Metabolite profiling of mutants for genes induced by ...

MetaboliteprofilingofmutantsforgenesinducedbyosmoticstressingerminatedArabidopsisseeds

Author: Brend Kemperman

[email protected]

MSc student Plant Biotechnology

Reg: 910319427030

Supervisors: Anderson Silva

Wilco Ligterink

Laboratory of Plant Physiology

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TableofContentsAbstract ............................................................................................................................................................................................. 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


Discussion ........................................................................................................................................................................................ 16

Genotyping .................................................................................................................................................................................. 16

Re‐establishment of DT by treating with PEG .............................................................................................................................. 16

Metabolomics .............................................................................................................................................................................. 16


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

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AbstractDesiccation 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

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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).

LEALEA’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).

HSPHeat 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).

ABAABA 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.

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MetabolomicsAmong 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 describesthe dismutation/disproportionation of superoxideradicals (O2

‐) by SOD associated with PSI. The H2O2 is thencatalysed by tAPX and sAPX into harmless H2O. Themonodehydroascorbate radicals (MDA) produced by theAPX are converted, with reactions with Fd ormomodehydroascorbate reductase (MDHAR), to ascorbicacid (AsA). Dehydroascorbate reductase (DHAR) iscatalyzing the DHA to AsA.

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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).

At3g20810 transcribes JMJD5 and JMJ30 which are jumonji domain

containing 5 and jumonji c domain‐containing protein 30 respectively,

Figure 3: Schematic drawing from Xu, Chen et al. (2015). VIP1 is affected by MPK6 which upregulates MYB44 which increases drought tolerance and sensitivity to ABA.

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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

for germinated A. thaliana seeds. A general trend

of the lines upon treatment with PEG was found.

Figure 1 Developmental stages of germinating Arabidopsisseeds; Testa Rupture (TR), Radical Protrusion (RP), Root Hair (RH),Cotyledons Greening (GC)

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Materialandmethods

SelectionofhomozygousmutantlinesOne 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‐establishmentofDTMutant 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.

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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 Function Expression during germination*

Expression of abiotic stress in the root**

Expression of abiotic stress in the shoot**

Osmotic Salt Drought Osmotic Salt Drought

ATCC At1g12520 Copper Chaperone for SOD1

O ‐/O ‐/O ‐/ O ‐ ‐ ‐/O

At1g61430 At1g61430

Protein phosphorylation (S‐Locus lectin protein kinase family protein)

O ‐/O ‐ ‐ O O O

DAA1 At1g64110 Gene coding for an ATPase

‐ +++ O O +++ +++ O

ASN1 At3g47340

Glutamine‐dependent asparagine synthetase1

‐ +++ +++ O ‐ ‐‐‐ O

DI19 At1g56280 Drought induced gene family

‐ ‐/O O O + O/+ +

At1g07728 At1g07728 Putativeexocytosis

ND ND ND ND ND ND ND

ATVIP1 At3g01310 Phosphate metabolism

‐ O O O O O O

JMJD5 At3g20810 Reverse histone methylation

‐ O O O O O O

Re‐establishmentofDTbytreatingwithPEGA 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.

MetabolomicsMetabolites 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).

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Dehydrationcurve;naturalre‐establishmentofDTTo 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‐establishmentofDTA 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.

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Results

GenotypingPlants 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‐establishmentofDTbytreatingwithandwithoutPEGAs 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.

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.

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.

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MetabolomicsMutant 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.

13

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).

