Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5'UTR that mediates ...cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5'UTR sequence (150 nts), since deletion of 17 nucleotides from the 5' end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5' end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Maize embryonic axes contain stored mRNAs, some of which are able to undergo cap-independent translation initiation during germination. The Hsp101 mRNA, encoding a heat shock protein, is essential ...for thermotolerance induction and is present among the stored transcripts. This research aimed to investigate whether the Hsp101 transcript is IRES-driven regulated upon heat stress. Hsp101 transcribed either in vitro or in vivo was efficiently translated via a cap-independent mechanism. This was observed either in an animal in vitro translation system containing proteolytically cleaved eukaryotic initiation factor eIF4G or in a plant system lacking both eIF4E and eIFiso4E initiation factors. Deletion of the 5' untranslated region (UTR) from the Hsp101 mRNA abolished its cap-independent translation indicating that this nucleotide sequence is required to confer cap-independent initiation. Bicistronic constructs containing the Hsp101 mRNA 5'UTR in sense and anti-sense directions between two reporter genes were translated in both cap-independent systems. A similar bicistronic construct containing a viral internal ribosome entry site (IRES) element between the reporter genes was used as control. Internal translation of the second reporter gene was observed when the Hsp101 5'UTR was in the sense but not in the anti-sense orientation in the bicistronic construct. Taken together, these data suggest that the 5'UTR of maize Hsp101, a plant cellular mRNA, functions as an IRES-like element accounting for its cap-independent translation during heat stress.
Intragenic modification of maize Almeraya, Erika V.; Sánchez-de-Jiménez, Estela
Journal of biotechnology,
11/2016, Letnik:
238
Journal Article
Recenzirano
•First use of intragenic system for targeting protein overexpression in corn plants.•Marker free strategy was used during the intragenic transformation.•Intragenic method was compared with a ...traditional breeding technique.•The intragenic procedure shows biosecurity advantages.•Intragenic application in maize could be directed to several improvement purposes.
The discovery of plant DNA recombination techniques triggered the development of a wide range of genetically modified crops. The transgenics were the first generation of modified plants; however, these crops were quickly questioned due to the artificial combination of DNA between different species. As a result, the second generation of modified plants known as cisgenic and/or intragenic crops arose as an alternative to genetic plant engineering. Cisgenic and/or intragenic crops development establishes the combination of DNA from the plant itself or related species avoiding the introduction of foreign genetic material, such as selection markers and/or reporter genes. Nowadays it has been made successful cisgenic and/or intragenic modifications in crops such as potato and apple. The present study shows the possibility of reaching similar approach in corn plants. This research was focused on achieve intragenic overexpression of the maize Rubisco activase (Rca) protein. The results were compared with changes in the expression of the same protein, in maize plants grown after 23 cycles of conventional selection and open field planting. Experimental evidence shows that maize intragenic modification is possible for increasing specific gene expression, preserving plant genome free of foreign DNA and achieving further significant savings in time and man labor for crop improvement.
Seed germination is a critical developmental period for plant propagation. Information regarding gene expression within this important period is relevant for understanding the main biochemical ...processes required for successful germination, particularly in maize, one of the most important cereals in the world. The present research focuses on the global microarray analysis of differential gene expression between quiescent and germinated maize embryo stages. This analysis revealed that a large number of mRNAs stored in the quiescent embryonic axes (QEAs) were differentially regulated during germination in the 24 h germinated embryonic axes (GEAs). These genes belong to 14 different functional categories and most of them correspond to metabolic processes, followed by transport, transcription and translation. Interestingly, the expression of mRNAs encoding ribosomal proteins (r)-proteins, required for new ribosome formation during this fast-growing period, remains mostly unchanged throughout the germination process, suggesting that these genes are not regulated at the transcriptional level during this developmental period. To investigate this issue further, comparative microarray analyses on polysomal mRNAs from growth-stimulated and non-stimulated GEAs were performed. The results revealed that (r)-protein mRNAs accumulate to high levels in polysomes of the growth-stimulated tissues, indicating a translational control mechanism to account for the rapid (r)-protein synthesis observed within this period. Bioinformatic analysis of (r)-protein mRNAs showed that 5′ TOP (tract of pyrimidines)-like sequences are present only in the 5′-untranslated region set of up-regulated (r)-protein mRNAs. This overall approach to the germination process allows an in-depth view of molecular changes, enabling a broader understanding of the regulatory mechanisms that occur during this process.
In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry ...and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.
Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to high concentrations during the late stages of seeds development, which are integral to desiccation tolerance. ...LEA proteins also play a protective role under other abiotic stresses. We analyzed in silico a maize protein predicted to be highly hydrophilic and intrinsically disordered. This prediction was experimentally corroborated by solubility assays under denaturing conditions. Based on its amino acid sequence, we propose that this protein belongs to group four of the LEA proteins. The accumulation pattern of this protein was similar to that of dehydrins during the desiccation process that takes place during seed development. This protein was induced by exogenous abscisic acid in immature embryos, but during imbibition was down-regulated by gibberellins. It was also induced in maize roots under osmotic stress. So far, this is the first member of the LEA proteins belonging to group four to be characterized in maize, and it plays a role in the response to osmotic stress.
In most non-photosynthetic eukaryotes it has been demonstrated a conserved signal transduction pathway, namely TOR-S6K, that coordinates growth and cell proliferation. This pathway targets the ...translational apparatus to induce selective translation of ribosomal mRNAs as well as stimulate the cell cycle transition through the G
1
/S phase. Thus, by activation of this pathway through environmental signals, nutrients, stress, or specific growth factors, such as insulin or insulin-like growth factors (IGF), this pathway allows organisms to regulate growth and cell division. In plants, evidence has shown that TOR protein has been highly conserved through evolution, being involved in growth and cell proliferation control as well. Particularly in maize, a peptide named ZmIGF has been found in actively growing tissues. It targets the maize TOR pathway at the same extent as insulin and, by doing so it induces growth, as well as ribosomal proteins and DNA synthesis. Thus, higher metazoans and plants seem to conserve similar biochemical paths to regulate cell growth through equivalent targets that conduce to activation of the TOR-S6K pathway. Recent research shows evidence that supports this proposal by uncovering the ZmIGF receptor in maize, providing further means for analyzing the role of the conserved TOR signaling pathway in this plant.
Insulin and 'insulin-like' growth factors (IGFs) are known to regulate cell growth in eukaryotes by stimulating a signal transduction pathway that exerts translational control. Intermediate kinases ...of this pathway, target of rapamycin (TOR) and ribosomal protein S6 kinase (S6K), have been reported in Arabidospsis thaliana and Zea mays. However, upstream signal inducers and downstream targets of the pathway are not well known in plants. The objective of this work is to inquire whether plant growth is regulated by a signal transduction pathway similar to the insulin/IGF-stimulated pathway in other metazoans. Insulin as well as Zea mays insulin-related peptide (ZmIGF), which is a maize, 20-kDa peptide fraction recognized by insulin antibody, were used as effectors to stimulate maize axes growth from germinating seeds. ZmIGF expression was identified in axes from germinating maize seeds and immunolocalized in the meristems of these tissues. Significant enhancement of specific de novo protein synthesis of the translational apparatus components was found in the stimulated axes. Reverse-transcription-polymerase chain reaction analysis of total and polysomal RNA pools in ZmIGF- or insulin-stimulated axes confirmed these data by revealing specific mRNA recruitment into polysomes. In addition, the same stimuli induced activation of S6 ribosomal protein kinase (ZmS6K) in germinating maize axes. All the above effects were inhibited by rapamycin, indicating that they depend on TOR activity. We conclude that a TOR-S6K signal transduction pathway is functional in maize germination, as that found for non-photosynthetic eukaryotes. The evolutionary implications of these findings are discussed.
The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has ...been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.
Establishment of somatic embryogenic cultures is highly influenced by the plant genotype and the explant type. In maize, immature embryos generate embryogenic callus (E), whereas quiescent embryos ...produce non-embryogenic callus (NE). E callus shows active growth and high capacity of plant regeneration, while NE callus shows slow growth and no regeneration capacity. Active growth is needed for the establishment of embryogenic cultures; therefore, lack of this characteristic pose a handicap for plant regeneration from NE callus. To correct the slow growth on NE callus, the Zea mays insulin-like growth factor (ZmIGF), a peptide that promotes growth by activating the target of rapamycin (TOR) pathway, was used as media supplement. Additionally, since the TOR pathway is connected to the auxin pathway, ZmIGF participation in cell differentiation was considered. To this end, this research explores ZmIGF effect, beyond growth and proliferation on a Mexican maize landrace, which has shown high somatic embryogenic capacity. Expression levels of reported genes involved in the embryogenesis and differentiation processes were evaluated in maize E, NE, and NE-ZmIGF (NE-Zm) growth-activated calluses. A tendency to upregulate messenger RNA (mRNA) expression was observed for genes encoding transcription factors and auxin transport. Some genes related to epigenetic control showed downregulation. Global DNA methylation and chromatin modifications results suggest an epigenetic activation state on E callus and a repression status on NE callus. ZmIGF induced modifications at DNA methylation and chromatin over NE callus, which changed its original repression state to an active one. Overall, results suggest that the expression of genes related to auxin signaling, mainly transport and efflux carriers, are essential for accomplishing plant regeneration through somatic embryogenesis (SE).
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