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Keywords:

  • Arabidopsis;
  • embryo;
  • maturation;
  • enzyme histochemistry;
  • glycolysis;
  • carbohydrate

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Arabidopsis as a molecular genetic model offers many advantages for the study of seed development, but these do not extend to biochemical and enzymatic studies, which are often compromised by the limited amount of material available from the small developing embryos. A set of assays based on the coupling of an enzymatic reaction to the reduction of NAD, NADP or FAD, and subsequent reduction and precipitation of a tetrazolium salt, have been adapted to investigate 18 enzyme activities associated with carbon metabolism in developing Arabidopsis embryos. The use of organelle-specific marker enzymes demonstrates the utility of the method for detection of activities in mitochondria, plastids and peroxisomes as well as the cytosol. The temporal staining patterns obtained allow classification of the activities into three main categories based on whether they peak in the early, intermediate or late stages of maturation. An interesting switch from ATP to pyrophosphate consuming pathways occurs at the onset of the maturation phase, which involves key steps in primary carbon metabolism such as phosphofructokinase. This spatiotemporal characterization of carbon metabolism has also been applied to various mutants disrupted in embryo development including gnom (gn), acetyl-CoA carboxylase1 (acc1), schlepperless (slp), and wrinkled1 (wri1). The data obtained demonstrate that the extent to which carbon metabolism is affected in mutants is not necessarily correlated to the severity of the mutation considered. Through the advanced characterization of trehalose-6-P synthase1 (tps1) embryos, this approach finally provides new insight into the regulatory role played by trehalose metabolism in embryo development.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

In the plant kingdom, the production of seeds enables the life cycle to temporarily pause, allowing survival in unfavourable conditions, and colonization of new territories. The process of seed development requires the coordinated growth of three tissues of distinct origins. The embryo and the endosperm are filial tissues, the development of which is initiated by the double fertilization of the embryo sac. The embryo and endosperm are protected by several maternally derived integuments, which constitute the seed coat. Seeds accumulate storage compounds in the form of starch, oils, or specialized storage proteins. These resources fuel post-germinative seedling growth and they also constitute the economic value of seeds in most field crops. In oleaginous species such as Brassica napus (rapeseed), one of the world's major oilseed crops, storage products consist of proteins and triacylglycerols (TAGs) synthesized in the embryo. The model plant Arabidopsis thaliana is closely related to rapeseed and has been developed as a model system to investigate oil synthesis in seeds.

Arabidopsis seed development can be divided into three main steps: early embryo morphogenesis, maturation, and late maturation. In the first phase, the embryo acquires the basic architecture of the plant through a series of programmed cell divisions (Mayer and Jürgens, 1998). Early embryo morphogenesis ends at the late heart stage (Mayer et al., 1991). The second stage, or maturation phase, begins with a period of rapid embryo growth (Goldberg et al., 1994). The embryo fills the seed sac while the endosperm is almost totally resorbed (Mansfield and Briarty, 1993). Maturation is characterized by a strong increase in seed DW: storage oils and proteins accumulate to high levels in the embryo, each accounting for approximately 40% of dry matter at the end of this stage (Baud et al., 2002). During late maturation, storage compound synthesis ends while the embryo becomes metabolically quiescent and tolerant to desiccation.

While TAGs represent the main form of carbon stored in Arabidopsis embryos, significant amounts of starch transiently accumulate during early maturation (Baud et al., 2002; Focks and Benning, 1998). Neither the function of this starch in the carbon economy of the developing embryo nor the factors controlling the shift from starch to oil accumulation have been elucidated (Eastmond and Rawsthorne, 2000; da Silva et al., 1997; Vigeolas et al., 2004). Both starch and oil are derived from sucrose, which is imported by the embryo from maternal tissues and metabolized in the cytosol, plastids, and endoplasmic reticulum (ER) (for a review, see Hills, 2004, Figure 1). Incoming sucrose can be cleaved via two distinct pathways involving either invertase or sucrose synthase (SUS) to produce hexose-phosphates. The hexose-phosphate pool fuels three main biochemical pathways: (i) the transient starch biosynthesis and degradation pathways in the plastids (da Silva et al., 1997), (ii) the oxidative pentose phosphate pathway (OPPP), the importance of which is still under debate as regards its role in the supply of plastidial NADP for fatty acid synthesis (Schwender et al., 2003), and (iii) the cytosolic and plastidial glycolytic pathways providing precursors for fatty acid biosynthesis. The final step of this network involves the assembly of TAGs through the sequential acylation of a glycerol backbone by the enzymes of the Kennedy pathway (Miquel and Browse, 1997). Although the overall biochemical pathways linking sucrose to oil have been well described, a number of important gaps in our knowledge still remain. First, the subcellular organization of enzymatic reactions and the proportions of carbon fluxes that pass through the various pathways described above are still poorly characterized. The 13C-metabolic flux analysis (MFA) system developed by Schwender and Ohlrogge (2002) recently shed some new light on the subject. For instance, in Brassica embryos cultured in vitro under low light, net flux of glucose through the OPPP was shown to account for 10% of the total hexose influx into the embryo (Schwender et al., 2003). This method has allowed the authors to describe a new metabolic route between carbohydrate and oil involving Rubisco (Schwender et al., 2004b). A second set of questions relate to factors regulating fatty acid synthesis and the control of total oil content in the seed. The regulatory potential of the various enzymatic reactions involved in the network has not been fully investigated (for reviews see Hills, 2004; Thelen and Ohlrogge, 2002). Data concerning one area of the network, from sugar entry into the sink cell to its conversion to acetyl-CoA, are particularly scarce. Furthermore, only one non-enzymatic component of the network has been isolated to date, namely the WRINKLED1 transcription factor, which appears to regulate several enzymatic activities of the glycolytic pathway in maturing seeds through an as yet undetermined mechanism. Finally, questions remain regarding the interplay between the metabolism of maturing embryos and the developmental progression of embryogenesis (Brocard-Gifford et al., 2003).

image

Figure 1. Scheme of the carbon metabolic network in maturing Arabidopsis oilseeds. The enzymatic activities investigated by in situ histochemistry in this study are shown. 6PGDH, 6-phosphogluconate dehydrogenase; ADH, alcohol dehydrogenase; AGPase, ADP-Glc pyrophosphorylase; FK, fructokinase; GK, glucokinase; G3PDH, NADP-dependent glyceraldehyde-3-P dehydrogenase; G6PDH, Glc-6-P dehydrogenase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; MS, malate synthase; Malic E, malic enzyme (both NAD and NADP dependent); PFK, ATP-dependent phosphofructokinase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucose isomerase; PGM, phosphoglucomutase; SDH, succinate dehydrogenase; SUS, sucrose synthase; UGPase, UDP-Glc pyrophosphorylase.

