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Plant Physiol. May 2006; 141(1): 257–270.
PMCID: PMC1459312

Transcriptome Analysis of Cold Acclimation in Barley Albina and Xantha Mutants1,[W]

Abstract

Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; ≥2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold.

Plants can increase their freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation or hardening. The molecular dissection of cold acclimation reveals a complex process characterized by the coordinated up- or down-regulation of hundreds of cold-regulated genes. The signal transduction pathways leading to expression of cold-regulated genes in Arabidopsis (Arabidopsis thaliana) involves a regulatory network (Shinozaki and Yamaguchi-Shinozaki, 2000; Thomashow et al., 2001; Fowler and Thomashow, 2002) where a few regulatory genes control many genes in the cold response. The C-repeat binding factor (Cbf) genes (also known as DREB1), regulators of the cold-induced transcriptional cascade, have been shown to increase freezing tolerance when overexpressed in transgenic plants (Jaglo-Ottosen et al., 1998; Liu et al., 1998). Yet, additional pathways are apparent from transcriptional profiling of plants overexpressing Cbf genes, where low temperature evoked elevated expression of additional genes despite the constitutive expression of Cbf regulators (Fowler and Thomashow, 2002). Similarly, in barley (Hordeum vulgare) and wheat (Triticum aestivum) many genes are cold regulated (Cattivelli et al., 2002), including Cbf-like sequences (Choi et al., 2002; Vágújfalvi et al., 2005) that transactivate cold-regulated genes (Xue, 2002, 2003). The Cbf locus on chromosome 5 cosegregates with a frost resistance quantitative trait loci in Triticum monococcum (Vágújfalvi et al., 2003) and barley (Choi et al., 2002; Francia et al., 2004). A number of adaptive mechanisms including accumulation of Pro and other osmolytes, alterations in carbon metabolism, and changes in lipid membrane composition, are part of the cold-hardening process (Iba, 2002). To what extent these varied processes are regulated by factors other than Cbfs has not yet been thoroughly explored.

The ability of plants to develop a frost-resistant phenotype is associated with the presence of light and photosynthetic activity during cold acclimation. Plants can perceive variations in day length, light quality, and light intensity. Chloroplasts utilize light as a source of energy and react to variations in light intensity by adapting metabolism to the redox state of the electron transport chain (Pfannschmidt et al., 1999, 2001). An increased PSII excitation pressure (the relative reduction state of Qa, the first stable electron acceptor of PSII; Huner et al., 1996) is one of the primary stimuli promoting expression of cold-regulated genes (Gray et al., 1997; Ndong et al., 2001). Consequently, exposure to cold in the absence of light reduces the induction of several cold-regulated genes (Crosatti et al., 1999; Kobayashi et al., 2004). Also, several morphological traits associated with cold acclimation, such as the development of a compact rosette growth habit in winter cereals (Fowler and Carles, 1979), are dependent on the PSII excitation pressure (Gray et al., 1997). Freezing tolerance was not detected in Arabidopsis and other plants acclimated in the dark, under low light intensity, or at normal light intensity in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (Wanner and Junttila, 1999; Bourion et al., 2003). Although light in combination with low temperature is an essential factor in promoting cold acclimation, low temperature alone can still promote part of the cold response. In addition, cold-regulated genes can be induced to high levels even in the absence of light, when no enhancement of freezing tolerance can be detected (Grossi et al., 1998; Wanner and Junttila, 1999).

Barley genetic stocks offer a unique collection of chloroplast-deficient mutants, most of them characterized at genetic and biochemical levels (Henningsen et al., 1993). Although these mutations are generally lethal, the large endosperm of barley seeds supports plant growth for several weeks, allowing analysis of the mutants at a seedling stage. In this work we analyzed four nonallelic albina (alb-e16 and alb-f17) and xantha (xan-s46 and xan-b12) mutants characterized by a block in subsequent steps of the chloroplast development (Henningsen et al., 1993). In the albina mutants the leaves are completely white. alb-e16 accumulates about 5% of protochlorophyllide present in wild type and the plastids are characterized by the presence of small crystalline prolamellar bodies. alb-f17 accumulates about 70% of protochlorophyllide present in wild type and most of it is converted to chlorophyllide; after illumination large crystalline prolamellar bodies are frequent but primary lamellar layers are absent. The mutant xan-s46 synthesizes carotenoids, chlorophyllide, and a small amount of chlorophyll; expanded and transformed prolamellar bodies with short profiles of lamellar layers are present. Leaves of xan-b12 contain carotenoids and up to 6% of the chlorophyll present in the wild type. Many small diameter grana with two to three discs are formed and most of the paired lamellar profiles occur in a few giant grana (Henningsen et al., 1993).

Several recent works have described the molecular response to cold, drought, heat, and salinity in Arabidopsis (Kreps et al., 2002; Seki et al., 2002; Rizhsky et al., 2004) and other species (Ozturk et al., 2002; Rabbani et al., 2003; Wang et al., 2003). The combination of transcriptional profiling with genetic mutants or transgenic plants represents a powerful tool for dissecting the molecular networks supporting cellular functions. The analysis of Arabidopsis mutants with chilling sensitive phenotype led to the identification of genes with a critical role in plant adaptation to low temperature (Provart et al., 2003). Mutants of Arabidopsis with defects in chloroplast function were employed to investigate plastid control over nuclear gene expression leading to the identification of a regulatory master switch (Richly et al., 2003).

Previously, we have shown that barley plants carrying mutations preventing chloroplast development, beside the expected albina or xantha phenotype, are completely frost susceptible as well as impaired in the expression of several cold-regulated genes (Baldi et al., 1999; Dal Bosco et al., 2003). The recent availability of a barley microarray (Close et al., 2004) provided a new tool to describe the barley stress response and the role of the chloroplast in cold acclimation. In this work we investigated four chloroplast barley mutants (albina e16, albina f17, xantha s46, and xantha b12; Henningsen et al., 1993) and the corresponding wild type with the Affymetrix Barley1 GeneChip to assess the effect of the chloroplast on the expression of cold-regulated genes.

