Metaboliteprofilingofmutantsfor
genesinducedbyosmoticstressin
germinatedArabidopsisseeds
Author: Brend Kemperman [email protected] MSc student Plant Biotechnology Reg: 910319427030 Supervisors: Anderson Silva Wilco Ligterink Laboratory of Plant Physiology TableofContents
Abstract ............................................................................................................................................................................................. 3 Keywords ........................................................................................................................................................................................... 3 Introduction ....................................................................................................................................................................................... 4 LEA ................................................................................................................................................................................................. 4 HSP................................................................................................................................................................................................. 4 ABA ................................................................................................................................................................................................ 4 Metabolomics ................................................................................................................................................................................ 5 Mutant lines ................................................................................................................................................................................... 5 Material and methods ....................................................................................................................................................................... 8 Selection of homozygous mutant lines .......................................................................................................................................... 8 Plant material and Re‐establishment of DT ................................................................................................................................... 8 Re‐establishment of DT by treating with PEG ................................................................................................................................ 9 Metabolomics ................................................................................................................................................................................ 9 Dehydration curve; natural re‐establishment of DT ................................................................................................................... 10 Natural re‐establishment of DT ................................................................................................................................................... 10 Results ............................................................................................................................................................................................. 11 Genotyping .................................................................................................................................................................................. 11 Re‐establishment of DT by treating with and without PEG ......................................................................................................... 11 Metabolomics .............................................................................................................................................................................. 12 Natural re‐establishment of DT ................................................................................................................................................... 15 Discussion ........................................................................................................................................................................................ 16 Genotyping .................................................................................................................................................................................. 16 Re‐establishment of DT by treating with PEG .............................................................................................................................. 16 Metabolomics .............................................................................................................................................................................. 16 Natural re‐establishment of DT ................................................................................................................................................... 17 Possible future projects ................................................................................................................................................................... 18 Acknowledgements ......................................................................................................................................................................... 18 References ....................................................................................................................................................................................... 19 Appendix .......................................................................................................................................................................................... 23 A. Protocol: DNA extraction ................................................................................................................................................ 23 B. Composition of Hyponex ................................................................................................................................................. 24 C. Mutant lines .................................................................................................................................................................... 25 D. Protocol: Extraction of metabolites (polar + apolar phase) for GC‐TOF‐MS .................................................................... 26 E. Batches greenhouse ........................................................................................................................................................ 27 F. Re‐establishment of DT with natural drying .................................................................................................................... 29 G. Metabolomics graphs ...................................................................................................................................................... 30 2 Abstract
Desiccation tolerance is the ability of an organism to survive a 0.1g H2O per gram of dry weight and be able to be rehydrated without any lethal damage to the organism itself. The aim of this study is to search for a metabolic profile in preselected Arabidopsis thaliana mutant lines which can explain desiccation tolerance. Mutant lines that showed a greater or lower establishment of desiccation tolerance were selected. Sod1, daa1 and di19 showed a greater establishment and atvip1 a lower establishment of desiccation tolerance. GC‐TOF‐
