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Plant Physiology 135:2379-2391 (2004) © 2004 American Society of Plant Biologists Gene Expression Profiling of the Tetrapyrrole Metabolic Pathway in Arabidopsis with a Mini-Array System1,[w]Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226–8501, Japan
Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.
Tetrapyrrole compounds play an essential role in all living organisms. They are involved in various metabolic processes such as energy transfer, signal transduction, and catalysis (Dailey, 1990  ). In plants, at least three structurally and functionally distinct classes of tetrapyrroles can be distinguished, Mg-porphyrins, Fe-porphyrins, and phycobilins. Mg-porphyrins, such as chlorophylls (Chls), are the most abundant tetrapyrrole compounds on earth. As part of the photosynthetic apparatus, the Mg-porphyrin rings of Chl a and Chl b are involved in light trapping and charge separation for photosynthesis. In addition, Mg-porphyrin intermediates have been shown to regulate the transcription of nuclear-encoded photosynthetic genes in plant systems (Susek and Chory, 1992; Rodermel, 2001). Fe-porphyrins, hemes, constitute the second class of tetrapyrroles. In particular, protoheme functions as a redox-active cofactor or prosthetic group in many proteins. For instance, protoheme is bound to the various cytochromes of the plastidic and mitochondrial electron transport chains and to soluble enzymes, such as catalase and peroxidase. Apart from these functions, heme and its derivatives have also been shown to regulate a variety of cellular processes in animal and bacterial systems, such as transcription (Guarente and Mason, 1983), translation (Chen et al., 1989; Joshi et al., 1995), and posttranslational protein translocation (Lathrop and Timko, 1993). Phytochromobilin belongs to the third class of plant tetrapyrroles, the phycobilins. In contrast to Chls and hemes, phycobilins are linear pigments. Phytochromobilin is covalently bound to the phytochrome apoprotein (Lagarias and Rapoport, 1980), and this holocomplex is known to act as a photoreceptor that controls various developmental processes (Furuya, 1993).
In plants and many bacteria, all of the tetrapyrroles originate from a common biosynthetic pathway (Beale, 1999
With the sequencing of the entire genome of Arabidopsis (Arabidopsis-Genome-Initiative, 2000 In this study, we developed a novel mini-array system to monitor the expression profile of the tetrapyrrole biosynthetic pathway in Arabidopsis. Using this system, we illustrate the profiles of changes in gene expression in terms of the onset of greening, endogenous rhythm, and developmental control. Our findings demonstrated the potential of our mini-array system for detection and analysis of the expression of genes involved in tetrapyrrole biosynthesis in a genome-wide fashion. With this system, we clearly showed that the genes corresponding to regulatory enzymes of tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated by endogenous factors to control the biosynthesis.
Development of the Mini-Array System
Our database search of the Arabidopsis genome identified 35 genes involved in the biosynthesis of tetrapyrrole (Table I) from numerous previous studies and a recent database search by Lange and Ghassemian (2003)
To enable quantitative analysis of the expression of each gene with high sensitivity, precautions were taken to produce long (150–300 bp) DNA probes immobilized on the membrane with high gene specificity. When coding sequences with high homology were found between isoforms, we chose gene-specific regions, such as the 3'-untranslated region, and amplified them by reverse transcription (RT)-PCR (Supplemental Fig. 1, available at www.plantphysiol.org). Sequences of primers used for amplification are listed in Supplemental Table I. The specific hybridization of each RT-PCR product was confirmed by genomic southern analysis to give a single band (Supplemental Fig. 2). Resultant DNA fragments were purified and used as DNA probes for the mini-array system. Probes for ubiquitin (UBQ10; Sun and Callis, 1997 ) and actin (ACT8; An et al., 1996) as housekeeping genes, the light-harvesting Chl a/b complex of photosystem II (Lhcb) as a marker gene of chloroplast development, and  DNA as negative control were also spotted. We also spotted various chloroplast-related and phytohormone-responsive genes (Table I). Consequently, a total of 48 probes were spotted in duplicate in a nylon membrane, in which one set of all probes was designed to spot in one grid area (Fig. 1A). Since the amount of probe material has a strong impact on the sensitivity and reproducibility (Bertucci et al., 1999), exactly 4 ng (equivalent to 1 x 1010 molecules) of each probe was immobilized to each spot to facilitate quantitative analysis.