RESULTS

Modification of the Transcriptome in Albina and Xantha Mutants at 20°C

For analysis of chloroplast mutants under nonchilling conditions, plants were grown at 20°C and the transcriptome of each mutant was compared to wild type using the 22 k Barley1 GeneChip (Close et al., 2004). Triplicate array data was analyzed using 2-fold change as the cutoff followed by analysis for statistically significant changes using t test and a false discovery rate correction (Benjamini and Hochberg, 1995) for multiple testing (Reiner et al., 2003). Microarray data was analyzed for differentially expressed genes by comparing mutants to wild type (Table I). For simplicity we refer to probe sets as genes. The number of differentially expressed genes was much higher in the three earliest mutants (alb-e16, alb-f17, and xan-s46; 2,391, 2,286, and 2,201, respectively) than in the genotype closest to wild type (xan-b12, 896; Table I). These genes represent between 19.6% (xan-s46) and 9% (xan-b12) of the transcriptome detected with the microarray. A detailed discussion of the changes in gene expression associated with normal chloroplast development will be the objective of a separate publication.

Table I.
Present calls on Barley1 GeneChip and number of genes (probe sets) detecting transcripts more than 2-fold up-regulated or 2-fold down-regulated in the indicated comparisons

Modification of Wild-Type and Mutant Transcriptome in Response to Cold Treatment

Exposure to low temperature is known to modify the plant transcriptome. Genes up- or down-regulated by cold treatment depend on the cultivar examined, light, temperature, and length of the treatment. When the mRNA population from 6-d-old, cold-treated wild-type plants was compared with the transcriptome of wild type grown at 20°C (control), 1,911 genes were more than 2-fold up-regulated by low temperature and 824 genes were more than 2-fold down-regulated by low temperature (Table I). Since wild-type plants exposed to 3°C for 6 d developed a cold-hardened phenotype, we can assume that these changes in the transcriptome were part of the low-temperature response leading to freezing tolerance. All together, these cold-regulated genes represent about 24.6% of the transcriptome.

As with wild type, in each mutant the exposure to cold promoted variations in gene expression. However, the number of cold-regulated genes was smaller in the mutants compared to wild type (Table I). The genes cold regulated in the mutants represent only between 8.8% and 11.5% of the mutant transcriptomes, much less than the 24.6% observed in wild type. There was a similar number of cold, up-regulated genes in alb-e16, alb-f17, and xan-b12 (798, 834, and 811, respectively), whereas the number of up-regulated genes was lower in xan-s46 (703). The number of cold, down-regulated genes was similar for xan-b12 and xan-s46 (316 and 331, respectively). A little less than twice as many cold-repressed genes were found in alb-e16 (542) and alb-f17 (591). Cold-regulated genes indicated in Table I are listed in Supplemental Table I.

Functional classification according to the gene ontology (GO) biological process (see “Materials and Methods”) of cold up-regulated genes showed a higher proportion of stress-related genes (response to abiotic and biotic stimuli and response to stress) in mutants compared to wild type. In contrast, the category protein metabolism had a higher proportion in wild type compared to mutants for induced genes (Table II, columns B–F; Supplemental Table II). These results suggest that the normal process of adjusting protein metabolism during cold acclimation might be limited in chloroplast-defective mutants, whereas genes involved in the general stress response are still regulated in response to stress. Similarly, a striking difference was also observed when analyzing GO cellular components. Among up-regulated genes the mutants had very few associated with ribosomes, whereas for wild type there were 6.5% up-regulated ribosome-related genes (Table II).

Table II.
Functional classification (biological processes and cellular components) of the genes cold regulated in mutants and wild type

Classification of Mutant and Wild-Type Cold-Responsive Genes

The lists of the cold-regulated genes identified in wild-type and chloroplast mutants were compared to resolve the component genes into classes described in Figure 1. Briefly, wild-type cold-regulated genes were subdivided into three classes: (1) chloroplast dependent; (2) chloroplast independent; and (3) chloroplast development dependent (Fig. 1; Table III). The chloroplast development-dependent class was further separated into three subgroups: xan-b12 dependent (3a), xan-s46 dependent (3b), and alb-f17 dependent (3c). In addition, we found a fourth class comprising cold-regulated genes that were expressed in all four mutants but not in wild type, which were termed albina/xantha dependent (Fig. 1; Table III). Other mutations affecting cold acclimation might also be present in the genetic background of albina and xantha mutants. It seems unlikely that genes affected by other mutations are included in classes 1 (chloroplast dependent), 2 (chloroplast independent), and 4 (albina/xantha dependent) since these mutations would have to be present in all four mutants to be included in one of those classes (Fig. 1). Similarly, for classes 3b and 3c, classification is based on two and three mutants, respectively. For class 3a, classification is only based on one mutant (xan-b12) and this class may contain genes affected by other mutations. Genes not fitting any of the above classes were grouped as other (class 5; Fig. 1; Table III). Genes belonging to this class are not further discussed in this work.