MS analysis of these four lines showed a general trend of increase upon treatment with polyethylene glycol. Specific plant metabolites such as valine, isoleucine and leucine which are metabolites that form a defence mechanism against drought stress were increased. This also applies for raffinose and proline which both act as a protectant against reactive oxygen species. Asparagine and threonine which are involved in salt tolerance are also increased for all lines. However other metabolites such as alanine, glutamine and glutamic acid which are expected to increase upon salt stress and tryptophan and phenylalanine which are expected to increase upon drought stress, were either not increased for all lines, no change occurred or were even decreased in abundance upon treatment with polyethylene glycol. In general, no clear metabolic profile has been found that could explain desiccation tolerance for germinated A. thaliana seeds. Furthermore a general trend of the lines upon treatment with polyethylene glycol was found and results partly correspond to previous reported results in literature. Keywords ABA Abscisic Acid DAP Days after pollination DW Dry Weight FW Fresh weight GC Green Cotyledon HSP Heat Shock Protein sHSP Small Heat Shock Protein LEA Late Embryogenesis Abundant PEG polyethylene glycol RH Relative humidity RH Root Hair ROS Reactive oxygen species RP Radicle Protrusion TR Testa Rupture WC Water content 3 Introduction Desiccation tolerance (DT) is the ability of a plant to be dehydrated until water content around molecules is lost and to be rehydrated without causing lethal damage to the cells (Hoekstra, Golovina et al. 2001). DT is different from drought tolerance. In drought tolerant conditions, cells have a minimal amount of water available to keep specific tissues hydrated to stay alive. Water content of cells that are drought tolerant is ~0.3g of water at minimum (H2O/g dry weight), whereas for DT it is ~0.1g (H2O/g dry weight). DT is based on the stability of tissues to replace water with sugars, metabolites and other molecules that form hydrogen bonds, which stabilize the proteins and membranes. During desiccation a glassy state of the cytoplasm is formed, which is a state where molecular diffusion and chemical reactions are reduced (Hoekstra, Golovina et al. 2001). Other macromolecules that play a role in establishing (DT) are: late embryogenesis abundant proteins (LEA’s), heat shock proteins (HSP’s) and abscisic acid (ABA). LEA
LEA’s are already studied for years and are not plant specific proteins (Hundertmark and Hincha 2008) because they have also been found in: bacteria (Bacillus subtilis) (Stacy and Aalen 1998) and nematodes (Aphelenchus avenae) (Browne, Tunnacliffe et al. 2002). Precise function, even when a LEA76 homologue shows that it is involved in establishing DT, is unknown (Battista, Park et al. 2001). In A. thaliana, the ABRE (ABA responsive element) core motif is present in the promoters of 82% of all LEA genes (Hundertmark and Hincha 2008). This high percentage of ABREs in LEA promoters suggest that ABA is also involved in activating LEA proteins. Furthermore LEA proteins have been shown to prevent aggregation of dehydration‐sensitive proteins for which the LEA proteins have a peptide profile that is shared with some chaperones (Wise and Tunnacliffe 2004, Goyal, Walton et al. 2005, Tunnacliffe and Wise 2007). A simpler explanation for the function of LEA proteins is that they improve glass stability (Wolkers, Tetteroo et al. 1999). This function is called a ‘molecular shield’ where the interaction between partially denatured proteins is decreased due to a physical barrier that is formed by the LEA proteins which have a very flexible structure (Wise and Tunnacliffe 2004, Goyal, Walton et al. 2005, Tunnacliffe and Wise 2007). HSP
Heat shock proteins (HSPs) are proteins ranging from 60 to 110 kDa in size. The HSPs family has subgroups of small heat shock proteins (sHSPs) (14‐43 kDa), which act as molecular chaperones (Hendrick and Hartl 1993, Sun and MacRae 2005). HSPs and sHSPs bind to a partially folded or denatured substrate of proteins and it can promote correct substrate folding and prevent irreversible aggregation (Sun, Van Montagu et al. 2002, Sun and MacRae 2005). HSP17.4, for instance, is expressed in the whole embryo in mature seeds (Wehmeyer and Vierling 2000), which might indicate that HSPs have an important role in protecting the whole embryo during a period of dehydration. Furthermore, abscisic acid (ABA) can induce the expression of HaHSP17.9‐CII in water stressed sunflowers (Coca, Almoguera et al. 1994, Coca, Almoguera et al. 1996). To strengthen this argument, in desiccation tolerant callus of the resurrection plant Craterostigma plantagineum treated with ABA, cytosolic sHSPs were found which are homologous to HaHSP17.9‐CII (Alamillo, Almoguera et al. 1995). ABA
ABA is involved in multiple pathways that are involved in establishment of DT in seeds. It is involved in enhancing transcription of HSP and LEA proteins, however function or mechanisms that LEA proteins and HSPs follow, are still unknown (Coca, Almoguera et al. 1994, Alamillo, Almoguera et al. 1995, Coca, Almoguera et al. 1996, Hundertmark and Hincha 2008). ABA itself is needed to develop DT in seeds and it inhibits germination of precocious seeds (Meurs, Basra et al. 1992). It was found that ABA is involved in establishing DT in the seeds 13 days after pollination (DAP) (Koornneef, Hanhart et al. 1989). ABA sensitivity correlates with the ability to re‐
establish DT during germination in A. thaliana and reduced re‐establishment of DT is observed for ABA‐
insensitive and ABA–deficient mutants (Maia, Dekkers et al. 2014). ABA therefore, is an important hormone involved in (re) establishment of DT. 4 Metabolomics
Among responses of plants to stress due to a change in environmental conditions, is a change in the accumulation of sugars, amino acids, betaines, organic acids and other osmolytes (compounds affecting osmosis) in the cytoplasm. Most amino acids show a linear increase when drought stress is induced and a decline of amino acids is observed upon rehydration (Delauney and Verma 1993). Branched chain amino acids (BCAA’s) like leucine, isoleucine and valine are amino acids that are involved in plant defence mechanisms against abiotic stresses and are actively involved in osmotic, drought and salt stress tolerance (Nambara, Kawaide et al. 1998). Free amino acids as leucine, isoleucine, proline, valine and phenylalanine are significantly increased in leaves, flowers and siliques in A. thaliana during drought stress (Nambara, Kawaide et al. 1998) similar results were found in (Joshi, Joung et al. 2010). Continuing, a recent study showed that a feedback‐
insensitive form of a key enzyme of methionine synthesis resulted in an increase of BCAA’s, sugars, proteins, starch and other amino acids, which resulted, in an increased germination rate of the transgenic seeds under salt and osmotic stresses. This also occurred when ethylene or abscisic acid was added to A. thaliana seeds (Cohen, Israeli et al. 2014). Sugars also play a role in establishing DT. It has been found in soybean (Glycine max L. Merr. Cv Williams) corn (Zea mays L. cv Merit) and pea (Pisum sativum L. cv Alaska) that oligosaccharides are present in the seeds during DT induction and DT is lost when the concentration of oligosaccharides are decreasing in the seeds (Koster and Leopold 1988). It also has been found that increases of stachyose, raffinose and sucrose in soybean (Glycine max L. Merril cv Chippewa 64) and red oak (Quercus rubra L.) seeds is correlated with establishment of DT (Blackman, Obendorf et al. 1992, Sun, Irving et al. 1994). A glassy state, or formation of intracellular glass found in corn seeds, occurs when the water content in the seeds is decreased and could help to protect the embryos from damage caused by DT (Williams and Leopold 1989, Koster 1991). The glassy state is a liquid solution with a viscosity of a solid present in the cells of the seed that consists of a high concentration of solutes, for instance soluble sugars (Williams and Leopold 1989, Koster 1991). The glassy state is lost again upon imbibition on which biochemical activity is resumed in the seed (Williams and Leopold 1989). Other compounds such as proline, glutamate, glycine‐betaine, carnitine, mannitol, sorbitol, fructans, polyols, trehalose and sucrose are also helping to keep proteins from denaturation and membranes from fusing together(Hoekstra, Golovina et al. 2001). Mutantlines
To investigate the role of genes induced by osmotic stress, re‐establishment of DT and metabolite profiling were performed in germinated A. thaliana seeds. Eight mutants with T‐DNA insertions were tested (atcc, at1g614304, daa1, asn1, di19, at1g07728, atvip1 and jmjd5) which showed greater expression in re‐establishment of DT. ATCC is responsible for CuZnSOD activity (Chu, Lee et al. 2005). CuZnSOD, or copper/zinc superoxide dismutase, found in the chloroplast is an enzyme that is responsible for catalysing reactive oxygen species (ROS) (fig 2). These ROS include superoxide, hydrogen peroxide and hydroxyl radicals. These molecules are generated as a by‐product during metabolism in cells and they might cause damage to the organism itself (Imlay and Linn 1988). There are two other SOD systems found in plants, namely manganese SOD (MnSOD) and iron SOD (FeSOD) which can catalyse the ROS (Bowler, Van Camp et al. 1994, Zelko, Mariani et al. 2002, Gao, Ren et al. 2003). Furthermore, other molecules as the organic compound ascorbate acid (AsA) which is oxidized Figure 2 image taken from Allen (1995), which describes