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A combination of existing approaches and techniques and development of new methodologies will be essential in order to answer these questions. The very small size of Arabidopsis embryos presents a major challenge for most biochemical and enzymatic approaches as the amount of tissue available is often limiting. When such experiments have been carried out (Baud et al., 2002; Focks and Benning, 1998; Gibon et al., 2002), whole seeds have had to be sampled, which is not ideal as the three tissues comprising the seed are very different and should if possible be analysed separately (Hill et al., 2003). In this paper we describe the development and application of in situ histochemistry approaches for the study of isolated Arabidopsis embryos that allow the spatial and temporal resolution of key enzymes of carbon metabolism. This quick and sensitive technique of high spatial resolution was used to monitor 18 distinct activities associated with carbon metabolism during embryogenesis of the wild type and various mutants, including several embryo lethals and wri1.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

In situ enzymatic histochemistry in Arabidopsis embryos

Most biological processes are controlled at multiple levels, including transcription, translation and post-translation. In the case of metabolism, it is well documented that enzyme activity can be influenced at all these levels, and as a consequence it can be much more informative to measure enzyme activities directly, rather than attempt to infer activities through measurement of associated mRNA or protein levels. Histochemical-based in situ localization of enzyme activities offers the possibility of detailed spatial and temporal information that would complement and extend other data sets such as reporter gene studies, mRNA in situ hybridization, and immunocytological methods. Histochemical-based in situ localization has been successfully used on various animal tissues (Van Noorden and Butcher, 1984) and plant organs (Sergeeva and Vreugdenhil, 2002; Wittich and Vreugdenhil, 1998). The material studied is fixed before staining to anchor the soluble enzymes under investigation in the tissue. If the material requires sectioning to aid visualization, the paraformaldehyde fixation procedure allows 120–200-μm sections to be obtained (Sergeeva and Vreugdenhil, 2002). We found that adequate visualization for spatial resolution could be achieved on intact embryos of Arabidopsis because of their small size, and thus it was not necessary to perform sectioning of the material.

Visualization of enzyme activities was based on the use of dehydrogenases to couple the oxidation of NADH, NADPH or FADH2 cofactors to the reduction of nitroblue tetrazolium (NBT). The method can be either direct or indirect depending on whether or not the enzymatic reaction under study results in the direct reduction of a particular cofactor; if it does not, then additional enzymes need to be introduced as coupling elements to transform the product of the reaction to the eventual reduction of one of the cofactors. Tetrazolium salts have been used successfully for decades for the histochemical localization of dehydrogenases. When the substrate of the dehydrogenase is present in the incubation medium the enzyme produces electrons by oxidation of its substrate. These electrons can then be picked up by water-soluble colourless NBT. Reduction of the tetrazolium salt generates a water-insoluble, intensely coloured formazan that precipitates at the site of dehydrogenase activity (Van Driel and Van Noorden, 1999). In Figure 1, the enzymatic reactions considered in our study are placed in the framework of carbon metabolism in maturing embryos. A specific protocol was designed for each of the enzymatic reactions analysed (Table 1). The distinct staining patterns obtained for the various enzymatic activities studied (see below) demonstrated the specificity of these protocols. To rule out the possibility of non-specific side reactions resulting in the reduction of NAD, NADP or FAD, control experiments were performed as follows: for each enzymatic activity embryos were incubated in the corresponding staining buffer with or without the substrate of the reaction under investigation. Non-specific activity was absent in all of the assays presented. Representative examples of control experiments are presented in Figure 2a. In embryos aged 8 days after flowering the phosphoglucomutase (PGM) activity was easily detectable, although confined to very specific regions of the embryo. A strong and very reproducible staining was observed both in the root tip and in the regions flanking the apical meristem. This staining appeared a few minutes after the beginning of incubation, and then grew darker where it had first appeared without spreading to the rest of the embryo (Figure 2b). This example clearly indicates that there was negligible, if any, diffusion of reduced NBT in the embryos. The staining observed therefore faithfully reflects the localization of the enzyme activities under study and the method gives good spatial resolution.

Table 1.  List of enzymatic activities considered in this study
EnzymeCoupling elements used
  1. Coupling elements when used are indicated. Enzymes exhibiting a dehydrogenase activity are in bold. 6PGDH, 6-phosphogluconate dehydrogenase; ADH, alcohol dehydrogenase; AGPase, ADP-Glc pyrophosphorylase; FK, fructokinase; G3PDH, NADP-dependent glyceraldehyde-3-P dehydrogenase; G6PDH, Glc-6-P dehydrogenase; GK, glucokinase; IDH, isocitrate dehydrogenase; Malic E, NAD-dependent malic enzyme; PFK, ATP-dependent phosphofructokinase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; SDH, succinate dehydrogenase; SUS, sucrose synthase; UGPase, UDP-Glc pyrophosphorylase. The coupling procedures used for AGPase, FK, HXK, PGM, UGPase, SUS and PGI are directly adapted from Sergeeva and Vreugdenhil (2002).

