SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

A primary role of β-amylase (BMY) is to produce maltose during hydrolytic starch degradation. In photosynthetic organs, BMY activity is present in the chloroplast, where substrate starch is localized. BMY activity is also found in the vacuole and cytosol, where starch is not known to be localized suggesting additional roles in glucan degradation. Transcript levels and activity of different isoforms are regulated by different stimuli including cold, heat and drought stress. However, little is known about the functional role of BMY during environmental stresses. Recently, the functionality of one of the chloroplastic forms of Arabidopsis, ct-Bmy/BMY8/BAM3, has been shown to be needed for the protection of PSII photochemical efficiency following freezing stress by presumably catalyzing the synthesis of maltose, which acts as a cryoprotectant compound and precursor of soluble sugar metabolism. Still, there remain important questions about how the mobilization of starch through maltose and glucose can lead to the accumulation of cryoprotectant soluble carbohydrates during cold acclimation.


Abbreviations – 
ABA

abscisic acid

BMY or BAM

β-amylase, ct-Bmy, Arabidopsis chloroplast-localized β-amylase

DPE2

disproportioning enzyme II

GA3

gibberellic acid

GWD

α-glucan/water dikinase

MEX1

maltose transporter

phyA

phytochrome A

phyB

phytochrome B

RNAi

RNA interference.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

The current view of cold acclimation and the induction of freezing tolerance in plants that can survive the stresses of a freeze-thaw cycle is based on hundreds of years of research and countless studies. Over the past 20 years, molecular approaches have served to advance a better understanding of the important role that differential gene expression plays during cold acclimation and the acquisition of freezing tolerance. During the past 5 years, genomic approaches have dramatically accelerated progress revealing the extent of genome-wide alterations in gene expression, and the omics revolution promises to further accelerate progress (de la Fuente et al. 2006, Renaut et al. 2006, Wang et al. 2006). It is widely accepted that the processes of signal perception and transduction, reprogramming of gene expression and metabolism, restructuring cellular structures, altering physiology and modifying growth and development in response to low temperature are vastly multigenic and complex (Chinnusamy et al. 2006, Moffatt et al. 2006, Murata and Los 2006, Nakashima and Yamaguchi-Shinozaki 2006, Suzuki and Mittler 2006, Uemura et al. 2006, Van Buskirk and Thomashow 2006). Yet, to better understand the whole, it is necessary to understand the specific. This review is specifically focused on an enzymatic function involved in starch breakdown, β-amylase (BMY), that is encoded by a small gene family of nine members in Arabidopsis. Following upon the repeated linking of BMY expression with cold stress/cold shock in transcriptome studies (Fowler and Thomashow 2002, Jung et al. 2003, Kreps et al. 2002, Seki et al. 2001, 2002, Sung 2001), we briefly describe its emerging function and relationship with plant cold stress responses.

Role of BMYs in starch degradation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

A primary role of BMY appears to be in hydrolytic transitory starch degradation (Chia et al. 2004, Lu and Sharkey 2004, Niittylèa et al. 2004, Scheidig et al. 2002, Sharkey et al. 2004, Smith et al. 2004, Weise et al. 2004). For example, in potato, downregulation of a chloroplast-localized BMY by antisense mRNA resulted in a starch-excess phenotype in leaves compared with wild-type plants (Scheidig et al. 2002). Such plants with reduced chloroplastic BMY activity could degrade only 8–30% of their total leaf starch in the dark, whereas wild-type plants could degrade 50% of leaf starch (Scheidig et al. 2002). In contrast, knockouts of two putative chloroplast-targeted BMYs (BMY6/BAM4 and ct-bmy/BMY8/BAM3) have been reported to exhibit a starch-excess phenotype (Kaplan and Guy 2005, Smith et al. 2004). A vacuolar form (ATβ-Amy/RAM1/BMY1/BAM5) also has been shown to exhibit a small increase in starch content (Table 1) (Kaplan and Guy 2005). Knockout lines for two other BMYs (BMY7/BAM1 and BMY9/BAM2) that each also encode a putative transit peptide, suggesting a stromal localization, did not influence starch metabolism (Table 1) when functional gene expression was reduced or knocked out (Kaplan and Guy 2005). These reports indicate that some members of the BMY gene family might be playing a major role and some members may be able to compensate for the absence of other BMYs during starch degradation in Arabidopsis leaves.