the dismutation/disproportionation of superoxide
radicals (O2‐) by SOD associated with PSI. The H2O2 is then
catalysed by tAPX and sAPX into harmless H2O. The
monodehydroascorbate radicals (MDA) produced by the
APX are converted, with reactions with Fd or
momodehydroascorbate reductase (MDHAR), to ascorbic
acid (AsA). Dehydroascorbate reductase (DHAR) is
catalyzing the DHA to AsA. 5 form dehydroascorbic acid (DHA), ascorbate peroxidase (APX) and glutathione reductase (GR) are active in chloroplasts. In the cytosol there are also hydrogen peroxide scavenging enzymes present, which help to protect the cells from damage caused by hydrogen peroxide and hydroxyl radicals (Smith, Vierheller et al. 1989, Asada 1992, Smirnoff and Wheeler 2000). No expression of ATCC is observed during germination and it is mildly down regulated during abiotic stress in the root and down regulated during osmotic and salt stress in the shoot (Table 1). At1g614304 is a S‐Locus lectin protein kinase family protein involved in protein amino acid phosphorylation. Not much is known about this protein family. It is expressed in the root during osmotic, salt and drought stress, but not during germination or abiotic stress in the shoot (Table 1). DAA1 an ATPase protein that is activated by the R2R3 MYB (a gene family in A. thaliana) (Stracke, Werber et al. 2001) transcription factor DUO1. Up regulation is observed in 14 day old seedlings upon drought, cold, heat and salt stress. Also in syncytia At1g64110 is upregulated (Ali, Plattner et al. 2013). Furthermore, this gene is induced in germ cells by DUO1 for differentiation (Borg, Brownfield et al. 2011). The gene is down regulated during germination but highly upregulated in the root during osmotic stress and in the shoot during both osmotic and salt stress (Table 1). ASN1 transcribes a glutamine‐dependent asparagine synthase 1, which is one of three ASN genes in A. thaliana. It is predominantly expressed in shoot tissues and asparagine plays an important role in transport and storage of assimilated nitrogen (Lam, Peng et al. 1994, Lam, Hsieh et al. 1998). Asparagine itself can enhance the tolerance of seedlings and plants, at least when added exogenous, to NaCl (Lehle, Chen et al. 1992). The gene is down regulated during germination but highly upregulated during osmotic and salt stress in the root, whereas in the shoot it is down regulated during salt and osmotic stress (Table 1). DI19 encodes the drought induced 19 protein which acts as a transcriptional regulator. DI19 regulates several pathogenesis related (PR) genes (PR1, PR2 and PR5) which are involved in plant responses to drought stress. DI19 is regulated by CPK11 which acts as a positive upstream regulator (Liu, Zhang et al. 2013) and CPK11 itself is again regulated by ABA (Zhu, Yu et al. 2007). DI19 is down regulated during germination and osmotic stress in the root. It is however upregulated during both osmotic and drought stress in the shoot and mildly upregulated during salt stress in the shoot (Table 1). AtG07728 transcribes a potential natural antisense gene, and the locus overlaps with At1G07725 which is a protein that is a member of the EXO70 gene family which is important for cell and organ differentiation (Synek, Schlager et al. 2006). No expression data was found in the Arabidopsis eFP Browser (Table 1). AtVIP1 transcribes a myo‐inositol hexakisphosphate kinase (AtVIP1) that, when overexpressed, shows hypersensitivity to ABA at seed germination and seedling stages is observed and significantly enhanced drought tolerance in transgenic plants (see fig 3). AtVIP1 is a regulator of the transcription factor AtMYB44(Xu, Chen et al. 2015). When AtMYB44 is overexpressed, an increased sensitivity to ABA for seeds germination and enhanced drought tolerance is observed. This drought tolerance is probably obtained by stomatal closure (Jung, Seo et al. 2008). Expression of the gene is down regulated during germination and no up or down regulation of the gene is observed during abiotic stress in both root and shoot (Table 1). Figure 3: Schematic drawing from Xu, Chen et al. (2015). VIP1 is affected by At3g20810 transcribes JMJD5 and JMJ30 which are jumonji domain MPK6 which upregulates MYB44 which containing 5 and jumonji c domain‐containing protein 30 respectively, increases drought tolerance and sensitivity to ABA. 6 which are histone demethylases that are involved in regulating chromatin structure and gene expression. JMJD5 is co‐regulated with evening‐phased clock components and it affects the expression of clock genes that are induced at dawn. JMJ30 is a protein that regulates the circadian clock in association with the central oscillator and it is repressed when it is bound with CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL, which are suggested to form a feedback loop in the circadian clock. (Jones, Covington et al. 2010, Lu, Knowles et al. 2011). As mutant line atvip1, expression of the gene is down regulated during germination and no differential regulation of the gene is observed during abiotic stress in both root and shoot (Table 1). The objective was to study genes that play a role in re‐establishment of DT upon polyethylene glycol (PEG) treatment. Re‐establishment of DT was tested on four developmental stages of germinated A. thaliana seeds: when seeds where at testa rupture (TR), radicle protrude (RP), first appearance of root hairs (RH) and when cotyledons turned green (GC). Metabolomics analysis of selected mutant lines atcc, daa1, di19 and atvip1 for the RH stage was performed to search for a metabolic profile with a correlation with DT. Mutant lines showed no clear metabolic profile that could explain desiccation tolerance Figure 1 Developmental stages of germinating Arabidopsis
seeds; Testa Rupture (TR), Radical Protrusion (RP), Root Hair (RH),
for germinated A. thaliana seeds. A general trend Cotyledons Greening (GC) of the lines upon treatment with PEG was found. 7 Materialandmethods
Selectionofhomozygousmutantlines
One leaf of the rosette or flower of each plant was used for DNA extraction. The DNA extraction protocol can be found in Appendix A. Primers were designed (http://signal.salk.edu/tdnaprimers.2.html) to identify the insertion in each line. Forward‐ plus reverse primer master mix and forward‐ ,reverse plus border (SALK or SAIL) primer master mix were used to check location of the insertions. The first combination was used to check if the WT allele was present in that specific line, whereas the second combination was to check if the SALK or SAIL T‐
DNA insertion was present. Each A. thaliana plant that showed only one amplicon smaller than the wild type amplicon was considered as being homozygous plant. Heterozygous plants would show two bands, one for the wild type and one band for the inserted T‐DNA. Heterozygous plants were discarded and a second check of the selected homozygous plants were performed by checking it on a gel with new extracted DNA from a leaf or flower. When homozygosis was again confirmed, the selected plants were grown until the seeds were fully matured. PlantmaterialandRe‐establishmentofDT
Mutant line were growth in the greenhouse on a table that has a flood feeding function. Relative humidity (RH) in the greenhouse was set at 80% with 16/8 hour light/dark controlled conditions. All plants received Hyponex (Appendix B) as nutrient solution. Each homozygote plant was harvested separately and the seeds were stored at room temperature. Eight homozygous mutants were selected (Table 1). Each of these eight mutant lines was tested for their ability to re‐establish DT upon polyethylene glycol (PEG) treatment. Other mutant lines of different genes also induced by osmotic stress were selected as being homozygous (Appendix C), but due to lack of plant material for these lines, they have not been used for experiments during this thesis. 8 Table 1: Mutant lines used in this thesis *Gene expression after 6‐24 hours of imbibition in water. +: increase, ‐: decrease, O: No increase or decrease in gene expression. ND: No Data. **Expression of abiotic stress, osmotic( 300mM Mannitol), salt (150mM NaCl) and drought stress (plants were exposed for 15 min with loss of app.10% fresh weight) in both root and shoot. O: No difference compared to the wild type (WT). ‐