IDHNone
NAD-Malic ENone
PFKAldolase –NAD-dependent G3PDH
G6PDHNone
6PGDHNone
AGPasePGM –G6PDH
SDHNone
G3PDHNone
NADP-Malic ENone
FKPGI –G6PDH
GKG6PDH
PGMG6PDH
UGPasePGM –G6PDH
SUSUGPase – PGM –G6PDH
PFPPGI –G6PDH
PGIG6PDH
ADHNone
MSMDH
image

Figure 2. Validation of the in situ histochemistry procedure in wild-type embryos of Arabidopsis (Col0 ecotype). (a) Specificity of the enzymatic reactions. To rule out the possibility of non-specific activities resulting in the reduction of NAD, NADP, or FAD leading to erroneous staining patterns, controls were run for all the enzymes considered in this study. For each activity, embryos were incubated with (+) or without (−) the substrate of the reaction under investigation. Four of these control experiments are presented. 6PGDH, 6-phosphogluconate dehydrogenase; G3PDH, NADP-dependent glyceraldehyde-3-P dehydrogenase; G6PDH, Glc-6-P dehydrogenase. Total image height = 1.8 mm. (b) High spatial resolution of the procedure. Embryos aged 8 days after flowering were stained for phosphoglucomutase (PGM) activity for various incubation times as indicated. Total image height = 0.6 mm. (c) Biological relevance of the approach. Comparative time-course analyses of starch content and AGPase activity in developing embryos. Starch values are the mean ± SE of five independent measurements. AGPase, ADP-Glc pyrophosphorylase. Total height of each image = 1.6 mm.

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To further evaluate this approach, we assayed ADP-Glc pyrophosphorylase (AGPase). In maturing embryos of oilseed rape that transiently accumulate high amounts of starch, AGPase is regarded as the key regulatory element of the starch biosynthetic pathway (Vigeolas et al., 2004). Starch measurements have also been performed on Arabidopsis seeds and embryos, and a similar transient accumulation to that reported in oilseed rape has been observed (Baud et al., 2002; Gomez et al., 2006). Time-course analyses of starch content and AGPase activity in developing embryos revealed that, between 8 and 10 DAF, starch content in the embryo exhibited a sharp rise, peaking at 147 ng per embryo at 10 DAF, and this was mirrored by an increase in the intensity of AGPase staining (Figure 2c). Both starch content and staining intensity then dropped between 10 and 15 DAF. These results are consistent with starch and AGPase activity measurements from developing embryos of oilseed rape (da Silva et al., 1997). Our data on AGPase thus firmly establish the biological relevance of this method of histochemical localization in Arabidopsis embryos and demonstrate that the procedure used is sensitive enough to detect changes in enzyme activity levels at different developmental stages.

Spatiotemporal analysis of carbohydrate metabolism in wild-type embryos

The histochemical localization method was first used to monitor carbohydrate metabolism in developing embryos of the Columbia (Col0) ecotype. Embryos were studied at 8, 9, 12 and 15 DAF. At 8 DAF morphogenesis is completed, and the embryo is torpedo-shaped and is just entering the maturation phase. At 9 DAF the embryo is upturned-U-shaped and is filling the seed sac. The 12-DAF embryo is at an intermediate stage of the maturation process, and the 15-DAF embryo is entering the late maturation phase. For each of these developmental stages we investigated 18 enzymatic activities covering the carbohydrate metabolic network as depicted in maturing Arabidopsis seeds (Ruuska et al., 2002; Schwender et al., 2004a; White et al., 2000; Figure 1).

Based on the staining throughout the developmental series, three distinct patterns were identified (Figure 3). In the first group, staining intensity steadily decreased during the maturation phase; in the second, staining intensity peaked between 9 and 12 DAF, falling sharply towards the end of the maturation phase; and in the third, there was a significant increase from 8–12 DAF, with staining intensity remaining high until the end of the maturation phase. Isocitrate dehydrogenase (IDH) and NAD-malic enzyme (NAD-malic E) activities clearly belonged to the first group, with staining being particularly intense during the first few days of the maturation phase and then gradually disappearing (Figure 3). Similarly, the ATP-dependent phosphosfructokinase (PFK) activity, which was very strong in the torpedo-shaped embryos, peaked while the embryo filled the seed sac before decreasing. The two dehydrogenases of the OPPP, Glc-6-P dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), belonged to the second group. At 8 DAF these activities were only detectable in the root and in the apical region of the embryo. The staining observed in the hypocotyl and in the cotyledons was barely detectable. At 9 DAF the heavy staining previously localized in very specific areas of the embryo spread to all tissues and remained intense until 12 DAF, but then fell away in late-maturing embryos. The AGPase activity similarly peaked at 9 DAF, while the maximum NADP-dependent glyceraldehyde-3-P dehydrogenase (G3PDH) and succinate dehydrogenase (SDH) activities were slightly delayed, not peaking until 12 DAF and again falling away thereafter. The third group consisted of enzymes associated with the SUS cleavage pathway, including SUS, UDP-Glc pyrophosphorylase (UGPase), and fructokinase (FK), together with enzymes of the glycolytic pathway such as glucokinase (GK), PGM, and pyrophosphate (PPi)-dependent phosphofructokinase (PFP). The alcohol dehydrogenase (ADH) activity, although present in the whole series, exhibited a steady increase in staining intensity throughout maturation. Finally, activity of the glyoxylate cycle marker enzyme malate synthase (MS) was only visible in late-maturing embryos, which is consistent with the recent report of an increase in MS transcript abundance in maturing embryos (Gomez et al., 2006).

image

Figure 3. Study of enzymatic activities associated with carbon metabolism in developing embryos of the wild type (Col0 ecotype). Embryos aged 8, 9, 12, and 15 days after flowering were excised, fixed, and stained for each of the 18 enzymatic activities considered in this study. For each enzyme, a picture of the stained embryos is presented together with a schematic summarizing the evolution of the relative enzyme activity during embryo maturation. At each developmental stage, four representative embryos are presented. The image of each embryo was analysed with Adobe Photoshop to determine the intensity of the staining and allow visualization of the enzyme activity pattern during embryo development; values presented are the means of independent measurements on four embryos. Because of the nature of the in situ staining, the data depicted in these graphs are only semiquantitative. 6PGDH, 6-phosphogluconate dehydrogenase; ADH, alcohol dehydrogenase; AGPase, ADP-Glc pyrophosphorylase; FK, fructokinase; G3PDH, NADP-dependent glyceraldehyde-3-P dehydrogenase; G6PDH, Glc-6-P dehydrogenase; GK, glucokinase; IDH, isocitrate dehydrogenase; MS, malate synthase; NAD-Malic E, NAD-dependent malic enzyme; NADP-Malic E, NADP-dependent malic enzyme; PFK, ATP-dependent phosphofructokinase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; SDH, succinate dehydrogenase; SUS, sucrose synthase; UGPase, UDP-Glc pyrophosphorylase. Total image height = 1.4 mm.