Figure 1.
Classification of mutant and wild-type cold-regulated genes. The four major classes are: (1) chloroplast dependent (cold-regulated genes induced or repressed only in wild type and not regulated by cold in any mutant); (2) chloroplast independent (cold-regulated ...
Table III.
Classification of the genes (probe sets) cold regulated in wild type and/or in the mutants

When functional categories of the chloroplast-dependent and chloroplast-independent up-regulated gene classes were compared, analysis of the GO biological processes showed that a higher proportion of stress-related genes (response to abiotic and biotic stimuli and response to stress) was present in the chloroplast-independent class, while genes belonging to the category protein metabolism were more frequent in the chloroplast-dependent cold-regulated genes. The analysis of GO cellular components pointed out a major difference for genes associated with ribosomes; all cold-regulated genes belonging to this category were classified as chloroplast dependent (Table II, columns G and H; Supplemental Table II). The class of chloroplast development-dependent cold-regulated genes is characterized by high proportions of stress-related genes (GO biological processes) and absence of ribosomes (GO cellular components), a profile similar to that one of the chloroplast-independent class (Table II, column I; Supplemental Table II).

The results of Table II point out that genes classified as stress related were distributed among both chloroplast-independent and chloroplast developmental-dependent classes, while genes related to ribosomes were all among the chloroplast-dependent genes.

The Chloroplast Controls the Regulation of Most Wild-Type Cold-Regulated Genes

A total of 1,332 and 498 genes were more than 2-fold up- or down-regulated, respectively, only in wild type and not in any mutant (chloroplast-dependent cold-regulated genes; Fig. 1, class 1). These genes represent 69.7% of the 1,911 up-regulated and 60.4% of the 824 down-regulated genes detected in wild type exposed to low temperature (Table III). These wild-type cold-regulated genes can be chloroplast dependent for either of two reasons; (1) One or more normally cold-activated signaling pathway(s) may be active at 20°C in the mutants, so there is no cold-induction/repression in the mutant above an already high/low basal level of expression, or (2) a low-temperature signaling pathway may be disrupted in the mutants, resulting in a constant expression level not modified neither by mutation nor by cold treatment.

After a quality threshold (QT) clustering analysis the genes up-regulated by cold in wild type were divided in 14 clusters (Fig. 2A) plus 198 genes not clustered and therefore assigned as unclassified. Genes in clusters 1 to 4 were more expressed in mutants than in wild type at 20°C. These genes were induced upon cold treatment in wild type but showed no or very little induction in cold-treated mutants. Only in cluster 2, a slight induction upon cold treatment was detected in the mutants. These expression profiles are in agreement with constitutive activation in the mutants at 20°C of signaling pathways activated by cold in the wild type. Genes in cluster 5 were also up-regulated in the mutants at 20°C, nevertheless after cold treatment their expression was slightly reduced in all mutants except in xan-b12 where the reduction of the expression level was much stronger. Genes in clusters 6 and 7 showed a significant induction at 20°C only in xan-b12 (cluster 5) or alb-e16 (cluster 6), while no significant induction was achieved by cold in any mutants. The expression pattern of genes in clusters 8 to 14 supports the hypothesis of a disrupted signaling pathway. Clusters 8, 9, and 10 contain genes with similar expression levels in wild type and mutants at 20°C, after cold treatment no change (clusters 8 and 9), or a minimal change (cluster 10) in the expression levels was detected in mutants, whereas higher expression (induction) in wild type was evident. Genes in clusters 11 to 14 showed small variations in their expression in the mutants compared to wild type at 20°C, but without reaching the expression level detected in wild type after cold treatment. The exposure of the mutants to cold did not affect the expression of these genes.

Figure 2.
QT clustering of chloroplast-dependent and chloroplast-independent genes. The y axis is the normalized signal intensities (log10) scale and in the x axis samples are ordered according to their involvement in the chloroplast developmental pathway. The ...

Cluster analysis of the chloroplast-dependent down-regulated genes identified seven clusters plus 132 genes not clustered, and therefore assigned as unclassified (Fig. 2B). Genes in cluster 1 were less expressed in mutants at 20°C compared to wild type at 20°C and no significant change in expression levels was observed in mutants upon cold treatment, whereas wild type showed a clear cold-dependent down-regulation, a behavior in agreement with constitutive repression in mutants at 20°C of signaling pathways that are normally repressed by low temperature in wild type. The genes in cluster 2 showed a small down-regulation in the mutants at 20°C and a further down-regulation after cold treatment. Cluster 3 indicates constitutive activation of some signaling pathways at 20°C, in the albina mutants but not the xantha mutants. The predicted subcellular location of proteins encoded by genes in cluster 3 indicated that 33 out of 61 of these peptides are targeted to the chloroplast. Clusters 4 to 7 contain genes with similar expression levels at 20°C in all genotypes, whereas after cold treatment their expression was down-regulated only in wild type; consistent with a hypothesis of a blocked signaling pathway in the mutants.

The wild-type cold-induced genes classified as chloroplast dependent included a high proportion of genes related to protein synthesis and no known stress-related genes. A total of 317 up-regulated genes were annotated as ribosomal protein, elongation factor, initiation factor, or component of the proteasome. We found 256 genes encoding various ribosomal proteins (241 up-regulated), 125 belonging to cluster 4, indicative of a constitutive activation at 20°C of a low-temperature signaling pathway; 25 and 43 to cluster 8 and 9, respectively, representing pathways disrupted by mutations. Furthermore, all key genes involved in lipid biosynthesis (acyl CoA thioesterase, monogalactosyl-diacyl-glycerol synthase, phosphatidylglycerolphosphate synthase, and acyl-[acyl carrier protein] thioesterase; Miege et al., 1999; Xu et al., 2002) were cold up-regulated in a chloroplast-dependent manner.

Among the wild-type cold-repressed genes classified as chloroplast dependent, there were many genes encoding enzymes involved in photosynthesis and components of the light-harvesting complex, which is consistent with the absence of photosynthetic activity in albina and xantha genotypes.

Along with these intuitively expected classes there were also 201 probe sets corresponding to novel genes without BLAST hits to the database (E-value cutoff e−10). Such genes may perhaps provide new insights about a general adaptation mechanism. All probe sets with a chloroplast-dependent behavior are listed in Supplemental Table III.