/O: 25‐50% negative difference compared to the WT. O/+:25‐50% positive difference compared to the WT. +: 50‐500% positive difference compared to the WT. ++: 500‐1000%: positive difference compared to the WT. +++:1000+ % positive difference compared to the WT. If expression is below a factor 100 a O (no change) will be noted. The ‐, ‐‐ and ‐‐‐ correspond to the same percentage as +, ++ and +++, but as a decrease in expression. Data obtained from: http://bar.utoronto.ca/efp/cgi‐bin/efpWeb.cgi Abbreviation ID code ATCC At1g61430 DAA1 ASN1 DI19 At1g07728 ATVIP1 JMJD5 Function Copper At1g12520 Chaperone for SOD1 Protein phosphorylation At1g61430 (S‐Locus lectin protein kinase family protein) Gene coding for At1g64110 an ATPase Glutamine‐
dependent At3g47340 asparagine synthetase1 Drought induced At1g56280 gene family Putative
At1g07728 exocytosis
Phosphate At3g01310 metabolism Reverse histone At3g20810 methylation Expression Expression of abiotic Expression of abiotic during stress in the root** stress in the shoot** germination* Osmotic Salt Drought Osmotic Salt Drought O ‐/O ‐/O ‐/ O ‐ ‐ ‐/O O ‐/O ‐ ‐ O O O ‐ +++ O O +++ +++ O ‐ +++ +++ O ‐ ‐‐‐ O ‐ ‐/O O O + O/+ + ND
ND
ND
ND
ND
ND
ND
‐ O O O O O O ‐ O O O O O O Re‐establishmentofDTbytreatingwithPEG
A duplicate experiment of four replicas of 30 seeds each in the stages RP and RH for the mutants with T‐DNA insertion; atcc, at1g614304, daa1, asn1, di19, at1g07728, atvip1, jmjd5 and a wild type (Col‐0) were incubated for 3d in a 1ml polyethylene glycol (PEG 8000) solution with an osmotic potential of ‐2.5 MPa on one layer of filter paper in a 6‐cm petri dish as described (Maia, Dekkers et al. 2011). After incubation, seeds were washed with distilled water to remove the remaining PEG and dried for 3d above a saturated CaCl2 solution on a new white filter paper inside a desiccator (42% RH at 20OC) (Maia, Dekkers et al. 2014). After drying, seeds were first pre‐humidified (relative humidity of 100%) for 24H at 220C under constant white light and further rehydrated on a Copenhagen table. Viability of the seeds was checked after 7 days. Seeds that continued their developmental program and turned into a seedling were considered as being DT tolerant. Metabolomics
Metabolites were measured of seeds at RH stage, treated in PEG for 3d. Four biological replicates for the mutant lines atcc, daa1, di19 atvip1 and wild type in the RH stage were used with 200 seeds for each biological replicate and treatment. Each line was treated with and without 1ml ‐2.5MPa polyethylene glycol (PEG 8000) solution. Metabolites were extracted from the seeds according to the Extraction protocol (polar + apolar phase) for GC‐TOF‐MS analysis (Appendix D). Samples were put in the GC‐TOF‐MS and obtained data was analyzed using MetaboAnalyst, a web‐based platform for comprehensive analysis of quantitative metabolomics data (Xia and Wishart 2011). 9 Dehydrationcurve;naturalre‐establishmentofDT
To access a more natural way to re‐establish DT in germinated seeds, four developmental stages were used to induce DT without PEG. Four developmental stages are: testa rupture (TR), radical protrusion (RP), first appearance of root hair (RH) and green cotyledons (GC). These stages occur after approximately 24, 28, 32 and 36 hours respectively, germinating in a 220C light chamber after stratification of at least 72h in a 4oC dark chamber. A dehydration curve was measured, for each developmental stage, during 72 hours in nine time points (0, 2, 4, 6, 8, 10, 24, 48 and 72 hours). Four replicas of Col‐0 with 36 seeds for each developmental stage and time point were selected and dried in a dark 22oC cabinet with a RH of 32% on a black membrane in a seed tray containing one blue filters and 25ml of demi‐water to represent a natural drying. The fresh weight (FW) of the seeds after drying for the different durations was measured. The seeds were thereafter placed in a 105oC oven to fully dry overnight. The seed dry weight (DW) was measured and the WC (water content) (g g‐1 dw) was calculated and put in a graph. Naturalre‐establishmentofDT
A duplicate experiment of four replicas of 30 seeds each in the stages RP and RH for the mutant lines atcc, at1g614304, daa1, asn1, di19, at1g07728, atvip1, jmjd5 and wild type (Col‐0) were selected, put on one black membrane and incubated in a seed tray with one blue filter and 25 ml of water. After drying, seeds were first pre‐humidified (100% RH) for 24H at 220C (Maia, Dekkers et al. 2014) in the light and subsequently further rehydrated on a Copenhagen table. Viability of the seeds was checked after 7 days. 10 Results
Genotyping
Plants were genotyped individually. Mutant lines At2G01100, At5G58720, At3G52180, NAKR3 and NTMC2T6.2 showed a homozygous genotype (mutant lines correspond to the numbers 6, 10, 22, 23 and 34 in appendix E). Other mutant lines showed heterozygous or wild type alleles. A few siliques of each mutant line were harvested and seeds were checked under a microscope in search for missing seeds that indicates a lethal allele, this was not found. Furthermore, mutant line 9, even after multiple attempts, did not germinate in the greenhouse. Re‐establishmentofDTbytreatingwithandwithoutPEG
As described in Maia, Dekkers et al. (2011), DT can be re‐established by treating the seeds using a PEG (‐
2.5MPa) solution. This experiment was a follow‐up of an experiment performed by the BSc student Josephine van Eggelen. Only mutant line atvip1 showed a significant decrease in re‐establishing DT for the RP stage (Appendix F). Whereas when the same mutants were treated with PEG (‐2.5 MPa ) solution to re‐establish DT in germinated seeds, each mutant was able to re‐establish DT at RP. Figure 4: Survival of ‐2.5 MPa PEG treated mutant lines after a 72H dehydration,
duplicate experiment of 4 replicates per line, 36 seeds per replicate. It was observed that the mutants atcc, daa1 and di19 show a greater DT compared with Col‐0, whereas mutant line atvip1 just as mutant At1g07728 showed no differential re‐establishment of DT. The same experiment with the same conditions was performed again and mutant line atvip1 did show a significant lower re‐establishment of DT. Mutant lines atcc, daa1, di19 and atvip1 were selected for metabolomics analysis. 11 Metabolomics
Mutant lines atcc, daa1, di19 and atvip1 were investigated further by performing a metabolomics analysis. A total of 50 different metabolites were measured. Figure 5 shows the strongest increase and decrease upon treatment in seeds treated with PEG. Xylose and 4‐hydroproline show both a significant decrease, whereas raffinose shows an increase. In table 2 a summary of each metabolite and the general response of the PEG treated mutant lines compared to the untreated mutant lines is shown. Figure 5. Metabolites of the Mutant lines without PEG treatment (coloured columns) and with PEG (‐2.5 MPa) treatment (striped columns), which show the highest change between with and without treatment. As shown in table 2, which summarized a comparison of mutant seeds treated with PEG (‐2.5 MPa) solution compared to their own non‐treated seeds. The abundance of 16 metabolites for the mutant seeds are increased, (asparagine, aspartic acid, benzoic acid, beta‐D‐methylfrutofuranoside, isoleucine, lysine, phophoric acid, proline, pyroglutamic acid, raffinose, serine, s‐methyl‐Lcysteine, sucrose, threitol, threonine and valine). 5 metabolites have no significant change (citric acid, methionine, phenylalanine, tryptophan, and tyrosine). 10 metabolites, are decreased (4‐hydroxyproline, alfa‐ketoglutaric acid, ethanolamine, fructose, galactinol, glyceric acid, malic acid, myo inositol succinic acid and xylose). 