Table 1. Arabidopsisβ-amylase (BMY or BAM) gene family and their influence on starch metabolism. RNAi, RNA interference. aChandler et al. (2001), bKaplan and Guy (2004), c2005, dLaby et al. 2001, eLao et al. (1999), fMita et al. 1997a, g1997b, hMonroe and Priess (1990), iSmith et al. (2004), jWang et al. (1995). kNA, not available. lPredicted subcellular location based on target P prediction.
NomenclatureMIPSOther nameTissue locationSubcellular locationMutant typeTranscriptStarch phenotype
BMY1bAt4g15210ATβ-Amyf, Ram1d, BAM5iPhloemjVacuolehT-DNA (SALK_032057)Not detectedStarch excessc
BMY2At5g45300BAM8i Cytosoll   
BMY3At5g18670BMY3a, BAM9bFlowersaCytosoll   
BMY4At2g45880BAM7i Cytosoll   
BMY5At2g32290BAM6i Cytosoll   
BMY6At5g55700BAM4i ChloroplastiT-DNANAkStarch excessi
BMY7bAt3g23920BAM1i ChloroplastlT-DNA (SALK_039895)Not detectedNonec
BMY8bAt4g17090ct-Bmye, BAM3i ChloroplasteRNAi (3 lines C5, C13, C14)ReducedStarch excessc
BMY9bAt4g00490BAM2i ChloroplastlT-DNA (SALK_086084)Not detectedNonec

It appears that maltose, the product of BMY, is exported to the cytosol during hydrolytic cleavage (Fig. 1). Several studies have shown that export of maltose and its breakdown compound, glucose, occurs from isolated chloroplasts (Neuhaus and Schulte 1996, Servaites and Geiger 2002, Weise et al. 2004). This is further supported by the recent discovery of a maltose translocator in the chloroplast membrane (Niittylèa et al. 2004). Mutations in the maltose translocator, a single copy gene, resulted in a starch-excess phenotype and an elevated maltose content (Niittylèa et al. 2004). Once maltose is exported to the cytosol, it is further metabolized to glucose and/or sucrose and maltodextrins by the activity of cytosolic glucosyltransferases during transitory starch degradation (Chia et al. 2004, Lu and Sharkey 2004). Proof for this model comes from knockouts of the cytosolic amylomaltase [disproportionating enzyme II (dpe2)] that transfers a glucosyl unit from maltose to glycogen in vitro (Chia et al. 2004). Consistent with the model, T-DNA insertion knockout plants for dpe2 contained high maltose (Chia et al. 2004, Lu and Sharkey 2004) maltodextrins and starch content (Lu and Sharkey 2004). Additional support for the model derives from an analysis of the anomeric forms of maltose during starch accumulation during the day and at night during starch mobilization (Weise et al. 2005). β-Maltose was found to be low during the day but high when transitory starch was being mobilized during the night. β-Maltose is the product of BMY. In mutants unable to utilize maltose, both anomeric forms were found to be present. Presumably, mutorotation was responsible for the presence of both anomeric forms as the two forms moved towards equilibrium, because the β-form could not be properly metabolized.

image

Figure 1. A simplified diagram of the components of hydrolytic breakdown of transitory starch showing the position of β-amylase (BMY) in the degradative process. LCG, long chain glucans; GWD, α-glucan/water dikinase; ISA, isoamylase; LDA, limit dextrinase; α-amylase; DPEp, disproportionating enzyme in the plastid; DPEc, disproportionating enzyme in the cytosol; HXK, hexokinase. BMY and its product maltose are shown in red.

Download figure to PowerPoint

Reaction mechanism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

BMY is an exoamylase that hydrolyses α-1,4 glycosidic linkages of polyglucan chains at the non-reducing end to produce maltose (4-O-α-d-glucopyranosyl-β-d-glucopyranose). Hydrolysis of the glycosidic bond is achieved by two conserved Glu residues using a general acid-base catalysis mechanism (Mikami et al. 1994). For the soybean enzyme, Glu186 plays a role as a general acid and Glu380 plays a role as a general base (Kang et al. 2004, Mikami et al. 1994). The carboxyl group of Glu186 is located on the hydrophilic surface of the glucose and donates a proton to the glycosidic oxygen. The carboxyl group of Glu380 lies on the hydrophobic face of the glucose residue at the subsite −1 and activates an attacking water molecule. The deprotonated Glu186 is stabilized by Thr342 after cleavage of the glycosidic bond (Kang et al. 2004, Mikami et al. 1994). The reducing glucose of the maltose product is in the β-form, hence the name BMY.

