Plants treated with sulfur starvation or OAS supplementation
In this study, we focused on early adaptive responses to relatively short-term sulfur deficiency. We intended to clarify the role of OAS in the regulatory mechanism of global gene expression profile in the context of sulfur deficiency stress. Three-week-old Arabidopsis plants were treated with sulfur deficiency and OAS supplementation for 48 h. It is known that under these growth conditions the expression of the genes encoding sulfate transporter isoforms were upregulated (Takahashi et al., 1997, 2000; Yoshimoto et al., 2002). The concentrations of sulfate and GSH decreased within 48 h after transfer to sulfur-deficient medium (Figure 1), indicating that during 48 h the internal nutrient status of sulfur had changed and plants initiated to change gene expression for adaptation. However, the concentrations of cysteine did not change significantly (Figure 1) and plant did not show any visible symptom in our case. Considering that cysteine content drastically decreased under longer sulfur-deficient condition (Nikiforova et al., 2003), 48-h treatment under sulfur deficiency seemed to be sufficiently short to elucidate the early response to sulfur deficiency.
Interestingly, cysteine and GSH contents increased in roots supplied with OAS where OAS content dramatically increased, which was consistent with our experience (Sogawa Y. and Fujiwara T., unpublished results). This result was also consistent with the reports that the availability of OAS is rate-limiting for cysteine biosynthesis (Harms et al., 2000; Saito et al., 1994).
Validity of data
In micro- and macroarray analyses, reliability of signals is a key issue. First, labeled target should be carefully prepared. We checked the quality of the target by calculating the ratio of the amount of longer cDNA to that of all cDNA synthesized. When the ratio is less than 90%, cDNA should not be used as a target (Sasaki et al., personal communication). Our target cDNA was synthesized using oligo(dT)12−18 primers and sometimes did not seem to be elongated to reach the 5′ end of template mRNA. This may be the basis for different results for several ESTs corresponding to the same gene in some of our experiments. For example, ESTs APD02b05 and APD19f09 cover the 3′ region and the 5′ region of APR2 gene, respectively. The former was upregulated significantly by sulfur deficiency in leaves and roots, whereas the latter did not change significantly, although similar tendency to the former was observed (Table 4). Alternative possibility is discussed below.
Second, the elimination of false-positives is the most important matter of array analyses. Although many useful normalization strategies have been developed to correct for the systematic biases, no single normalization method has become a standard (Finkelstein et al., 2002). SAM, developed by Tusher et al. (2001), assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements and identifies genes with statistically significant changes in expression. A number of papers were published in which SAM was successfully used for data analyses (Blanchard et al., 2001; Detweiler et al., 2001; Furlong et al., 2001).
We also analyzed the data with Student's t-test using normal (i.e. not logarithmic) value data (local background subtracted, or not subtracted), and with SAM using the normal value data, to identify the clones induced/repressed significantly. The global induction and repression patterns obtained by three different analyses led to the same conclusions that: (i) there was a positive correlation between the degrees of induction/repression by sulfur deficiency and by OAS application both in leaves and roots; and (ii) there was no correlation between the degrees of induction/repression by sulfur deficiency in leaves and roots (see below).
Possibility of cross-hybridization
We observed that the degrees of induction/repression calculated from the signal intensities for several ESTs corresponding to a single gene were different to each other. This might result from cross-hybridization between the isoforms of gene families. As mentioned above, in case of APR2, APD02b05 was upregulated significantly by sulfur deficiency in leaves and roots, whereas APD19f09 did not change significantly (Table 4). The former clone includes the 3′ untranslated region which has low homology between the isoforms, whereas the latter covers only the coding region conserved in three APR genes to cause obscure result.
