A mutant of rice lacking the leaf large subunit of ADP-glucosepyrophosphorylase has drastically reduced leaf starch contentbut grows normally
Sandrine RostiA, Brendan FahyA and Kay DenyerA,B
AJohn Innes Centre, Norwich Research Park, Norfolk NR4 7UH, UK.BCorresponding author. Email: [email protected]
Abstract. A mutant of rice was identified with a Tos17 insertion in OsAPL1, a gene encoding a large subunit (LSU)of ADP-glucose pyrophosphorylase (AGPase). The insertion prevents production of a normal transcript from OsAPL1.Characterisation of the mutant (apl1) showed that the LSU encoded by OsAPL1 is required for AGPase activity in riceleaf blades. In mutant leaf blades, the AGPase small subunit protein is not detectable and the AGPase activity and starchcontent are reduced to <1 and <5% of that in wild type blades, respectively. The mutation also leads to a reduction instarch content in the leaf sheaths but does not significantly affect AGPase activity or starch synthesis in other parts of theplant. The sucrose, glucose and fructose contents of the leaves are not affected by the mutation. Despite the near absenceof starch in the leaf blades, apl1 mutant rice plants grow and develop normally under controlled environmental conditionsand show no reduction in productivity.
Starch is synthesised in many plant organs as a storagecompound. It is the main storage compound in cereal seeds,accounting for example, for ∼60% of the weight of a mature ricegrain. In many plants including rice, starch is also synthesised inleaves during the day; in leaf sheaths and stems as a temporaryreserve before heading (grain-filling), in pollen grains beforetheir germination and in root cap cells, where it is involved ingravity perception. Starch is a polymer of glucose, the glucanchains being elongated by the addition of glucose units fromADP-glucose. ADP-glucose is synthesised by the enzyme ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27). In plants,this enzyme consists of two types of subunit called the smalland large subunits. Mutational analysis shows that both subunitsare required for normal AGPase activity in vivo (Tsai and Nelson1966; Dickinson and Preiss 1969; Bhave et al. 1990). In vitro,the small subunit (SSU) of AGPase from barley (Doan et al.1999) and potato (Ballicora et al. 1995) is active in the absenceof the large subunit (LSU), whereas the potato LSU alone has noactivity (Ballicora et al. 2005). This is consistent with the ideathat the SSU is the catalytic subunit and that the LSU may have arole in modifying the allosteric properties of the SSU (Ballicoraet al. 2005).
The relationships between genes, proteins and functionsof AGPase subunits have been extensively studied in cereals.However, knowledge of LSU of AGPase lags behind that ofthe SSU. In many plants, the LSU of AGPase are encoded bymultiple genes. For example, four LSU genes per species have
been identified in rice, maize and Arabidopsis thaliana L. Thosein rice and maize are orthologous but the Arabidopsis genesare distinct from those in cereals. This suggests that the multipleLSU genes in monocots and eudicots evolved independently. Thefunction of one of the AGPase LSU genes of maize, Shrunken-2,was shown by mutations that drastically reduce the starchcontent of the endosperm. These showed that the Shrunken-2gene encodes the LSU of the major isoform of AGPase in theendosperm (Tsai and Nelson 1966; Bhave et al. 1990), which islocated in the cytosol (Denyer et al. 1996).
In rice, no mutant for any of the four rice LSUs (OsAPL1to OsAPL4; Table 1) has previously been identified. Expressionanalysis (northern blots; Akihiro et al. 2005; quantitative real-time PCR; Ohdan et al. 2005) of the LSU genes and proteomicanalysis (Koller et al. 2002) together suggest that in rice,OsAPL1 probably encodes a LSU found predominantly in theleaf, OsAPL2 and OsAPL3 probably encode seed isoforms andOsAPL4 probably encodes a LSU, which might have a minor rolein the leaf and/or seed. The orthologue of Shrunken-2 in maize,OsAPL2, does not encode an obvious transit peptide for importinto the plastids suggesting that, such as Shrunken-2, it encodesa cytosolic LSU (Akihiro et al. 2005; Ohdan et al. 2005). Theproteins encoded by OsAPL1 and OsAPL4 both have predictedplastid-targeting sequences and are assumed to be plastidialbut the location of the protein encoded by OsAPL3 cannot beunambiguously predicted from its sequence.
