UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE

UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE FACULTY OF HORTICULTURE OANA CIUZAN ROLE OF THE GLYCINE-RICH RNA-BINDING PROTEINS IN PLANT EARLY DEVELOPMENT AND ABIOTIC STRESS RESPONSE ABSTRACT OF PhD THESIS SCIENTIFIC COORDINATOR Prof. Dr. DORU PAMFIL Cluj-Napoca 2013

TABLE OF CONTENTS INTRODUCTION, AIM AND OBJECTIVES... 3 MATERIAL AND METHODS... 5 BIOLOGICAL MATERIAL... 6 METHODS... 9 RESULTS... 11 INVOLVMENT OF THE GLYCINE-RICH RNA-BINDING PROTEINS IN EARLY DEVELOPMENT... 11 Analysis of the phenotype of the AtGRP7 knock-out mutant... 12 Analysis of the phenotype of the AtGRP2 knock-out mutant... 13 Analysis of the phenotype of the Arabidopsis thaliana abi mutants... 17 Summary of results... 21 INVOLVMENT OF THE GLYCINE-RICH RNA-BINDING PROTEINS IN ABIOTIC STRESS. 22 Germination capacity of Arabidopsis thaliana mutant seeds on control culture media... 22 Salt tolerance of the Arabidopsis thaliana mutants... 23 Drought tolerance of the Arabidopsis thaliana mutants... 25 Germination capacity of the Arabidopsis thaliana mutants under abscisic acid stress... 26 Oxidative stress tolerance of the Arabidopsis thaliana mutants... 27 Influence of ethylene on the germination capacity of the Arabidopsis thaliana mutants... 30 Influence of sodium nitroprusside (SNP) on the germination capacity of the Arabidopsis thaliana mutants... 32 The expression of GRP2 and GRP7 genes in control and salt treated wild type Col0 and abi3 mutant seeds... 34 The expression of ABI3 gene in control and salt treated wild type Col0, grp2 and grp7 mutant seeds... 37 Summary of results... 39 CONCLUSIONS... 40 REFERENCES... 41 2

INTRODUCTION, AIM AND OBJECTIVES The crop yield and production is more and more affected by several factors such as abiotic and biotic stresses. As the population is increasing day by day, being estimated to reach 9 billion by the year 2050, it is of vital importance to find solutions in order to remove all causes leading to lower yields of cereals and crop plants. The main causes that lead to almost 70% loss of the crop plants are produced by abiotic stressors such as salinity, drought, and very high or very low temperatures. These have also increased in the last decade due to the global climate change problems. As living organisms are incapable of controlling the environmental factors, the majority developed mechanisms in order to avoid (organisms with mobility) or to adapt (organisms lacking mobility) to these types of stressors. In the case of plants, the adaptation strategies developed undergo biochemical, physiological and molecular processes. Gene activation to environmental stimuli plays an important role in plant adaptation to adverse conditions and favors the appearance of specific proteins. The most researches regarding the adaptability of plants to several abiotic stress conditions were focused on studying the involvement of genes and proteins in the plant genome using the model plant Arabidopsis thaliana. This plant was chosen as a model due to some advantages like small genome size (only five chromosomes) or rapid life cycle (only eight weeks from seed germination to seed production). The most important advantage also from the economical point of view is the similarity of Arabidopsis thaliana to other higher species including valuable crop plants (rice, maize). The glycine-rich proteins (GRP) are associated with multiple independent physiological processes because of their diverse sub-cellular localization. The expression patterns of the GRP genes are both diverse and highly tissue specific. A subset of the GRP genes encodes proteins that bind nucleic acids. The Arabidopsis thaliana genome encodes eight glycine-rich RNA-binding proteins (GR-RBP1 to GR-RBP8). These proteins are characterized by containing an RNA recognition motif (RRM) or an RNA/DNA-binding cold shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus. They are thought to play a prominent role in germination and seedling development of Arabidopsis thaliana under freezing conditions (temperature lower than 0 degrees). However, their normal developmental roles have not been studied in detail. 3

The aim of this PhD thesis was to investigate the role of the glycine-rich proteins GRP2 and GRP7 in the germination processes and abiotic stress tolerance using the model plant Arabidopsis thaliana, in order to find possible solutions to the problems of crop loss caused by external factors such as salinity, drought and temperature. The main objectives were: 1. Obtaining the Arabidopsis thaliana knock-out lines of interest and assessing their individual phenotypes in detail. 2. Assessment of the role of GRP proteins in abiotic stress conditions and ABA-mediated pathways in the germination process. 3. Evaluation of the gene expression levels for the genes of interest. Thesis structure: the thesis is structured into two main parts, namely the state of the art (I) and original contributions (II) including the experimental conditions, measurements, results and discussions. The first part includes the first chapter of the thesis focusing on the characterization of the most important abiotic stress factors, the model plant Arabidopsis thaliana and the description of the glycine rich proteins and their involvement in several biological and biochemical activities. The second part includes the other four chapters, namely the presentation of the used biological material and the description of the techniques and protocols. Two chapters regarding the results obtained in the studies of the involvement of the glycine-rich RNA-binding proteins in plant early development and abiotic stress response. And the last chapter related to the discussion of the results and their comparison to other related studies and the further perspectives of this studies. 4