To evaluate the quality of this mini-array, we carried out a pilot hybridization experiment with two independent samples: total RNA isolated from 3-d-grown etiolated seedlings and subsequently 9-h-illuminated seedlings. In both samples, two mini-array membranes were hybridized simultaneously with 33P-labeled cDNA probe synthesized from each sample. Consequently, four images were obtained from each sample, which were almost indistinguishable either in the same or in another mini-array membrane (Fig. 1A). To make quantitative evaluations, the filter background, which is 0.8 times the lowest signal, was subtracted from the hybridization value, and then normalized by division with the signal intensity of the housekeeping ACT. This lower limit was selected since at levels closer to the background, a slight variation in background levels across the filter artificially affects the profile (see "Materials and Methods"). Figure 1, B and C, shows plots of two representative sets of data on row signal intensity from two samples. A good correlation was obtained in all data sets. We observed a linear correlation with a mean correlation factor of more than 0.98 and mean slope of 1.03 to 1.04 with a SD of less than 0.09. Thus, the developed mini-array system has a high degree of reproducibility and is valid for linear quantitative determination of the expression of genes of tetrapyrrole biosynthesis in a genome-wide fashion. When averaged data from two samples were compared, most of the spots showed significant changes in their expression level (Fig. 1D).
During chloroplast development, higher plants need to synthesize Chl synchronously with Chl-binding proteins. In fact, it has been demonstrated that the expression of Chl-binding proteins and key enzymes of tetrapyrrole biosynthesis, such as glutamyl-tRNA reductase (Tanaka et al., 1996
After subsequent data processing, normalized expression (NE) values (see "Materials and Methods") were obtained for each gene. The NE values were further transformed into values relative to the NE values of a dark control (0 h), since these relative values are generally favorable for categorizing expression profiles, while the NE is valuable for evaluating absolute expression levels. Accuracy of resultant expression profiles was confirmed by northern-blot analysis (Supplemental Fig. 3). Subsequently, using the profile of the relative value from each gene, we performed clustering for all the genes using self-organizing mapping (SOM; Tamayo et al., 1999
Rhythmic Regulation of the Genes of Tetrapyrrole Biosynthesis in Mature Plants
In mature leaves that possess developed chloroplasts, the synthesis of Chl and Chl-binding proteins is closely coordinated and regulated by light and the endogenous clock for the assembly of a functional photosynthetic apparatus (Beator and Kloppstech, 1993 After data processing, the SOM analysis clustered genes into three categories: the first group is regulated by both diurnal and circadian rhythms, the second is only regulated by diurnal rhythm, and the third is nonrhythmic (Fig. 3). In these plots, to compare the relative changes in gene expression throughout the tested periods, the NE value from each gene was normalized by dividing by the average NE value of each gene. In this case, the resulting relative ratio gives the relative fluctuation of gene expression and the average of NE is the average expression level relative to that of ACT.