The development in rice of several insertional mutagenesissystems has made functional analysis of large numbers of
Characterisation of a rice mutant lacking leaf starch Functional Plant Biology 481
Table 1. Gene nomenclatureThe genes (and corresponding full-length cDNAs) encoding large subunits of AGPase in rice, their subcellular locations and their spatial patterns of expressionas reported by Akihiro et al. (2005) [1] and Ohdan et al. (2005) [2]. The abbreviated gene names suggested by Akihiro et al. (2005) have been used in the
rest of this paper. LSU, large subunit
Gene name (cDNA) Abbreviated gene names Predicted subcellular location Expression
Os03 g52460 (AK069296) OsAPL1 [1], OsAGPL3 [2] Plastid [1, 2] Predominant LSU transcript in leaf blade [2],also present in grain and leaf sheath [1]
Os01 g44220 (AK071497) OsAPL2 [1], OsAGPL2 [2] Cytosol [1, 2] Transcript present in grain [1, 2],scarce in leaf blade [2]
Os05 g50380 (AK100910) OsAPL3 [1], OsAGPL1 [2] Cytosol [1] plastid [2] Transcript abundance relatively low in grainand scarce in leaf blade [2],also present in leaf sheath [1]
Os07 g13980 (AK121036) OsAPL4 [1], OsAGPL4 [2] Plastid [1, 2] Transcript scarce in grain and leaf blade [2],also present in leaf sheath [1]
specific genes possible. One of these systems makes use ofan endogenous transposable element of rice called Tos17. Thetransposition of Tos17 is activated by tissue culture and becomesinactivated in regenerated plants. Large-scale sequencing andcataloguing of Tos17-flanking sequences has been initiated andthe mutant materials and information have been made publiclyavailable. To further investigate the function of the LSUs ofAGPase in rice, we searched for rice insertional mutants affectingany of the four LSUs of AGPase in the Tos17 mutant database,and in other databases. We identified a single mutant line witha Tos17 insertion in the OsAPL1 gene. Here we use this mutantto show that OsAPL1 encodes a leaf LSU that is required forat least 99% of the AGPase activity in rice leaf blades. Inthe leaves of the mutant, the starch content is <5% of thatin the wild type leaves. We have used this rice mutant toinvestigate the importance of starch in the leaf for normal growthand productivity.
Materials and methodsPlants and growth conditionsMutant line NG7525 was developed from Oryza sativa L.cultivar Nipponbare by the Rice Genome Project of theNational Institute of Agrobiological Sciences, Japan (Miyaoet al. 2003). T2 rice seeds that were segregating for theTos17 insertion (T36075T) were obtained from the RiceGenome Resource Centre, Japan. Seeds were washed threetimes in sterile water, transferred to moist sterile filter paper,sealed in a petri dish and incubated at 25◦C until the rootand shoot were established. Seedlings were transferred toindividual 300-mL pots containing 2 parts sand : 1 partpouzzolane : 4 parts loam (pH 5.6). When established,plants were transferred to 1-L pots in the same growthmedium containing 0.8 g kg−1 NPK/Mg fertiliser (Scotts ExactStandard 3–4 months; Scotts Professional, Geldermalsen,The Netherlands). Seedlings and plants were grown in acontrolled environment room with 12 h light (28◦C)/12 hdark (24◦C) and 90% humidity. The quantum irradiancewas 300–350 µmol quanta m−2 s−1 at pot height and400–500 µmol quanta m−2 s−1 at panicle height. For thesegregation experiments, seeds were sterilised in 2% (v/v)aqueous sodium hypochlorite for 30 min and transferredto petri dishes containing sterile 1% agar. The petri dishes
were incubated in a growth cabinet with continuous light(100 µmol quanta m−2 s−1) at 25◦C.
DNA acid isolation and genotypingLeaf samples were ground in DNA extraction buffer (200 mM
TRIS-HCl (pH 7.5), 25 mM EDTA, 0.5% (w/v) SDS, 250 mM
NaCl). After centrifugation at 20 000g for 3 min, the supernatantwas mixed by inversion with one volume of isopropanol. TheDNA was precipitated for 10 min at room temperature and thencollected by centrifugation at 20 000g for 7 min. The DNA pelletwas air-dried and re-dissolved in 100 µL 100 mM TRIS-HCl(pH 8.0).
A 1-µL aliquot of DNA was used as a template for PCRamplification with Taq polymerase (Roche Diagnostics Ltd,Lewes, UK) and 35 cycles of 94◦C for 60 s, 55◦C for 30 sand 68◦C for 60 s. The primers were P1 (5′-GAGATGGATTCGTCGAGGTG-3′), P2 (5′-ATTGTTAGGTTGCAAGTTAGTTAAGA-3′) and P3 (5′-TCCATGTAGTCCATGCGGTA-3′).
