infrared Spectroscopy (FTIR) also supported observations in cuticular wax deposition under cold acclimation. Our data indicate cuticular alkane waxes along with alcohols and fatty acids can facilitate avoidance of both ice formation and leaf water loss under dehydration stress and are promising genetic targets of interest

An activated form of UFO alters leaf development and produces ectopic floral and inflorescence meristems

Plants are unique in their ability to continuously produce new meristems and organ primordia. In Arabidopsis, the transcription factor LEAFY (LFY) functions as a master regulator of a gene network that is important for floral meristem and organ specification. UNUSUAL FLORAL ORGANS (UFO) is a co-activator of LEAFY and is required for proper activation of APETALA3 in the floral meristem during the specification of stamens and petals. The ufo mutants display defects in other parts of the flower and the inflorescence, suggestive of additional roles. Here we show that the normal determinacy of the developing Arabidopsis leaves is affected by the expression of a gain-of-function UFO fusion protein with the VP16 transcriptional activator domain. In these lines, the rosette and cauline leaf primordia exhibit reiterated serration, and upon flowering produce ectopic meristems that develop into flowers, bract leaves and inflorescences. These striking phenotypes reveal that developing leaves maintain the competency to initiate flower and inflorescence programs. Furthermore, the gain-of-function phenotypes are dependent on LFY and the SEPALLATA (SEP) MADS-box transcription factors, indicative of their functional interactions with UFO. The findings of this study also suggest that UFO promotes the establishment of the lateral meristems and primordia in the peripheral zone of the apical and floral meristems by enhancing the activity of LFY. These novel phenotypes along with the mutant phenotypes of UFO orthologs in other plant species suggest a broader function for UFO in plants.Peer reviewed: YesNRC publication: Ye

UBC13, an E2 enzyme for Lys63-linked ubiquitination, functions in root development by affecting auxin signaling and Aux/IAA protein stability

Unlike conventional lysine (K) 48\u2013linked polyubiquitination, K63-linked polyubiquitination plays signaling roles in yeast and animals. Thus far, UBC13 is the only known ubiquitin-conjugating enzyme (E2) specialized in K63-linked polyubiquitination. Previous identification of Arabidopsis genes encoding UBC13 as well as its interacting partner UEV1 indicates that the UBC13-mediated ubiquitination pathway is conserved in plants; however, little is known about functions and signaling mediated through K63-linked polyubiquitination in plants. To address the functions of UBC13-mediated ubiquitination in plants, we created Arabidopsis ubc13 null mutant lines in which the two UBC13 genes were disrupted. The double mutant displayed altered root development, including shorter primary root, fewer lateral roots and only a few short root hairs in comparison with the wild type and single mutant plants, indicating that UBC13 activity is critical for all major aspects of root development. The double mutant plants were insensitive to auxin treatments, suggesting that the strong root phenotypes do not simply result from a reduced level of auxin. Instead, the ubc13 mutant had a reduced auxin response, as indicated by the expression of an auxin-responsive DR5 promoter-GFP. Furthermore, both the enzymatic activity and protein level of an AXR3/IAA17-GUS reporter were greatly increased in the ubc13 mutant, whereas the induction of many auxin-responsive genes was suppressed. Collectively, these results suggest that Aux/IAA proteins accumulate in the ubc13 mutant, resulting in a reduced auxin response and defective root development. Hence, this study provides possible mechanistic links between UBC13-mediated protein ubiquitination, root development and auxin signaling.Peer reviewed: YesNRC publication: Ye

Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis

Embryogenesis is central to the life cycle of most plant species. Despite its importance, because of the difficulty associated with embryo isolation, global gene expression programs involved in plant embryogenesis - especially the early events following fertilization are largely unknown. To address this gap, we have developed methods to isolate whole live Arabidopsis thaliana embryos as young as zygote and performed genome-wide profiling of gene expression. These studies revealed insights into patterns of gene expression relating to: maternal and paternal contributions to zygote development; chromosomal level clustering of temporal expression in embryogenesis; and embryo-specific functions. Functional analysis of some of the modulated transcription factor encoding genes from our datasets confirmed that they are critical for embryogenesis. Furthermore, we constructed stage-specific metabolic networks mapped with differentially regulated genes by combining the microarray data with the available KEGG metabolic datasets. Comparative analysis of these networks revealed the network-associated structural and topological features, pathway interactions and gene expression with reference to the metabolic activities during embryogenesis. Together, these studies have generated comprehensive gene expression datasets for embryo development in Arabidopsis and may serve as an important foundational resource for other seed plants.Peer reviewed: YesNRC publication: Ye