Journal of Experimental Botany, Vol. 54, No. 391, pp. 2343-2349,
October 1, 2003
© 2003 Oxford University Press
Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR
Received 16 January 2003; Accepted 15 June 2003
Zentrum für Molekularbiologie der Pflanzen, Allgemeine Genetik, University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
* To whom correspondence should be sent. Fax: +49 7071 295042. E-mail: friedrich.schoeffl{at}zmbp.uni-tuebingen.de
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Abstract |
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The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 °C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 °C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.
Key words: Actin, heat-shock, internal standard, mRNA quantification, real-time PCR, ribosomal protein.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Transcriptional reprogramming is the central mechanism of the heat shock response that is characterized by the synthesis of heat shock proteins (HSP) and the acquisition of thermotolerance. Although, the induction of transcription of heat shock genes by heat shock transcription factors HSF is well understood, there is much less known about of the effects of heat stress on the expression of other genes, not encoding HSP. In plants, the transcriptional activation of heat shock genes is concomitant with a general decline of the transcriptional activity of other genes. Kinetic hybridizations have shown that, in soybean seedlings, the complexity and abundance of mRNAs is significantly reduced after heat stress (Schöffl and Key, 1982), and that for some randomly selected, constitutively expressed non-heat shock genes transcription was down-regulated during heat stress (Schöffl et al., 1987), while under these conditions the transcription of heat shock genes was strongly activated. For an evaluation of quantitative changes in gene expression, the mRNA levels of ‘house-keeping’ genes are taken as internal references (Panchuk et al., 2002), as for example, ribosomal protein genes (Gao et al., 1994), or Actin 2 (ACT2), a member of a total of 10 actin genes in Arabidopsis thaliana (McDowell et al., 1996). However, their exact expression profiles and that of orthologues in other plant species have not been determined under changing environmental conditions, although significant rearrangements of the actin cytoskeleton in response to different extracellular stimuli are well documented (Staiger, 2000). By comparison with ribosomal proteins (Gao et al., 1994; Barakat et al., 2001), the actin gene family is much more complex (Baird and Meagher, 1987; McDowell et al., 1996). The ACT2 gene of A. thaliana, a popular standard for the analysis of RNA expression, is strongly expressed in vegetative tissues (An et al., 1996; Kandasamy et al., 2002). By contrast with the much-studied actin genes of A. thaliana, tissue specificity was described for only two actin genes of Nicotiana tabacum: Nt-ACT9 expressed in root, leaf, stigma, and, especially in pollen, whereas the transcripts of Nt-ACT25 were detected in pollen only (Thangavelu et al., 1993).
However, for conclusive interpretations of mRNA expression profiling it is necessary to know whether the mRNA standards are changed under the respective environmental conditions or in different organs that have been shown to be relevant for the analysis of HSP expression.
In this study real-time PCR was used to quantify precisely the relative abundances of mRNAs of A. thaliana and N. tabacum for several actin and ribosomal protein genes with respect to changes upon heat stress and in different organs. These data show that at the mRNA level the expression of actin and ribosomal proteins is negatively affected by heat stress in both species, but to a different extent. The results suggest that depending on the severity of the stress treatment, mRNA expression is differentially influenced, involving transcriptional and post-transcriptional mechanisms.
The modern methods such as microarray hybridization and real-time RT-PCR both allow precise measurements of mRNA steady-state levels. However, the expression profiling of complete plant genomes using microarray analysis is still limited to A. thaliana and appears to be especially useful for monitoring global changes of gene expression. By contrast, the advantage of real-time RT-PCR is a precise quantification of mRNA levels of genes of interest when expression levels are compared under different conditions or treatments. Another advantage of real-time RT-PCR is a very high sensitivity and specificity by using appropriate primer design, that allows discrimination of closely related genes or even allelic variants: for example, the heat-dependent expression of some genes of the APX-family, detected by real-time RT-PCR (Panchuk et al., 2002) remained undetectable in high density microarray analysis, probably due to the relatively low expression level of these genes (RA Volkov, F Schöffl, unpublished data).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material, growth conditions and stress treatment
A. thaliana plants (ecotype Columbia 24) were grown on soil in a 16/8 h light/dark cycle at 20 °C for 6 weeks, then either kept under theses conditions or the growth temperature was elevated to 28 °C or 34 °C for 3 d to evaluate the long-term heat shock effects. Twenty-five leaves of the same developmental state (from the middle of the rosette) were collected, subjected to treatments as indicated, frozen in liquid nitrogen and used for isolation of total RNA. Whole flowers, stems and young siliques of 8-week-old plants were also used for RNA extraction.