RNA extraction and transcript analysisTotal RNA was isolated from ∼50 mg leaf tissue byhomogenisation in 1 mL TRIzol Reagent (Invitrogen Ltd,Paisley, UK) according to the manufacturer’s instructions. Firststrand cDNA was synthesised with the Superscript II reversetranscriptase (Invitrogen) according to the manufacturer’sinstructions.
A 1-µL aliquot of cDNA was used as a templatefor PCR amplification with the Pfu Turbo (Stratagene,Amsterdam, The Netherlands) high-fidelity polymerase.Reactions included 10% DMSO. The primers used were P4(5′-GAAGGATCACCAGCTCACCAC-3′) and P3. Cyclingconditions were 35 cycles of 94◦C for 30 s, 55◦C for 30 s and72◦C for 60 s.
Extraction and assay of AGPase activityAll steps were performed at 4◦C or on ice. Tissue washomogenised in extraction buffer containing 50 mM HEPES(pH 7.4), 2 mM MgCl2, 1 mM EDTA and 1 mM DTT. Aftercentrifugation for 15 min at 20 000g, the supernatant was assayedfor AGPase activity as follows:
Spectrophotometric assay. The activity was assayed in thepyrophosphorolysis direction by the method described by Smith(1990) and modified by Burton et al. (2002).
482 Functional Plant Biology S. Rosti et al.
Radioactive assay. The activity was measured in the directionof ADP-glucose synthesis as in Rosti et al. (2006) except thatthe assay buffer was 100 mM HEPES-KOH (pH 7.4).
Extraction and assay of starch and sugarsLeaf blades, leaf sheaths and the stems supporting the panicleswere weighed, immediately frozen and later, ground in liquid N2.The frozen samples were ground in 2 mL of 0.77 M perchloricacid and allowed to thaw to 4◦C on ice for 30–45 min. Theinsoluble material, including starch, was separated from thesoluble metabolites including sugars by centrifugation at 12 000gfor 10 min at 4◦C. The pellet was washed by resuspensionfollowed by re-centrifugation in 2 mL of 0.77 M perchloric acidand the resulting second supernatant was pooled with the first.The pellet was washed with ethanol, resuspended in 5 mL waterand assayed for starch according to Rosti et al. (2006). Thesupernatant was neutralised with KOH/MES/KCL (2 M KOH,0.4 M MES, 0.4 M KCL), centrifuged at 2000g for 5 min at 4◦Cto remove insoluble potassium perchlorate and four 0.2-mLreplicate aliquots of the resulting supernatant were assayedfor sugars as follows. Sucrose was digested to glucose andfructose by the addition of 0.1 mL 0.22 M sodium acetate (pH 4.8)containing 3.7 U invertase (β-fructosidase, Roche DiagnosticsLtd, Burgess Hill, West Sussex, UK) to two of the aliquotsand incubation at 37◦C for 2 h. The other two aliquots wereincubated as above but with no invertase (undigested controls).Glucose and fructose were assayed in all four samples withstandard spectrophotometric techniques employing hexokinase,glucose 6-phosphate dehydrogenase (NADP-dependant) andphosphoglucose isomerise as coupling enzymes. Free glucoseand fructose contents were those measured in the undigestedcontrols. To calculate the sucrose content, the glucose andfructose content in the undigested controls was subtracted fromthose in the digested samples.
Iodine stainingLeaves were destained by heating in 80% ethanol to removechlorophyll and then stained with iodine solution (Lugol’ssolution; Sigma-Aldrich Co. Ltd, Gillingham, UK) diluted 1 : 4with deionised water.
ImmunoblottingSamples of leaves (200 mg) from young plants before floweringwere homogenised in gel sample buffer containing SDS. After
centrifugation for 10 min at 20 000g, the supernatant was heatedto 100◦C for 5 min before loading onto an SDS polyacrylamidegel (7.5% acrylamide, 0.75 mm thick). After electrophoresis,proteins were transferred to a nitrocellulose membrane that wasprobed with an antiserum to the maize endosperm AGPase SSUBrittle-2, at a dilution of 1/5000.
Results
Nomenclature of LSU genes
The genome sequence of Oryza sativa ssp. Japonicacv. Nipponbare (http://rgp.dna.affrc.go.jp/IRGSP/, accessed14 March 2007) shows four genes encoding LSUs of AGPase(Table 1). Different abbreviated names have been assigned tothese genes by different groups. Here, we will use the originalnomenclature proposed by Akihiro et al. (2005) (Oryza sativaAGPase LSU genes 1 to 4: OsAPL1 to OsAPL4).
