Physiological response of rice to drought stress
The aim of this study was to identify mechanisms with a general relevance for drought tolerance in rice by comparing cultivars that differ in tolerance to long-term drought stress. We focused on long-term stress, as we were most interested in mechanisms that contribute to performance of rice in an agronomic environment under upland growth conditions where drought stress often persists for a considerable time of the plant’s life cycle. For the varieties used in our study, life cycle is three to four months. A stress treatment of more than two weeks in the juvenile phase, which is an especially drought-sensitive growth stage (Banoc et al. 2000; Kamoshita et al. 2004), can thus be considered long-term. Seedling vigor, the ability to keep a high biomass alive during drought stress, has been shown to be essential for recovery and final yield in field and greenhouse experiments (Kamoshita et al. 2004). Mechanisms identified to keep the plant vital during drought stress in the juvenile stage are thus relevant for performance in a drought-prone environment.
The response of plants to stress will depend not only on the duration, but also on the degree of stress imposed. We used the parameters leaf water potential, growth reduction and drought score to characterize the degree of stress and to allow comparison with results from other experiments. The water potentials observed under drought stress in our experiments were comparable or higher (less negative) to those found in drought stress experiments under field conditions (Turner et al. 1986; Jongdee et al. 2002; Kamoshita et al. 2004). The reduction of shoot biomass by about 75% was more severe than in moderate drought stress trials that resulted in 25–50% yield loss (Babu et al. 2003; Fischer et al. 2003; Lanceras et al. 2004), but less severe than in terminal drought stress trials (Lafitte et al. 2006). Based on the drought score, the stress imposed in our experiments yielded less or similar damage than the stress treatment in field trials (Babu et al. 2003). Thus, the stress imposed can be classified as moderate to strong long-term drought stress comparable to stress under field trial conditions.
The relevant parameter for a stress tolerant crop is yield: varieties that produce more grain under stress than sensitive cultivars are considered tolerant (Fischer et al. 2003). The parameter yield cannot be determined in a short-term test like ours. We therefore used so-called secondary traits to estimate tolerance. The parameter absolute biomass at the end of the drought stress was chosen as it is associated with superior recovery ability after stress release (Fukai and Cooper, 1995; Kamoshita et al. 2004). The parameter drought score, which is based on leaf survival, was used as it correlates to yield and shows the best heritability of those secondary traits that can be scored in the vegetative stage (Fischer et al. 2003). Furthermore, we found a higher reproducibility of a tolerance classification based on these parameters compared to other parameters (e.g. PAM measurements, height, tiller numbers; data not shown).
Based on the secondary traits absolute biomass and drought score, 21 cultivars, including 17 Vietnamese cultivars from a breeding program for drought stress resistance, were characterized for drought tolerance in our experimental system. The two sensitive cultivars (NB and TP) and the two tolerant cultivars (LC and IR) were chosen as they showed the most stable response over three independent experiments. The characterization of drought tolerance was done in an experimental system with a low soil depth, in which water was supplied from above. This system mimics an upland field with a shallow soil layer and insufficient water supply by rain or irrigation. The effect of differences in rooting depth on the tolerance assessment, which is often linked to superior performance under drought conditions (Kamoshita et al. 2000; Wade et al. 2000), was reduced in the experimental system. Indeed, shoot:root ratios under drought stress did not differ significantly between cultivars. In spite of that, both tolerant cultivars depleted the soil water more than the sensitive cultivars. At the same time, the higher (less negative) mid-day water potentials in the tolerant cultivars suggest a lower degree of stress compared to the sensitive cultivars. This is confirmed by the higher harvest biomass and significantly higher water use efficiency in the tolerant compared to the sensitive cultivars. Thus, the tolerant cultivars were able to use more of the available water and use it more efficiently for dry matter production. Maintenance of a high transpiration rate during periods of severe drought correlates with a superior recovery of young plants when drought is released (Wade et al. 2000). Within a group of closely related double-haploid rice lines, not only high transpiration rates during drought stress were linked to drought tolerance, but also high water use efficiency (Siopongco et al. 2006). The adaptive mechanisms of LC and IR, that both show high water uptake and water use efficiency, are thus relevant for the selection of improved cultivars within the ‘more crop per drop’ strategy.