The effects of short-term heat stress were determined on 7-week-old plants, including the final cultivation for 3 d at 28 °C. The cultivation at 28 °C was chosen because it is not sufficient to induce the expression of HSP in wild-type Arabidopsis, but at this temperature the heat-stress response is clearly detected in HSF3-transgenic plants (Panchuk et al., 2002). Leaves were collected and incubated in section incubation buffer (SIB: 1 mM potassium phosphate, pH 6.0, 1% (w/v) sucrose) in a shaking water bath (60 strokes min–1) at 37 °C, 44 °C or at room temperature for 1, 2 or 4 h in the dark. The effects of post-stress recovery on gene expression were examined after immediate incubation of heat-shocked leaves at room temperature for 1 or 2 h, respectively, as indicated.
Tobacco plants were grown on soil in a 16/8 h light/dark cycle at 24 °C. For heat stress treatments, leaf discs were prepared from the middle portion of 14–16 cm long leaves of 4–5-month-old flowering plants and incubated in SIB on filter paper in Petri dishes for 2 h at 42 °C or at room temperature.
mRNA isolation and cDNA preparation
Poly(A)+-mRNA and cDNA were prepared as described by Panchuk et al. (2002). The amount of poly(A)+-mRNA/cDNA double-stranded products obtained after reverse transcription was measured using PicoGreen dsDNA Quantitation reagent (Molecular Probes). Using this method of template quantification improved the reproducibility of data of subsequent real-time PCR.
For monitoring the degree of potential template degradation during the preparation of poly(A)+-mRNA/cDNA, two primer pairs spanning proximal and distal parts of the At-ACT2 mRNA with respect to the translation stop-codon were used. Identical threshold cycles with both pairs of primers indicated the integrity of mRNA/cDNA.
Primer design and PCR-products identity
Primer pairs were designed using Primer 3 Software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) and gene sequences available in Genebank (Table 1). Gene-specific primers were chosen so that the resulting PCR product had approximately the same size of 300 bp. The quality of PCR products was visually inspected by electrophoresis, the generation of only one single band of the expected size was taken as a criterion for specificity. The identity of PCR products was confirmed by direct DNA-sequencing.
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Real-time PCR
Quantification of gene-specific cDNA was performed by the real-time PCR essentially as described by Panchuk et al. (2002). Two concentrations of cDNA (1 ng and 0.1 ng) were routinely measured in parallel and duplicate samples were run for each concentration. All experiments were repeated at least twice for cDNA prepared for two batches of plants. Using standardized conditions, deviations of threshold values were less than 1.0 cycle for independent cDNA preparations and less than 0.5 cycle for replicates of the same cDNA. The mRNA levels were normalized with the respect to the level of mRNA for ribosomal proteins in non-treated leaves, which was defined as 100%. Changes in the relative concentrations of PCR-products/steady-state mRNA levels were checked for statistical significance according to t-test (Engel, 1997).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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House-keeping gene expression standards
The database for orthologues of house-keeping genes, actin and ribosomal proteins genes, that are frequently used as standards for RNA expression analysis was screened. The sequence of the tobacco L25 ribosomal protein, which is encoded by a small gene family (Gao et al., 1994) was used for the search, but only two similar genes in A. thaliana: At-L23a (accession number AF034694 [GenBank] ) and At-L23a-like (accession number AL132954 [GenBank] ) were identified. At-L23a was chosen for further analysis.
In a search using the A. thaliana actin gene At-ACT2, several genes were found with sequence similarity in tobacco, however, it was not possible to define unambiguously one of them as an orthologue of At-ACT2. In tobacco, the actin multigene family comprises 20–30 genes, only 10 of which are completely or partially sequenced (Thangavelu et al., 1993; Moniz de Sa and Drouin, 1996). The deduced amino acid sequences of Nt-ACT9 and Nt-ACT66 show the lowest similarity (not shown), therefore both genes were included as representatives of different actin subfamilies of tobacco in further analyses.