Expression of Previously Known Cold-Regulated Genes Is Chloroplast Independent

A total of 243 up-regulated and 59 down-regulated probe sets were cold regulated in all genotypes. These genes were termed chloroplast independent (Fig. 1; Table III, class 2). This is only 12.7% and 7.2% of up- and down-regulated wild-type cold-regulated genes. Among the up-regulated genes, there were 39 sequences coding well-known stress-related proteins, including one Cbf-like (Choi et al., 1999), five Dehydrins/late-embryogenesis abundant (Dhn5, Dhn7, and Dhn8; Choi et al., 2002), two abscisic acid responsive, nine low-temperature responsive proteins, four ice recrystallization inhibitors, three Blt14, one Blt101, one Cor14, one amino acid selective channel protein (for review, see Cattivelli et al., 2002), two heat-shock proteins, seven early light inducible proteins (Shimosaka et al., 1999), and several enzymes involved in the accumulation of compatible solutes (e.g. pyrroline-5-carboxylate synthetase; Delauney and Verma, 1993). All probe sets with a chloroplast-independent behavior are listed in Supplemental Table IV.

QT clustering of the chloroplast-independent genes identified three up-regulated (1–3; Fig. 2C) and one down-regulated (Fig. 2D) cluster underlining some differences in the expression profile. Twenty chloroplast-independent genes (all down-regulated) were not clustered and were assigned as unclassified. Analysis of the three up-regulated clusters showed that only genes in cluster 1 were expressed to a similar extent in all genotypes at both 20°C and after cold treatment. Up-regulated cluster 1 contains almost all the previously known stress-related genes cited above. Genes in cluster 2 were less expressed in mutants compared to wild type at 20°C and this difference persisted after cold treatment. Cluster 3 shows an opposite trend to cluster 2; genes were expressed more in mutants compared to wild type both at 20°C and after cold treatment. Interestingly, the expression levels of these genes in xan-s46, alb-f17, and alb-e16 at 20°C were similar to the levels in cold-treated wild type. Most of the chloroplast-independent down-regulated genes showed already at 20°C a reduced expression in the mutants compared to wild type. This reduction was progressively stronger moving from xan-b12 (the genotype closest to wild type) to alb-e16 (the most extreme mutant). In all genotypes the cold treatment promoted a further reduction of the expression level of the genes in cluster 1 (Fig. 2D).

Some Wild-Type Cold-Regulated Genes Are Regulated by a Specific Chloroplast Developmental Stage

The chloroplast-defective mutants reported in this work represent four subsequent steps in plastid biogenesis. We asked whether up- or down-regulation of wild-type cold-regulated genes could be linked to progress in chloroplast development (Fig. 1; Table III, class 3). Ninety up- and 38 down-regulated genes behaved in a similar manner in wild type and mutant xan-b12 (the last step in plastid biogenesis considered in this work), while they were not induced nor repressed in the mutants representing earlier steps of chloroplast biogenesis. Cold regulation of these genes can therefore be associated with the xan-s46 to xan-b12 transition. Seventeen up- and seven down-regulated genes were found associated with the alb-f17 to xan-s46 transition since they behaved in a similar manner in wild type, xan-b12, and xan-s46 but not in alb-f17 and alb-e16. Finally, 38 up- and 33 down-regulated genes were found associated with the alb-e16 to alb-f17 transition. Among the cold up-regulated chloroplast development-dependent genes we found several previously described stress-responsive genes such as lipocalin (Charron et al., 2002), Cor14b (Dal Bosco et al., 2003), and Wcor518. Many of the cold down-regulated chloroplast development-dependent genes encode for chlorophyll a/b-binding (Cab) proteins and other components of the photosynthetic apparatus: four Cab genes were repressed in wild type and xan-b12, one Cab gene was repressed in wild-type xan-b12 and xan-s46, while 11 Cab genes were repressed in all genotypes except alb-e16. Since the mRNAs corresponding to all these genes were not detected in alb-e16 at 20°C, the ability of the other mutants to down-regulate these genes at low temperatures mainly reflects the ability of the plant to express these genes at 20°C. All probe sets with a chloroplast-development dependent behavior are listed in Supplemental Table V.

Albina-/Xantha-Dependent Non-Wild-Type Cold-Regulated Genes

Chloroplast mutants induced or repressed a common set of genes after cold treatment regardless of the developmental stage of the plastids. We found 123 up-regulated and 44 down-regulated genes responsive to cold in all four mutants but not in wild type. These genes were termed albina/xantha dependent (Fig. 1; Table III, class 4). Among the up-regulated, there were numerous known stress-responsive genes such as Dhn3, Dhn4, and Dhn9 (Choi et al., 1999) and genes encoding heat-shock proteins, early light-induced proteins (Shimosaka et al., 1999), Suc phosphate synthase (Guy et al., 1992), and water stress-induced proteins. This suggests a higher cold susceptibility of the chloroplast mutants compared to wild type. The probe sets regulated by cold in all mutants but not in wild type are listed in Supplemental Table VI.

