Arabidopsis and Auxin, a Perfect Pair to Engineer Gene Expression Reporter genes can be manipulated to study the expression

of transcription factors involved in the signaling cascade of the Arabidopsis thaliana plant (Figure 1). Several features make the Arabidopsis an ideal species for molecular genetic studies: small size, quick generation time, ability to thrive in a controlled environment, high fertility rate, simplicity with which a mutant line can be maintained by self-fertilization, and the ease with which a line can be outcrossed. In addition to having all the key features of a model system for the study of genetics, Arabidopsis has the smallest known plant genome with the least amount of repetitive sequences than any other known higher plant species1, significantly enhancing their ability to contribute to molecular research. In order to induce genetic signaling cascades in Arabidopsis, the plant hormone auxin (Figure 2) is utilized. Auxin is a vital and fundamental plant hormone that synchronizes diverse processes such as cell division, embryogenesis, meristem formation, root and leaf patterning, tropism, and reproduction. A vast amount of molecular research has isolated a substantial set of auxin-inducible genes and the auxin-responsive cis-elements and the relevant trans-acting factors2. Furthermore, genetic methods have led to the identification of genes involved in auxin responses and transport, and the unraveling of protein degradation control in auxin signaling3. In this experiment, the molecular and biochemical mechanisms essential to the auxin signaling transduction pathways controlling gene transcription and plant morphogenesis will be distinguished and characterized. The goal of this experiment is to study the regulatory genes important in auxin-inducible transcription and morphogenesis by using Arabidopsis as a model system. To achieve this, the various steps of the auxin-dependent transcription and morphogenesis, including cell proliferation, elongation, and differentiation, will be investigated.

Figure 1: Arabidopsis thaliana On the left, the vegetative stage, before flowering and growth of the floral stalk (bottom left). In the center, an adult plant at full flowering/seed set. On the right, flower, floral stem and seeds. White bars are 1 cm, except for the flower and seeds, 1 mm 4.

Figure 2: Auxin 5

Material and Methods During the first week of this experiment, sterilized Arabidopsis seeds with transgenic marker lines were planted onto a plant growth media (MS salts) of agar. The plants were then grown under artificial lighting for a full week, allowing the seeds to germinate and produce leaves and roots (Figure 3). Approximately 45 seeds of each transgenic line were plated, including CyclinB1, Columbia (control group), LBD16, and LBD33. Cyclin is a promoter in cell division and is also the regulatory subunit of the M-phase promoting factor. Adequate adjustment of Cyclin

within the cell is fundamental in the initiation of mitosis. Lateral Border Domain 16 (LBD16) is involved in lateral root formation and is regulated by the transcriptional activators ARF7 and ARF19. Without LBD16, there will be less lateral root formation in the Arabidopsis plant. Similarly, LBD33 is an activator of lateral root formation and is closely associated with LBD16.

Figure 3: The development of Arabidopsis thaliana. a) The egg cell far within the ovule. b) After fertilization, the egg goes through an asymmetric division and produces an apical cell and a bigger basal cell. The basal cell distributes the auxin and in return, the apical cell reacts. c) The basal cell splits to develop the suspensor, while the apical cell that received the auxin divides to produce the two celled proembryo. d) Once the 32 celled globular embryo develops, the stream of auxin is swiftly inverted to collect in the hypophysis. This is where the basal regions of the embryonic root originate. e) Now, the regions that generate the apical, central and basal parts of the seedling can be differentiated 6.