Current models of BMY function explain its mode of action on polyglucan chains. BMY appears to degrade amylose chain substrates by a random encounter-binding mechanism but can also sequentially catalyse maltose production after an initial substrate binding. This combined hydrolytic process is referred to as ‘multiple attack’ (Adachi et al. 1998). The estimated Kcat for the major soybean enzyme is between 1260 and 1280 (s−1), and the optimum pH is 5.4–6.0 (Adachi et al. 1998, Hirata et al. 2004).

Subcellular localization of BMYs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

BMYs are localized (Table 1) to the stroma of mesophyll cell chloroplasts (Lao et al. 1999, Scheidig et al. 2002), the vacuole (Datta et al. 1999, Ziegler and Beck 1986) and the cytoplasm. One Arabidopsis BMY, designated ct-Bmy (At4g17090, ct-bmy/BMY8/BAM3), has been biochemically localized to the chloroplast stroma (Lao et al. 1999) based on import studies with isolated pea chloroplasts and confirmed by accumulation of a BMY–green fluorescent protein fusion protein in Arabidopsis chloroplasts (Lao et al. 1999). Arabidopsis contains three additional highly homologous BMY genes that, based on sequence analyses (Table 1), are predicted to be plastid localized. A chloroplast localization of at least one BMY was also shown for potato (Scheidig et al. 2002). In contrast, other subcellular fractionation studies with pea have demonstrated a vacuolar localization for BMY (Ziegler and Beck 1986). Isolated vacuoles from pea and wheat leaf protoplasts were found to contain detectable amylolytic activity, which was identified as BMY activity (Ziegler and Beck 1986). Somewhat surprising, the majority of BMY activity in leaves of maize and pearl millet was localized in the vacuole, 94 and 80%, respectively (Datta et al. 1999). Further support for multiple subcellular localizations of BMY in plant cells comes from bioinformatic analysis of the Arabidopsis genome, where five of the nine (currently released sequences from GenBank) probable BMY genes encode proteins predicted to localize to at least two extraplastidic compartments (Table 1).

Relationship of starch-sugar interconversions during temperature stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

It has long been known that starch is converted to soluble sugars during temperature stress and soluble sugars are converted back to starch when temperature stress is removed (Li et al. 1965, Parker 1962, Sakai and Yoshida 1968, Siminovitch et al. 1952). On the basis of numerous studies with perennials during seasonal changes, starch content declined with the onset of low temperature in the late Fall, whereas soluble sugar content increased in overwintering tissues (Li et al. 1965, Sakai and Yoshida, 1968, Siminovitch et al. 1952). In many studies, the increased soluble sugar content was highly correlated with enhanced in cold hardiness (Li et al. 1965, Parker 1962, Sakai and Yoshida 1968, Siminovitch et al. 1952). In the early spring, with the onset of warm weather, sucrose and free sugars were rapidly converted back to starch (Sakai and Yoshida 1968, Siminovitch et al. 1952). Therefore, it seemed likely that such starch-sugar interconversions might be mediated, in part, by BMY and through its product, maltose, during temperature stress based on recent studies of temperature regulation of BMY and maltose content (Kaplan and Guy 2004, 2005, Seki et al. 2001, Sung 2001). This seems ever more probable given the recently emergent prominent role of BMY and maltose in transitory starch degradation (Chia et al. 2004, Lu and Sharkey 2004, Niittylèa et al. 2004, Scheidig et al. 2002, Sharkey et al. 2004, Smith et al. 2004, Weise et al. 2004).

The importance of starch to sugar conversion and β-amylase during the early stages of cold shock and cold acclimation of Arabidopsis was recently demonstrated with a mutant for α-glucan/water dikinase (GWD) mutant. This enzyme phosphorylates α1[RIGHTWARDS ARROW]4 glucan chains at the C6 and C3 position (Ritte et al. 2002). For reasons not yet fully understood, phosphorylation of starch is an apparent requirement for hydrolytic transitory starch breakdown (Lloyd et al. 2005) by, presumably, facilitating hydrolytic attack by β-amylase. Knockout mutants for GWD (also known as SEX1) showed impaired ability to develop increased freezing tolerance during the first 24 h of low temperature exposure (Yano et al. 2005). In the mutants, starch degradation during cold shock was reduced and maltooligosaccharides were not accumulated. As a result, accumulation of free glucose and fructose was delayed in the mutants during the early stage of cold acclimation but reached wild-type levels after 7 days when the freezing tolerance of the mutants equalled that of wildtype (Yano et al. 2005). Still, a number of open questions remain regarding the details of the biochemical mechanisms that control and influence the flow of carbon from starch into soluble sugars and, subsequently, other low molecular weight compounds characteristic of acclimating or acclimated plants (Kaplan et al. 2004).

