To achieve a global economic growth, there must be a sustained increase in agricultural production; seeds constitute the main propagule for plant growth and survival hence seed quality and its performance remain vital in crop production. The use of model system to study traits associated to seed development, dormancy and germination for example Arabidopsis thaliana, has become very important in understanding seed developmental processes and identify problems associated with poor seedling establishment and pre-harvest sprouting. Most importantly is that this information can be transferred to different crops species to improve their seed quality and performance.
Arabidopsis thaliana: A model plant
Arabidopsis is a small flowering plant which belongs to the family Brassicaceae. It is dicotyledonous plant indigenous to Asia, Europe and some part of Africa (Price et al., 1994) and has become the model plant of choice for research in plant biology and genetics (Meinke et al., 1998). Although Arabidopsis is related to crops such as oil seed rape, cabbage and cauliflower, it has no economic value and therefore regard as a weed. The advantage Arabidopsis offers as a model plant to crop research is that it has a rapid life cycle (8 weeks), small stature and produces many seed (prolific). In addition, it has a small genome size (125Mb) and chromosome number (2n=10) making it ideal for genetic and mutational analysis (Slater et al., 2003; Rensink and Buell, 2004). Recently Arabidopsis genome became the first plant genome to be fully sequenced (Arabidopsis Genome Initiative, 2000) this has enhanced the value of Arabidopsis as a model plant and most importantly broadening and enhancing crop research as complex developmental processes are now better understood.
1.2 Seed germination and dormancy
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Germination is a vital process in crop production; it is a progression of discrete changes that occurs in quiescent seed, changing it into a growing seedling. Conversion of this dry dispersal unit into a new plant is one of the important survival tactics employed by plants. It is an exciting event because it represents several complex developmental and physiological changes which occur in the seed after its shed from the mother plant. Germination begins with imbibition (water up take) by a seed and ends when the embryonic axis begins to elongate (Bewley, 1997). In most plant species, for example Arabidopsis, the end of germination is marked by the protrusion of the embryonic root precursor, called radicle. It is important to note that appropriate environmental conditions are required for germination to occur, however, many plant species produce seeds that do not actually germinate even when they are placed into completely appropriate conditions. This is referred to as seed dormancy (Johnson, 2004).
Seed dormancy is an innate property that provides a mechanism for plants to delay germination pending when conditions are favourable. It is controlled by external environmental signals, and partly, by intrinsic hormones and metabolic pathways (Garjets et al., 2010). The biochemical and physiological processes involved in seed dormancy and germination have been widely studied (Hilhorsta et al., 1997; Kozowski and Pallardy, 1997; Koornneef et al., 2002 and Holdsworth et al., 2008). Most recently the use of molecular genetics has provided more light about the transcriptional and translational signals that lead to the physiological changes that occurs from seed maturation through dormancy to germination (Holdsworth et al., 2008).
Dormancy and germination are controlled by the antagonistic actions of the plant hormones Abscisic acid (ABA) and Gibberellin (GA), and ABA has been shown to be a negative regulator of germination, thereby promoting dormancy (Finkelstein et al., 2002), while GA antagonizes the ABA role and acts a positive regulator of germination by counteracting the effect of ABA (Kucera et al., 2005). Other plant hormones such as ethylene, brassinosteroids, auxin and cytokinin have also been shown to be involved in the regulation of germination and dormancy processes. Holman et al., (2009) in their work identified two components (PROTEOLYSIS6 PTR6 and ARGINYL-TRANSFARASE ATE ) of the N-end rule pathway of targeted proteolysis that promote seed germination and establishment in Arabidopsis thaliana through the removal of ABA sensitivity. The implication of this is that N-end rule pathway plays a crucial role in the process of seed dormancy and germination. Although how this pathway promotes germination is poorly understood.
N-end rule pathway of targeted proteolysis
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Fig. 1 Diagrammatic representation of the N-end rule pathway associated with PRT6 function. Components for which orthologous Arabidopsis genes have been identified are highlighted (Holman et al., 2009).
The N-end rule state that the half life of a protein is determined by the nature of its N-terminal residue; which therefore defines the stability and instability of a protein (Mogk et al., 2007). Amino acids are classified as stabilizing or destabilizing residues, which serve as recognition determinants for targeted protein degradation. Studies have shown that the N-end rule operates in all organisms from mammal to fungi and bacteria (Varshavsky, 1997; Mogk et al., 2007; Tasaki and kwon, 2007). The analysis of the N-end rule pathway reveal that prokaryotes and eukaryotes utilize separate protolytic system for degradation of targeted proteins; however recent findings have shown that they share similar substrate recognition signal (Mogk et al., 2007). Also interesting is the recent structural identification of the N-end rule pathway in plants, showing the complete set of the destabilizing and stabilizing N-terminal residues and their hierarchical organisation which indicates that N-end rule pathway in plants is similar to that of mammals (Graciet et al., 2010).
Target proteolysis is an important mechanism in plant development. A vital proteolytic pathway in eukaryotes is the Ubiquitin-(Ub)-dependent proteolytic system. In Ub-dependent proteolysis, substrates are ubiquitylated by the action of three enzymes namely, the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2) and the Ub-ligase (E3) (Tasaki and kwon, 2007). The selectivity for ubiquitination is determined mainly by the E3 Ub-ligase which recognizes a degradation signal (degron) of the target protein (Tasaki and kwon, 2007).
