Issue 1, August 2003

Biological & Biomedical Sciences

Expression analysis of Amidohydrolase Homologs from Arabidopsis thaliana and Arabidopsis suecica

Vinela Bakllamaja
Montclair State Univeristy
Advisor: James J. Campanella, Ph.D.
Montclair State University

Introduction

In higher plants, the growth hormone indole-3-acetic acid (IAA or auxin) (Figure 1a) is stored conjugated to sugar moieties via an ester linkage or to amino acids or peptides via an amide linkage (Cohen and Bandurski 1982; Bandurski et al. 1995; Walz et al. 2002). More than 95% of the hormone in a plant can be found in the conjugated form, leaving only a small amount of free hormone available to stimulate and control cellular growth (Hangarter and Good 1981; Campell and Town 1991; Bandurski et al. 1995; Campanella et al. 1996; Leclere et al. 2002).

Figure 1. Chemical structures of auxin and auxin amino conjugates. a) IAA-3-Acetic Acid, b) IAA-aspartate, c)IAA-glycine.

Amide conjugates account for the bulk of conjugated IAA in dicots. IAA-Aspartate and IAA-Glutamate have been identified as natural conjugates in cucumber (Sonner and Purvis 1985) and soybean (Cohen 1982). IAA-Alanine has been detected in Picea abies (Ostin et al. 1992). IAA-Ala, IAA-Asp, IAA-Leucine, and IAA-Glutamate have been detected in the important model plant Arabidopsis thaliana (Barratt et al. 1998; Tam et al. 2000; Kowalczyk and Sandberg 2001), although recent data suggest that these conjugates are present in very low abundances while IAA-peptides probably account for the majority of IAA-conjugates (Walz et al. 2002) (Figures 1b and 1c).

The overall levels of active IAA in a plant can be controlled not only by the amount of IAA synthesized, but also by the quantity of IAA that is released from the conjugated state into the "free" state (Cohen and Bandurski 1982; Bandurski et al. 1995). In dicots, the active IAA hormone is released by an amidohydrolase enzyme that cleaves the amide bond between the auxin and the amino acid. Several IAA amidohydrolases have been isolated from A. thaliana (Bartel and Fink 1995; Davies et al. 1999; Leclere et al. 2002). Each of these enzymes has different substrate specificities. The A. thaliana IAA amidohydrolase, IAR3, is able to cleave IAA-Alanine (Davies et al. 1999; Leclere et al. 2002), while products of the other amidohydrolase genes, known as the ILR1-like family of hydrolases (ILR1, ILL1, ILL2, ILL3, and ILL5), cleave primarily IAA-Phenylalanine and IAA-Leucine (Bartel and Fink 1995).

In order to examine molecular evolution across species, we have isolated genomic and cDNA sequences of an IAA amidohydrolase gene (sILR1) from the species Arabidopsis suecica, which is closely related to A. thaliana (Campanella et al. 2003). The cloned sILR1 genes were sequenced, and the DNA homology between sILR1 (GenBank Accession #AF468012)and ILR1(GenBank Accession #AY081499) was determined to be 98% at both the genomic and cDNA levels. Although the gene sequences are highly homologous (Campanella et al. 2003), one question that arises is whether the regulation of these genes differs. Understanding of ILR1 and sILR1 regulation may lead us to better understand how the functions of these genes have changed or remained the same in these species. One way to establish these differences is to examine how the gene products are developmentally regulated over time in the plant tissues of the two species. This present study examines the differences in expression between ILR1 and sILR1 at a whole seedling level up to 15 days of age after germination.

Materials and Methods

Plants and Plant Growth

The A. thaliana and A. suecica seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, Ohio, USA).