For the gradation of different expression levels, the steady-state level of At-L23a mRNA in leaves of plants cultivated at 28 °C was defined as 100%. Taking into account that in leaves the genes of ribosomal proteins are moderately expressed (Gao et al., 1994; Moran, 2000) whereas the level of At-ACT2 mRNA is considered to be high (An et al., 1996), the following scale is proposed: (1) below 1%: very low expression; (2) 1–10%: low expression; (3) 10–100%: moderate expression; (4) 100–1000%: high expression; (5) more than 1000%: very high expression.
Changes of gene expression after heat stress in Arabidopsis
For the evaluation of heat-induced changes in gene expression in leaves of A. thaliana, mRNA levels were determined of two small HSP (sHSP) genes, At-HSP17.6 and At-HSP18.2, and of two house-keeping genes, At-L23a and At-ACT2, using equal amounts of poly(A)+-mRNA/cDNA as the template for real-time PCR (Fig. 1A). Both sHSP genes were expressed at very low levels in leaf tissue. After 1 h heat treatment at 37 °C (the temperature optimum for expression of sHSP in Arabidopsis; RA Volkov, F Schöffl, unpublished data), compared with incubation at room temperature, mRNA levels of these genes increased up to very high levels, i.e. about 1000-fold (Fig. 1A). Interestingly, after 4 h treatment the level of At-HSP18.2 mRNA still remains at a very high level, whereas the level of At-HSP17.6 mRNA declines. Both sHSP genes were also transiently induced by the incubation of the cut plant material in buffer at room temperature, up to 24-fold for At-HSP17.6 after 1 h, as compared witho non-treated leaves. By contrast with sHSP genes, the mRNA levels of At-L23a and At-ACT2 gradually increased up to 6-fold during incubation at room temperature, whereas heat-stress treatment caused a down-regulation of mRNAs to about the levels observed in untreated, fresh cut leaves.
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Treatments of heat stress followed by incubation at room temperature were combined in order to test whether mRNA levels are changed during post-stress recovery. After 1 h recovery the levels of all four mRNAs studied were not significantly changed, but the levels of sHSP mRNA became remarkably diminished after 2 h recovery: the level of At-HSP17.6 mRNA declines about 22-fold indicating that heat-inducible transcripts are subjected to rapid degradation under non-heat stress conditions. The reduction of sHSP-mRNA during recovery, with a half-life of approximately 1 h, has been originally described for soybean (Schöffl and Key, 1982). This decline was attributed to the rapid decay of sHSP-mRNA at normal temperature without replenishing the pool by transcription. Provided transcription of sHSP genes is not continued during recovery from heat stress, these data suggest that At-HSP18.2 mRNA has a half-life of approximately 1 h, whereas At-HSP17.6 mRNA disappeared with a half-life of approximately 25 min.
To test whether the severity of heat stress affects gene expression, the effects of heat shock at 44 °C were tested, which is, upon prolonged exposure, lethal for A. thaliana. Compared to the incubation at room temperature the mRNA levels of both sHSP genes increased, but only up to 15-fold for At-HSP18.2 after 4 h treatment (Fig. 1A). These data indicate that under severe heat treatment, the heat shock response is primarily blocked at the mRNA level. The mRNA levels of the house-keeping genes, At-L23a and At-ACT2, remain unchanged after 1 h treatment at 44 °C compared to the incubation at room temperature, i.e. approximately 3-fold higher than in non-treated leaves (Fig. 1A). Contrasting the incubation at room temperature, the mRNA level of At-L23a, but not that of At-ACT2, declines during the incubation at 44 °C. These data indicate that the transcription and probably also the decay of non-heat shock genes is inhibited by severe heat-stress.
Effects of growth at elevated temperatures
Both long-term acclimation at elevated temperature and short-term heat stress can induce heat tolerance in plants, but to different levels and probably by different mechanisms (Wu and Wallner, 1984). In order to compare the effects of short-term heat treatments described above with long-term acclimation at elevated temperature, mRNA levels were evaluated in leaves after 3 d cultivation of plants at 28 °C or 34 °C, respectively, compared with plants continuously grown at 20 °C (Fig. 1B). The levels of sHSP mRNA slightly increased during cultivation at 28 °C and 34 °C, whereas the levels of At-L23a and At-ACT2 mRNA remained unchanged at 28 °C and decreased (approximately 2-fold) in plants grown at 34 °C. These data indicate that the threshold for inducing opposite effects on HSP and actin gene expression is coincident with the onset of the heat shock response.