To plate Arabidopsis seeds, a seed pipette must first be assembled. A sterile transfer pipette was used and the bulb of the pipette was attached to the end of a p-1000 tip using parafilm. With this newly constructed seed pipette, water containing a cluster of seeds from an Eppendorf tube was manipulated and each individual seed was placed on the agar plate in three distinct rows. Every seed was placed within equal distance from each other, approximately fifteen seeds per row, and only one genotype was used per plate. To provide more room for the roots to grow, extra space was left at the bottom of the plate between the plastic rim and the seeds than was left at the top. At the end of the first growing period, each plant was observed based on its general stature (Table 1). For Columbia (control group), one plant per square was planted on the agar plate. 3

Almost all of the seeds germinated, with only two seeds not germinating and 86.6% actively growing (only had 15 seeds in total). There was no variation in the length of the roots for the Columbia line, but the lengths of the shoots did vary slightly. For LBD16, two plants per square were planted, and 32 out of 45 seeds germinated. With 71% of the seeds germinating, the seeds that did not germinate were possibly planted too deep into the agar or too close to the edge of the plate. There was no variation in roots, but again, there were differences in the lengths of the shoots. One to two plants of LBD33 were planted per square because only 23 seeds were provided. One lone seed did not germinate, with 95.7% actively growing, and that one seed that did not germinate was most likely due to being on the edge of the plate. The only variation in the plants was the length of the shoots. For Cyclin, three plants per square were planted and 85.7% were actively growing with 36 out of 42 plants germinating. The un-germinated seedlings could have been due to being planted too deep into the agar or possibly just not growing. The Cyclin line had slight variances in the lengths of the roots and no differences in the lengths of the shoots. There were minor differences in length between the genotypes, as was suspected since each line has a different transgenic marker.
Plant Type # Of Seeds Planted per Square 1 plant per square plate 2 plants per square 1 to 2 plants per square 3 plants per square Fraction of Seeds that Germinated 13/15 Reasons for UnGerminated Plants Yes, maybe just did not grow or was put too deep into the agar. Yes, the ones on the edge didnt germinate. Yes, the one that did not germinate was on the edge. Yes, maybe some just didnt grow or were put too deep into the agar. Variations in the Roots No. Differences in the Shoots

Columbia

Yes, the lengths of the shoots in the plants vary. Yes, the lengths of the shoots have some variations. Yes, the lengths of the shoots are different lengths. No, the lengths of the shoots do not vary.

LBD 16

32/45

No.

LBD 33

22/23

No.

Cyclin

36/42

Yes, slight variance in the roots. Different plants grow at different rates.

Table 1: Results from growing Columbia, Cyclin B1, LBD 16, and LBD33 seed lines on agar for one week, under artificial lighting.

After assessing the plants, each line was treated with auxin as the inducer. Three different concentrations of synthetic auxin (2,4-D) dissolved in water were used to treat each group of plants; one group was left untreated as the control group. To achieve this, four petrie dishes were divided into four sections, one section for each gene line. Then, a 1.2% solution of agarose was made, which was later added 1:1 to the auxin solutions to produce a final concentration of 0.6% agarose. To make the induction media, 0.5g of Agar was weighed out and transferred into a 500mL Erlenmeyer flask and 40mL of deionized water was added. The mixture was swirled together and placed in the microwave for one minute. When the flask was carefully removed from the microwave, the sample was swirled again and all agar particles had melted into the water. The flask cooled for four minutes and then the auxin was added to produce the final concentration. For the auxin concentrations, 0.5mL was added directly to petrie dish one, 1.0mL of auxin was added to dish two, 1.5mL was added to dish three, and the fourth dish did not have any auxin added as it was the negative control. Using a serological pipette, 5mL of the melted agar was slowly added to the auxin while swirling the plate to thoroughly mix the two. Then the plants were carefully transferred from the agar to the appropriate section on each of the pre-labeled petrie dishes, corresponding to the specific genotype and final concentrations of auxin. When the transfers were complete, the plants were left to grow for another week in a growth chamber with sixteen hours of fluorescent light followed by eight hours of darkness. After the plants were treated for one week with auxin at room temperature, each plant was assessed to compare and contrast any physical changes in comparison to the plants structure prior to the auxin treatment. The control group was not treated with any auxin. In this group, the LBD33 plants had roots that were growing towards the lid, the LBD16 stalks grew a small amount, the