For expression studies, A. suecica and A. thaliana seeds were germinated in 250 ml flasks with 50 ml of liquid Murashige-Skoog medium (Sigma Corporation). The flasks were agitated at ~100 rpm at 23° C in constant light (cool white, fluorescent, ~100 mmol/s/m²) in a plant growth chamber (Percival Scientific, Model E-30B). Seedlings were collected 1, 2, 3, 4, 5, 10, and 15 days after germination and stored frozen at –80° C until RNA extraction.

RNA Extraction

Since RNA is sensitive to degradation, all pertinent glassware was soaked in Diethylpyrocarbonate-treated water and autoclaved. Total RNA was extracted from ~0.2 g of A. suecica and A. thaliana, liquid-grown plant tissue (1, 2, 3, 4, 5, 10, and 15 days post-germination) using the RNeasy RNA extraction kit (Qiagen Corporation). Before extraction, micropestles and all microfuge tubes were treated with an 8% solution of RNA Secure (Ambion Corporation) for 10 minutes at 65° C. RNA samples were stored as aliquots at –80° C until analysis.

Semi-quantitative RT-PCR

We employed Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to perform our expression studies (Wang and Brown 1999). In the RT-PCR reaction, amplification starts with mRNA as a template. This template is treated with reverse transcriptase and a specific forward amplifying primer. A cDNA product is made from this RT reaction. This cDNA product then becomes the target of the PCR reactions that follow using forward and reverse primers. In semi-quantitative RT-PCR, the amount of amplified product is detected during successive cycles of the PCR portion of the reaction. This detection is performed to establish when the initial cycle of logarithmic amplification begins. One employs this system to determine relative expression of genes, since there is an indirect relationship between the number of mRNA transcripts expressed from a particular gene and the first cycle at which amplification can be observed. The technique is very sensitive to detecting even modestly expressed transcripts since it employs PCR amplification. Errors can be potentially introduced into the results if the RT-PCR is not done in a most careful manner. The most common sources of error are post-PCR manipulations while pipetting samples, loading electrophoretic gels, or quantitating by densitometry.

The semi-quantitative RT-PCR protocol employed in this study was based on that of Nakayama and Fujita (1992). Total RNA from 1, 2, 3, 4, 5, 10, and 15 day-old A. suecica and A. thaliana plants was used for semi-quantitative RT-PCR to examine the expression of the sILR1 and ILR1 hydrolase genes. The transcript specific primers used to amplify sILR1 and ILR1 were

5'-ATTCATGAGAACCCAGAGACA-3' (ILR1F) and 5'-CAACCCGAAACCTAACCTCA-3'(ILRR) (Campanella et al. 2003).

As an expression control for use in quantification, universal 18S primers (Ambion Corporation) were included in the same reaction mixes. This mixture of two sets of primers constituted a duplexed RT-PCR reaction in which the primers were able to amplify two different transcripts without interfering with each other. The use of such a multiplexed reaction assumes identical reaction efficiencies, no common primer recognition sites, and no interference between the experimental and control primers.

The RT-PCR was combined in a single-tube reaction using 1370 ng of A. thaliana or A. suecica mRNA with components of a One-Step RT-PCR kit (Qiagen Corporation). This single 50 ml mixture was reacted in an RNase-free 0.5 ml microfuge tube using a Mastercycler gradient thermocycler (Eppendorf, Inc.). The reverse transcriptase reaction was incubated at 50° C for 1 h, then at 95° C for 10 min.

At the end of the RT reaction, 18S competimer primers (Ambion Corp.) were added to the reaction tube to ensure that the abundant 18S transcript did not overwhelm the hydrolase transcripts in amplification. The ratio of 18S primers to 18S competimers in the reaction was 3:7. The 18S competimers are synthesized by Ambion Inc. in a paired fashion with 18S primers. The required ratios of the 18S competimers to 18S primers will vary for each species and each transcript with which they are multiplexed. The 3:7 ratio is used when the experimental transcript being examined is moderately expressed. The lower the expression level of the experimental transcript being expressed, the more 18S competimer is needed to limit the 18S amplification.