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Detection of the mitochondria-localized succinate dehydrogenase and NAD-dependent isocitrate dehydrogenase, the plastid-localized NADP-dependent malic enzyme and the peroxisome-localized malate synthase demonstrated that enzymes from these major cell compartments were accessible for analysis by the in situ histochemical method.

Enzymatic activities associated with carbon metabolism in Arabidopsis embryos therefore change markedly during the maturation process. Our methodology allows an evaluation of the relative incidence of the different aspects of metabolism covered by the 18 selected enzymes during embryo development. For instance, enzymes known as key regulators of the starch biosynthetic pathway (AGPase; Vigeolas et al., 2004) and of the OPPP (G6PDH and 6PGDH; Plaxton, 1996) were specifically detected during early maturation. The coordinate induction of these two pathways, which both use Glc-6-P as a precursor, is consistent with reports that Glc-6-P transport activity between the cytosol and plastids is particularly elevated at this developmental stage (Eastmond and Rawsthorne, 2000; Ruuska et al., 2002). During the progression of the embryo through the maturation phase, activities associated with the SUS cleavage pathway and glycolysis became more pronounced. At least some of the changes in staining intensity, and thus enzymatic activity, are likely to be controlled at the transcriptional level. This is the case, for instance, for ADH, the relative enzymatic activity of which is tightly correlated with transcript levels for the ADH gene (At1g77120) in a transcriptomic data set obtained from developing Arabidopsis seeds (Figure 4a; Schmid et al., 2005). Several other activities also correlated with transcript levels of one or more of the genes designated to encode the respective enzymes. UGPase activity in embryos seemed to be strongly associated with At5g17310 transcription level (Figure 4b). NAD-malic E activity correlated with the transcript levels of one malic enzyme gene in particular (At2g13560; Figure 4c), while the GK/FK activity pattern appeared to be more closely associated with At2g19860 and At1g50460 than with the other HXK-encoding genes (Figure 4d). The case of SUS is somewhat intriguing. In the Col0 ecotype (Figure 4e), the increase in SUS activity during maturation paralleled the AtSUS3 (At4g02280) transcription profile, while previous reports (Baud et al., 2004) seemed to indicate that SUS activity in the Wassilewskija (Ws) ecotype was more closely correlated to the AtSUS2 (At5g49190) expression level. However, for most of the enzymatic activities investigated in this study, no correlation could be observed between relative enzymatic activities and transcription levels of the corresponding genes. In the case of 6PGDH, for instance, the enzyme activity steadily increased between 8 and 9 DAF while the transcript levels of all 6PGDH-encoding genes fell sharply. The discrepancies between microarray and enzyme activity data could be the consequence of post-transcriptional or post-translational control of enzyme activity (Gibon et al., 2004). Alternatively, the different sampling procedures employed in the different analyses could account for at least some of the differences, as the microarray study was performed on mRNA isolated from whole seeds, whereas our analysis of enzyme activities was performed on developing embryos. In young seeds, in particular, embryos represent a minor fraction of the seed and the high transcript levels detected on arrays may originate from the testa and/or the endosperm. A particularly striking finding of this study is the major switch in phosphofructokinase activity during the progression of embryos through the maturation phase. The ATP-dependent PFK activity was progressively replaced by the PPi-dependent activity (PFP; Figure 3). Taken together with the induction of the SUS cleavage pathway, this switch illustrates the importance of PPi and associated pathways during the maturation process. PPi has been presented as a possible coordinator of carbon metabolism within starch-storing potato tubers (Solanum tuberosum; Geigenberger et al., 2000). The switch from an invertase-dependent and ATP-consuming to a SUS-dependent and PPi-consuming sucrose breakdown process during tuber development is well documented and has been associated with the stolon-to-tuber transition (Appeldoorn et al., 1997). Here we demonstrate that storage compound accumulation in Arabidopsis embryos is similarly associated with the use of PPi, not only for the cleavage of sucrose, but also for the synthesis of Fru-1,6-P, in downstream glycolysis. This switch to pathways that consume less ATP and thus utilize oxygen more efficiently may constitute a response to the low-oxygen conditions prevailing inside developing oilseeds (Porterfield et al., 1999). These conditions were shown to limit storage metabolism (Vigeolas et al., 2003). The proposal that PPi plays an important role in recycling waste energy to fuel important central metabolic and cellular functions under low oxygen in the maturing embryo in order to avoid anoxia is supported by the observation that fermentative enzymes such as ADH are also clearly on during this period of development.

image

Figure 4. Comparative analysis of relative enzyme activities and transcript levels of corresponding genes. The images of embryos stained by in situ histochemistry were analysed with Adobe Photoshop to quantify the intensity of the staining and thus evaluate the pattern of enzyme activity during embryo development; values presented at each developmental stage, namely 8, 9, 12, and 15 days after flowering, are the means of independent measurements on four embryos. The relative transcription levels of genes putatively encoding these enzymes were isolated from AtGenExpress Developmental series. Values presented are the means of three independent expression estimates coming from triplicate Affymetrix ATH1 arrays (Schmid et al., 2005). (a) Alcohol dehydrogenase (ADH). (b) UDP-Glc pyrophosphorylase (UGPase). (c) Malic enzyme (Malic E). (d) Glucokinase (GK) and Fructokinase (FK). (e) Sucrose synthase (SUS). (f) 6-phosphogluconate dehydrogenase (6PGDH).