9 metabolites showed no significant increase, but for some mutants these metabolites were upregulated (alanine, ascorbic acid GABA, glutamine, glycine, glycolic acid, leucine, monomethylphosphate and nonanoic acid). The last 10 metabolites showed no significant increase in the WT, but for some mutants these metabolites were down regulated. (urea, beta alanine, gumaric acid, glucopyranose, glucose, glucose‐6‐phosphate, glutamic acid, N‐acetylglutamic acid, putrescine, and pyruvic acid) Some lines showed a significant difference for some metabolites in a treatment compared to the wild type (Col‐0) for the same treatment (see Appendix G). Mutant line atcc showed an increase of monomethylphosphate for the untreated seeds. Mutant line daa1 showed a decrease in both urea and putrescine for the treated seeds and for the untreated seeds an increase in glucose . Mutant line di19 showed for treated seeds an increase in threitol and a decrease in myo inositol. The untreated seeds for this line showed only an increase in glucose. Most significant changes were found for mutant line atvip1. The treated seeds show an increase in sucrose and an decrease for GABA, putrescine and myo inositol. Untreated seeds showed an increase in abundance in phosphoric acid and glutamine. A decrease in abundance, for untreated seeds, was found for in putrescine, beta‐D‐methylfructofuranoside, beta‐alanine, 4‐hydroxyproline and myso inositol. Metabolites, that are important or are involved in establishing DT are shown in figure 6 (CHIANG and Dandekar 1995, Nambara, Kawaide et al. 1998, Joshi, Joung et al. 2010). The metabolites valine, isoleucine, proline, lysine, asparagine and threonine are all increased for all mutant lines upon treatment with PEG (‐2.5 MPa) solution. All mutant lines have increased levels for leucine, except atvip1 which does not show an increase or decrease. Tryptophan is increased for mutant line atvip1 and not increased or decreased for the other mutant lines. Alanine shows for both daa1 and atvip1 no increase or decrease, but for sod1 and di19 levels are higher. Glutamine is not increased in mutant line sod1, while the other mutant lines show a higher level. Finaly, glutamic acid shows a decrease for sod1 and atvip1 but this is not found for the other mutant lines. 12 Figure 6: Measured metabolites that are important or involved in establishing DT or are found to be increased upon drought or salinity stress. Mutant lines without PEG treatment (coloured columns) and with PEG (‐2.5 MPa) treatment (striped columns). 13 Table 2: An schematic overview of 50 measured metabolites which show the trend that the mutant lines atcc, daa1, di19 and atvip1 follow when they are treated with PEG. A + indicates that all 4 lines show a significant increase for that particular metabolite when they are treated with a ‐2.5 MPa PEG solution compared to their own non treated mutant wild type. A – indicates a decrease and a O indicates no significant increase was found. The combination of ‐/O and O/+ indicates that both an increase or decrease together with no significant change was found for that particular metabolite. Metabolite Asparagine + Serine
Metabolite + Metabolite
Glycine 3tms
O/+ Metabolite
Urea
Benzoic Acid + Sucrose + Leucine 2tms
O/+ Fumaric acid ‐/O Beta‐D‐
Methylfructof
uranoside Isoleucine + Threitol + Monomethylphosph
ate 2tms O/+ Glucopyrano
se ‐/O Metabolite
4‐
Hydroxyproli
ne Alfa‐
Ketoglutaric Acid Ethanolamin
e Fructose
Aspartic Acid + S‐Methyl‐L‐
+ Glycolic acid
O/+ Beta alanine ‐/O + Threonine + Nonanoic acid
O/+ Glucose
‐/O Galactinol
‐
3tms + Valine
+ Citric acid
‐/O Glyceric Acid
‐
‐/O Malic Acid
‐
‐/O Myo Inositol
‐
‐/O Succinic Acid
‐
‐/O Xylose
‐
‐/O cysteine Lysine Phosphoric Acid Proline + Alanine O/+ Methionine
O + Ascorbic O/+ Phenylalanine
O + GABA
O/+ Tryptophan
O Glucose‐6‐
phosphate Glutamic acid N‐
acetylgluta
mic acid Putrescine
+ Glutamine O/+ Tyrosine 3tms
O Pyruvic Acid O Acid Pyroglutamic acid Raffinose 14 ‐
‐
‐
‐
Naturalre‐establishmentofDT
To perform a more natural way of re‐establishment of DT, a dehydration curve has been made for each stage: TR, RP, RH and GC. Dehydration curves are shown in figure 7 . Figure 7: Dehydration curve of the different stages TR, RP, RH and GC altogether and each of them separately. Inside the combined dehydration curve, another curve is shown that represents the survival rate of the different developmental stages when dried in natural conditions. TR and RP stage survival ration is 100% upon re‐hydration whereas for the RH a 20% and GC stage a 0% survival rate is observed. All stages have reached the same water content after 72H of drying. The dehydration curves show a similar decrease of moisture during a drying period of 72H in a 220C 32% RH environment for all developmental stages. Within 6h all stages, except TR, have lost more than 90% of their moisture. After 10h the slope of all the stages has stabilized. The stages reached the same water content after 72H of drying and only TR and RP have a 100% survival rate upon re‐hydration. The stages lost 90 to 96 % of their total weight after 72H of drying. 15 Discussion
Genotyping
Most of the mutant lines showed heterozygosity. Even after multiple attempts and several batches, no homozygous genotype was found for these lines but only heterozygous or wild type genotypes. It is a strange occurrence that out of 25 plants and for the second batch which was grown out of heterozygous seeds, not a single homozygous plant was found when mendel’s law of segregation suggest that 25% of the 25 plants should be homozygous. This raised the question if, for at least one line, a lethal allele could exist. This was not the case when the siliques were checked; all seeds were present for all lines. For mutant line 9, the knocked‐out gene encodes a RING‐finger type E3 ligase. This ligase is necessary for mitotic cell cycle progression during gametogenesis in A. thaliana (Liu, Zhang et al. 2008). This could explain why none of the seeds germinated in the greenhouse. Re‐establishmentofDTbytreatingwithPEG
Lines that showed an increased or decreased survival rate with PEG at RH, were chosen to continue research by performing a MS‐TOF‐GC analysis. Mutant line atcc, daa1, di19 and atvip1 were chosen to investigate more thoroughly. Mutant lines asn1 and Atg07728 were not chosen, since they did not show a significant difference when compared to col‐0. Mutant line atvip1 was chosen, due to a significant score in the second experiment and due to the fact that it showed a lower establishment of DT. It was preferred to use the phenotypes that showed greater or lower re‐establishment of DT, so a possibility to check if a specific metabolic profile is present that could explain the re‐establishment of DT in the seeds. Metabolomics
To check which metabolites would have a role in re‐establishment of DT in A. thaliana germinated seeds, mutants with greater re‐establishment at RH (atcc, daa1 and di19) and lower (atvip1) were used for metabolite analysis. Change in valine, leucine and isoleucine are observed upon treatment with PEG (fig 6). These free amino acids are responsible to act as a defensive mechanism against abiotic stresses, in this case drought stress (Nambara, Kawaide et al. 1998, Joshi, Joung et al. 2010). This is seen in the results except for mutant line atvip1, which does not show an increase in leucine. In plant material these three amino acids, together with phenylalanine, proline, tryptophan and lycine (fig 6), are increased several fold during dehydration (Nambara, Kawaide et al. 1998). This trend is also seen for threonine, asparagine, glutamate, glutamine, proline and alanine at increasing NaCl concentrations with 2 week old plant material (fig 6) (CHIANG and Dandekar 1995). Proline, lycene, asparagine and threonine are increased for all treated seeds. In A. thaliana seeds proline can enclose 17% of the total free amino acids. The same accounts for asparagine, which encloses 22%. However in tissues with a higher water content, a lower level of proline and asparagine is found (CHIANG and Dandekar 1995). In general proline acts as a protectant and can scavenge ROS. In many species it can be activated by ABA (KISHOR, POLAVARAPU et al. 2014). Alanine, tryptophan, phenylalanine and glutamate (fig 6), however, do not show an increase in most of the mutants and for some mutant lines even a decrease is observed compared to col‐0. For instance, tryptophan and phenylalanine could both be involved in providing protein stability (Costa, Righetti et al. 2015). Tryptophan is increased for mutant line atvip1 which shows a negative effect on re‐
establishment of DT, whereas phenylalanine shows a decrease for mutant line daa1 which shows a greater re‐
establishment of DT at RH. So a change in one metabolite does not have to correlate with the phenotypical behaviour of that specific line. Studies correlate in most cases with the obtained metabolic results. However most metabolite results in other studies are obtained from 2 week old plant material and not from the seed itself. It is possible that therefore results obtained in this thesis do not correlate with results from other studies. Three metabolites, raffinose, xylose and 4‐hydroxyproline showed the most pronounced change in abundance. Raffinose together with galactinol and stachyose is accumulated in the seeds of A. thaliana. Raffinose and galactinol are detected in stressed plants, whereas stachyose is not (Taji, Ohsumi et al. 2002). Two of seven 16 genes for galactinol synthase AtGolS1 and AtGolS2 are induced during drought or salt stresses (Taji, Ohsumi et al. 2002). Overexpression of AtGOLS2 showed an increase in the accumulation of galactinol and raffinose together with an increase of drought‐stress tolerance (Taji, Ohsumi et al. 2002). It is suggested that both galactinol and raffinose are acting as a protectant just as proline (Taji, Ohsumi et al. 2002, Stoyanova, Geuns et al. 2011). Xylose is important for a plant, because plant N‐linked glycan’s which contain β1.2‐xylose and α1,3‐