Outlook: As future plans it may be useful to keep studying the relative gene expression under other abiotic stress conditions and also to extrapolate the research from the germination process to other important processes in the development of plants. Part of this PhD thesis was achieved at the Department of Molecular Cell Physiology, Bielefeld, Germany and at the University of the West of England, Bristol, UK in collaboration with our University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania. The financial support for these experiments was provided by the University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania and a Deutsche Bundesstiftung Umwelt grant. MATERIAL AND METHODS The plant glycine-rich proteins (GRP) belong to a superfamily that is characterized by a high content of glycine (from 20% up to as much as 70%) generally found in the form of (Gly)n-X repeats. The first GRP gene was identified by Condit and Meagher in 1986 in the process of attempting to isolate petunia oncogenes using Epstein-Barr virus (Carol and Richard, 1986). After the discovery of grp-1, several other GRP genes were identified. However it became clear that there is significant heterogeneity in the superfamily; and the highly variable expression pattern and subcellular localization of these proteins suggested that they are involved in several and distinct physiological processes. A subset of GRP proteins known as class IV contains RNA-binding domains suggesting an involvement in posttranscriptional processes in gene regulation. Increasing evidence suggests that the class IV GRP proteins are of key importance in the ability to respond to severe environmental stress. There are thought to be four classes of GRPs. Class I GRPs include a signal peptide that is followed by a region high in glycine-content, consisting of (GlyGlyX)n repeats. Most of the GRPs included in this class are associated with a structural function as a result of their cell wall localization (Cassab, 1998). Class II GRPs are proteins with a glycine-rich region followed by a C-terminal cysteine-rich region. They are characterized by the presence of (GlyGlyXXXGlyGly)n repeats and can also possess a signal peptide. The class III GRPs are proteins with the lowest glycine content in comparison to the other groups. These proteins are characterized by a high rate of structural diversity and by the presence of (GlyXGlyX) repeats. They can also carry a signal peptide often at the amino 5

terminus. Class IV consists of the RNA binding GRPs which contain the widely studied and widespread RNA recognition motif (RRM) or a cold shock domain (CSD). The CSD has the ability to bind both DNA and RNA, and is present in a diverse range of proteins including small, prokaryotic cold shock proteins and eukaryotic transcription factors/mrna masking proteins known as the Y-box proteins (Sommerville and Ladomery, 1996). Some GRPs also possess CCHC zinc-fingers (Kar et al., 2012) )(Figure 1). The genes of interest studied during this PhD thesis belong to the fourth class of glycine-rich proteins. Fig.1. Schematic representation of the most important classes of plant glycine-rich proteins (GRPs) After (Bocca et al., 2005) BIOLOGICAL MATERIAL The studies examined wild-type Arabidopsis thaliana, five mutant knock-out lines and one over expression line. The Col0 strain was selected as wild-type, as it is the most widely used. Three of the mutants studied labeled abi3 (N6131), abi4 (N3836) and abi5 (N8105) were obtained through EMS (Ethyl methansulfonate) mutagenesis (provided by the University of the West of England). These abi mutants are insensitive to very high concentration of the plant hormone abscisic acid (ABA) (Koornneef et al., 1984). The other plants used in this study are labeled grp2 (SALK_048476C bought from the Nottingham Arabidopsis Stock Center NASC), grp7-1 and WS7ox (obtained from 6

Prof. Dr. Dorothee Staiger, University of Bielefeld, Germany). These mutants were obtained through the T-DNA insertion technique and are verified knock-out lines for AtGRP2 and AtGRP7. The last mutant taken into consideration was a line that over expressed the GRP7 protein and was labeled Ws7ox, also obtained from the group of Prof. Dorothee Staiger (Figure 2). The abi mutants were obtained from the wild-type plants through the use of the EMS mutagenesis. This type of mutagenesis produces random point mutations in the genetic material mostly through substitution of nucleotides. EMS most frequently interacts with guanine changing the original base pair G:C into A:T producing a transition mutation. The weak point of this mutagenesis process is the fact that there is no certain knowledge of were the mutation point is located on the gene unlike in the use of the T-DNA insertion technique which generates mutation at a known genetic locus. The abi3 mutant is resistant to the phytohormone abscisic acid presenting a strong allele and reduced sensitivity to ABA inhibition of germination. The germination of the seeds is hardly inhibited by 1 mm ABA and the seedlings show a normal growth. Produced seeds fail to denaturate chlorophyll during the maturation process keeping seeds green until maturity showing also lack of seed dormancy. Other features presented by the seeds produced by this type of mutant are: reduced desiccation tolerance; reduced longevity; loss of viability within five weeks of storage at room temperature. The ABI3 gene encodes a protein presenting regions of homology to the maize VP1 product (Ooms et al., 1993). The abi4 mutant is abscisic acid resistant and shows reduced sensitivity to ABA inhibition of germination. The generated seeds germinate in the presence of 5 µm ABA and seed dormancy response appears to be affected. The plants also show reduced sensitivity to the inhibitory effects of sugars on early seedling development stage. The seeds germinate and form seedlings with expanded cotyledons and true leaves on large concentrations of glucose (0.3 M), sucrose (0.3 M), mannose (2 mm) or sorbitol (0.4 M). These substances seem to have an inhibitory effect during these processes in wild type plants. The seedlings furthermore exhibit decreased sensitivity to the presence of the gibberellic acid biosynthesis inhibitor paclobutrazol (35 mg/l). The seedlings lack trichomes on stems and leaves but the presence of some trichomes at the leaf margins can be noticed (Adie et al., 2007). The abi5 mutant is abscisic acid resistant and presents reduced sensitivity to ABA inhibition of germination. The seeds germinate in the presence of 3 µm ABA. The seedlings do not exhibit compromised water relations, have normal stomatal regulation and do not display a wilty phenotype. 7