The first group contained six genes. Interestingly, this group was composed of the four genes in cluster c1 and the two genes in cluster c4 (Fig. 3, A and B). The phase and amplitude of the four genes in c1 were synchronized and coincided with that of Lhcb, whereas those of the two genes in c4 (PORA and PORB) were synchronized to each other, but the peak of oscillation was somewhat delayed compared to that of c1. These results suggest that four genes in c1, which are dramatically induced by illumination at the onset of greening, are also important for coordinating the assembly of a functional photosynthetic apparatus with the endogenous clock, and two genes in c4 may be differentially regulated by the endogenous clock to maintain Chl biosynthesis. As shown in Figure 3, C and D, 19 genes were clustered into the second group, which was regulated only by diurnal rhythm. This group was composed of the c2 and c3 clusters and 16 genes corresponding to the earlier steps of tetrapyrrole biosynthesis up to the metal insertion stage. Moreover, the phase and amplitude of all genes coincided well under diurnal light/dark cycles (Fig. 3C). When plants were transferred to continuous light, the rhythmic expression of these genes disappeared, although some fluctuations remained (Fig. 3D). The remaining genes involved in the c2 and c3 cluster did not show a pronounced change in expression under diurnal or circadian growth conditions (Fig. 3, E and F).
Finally, we determined the development-dependent control of the expression of these genes. Whole plants were sampled every week after sowing and analyzed using the mini-array system (Supplemental Table IV). The relative expression level in each developmental stage was calculated by dividing the NE value of each developmental stage by the average NE value for all the stages. In this experiment, regardless of the clusters of light responsiveness, all genes basically showed a similar profile of expression (Fig. 4). The highest level of gene expression was observed after 1 week of germination, after which there was a decline to the average level, which was retained until week 4. Although several genes showed a peak of expression after 3 weeks of germination, we could not find definite correlation of these genes with the gene clusters. The level then declined after 5 weeks of germination. Thus, it is likely that the genes of tetrapyrrole biosynthesis tested with this mini-array system are similarly controlled by development, although they markedly differed in the light-responsive and rhythmic control of their expression.
In this study, we developed a mini-array system to monitor the genes of tetrapyrrole biosynthesis in Arabidopsis. While the technical principle is not new, owing to precautions taken to prepare DNA probes of 150–350 bp with high gene specificity and to immobilize an exact quantity (4 ng) of highly purified probe to nylon membranes, the data presented here show that this mini-array method has good reproducibility and sensitivity (Fig. 1). It has been reported that the sensitivity of mini-arrays was dependent on the concentration of probe on the filter (Bertucci et al., 1999 ), and 4 x 109 molecules/spot are necessary for a quantitative analysis using the mini-array method with colorimetrical detection (Quere et al., 2002). In this study, 4 ng of probe DNA is equivalent to 1 x 1010 target molecules/spot, if one assumes that the average probe size is 300 bp and all the materials spotted bind to the membrane and are available for hybridization. Thus, this mini-array system is considered to have a sensitivity comparable to that of other macro- and microarray systems. Furthermore, a significant advantage in cost enabled gene expression analysis of various physiological events, such as greening, rhythmic control, and developmental control. We have confirmed that it is possible to reuse membranes several times after stripping the target DNA (data not shown). Thus, this method is effective and inexpensive for quantitative analysis of the expression of each gene involved in tetrapyrrole biosynthesis in Arabidopsis.
Using this system, we obtained information on the absolute value (NE) and the value relative to appropriate control levels of gene expression, which is important to evaluate the exact contribution of each gene and the profiles of each expression, respectively. The calculation of NE value comprises two steps, subtraction from an appropriate background value and normalization to that of ACT. The choice of background was critical as the profiles of genes expressed at very low levels were greatly affected by slight variations in the background level. We found that a background 0.8 times the level of the lowest signal usually gave a linear correlation and high reproducibility, as in the case of cDNA macroarray experiments (Obayashi et al., 2004
So far, several algorithms are available for clustering array data, such as one-dimensional and two-dimensional SOM. In this study, we use one-dimensional SOM for clustering, since this procedure is simple to understand and appropriate for categorizing small groups of genes. In clustering, a smaller number of categories is better for understanding the physiological characteristics in each cluster. In this study, the genes of tetrapyrrole biosynthesis were clustered into four major groups based on their expression profiles during greening. Genes in c1 and c4 were clearly distinguishable as having pronounced changes in expression, although the classification of genes in c2 and c3 was somewhat obscure. We carried out repetitive SOM analyses by modifying the number of categories, and finally determined four gene clusters. These four categories showed a good correlation with the maximum expression ratio. Moreover, genes in the same group were similarly controlled in an endogenous rhythm. A summary of gene clustering and the NE value of each gene in 3-d-old etiolated and 3-week-old mature seedlings are shown in Figure 5.