Overlap between Cold-Dependent and Photooxidative-Dependent Signaling Pathways

Albina and xantha mutants are characterized by the lack of carotenoids (albina) and disruption of the assembly of photosynthetic membranes (albina and xantha). These conditions might cause severe photooxidative damage during exposure to light. Since oxidative signaling is a part of the cold response (Prasad et al., 1994) the constitutive activation in the mutants of signaling pathways normally induced by low temperature in wild type might be explained by the overlap between cold-dependent and photooxidative-dependent signaling pathways. To explore this hypothesis we used the nucleotide sequences of genes described as induced by oxidative stress in Arabidopsis (Desikan et al., 2001; Rossell et al., 2002) to identify the homologous barley genes. These putative barley oxidative responsive genes were then cross referenced with the lists of the cold-regulated genes described in this work. Fourteen common sequences were used in real-time quantitative reverse transcription (qRT)-PCR to assess gene expression in wild type and mutants treated with norfluorazon, an herbicide that blocks carotenoid biosynthesis, in comparison to control and cold-treated samples. With one exception only, all genes induced by norfluorazon and by cold in wild type were also constitutively expressed in the alb-e16 and alb-f17 mutants at 20°C, suggesting that albina plants were exposed to photooxidative stress even at 20°C. In contrast, only a subset of the genes induced by norfluorazon in wild type were constitutively expressed in xan-s46 (nine genes) and xan-b12 (two genes), a behavior that can be attributed to the protective activity of the carotenoids against photooxidation (white bars in Fig. 3).

Figure 3.
Real-time qRT-PCR analysis of 18 sequences involved in the response to photooxidative and cold stress. A, Wild-type and mutants plants grown at 20°C for 8 d (C, Control) were treated with norfluorazon (NF; see “Materials and Methods”) ...

During the search for genes up-regulated by oxidative stress and by cold, two genes induced by oxidative stress and not by cold in wild type (isocitrate lyase and glycerophosphoryl diester phosphodiesterase) as well as two genes up-regulated only by cold in wild-type plants and not in any mutants (60S ribosomal protein and ribosomal protein L7A) were also identified (gray and black bars, respectively, Fig. 3).

The real-time qRT-PCR experiments also provided validation of the array data. The correlation coefficient between expression values detected in real time and the corresponding values obtained from the array analysis was r = 0.71 (significant at 0.01%).

The Expression of Some Early Cold-Induced Transcription Factors in Albina and Xantha Mutants Is Chloroplast Independent

Present knowledge suggests that transient induction of Cbf proteins has an important role in the control of the hardening process (Fowler and Thomashow, 2002; Francia et al., 2004; Vágújfalvi et al., 2005). The analysis of the barley mutants described above was performed after 6 d of cold acclimation, which is not suitable for detecting early and transient responses. Cbf transcripts accumulate rapidly and transiently within 15 min of transfer to low temperature and continue to increase over the next 2 h. Later, they begin to decline and return to near the initial level by about 24 h (Liu et al., 1998; Choi et al., 2002). The mutants were therefore examined for transient Cbf expression during the early phases of the cold response. A cDNA containing the AP2 and Cbf-signature conserved domains (Jaglo et al., 2001) was used to probe RNA samples in northern blots from wild type and mutants grown at 20°C or exposed at 3°C for 4 h. The barley Cbf gene family was vigorously induced by cold in wild-type and chloroplast mutants (Fig. 4A). Further analyses were carried out with RT-PCR on specific Cbf-like sequences. BCbf1, HvCbf1, and the Cbf-like sequence corresponding to the accession number BG367653 were cold induced to a similar extent in mutants and wild-type plants after 4 and 8 h of cold treatment, and expression was greatly reduced after 24 h of cold treatment. Beside Cbfs, a barley sequence (HvICE1; Tondelli et al., 2006) highly homologous to the Arabidopsis ICE1 (a constitutively expressed activator of Cbf induction; Chinnusamy et al., 2003) and the cold-regulated barley gene Hv-WRKY38 (a cold-induced transcription factor belonging to the ICE1 regulon; Marè et al., 2004) were also investigated and their expression profile was identical in mutants and wild-type plants (Fig. 4B).

Figure 4.
Analysis of Cbf expression in wild-type and chloroplast mutants of barley. A, Northern-blot analysis, each lane contained 15 μg of total RNA from the samples indicated (20°C, control; 3°C, cold treated for 4 h) and probed with ...

Many studies have recently investigated the activation of transcription, during cold acclimation, due to Cbf action in Arabidopsis. Arabidopsis sequences ascribed to the Cbf regulon were downloaded from GenBank (dehydrins, P5CS, genes involved in sugar metabolism, transporter, proteolysis, and other transcription factors; Fowler and Thomashow, 2002; Vogel et al., 2005) and used in homology searches to identify the corresponding barley genes. A total of 19 barley sequences corresponding to Arabidopsis Cbf-regulated genes (E-value cutoff = ≤e−10) were found. Seven of these sequences that could be analyzed in the expression data identified 10 probe sets present on the barley array, all but two belonging to the chloroplast-independent cold-regulated gene class. This means that expression of the ICE1-Cbf cold signaling pathway is unaffected by chloroplast mutations.

DISCUSSION

We used the Barley1 GeneChip (Close et al., 2004) to compare the molecular response to cold of a wild-type barley and four chloroplast-defective mutants. The effect of the chloroplast on the expression of genes cold regulated in wild type was much higher than the effect of the chloroplast on the transcriptome overall. A total of 66.9% of the 2,735 wild-type cold-regulated genes were not cold regulated or not correctly cold regulated in the mutants. These genes were classified as chloroplast dependent (Fig. 1; Table III, class 1). At 20°C, 896 to 2,391 genes, depending on the mutant analyzed, had altered expression levels relative to wild type. This was 9.0% to 19.6% of the transcriptome (Table I). Together, these results demonstrate that the chloroplast has a major role in the control of molecular adaptation to cold. Furthermore, while the differences between wild type and mutants were significantly reduced in xan-b12 (the mutant closest to wild type), the effect of some progress in chloroplast development on the expression of genes identified as cold regulated in wild type was much smaller. The xan-b12 mutation still affects the expression of 1,830 genes (about 66.9% of the wild-type cold-regulated genes) and only 223 genes (8.2% of the wild-type cold-regulated genes) are cold regulated in a chloroplast development-dependent manner. We conclude that a fully operational chloroplast rather than a specific step in chloroplast development is required for normal cold-dependent regulation of the transcriptome.

