Signaling in the Nitrogen Assimilation Pathway of Arabidopsis Thaliana

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1 Biochemistry: Signaling in the Nitrogen Assimilation Pathway of Arabidopsis Thaliana 38 CAMERON E. NIENABER ʻ04 Abstract Long recognized as essential plant nutrients and metabolites, inorganic and organic forms of nitrogen such as nitrate, ammonia, and amino acids, are now being examined as possible signaling molecules, important in gene expression regulation. Here quantitative-pcr was used to examine the regulatory effects of inorganic (nitrate and ammonia) and organic (glutamine and glutamate) forms of nitrogen on the expression of ASN1 and ASN2, two genes known to encode asparagine synthetase (AS), a crucial enzyme in the nitrogen assimilation pathway. In order to differentiate between induction/repression due to inorganic nitrogen sources and changes in expression due to organic forms of nitrogen, methionine sulfoximine (MSX) was employed. MSX inhibits glutamine synthetase (GS) thereby blocking the conversion of ammonia to glutamine (Gln). Two week old Arabidopsis plants were treated for two hours with one of six treatments containing a combination of MSX, inorganic and organic nitrogen forms. In plants treated with inorganic nitrogen (K and NH 4 ) both ASN1 and ASN2 expression was induced when compared to no treatment plants. However, in plants given both inorganic nitrogen and MSX, ASN1 expression fell back to no treatment level whereas ASN2 expression remained induced. These results indicate that ASN1 regulation relies on a molecule in the nitrogen assimilation pathway downstream from ammonia (i.e. an organic form of nitrogen). This hypothesis is supported by the finding that ASN1 expression is induced in plants given inorganic nitrogen, MSX, and Gln, as the addition of Gln bypasses glutamine synthetase (GS) and allows the synthesis of downstream organic nitrogen molecules. Conversely, the ASN2 data suggest that ASN2 expression is regulated by an inorganic nitrogen source, as no significant effect is seen by blocking the production of organic nitrogen molecules using MSX. Introduction Nitrogen assimilation is crucial for plant growth and development and has therefore been a well-studied biochemical pathway for many years. The assimilation of inorganic nitrogen, in the form of nitrate, nitrite, and ammonia, into carbon-based amino acids is performed by a variety of enzymes including glutamine synthetase (GS), asparagine synthetase (AS), and glutamate synthase (GOGAT) (Fig. 1). Additionally inorganic nitrogen is converted from one form to another by nitrate reductase (NR) and nitrite reductase (NiR) (Fig. 1). Studies have found multiple isoenzymes for each of these enzymes and genetic analyzes have revealed gene families in which specific members encode distinct isoenzymes. Each isoenzyme-encoding gene is specifically regulated by a variety of environmental and metabolic signals allowing for a diversity of transcriptional responses (1). In this paper, I will examine the regulation of two isoenzyme-encoding genes for asparagine synthetase (AS), the enzyme responsible for catalyzing the conversion of glutamine to asparagine. In Arabidopsis, the AS gene family has three members: ASN1, ASN2, and ASN3 (2). Here I will look at regulation of ASN1 and ASN2 expression as measured by mrna levels. Much work has been done to understand the regulation of these AS genes as well as the regulation of other isoenzyme-encoding genes important in the nitrogen assimilation pathway. Specifically, it has been found that the gene expression of ASN1 and ASN2 are regulated in an opposite and reciprocal manner in response to carbon, light and various metabolites (2). From these findings it has been postulated that the two isoenzymes encoded by these genes play very different roles in plant physiology. Asparagine, the end product of the AS catalyzed reaction, is an important nitrogen carrier, used to transport nitrogen to various plant tissues as well as to create nitrogen stores (3). As its expression is induced in dark-adapted and carbon-limited plants, ASN1 has been identified as the AS gene responsible for AS activity directed towards nitrogen transport and storage (4). ASN2 expression, on the other hand, is induced by light, indicating that its gene product does not play the same physiological role as the ASN1 gene product (2). Recent