Cyclin plants had roots that were also growing towards the lid but were not as long as the LBD33 plants, and the Columbia plants did not exhibit any change or growth. For the petrie dish that was treated with 0.5mL of auxin, two out of the three LBD33 plants turned white and the stalks and roots also grew a small amount. The LBD16 plants did not grow at all and all of the plants were almost completely white. For Cyclin, two out of the three plants turned white and the roots grew only on the plant that was still green. In the Columbia plants, there was no growth, one plant turned completely white, and the other two plants were half white. The next petrie dish was treated with 1.0mL of auxin. The LBD33 plants did not exhibit any growth with one plant remaining green and the other two turned white. For the LBD16 plants, there was minimal growth, the stalks started to move towards the lid, and all of the plants remained green. There was no change in the Cyclin group and all of the plants were green. For Columbia, two of the plants exhibited no growth, all of the plants remained green, and one plants roots started to grow towards the lid. The last petrie dish was treated with 1.5mL of auxin. There was no growth in any of these plants and they all turned completely white. The levels of auxin in this dish could have been too high and this dosage may not have a beneficial affect on these plants. During week three, the plants were stained for GUS gene expression using X-Gluc in Na2HPO4 buffer with the detergent X-100. The objective of this method is to examine and evaluate the functioning of the promoter through the analysis of the plants tissues. GUS gene expression will be seen in actively dividing cells of the Arabidopsis plant. Therefore, each plant was carefully removed from the agar containing auxin with forceps and individually put into Eppendorf tubes containing GUS staining buffer. Using only enough buffer to cover the plants, the whole plant was placed into a tube and completely immersed in the buffer. Each tube was labeled with the auxin treatment concentration, the plant genotype, and the Eppendorf tubes were wrapped with parafilm. The plants were then left to incubate for another week at 37C. 6

During week four, destaining the plants and imaging them for GUS expression enabled the GUS staining patterns to be analyzed. First, to destain the plants, a transfer pipet was carefully placed into the tube containing the Arabidopsis plants that were in the staining buffer. Then, as much buffer was removed as possible without disrupting the plants. Each sample was washed with 70% ethanol four times over the span of one hour. To wash each plant, ethanol was added until the Eppendorf tube was filled, the tube was closed and inverted several times to thoroughly clean the sample, and then the ethanol was removed with a transfer pipet. The pigment started to fade with each wash cycle and all plants remained in the ethanol for fifteen minutes at a time. After the washing was complete, each stained plant was placed back in a clear petrie dish and a destain buffer of 50% ethanol and 15% glycerol was added to each sample with a dropper. The leaves, shoots and roots of the plants were spread out to allow for examination under the microscope. Results While under the microscope, each plant was dissected to examine the gene expression. In the control group, the Cyclin plant (Figure 4) exhibited staining only in the leaves, the LBD16 plant (Figure 5) turned completely blue, the LBD33 plant (Figure 6) displayed staining only in the roots, and the Columbia plant (Figure 7) presented staining throughout the shoot and the roots. In addition, the Columbia sample had the most lateral root formation out of all four plants. The other plants had very minimal lateral root growth. For the group with 0.5mL of auxin added, the Cyclin plant went missing at some point during the washing process, therefore, we were unable to stain and analyze it. All of the other plants had a small amount of lateral root growth with Columbia having the most and LBD16 having the least. The Columbia plant (Figure 8) was not stained at all, LBD33 (Figure 9) displayed a little bit of staining in the lateral roots, and LBD16 (Figure 10) had a hint of blue in the shoot. From the petrie dish that 1.0mL of auxin was added, the LBD33 7