The c1 Cluster Comprises the Most Important Regulatory Genes in Tetrapyrrole Biosynthesis Four genes in the c1 cluster were rapidly induced by light at the onset of greening and synchronously fluctuated under diurnal and circadian rhythms. It should be noted that the oscillation and amplitude of the rhythmic regulation of these genes are synchronized to those of Lhcb. The pronounced regulation of these genes suggests that they are the most important regulatory gene group for the biosynthesis and flow into the Chl branch pathway of tetrapyrroles.
The first gene in c1 was HEMA1 encoding glutamyl-tRNA reductase, which is the first committed enzyme of ALA biosynthesis. The formation of ALA is the rate-limiting step for tetrapyrrole biosynthesis and is the primary determinant of the rate of Chl synthesis in light-grown plants (Beale and Weinstein, 1991
The second gene in c1 was CHLH encoding a subunit of Mg-chelatase, which is the branch point of Chl and heme biosynthesis. Mg-chelatase is composed of three subunits, which are commonly referred to as ChlI, ChlD, and ChlH. CHLH is demonstrated to be the protoporphyrin IX-binding subunit (Gibson et al., 1995
The third gene in c1 was CRD1, which had the highest NE value among all the genes tested. This gene was first isolated from pea as a mesophyll-specific cDNA that is regulated by a phytochrome and circadian rhythm (Zheng et al., 1998
The fourth gene in c1 is CAO encoding chlorophyll(ide) a oxygenase, which is responsible for Chl b biosynthesis (Tanaka et al., 1998
Most genes in the biggest cluster c2 were involved in the earlier steps of tetrapyrrole biosynthesis up to the insertion of metal. All genes in c2 were induced by light at the onset of greening and showed a diurnal fluctuation in mature leaves. Thus, it is likely that the genes in c2 encoding the enzymes of tetrapyrrole biosynthesis are similarly controlled to provide tetrapyrrole intermediates for the production of Chl and heme in photosynthetic tissues. As these genes were not regulated by circadian rhythm, light may be the primary determinant for the gene expression. A POR isoform, PORC, and CHLG encoding Chl synthetase were in this group. The light-induced induction of PORC was previously reported (Oosawa et al., 2000
The cluster c3 comprised genes that were constitutively expressed with high NE values, such as HO1, CPO1, and CPO2, and with very low NE values, such as HEMA3, HO3, and HO4. It is probable that some genes in this group are pseudogenes or their functional contribution is not significant. Alternatively, it is possible that they are induced under particular environmental conditions, such as stress. In fact, the expression of HEMA2 and FC1 in Arabidopsis is known to be induced by a cytoplasmic protein synthesis inhibitor, cycloheximide (data not shown), as well as cucumber (Suzuki et al., 2002
Two genes in the cluster c4 were well-known isoforms of PORA and PORB, which were negatively regulated by light. The proteins encoded by these two genes are known to be the major components of prolamellar bodies in etioplasts of dark-grown seedlings (Masuda et al., 2003
In summary, we have developed a mini-array of the genes involved in the biosynthesis of tetrapyrrole in Arabidopsis. The system is reasonably cheap, is highly sensitive, and can be used to monitor the expression profiles of all genes specifically and simultaneously. All these parameters will contribute to the study of other metabolic or regulatory genes of interest. With this system, we could group genes into four categories based on their expression profile during greening, although c2 and c3 have insufficient features to classify further. Clustering of c1 and c2 was also applicable to the rhythmic expression of the genes of tetrapyrrole biosynthesis. Interestingly, a good coordination of gene expression was observed in each group, suggesting that each group of genes is regulated by common regulatory machinery. Among them, the c1 cluster contained the most important genes that control the biosynthesis and efflux of tetrapyrroles in Arabidopsis. Further analysis with the aid of this system will contribute to the elucidation of the complete regulatory circuit of tetrapyrrole biosynthesis in Arabidopsis.