Several chloroplast signals of different origin are known to influence gene expression. Redox status of the electron transport chain, accumulation of reactive oxygen species, intermediates of the chlorophyll biosynthesis, and accumulation of sugars all can provide signals for the regulation of nuclear genes coding for plastid and nonplastid proteins (Escoubas et al., 1995; Jarvis, 2003). Some of these signaling mechanisms are also involved in the response to low temperature. Exposure to cold promotes changes in the redox state (Huner et al., 1996) and accumulation of reactive oxygen species (Foyer et al., 1994). There are two plausible explanations for the expression characteristics of chloroplast-dependent cold-regulated genes. Signaling pathways activated by cold in wild type might be constitutively active in the mutants at 20°C leading to the constitutive expression/repression of wild-type cold-regulated genes. Alternatively, if no change is observed after cold treatment it may reflect disruption of low-temperature signaling pathways. We have found evidence for both factors. About one third of the chloroplast-dependent cold-regulated genes were already expressed in the mutants at 20°C at levels similar to cold-treated wild type, and cold treatment did not significantly change the expression of those genes in the mutants (clusters 1–4 in Fig. 2A; cluster 1 in Fig. 2B). This indicates a constitutive activation of signaling pathways in the mutants at 20°C. Conversely, clusters 8 to 14 in Figure 2A and clusters 5 to 7 in Figure 2B contain genes that were cold regulated in wild type but not significantly regulated by cold in the mutants. These examples seem to correlate with disruption of low-temperature signaling pathways in the mutants. Therefore, the absence of a fully functioning chloroplast either leads to a lack of induction/repression of wild-type cold-regulated genes or causes a shift of the transcriptome (genes normally regulated by cold treatment are induced/repressed at 20°C).

The mutants respond to cold treatment by activating a set of albina-/xantha-dependent cold-regulated genes (123 up- and 44 down-regulated) whose expression was not induced or repressed in wild type (Table III). Some of these up-regulated genes encode known stress-responsive proteins, for example three members of the Dhn family (Dhn3, Dhn4, and Dhn9), previously found to be induced by freeze/thaw (not induced at 4°C) and by dehydration treatment (Zhu et al., 2000). In fact, 63 (51%) out of 123 up-regulated genes were also induced in drought-treated barley (E. Rodriguez, J. Svensson, and T.J. Close, unpublished data). This suggests that cells without active chloroplasts tend to activate stress-signaling pathways more readily than does wild type upon exposure to low temperature. Apparently, plants with defective chloroplasts are more predisposed to stress. Functional classification of all mutant and wild-type cold-regulated genes supports this observation. A higher proportion of stress-related genes were found in mutants than in wild type (Table II).

Among cold-regulated genes identified in wild type, the largest group encodes products related to protein synthesis, particularly ribosomal proteins. Among mutant cold-regulated genes there are relatively fewer involved in protein metabolism and none classified as ribosomes (Table II). Induction of genes involved in protein synthesis has been one of the major changes previously reported during chilling response in Arabidopsis. When 12 chilling sensitive mutants were analyzed under normal (22°C) and chilling (13°C) conditions, all mutants failed to up-regulate these genes in response to chilling (Provart et al., 2003). As in our study, Provart et al. (2003) found a set of genes that were induced or expressed only in the mutants and not in wild type after chilling treatment, however no functional classification was given for those genes. We found numerous genes involved in protein biosynthesis that were expressed to a similar extent in the mutants at 20°C as by low temperature in the wild type. This is consistent with Baldi et al. (2001), who reported that two ribosomal protein genes and a gene encoding elongation factor 1Bβ were cold induced in green barley leaves but constitutively expressed at high level in albina and etiolated leaves. The data reported in our work further support a role of the chloroplast in the regulation of protein synthesis machinery through a signal activated by cold.

Previously described cold-regulated genes (Cattivelli et al., 2002) were almost all included in the chloroplast-independent cold-regulated gene class (Fig. 1, class 2). The Cbf-like genes (Fig. 4) and certain Dhn genes were among these chloroplast-independent genes. Barley Cbf genes interact with the C-Repeat/DRE motif (G/a)(C/t) CGAC (Xue, 2002). A search for this cis-element (http://intra.psb.ugent.be:8080/PlantCARE/) in promoters of barley cold-regulated genes found several copies of C-Repeat/DRE in the promoters of Dhn5 and Dhn8 (3 and 5, respectively), two genes classified as chloroplast independent using our work. In Arabidopsis, cold induction of genes coding for DHNs and pyrroline-5-carboxylate synthetase (also chloroplast independent in our experiment) was under the control of CBF transcription factors (Jaglo-Ottosen et al., 1998; Fowler and Thomashow, 2002). The induction of Cbf-like genes and of putative Cbf target genes in albina and xantha plants suggest that the Cbf transduction pathway is active in the mutants and, therefore, independent of a fully activated chloroplast. The fact that the majority of the wild-type cold response was impaired in the mutants suggests the requirement of other factors for the activation of most cold-mediated responses. When the transcriptome of wild type and Cbf-overexpressing Arabidopsis plants was investigated at normal and low temperature, only 12% of the cold-responsive genes were ascribed to the Cbf regulon, suggesting likewise that the majority of the cold-regulated genes are partially or completely independent from the Cbf system (Fowler and Thomashow, 2002). Our results parallel this observation. We show that a fully functional chloroplast is required to promote the molecular changes associated with cold acclimation.