Drought effects on gene expression
Transcript profiles of leaf samples from control and drought stressed plants were generated to identify genes and pathways that may contribute to the higher tolerance and water use efficiency of LC and IR compared to NB and TP. The sequence data from one of these cultivars, Nipponbare (NB) are the basis of the gene models from the TIGR Rice Annotation, that were used to design the NSF oligonucleotide microarray. This array contains about 50% of the rice predicted genes models. As the oligonucleotides on the array are short (50–70 bases) and only a single oligonucleotide has been spotted per gene, sequence differences between the cultivars could result in a stronger hybridization of labelled cDNA from the japonica cultivars compared to the indica cultivar IR. Obviously, also the expression of genes in the indica cultivars that are not present in the japonica genome could not be detected with the arrays used in our study. We did not optimize the design of the experiments and data evaluation to identify constitutive differences in gene expression between tolerant and sensitive cultivars, although they could also be a source of increased stress tolerance.
We focused on genes that differed in their response to drought stress between two tolerant cultivars on the one hand and two sensitive cultivars on the other hand. In statistical terms, this means that we searched for genes showing a significant interaction effect between condition and tolerance group. To validate our method, we checked, whether genes that had previously been described as drought induced in rice or other monocots can be found among those that showed a significant effect of condition on expression in our experiments. Among the genes that were significantly drought induced, we indeed found metallothioneins and late embryogenesis abundant proteins that had previously been found to be induced in young rice plants under long-term drought stress (Reddy et al. 2002; Hazen et al. 2005; Markandeya et al. 2005, 2007) and in barley and Arabidopsis thaliana (Ozturk et al. 2002; Seki et al. 2002; Talame et al. 2007) under drought stress. Also, cytochrome P450 family proteins and serine/threonine protein kinases that were prominent among genes in EST libraries from drought-stressed rice plants (Reddy et al. 2002) showed a significant effect of condition in our study.
To facilitate a functional interpretation of the changes in gene expression of rice in response to drought stress, we used the published sequence of Oryza sativa cv. Nipponbare (Matsumoto et al. 2005) for a homology search to the Arabidopsis genome and sort the genes that we found expressed on the NSF array into functional categories, using the established MapMan bins. We used two statistical methods to identify those bins in which gene expression was strongly affected by drought. In the first approach, the mean induction factor for all genes in a bin was calculated and compared to the mean induction factors of all other bins. In the second approach, the percentage of genes with significantly changed expression in a bin was compared to the overall percentage of genes with significantly altered expression. Both approaches can lead to completely different but biologically meaningful results. If half of the genes in a bin are strongly repressed and the other half is strongly induced, the average induction factor will not be significantly different from zero. However, the percentage of differentially expressed genes will be 100% and therefore significantly different from the overall percentage of regulated genes. Such a pattern might be expected if expression of genes within a large family switches from a set of genes coding for nontolerant isoenzymes to stress tolerant isoenzymes. On the other hand, most of the genes in a family could be induced just below the set threshold and only a few above it. In this case, the percentage of significantly induced genes would not be different from the general mean, but the average induction factor for the bin could be significantly higher than the average over all other bins. As both situations, switch to different genes of a family and weak but concordant induction of many genes in a functional group, could be important for the identification of functional categories relevant for drought stress responses, we used both approaches.
Like other authors (Munne-Bosch and Alegre 2004; Hazen et al. 2005), we found strong evidence that drought stress causes a transition of metabolism from protein synthesis to degradation in rice. Amino acid activation and synthesis of ribosomal proteins were down-regulated, and amino acid and protein degradation, especially by the ubiquitin pathway, were up-regulated. Together with the general down-regulation of protein synthesis, genes coding for proteins of the photosynthetic light reactions were repressed as well, especially those of photosystem II. This corresponds to the visible bleaching of drought-stressed leaves and a decrease in photosynthetic activity (Do and Zuther, unpublished data). Photosystem II activity and its main regulatory mechanisms are severely affected by drought (Pieters and El Souki 2005). Down-regulation of photosynthesis genes under drought stress has been observed before in rice and barley under moderate long-term drought-stress in the field (Ozturk et al. 2002; Hazen et al. 2005) and under controlled conditions (Talame et al. 2007).
Differential response of tolerant and sensitive cultivars to drought stress
To identify genes that may be relevant for the differential drought tolerance of rice cultivars, we looked for genes that showed differences in expression between the tolerance groups identified by our physiological measurements. This search strategy implies that genes contributing to tolerance show different expression patterns in the tolerant compared to the sensitive cultivars.
To find such genes, we identified those that showed a significant t-test for the condition × tolerance group term and an interaction factor higher than 1.5. To identify the source of the interaction, we compared the expression in sensitive cultivars under control (cS) and under drought conditions (dS), and in tolerant cultivars under control and drought conditions (cT, dT). The number of genes that were significantly drought-induced was much higher in the group of sensitive than in the group of tolerant cultivars. (Hazen et al. 2005) also found large differences between cultivars in the number of drought affected genes. In controlled environment experiments, moderate and severe drought stress induced a higher number of genes in IR62266, which is considered to be tolerant under these conditions, than in CT9993, which is considered to be sensitive to drought (Hazen et al. 2005).