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Finally, it is interesting to note that the increase in staining intensity observed for most of the enzymatic activities peaking at 9–12 DAF or increasing throughout maturation was non-homogeneous throughout the embryo. An intense coloration first appeared in the meristematic regions before spreading to the hypocotyl and cotyledons. In the apical meristem the appearance of a deep staining was first restricted to peripheral regions of the meristem. Such a spatial regulation of carbohydrate metabolism within the apical meristem has already been reported during early leaf development in tomato plants (Lycopersicon esculentum; Pien et al., 2001). Data such as these begin to reveal the complexity of the concerted regulation of developmental and metabolic processes. In Arabidopsis embryos it is tempting to speculate that signals originating from the meristematic areas are transported to the adjacent tissues, generating gradients and gradually triggering metabolism associated with the maturation process.

Localization of enzymatic activities in mutant embryos

The putative AP2/EREBP transcription factor WRINKLED1 (WRI1) is involved in the regulation of seed storage metabolism in Arabidopsis (Cernac and Benning, 2004). A splicing mutant allele, wri1-1, which exhibits an 80% reduction in seed oil content, has been described (Focks and Benning, 1998). Interestingly, the wri1-1 mutation is not embryo lethal and the activities of several glycolytic enzymes could be determined spectrophotometrically on extracts prepared from homozygous seeds. Several of these activities were found to be significantly decreased in wri1-1 and we have used this mutant to establish if our in situ enzymatic assay methodology reflected these results. Seven enzymatic activities representative of the various carbon pathways involved in maturation were investigated in wri1-1 embryos 9, 12 and 15 DAF (Figure 5a). The pattern observed for G6PDH activity was unmodified in wri1-1 compared with the wild type and the increase in AGPase activity occurring during early maturation was only slightly delayed in the mutant. In contrast, both GK and FK activities were almost completely absent in wri1-1 embryos throughout the maturation process. In the case of PGM and PFP activities, the decrease in staining intensity observed in the mutants was less pronounced. Finally, the ADH activity appeared stronger in early-maturing wri1-1 embryos than in the wild type. These observations confirm at the embryo level measurements performed by Focks and Benning (1998) on whole seed extracts of several glycolytic activities, and thus demonstrate the validity and utility of our in situ procedure for the characterization of mutant embryos. This analysis also demonstrates that, while key enzymes of glycolysis are downregulated in wri1-1 embryos, enzymes of other pathways such as the OPPP and starch biosynthetic pathways are largely unaltered. Changes in starch content in the wri1-1 seeds (see figure 4 in Focks and Benning, 1998) are consistent with this proposal. The up-regulation of ADH activity much earlier in wri1-1 than in the wild type may indicate a response to anoxic conditions appearing earlier in the mutant, because of perturbation of glycolysis or an as yet uncharacterized role of the WRI1 transcription factor.

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Figure 5. Study of enzymatic activities associated with carbon metabolism in mutant embryos. (a) Altered carbon metabolism in wrinkled1 (wri1) embryos. Mutant and wild-type (WT) embryos (of the Col2 ecotype) aged 9, 12, and 15 days after flowering were excised, fixed, and then stained by in situ histochemistry. Total image height = 0.75 mm. (b) Enzymatic activities in three embryo lethals: gnom (gn), acetyl-CoA carboxylase1 (acc1), and schlepperless (slp). For each mutant considered, embryos with or without phenotype were harvested from siliques of heterozygous plants, fixed, and then stained by in situ histochemistry. 6PGDH, 6-phosphogluconate dehydrogenase; ADH, alcohol dehydrogenase; AGPase, ADP-Glc pyrophosphorylase; FK, fructokinase; G6PDH, Glc-6-P dehydrogenase; GK, glucokinase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase. Total image height = (WT) 0.7 mm, (gn) 0.7 mm, (acc1) 0.35 mm, (slp) 0.7 mm.

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Unlike wri1-1, the majority of mutants known to affect embryogenesis are embryo lethal (Tzafrir et al., 2004), which means that it is virtually impossible to perform conventional biochemical analysis because of a lack of material. The in situ methodology could therefore provide a first opportunity to gain an insight into biochemical processes operating in embryo-lethal mutants. Three embryo lethals exhibiting various phenotypes were chosen and enzymatic activities associated with carbon metabolism characterized by in situ histochemistry. The GNOM gene, also called EMB30, encodes a GDP/GTP exchange factor for small G-proteins of the ADP ribosylation factor (ARF) class, and can be viewed as a regulator of intracellular vesicle trafficking essential for auxin transport in particular (Geldner et al., 2003). Loss-of-function alleles lead to severe defects in the establishment of the embryonic axis, resulting in early developmental arrest (Mayer et al., 1993). Despite their aberrant shape, gn mutant embryos exhibited wild-type-like staining patterns for most of the activities analysed (Figure 5b). Disruption of the ACC1 gene, which codes for acetyl-CoA carboxylase 1, also results in embryo lethality (Baud et al., 2003). Null mutations lead to cucumber-like structures lacking cotyledons. In acc1 as in gn, most of the activities studied were similar to those in the wild type (Figure 5b). The only alteration detected in acc1 consisted of a slight increase in G6PDH staining at 12 DAF. This was specific to the first dehydrogenase of the OPPP, as the 6PGDH activity was unmodified. Taken together, the results obtained for gn and acc1 demonstrate at the enzymatic level that maturation processes can take place even when embryo morphogenesis has failed. This has already been demonstrated at the biochemical level for acc1-1, as mutant embryos accumulate the usual complement of storage proteins, while TAGs were stored in oil bodies but exhibited an altered fatty acid composition (Baud et al., 2003). Similar observations of mutants arrested at the globular (Yadegari et al., 1994) or heart stage (Boisson et al., 2001; Nickle and Meinke, 1998) also indicate that established maturation can progress even if embryo morphogenesis is altered.