Regulation of BMY

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

For a rather mundane enzyme, regulation of BMY expression and activity is unexpectedly rather complex. BMY expression and activity are regulated by developmental programming (Gana et al. 1998), light (Chandler et al. 2001, Harmer et al. 2000, Sharma and Schopfer 1982, Smith et al. 2004, Tepperman et al. 2001), sugars (Nakamura et al. 1991, Maeo et al. 2001, Mita et al. 1995), phytohormones (Ohto et al. 1992, Wang et al. 1996), abiotic stresses (Datta et al. 1999, Dreier et al. 1995, Kaplan and Guy 2004, Nielsen et al. 1997, Seki et al. 2001, Sung 2001) and proteolytic cleavage (Hara-Nishimura et al. 1986, Sopanen and Lauriere 1989). Furthermore, different isoforms or gene family members of plastid and non-plastid BMYs are regulated by different stimuli. Table 2 summarizes the regulation of different isoforms in Arabidopsis.

Table 2.  Regulation of Arabidopsisβ-amylases (BMYs). I, induced; R, repressed; N, no response; D, diurnal; C, circadian clock. aChandler et al. (2001), bHarmer et al. (2000), cKaplan and Guy (2004), d2005, eKaplan et al. (unpublished), fKreps et al. (2002), gMartzivanou and Hampp (2003), hMita et al. (1995), iRizhsky et al. (2004), jSmith et al. (2004).
BMYSubcellular locationCold shockHeat shockLightSugarHyper-gravity
BMY1VacuoleRcIcNjIhIg
BMY2CytosolNdNeNj  
BMY3CytosolRdNeDj, Ca  
BMY4CytosolNdNeNj  
BMY5CytosolNdReNj  
BMY6ChloroplastNdNeDj  
BMY7ChloroplastNc,dIc,iDj  
BMY8ChloroplastIc,d,fNcDj, Cb  
BMY9ChloroplastNcRcNj  

Abiotic stress

Regulation of BMY expression and activity by abiotic stress includes osmotic (Datta et al. 1999, Dreier et al. 1995), drought (Yang et al. 2001), salt (Datta et al. 1999, Dreier et al. 1995), cold (Nielsen et al. 1997, Seki et al. 2001, Sung 2001) and heat stress (Dreier et al. 1995, Sung 2001). More specifically, regulation of BMY activity by osmotic stress appears to be a general response for several plant species (Datta et al. 1999, Dreier et al. 1995). Exposure of barley (Dreier et al. 1995), pearl millet and maize (Datta et al. 1999) to osmotic stress with 300 mM sorbitol for 4 days results in the increase of vacuolar BMY activity, which is correlated with an increase in BMY protein. Similarly, when cucumber cotyledons are treated with 30 or 50% polyethylene glycol for 0.5 and 1 day, BMY activity increases followed by increases in the free sugars' sucrose and maltose (Todaka et al. 2000). Not surprisingly, salt stress also stimulates induction of BMY protein accumulation and activity (Datta et al. 1999, Dreier et al. 1995) in maize, pearl millet (Datta et al. 1999) and barley (Dreier et al. 1995).