Intuitively, one might expect more changes in the tolerant cultivars, which should carry those genes that contribute to increased tolerance. In fact, this pattern has recently been observed in Arabidopsis accessions differing in freezing tolerance (Hannah et al. 2006). However, the sensitive genotypes could show more changes if the imposed degree of stress evoked additional, damage related responses that were not yet induced in the tolerant genotypes. This pattern has been observed in salt- stressed rice, where salt stress changed expression of many more genes in the sensitive than in the tolerant cultivars (Walia et al. 2005, 2007). These differences were attributed to the higher Na+ accumulation in the sensitive cultivars that required more adjustments of metabolism. For these damage related genes, the tolerant cultivars should show low expression levels under both control and stress conditions, whereas the sensitive cultivars should show increased expression under stress. The resulting interaction factor [(dT−cT)−(dS−cS)] for these genes would then be negative.
Alternatively, genes that contribute to drought tolerance could be constitutively highly expressed in the tolerant group. If these genes are not (or very lowly) expressed in the sensitive cultivars, they will not be reliably identified with our search strategy. If these tolerance genes are drought-induced in sensitive cultivars, a negative interaction factor will result. A negative interaction factor can thus result from both stress-damage induced gene expression and stress-induced expression of tolerance genes that are constitutively expressed in tolerant cultivars. In the former case, expression levels will be low in the tolerant cultivars, in the latter case high.
Most genes with a negative interaction factor code for enzymes involved in degradation pathways, namely of lipids and proteins, especially cysteine proteases. For these genes, expression levels were generally low under control conditions for all cultivars and increased in the sensitive cultivars under drought stress. This expression pattern indicates that genes are most likely associated to damage-related responses. A similar response has been found in Fabaceae, where the activity of proteolytic enzymes increases more under drought stress in sensitive than in tolerant species (Roy-Macauley et al. 1992). In addition to lipid and protein degradation, downstream catabolic pathways of degradation products were induced in sensitive cultivars. This expression pattern was found for genes coding for enzymes of amino acid degradation pathways and of the TCA cycle that may contribute to metabolizing products of lipid degradation and fumarate produced by the urea cycle during amino acid degradation. This suggests that up-regulation of many of these genes is related to stress induced damage in the sensitive group rather than a tolerance conveying response. This is emphasized by findings in wheat (Gregersen and Holm 2007) that genes coding for enzymes involved in protein degradation as well as fatty acid and carbohydrate breakdown are induced during leaf senescence. Induction of cysteine proteases and lipid degrading enzymes were reported as part of programmed cell death in senescing leaves (Lim et al. 2007). The same authors report a down-regulation of anabolic pathways, especially of protein synthesis, rRNA and tRNA during senescence. We also found a down-regulation of many genes coding for components of the protein synthesis pathway, especially ribosomal proteins, under drought stress in the sensitive cultivars. The tolerant cultivars were much less affected, as indicated by the significantly positive interaction term. The majority of genes that were induced by drought stress in sensitive but not tolerant cultivars are thus related to senescence rather than to stress tolerance mechanisms. This interpretation is in accordance with the visual phenotype of the plants: sensitive cultivars showed yellowing and partial leaf death under drought stress, whereas the leaves of tolerant cultivars remained green. Recently, a remarkable increase of drought tolerance has been shown in plants, in which drought-induced leaf senescence was suppressed by the overexpression of isopentenyltransferase under the promoter of a senescence associated receptor protein kinase (Rivero et al. 2007). This stresses that the difference in the expression of senescence related genes between sensitive and tolerant cultivars is more than a side effect and may actually actively contribute to drought sensitivity.
As a case study for constitutively expressed tolerance genes, we compared gene expression in LC, which had a constitutively low leaf water potential, to the other cultivars by contrast analysis. Only 17 genes were significantly higher expressed in LC than in the other cultivars and showed a significant induction under drought in the latter. As especially the first comparison has a high type II error risk, the number of genes that show this expression pattern may be considerably higher. With the exception of an amino acid transporter, none of these 17 genes was involved in the synthesis or transport of known compatible solutes, although genes for trehalose, inositol and proline metabolism and 36 amino acid transporters were represented on the chip and expressed in the leaf tissues of the cultivars.