In contrast to the gn and acc1 mutations, a mutation in the SCHLEPPERLESS (SLP) gene encoding the plastid chaperonin-60 alpha subunit does not cause aberrant tissue organization of the embryo, but it does delay embryo development so that growth of the cotyledons is severely impaired (Apuya et al., 2001). Although all of the enzyme activities assayed could be detected in slp embryos, most of the staining patterns obtained were aberrant. Some activities, such as that of GK, were detected in the entire embryo at 12 DAF, but the intensity of the staining was decreased compared with the wild type (Figure 5b). G6PDH and 6PGDH patterns at 12 DAF, and PGI and ADH patterns at 15 DAF were non-homogeneous, with some regions of the embryo exhibiting a strong wild-type-like staining while other regions were not stained at all. Finally, PGM activity at 15 DAF was only detected in small areas of the apical region of the embryo. Apuya and coworkers (2001) suggested that the ability to transcribe maturation genes is simply delayed in slp embryos. The data presented in this study are not consistent with a mere delay in the onset of maturation metabolism, but more likely reflect, at a post-translational level, profound alterations affecting carbon metabolism in response to the aberrant plastid production described in mature slp embryos. This stresses the importance of, and the central role played by, plastids in Arabidopsis seed metabolism (for a review, see Neuhaus and Emes, 2000).

Characterization of carbon metabolism in trehalose-6-P synthase1 (tps1) embryos

Finally, we took advantage of the in situ histochemistry approach to gain further insight into carbon metabolism in tps1 embryos. TPS1 is required for progression past the torpedo stage of embryo development (Eastmond et al., 2002). The development of the tps1 embryo follows a normal growth and patterning process, but the rate of growth is decreased (Gomez et al., 2005). Consequently, tps1 embryos only reach torpedo or early bent-cotyledon stage when wild-type embryos in the same silique reach the end of the maturation phase. Enzymatic activities were thus analysed at 12 and 15 DAF, the dissection of mutant embryos being impossible from younger seeds (Figure 6). Activities that typically peak during early maturation of wild-type embryos were also detected in tps1 at 12 DAF. Of these, both NAD-malic E and IDH activities then decreased significantly at 15 DAF in tps1 as in the wild type, but the expected drop in PFK activity was delayed in tps1. The G6PDH, 6PGDH, AGPase, and GK activities, which typically peak 9-12 DAF in the wild type, all generally showed a reduction at the equivalent temporal stage in tps1. With the notable exception of ADH, which showed an early elevation of activity in young tps1 embryos and remained stable throughout maturation, other enzymatic activities that typically increase with the onset of maturation appeared to be reduced in the mutant background. The decrease in staining intensity compared with the wild type could affect either the entire embryo (see for instance PFP and SUS activities at 12 DAF) or the hypocotyl only (see for instance PGI activity). The altered enzymatic patterns observed in tps1 embryos were not consistent with a mere delay in embryo development. Similarly, transcriptomic, metabolite, and electron microscope-based studies showed that tps1 torpedo-shaped mutants aged 12-15 DAF were different from wild-type torpedos aged 8 DAF (Gomez et al., 2006). In tps1, as in slp, the in situ histochemical data set suggests a significant impact of the mutation on carbon metabolism. It is interesting to note that microarray data published to date have revealed very few genes encoding enzymes of primary metabolism, the expression of which was putatively regulated by trehalose-6-P (Gomez et al., 2006; Schluepmann et al., 2004). Among transcriptomic data obtained from tps1 embryos, apart from several genes associated with starch degradation, including several amylases, glucan phosphorylase, and glucan water dikinases, which were repressed, only two sucrose-P synthases (At1g04920 and At5g20280) and two weakly expressed SUS genes (At1g73370 and At5g20830) were found to be downregulated (Gomez et al., 2006).

image

Figure 6. Study of enzymatic activities associated with carbon metabolism in developing trehalose-6-P synthase1 (tps1) embryos. Embryos with or without phenotype were harvested from siliques of heterozygous plants at 12 and 15 days after flowering, fixed, and then stained by in situ histochemistry. 6PGDH, 6-phosphogluconate dehydrogenase; ADH, alcohol dehydrogenase; AGPase, ADP-Glc pyrophosphorylase; FK, fructokinase; G3PDH, NADP-dependent glyceraldehyde-3-P dehydrogenase; G6PDH, Glc-6-P dehydrogenase; GK, glucokinase; IDH, isocitrate dehydrogenase; Malic E, NAD-dependent malic enzyme; PFK, ATP-dependent phosphofructokinase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; SDH, succinate dehydrogenase; SUS, sucrose synthase; UGPase, UDP-Glc pyrophosphorylase. Total image height = (WT) 0.75 mm, (tps1) 0.55 mm.

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This limited transcriptional control of primary carbon metabolism by trehalose-6-P in embryos strongly suggests that direct or indirect post-transcriptional regulation occurs which explains the altered enzymatic patterns observed in the mutant background. Through the phosphorylation of enzymes of carbohydrate metabolism, kinases such as AKIN11, whose expression actually correlates with trehalose-6-P levels (Schluepmann et al., 2004), could participate in this complex regulatory process. Regulation of SUS activity is mediated to an extent by phosphorylation (Hardin et al., 2004). Interestingly, recent promoter–reporter gene-based expression studies showed that, from the torpedo stage onwards, TPS1 expression extends throughout the hypocotyls of maturing embryos, with very little expression in cotyledons (Van Dijken et al., 2004). This spatial expression pattern in maturing embryos correlates with the hypocotyl-specific reduction in activity observed for several activities in tps1 embryos, and implicates TPS1 in regulating enzyme activity. Kolbe et al. (2005) recently demonstrated that trehalose-6-P regulates starch synthesis in leaves via post-translational redox activation of AGPase. However, given the more general effect of the tps1 mutant phenotype on growth of the whole embryo, it is likely that TPS1 also has other roles beyond carbon metabolism.