The description of the cold response in wild-type barley and in four independent chloroplast mutants allowed the identification of three main pathways containing more than 80% of the wild-type cold-regulated genes: (1) cold-regulated genes unaffected by any mutations, including Cbf genes and many genes known to be under Cbf control; (2) cold-regulated genes constitutively induced, although to different levels in all mutants, including those activated in response to photooxidative stress; and (3) cold-regulated genes belonging to signaling pathway(s) disrupted in all mutants, whose expression consequently was not, or was only marginally responsive to cold. In addition, we also found several other expression profiles grouping genes regulated by cold in a mutant-dependent manner. Since only a minor portion of cold-regulated genes belong to the same regulatory pathway as Cbf, we conclude that factors deriving from the chloroplast in addition to Cbf are required to promote the full suite of molecular changes associated with cold acclimation.

MATERIALS AND METHODS

Genetic Materials

A spring barley (Hordeum vulgare cv Bonus) and four nonallelic albina (alb-e16 and alb-f17) and xantha (xan-s46 and xan-b12) mutants obtained by chemical or physical mutagenesis in the genetic background of cv Bonus (Henningsen et al., 1993) were used. All mutants were maintained as heterozygous stocks and after germination a 3:1 segregation was observed. These mutations affecting chloroplast development have been located in the chloroplast biogenesis pathway as described by Henningsen et al. (1993).

Growth Conditions

Wild type and mutants were germinated in peat pots and grown in a controlled-environment chamber for 8 d at 20°C with 12-h photoperiod (300 μmol m−2 s−1). When the first leaf was fully emerged the plants were treated for 6 d at 3°C, 12-h light (150 μmol m−2 s−1)/1°C, 12-h dark. Plants were harvested into liquid nitrogen. Control plants were harvested after 8 d at 20°C. All samples were collected in the middle of the light period. This experiment was conducted three times to yield three independent biological replicates. To analyze the involvement of the photooxidative stress, seeds of wild type and mutants were imbibed for 3 h in water containing 0 (control) or 50 μm norfluorazon. The hydrated seeds were grown in a growth-controlled environment chamber as above except that light intensity was 10 μmol m−2 s−1 for the first 8 d and 200 μmol m−2 s−1 for the next 6 d. During the growth, treated plants were watered two times with 50 μm norfluorazon.

For analysis of Cbf expression, treated samples were collected after 4, 8, and 24 h of cold treatment in the light.

RNA Isolation and Array Hybridization

Total RNA was prepared using TRIZOL reagent according to the method published at the Arabidopsis (Arabidopsis thaliana) functional genomics consortium Web site (www.Arabidopsis.org/info/2010_projects/comp_proj/AFGC/RevisedAFGC/site2RnaL.htm#isolation) and further cleaned using RNeasy columns (Qiagen) following the manufacturer's instructions. Purified RNA was adjusted to a final concentration of 1 μg/μL in diethyl pyrocarbonate-treated water. All RNA samples were quality assessed prior to beginning the labeling procedure by running a small amount of each sample (typically 200–250 ng) on a RNA Lab-On-A-Chip (Caliper Technologies) using an Agilent Bioanalyzer 2100 (Agilent Technologies).

Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (10 μg total RNA starting material each sample reaction) using the SuperScript double-stranded cDNA synthesis kit (Invitrogen) and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting double-stranded cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the BioArray high-yield RNA transcript labeling kit (T7; Enzo Diagnostics). The resulting biotin-tagged cRNA (15 μg) was fragmented to strands of 35 to 200 bases in length following Affymetrix protocols. Fragmented target cRNA (10 μg) was hybridized at 45°C with rotation for 16 h (Affymetrix GeneChip hybridization oven 320) to probe sets present on Affymetrix Barley1 GeneChip arrays. The arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 400, followed by scanning on a Hewlett-Packard GeneArray scanner. These steps were performed at the DNA and Protein Microarray Facility, University of California, Irvine (http://sense.ucicom.uci.edu/dmaf/).

Data Analysis

Scanned images were analyzed using the software MicroArray Suite 5.0 (MAS 5; Affymetrix). Expression analysis was done using default values. Scaling (global normalization) was done to a target signal of 500 using data from all probe sets. Quality control values, present calls, background, noise, scaling factor, spike controls, and the 3′/5′ ratios of GAPDH and tubulin showed low variation. Two of the control probe sets (TIF 5A and actin) showed high 3′/5′ ratio variation, similar results were obtained in other experiments and in other labs (Close et al., 2004).

MAS 5.0 data were imported to the software GeneSpring 7.0 (Silicon Genetics) for analysis. Each chip was normalized to the median of the measurements taken from that chip, probe sets with normalized signal value below 0.35 were transformed to 0.35, and probe set normalization was done to the median value of each probe set or in some cases to specific samples. The data transformation of normalized values below 0.35 to 0.35 was done to floor absent genes. The 75th percentile of absent calls was 80 (prenormalization), which corresponded to a normalized value of 0.35.

Two comparisons were done: (1) wild-type control compared to each mutant control and (2) control compared to cold treated for each genotype. Baseline was set as wild-type control in 1 and as each genotype control for 2. The experiment was repeated three times and each replicate was initially analyzed separately. Each probe set contained 11 paired perfect match and mismatch 25-mer probes that are used to calculate the detection call and the signal. The MAS 5.0 algorithm uses a nonparametric statistical test (Wilcoxon signed rank test) of whether significantly more perfect match probes have higher signal than their corresponding mismatch probes to produce a detection call; present, marginal, or absent for each probe set. Data for genes not actually expressed (absent) represent experimental noise and can generate false positives. For this reason we used detection calls (present) as an initial filtering step. For decreasing probe sets, those with a detection call of present in the baseline samples and at least a 2-fold change were further considered. For increasing probe sets, those with a present call and 2-fold change were further considered. Replicate datasets were analyzed by Boolean searches such that only genes found in intersections were further analyzed. For example an up-regulated gene had to be 2-fold or more up-regulated in all three replicates. These genes were further analyzed for statistically significant changes by a Welch t test and the Benjamini and Hochberg false discovery rate correction for multiple testing (Reiner et al., 2003); the false discovery rate adjusted p-value cutoff was set to 0.05.