The seed dormancy is similar to wild type. This type of mutants manifest reduced sensitivity to salt and osmotic stress during germination (Finkelstein and Lynch, 2000). The AtGRP7 knock-out mutants are less-sensitive to osmotic stress as shown by the germination assays, and more sensitive to cold stress based on freezing tolerance analyses, than the wild type plants. These mutants also exhibit altered stomatal responses. ABA-induced stomatal aperture is decreased, cold-induced stomatal aperture is increased, and NaCl and mannitol-induced stomatal aperture is also increased in this type of plants in comparison to the wild type. (Kim et al., 2008). The plantlets also show late flowering in both short and long day conditions (Streitner et al., 2008) and increased susceptibility to Pseudomonas syringae infection. The reduced callose deposition suggests defect in innate immune response in this type of plants (Fu et al., 2007). The AtGRP2 knock-out mutants are more sensitive to salt stress but do not show any sensitivity to this type of stress during the growth stage. The germination rate of the seeds resulted from this type of mutant was severely affected by cold temperatures in contrast to the wild type plants and also the seedling development was influenced. The seed germination rate of these knock-out mutants was not altered by the use of abscisic acid or glucose in the culture media (Kim et al., 2007b). The flowering period is earlier then in their wild type counterparts but the plants show some defects in anther and seed development (Nakaminami et al., 2009). Fig.2. Representative images of wild type and knock-out strains after 4 weeks from germination. (A) Wild-type Arabidopsis thaliana, Col0 strain; (B) grp2 knockout; (C) grp7-1 knockout; 8

Fig.2. Representative images of wild type and knock-out strains after 4 weeks from germination. (D) abi3 knockout; (E) abi4 knockout; (F) abi5 knockout. There are very little results relating to the phenotypic appearance of the mutant taken into this study. The Ws7ox is a gain of function mutant primarily considered as being in the Wassilewskija background (the T-DNA insertion was induced in wild type plants belonging to the Wassilewskija ecotype). After some tests run by the group from Germany they concluded that the mutation is actually in the Columbia background as the plants had a Col0 like phenotype. The gain-of-function mutants are obtained through the same technique as the loss of function mutants, the only difference being that the expression of the T-DNA fragment is increased via the 35S enhancer of the cauliflower mosaic virus (CaMV). METHODS For the phenotype analysis Arabidopsis thaliana mutants homozygous for the mutation of interest were grown for eight weeks in pots in the green house under controlled climatic conditions (22 C, 70 % moisture and 16/8 h light/dark cycle). After the development of the first two cotyledons the plants were monitored each day. Quantitative traits like: number of leaves, rosette diameter, bolting day, number of flowers on the primary inflorescence, length of the inflorescence, number of internodes on the primary inflorescence, distance between the internodes and the ratio between the length and width of the 6th leaf were assessed. In order to test the Arabidopsis thaliana mutant lines for the glycine-rich protein 2 (GRP2) and glycine-rich protein 7 (GRP7) the polymerase chain reaction (PCR) technique was used. The DNA of 9

the two tested mutant lines labeled grp2 and grp7-1 was extracted using two protocols one provided by the group from Germany and the other obtained from the Department of Defense Genetics, KVL University, Denmark. The DNA isolated was quantified using the NanoDrop device which also provides information regarding the quality of the obtained DNA samples. The amplification of the DNA was done using two ready to use master mixes bought from Bioline and from Rovalab. The program used for the PCR amplification process was as follows: 1 minute at 95 C initialization step; 35 cycles at the following temperature profile: 15 seconds at 95 C denaturation, 15 seconds at 48 C primer annealing, 0 seconds at 72 C elongation and 10 minutes at 72 C final elongation. The resulted amplification products were migrated electrophoretic in a 1.4 % agarose gel and the visualization was performed under UV light. The images were taken with the help of the Vision Works LS program. For the identification of the AtGRP7 protein in the lines of interest the western blot technique was used. Western blot is often used in scientific research in order to separate and identify proteins. In this technique a protein mixture is separated according to molecular weight through gel electrophoresis. The products are then transferred to a membrane producing a band for each protein of interest. The membrane is then incubated with specific antibodies for the protein of interest. The unbound antibody is washed away leaving only the antibody bound to the protein of interest. Bound antibodies are then detected through a film development or through chemiluminescence. Since antibodies bind only to proteins of interest just one band should become visible. Band thickness corresponds to the amount of protein present in the sample processed and the use of a standard marker like bovine serum albumin (BSA) can help identify the correspondent protein of interest (Bers and Garfin, 1985). In order to assess the role of the glycine-rich proteins in the different functions in plant and in their adaptability to several abiotic stress factors we focused on observing the germination rate of the seeds collected from the wild type Col0 and also from the six mutants taken into consideration (abi3, abi4, abi5, grp7-1, grp2 and Ws7ox). Fifty seeds were used for each germination test in three independent replicates. The experiment was repeated for three times in order to certify the obtained results. Even though the UPOV legislation regarding seed germination tests implies the use of batches containing 100 seeds per replicate the motivation of using batches of only 30 or 50 seeds per replicate was that our study interest was focused more on the evaluation of the impact of the glycine-rich 10