The PCR step was performed for 36, 40, or 42 cycles at the following times and temperatures for ILR1 and sILR1: 45 s at 95° C, 45 s at 58° C, and 1 min at 72° C. A 5 ml aliquot was removed from the main sILR1 reaction tube for 10 alternating cycles starting at cycle #18. The ILR1 reaction tubes were sampled for 10 alternating cycles starting at cycle #18, 22, or 24. The starting cycle for sampling of each ILR1 reaction was established by initial test-runs of RT-PCR to determine when visible amplification commenced. Since ILR1 did not amplify until later cycles than sILR1, the first ILR1 aliquots were withdrawn at later cycles. After sampling, the reaction tube was placed back on the thermocycler to proceed. Samples were always withdrawn during temperature ramping between cycles. Each sample tube was frozen at –20° C when removed from the thermocycler and stored for later analysis.

The aliquots were separated and analyzed by agarose gel electrophoresis on a 1% gel and stained with ethidium bromide. The RT-PCR products were imaged using an Ultralum gel documentation system (Ultralum, Inc.) and Scion computer software (Scion, Inc.). Densitometry was performed on each cDNA band by application of the ImageTool Analysis program (University of Texas Health Science Center in San Antonio). These experiments were repeated three times and averaged for each age of tissue.

Results

At 1, 2, and 3 days after germination, expression could not be detected for sILR1 (data not shown). At the same time, the 18S expression control could be clearly detected at those growth stages. This result implies that either the transcript is not being produced at these early ages or our system of detection is not sensitive enough to detect the tiny amounts of transcript produced.

Figure 2. a) sILR1 expression examined by semi-quantitative RT-PCR from 4, 5, 10, and 15 day old seedlings. 5 ?l aliquots of the RT-PCR reaction mixtures were taken for 10 alternating cycles starting at cycle #18. Analyses of plants that were 1, 2, and 3-days-old were not included here because no expression was evident. b) ILR1 expression examined by semi-quantitative RT-PCR from 1, 2, 5, 10, and 15 day old seedlings. 5 ?l aliquots of the RT-PCR reaction mixtures were taken for 10 alternating cycles starting at cycle #18, 22, or 24. c) Amplification of the 18S control. The control was expressed at the same level for all samples examined. All samples were analyzed on 1% agarose gels stained with ethidium bromide.

The expression sILR1 was first detected at day 4 (Figures 2a and 3). The important part of the curve is not the plateau but rather the linear portion. The lower the number of cycles at which the linear portion of the curve is attained, the faster the amplification occurred and the more mRNA was present for RT-PCR amplification. At day 5, sILR1 expression peaks to its highest level. At day 10, slightly less sILR1 transcript is being made, and at day 15 slightly less is being synthesized (Figures 2a and 3). The 18S expression control is consistently expressed at the same level across all the tissues (Figure 2c).

Figure 3. Semi-quantitative, coupled one-step RT-PCR (Qiagen Corp.) amplification of sILR1 cDNA from A. suecica. Total RNA was used to determine expression for each staged plant examined. Semi-quantitative RT-PCR protocol was performed by the method of Nakayama and Fujita (1992). 18S RNA (Ambion Corp. 18S primers) was used as an expression control in multiplexed reactions. The 18S cDNA was expressed consistently across the samples (data not shown). No sILR1 expression could be detected in 1, 2, or 3-day-old seedlings (data not shown).

The ILR1 transcript is initially expressed in A. thaliana at day 1 after germination. The transcript is then down-regulated over the next few days until it can be detected at a lower, but consistent level over days 5, 10, and 15 (Figures 2b and 4). The total amount of mRNA transcript does not change over that later 10-day period and remains at a mostly steady state concentration that rises a slight amount by day 15. Based on the time of emergence of the linear portion of the graph, there is a quantitatively lower level of ILR1 transcript made than that of sILR1, which can be seen when the expression graphs are directly compared (Figures 3 and 4).