(Figure11) and LBD16 sample (Figure 13) turned completely blue and very little lateral root growth was prevalent. The Cyclin plant (Figure 12) had an entirely stained blue seed, a lot of lateral root growth, and there was also staining in the leaves and a small amount in the shoot. The Columbia sample (Figure 14) did not display any staining. The last group had 1.5mL of auxin added to the petrie dish. The Columbia plant (Figure 15) did have a good amount of lateral root growth and there was a hint of blue at the end of the main root. For the Cyclin sample (Figure 16), the staining was prevalent in the top portion of the shoot and in the leaves. The LBD33 plant (Figure 17) displayed a little bit of blue in one of the leaves and there was a blue hint in the bottom portion of the main root, but there was minimal lateral root formation. LBD16 (Figure 18) had a lot of lateral root formation and there was staining towards the end of the roots. From the results listed above, there is a correlation between the lateral root growth and the concentration of auxin. The control group did have some growth, but the growth was consistent with the average expected development of a plant and Columbia (the control gene line) had the greatest amount of growth. In the 0.5mL group, there was a little more growth than the control group, but not enough yet to make a significant difference. At this small amount, the auxin did have an effect, yet the Columbia plant still had the most progression. The Cyclin sample in the 1.0mL group had the largest amount of lateral root growth, revealing that 1.0mL of auxin is a possible ideal concentration for the Cyclin gene line. In the 1.5mL group, LBD16 had the greatest amount of lateral root progression, suggesting that 1.5mL of auxin might be an ideal concentration for the LBD16 gene line. In the instances where auxin was added and the roots did not display a clear progression of lateral growth, the plants could have possibly received too much or too little concentration of auxin. Discussion Research and data concerning identifiable changes in gene expression are vital for the 8

understanding of the genetic regulatory networks fundamental to the development of all living organisms. In Arabidopsis, gene expression is portrayed in a spatial and a temporal manner, resulting in the activation of genes within specific tissues of the plant at specific times during development. For example, the Cyclin and GUS expression pattern is demonstrated in the actively dividing cells of the sample. The aerial parts of the plant, or the parts of the plant that are generally above ground, are created by rapidly dividing cells. Therefore, it follows that gene expression would most definitely be evident in the aerial portions of the Cyclin plants. On the other hand, LBD16 and LBD33 are active in plant tissue involved in the growth of lateral roots. Consequently, staining will be present in areas where lateral roots are developing. LBD genes do not have the same expression patterns even though they are both associated with lateral root growth. For instance, the LBD33 plant in the 1.5mL group displayed staining in the roots just as the LBD16 sample did, but LBD33 did not have as much lateral root development as the LBD16 plant. The triton-X-100 that was added to the staining buffer in this step of the experiment was used as a detergent in order to permeabilize the fixed cells, which allows for increased penetration through the cell membrane and better analysis. Furthermore, the gene expression patterns resulting from this experiment are also quantifiable. This is due to the fact that reporter genes generates a specific phenotype which allows for differentiation of cells containing this gene from other plants without that particular gene. Reporter genes can be used to track and study related genes within a plant. Each reporter gene produces a particular protein product, which is easily detected and quantified without destroying the plant tissue. Then, one can easily discover the expression patterns of a gene within the cell by combining its promoter with a reporter gene and transforming the inside of the cells. Transformed cells turned blue in the presence of substrate, X-gluc. The auxin gene cascade induced lateral root growth in the LBD16 plants of this experiment, although the LBD33 plants did 9

not have an increase in the overall development of lateral roots. The Cyclin plants also portrayed a general increase in growth and development. Quantitative research is extremely beneficial in the examination and discovery of plant transfection, enabling the analysis of the activities of the regulatory elements in Arabidopsis such as the promoter. GUS expression in a transgenic plant is dominant, therefore, its phenotypes are dominant and do not need additional rounds of sexual recombination. Expression levels in different transgenic lines of Arabidopsis carrying the same construct can vary. Measuring the mRNA or protein levels in various plant tissues generally depicts the expression of plant genes. Analysis of gene expression can be expedited by the use of plant transformation techniques and the introduction of reporter genes such as Glucuronidase (GUS). In spite of this, research suggests that the level of transgene expression differs between single transformants with the same transgene copy number. This quantitative discrepancy depends on the site of integration and it is referred to as the position effect7. For example, genes translocated to regions of heterochromatin are often not expressed. Discovery of the starting point of a transcription site leads to the location of the promoter of a gene. A promoter is an area of DNA that enables the transcription of a specific gene. Promoters are positioned near the genes they regulate, on the same strand, and typically upstream towards the 5' region of the sense strand. Promoters can be derived directly from naturally occurring genes, or they may be synthesized to combine regulatory sequences from different promoter regions. The promoters interact with other regulatory sequences, such as enhancers or silencers, and regulatory proteins (i.e. transcription factors) to influence the amount of gene transcription and expression. A transgenic plant has a gene or multiple genes that have been synthetically inserted as an alternative to the plant acquiring them through pollination. The implanted gene sequence, called a 10