proteins in the germination process than the evaluation of the seed germination regarding the culture process. We used different substances that cause a variety of abiotic stresses in plants such as: NaCl (salt stress), mannitol (drought), ABA (germination inhibitor), N,N -dimethyl-4,4 -bipyridinium dichloride/ paraquat (photosynthesis inhibitor producing oxidative stress), 4,5,6,7-tetrachloro-2',4',5',7'- tetraiodofluorescein/ rose bengal (oxidative stress), 1-aminocyclopropane-1-carboxylic acid (ACC) (roll in biosynthesis of the plant hormone ethylene ) and sodium nitroprusside (SNP) (to enhance seed germination). For the evaluation of the phenotype related quantitative characters the Student s t test was used. The statistic interpretation was generated using GraphPad Prism 6.01 trial version a commercial scientific 2D graphing and statistics software published by GraphPad Software, Inc., California. The statistical evaluation regarding the germination rate was made using the analysis of variance (ANOVA) test. The statistic interpretation was developed using SPSS version 18 (SPSS Inc. Chicago, IL, USA). RESULTS INVOLVMENT OF THE GLYCINE-RICH RNA-BINDING PROTEINS IN EARLY DEVELOPMENT From the several Arabidopsis mutant lines used during the study only two mutants grp2 and grp7-1 were created through the use of a T-DNA insert which was able to be detected using the primer combination mentioned in the materials and methods chapter. In case of the AtGRP7 knock-out mutants some of the amplified DNA generated a single product of 500 bp in length present in the electrophoresis gel proving the fact that the plants from which the DNA was extracted were homozygous for the T-DNA insert. In other cases the amplified DNA generated two products, one between 900 and 1100 bp in length and the other one of 410bp long establishing the fact that the plants were heterozygous. Only plants which generated single products in 11

the electrophoresis gel were further analyzed. In case of the AtGRP2 plants the whole DNA extracted generated only a product of 510 bp showing the fact that all plants were homozygous for the T-DNA insert. As the plants resulted from the seeds of the AtGRP7 knock-out (grp7-1) and overexpressor (Ws7ox) were already tested for their homozigosity for the T-DNA insert the study using the western blot technique was focused on assessing the level of GRP7 in the other Arabidopsis thaliana mutants (abi3, abi4, abi5 and grp2) take under observation in comparison to the level of protein present in the wild type Col0 plants. The results obtained in regard to the level of GRP7 protein present in the abi3 mutant plants compared to that of the wild type plants were very similar. The GRP7 protein seems not to be affected by the initial mutation encountered in the phenotype of the abi3 plants. The level of GRP7 protein encountered in the abi4 and abi5 mutant plants was also similar to that present in the wild type Col0 plants. Even though in two abi4 and three abi5 mutant plants there was a stronger signal observed the difference was not significant. The knock-out mutants for AtGRP2 showed also no significant differences in comparison to the wild type Col0 in regard to the level of GRP7 protein extracted. The GRP2 knock-out mutation seems not to interact with the level of this other protein even though they are part of the same family of glycine-rich proteins. Analysis of the phenotype of the AtGRP7 knock-out mutant The results obtained regarding the bolting day of the plants taken into consideration revealed the fact that the grp7-1 mutant flowers significantly later than the wild type Col0. Most of the wild type plants started to flower between the 25 th and the 28 th day after germination in contrast to the grp7-1 mutant plants which only started flowering after the 28 th day from germination start of the seeds (Figure 3). A statistically significant difference could be observed also between the number of leaves of the two plant groups studied. The wild type Col0 showed in the fourth week from germination start a number of leaves between 16 and 21 compared to the mutant grp7-1 where the plants showed a leaf number between 10 and 17 during the same period (Figure 4). 12

In case of the other quantitative features assessed there were no significant differences observed between the ecotype and the mutant. Similar results were obtained by Streitner and her coworkers (2008) who concluded the fact that the AtGRP7 knock-out mutants were flowering later then their corresponding wild type plant under long day and also short day conditions. *** Col0 grp7-1 Col0 grp7-1 Fig.3. Bolting day of the Arabidopsis thaliana mutant grp7-1 compared to the wild type Col0. The statistical significance is shown for a P value <0.05. Fig.4. Number of leaves of the Arabidopsis thaliana mutant grp7-1 compared to the wild type Col0 after four weeks from germination start. The statistical significance is shown for a P value <0.05. Analysis of the phenotype of the AtGRP2 knock-out mutant The observations made regarding the bolting day of the two plant groups taken into consideration pointed out that there is a significant difference between the wild type Col and the mutant labeled grp2. The wild type plants taken under observation flowered earlier compared to the mutant grp2 where the bolting occurred between the 26 th and the 29 th day after germination start (Figure 5). 13