For clustering we normalized each probe set to the median of that probe set. Clustering was done using the QT clustering function in GeneSpring. The minimum clusters size was set at 20 genes and the minimal correlation coefficient (Pearson) to 0.7.

The Arabidopsis International Resource GO Web site (http://www.arabidopsis.org/tools/bulk/go/index.jsp) was used for functional classification. The classification is based on Arabidopsis gene identifiers, therefore only genes with a homolog (cutoff E-value = e−10) in Arabidopsis were classified. BLAST search results were exported from HarvEST 1.35 (www.harvest.ucr.edu).

Expression Analysis of Early Cold-Induced Transcription Factors

Fifteen micrograms of total RNA for each sample were separated on an agarose formaldehyde gel and transferred to a positively charged nylon filter (Millipore). A XbaI-HindIII DNA fragment of 382 bp encompassing the conserved Cbf regions contains an AP2 domain and Cbf signature (Jaglo et al., 2001) was cleaved from BCbf1 (accession AF298230), labeled with [α-32P] dCTP using random priming, and used as probe. Identity between the probe and the corresponding region of other known Cbf-like barley genes is >68%. The hybridization was performed using UltraHyb buffer (Ambion) at 42°C. Detection was performed with a phosphor-imager system, Typhoon 9210 (Amersham Biosciences) and data quantified with ImageQuant software (Amersham Biosciences).

For RT-PCR, first-strand synthesis was done using 200 units of SuperScriptII RNase H reverse transcriptase (Invitrogen) and a poly(T) primer from 4 μg of total RNA following the manufacturer's recommendations, except for the use of the RNaseH (final volume of 20 μL). Three Cbf-like transcripts were amplified using the following primers: BCbf1 (AF298230, probe set contig15617_at), 5′-TACATCTCGTCCGGCGACCTGTTGGAGC-3′ and 5′-AACTTAGCACAATTGAATCGGATGAGATC-3′ (annealing 60°C); HvCbf1 (AF418204, probe set contig13523_at), 5′-GGATGCTCATTGCCCCTCCT-3′ and 5′-AGCCCCAACACTCCTTCGGA-3′ (annealing 55°C); BG367653 (probe set: contig2479_at), 5′-AGCTGGACGTCCTGAGCGACATG-3′ and 5′-GCTCTGTTTCCCCAATTTGCAC-3′ (annealing 58°C). Two additional sequences coding Hv-WRKY38 (Marè et al., 2004), 5′-CCGTCAAAGCCTCGCAGACAAAGC-3′ and 5′-TATGGAACGGAACATTTGAATGA-3′ (annealing 58°C) and HvICE1, a barley homolog of the Arabidopsis ICE1 gene (Tondelli et al., 2006), 5′-CTGAGCAATGCAAGGATGG-3′ and 5′-GACACAGGGTAGTGACATCAGG-3′ (annealing 55°C) were also investigated. The transcripts were amplified from 400 ng of cDNA as template and 2 units of Taq DNA polymerase (Invitrogen) using 28 cycles (94°C for 1 min, annealing for 30 s, 72°C for 30 s) followed by a final extension step at 72°C for 7 min. RT-PCR reactions performed with primers designed on the gene coding for β-actin protein was used as standard. Amplification products were separated on 2% agarose gel.

Real-Time qRT-PCR

Real-time qRT-PCR was performed with SYBR Green fluorescence detection in a real-time PCR thermal cycler (GeneAmp 5700, Perkin-Elmer). PCR mix was prepared with 100 ng of cDNA, 10 μL of Platinum SYBR Green qPCR Supermix UDG (Invitrogen), 1 μL of ROX Reference dye, MgCl2 (final concentration 3 mm), forward and reverse primers (final concentration 0.2 μm) in a total volume of 25 μL. The cycling conditions were: 2 min at 50°C and 2 min at 95°C, followed by 40 cycles of 95°C for 15 s/60°C for 60 s. Melting curve analysis was performed after PCR to evaluate the presence of nonspecific PCR products and primer dimers. Normalization was carried out with β-actin constitutively expressed gene. The probe sets and the corresponding primer pairs used for the analysis are reported in Supplemental Table VII.

The real-time qRT-PCR data were plotted as the ΔRn fluorescence signal versus the cycle number. The PE Biosystems 5700 detection system software calculates the ΔRn using the equation ΔRn = (Rn+) − (Rn), where Rn+ is the fluorescence signal of the product at any given time and Rn is the fluorescence signal of the baseline emission during cycles six to 13. An arbitrary threshold was set at the midpoint of the log ΔRn versus cycle number at which the ΔRn crosses the threshold (Ct). The Ct value was used to calculate the fold changes (FC) in each sample with respect to the expression level found in wild-type plants at 20°C (baseline) with the following formula:

equation M1

The mean concentration of the β-actin gene was used as a control for input RNA. The data are expressed as log2 of the average FC of three independent experiments; standard variation in all samples was lower than 20%.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Dr. J.D. Heck and K. Nguyen at the University of California, Irvine, DNA Array Core Facility for excellent services, and Professor D. von Wettstein (Washington State University, Pullman, WA) and Dr. David Simpson (Carlsberg Laboratory, Copenhagen) for the kind gift of barley mutants.

Notes

1This work was supported by the GENEFUN (functional genetics) program, the Fondo Investimenti Ricerca di Base programs (nos. RBNE01LACT [plant stress] and RBAU01E3CX), and the U.S. Department of Agriculture/Cooperative State Research, Education and Extension Service/Initiative for Future Agriculture and Food Systems (2001–52100–11346).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Luigi Cattivelli (ti.arcetne@illevittac.igiul).

[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.105.072645.

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