2 findings indicate that the ASN2 product may play a role in ammonia metabolism however further research is needed in this area (5). This paper aims to further elucidate how ASN1 and ASN2 regulation may differ by looking at the influence of inorganic and organic nitrogen on mrna levels. Specifically, my aim is to distinguish signaling by inorganic nitrogen from signaling by organic nitrogen through the use of a GS inhibitor, MSX. A better understanding of the gene regulation involved in the nitrogen assimilation pathway may lead to a better understanding of the role and regulation of each isoenzyme. Methods Growth and Treatment of Plants Tissue Culture Arabidopsis thaliana plants (ecotype Columbia) were grown vertically in a controlled environment chamber at 23 o C under 16 h light/8 h dark cycles after the seeds had been surface-sterilized and vernalized for 48 h at 8 o C. Plants were grown for 14 d on plates containing 50 ml of media containing 1X basal media, 0.8% bactoagar, 1 mm K, sucrose (5g/L), and ph adjusted to 5.7 with KOH. On day 14 of growth plants were transferred to treatment plates at 9 am. Nine plates of each treatment were grown (3 plates x 3 replicates). Treatments were as follows: No treatment = No N, basal media; 1XN = 20 mm NH 4 and 20 mm K, basal media; 1XN mm MSX; 1XN mm MSX + 10 mm Gln; 1XN mm MSX + 10 mm Glu; 1 mm K mm MSX. After two hours of treatment whole plants were harvested, immediately frozen in liquid nitrogen and stored at - 80 o C. Hydroponics (Magenta Boxes) Same growth and treatment procedure as for tissue culture except plants were grown and treated in 60 ml of media containing no agar (liquid media) and plants were gently agitated on a gyrotory shaker throughout growth. Five boxes of each treatment were grown (2 plates x 2 replicates, 1 plate x 1 replicate). Upon harvesting, plants were cut at the mesh float, yielding the roots samples (consisting of only root tissue) and the shoots samples (consisting of all non-root tissue). These samples were immediately frozen in liquid nitrogen, stored at - 80 o C, and processed separately. RNA isolation and Quantitative PCR RNA was isolated from whole plants by grinding in liquid nitrogen, extracting with phenol and chloroform, extracting with 3M NaOAc (ph 5.2), extracting with 4M LiCl, and redissolving in sterile water. cdna synthesis from total RNA was carried out according to Invitrogen (cat. no ). Real-time quantitative PCR was done using a LightCycler (Roche Diagnostics, Mannhein Germany). A 20-µL reaction volume for PCR amplification contained a master mixture containing Taq DNA polymerase, dntp mixture and buffer (LightCycler DNA Master Hybridization probes, Roche Diagnostics), 4 mm MgCl µm of each primer, 0.2 µm of each hybridization probe, and cdna in a glass capillary tube. Primers and probes for ASN1 and ASN2 were as in Thum et al. (6). Analysis Data was normalized to EIF4 (A control gene whose expression is known to remain constant with varying nitrogen treatments) using an equation derived from a standard curve of serial dilutions. Statistical analysis consisted of one-tailed distribution, paired t- tests (unpaired equivalent t-tests when there was uneven sample sizes) taking p-values of < 0.05 as significant. Results Quantitative real-time PCR was used to measure the levels of mrna for two genes (ASN1 and ASN2) in whole plants (tissue culture) and roots and shoots (hydroponics) under six treatments (Table 1). Table 1. ASN1 Tissue Culture - Significant induction of ASN1 expression was seen in the 1XN treatment as compared to the no treatment control (Figure 2). This induction was blocked by the addition of MSX (treatment 3). Induction was rescued and supplemented by the addition of glutamine (treatment 4) however addition of glutamate did not significantly induce gene expression (treatment 5). Treatment 6 showed significant repression of ASN1 expression as compared to no treatment. Hydroponics - Although error was much greater in roots and shoots samples than in tissue culture, averages of expression in three replicates in both roots and shoots FALL

3 demonstrated a similar pattern as seen in tissue culture (Figures 3 & 4). Rescue of induction with the addition of Gln was not as pronounced in shoots as in tissue culture. In the roots no significant rescue of induction by Gln or Glu was seen. ASN2 Tissue Culture - Q-PCR analysis of ASN2 expression showed significant induction as compared to no treatment in all five treatments (Figure 5). Significant super induction was seen with the addition of amino acids Gln and Glu. Treatment 6 showed only slight induction of ASN2 expression from no treatment. Hydroponics - Shoots samples displayed the same ASN2 expression pattern as seen in tissue culture. However, significant super induction was not seen with the addition of amino acids (Figure 6). Root samples showed no significant change in ASN2 expression across all treatments (Figure 7). Figure 2: Statistical significance is denoted by an asterisk followed by the treatment number to which that treatment is significant. Figure 5. Figure 3. Figure Figure 4. Discussion By examining the expression of ASN1 and ASN2 in plants treated with a variety of nitrogens and a GS inhibitor, we have come a step closer to understanding how these genes are regulated as part of the nitrogen assimilation pathway. MSX is known to inhibit the production of organic nitrogen forms, specifically glutamine by inhibiting the activity of GS. Thus, MSX