Statistically significant differences were also observed regarding the number of leaves. It could be observed that the grp2 mutant presented a higher number of leaves with values between 16 and 31 compared to the wild type Col0 that only showed a number of leaves between 13 and 25 (Figure 6). Bolting day Number of leaves 32 30 30 ** 28 20 26 10 24 22 Col0 grp2 0 Col0 grp2 Arabidopsis genotype Arabidopsis genotype Fig.5. Bolting day of the Arabidopsis thaliana mutant grp2 compared to the wild type Col0. The statistical significance is shown for a P value <0.05. Fig.6. Number of leaves of the Arabidopsis thaliana mutant grp2 compared to the wild type Col0 after four weeks from germination start. The statistical significance is shown for a P value <0.05. The measurements made on the length of the primary inflorescence indicate statistically significant dissimilarities between the wild type Col0 and the grp2mutant plants. The wild type Col0 showed a length of the primary inflorescence that varied among the values of 0.1 cm and 2.3 cm in contrast to those of the grp2 mutant were the length varied between the values of 0.1 cm and 17 cm (Figure 7). Regarding the number of flowers present on the primary inflorescence the results obtained were also statistically significant. Whereas in the wild type group only a number of flowers between 1 and 5 were present, in the group of the grp2 mutants a significant larger number of flowers were observed which ranged between 1 and 10 flowers/ primary inflorescence (Figure 8). 14

*** *** Col0 grp2 Col0 grp2 Fig.7. Length of the primary inflorescence of the Arabidopsis thaliana mutant grp2 in comparison to the wild type Col0. Statistically significance shown for a P value< 0.05. Fig.8. The number of flowers present on the primary inflorescence in case of the Arabidopsis thaliana mutant grp2 compared to the wild type Col0. Statistically significance shown for a P value0.05. The counting of the internodes on the primary inflorescence revealed also significant differences between the wild type Col0 and the grp2 mutant. The mutants showed a larger number of internodes present on the primary inflorescence in contrast to the wild type Col0. The number of internodes observed in the grp2 plant group ranged between 0 and 4 (Figure 9). The difference was maintained also when the distance between the internodes was taken under consideration. Here the values in the grp2 plant group varied among 0.1 cm and 3.1 cm (Figure 10). 15

No. of internodes *** cm *** Col0 grp2 Col0 grp2 Fig.9. Comparison of the wild type Col0 and Arabidopsis thaliana mutant grp2 regarding the number of internodes present on the primary inflorescence. Statistical significance shown for a P value < 0.05. Fig.10. Comparisons of the distance between the internodes in case of the wild type Col0 and the Arabidopsis thaliana mutant grp2. Statistical significance shown for a P value< 0.05. These results are contradictory to those obtained by Fusaro and his team (2007) which showed an early flowering time of the AtGRP2 mutants. In regard to the stamen number and the seed development our results also contradict these group outcomes which report an altered stamen number and affected seed development (Fusaro and Sachetto-Martins, 2007). The stamen number was similar to that of the wild type and also the seeds showed no developmental problem. 16

Analysis of the phenotype of the Arabidopsis thaliana abi mutants As far as the analysis of the three ABI mutants bolting day in comparison to the wild type Col0 the results obtained pointed out that all of the mutants flower much earlier resulting in a significant difference between the plant groups taken into consideration. The abi5 were the first ones that started bolting during the 18 th day from the germination start, followed by the abi3 plants which started bolting during the 20 th day and the abi4 mutants which started bolting during the 23 th day from germination start (Figures 11, 12 and 13). Col0 abi3 Col0 abi4 Fig.11. Bolting day of the Arabidopsis thaliana mutant abi3 compared to the wild type Col0. The statistical significance is shown for a P value <0.05. Fig.12. Bolting day of the Arabidopsis thaliana mutant abi4 compared to the wild type Col0. The statistical significance is shown for a P value <0.05. Fig.13. Bolting day of the Arabidopsis thaliana mutant abi5 compared to the wild type Col0. The statistical significance is shown for a P value <0.05. 17

The comparison of the length of the primary inflorescence of the several ABI mutants with that of the wild type Col0 showed also significant differences between these plant groups. All three ABI mutants taken into the study developed a much longer inflorescence in contrast to the wild type plants. The length of the primary inflorescence varied between 0.1 cm and 7 cm for the abi3 mutants, 0.1 cm and 7.5 cm for the abi4 mutants and 1.5 cm and 18.2 cm for the abi5 mutant (Figures 14, 15 and 16). * * Col0 abi3 Col0 abi4 Fig.14. Length of the primary inflorescence of the Arabidopsis thaliana mutant abi3 in comparison to the wild type Col0. Statistically significance shown for a P value< 0.05. Fig.15. Length of the primary inflorescence of the Arabidopsis thaliana mutant abi4 in comparison to the wild type Col0. Statistically significance shown for a P value< 0.05. *** Col0 abi5 Fig.16. Length of the primary inflorescence of the Arabidopsis thaliana mutant abi5 in comparison to the wild type Col0. Statistically significance shown for a P value< 0.05. 18

Also the number of flowers present on the primary inflorescence determined significant differences in the development of the ABI mutants compared to the wild type Col0. The number of flowers developed on the primary inflorescence varied between one and seven in case of the abi3 mutant, one and 11 for the abi4 mutants and one and nine in case of the abi5 mutant (Figures 17, 18 and 19). *** *** Col0 abi3 Col0 abi4 Fig.17. The number of flowers present on the primary inflorescence in case of the Arabidopsis thaliana mutant abi3 compared to the wild type Col0. Statistically significance shown for a P value0.05. Fig.18. The number of flowers present on the primary inflorescence in case of the Arabidopsis thaliana mutant abi4 compared to the wild type Col0. Statistically significance shown for a P value0.05. Number of flowers on the primary inflorescence 8 *** 6 4 2 0 Col0 abi5 Arabidopsis genotype Fig.19. The number of flowers present on the primary inflorescence in case of the Arabidopsis thaliana mutant abi5 compared to the wild type Col0. Statistically significance shown for a P value0.05. 19