14

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 Metabolite Metabolite Metabolite Metabolite

Asparagine + Serine + Glycine 3tms O/+ Urea ‐/O 4‐Hydroxyproline

‐

Aspartic Acid + S‐Methyl‐L‐cysteine

+ Glycolic acid O/+ Beta alanine ‐/O Alfa‐Ketoglutaric Acid

‐

Benzoic Acid + Sucrose + Leucine 2tms O/+ Fumaric acid ‐/O Ethanolamine

‐

Beta‐D‐Methylfructofuranoside

+ Threitol + Monomethylphosphate 2tms

O/+ Glucopyranose

‐/O Fructose ‐

Isoleucine + Threonine 3tms

+ Nonanoic acid O/+ Glucose ‐/O Galactinol ‐

Lysine + Valine + Citric acid O Glucose‐6‐phosphate

‐/O Glyceric Acid ‐

Phosphoric Acid

+ Alanine O/+ Methionine O Glutamic acid

‐/O Malic Acid ‐

Proline + Ascorbic Acid

O/+ Phenylalanine O N‐acetylglutamic acid

‐/O Myo Inositol ‐

Pyroglutamic acid

+ GABA O/+ Tryptophan O Putrescine ‐/O Succinic Acid ‐

Raffinose + Glutamine O/+ Tyrosine 3tms O Pyruvic Acid ‐/O Xylose ‐

15

Naturalre‐establishmentofDTTo 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.

16

Discussion

GenotypingMost 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‐establishmentofDTbytreatingwithPEGLines 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.

MetabolomicsTo 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

17

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.

18

PossiblefutureprojectsThere 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.

AcknowledgementsFirst 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.

19

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23

Appendix

A. Protocol:DNAextraction1. 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)

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B. CompositionofHyponexMain Elements Concentration (mM/L) Trace Elements Concentration (μM/L)

NH4 1.7 Fe 21.0

K 4.131 Mn 3.4

Ca 1.97 Zn 4.7

Mg 1.239 B 14.0

NO3 4.141 Cu 6.9

SO4 3.411 MO 0.1

P 1.287

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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 Salt Drought

3 AT1G33970 1‐6h + 12‐24h ‐

O/+ O O ++ O O

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

25 AT5G03240 1‐6h + 12‐24h ‐

O O ‐/O + + O

29 AT5G57110 ‐ O O O O O O

30 AT1G53780 ‐ O O O + O/+ O

31 AT2G47850 ‐ O O O O O O

34 AT3G14590 ND ND ND ND ND ND ND

36 AT4G16160 1h + 3‐24h ‐

O O O O O O

38 AT5G04750 1h + 3‐24h ‐

+ O O + + O/+

39 AT5G52300 ‐ +++ +++ O +++ +++ O

40 AT1G30500 ‐ O O O + O/+ O

41 AT2G41070 ‐ O O O O O O

42 AT2G41710 ‐ O O O O O O

43 AT2G33830 1h + 3‐24h ‐

+ O O ++ O O

44 AT3G62090 ‐ O O O O O O

45 AT1G01240 ‐ + + O + O/+ +

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D. Protocol:Extractionofmetabolites(polar+apolarphase)forGC‐TOF‐MSmodified 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


‐ 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


‐ keep on ice ~10min

‐ add 200 μL H2O


‐ 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

27

E. Batchesgreenhouse

Table 1 Selected Mutant lines

Line Batch

T‐DNA (agi) Name/Code Function T‐DNA line NASC code

Genotyping

1 2 3

3 x AT1G33970 P‐loop containing nucleoside triphosphate hydrolases superfamily protein

response to bacterium/GTP binding SAIL_534_B01 N866206 Wild type

6 x x x AT2G01100 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 x x AT5G58720 smr (Small MutS Related) domain‐containing protein

Smr protein/MutS2 C‐terminal SALK_065495C N656593 Homozygous

12 x AT5G66400 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 Encodes a plant‐specific glucan phosphatase that contains a noncatalytic carbohydrate‐binding module as well as a dual specificity protein phosphatase domain.