4 Figure 7. provides a useful way to distinguish between gene regulation due to organic vs. inorganic nitrogen. The tissue culture samples, consisting of whole plants, give an overall picture of gene regulation, while the roots and shoots samples allow for a more in depth look at regulation in various plant tissues. In treatment 2 gene expression induction or repression due to any form of nitrogen can be observed because the plants can make organic forms of nitrogen from the inorganic K and NH 4 given in the media. However, in treatment 3, any variation from no treatment in gene expression is hypothetically due to only inorganic nitrogen as MSX inhibits the production of all organic forms. Treatments 4 and 5 provide inorganic and organic forms of nitrogen in the media so although the plants cannot make organic nitrogen molecules from inorganic ones due to the inhibition of GS by MSX, induction or repression of gene expression could be due to the given organic nitrogen or a downstream molecule. Treatment 6 is an attempt to see any effect of MSX alone, however since there is still a small level of nitrogen given there may be affect on gene expression. In further experiments, it would be useful to run a control in which only MSX is given, with no nitrogen present. The suppression of ASN1 induction by MSX seen in treatment 3, Figure 2 indicates that ASN1 induction is regulated by an organic molecule synthesized by or downstream from GS (Figure 8). This claim is further supported by the reinduction seen in treatment 4, in which organic nitrogen (Gln) is available. No reinduction is seen with Glu however, supporting a hypothesis that Glu is not the ultimate signaling molecule for ASN1 expression. Instead, Gln or a further downstream product acts as the signaling molecule. Functional GS is needed to convert Glu into Gln, therefore in treatment 5, where both MSX and Glu are present, the plants cannot produce Gln or any other downstream products and therefore, I hypothesize, ASN1 expression is not induced. Unsuppressed induction of ASN2 in treatment 3, Figure 5 indicates that regulation of ASN2 expression is not dependent on amino acid synthesis. My hypothesis is that ASN2 expression is primarily regulated by an inorganic nitrogen molecule (Figure 8). Secondary regulation may involve organic molecules as super induction in treatment 4 indicates additional induction by Gln. A smaller secondary induction may also be present with Glu however my data is inconclusive on this point. A similar expression pattern in shoots samples further supports my hypothesis that ASN2 expression is regulated by an inorganic form of nitrogen. Low, uniform expression across all treatments in the roots may indicate that ASN2 expression is not as closely regulated in the roots as in the shoots. Based on the findings discussed here it would be interesting to use MSX to identify and classify all the genes in the Arabidopsis genome that are regulated by inorganic vs. organic forms of nitrogen. Gene expression chips could be run using the tissue culture samples from treatments 1-3. Criteria for genes regulated by inorganic forms of nitrogen would be induction in both treatments 2 and 3 as compared to treatment 1 or repression in both treatments 2 and 3 as compared to treatment 1. Criteria for genes regulated by organic forms of nitrogen would be induction or repression in treatment 2 as compared to treatment 1 but no significant difference from treatment 1 in treatment 3. Additionally, treatments 4 and 5 could be used to verify regulation by organic nitrogen molecules and help to narrow down possible signaling molecules, as was done in the analysis of ASN1 expression. Genes identified by this analysis could then be grouped into pathways and used to understand signaling on a organismal level. In this study, I have identified another difference in the regulation of the isoenzyme-encoding genes, ASN1 and ASN2, which code for AS. My findings indicate that ASN1, which codes for an AS responsible for nitrogen transport and storage, is regulated by an organic nitrogen FALL

5 form, possibly Gln or a further downstream molecule. ASN2 expression, conversely, is regulated by inorganic nitrogen forms, lending further support to the idea that ASN2-encoded AS plays a different physiological role than AS encoded by ASN1. Through this study and others of similar nature we are coming to a greater understanding of gene expression regulation within metabolic pathways such as the nitrogen assimilation pathway of Arabidopsis. Acknowledgements I thank Dr. Gloria Coruzzi for her instruction and guidance on this project. Also, many thanks to Alexis Cruikshank, Dr. Laurence Lejay and Dr. Peter Palenchar. REFERENCES 1. Lam H.-M., Coschigano K. T., Oliveira I. C., Melo-Oliveira R., Coruzzi G. M. The Molecular-Genetics of Nitrogen Assimilation into Amino Acids in Higher Plants. Annu. Rev. Plant Physiol. and Plant Mol. Biol 47, (1996). 2. Lam H-M, Hsieh M-H, Coruzzi G. Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J 16, (1998). 3. Sieciechowicz, K.A., Joy, K.W., Ireland, R.J. The metabolism of asparagine in plants. Phytochemistry 27, (1988). 4. Lam, H.-M., Peng, S.S.-Y., Coruzzi, G.M. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana. Plant Physiol. 106, (1994). 5. Wong HK, Chan HK, Coruzzi GM, Lam HM. Correlation of ASN2 gene expression with ammonium metabolism in Arabidopsis. Plant Physiol. 134, (2004). 6. Thum E., Shasha D., Lejay L., Coruzzi G. Light- and Carbon- Signaling Pathways. Modeling Circuits of Interactions. Plant Physiol. 132, (2003). Faculty: have an undergraduate working in your lab? Has a student of yours produced an especially well-written class paper? Encourage him or her to submit to the DUJS. 42