The number of internodes present on the primary inflorescence also determined significant differences between the three ABI mutants taken into the study and the wild type Col0 plants. The number of internodes ranged between zero and four for the abi3 mutant, zero and five for the abi4 mutant and zero and four for the abi5 mutant (Figures 20, 21 and 22). ** *** Fig.20. Comparison of the wild type Col0 and Arabidopsis thaliana mutant abi3 regarding the number of internodes present on the primary inflorescence. Statistical significance shown for a P value < 0.05. Fig.21. Comparison of the wild type Col0 and Arabidopsis thaliana mutant abi4 regarding the number of internodes present on the primary inflorescence. Statistical significance shown for a P value < 0.05. *** No.of internodes Col0 abi5 Fig.22. Comparison of the wild type Col0 and Arabidopsis thaliana mutant abi5 regarding the number of internodes present on the primary inflorescence. Statistical significance shown for a P value < 0.05. 20

Regarding the ABI mutants the results obtained through our research were in concordance to those obtained by Koornneef and his coworkers (1984). The seeds had reduced dormancy, were green until the maturity and the plantlets did not show a wilty phenotype. The same results were obtained also by Ooms (1993). Still some contradictions were detected in regard to the appearance of the mutants. Our research showed that the abi3 and abi5 are less developed in regard to the wild type and abi4 which displayed a normal growth. Finkelstein and Lynch (2000) concluded in their research that the seeds of the abi5 mutant exhibit similar seed dormancy with the wild type. These results are also contradictory to our outcomes which showed that the wild-type Col0 exhibited difficulties in germination when the seeds were not before vernalized. Take together we can conclude that abscisic acid is a very important hormone in the healthy development of plant species. Summary of results The analysis of phenotypes revealed significant differences in the development of the knockout lines compared to wild-type. We observed that the ABI mutants have a relatively faster development and confirmed their typical early flowering period and seed production. The GRP knockouts displayed slower development suggesting that these glycine-rich RNA-binding proteins are involved in early developmental processes. However the phenotypes of the grp2 and grp7-1 knockouts were not identical; grp2 knock-outs had more leaves, longer inflorescence, more flowers and earlier flowering compared to grp7-1 suggesting that the proteins have overlapping but distinct, nonredundant roles in development. Future research will consider in more detail the developmental roles of these proteins, and to determine how their developmental roles intersect with their involvement in biotic and abiotic stress adaptation. 21

INVOLVMENT OF THE GLYCINE-RICH RNA-BINDING PROTEINS IN ABIOTIC STRESS Several studies were undertaken looking at the response of the glycine-rich proteins to abiotic stress conditions in the last years. Many of them were focused on determining the resistance of some Arabidopsis thaliana mutants created to overexpress or not express proteins from this family. The research was focused more on the germination and development of these mutants in comparison to the wild type when submitted to three of the most commune abiotic stressors like salt, drought and temperature (Cao et al., 2006); (Kim et al., 2007a). Another interest was the response of these mutants in regard to the plant hormone abscisic acid which is known to be involved in several physiological processes like inhibition of germination or the determination of seed dormancy under abiotic stress as an adaptive response (Leung and Giraudat, 1998). Other researches using Arabidopsis thaliana mutants were focused on their response to oxidative stresses. Some of them had even as main focus the response of germination to paraquat (Zhu et al., 2010) or rose bengal (Kurepa et al., 1998) but none of them used the mutants involved into our study. The main purpose of this research was to find out more regarding the germination capacity of the several Arabidopsis thaliana mutants (grp2, grp7-1, Ws7ox, abi3, abi4 and abi5) taken under observation in relation to abiotic stress. Another point was to determine if the expression of GRP2 and GRP7 undergoes changes during the imbibition process in wild type and abi3 mutant plants and also if ABI3 expression changes during the same process in wild type, grp2 and grp7-1 mutant plants. These studies being able to help in resolving the question related to ABA signaling. Germination capacity of Arabidopsis thaliana mutant seeds on control culture media In order to evaluate the germination capacity of the several mutant seeds taken into the study in comparison to those obtained from the wild type Col0 batches of 30 seeds were set to germinate on ½ Murashige-Skoog (MS) culture media without sugar. The seed were counted for seven days. 22

There were no significant differences observed regarding the germination rate of the several mutants compared to the wild type Col0. By the end of the experiment all seeds germinated to full percentage except the ones obtained from the overexpressor (WS7ox) were only 80% of them managed to germinate (Figure 23). Fig. 23. Seed germination capacity on control media. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. Salt tolerance of the Arabidopsis thaliana mutants To test the response to salt stress of the several Arabidopsis thaliana mutants (abi3, abi4, abi5, grp7-1, grp2 and Ws7ox) and wild type Col0 taken into the study the germination capacity of the seeds on ½ MS culture media supplemented with three different concentrations of NaCl ranging between 100 mm and 200 mm was observed. Even though the effects of salinity on seed germination were observed from the smallest concentration of NaCl (100 mm) the most obvious differences were observed as expected at the 23