Heat-stress-dependent changes of actin and L25 gene expression in tobacco
Although the induction of expression of HSP in tobacco has been demonstrated (Park and Hong, 1998), very little is known about the effects of heat shock on other genes. To shed light on the problem, the mRNA levels of a ribosomal protein gene (Nt-L25) and two actin genes (Nt-ACT9 and Nt-ACT66) were tested prior to and after 2 h heat stress at 42 °C (the temperature optimum for expression of sHSP in tobacco; RA Volkov, F. Schöffl, unpublished results). By contrast to A. thaliana, the mRNA levels of all three genes markedly decreased after heat shock (10-fold reduction) but there was only a slight increase by incubation at room temperature (Fig. 2).
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These data show that (1) incubation of isolated leaves of A. thaliana at room temperature leads to a gradual increase of At-L23a and At-ACT2 mRNA levels. By contrast, mRNAs of sHSP were induced only transiently after 1 h treatment. The molecular mechanisms causing these changes are not known, although it is assumed that several factors such as wounding, water-logging, and/or the composition of the incubation buffer may be responsible. In tobacco, the wounding-dependence of Nt-L25 expression (Gao et al., 1994) is confirmed by these experiments, but the extent is much lower than the induction of non-heat shock genes in Arabidopsis. (2) Heat treatment at the temperature optimum for heat stress response induces very high levels of sHSP gene mRNAs, whereas the mRNA levels of house-keeping genes decrease in both A. thaliana and N. tabacum, but to a different extent. In soybean, heat-stress-dependent transcriptional reprogramming results in the transcription stimulation of HSP genes and repression of other non-heat shock genes (Schöffl and Key, 1982; Schöffl et al., 1987). Regarding the rapid decrease of mRNA of non-heat shock genes under heat shock conditions, three different mechanisms may be involved: (i) transcriptional repression/lack of induction of mRNAs, (ii) decay of specific mRNA and (iii) heat stress-dependent ‘dilution’ of certain mRNAs by changes in abundance within the pool of total mRNA. In heat-stressed soybean seedlings, the mRNA levels of individual HSP reach up to 19 thousand molecules per cell and the total amount of novel mRNA increased up to 20% (Schöffl and Key, 1982). Hence, the high proportion of HSP mRNA causes an under-representation of the other non-heat shock mRNAs, for example, house-keeping genes. However, a 10-fold decrease of ribosomal protein and actin mRNA levels observed in tobacco can not be attributed to this passive effect; it suggests that active inhibition of transcription and/or decay of these mRNA occurs upon heat stress.
Tissue-specific changes in gene expression
In order to evaluate tissue-specific changes in the expression, mRNA levels of ribosomal proteins and actin genes were measured in different organs of A. thaliana grown at 28 °C. Compared to leaves, the level of At-L23a mRNA was increased in flowers and reduced in stems and siliques (Fig. 1B). These observations are in accordance with the idea that ribosomal protein genes in plants are transcriptionally up-regulated in actively growing tissue and down-regulated in metabolically inactive tissues (Marty et al., 1993; Gao et al., 1994; Moran, 2000).
At-ACT2 is highly expressed in leaves and stems, only slightly lower in flowers and significantly lower (20-fold) in young siliques (Fig. 1B), which confirms the data of An et al. (1996), showing a strong and constitutive expression of the gene in vegetative tissues of A. thaliana. This study’s data show that the mRNA levels of ribosomal protein and actin genes are differentially modulated in different vegetative tissues. For instance, in stems as compared with leaves, At-L23a is expressed at a lower level whereas At-ACT2 remains at the same level.
To summarize, these data show that the mRNA levels of neither actin, nor ribosomal protein genes remain at an equal level under the different conditions tested. Upon heat stress, not only the activation of HSP genes, but also the down-regulation of the mRNA of non-HSP genes occur. This may reflect the necessity to save metabolic resources (energy), which have to be preferentially directed towards the synthesis of proteins with protective functions. The magnitude of changes at the RNA level of house-keeping genes (ribosomal protein L23/25, actins) appears significantly lower than for heat shock protein genes, but this can not be ignored if corresponding mRNA/cDNA is used as an internal standard in expression studies. The house-keeping genes of two model plants, A. thaliana and N. tabacum, show different responses under heat stress, demonstrating that the results obtained for one species can not be extrapolated to another one.
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