Plant Materials and Growth Conditions
Seed stocks of Arabidopsis L. Heynh from the Columbia (Col) ecotype were used for all experiments. Surface-sterilized seeds were plated on a 0.8% (w/v) agar medium containing Murashige and Skoog (1962)
Total RNA was prepared from 3-d-old etiolated or 2- to 4-week-old mature plants with an RNeasy Mini Kit (Qiagen, Chartsworth, CA) as recommended by the manufacturer. RT-PCR was carried out with RNA PCR kit (Avian Myeloblastosis Virus) version 2.1 (Takara, Otsu, Japan) with 1 µg of total RNA according to the manufacturer's instruction. RT-PCR amplification was carried out with cognate gene-specific primers (Supplemental Table I) as follows: 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The DNA fragments were checked by agarose electrophoresis (Supplemental Fig. 1), cloned into a TA-cloning vector, and subsequently sequenced using a Thermo Sequenase Sequencing kit (Amersham Bioscience, Piscataway, NJ). The inserted DNA fragment of each clone was amplified by PCR with universal M13 forward and reverse primers. PCR products were purified with a Qiaquick PCR Purification kit (Qiagen, Valencia, CA), and their DNA concentration was determined by spectrometry and agarose electrophoresis. Using the spotting machines, the Hydra Microdispenser and Tango Liquid Handling system (Shieh et al., 2002
The gene specificity of the DNA probes was confirmed by genomic southern hybridization analysis (Supplemental Fig. 2). Genomic DNA was isolated from Arabidopsis seedlings according to Murray and Thompson (1980)
Total RNA was prepared from samples as described above. With the resultant RNA sample (10 µg) as a template, the labeling of the target DNA was carried out by RT in the presence of [
Radioactive images were obtained with a high-resolution scanner (Storm, Amersham Bioscience), and the signal intensity was quantified with Array Vision software (Amersham Bioscience). Quantitative analysis of hybridization with the same probe resulted in high degree of reproducibility (Fig. 1). To normalize the hybridization signal intensities of each membrane, it was first necessary to subtract the appropriate background value. The subtraction of a real value, such as the signal intensity of nonspotted area or
For the clustering of gene expression profiles, one-dimensional SOM was carried out using GeneCluster2 released by the Whitehead Institute (http://www-genome.wi.mit.edu/cancer/software/software.html). As a result of a trial altering the number of categories, four major clusters were obtained by the SOM of gene profiles during greening. Similarly, analysis was carried out in terms of profiles of rhythmic and development-dependent expression.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
We are grateful to Hiroshi Shimada for valuable discussions. We would like to thank Nozomi Taki for technical assistance in preparing the radio-labeled cDNA targets for hybridization. Received March 10, 2004; returned for revision April 30, 2004; accepted May 3, 2004.
1 This work was supported by a grant from the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology, and by a project titled Development of Fundamental Technologies for Controlling the Process of Material Production of Plants based on funds provided by the Ministry of Ecology, Trade and Industry in Japan. 
2 Present address: Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo, 3–8–1 Komaba, Meguro, Tokyo 153–8902, Japan.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.042408. * Corresponding author; e-mail tmasuda{at}bio.titech.ac.jp; fax 81–45–924–5823.
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ABSTRACT
RESULTS
-33P] dCTP and 1 µg of oligo(dT)16–18 primer with a SuperScript First-Strand Synthesis system (Invitrogen) according to the manufacturer's directions. The labeled cDNA was denatured and used as target DNA for hybridization. Hybridization with the labeled target was performed in the presence of 0.5 M Na2HPO4, 1 mM EDTA, and 7% SDS (Church and Gilbert, 1984