Global expression profile in response to sulfur deficiency
The main purpose of this study is to profile the global gene expression in response to relatively short-term sulfur deficiency, and to describe the trends of gene expression in response to sulfur deficiency and OAS. For this purpose, we plotted the degree of induction/repression by sulfur deficiency against that by OAS treatment. The positive correlation between them suggests that OAS mediates the expression of not only sulfur-assimilatory genes but also other genes in response to sulfur deficiency. However, this does not necessarily mean that OAS is the only compound important for regulation. Cysteine and GSH are known to be negative regulators of gene expression (Bolch et al., 1999; Lappartient et al., 1999; Vauclare et al., 2002; Vidmar et al., 1999, 2000). In our experiments, cysteine and GSH contents in roots treated by OAS increased (Figure 1). As shown in Table 3, some ESTs were upregulated by both sulfur deficiency and OAS treatment, whereas others were upregulated under sulfur deficiency and downregulated by OAS treatment. In the latter case, increased GSH might overcome the positive effect of OAS on gene expression. It is interesting that there is no EST in Table 3, which was repressed significantly under sulfur deficiency and induced significantly by OAS. Such an expression pattern could not be explained simply by OAS and GSH contents. We assume the several regulatory pathways of gene expression under sulfur deficiency. Some genes may be regulated via OAS, some may be via cysteine and/or GSH, and others may be regulated by the combinations of OAS, cysteine and GSH or by unknown factor(s). For example, the gene encoding the β subunit of β-conglycinin is shown to be regulated by the ratio of OAS and methionine levels (Hirai et al., 2002).
In OAS-supplemented leaves, the increase in OAS content was little compared to that in sulfur-starved leaves, although the change was statistically significant (Figure 1). However, the global transcript profile changed similarly to the case of sulfur-starved leaves. This observation depicts a possibility that OAS content in particular organelles might increase sharply. To better understand the regulatory mechanism, concentrations of metabolites should be determined in specific organelles or cell types where the regulatory mechanism works.
Putative functions of the genes regulated by sulfur deficiency and/or OAS
For identification of the genes by comparison of two or more experimental conditions, we used a statistical analysis SAM (see Experimental procedures) and identified a list of genes whose expression was regulated (Tables S1 and 3).
More than half of identified genes were downregulated under short-term (2 days) sulfur deficiency (Table 2). It is in contrast to the report by Nikiforova et al. (2003) where more genes were upregulated under relatively long-term deficiency. They also observed that the ratio of the number of the upregulated genes to that of the downregulated genes increased with increasing duration of sulfur deficiency (6–10 days). Drastic decrease in the environmental sulfate concentration resulted from medium exchange might cause the general and temporal downregulation of gene expression, and then specific genes might be upregulated afterward.
In our experiments, the genes involved in JA biosynthesis and those induced by JA or MeJA treatment were upregulated (Table 3). It is quite interesting that under different experimental conditions, Nikiforova et al. (2003) also found that JA biosynthetic pathway was induced. Recently, He et al. (2002) reported that exogenous application of JA caused premature senescence in the wild-type Arabidopsis. JA levels increased in senescing Arabidopsis leaves and genes encoding the enzymes that catalyze most of the reactions of the JA biosynthetic pathway were differentially activated during leaf senescence, suggesting that JA may play a role in the senescence program (He et al., 2002). Although our plant samples subjected to sulfur deficiency did not show any changes in appearance, it is reasonable to consider that senescing program mediated by JA had just started in these plants for redistribution of internal sulfur. In addition, OAS induced the same response. Interestingly, it is reported recently that sulfur deficiency-responsive APR1 gene is upregulated by MeJA and its precursor 12-oxo-phytodienoic acid (OPDA) (Sasaki-Sekimoto et al., 2003).
Almost all genes classified to protein degradation category are components of ubiquitin-dependent proteolytic pathway. Proteins destined for degradation are tagged by conjugation to ubiquitin, whose process is carried out by an enzyme complex consisting of an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin-ligating enzyme (E3). E3 and other proteins assemble to form SCF (Skp1-Cullin-F-box protein) complex (Spremulli, 2000). Recently, SCFCOI1 ubiquitin-ligase complexes are shown to be required for the response to JA in Arabidopsis (Xie et al., 1998; Xu et al., 2002). SCF complex also plays an essential role in auxin response (for review, see Gray and Estelle, 2000). In our study, nitrilase 1 and auxin response factor 1 were also regulated (in others category). Nitrilases (NIT1, NIT2, and NIT3) are involved in auxin biosynthesis, and NIT3 promoter activity is strongly induced by sulfur starvation and OAS, suggesting that NIT3 plays a role in the regulation of root morphology in sulfur-starving Arabidopsis (Kutz et al. 2002). Nikiforova et al. (2003) also suggested the involvement of auxin in the sulfur starvation response. SCF complex-mediated JA and/or auxin responses might occur under sulfur deficiency. In relation to this, genes involved in defense response to pathogens, which is known to be mediated partly by JA, were identified among the clones regulated by sulfur deficiency and OAS.