Conclusion

In situ staining of enzymatic activities has been successfully adapted for the study of carbohydrate metabolism in Arabidopsis embryos. This technique enables the collection of temporal and spatial data for a wide set of enzyme activities using very limited amounts of material. The procedure used is specific, sensitive, relatively quick, and constitutes a significant technical advance in the field. It allows enzymatic information to be obtained from specific regions of Arabidopsis embryos rather than from whole seeds which also contain endosperm and integuments, the metabolism of which differs significantly from embryo metabolism. Spatial localization of enzymatic activities with high resolution is complementary to data obtained from in situ hybridization techniques, reporter gene approaches, and immunocytological methods now routinely used in Arabidopsis. The results obtained in this study raise interesting questions concerning the spatial and temporal regulation of enzymatic activities associated with carbohydrate metabolism. The results also provide new insight into the relative importance of ATP and PPi during the maturation process, raising important questions regarding the impact of oxygen availability on oilseed metabolism that need further investigation. Finally, we have shown that the spatial localization methodology can provide new insight into metabolic processes in a variety of mutants perturbed or blocked in embryo development, thus providing an important tool for the further characterization of the large number of mutants that fall into this class.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Plant material and growth conditions

To characterize carbon metabolism in a wild-type background, Arabidopsis seeds of the Col0 ecotype were chosen. Wri1-1 (Cernac and Benning, 2004), gn [N8146 line from the Nottingham Arabidopsis Stock Centre (NASC)], acc1-1 (Baud et al., 2003), slp (N3719 line from the NASC) and tps1-1 (Eastmond et al., 2002) were used to investigate carbon metabolism in various mutant backgrounds.

Seeds were surface-sterilized and germinated on MS medium solidified with 0.7% (w/v) agar. After a cold treatment of 48 h at 4°C in the dark, plates were transferred to a growth chamber at 20°C, with a 16-h photoperiod and 50–70 μm m−2 sec−1 light from fluorescent bulbs. After 10 days, the plantlets were transferred to compost and grown in a glasshouse under similar conditions. Only primary shoots were used and secondary shoots were removed. To harvest siliques of defined developmental stage, individual flowers were tagged using coloured tape on the day of flowering.

Starch determination

For starch extraction, 50 embryos were excised from seeds, immediately frozen in liquid nitrogen, and then ground twice in 2 × 500 μl of 80% (v/v) ethanol at 4°C. After centrifugation (15 000 g for 10 min at 4°C), supernatant containing the soluble carbohydrates was discarded, and the pellet left from the extraction was used for starch determination, dried at 50°C for 30 min and then homogenized in 150 μl of 50 mm MOPS buffer (pH 7.0) containing 15 U of heat-stable alpha-amylase (EC 3.2.1.1). The suspension was incubated at 100°C for 6 min. Then, 200 μl of 0.2 m sodium-acetate buffer (pH 4.8) containing 35 U of amyloglucosidase was added and the suspension was incubated at 50°C for 30 min with regular shaking. Following centrifugation the glucose in the supernatant was used for starch quantification. This method was adapted from that of McCleary et al. (1994). Glucose measurements were performed with a kit (R-Biopharm, Glasgow, UK), based on the enzymatic method of Bergmeyer and Bernt (1974).

Handling and fixation of the embryos for in situ assays

Embryos were fixed immediately after excision in 1 ml of 2% parformaldehyde with 2% polyvinylpyrrolidone 40 and 1 mm dithiothreitol (DTT), pH 7.0, at 4°C for 1 h in a microtube. After fixation the microtube was centrifuged briefly at low speed. The supernatant was discarded and the embryos rinsed five times with 1 ml of distilled water and stored overnight at 4°C.

Histochemical enzyme assays

Fixed embryos were incubated in 250 μl of staining medium in microtubes placed in a dry heating block for 1–3 h, depending on the enzymatic activity being assayed. For any one enzymatic activity, all the samples analysed were incubated for exactly the same period. After incubation the staining medium was replaced with distilled water to stop the enzymatic reaction. The embryos were stored at 4°C. Staining buffers and incubation conditions for each enzyme assayed are detailed below.

NAD-dependent malic enzyme (EC 1.1.1.39; NAD-malic E): 100 mm HEPES (pH 7.2), 0.1 mm ethylenediaminetetraacetic acid (EDTA), 2 mm nicotinamide adenine dinucleotide (NAD), 0.1 mm Co enzyme A (CoA), 4 mm MnCl2, 5 mm malate, 0.8 mm nitroblue tetrazolium dye (NBT), and 0.4 mm phenazine methosulfate (PMS) (adapted from Outlaw and Manchester, 1980). A 1-h incubation was carried out at 30°C.

NAD-dependent isocitrate dehydrogenase (EC 1.1.1.41; IDH): 100 mm HEPES (pH 7.4), 4 mm MnCl2, 2 mm NAD, 2 mmdl-isocitrate, 0.8 mm NBT, and 0.4 mm PMS (adapted from Hathaway and Atkinson, 1963). A 2 h 35 min incubation was carried out at 30°C.

ATP-dependent phosphofructokinase (EC 2.7.1.11; PFK): 200 mm bicine (pH 8.0), 5 mm MgCl2, 3 mm ATP, 2 mm NAD, 4 mm Fru-6-P, 5 mm Na2HAsO4, 1.2 U ml−1 aldolase, 2.4 U ml−1 glyceraldehyde-3-P dehydrogenase, 0.8 mm NBT, and 0.4 mm PMS (Técsi et al., 1996). A 40-min incubation was carried out at 30°C.

Glc-6-P dehydrogenase (EC 1.1.1.49; G6PDH): 100 mm HEPES (pH 7.8), 0.5 mm NADP, 1.2 mm Glc-6-P, 5 mm MgCl2, 4 mm maleimide, 0.8 mm NBT, and 0.4 mm PMS (Técsi et al., 1996). A 2 h 40 min incubation was carried out at 30°C.

6-Phosphogluconate dehydrogenase (EC 1.1.1.44; 6PGDH): 100 mm HEPES (pH 7.8), 0.5 mm NADP, 1.2 mm 6-phosphogluconate, 5 mm MgCl2, 0.8 mm NBT, and 0.4 mm PMS (Técsi et al., 1996). A 2-h incubation was carried out at 30°C.

ADP-Glc pyrophosphorylase (EC 2.7.7.27; AGPase): 75 mm HEPES (pH 8.0), 0.44 mm EDTA, 5 mm MgCl2, 0.1% bovine serum albumin (BSA), 1 mm NAD, 2 U ml−1 phosphoglucomutase (PGM), 6 U ml−1 G6PDH, 20 μm Glc-1,6-P, 10 mm NaF, 1.4 mm PPi, 0.37 mm NBT, and 2 mm ADP-Glc (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1999). A 3-h incubation was carried out at 30°C.