fucose have the function to affect the co and post‐translational folding of proteins (Rayon, Lerouge et al. 1998). The N‐glycan proteins, present in the Golgi apparatus, are essential for root salt tolerance and both β1.2‐xylose and α1,3‐fucose might have an overlapping function in salt tolerance (Kang, Frank et al. 2008) and if one of the two is disturbed, salt sensitivity is increased (Kaulfürst‐Soboll, Rips et al. 2011). In this study, interestingly, xylose drops upon treatment with PEG, but a higher re‐establishment of DT is observed for mutant lines atcc, daa1 and di19. A lower amount of xylose is observed for mutant line atvip1 in non‐treated seeds. An amino acid (4‐Hydroxyproline) is used by the cell to produce hydroxyproline‐rich glycoproteins , which is necessary for a normal embryo development in A. thaliana (Hall and Cannon 2002). Without this protein, the developing cells of for instance the root will show abnormal shapes, cell differentiation occurs to some degree but cell expansion does not occur and cell growth is in random directions (Tierney and Varner 1987, Hall and Cannon 2002). It is a logical result that this metabolite is decreased in abundance since no cell differentiation is necessary during periods of drought stress in A. thaliana seeds. It is noteworthy to mention mutant line atvip1, which shows a phenotype with a lower re‐establishment of DT. This line has the most differences in metabolites between treatments compared to the three other mutants (atcc, daa1 and di19). There are 12 differences, of which eight for untreated and four for treated seeds. Two metabolites, putrescine and myo‐inositol, show for untreated and treated seeds a decrease in abundance. There are indications that putrescine can influence the transpiration rate and stomatal conductance as response for dehydration. This was shown in transgenic A. thaliana plants where Arginine decarboxylase 2 gene was overexpressed and an overproduction of putrescine was observed (Alcázar, Planas et al. 2010). Results showed that putrescine plays a role in re‐establishment of DT in germinated seeds. Myo‐inositol is an amino acid which is used as a substrate for rafinose, which is a oligosacharide that can protect a plant against drought stress (Taji, Seki et al. 2004). That a decrease of both metabolites would cause a drastic lower re‐establishment of DT for mutant line atvip1 is unlikely. Sucrose is increased for mutant line atvip1 when treated with PEG which has also been shown for the sucrose content of both cucumber and Impatiens when treated with PEG (Bruggink and van der Toorn 1995). This is probably due to breakdown of food reserves, which might also have a function in stabilizing cell membranes and proteins in the absence of water (Bruggink and van der Toorn 1995). It is important to mention that not only the T‐DNA insertion in the genome of these lines are the cause of the metabolic profiles that were found. Point mutations, insertions, deletions or other T‐DNA fragments could have altered the genome and therefore altered the accumulation of amino acids and sugars during DT. Naturalre‐establishmentofDT
A dehydration curves show a similar decrease of moisture during a drying period of 72H in a 220C 32% RH environment for all developmental stages. Within 6h all stages, except TR, have lost more than 90% of moisture content. This is caused by a lower influence of the seed coat which encloses the embryo in the TR stage almost completely and therefore inhibits the evaporation of water present in the embryo at a higher degree (Debeaujon, Léon‐Kloosterziel et al. 2000). For the GC 0h the highest FW has been found, which is a logical result of a germinating seed that grown longer in favourable conditions. After drying the samples for 72H the DW of the seeds is <0.1 mg. This curve can be used as a standard to search for mutant lines that show a different trend in losing moisture. 17 Possiblefutureprojects
There are multiple possible future projects that could help to continue the study on desiccation tolerance. First of all, an ABA measurement could be performed. Seeds are already treated and stored a the ‐800C freezer. It is expected that upon drying an increase of ABA is shown and upon rehydration of 24 hours after 72 hours drying an decrease of ABA is expected to be seen. This curve can be used as a standard for the behaviour of seeds upon germination. Similar ABA measurements can be made to study ABA in the mutant lines. If an alteration of ABA behaviour is observed, further investigation could be advised. Second, a sugar measurement in germinated A. thaliana seeds is another experiment that is going to be performed. The seeds were prepared and are stored in a ‐800C freezer until they can be put into the machine. It is interesting to see if treatment with PEG alters the amount of specific sugars. That alteration happens has already been shown for raffinose, fructose and sucrose in the metabolomics analysis for different treatments. However, there are more sugars, for instance trehalose, that are involved in desiccation tolerance and can therefore be investigated. Trehalose is necessary for the survival of desiccation over a long period of time in Saccharomyces cerevisiae (Tapia and Koshland 2014). Overexpression of yeast trehalose‐6‐phosphate in tobaco plants increases its tolerance to drought (Romero, Bellés et al. 1997). In A. thaliana, 11 trehalose‐6‐phosphate synthase genes were found which synthesize UDP glucose and glucose‐6‐phosphate to trehalose‐6‐
phosphosphate and Tre6P phosphatase (TPP) can dephosphorylate it to trehalose (Leyman, Van Dijck et al. 2001). Sugars are, as introduced, important in establishing DT. It would be interesting to see the ratios of sugars that are present in the mutant lines and it would be interesting if differences in sugar ratios between the mutant lines can be found. Third, as introduced, LEA proteins and HSPs are also important in helping a seed to establish DT. It would be interesting to check which LEA and HSP proteins are transcribed for the developmental stages RP and RH and how ABA is correlated to the transcription of these proteins. Finally as mentioned in the discussion, the RH stage of the ‘natural’ drying experiment performed by Josephine van Eggelen should be repeated (appendix H). Results show a low survival rate, below 10% in average, for the developmental stage RH, whereas figure 7 shows that survival of at least 20% is expected for the wild type. Furthermore, line atvip1 shows an positive establishment of DT, whereas for RP (appendix G) and for RH when treated with PEG a negative establishment of DT is shown. There is a possibility that the seeds were not picked at the right moment of germination. If the seeds show even the tiniest bit of green, there is no chance of survival of that seed. The time frame of picking the seeds is roughly 3 to 4 hours. Which can be difficult if a high number of seeds have to be picked. Acknowledgements
First of all, I would like to thank my direct supervisor Anderson Silva, who guided me through my thesis, helped me in the laboratory and reviewed this report. Second I would like to thank my other supervisor Wilco Ligterink who gave me the opportunity to work in the Laboratory of Plant Physiology. I would like to thank the technicians Leo Willems for helping me with the GC‐TOF‐MS and Juriaan Rienstra for answering my questions in the laboratory. Finally, I would like to thank Josephine van Eggelen and Adane Demissie for their help in the laboratory and greenhouse. 18 References
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dependentproteinkinases,CPK4andCPK11,regulateabscisicacidsignaltransductioninArabidopsis."ThePlantCell
Online19(10):3019‐3036.
22 Appendix
A. Protocol:DNAextraction
1. 1 leaf per tube 2.
Add 200 µL of extraction buffer, one stainless steel bullet and grind (1 min at 30 Hz) 3.
60 minutes at 60 ºC 4.
Spin 15 minutes at 2.800 g 5.
Take 75 µL of supernatant to new tube (Microtiter plate) 6.
Add 75 µL of iso‐propanol and 30 µL of 10M of NH4Ac 7.
15 minutes at Room Temperature/ or over‐night 8.
Spin 15 at room temperature at 2.500 g 9.