Figure 4. Semi-quantitative, coupled one-step RT-PCR (Qiagen Corp.) amplification of ILR1 cDNA from A. thaliana. The 18S cDNA was expressed consistently across the samples (data not shown).

Discussion

We have isolated a homolog of the ILR1-like family of hydrolases from A. suecica. The A. suecica sILR1 gene sequence resembles the primary sequence of A. thaliana ILR1 to a high degree, and the predicted sILR1 protein also closely resembles the ILR1 gene product (Campanella et al. 2003). These two genes are expected to be structurally similar since it has been shown that A. suecica arose from a genetic hybridization thousands of years ago between the species A. thaliana and Arabidopsis arenosa (O’Kane et al. 1996). The present expression data suggest that despite this genetic closeness, the ILR1 and sILR1 enzymes may now be functioning in a different manner in the two species.

The gene product of sILR1 is clearly needed early at day 4 in the developmental process of A. suecica growth to cleave IAA-conjugates into activity. At day 5, there is a large increase in the amount of sILR1 transcript that is present. The amount of sILR1 transcript declines slightly over the next 10 days of growth, but does not decline as low as the initial expression at day 4 (Figures 2a and 3). These results suggest that a slightly greater amount of auxin is required to stimulate growth earlier (day 5) in the process than later (day 15), but that the overall levels of auxin must remain high up to day 15 of growth.

We have been unable to detect expression of sILR1 at days 1, 2, or 3. At the same time, the 18S control can be easily detected in 1, 2, or 3-day-old plants. This may suggest that our RT-PCR system is not sensitive enough to detect the low levels of transcript present, or alternatively, it may mean that sILR1 is actually not expressed until day 4 after germination. We are presently examining what occurs in sILR1 expression after day 15. For example, does expression start to decline in later development? Is sILR1 expression maintained at all in adult plants?

The ILR1 expression is first observed with a big burst at day 1 after germination, much earlier than sILR1. The ILR1 transcript starts to decline by day 2, and stays at a low, but steady level in the tissues between days 5 and 15 (Figures 2b and 4). There does not appear to be a big increase in ILR1 transcript. There are several possibilities to explain the relatively low levels of expression observed. It may be that the A. thaliana tissues do not need a burst of ILR1 because not as much auxin is required for growth compared to A. suecica tissues. Alternatively, since sILR1 and ILR1 have different substrate specificities (Campanella et al. 2003), it may be that not as much ILR1 enzyme is needed to cleave the specific IAA-conjugate forms found in A. thaliana, and so not as much enzyme is produced. A third possibility is that since ILR1 is a member of a family of related enzymes with related functions, ILR1 may be of less importance functionally in the A. thaliana ILR1-like family than sILR1 is in A. suecica. A final possibility is that we are observing differences in the level of post-transcriptional regulation in ILR1.

Our next experiments will determine if ILR1 continues to stay at a low level of expression after 15 days of age. It is certainly of interest as to whether this enzyme is important enough to be produced into adulthood in the Arabidopsis tissue. The obvious declines in ILR1 expression up to day 15 suggest that ILR1 may not be of the greatest physiological importance in later growth. Additionally, we are interested in whether there is a spatial regulation of ILR1 and sILR1, creating a difference in expression between the plant tissues. LeClere et al. (2002) have demonstrated that the ILR1-like family member IAR3 is differentially expressed between tissues; we are intrigued whether this is also the case for ILR1 and sILR1.

Our studies of enzymatic homologs give us a unique opportunity to observe molecular evolution’s direct effect on enzymatic activity and function. Our further analysis of ILR1 and sILR1 gene expression and protein products will provide a more complete picture of IAA-conjugate function and metabolism.

Acknowledgements

We wish to thank the Arabidopsis Biological Resource Center at Ohio State University for their quick response to our seed requests. We also would like to thank Mrs. Lisa Campanella for her help in editing. This work was supported primarily by a Sokol grant for undergraduate research from Montclair State University.