SALK_102567C N663517 Homozygous

23 x x x AT3G53530 SODIUM POTASSIUM ROOT DEFECTIVE 3 metal ion transport SALK_000691C N673581 Homozygous

24 x x AT4G25800 Calmodulin‐binding protein SALK_040280C N655462 Heterozygous

25 x x AT5G03240 POLYUBIQUITIN 3 encodes ubiquitin that is attached to proteins destined for degradation


29 x x x AT5G57110 AUTOINHIBITED CA2+ ‐ATPASE SALK_057877C N662571 Homozygous

30 x x AT1G53780 peptidyl‐prolyl cis‐trans isomerases ATPase activity/protein catabolic process/nucleoside‐triphosphatase activity/peptidyl‐prolyl cis‐trans isomerase activity/nucleotide binding


31 x x AT2G47850 Zinc finger C‐x8‐C‐x5‐C‐x3‐H type family protein

zinc ion binding SALK_054523C N662511 Heterozygous

28

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 AT5G04750 F1F0‐ATPase inhibitor protein SALK_023158C N679430 Heterozygous

39 x x AT5G52300 73A HM Primers 926/927 Homozygous

40 x x AT1G30500 CBF/NF‐yA7 SALK_020063 Homozygous

41 x x AT2G41070 Bzip12/eel SALK_021965 Homozygous

42 x x AT2G41710 N658851 Homozygous

43 x x AT2G33830 dormancy/auxin associated family protein SALK_054451.53.45.x N554451 Homozygous

44 x x AT3G62090 PIL2 (PHYTOCHROME INTERACTING FACTOR 3‐LIKE 2); protein binding / transcription factor

SALK_090239C N661012 Homozygous

45 x x AT1G01240 unknown protein SALK_104275C N653407 Homozygous

29

F. Re‐establishmentofDTwithnaturaldrying

Figure 8: Re‐establishment of desiccation tolerance innatural drying conditions for the developmental stages RPand RH. After 72 hour drying seeds were put for 1 week onthe Copenhagen table. Seeds which germinated werecounted and are shown as a survival percentage.

30

G. Metabolomicsgraphs

c

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Xylo se

abc c abc abc abc a ab bc abc abc

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Urea

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Valine

abc c a abc ab abc ab abc abc bc

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Tyrosine 3tms

ab

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Th reonine 3tms

abc de bc

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Threito l

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Sucrose

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Su ccinic acid

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S-M eth yl-L-cysteine

ab

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Ser ine

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Raffinose

dabcd

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Pyru vic acid

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Pyro glu tamic acid

bde df bc def

bcde c bc e c def

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Putrescine

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Proline

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Pho sphoric acid

bcab

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aba

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Ph enylalan ine

abc bc ab aabc

abc abc abc a c

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No nanoic acid

bc

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N-Acetylglu tamic acid

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Myo ino sitol

a c bc bcab

abcabc

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Mono methylpho sp hate (2TM S)ab

ab ab bb

aba ab

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Methio nin e

b

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M al ic acid

abc

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Lysine

a bc abc a bc a c

abbc

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Leucine 2tms

ab

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isoleu cine

acde

abd

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cea

cab bde

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Glycolic acid

abc abcd abd b c d a bc d ab

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G lycin e (3tms)

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Glyceric acid

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Glutamine

bcdabc

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Glu tamic acid

cabc

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G lucose-6-pho sphatebc

abc bc abc

abcbc

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Glucose

abab

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Glu copyrano se

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Galactinol

a deab bc de abc d abc e c de

abcdeab

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GABA

bc dab

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abbc

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Fu maric acid

c d

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Fructose

c d

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Ethano lamin e

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Citric acid

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beta-D-Methyl fr uctofuranoside

cabc

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Beta alan ine

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Ben zo ic acida b a b a b a b a b

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aspartic acid

ab c da

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Asp ar agin e

abc abd bc d a bc d

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ascorb ic acid

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alfa-ketoglu tar ic acidc

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4-Hydroxypr olineab

b a b ab ab a bab

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Alan ine

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Tryp to phan

Metabolite profiling of mutants for genes induced by ...

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