Wash pellet with 70% of ethanol 10. Spin 5 minutes at 2.500 g, remove the ethanol 11. Quick spin on the plate upside‐down 12. Dissolve the pellet in 50 µL Milli‐Q water 13. Use up to 4 µL for the PCR Extraction buffer 2M of NaCl (116.88 g per Litre) 200mM of Tris‐HCl pH8 (31.520 g per 1 Litre) 70mM EDTA (29.134 g per Litre) 20mM Na2S2O5 (3.802 g per Litre) 23 B. CompositionofHyponex
Main Elements Concentration (mM/L) NH4 1.7 K 4.131 Ca 1.97 Mg 1.239 NO3 4.141 SO4 3.411 P 1.287 Trace Elements
Fe
Mn
Zn
B
Cu
MO
Concentration (μM/L)
21.0
3.4
4.7
14.0
6.9
0.1
24 C. Mutantlines
Table 4: Mutant lines used in this thesis *Gene expression after 6‐24 hours of imbibition in water. +: increase, ‐: decrease, O: No increase or decrease in gene expression. ND: No Data. **Expression of abiotic stress, osmotic( 300mM Mannitol), salt (150mM NaCl) and drought stress (plants were exposed for 15 min with loss of app.10% fresh weight) in both root and shoot. O: No difference compared to the wild type (WT). O/‐: 25‐50% negative difference compared to the WT. O/+:25‐50% positive difference compared to the WT. +: 50‐500% positive difference compared to the WT. ++: 500‐1000%: positive difference compared to the WT. +++:1000+ % positive difference compared to the WT. If expression is below a factor 100 a O (no change) will be noted. The ‐, ‐‐ and ‐‐‐ correspond to the same percentage as +, ++ and +++, but as a decrease in expression. Data obtained from: http://bar.utoronto.ca/efp/cgi‐bin/efpWeb.cgi Number T‐DNA (agi) Expression during germination* Expression of abiotic stress in the root**
Expression of abiotic stress in the shoot**
Osmotic Salt Drought Osmotic O/+ O O ++ O O Salt Drought 3 AT1G33970 1‐6h + 12‐24h ‐ 6 AT2G01100 ‐ O O O + + O 8 AT5G01520 ‐ + + O +++ ++ O 9 AT5G22000 ‐ O/+ + O + + O 10 AT5G58720 ‐ O/+ O O O/+ O O 12 AT5G66400 1h + 3‐24h ‐ +++ +++ O +++ +++ + 13 AT4G01450 + + O O/+ O + O/+ 18 AT1G04300 ‐ O/+ O O + O O 20 AT2G04240 1h,6h + 3,12,24h ‐ O O O + + O 21 AT3G14880 ‐ O O O O O O 22 AT3G52180 + O O O + + O 23 AT3G53530 ND ND ND ND ND ND ND 24 AT4G25800 O O O O O O O O ‐/O + + O O 1‐6h + 12‐24h ‐ 25 AT5G03240 29 AT5G57110 ‐ O O O O O O 30 AT1G53780 ‐ O O O + O/+ O 31 AT2G47850 ‐ 34 AT3G14590 ND 36 AT4G16160 38 AT5G04750 39 AT5G52300 40 O O O O O O ND ND ND ND ND ND O O O O O O + O O + + O/+ ‐ +++ +++ O +++ +++ O AT1G30500 ‐ O O O + O/+ O 41 AT2G41070 ‐ O O O O O O 42 AT2G41710 ‐ O O O O O O + O O ++ O O 1h + 3‐24h ‐ 1h + 3‐24h ‐ 1h + 3‐24h ‐ 43 AT2G33830 44 AT3G62090 ‐ O O O O O O 45 AT1G01240 ‐ + + O + O/+ + 25 D. Protocol:Extractionofmetabolites(polar+apolarphase)forGC‐TOF‐MS
modified from Proteomics 2004, 4, 78-83
Internal standard stocks: ribitol (adonitol) 1 mg/ml in water (polar phase) nonanedecanoic acid methylester 1 mg/ml CHCl3 (organic phase) Extraction mixes: ‐ Mix A: MeOH:CHCl3:internal standard (nonanedecanoicacid me 1 mg/ml): 400µl:280µl:20µl ‐ Mix B: Water ribitol: 130 µl water + 20 µl ribitol (1 mg/ml) ‐ Mix C: MeOH:CHCl3, 1:1 (v/v) All steps kept on ice! Extraction ‐ weigh ~20mg FW material in 2 ml Eppendorf tube and keep everything frozen! ‐ add 700 µl mix A ‐ vortex briefly ‐ add 150 µl mix B ‐ vortex thoroughly ‐ 10 min sonication in Ultrasonic bath ‐ add 200μL H2O ‐ vortex thoroughly ‐ centrifuge 5 min 15000 rpm ‐ transfer 90% of the upper phase to a 2mL Eppendorf tube ‐ add 0.5 mL mix C to the remaining organic/inter phase ‐ vortex thoroughly ‐ keep on ice ~10min ‐ add 200 μL H2O ‐ vortex thoroughly ‐ centrifuge 5 min 15000 rpm ‐ collect 90% of the upper phase and add to the previously collected polar phase ‐ collect the (lower) organic phase and transfer to a glass vial ‐ store the organic phase at ‐20°C until further processing ‐ transfer 25 µl of 10x diluted and 25‐100‐200 µl of the undiluted polar phase to inserts in 1.5 ml glass GC‐vials and dry over night ‐ analyse on the GC‐TOF‐MS using the online derivatisation protocol. 100µl in insert 26 E. Batchesgreenhouse
Table 1 Selected Mutant lines Line T‐DNA (agi) Name/Code
Function
T‐DNA line
NASC code Genotyping 3 Batch
1 2
3
x AT1G33970 response to bacterium/GTP binding
SAIL_534_B01
N866206
Wild type 6 x AT2G01100 P‐loop containing nucleoside triphosphate hydrolases superfamily protein unknown protein
SALK_009782C
N681000
Homozygous 8 x AT5G01520 ABA INSENSITIVE RING PROTEIN 2
zinc ion binding
SALK_062995
N562995
Wild type 9 x AT5G22000 RING‐H2 GROUP F2A
regulation of cell cycle
SALK_128063C
N659554
‐
10 x AT5G58720 Smr protein/MutS2 C‐terminal SALK_065495C
N656593
Homozygous 12 x AT5G66400 smr (Small MutS Related) domain‐containing protein RESPONSIVE TO ABA 18
SAIL_244_F10
N866138
Wild type 13 x x
AT4G01450 UMAMIT30
nodulin MtN21‐like transporter family protein
SALK_146977
N646977
Heterozygous 18 x x
AT1G04300 TRAF‐like superfamily protein
molecular function unknown SALK_026088
N678276
Heterozygous 20 x x
AT2G04240 XERICO zinc ion binding/abscisic acid metabolic process/response to osmotic stress/response to chitin/protein binding/response to salt stress/response to gibberellin stimulus SALK_075188C
N675804
Heterozygous 21 x AT3G14880 transcription factor‐related
SALK_056670
N556670
Wild type 22 x x
x
AT3G52180 STARCH‐EXCESS 4
SALK_102567C
N663517
Homozygous 23 x x
x
AT3G53530 SODIUM POTASSIUM ROOT DEFECTIVE 3
SALK_000691C
N673581
Homozygous 24 x x
AT4G25800 Calmodulin‐binding protein
SALK_040280C
N655462
Heterozygous 25 x x
AT5G03240 POLYUBIQUITIN 3
SALK_148831C
N683036
Heterozygous 29 x x
AT5G57110 AUTOINHIBITED CA2+ ‐ATPASE
SALK_057877C
N662571
Homozygous 30 x x
AT1G53780 peptidyl‐prolyl cis‐trans isomerases
SALK_106176C
N654821
Heterozygous 31 x x
AT2G47850 Zinc finger C‐x8‐C‐x5‐C‐x3‐H type family protein SALK_054523C
N662511
Heterozygous x
x
x
x
x
Encodes a plant‐specific glucan phosphatase that contains a noncatalytic carbohydrate‐binding module as well as a dual specificity protein phosphatase domain. metal ion transport
encodes ubiquitin that is attached to proteins destined for degradation ATPase activity/protein catabolic process/nucleoside‐
triphosphatase activity/peptidyl‐prolyl cis‐trans isomerase activity/nucleotide binding zinc ion binding
27 34 x x
x
AT3G14590 NTMC2T6.2
Calcium‐dependent lipid‐binding SALK_131027
N631027
Homozygous 36 x x
AT4G16160 ATOEP16‐S
plastid outer membrane/mitochondrial inner membrane presequence translocase complex/P‐P‐bond‐hydrolysis‐driven protein transmembrane transporter activit SALK_025109
N525109
Heterozygous 38 x x
39 x
x
AT5G04750 F1F0‐ATPase inhibitor protein
SALK_023158C N679430
AT5G52300 73A HM 40 x
x
AT1G30500 CBF/NF‐yA7
SALK_020063
Homozygous 41 x
x
AT2G41070 Bzip12/eel
SALK_021965
Homozygous 42 x
x
AT2G41710 43 x
x
AT2G33830 dormancy/auxin associated family protein
44 x
x
AT3G62090 45 x
x
AT1G01240 PIL2 (PHYTOCHROME INTERACTING FACTOR 3‐LIKE 2); protein binding / transcription factor unknown protein
Primers 926/927
Heterozygous Homozygous N658851
Homozygous SALK_054451.53.45.x
N554451
Homozygous SALK_090239C
N661012
Homozygous SALK_104275C
N653407
Homozygous 28 F.