In contrast, there are some genes and gene groups, for which tolerant cultivars show more change and these are the interesting candidates for tolerance related processes. One candidate process whose regulation may contribute to drought tolerance is photosynthesis. Amounts of thylakoid membrane proteins were reduced (data not shown) and genes coding for PSI and PSII subunits were down-regulated by drought stress in all cultivars. The reduction of gene expression suggests that the observed decrease of photosynthetic capacity was not only due to drought induced damage of the photosynthetic apparatus, but may be a regulatory response. The number of significantly down-regulated photosynthesis-related genes is indeed higher in the tolerant than in the sensitive group, indicating a role for this regulation in drought tolerance. It is nevertheless unexpected, as the tolerant cultivars produced more biomass (Fig. 1) and had a higher photosynthetic capacity (data not shown) under drought conditions than the sensitive cultivars. A down-regulation of photosynthetic genes in the tolerant cultivars may therefore indicate an adaptive response to prevent photodamage during times of reduced CO2 availability in the mesophyll when stomata are closed due to water shortage. Reduction of photosynthesis is by no means drought specific, but is observed under heat, salt and chilling stress as well (Sayed 2003; Yan et al. 2006). In fact, photosynthesis-related genes are found to be massively repressed in Arabidopsis after a shift to low growth temperature (Hannah et al. 2005) and the magnitude of this repression is positively correlated with the freezing tolerance of different accessions (Hannah et al. 2006). However, drought stress seems to specifically act on proteins of the light harvesting complex of photosystem II (Sayed 2003). In agreement with this, additional photosynthesis measurements on the four cultivars (results not shown) revealed specific changes in the photosynthetic electron transport chain in response to drought. Thus, investigating the regulation of photosynthesis under drought stress may yield important insights into drought tolerance mechanisms.
Within the second candidate group, the cytochrom P450 genes, two cyp86A2 genes were induced under drought in the tolerant but not in the sensitive cultivars. In Arabidopsis, CYP86A2 catalyze the oxidation of fatty acids and are involved in the biosynthesis of extracellular lipids and cuticule development (Xiao et al. 2004). CYP86A2 transcripts are increased under various stress conditions including drought (Duan and Schuler 2005) and co-expressed among others with genes encoding enzymes involved in the TCA cycle, fatty acid elongation, wax and cutin metabolism (Ehlting 2006; Ehlting et al. 2008). In rice, epicuticular wax content is low but genetic variation of the amount exists (O’Toole and Cruz 1983). Induction of cuticula biosynthesis under drought could thus reduce non-stomatal water loss in the tolerant cultivars and thereby contribute to the observed increased water use efficiency. In Arabidopsis, cyp86A2 is furthermore coexpressed with genes coding for chlorophyll biosynthesis and photosystems, which suggests a link to the second process that has been identified as relevant for rice drought tolerance (Ehlting 2006).
Cytochrom P450 76C2, which is more highly induced in the sensitive than in the tolerant cultivars, is known to be induced during hypersensitive and developmental cell death, senescence and also under drought stress (Godiard et al. 1998; Narusaka et al. 2004), stressing the significance of both differences in P450 protein regulation and senescence associated processes for the drought-tolerance of rice.
Candidate selection by comparison with known QTL
In contrast to the drought-induced genes, many of which are functionally annotated, the genes with the highest repression factors were mostly of unknown or putative function. These genes could be as relevant for drought-tolerance as the highly induced genes, however, they are obviously much more difficult to interpret and much more time consuming to study functionally. To narrow down the list of genes with a significant G × E interaction to those that could be relevant in an agronomical environment, we compared their positions with published QTL, a strategy that has been successfully used before (Wayne and McIntyre 2002; Hazen et al. 2005). Indeed, four of the six cytochrome P450 genes that showed a significant G × E effect and the most highly induced gene encoding a late embryogenesis abundant protein are located in QTL. Among the five metallothionein-like protein genes represented on the chip, four, including the most highly drought induced gene, co-locate with drought QTL. Thus, the approach may yield interesting candidates for further functional studies, e.g. through transgenic approaches. The candidate list could be further narrowed down by checking candidate gene expression in DH or RIL lines characterized for their contrasting drought tolerance in the QTL region of interest. This strategy could also include genes of unknown function and thus open up the chance to discover truly unknown genes that are relevant for drought stress tolerance. The feasibility of confirming the expression pattern of such genes identified by array experiments in independent plant material has been shown in our study using qRT-PCR. In spite of the false positive risk in the array study and the high type II error in the qRT-PCR study, a significant interaction was confirmed for half of the genes. Furthermore, interaction coefficients calculated from microarray data and qRT-PCR correlated closely (data not shown). In further studies (Degenkolbe et al., manuscript in preparation), we tested the relevance of these candidate genes in an association-type approach by measuring their expression in a range of more than 20 rice cultivars of varying drought tolerance from different genetic backgrounds. Furthermore, this approach will unravel potential associations between the candidate gene and the parameters used for tolerance determination (MacNair 1993).