highest one. The analysis of the germination capacity on ½ MS culture media supplemented with 200 mm NaCl revealed the fact that the germination of the ABI mutants is not affected the seeds being able to germinate to a full percentage in comparison to the wild type Col0 whose seed germination potential started to be affected (Figure 24). The grp2 seed germination capacity was not very affected, 80% of the tested seeds being able to fully germinate by day seven from the start of the experiment. The grp7-1 mutant and the Ws7ox mutant germination potential was in contrast severely affected by this concentration of NaCl, only 20% respectively 10% of the seeds being able to fully germinate by day seven. The results obtained during this thesis confirm the affirmation done by Cao and his group (2006) in regard to the germination capacity of the AtGRP7 knock-out mutant and also with those suggested by Kim and his coworkers (2008) relating to the response of the GRP7 overexpressor to osmotic stress. Still there are contradictions with Kim s provided outcomes regarding the knock-out mutant but this may be due to the fact that they worked with a different mutant line than that used during our research and also due to the different concentrations of NaCl tested. Fig.24. Seed germination capacity on 200 mm NaCl. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 200 mm NaCl. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. 24

Drought tolerance of the Arabidopsis thaliana mutants The response of the Arabidopsis thaliana mutants and wild type Col0 to drought stress was tested by observing the germination capacity of the seeds on ½ MS culture media supplemented with three different concentrations of mannitol varying among 100 mm and 300 mm. The higher concentration of mannitol used in this type of culture media influenced in a negative way the germination capacity of the wild type Col0 seeds resulting in a significant lower germination rate in contrast to that of the mutants abi3 and abi5. Only 50% of the grp7-1 mutant seeds were able to germinate by day seven. The seeds of the Ws7ox mutant were severely affected resulting in a germination rate lower than 20%. The grp2 mutants were in contrast not very affected by the high concentration of mannitol used being able to germinate up to 80%. The ABI mutant seed germination rate was not at all affected by the use of mannitol (Figure 25). Fig.25. Seed germination capacity on 300 mm mannitol. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 300 mm mannitol. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. 25

Germination capacity of the Arabidopsis thaliana mutants under abscisic acid stress In order to test the germination capacity under abscisic acid stress of the Arabidopsis mutants and wild type taken into the study seeds were set to germinate on ½ MS culture media supplemented with three different concentrations of abscisic acid. Three of the Arabidopsis mutants (abi3, abi4 and abi5) tested are known to be abscisic acid insensitive so their germination capacity should not be affected by any of the concentrations used in the experiment. The concentrations used in the experiment, ranged between 0.5 µm and 1 µm ABA (abscisic acid). The results obtained pointed out a significant higher germination rate of the ABI mutants in comparison to the wild type Col0 and the AtGRP7 knock-out mutant and overexpressor. The grp7-1 mutant seeds started to germinate only by day four and by day seven only 40% of these completed the germination. The Ws7ox mutant seeds were even more affected by the concentration of ABA used in this experiment even though the germination started also by day four, by day seven less than 15% of them fully germinated. A significant difference between the germination rate of the grp2 mutant and the grp7-1 was also observed. More than 80% of the seeds of the grp2 mutant were fully germinated by day seven (Figure 26). The correlation between the results obtained by Karlson and Imai, 2003, Nakaminami et al., 2009, Fusaro et al., 2007 and Park et al., 2009 in regard to the response of the GRP7 protein to temperature and our results obtained regarding the other two major abiotic stressors namely salt and drought conclude the fact that there is a positive effect of this protein in response to low temperatures in plants and a negative effect in the sensitivity matter related to the response to salt and drought. When correlating the results obtained through our research with the ones regarding the temperature tolerance of the AtGRP2 mutants done by Kim and his coworkers (2008), the conclusion obtained was similar to that of the AtGRP7. There seems to be a negative influence of the GRP2 protein in seed germination capacity under salt, drought and ABA stress in contrast to the positive one related to the cold temperature influence. 26

Fig.26. Seed germination capacity on 1µM ABA. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 1 µm ABA. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. Oxidative stress tolerance of the Arabidopsis thaliana mutants To assess the response of the Arabidopsis thaliana mutants and the wild type Col0 in terms of oxidative stress the study was focused on observing the germination capacity of the seeds on ½ MS culture media supplemented with three different concentrations of N,N -dimethyl-4,4 -bipyridinium dichloride (paraquat) ranging between 1 µm and 3 µm and three different concentrations of 4,5,6,7- tetrachloro-2',4',5',7'-tetraiodofluorescein (rose bengal) varying among 1 µm and 5 µm. The results obtained during this experiment were similar to those obtained on the ½ MS culture media supplemented with 2 µm paraquat in regard to the germination rate of the ABI mutants and the grp2 mutant. The only difference observed was that the germination rate of the grp7-1 mutant was not statistically significant higher than that of the Ws7ox mutant even though more than 50% of the first 27

mutant germinated in contrast to the seeds of the second mutant mentioned where the germination percentage was under 25% (Figure 27). Fig.27. Seed germination capacity on 3 µm Paraquat. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 3 µm paraquat. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. The use of a 5 µm concentration of rose bengal into the culture media had a negative effect on the germination potential of the wild type Col0 in comparison to that of the seeds originated from the ABI and the grp2 mutant plants. There was also a significant lower germination potential of the seeds obtained from the grp7-1 mutant plant in contrast to those produced by the ABI mutants. Less than 20% of the seeds obtained from the Ws7ox mutant plants were able to germinate on this type of culture media used (Figure 28). 28