AT1g13930, a putative hydroxyproline-rich glycoprotein (HRGP) gene was upregulated in leaves and downregulated in roots (Table 3, cell wall synthesis category). In Chlamydomonas reinhardtii, two cell wall-localized HRGPs, which are very poor in sulfur-containing amino acid, accumulated under sulfur deficiency as the substitutes for sulfur-rich cell wall components (Takahashi et al., 2001). Database search against GenBank and MAtDB (MIPS Arabidopsis thaliana database; http://mips.gsf.de/proj/thal/db/) revealed at least four other putative HRGP genes of Arabidopsis. Peptide for AT1g13930 has second least sulfur-containing amino acids (1.9%) among the five, implying the similar adaptive strategy in leaves to C. reinhardtii.
When C. reinhardtii was starved of sulfur, there was a dramatic reduction in the level of photosynthesis due to loss of functional photosystem II reaction centers (Wykoff et al., 1998). It may be the case with Arabidopsis. In our study, however, sulfur-sufficient plants which were grown in plastic plate did not seem to have sufficient photosynthetic activity, judging from the fact that they could not grow to maturity without exogenous sucrose supply (data not shown). It may be the reason why the genes for photosynthesis were downregulated mainly in roots.
Genes involved in sugar metabolism were also regulated. In leaves, At5g17310 (UDP-glucose pyrophosphorylase) was downregulated, and two glycolysis/gluconeogenesis-related genes AT3g04120 and AT2g21330 (glyceraldehyde-3-phosphate dehydrogenase C subunit and putative fructose bisphosphate aldolase, respectively) were upregulated. It may suggest that sucrose synthesis for carbon storage was repressed and glycolysis occurred to generate energy. However, Arabidopsis has a number of isoforms for each step of sugar metabolism, and each isoform may express in specific organs, tissues and organelles under specific regulation. In fact, the isoforms were regulated differentially by sulfur deficiency (data not shown), suggesting more complicated and fine regulation.
In our study, AT1g75280 (NADP oxidoreductase, putative) was upregulated in leaves and roots. This is one of the highest over-expressers in the study of Nikiforova et al. (2003) and they gave a good speculation on its function. Several other genes (AT1g19570, AT2g34420, AT5g54270, AT4g16190, and AT5g34850) were shown to be regulated significantly both in Nikiforova et al. (2003) and our (Table 3) experiments.
Compared to Nikiforova et al. (2003), the degrees of induction/repression were generally lower in our study (Table 3). There are several possible reasons for this fact. First, as mentioned above, concentrations of metabolites in the sulfur assimilation pathway were not so severely influenced in our plants compared to the case of Nikiforova et al. (2003) possibly resulting in mild change in gene expression. Second, lower fold change was due to our analytical method in which the data were converted to logarithm for normalization. We normalized the raw signal intensities after conversion to logarithmic values according to Equation (1) (see Experimental procedures). As MED was greater than 10, relative intensity(1) became smaller than relative intensity(2) calculated without conversion to logarithmic value according to:
Then, the degree of induction/repression, calculated as the ratio of the average relative intensities, became smaller when using Equation (1). For example, the degrees of induction of AT3g45140 (AtLOX2) and At1g76680 (OPR1) in sulfur-starved leaves calculated by Equation (1) were 1.77 and 1.52, respectively (Table 3). On the other hand, when calculated by the Equation (2), the degrees were 2.95 and 2.24, respectively.