Re‐establishmentofDTwithnaturaldrying
Figure 8: Re‐establishment of desiccation tolerance in
natural drying conditions for the developmental stages RP
and RH. After 72 hour drying seeds were put for 1 week on
the Copenhagen table. Seeds which germinated were
counted and are shown as a survival percentage. 29 G. Metabolomicsgraphs
Xylo se
Va line
16
15
c
c
c
c
b
a
a
bc
d
c
ab
8
12
c abc abc abc
a
ab
abc
bc abc abc
c
a
abc ab abc ab abc abc bc
12
12
8
8
4
4
abc
ab
bc
ab c
a
bc
ab
b
ab c
c
9
6
6
3
4
16
16
abc
Tryp to phan
Tyrosine 3tms
15
9
a
a
Urea
d
d
a
12
12
a
d
d
abc
3
0
0
0
Th reoni ne 3tm s
16
b
b
a
a
Threi to l
b
b
a
a
0
a
20
12
c de
12
ab
bc
ab
c
c
b
b
Su ccini c aci d
c
b
c
a
16
12
b
b
b
15
a
12
a
a
a
a
10
8
5
4
0
0
a
0
Ser ine
Raffinose
c
c
c
c
ab
a
ab
12
b
b
b
b
d
a b cd
b
d
cd
ab c d
Pyro glu tam i c aci d
bc d
16
cd
abc
a
c
c
ab
ab
c
b
ab
c
a
a
a
Putresci ne
b
c
12
ab
bde
12
a
8
a
0
Pyru vic acid
16
b
12
a
4
4
4
c
b
8
8
16
b
b
a
8
0
b
b
de
abc d
bc
b
b
ef
S -M eth yl-L-cystei ne
d
f
ef
0
S ucrose
b
9
12
9
6
8
6
df
f
bcd e
bc de
c
bce c def
a
ab
a
a
a
8
a
4
4
3
4
3
0
0
0
0
0
P roli ne
20
bc
bc
a
Pho sphori c aci d
a
c
bc
b
a
Ph enylal an ine
16
20
a
a
a
c
c
ab
ab
c
ab
c
b
bc
bc
ab
c
c
ab c
No nanoi c aci d
ab
ab
N-Acetyl glu tam i c aci d
16
12
bc
a
ab
abc
bc
ab
abc
a
abc
abc
ab c
a
c
bc
15
15
12
9
12
10
10
8
6
8
5
5
4
3
4
0
0
0
0
0
Myo ino sitol
Mono methyl pho sp hate (2TM S)
20
Methio nin e
10
12
d
d
d
b
ab
cd
ab
a
15
a
c
a
c
bc
bc
ab
abc
ab c
c
ab
ab
ab
ab
b
abc
M al ic acid
c
abc
c
ab
a
Lysine
b
b
ab
a
ab
ab
20
b
a
a
8
b
b
b
abc
9
c
c
ab
16
b
a
a
a
a
15
12
10
8
5
4
0
0
c
bc
a
a
bc
a
c
a
b
6
10
6
5
3
0
0
4
2
0
Leuci ne 2tm s
i soleu cine
16
16
a
bc
a
bc
a
bc
a
c
ab
a
bc
12
b
b
a
a
b
Gl ycoli c aci d
a
b
G lycin e (3tms)
16
a
12
a b c a bcd
12
Glyceric acid
16
16
b
a
c
cd e
ab
abd
ce
c
a
ab
ab
d
b
cd
a
bc d
ab
b
12
12
8
8
8
8
4
4
4
4
4
0
0
0
0
0
Gl utami ne
Glu tam i c aci d
G lucose-6-pho sphate
20
a
c
a
a bc
a
bc
a
c
b
16
b cd
c
ab c
d
cd
ab
bc d
ab c d
a
c
15
15
12
10
10
5
0
a bc
Glucose
abc
bc
c
ab
abc
abc
bc
a
8
5
4
5
4
0
0
a
0
GABA
b c de
ab
a bc
d
abc e cde
c
bc
ab
ab
a
bc
c
c
d
bc
ab
a
a
cd
bd
a
a
12
8
8
8
10
10
4
4
4
5
5
0
0
0
0
0
a
a
a
a
Ci tri c aci d
20
a
ab
a
ab
a
beta-D-Methyl fr uctofuranosi de
ab
ab
ab
ab
c
a bc
c
c
c
15
Beta alan ine
12
16
b
c
ab
c
ab c
c
abc
10
b
12
a
b
a
a
b
a
b
a
10
8
6
5
4
3
0
0
0
ab
ab
a
c
a
bd
a
a
aspartic acid
b
a
b
16
a
b
a
b
a
b
a
b
a
b
12
9
b
ab
8
15
b
ab
15
c
c
Ben zo ic aci d
bc
a bc
c
bc
ab
Ethano lam in e
c
12
a
12
ab
20
cd
bc d
a
Fructose
20
a b cd e
ab
a
0
Fu mari c aci d
16
de
b
a
d
cd
abc d
ab
10
b
b
a
16
bc
ab
a
Glu copyrano se
bc
8
a
b
a bc
ab c
bc
ab
c
12
16
b
b
c
c
15
Gal acti nol
16
b
20
cd
ab
a
b de
8
20
b
b
bc
a
6
8
4
4
2
Asp ar agin e
16
ab
cd
a
cd
a
cd
ab
0
ascorb ic acid
cd
bc
d
ab
c
abd
bc d
a
bc d
abc d
0
alfa-ketoglu tar ic acid
Alan ine
16
16
16
cd
ab
bc d
b
12
12
12
8
8
4
4
b
b
ab
b
a
b
ab
ab
4-Hydroxypr oli ne
a
b
ab
ab
16
12
12
8
8
8
4
4
4
ac
ac
a
c
c
c
c
b
b
bc
a
a
a
a
a
a
a
30 31