Fig.28. Seed germination capacity on 5 µm Rose Bengal. Comparison of the germination capacity of the Arabidopsis thaliana mutants abi3, abi4, abi5, grp2, grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 5 µm rose bengal. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. Similar results regarding the resistance to paraquat of knock-out mutants were observed in the research done by AL-Quraan and coworkers (2011), were through assessing the involvement of the CAM (calmoduline) genes in response to the oxidative damage caused by paraquat discovered that from seven knock-out lines tested only two cam5-4 and cam6-1 showed high sensitivity to this type of substance regarding seed germination and seedling growth and oxidative damage implying the direct involvement of the CaM5 and CaM6 genes in seed germination and early development especially in the acquisition of tolerance in reaction to abiotic stresses. 29

The research conducted by Li and collaborators (2013), in describing the Arabidopsis paraquat resistant 1 revealed the fact that the par1 mutation confers a reduction in the accumulation of paraquat in the chloroplast. The same group discovered a rice (Oryza sativa) gene OsPAR1, where the overexpression resulted in hypersensitivity to paraquat, the knock-down of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. In contrast to the similarities obtained in case of the knock-out mutant grp2 with the current literature on paraquat research, in case of the overexpressor for GRP7 the results were more in contradictory to those obtained by other groups in the assessment of overexpressing plants for different genes. A study conducted by Gaber and his group (2012) revealed that the knock-out mutation of a glutathione peroxidase (GPX) isoenzyme namely AtGPX8 increased the plants sensitivity to oxidative stress damage produced by paraquat. In contrast when the isoenzyme was overexpressed the plants tolerance to this type of damage increased suggesting a protective role of the AtGPX8 for cellular components in oxidative stress damage. Influence of ethylene on the germination capacity of the Arabidopsis thaliana mutants To assess the influence of ethylene on two of the Arabidopsis thaliana mutants namely the knock-out and the overexpressor mutant for the AtGRP7 protein and the wild type Col0 the study was focused on observing the differences in germination and also on their root development. Three different concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) with roll in biosynthesis of the plant hormone ethylene were chosen to supplement the simple ½ MS culture media. The concentrations ranged between 10 µm and 100 µm ACC. The enlarged concentration of ACC used during this study had a negative effect on the germination rate of the wild type seeds in comparison to those originated from the AtGRP7 mutants. Only 50% of the wild type Col0 seeds were able to germinate by day seven from the start of the experiment in contrast to the 95% of the grp7-1 mutant and the 80% of the Ws7ox mutant seeds (Figure 29). The effect of ACC on the germination of the seeds originated from the AtGRP7 knock-out mutant and overexpressor was a positive one, enhancing in both cases the seed germination capacity 30

and rate in comparison to the wild type Col0. Similar effects of this compound on seed germination were registered also in case of cucumber seeds set under saline stress. The effect of NaCl was alienated through the use of ACC and Ethephon (the most widely used plant growth regulator) (Chang et al., 2010). The research conducted by Siddikee and collaborators (2011), proved that the use of ACC deaminase-producing halotolerant bacteria decreases the effect of salt stress on growth of red pepper plants by reducing salt stress-induced ethylene production. Through the inoculation of this type of bacteria the plantlets increased in growth, the nutrient up take improved and also the ethylene production was reduced. The correlation of our results with other studies regarding the effect of ACC on seed germination leads to the conclusion that this type of compound has a positive impact on the enhancement of the germination capacity and also on the development of seedlings. Fig.29. Seed germination capacity on 100 µm ACC. Comparison of the germination capacity of the Arabidopsis thaliana mutants grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 100 31

µm ACC. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days Influence of sodium nitroprusside (SNP) on the germination capacity of the Arabidopsis thaliana mutants The influence of sodium nitroprusside (SNP) on the Arabidopsis thaliana mutants grp7-1 and Ws7ox and on the wild type Col0 was studied through the observations made on the germination rate of the seeds originated from the above mentioned plants. The effect of this chemical compound should be the enhancement of the germination capacity of the seeds taken into consideration. Three different concentrations varying among 10 µm and 100 µm were used to supplement the simple ½ MS culture media. The results obtained on this type of culture media pointed out no significant differences between the germination rates of the three Arabidopsis lines taken into the experiment. Both the AtGRP7 mutants and the wild type reached a high germination percentage by the end of the experiment (Figure 30). Sodium nitroprusside is known to have a protective role in plants during the exposure to abiotic and biotic stress conditions. Our focus during the research was to evaluate the effect of this widely used NO (nitric oxide) donor, which acts as a signal molecule in plants and is responsible for the expression regulation of many antioxidant enzymes (Antoniou et al., 2013), in the enhancement of seeds germination capacity of the AtGRP7 knock-out mutant and overexpressor. The results obtained by Liu and coworkers (2009), in regard to the germination capacity of Col0 Arabidopsis seeds were alike to our outcomes which showed an increased germination capacity of the seeds taken under observation when set to germinate on MS culture media supplemented with SNP. The pretreatment with SNP of winter wheat (Triticum aestivum L., cv. Huaimai 17) seeds resulted in a higher germination rate under salt stress in contrast to the seeds that were not soaked in SNP solution (Zheng et al., 2009). 32

Fig.30. Seed germination capacity on 100 µm SNP. Comparison of the germination capacity of the Arabidopsis thaliana mutants grp7-1, Ws7ox and wild type Col0 on ½ MS culture media supplemented with 100 µm SNP. Mean germination is represented by the mean of the seed germination capacity of three independent replicates scored for seven days. 33