Temperature stress has been shown to lead to the induction of either transcripts and/or activity of BMY (Datta et al. 1999, Dreier et al. 1995, Fowler and Thomashow 2002, Jung et al. 2003, Kreps et al. 2002, Seki et al. 2001, Seki et al. 2002, Sung 2001). More recently, the increased BMY transcript and/or activity has been linked with the increased maltose content (Kaplan and Guy 2004, Nielsen et al. 1997). For example, when Arabidopsis plants grown at 20° C were exposed to 40° C for 1 h, the BMY7/BAM1 transcript, encoding a putative chloroplast-targeting peptide, increased about five-fold (Kaplan and Guy 2004, Sung 2001). This was also found to be accompanied by maltose accumulation (Kaplan and Guy 2004). Similarly, raising barley growth temperature from 25 to 35° C resulted in induction of BMY activity (Dreier et al. 1995). Intuitively, if heat stress induced BMY expression and enzymatic activity, one might expect low temperature to repress expression and activity. Yet, cold stress also induces BMY transcript level or activity (Fowler and Thomashow 2002, Kaplan and Guy 2004, 2005, Kreps et al. 2002, Nielsen et al. 1997, Seki et al. 2001, Sung 2001). For example, when Arabidopsis was cold-stressed at 4° C for 12 h, chloroplast-localized BMY (ct-Bmy/BMY8/BAM3) (Table 2) transcript levels increased (Kaplan and Guy 2004, Sung 2001), and induction was found to occur as early as 2 h of exposure to cold stress (Seki et al. 2001). The increased BMY8 transcript level was associated with maltose accumulation (Kaplan and Guy 2004). Furthermore, BMY8 RNA interference (RNAi) lines with reduced BMY transcript levels were found to have less maltose accumulation in response to cold shock compared with wildtype (Kaplan and Guy 2005). Similarly, reducing potato tuber storage temperature from 20 to 5 or 3 °C resulted in increased BMY activity (four–five-fold) over a 10-day period (Nielsen et al. 1997). The increased BMY activity was followed by maltose accumulation, whereas the activities of α-glycosidase and endoamylase remained unchanged (Nielsen et al. 1997). Given the rapid accumulation, it has been suggested that increased maltose content during temperature shock might contribute to the protection of photosynthetic electron transport chain and proteins in chloroplast stroma. Kaplan and Guy (2005) demonstrated in vivo that ct-Bmy/BMY8/BAM3 RNAi lines with reduced maltose content had reduced chlorophyll fluorescence parameters following freezing stress and concluded that stress-induced BMY results in maltose accumulation, which, by itself or with glucose and fructose, can contribute to the protection of the photosynthetic electron transport chain, stromal proteins and membranes inside the chloroplast during temperature shock.

Light

BMYs are under the control of the circadian clock and diurnal regulation in Arabidopsis (Chandler et al. 2001, Harmer et al. 2000, Lu et al. 2005, Smith et al. 2004). Gene expression-profiling studies have indicated that the transcript levels of at least one chloroplastic (ct-Bmy/BMY8/BAM3) and one cytosolic (BMY3/BAM9) BMY to be regulated by the circadian clock (Chandler et al. 2001, Harmer et al. 2000, Lu et al. 2005). BMY transcript levels increased during the night and decreased during the day and continued this pattern of expression even following transfer of plants to continuous light conditions. Transcripts for other genes involved in starch mobilization showed very similar profiles as well (Harmer et al. 2000, Lu et al. 2005). Furthermore, a recent study has also shown that transcripts of four BMYs (BMY3/BAM9, BMY6/BAM4, BMY7/BAM1 and ct-Bmy/BMY8/BAM3) were diurnally regulated. Three of the four, BMY6/BAM4, BMY7/BAM1 and ct-Bmy/BMY8/BAM3, encode putative transit peptides for chloroplast stromal targeting. Transcript levels of BMY6, BMY7 and BMY8 (Table 2) showed a steady increase until dawn and then decreased after sunrise (Smith et al. 2004). Another study has shown that light is necessary to maintain cycling of gene expression and maltose concentration on a diurnal basis (Lu et al. 2005). Such an expression profile is consistent with the recently demonstrated role of plastid-localized BMYs in starch degradation (Kaplan and Guy 2005, Scheidig et al. 2002, Smith et al. 2004).

Light regulation of BMY is further accomplished through red/far-red photoreceptor phytochromes, which are regulated by circadian clock and feed back to modulate light input to the clock (Sharma and Schopfer 1982, Tepperman et al. 2001, Tepperman et al. 2004). During cotyledon photomorphogenesis in mustard, BMY induction is photoreversible by far-red light during the first 36 after sowing, but mRNA synthesis does not increase until about 46 h after sowing (Sharma and Schopfer 1982). This timing difference of phytochrome-mediated induction of BMY activity was suggested to involve a stable regulatory element (Sharma and Schopfer 1982) in the signal-transduction pathway. In Arabidopsis, at least one member of the BMY family is under the control of phytochromes. Transcript levels of ct-Bmy/BMY8/BAM3 in wild-type Arabidopsis were increased within 1 h of continuous far-red light exposure, whereas the transcript levels of ct-Bmy/BMY8/BAM3 remained unchanged in a phytochrome A (phyA) null mutant, hence this gene was classified as a phyA early-induced gene (Table 2). The induction of ct-Bmy/BMY8/BAM3 transcript levels in response to the continuous red light was also dependent on phytochrome B (phyB) (Tepperman et al. 2004).