transgene, may come from another unrelated plant or from a completely different species. Plants compromised of transgenes are often called genetically modified, although in reality, almost all plants have been genetically modified from their original state by domestication, selection, and controlled breeding over long periods of time. When breeding plants, a breeder tries to assemble a combination of genes in a plant that will make it as useful and productive as possible. Depending on where and for what purpose the plant is grown, desirable genes may provide features such as improved quality, disease resistance, and tolerance to heat or cold. Combining the best genes in one plant is a long and difficult process, especially as traditional plant breeding has been limited to artificially crossing plants within the same species or with closely related species to bring different genes together. Transgenic technology enables plant breeders to bring together in one plant useful genes from a wide range of living sources, not just from within the crop species or from closely related plants. This technology provides the means for identifying and isolating genes controlling specific characteristics in one kind of organism, and for moving copies of those genes into another quite different organism, which will then also contain those characteristics. This effective tool enables plant breeders to do what they have always done, to create more useful and productive crop varieties containing new combinations of genes. This technology expands the possibilities beyond the limitations imposed by traditional cross-pollination and selection techniques. An example of how this technique is used today is how a plant breeder might cross-pollinate corn plants. Transgenic technology is also an expansion of agricultural practices that have been used for hundreds of years, such as in selective breeding and special feeding or fertilization programs. It may reduce or even replace the large-scale use of pesticides and long-lasting herbicides. When fully developed, it would offer a number of advantages over traditional methods. In comparison to traditional methods, the benefits of transgenic breeding are: 11

1) More Specific: scientists are able to choose with greater accuracy and precision the trait they want to establish and the number of unwanted traits can be kept to a minimum. 2) Faster: establishing the trait takes only one generation compared with the many generations frequently needed for traditional selective breeding, where much is left to chance. 3) More Flexible: traits that would otherwise be unavailable in some animals or plants may be achievable using transgenic methods. 4) Less Cost: a large portion of the cost and labor involved in administering feed supplements and chemical treatments to animals and crops could be avoided.

However, modifications to a desired organism may result in the simultaneous genetic modification to an undesired organism. For instance, when modifying a plant to be resistant to a particular disease-causing organism, this process may acquire natural variations that could possibly enable it to infect other plant species not previously infected8. In conclusion, Auxin is an important plant hormone, which regulates plant growth and development including embryogenesis, seedling growth, vascular patterning, and flower development. Auxin exerts its biological functions by activating signal transduction pathways. These pathways ultimately regulate the expression of downstream target genes that control particular developmental processes. In conjunction with an ideal species for molecular genetic studies, auxin and Arabidopsis make a perfect pair to study gene expression. The knowledge and information being gathered from studying the effects of manipulating reporter genes and the expression of transcription factors will ideally bring improvement to a better future.

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A novel transcription factor involved in plant defense endowed with protein phosphatase activity. The EMBO Journal Vol. 22, 3376 - 3384 (2003).
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Auxin Signaling: Homing in with Targeted Genetics. Plant Cell Vol. 10, 1581-1584 (1998).

Arabidopsis thaliana: a model species for plant biology. INRA (2010). Retrieved November 13th, 2011, from http://www-ijpb.versailles.inra.fr/en/arabido/arabido.htm Indole Acetic Acid the Plant Hormone made with freeware, Chemsketch, version 8.17, Advanced Chemistry Development, Inc., Toronto ON, Canada, www.acdlabs.com (2005).
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Plant development: An axis of auxin. Nature Vol. 426, 132-135 (2003).

Position Effects on Eukaryotic Gene Expression. Annual Review of Cell Biology Vol. 6, 679-714 (1990).
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Transgenic Crops: How Genetics is Providing New Ways to Envision Agriculture. The Science Creative Quarterly. Issue 6 (2003).

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