Glucokinase (EC 2.7.1.1; GK): 50 mm 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol (Bistris) buffer (pH 8.0), 6.25 mm MgCl2, 2.5 mm ATP, 1 mm NAD, 1 U ml−1 G6PDH, 12.5 mm HEPES (pH 7.4), 0.25 mm EGTA, 0.025% BSA, 0.37 mm NBT, and 0.5 mm Glc (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1997). A 2-h incubation was carried out at 30°C.

Succinate dehydrogenase (EC 1.3.5.1; SDH): 100 mm phosphate buffer (pH 7.6), 9.3 mm succinate (fresh), 5 mm EDTA, 1.2 mm NBT, 10 μm sodium azide and 0.4 mm PMS (Levine et al., 2002). A 3-h incubation was carried out at 30°C.

Fructokinase (EC 2.7.1.4; FK): 50 mm 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol (Bistris) buffer (pH 8.0), 6.25 mm MgCl2, 2.5 mm ATP, 1 mm NAD, 1 U ml−1 G6PDH, 1 U ml−1 phosphoglucoisomerase (PGI), 12.5 mm HEPES (pH 7.4), 0.25 mm EGTA, 0.025% BSA, 0.37 mm NBT, and 0.5 mm Fru (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1997). A 2-h incubation was carried out at 30°C.

NADP-dependent glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13; G3PDH): 100 mm Bicine (pH 8.0), 0.5 mm NADP, 1 mm dihydroxyacetone phosphate, 5 mm Na2HAsO4, 20 U ml−1 triose-phosphate isomerase, 0.8 mm NBT, and 0.4 mm PMS (Técsi et al., 1996). A 1-h incubation was carried out at 30°C.

NADP-dependent malice enzyme (EC 1.1.1.40; NADP-malic E): 50 mm HEPES-KOH, 1 mm NADP, 5 mm malate, 10 mm MgSO4, 0.4 mm PMS, and 0.8 mm NBT, pH 7.65 (adapted from Detarsio et al., 2004). A 1 h 30 min incubation was carried out at 30°C.

Phosphoglucomutase (EC 5.4.2.2; PGM): 42 mm HEPES (pH 7.4), 4.2 mm MgCl2, 0.84 mm EDTA, 0.84 mm EGTA, 0.084% BSA, 1.4 mm NAD, 1 U ml−1 G6PDH, 0.37 mm NBT, and 4.35 mm Glc-6-P (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1999). A 2-h incubation was carried out at 30°C.

PPi-dependent phosphofructokinase (EC 2.7.1.90; PFP): 50 mm HEPES (pH 6.8), 0.5 mm Fru-1,2-P, 5 mm PPi, 5 μm Fru-2,6-P, 0.5 mm NAD, 2 U ml−1 PGI, 1 U ml−1 G6PDH, 0.8 mm NBT, 0.4 mm PMS and 5 mm MgCl2 (adapted from Theodorou and Plaxton, 1996). A 1 h 40 min incubation was carried out at 30°C.

Phosphoglucose isomerase (EC 5.3.1.9; PGI): 42 mm HEPES (pH 7.4), 4.2 mm MgCl2, 0.84 mm EDTA, 0.84 mm EGTA, 0.084% BSA, 1.4 mm NAD, 1 U ml−1 G6PDH, 0.37 mm NBT, and 4.35 mm Fru-6-P (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1999). A 2-h incubation was carried out at 30°C.

UDP-Glc pyrophosphorylase (EC 2.7.7.9; UGPase): 100 mm HEPES (pH 7.5), 1 mm EDTA, 2 mm Mg-acetate, 1 mm NAD, 1 U ml−1 PGM, 1 U ml−1 G6PDH, 20 μm Glc-1,6-P, 0.9 mm PPi, 0.37 mm NBT, and 5 mm UDP-Glc (Sergeeva and Vreugdenhil, 2002, modified from Appeldoorn et al., 1997). A 3-h incubation was carried out at 30°C.

Sucrose synthase (EC 2.4.1.13; SUS): 50 mm HEPES (pH 7.4), 5 mm MgCl2, 1 mm EDTA, 0.1% BSA, 1 mm EGTA, 1 mm NAD, 1 U ml−1 PGM, 1 U ml−1 G6PDH, 20 μm Glc-1,6-P, 1 U ml−1 UGPase, 0.37 mm NBT, 3.6 mm sucrose, 71 μm UDP, and 71 μm PPi (Wittich and Vreugdenhil, 1998). A 3-h incubation was carried out at 30°C.

Alcohol dehydrogenase (EC 1.1.1.1; ADH): 100 mm Tris (pH 8.0), 10% ethanol, 1.5 mm NAD and 0.37 mm NBT (Dolferus et al., 1994). A 3-h incubation was carried out at 30°C.

Malate synthase (EC 4.1.3.2; MS): 85 mm Tris–HCl (pH 8.0), 8 mm MgCl2, 0.2 mm acetyl-CoA, 2 mm glyoxylate, 6 U ml−1 malate dehydrogenase, 1 mm NAD, and 0.37 mm NBT. A 3 h 30 min incubation was carried out at 30°C.

Microscopy and image analysis

Embryos were photographed on agar medium using a Leica MZ6 dissecting microscope and a SPOT Advanced image capture system (Diagnostic Instruments, Sterling Heights, MI, USA). Images were then prepared and/or analysed with Adobe Photoshop (Mountain View, CA, USA). To analyse the relative changes in enzyme activities throughout a developmental series, images of embryos were analysed with the pipette instrument of Adobe Photoshop; CMYB values were measured in three regions of the stained embryo, namely cotyledon, hypocotyl, and root, and then averaged. The B (black) value (as a percentage) was used as a relative indicator of staining intensity.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

We gratefully acknowledge Yi Li and Oliver Thimm for their precious help in utilizing publically available transcriptomic data sets, and Steve Penfield for his advice on assaying malate synthase activity. We are grateful to A. Cernac and C. Benning for the gift of wri1-1 seeds, and to C. Rochat for the gift of acc1-1 seeds. David Meinke, Syngenta, the SeedGenes Project, and the NASC are acknowledged for providing us with the other embryo lethals used in this study.

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  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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