Sugars

Sugars function as metabolites, as osmolytes and as regulators of gene expression. Sugar signalling regulatory activity is thought to be accomplished through hexokinase (HXK), hexose and sucrose-dependent pathways (Smeekens 2000). Thus, treatment with 3 and 6% fructose, glucose and sucrose induced BMY transcript, protein accumulation and activity in sweet potato leaf petiole cuttings (Nakamura et al. 1991) and Arabidopsis plants (Table 2) (Mita et al. 1995). In Arabidopsis, sugar-regulated BMY appears to involve the vacuolar form. Sugar signalling and regulation is also supported by studies with transgenic tobacco plants harbouring a construct for β-amy : β-glucuronidase (GUS), where GUS expression is driven by a sweet potato BMY promoter (Maeo et al. 2001). The GUS activity shows induction in leaf tissues treated with glucose, fructose, mannose and sucrose but not the non-metabolizable 2-deoxyglucose that can be phosphorylated by HXK. Use of glucosamine, an inhibitor of HXK that also inhibits sucrose-induced GUS activity further supported a role for HXK-mediated regulation (Maeo et al. 2001). Thus, sugar regulation for BMY appears to be accomplished through hexose- and sucrose-dependent pathways (Maeo et al. 2001, Mita et al. 1995, Nakamura et al. 1991) requiring HXK activity (Maeo et al. 2001).

Phytohormones

Phytohormones regulate BMY at the levels of transcript abundance and activity (Ohto et al. 1992, Wang et al. 1996). One example of this has been shown in germinating rice seeds, where BMY activity was inhibited by exogenous supply of 10 µM abscisic acid (ABA). This inhibition was fully reversible by adding 1–10 µM gibberellic acid (GA3). The ABA inhibition of BMY activity was exerted at the mRNA level, either by inhibiting transcription or by destabilizing the BMY mRNA (Wang et al. 1996). On the other hand, treatment of sweet potato leaf petioles with 100 µM ABA led to induction of BMY mRNA, but this induction was repressed by 50 µM GA3 supply (Ohto et al. 1992). These opposite responses to ABA and GA3 could result from species-specific processes, differences between petioles and seeds or different phytohormone treatment levels.

Proteolytic cleavage

The seeds of some species contain stored BMY that is bound to starchy endosperm possibly through S–S bridges (Hara-Nishimura et al. 1986). During the germination of barley seeds, BMY is not synthesized de novo, but instead, is released from a bound form by proteolytic cleavage to produce a smaller sized free form (Sopanen and Lauriere 1989). Western blot analysis showed that the free (59 kDa) and the bound (64 kDa) form of BMY could be isolated from germinated and ungerminated seeds, respectively (Sopanen and Lauriere 1989). The uncleaved form has lower activity compared with the free form; thus, proteolytic cleavage is thought to relieve steric hindrance that prevent substrates from reaching the active site (Sopanen and Lauriere 1989).

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References

In conclusion, genetic studies with potato and Arabidopsis have shown that BMYs play a major role in starch degradation and in the daily turnover of transitory starch in photosynthetic organs. In Arabidopsis, there are nine members of BMY with different subcellular and tissue localization and regulation. Four members (BMY6, BMY7, BMY8 and BMY9) encode a putative targeting signal for chloroplast localization, where substrate starch is located. BMY6 and BMY8 appear to have a larger role in transitory starch degradation than the other members. Besides their role in starch degradation, BMYs have been implicated to have a role in stress tolerance during temperature stress by increasing maltose and other soluble sugars that can act as emergency compatible solutes. The biochemical details of how hydrolytic starch mobilization contributes to carbohydrate metabolism during cold stress remain to be understood. BMY transcript levels and activity have been found to be induced by a variety of stresses; however, the complete picture of the function of BMY induction in response to these stresses remains to be discovered.

Acknowledgements –  We thank Dale Haskell for critical reading and comments. We thank the Arabidopsis Biological Resource Center (Ohio) for providing seed of T-DNA insertion lines and USDA NRI (no. 2002-35100-12110) for funding.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Role of BMYs in starch degradation
  5. Reaction mechanism
  6. Subcellular localization of BMYs
  7. Relationship of starch-sugar interconversions during temperature stress
  8. Regulation of BMY
  9. Conclusion
  10. References