In the N-end rule pathway, for instance in Arabidopsis where components of this pathway have been identified, the Amino-terminal destabilising residues (N-degron) are recognised by the N-recognin E3 ligase and are targeted for protolysis via the 26S proteosome (Holman et al., 2009). Also in yeast and mammals two classes of N-recognins(type I and II) recognizes either basic (Arginine, Lysine and Histidine) or hydrophobic (Phenylalanine, Tyrosine, Tryptophan, Leucine and Isoleucine) amino acid terminals. In yeast, the UBR1 was identified as an E3 ligase with N-degron activity and there are at least seven proteins containing the characteristic UBR box in mammals (UBR1-UBR7) (Mogk et al, 2007).
In eukaryotic cells, during post-translational modification the cleavage of newly synthesized protein will create either stabilizing or destabilising residues. Destabilising residues consist of tertiary (30), secondary (20) and primary (10) residues. Primary residue target the protein for degradation, while 20 and 30 residues must be first converted to 10. Tertiary destabilising residues can be created after N-terminal cleavage or by the action of endopepetidases. Secondary destabilizing residues can be created from tertiary residues through deamidation by N-terminal amidohydrolase (NTAN) (Asn, Gin) or by Cys oxidation before arginylation by arginyl-tRNA protein arginyltransferase (ATE). It has been shown that Arginylation require nitric oxide (NO) in addition to oxygen (Tasaki and kwon, 2007; Holman et al, 2009). A Primary residue can be created from the secondary residue through arginylation by ATE. In plants, ATE has been shown to influence the transition of the seed to seedling due to it provide arginylation of secondary destabilizing residue thus creating primary destabilizing residue which are targeted for degradation by the PTR6. This targeted proteolysis has been suggested to be responsible for removing ABA sensitivity (Holman et al., 2009).
Many studies have analysed the expression of the genome in Arabidopsis seeds at both the proteome and transcriptome levels (Cadman et al., 2006; Catusse et al., 2008; Holdsworth et al., 2008) These studies have compared different identified mutant and wild type plants, analyse patterns of seed germination at different conditions and the influence of environment on developmental state (dormant and after-ripened) in relation to genome expression (Holdsworth et al, 2008). The use of different genetic technologies such as PCR, molecular markers, and Qrt PCR in these studies provides an insight, allowing an understanding of different gene expression levels at different conditions thus giving the opportunity to compare these variations especially during dormancy cycling, after-ripening and imbibition, and during post-germination (Holdsworth et al., 2008). As transcription analyses have been used widely for seed transcriptome profiling by many authors (Nakabayashi et al, 2005; Cadman et al, 2006 and Holdsworth et al, 2007), the same approach will be used to study the genes that promotes seed germination which are regulated by the N-end rule pathway.
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Fig. 2 Diagram showing N-end rule pathway (NERP) desensitization of seed to ABA, during ABA response window through the ABA signal pathway, Experimental Hypothesis and Null hypothesis
Holman et al (2009) in their work have identified the N-end rule component PRT6 and ATE playing a key role in the removal of ABA sensitivity in the Arabidopsis seed to seedling translation process. This implies that N-end rule pathway may well be responsible for desensitization of seed to ABA. However these authors could not state whether the N-end rule pathway targets ABA signal directly or if it functions upstream or downstream of the pathway during desensitization of seeds to ABA. In a related study (Md isa et al., 2009 unpublished) identified Methionine aminopeptidase (MetAP) as the endopeptidase substrate of the above pathway as well as many downstream regulated genes. They employed a transcriptomic approach in analysing the role of the N-end rule pathway germination potential of seeds and concluded that N-end rule pathway functions by regulating genes downstream of the pathway.
The aim of this work is to genetically characterise downstream gene which are regulated by the N-end rule pathway.
Seeds (Mutant and wild type) for this experiment are from the Nottingham Arabidopsis Stock Centre. (NASC)
Seed sterilization and plating: this is to remove any form of contamination in the seed lot. Seed are plated onto media for germination analysis.
Germination media: germination media containing a combination of agarose and MS salts and a regulated ph will be used.
Identification of homozygous mutant plant using PCR with primers specific to wild type and mutant: for the purpose of comparing variation in germination potential the use of pure mutant and wild lines is essential. However more often than not seeds from stock centres are usually a mixed population of both homozygous and heterozygous hence the need for proper identification.
Analysis of germination behaviour of mutant and wild type in response to abiotic stress such as: Abisisic acid concentration, Temperature, Salt concentration and Osmoticum (Poly ethylene glycol).
It is important measure the levels of seed sensitivity to the above stresses and compare how both the wild type and mutant respond to such stress as well as how the stress affects germination in order to understand germination pattern .
Creation of double mutant and analysing germination behaviour.
Quantitative-PCR: to analyse the amount of RNA expressed by genes during seed germination.
This will be in two phases
Phase I: Using wild type and prt6-1 mutant
Phase II: Using mutants generated from transcriptomics dataset.
3.7 Time scale.
Preparation of Germination media: One week
Seed sterilization and plating: One weeks
Seed growing period: two months
Identification of homozygous plants: Two weeks
Creation of double mutant and analysis: two months
Analysis of germination behaviour: two weeks
Qrt PCR : three weeks.