Identification and genotyping of the apl1 mutant line
A search of the Tos17 insertion mutant database (http://tos.nias.affrc.go.jp/∼miyao/pub/tos17/, accessed 14 March 2007) forTos17-flanking sequences matching any of the four rice LSUsequences identified a single line, NG7525, with a Tos17insertion (T36075T) in exon 4 of the OsAPL1 gene. T2 seeds thatwere segregating for the insertion were obtained from the RiceGenome Resource Centre, Japan. To genotype the T2-generationseedlings, primers were designed to amplify fragments of thewild type and mutant OsAPL1 genes (Fig. 1). Sequencing ofthe P2/P3 PCR product confirmed the insert position in exon 4.The mutant allele was named apl1 and the wild type alleleApl1. Homozygous mutant plants (apl1/apl1), referred to as apl1plants, were used in subsequent experiments and homozygouswild type plants (Apl1/Apl1) were used as controls.
Amplification and sequencing of the apl1mutant transcript
To determine whether a transcript was produced from themutant apl1 allele, we used RT-PCR to amplify a region of thetranscript spanning the Tos17 insertion site. First strand cDNAwas prepared from wild type and apl1 mutant seedling leavesand PCR-amplified from primers in exons 1 and 5 of OsAPL1(Fig. 2). A fragment was amplified from apl1 mutant cDNA thatwas ∼100 bp shorter than that obtained from wild type cDNA(Fig. 2A). Sequencing showed that the mutant OsAPL1 transcriptlacked the sequence encoded by exon 4. Thus, the Tos17 insertion
M
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Fig. 1. Plants were genotyped with PCR. Primers P1 and P3 produce a 587 bp product with wild type (WT) DNA. Primers P2 and P3produce a 444 bp product with apl1 mutant (MUT) DNA. Both products are generated with DNA from Apl1/apl1 heterozygous (HET) plants.(A) A schematic representation of the OsAPL1 gene showing the position of insertion of the 3′. Exons are shown as boxes, introns as lines andprimers as arrowheads. (B) PCR products on an agarose gel. The sizes of molecular weight markers (track M) are indicated.
Characterisation of a rice mutant lacking leaf starch Functional Plant Biology 483
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Fig. 2. The OsAPL1 transcripts from apl1 mutant (MT) and wild type(WT) leaves were analysed with RT–PCR. Primers P3 and P4 were designedto amplify a region of the transcript (T) spanning the Tos17 insertion site(T36075T) in the OsAPL1 gene (G). P3 is in exon 5 and P4 in the 5′UTR ofexon1. (A) PCR products on an agarose gel. The sizes of molecular weightmarkers (track M) and products are indicated. (B) A schematic representationof the splicing part of the OsAPL1 gene in the wild type compared to thatin the apl1 mutant. Exons are shown as boxes numbered 1 to 5, introns areshown as lines numbered i1 to i4 and primers as arrowheads. (C) The deducedamino acid sequence of the OsAPL1 protein. The 31 amino acids encodedby exon 4 and missing in the predicted mutant protein are underlined. The54 amino acids encoded by the putative transit peptide are in italic.
in exon 4 of OsAPL1 results in the exclusion of this exon fromthe mature transcript by abnormal splicing between the donor ofthe third intron and the acceptor of the fourth intron (Fig. 2B).No normal transcript was detected in the mutant.
The effect of the mutation in OsAPL1on the AGPase protein
Abnormal splicing of exon 4 in the apl1 mutant transcript doesnot create a frameshift and so the apl1 mutant transcript encodesa predicted LSU protein lacking the 31 amino acids encoded byexon 4 but is otherwise identical to the LSU encoded by the wildtype allele (Fig. 2C). Exon 4 encodes a domain that is conservedin both the SSU and LSU of plant AGPases (see Akihiro et al.2005). In the SSU, an aspartate residue within this domain hasbeen shown to be essential for activity (Frueauf et al. 2003). Theimportance of this conserved domain for LSU functionality hasnot been established experimentally but it is unlikely that a LSUprotein lacking the conserved domain encoded by exon 4 wouldbe functional.
To discover whether the abnormal LSU protein predicted bythe apl1 mutant transcript is present in mutant rice leaves, weused antisera raised to the LSU of maize endosperm, spinachleaf and pea embryos to probe western blots of leaf extracts.Unfortunately, these antisera failed to identify the LSU proteinin either the wild type or apl1 mutant leaves (data not shown).We cannot therefore determine whether the leaves of apl1accumulate an abnormal LSU protein, a normal LSU encodedby another gene or no LSU protein.
In wild type leaves, the LSU of AGPase forms a complex witha SSU protein. Previous work on the adg2 mutant of Arabidopsissuggested that, in the absence of a functional LSU, the amount ofthe SSU protein is reduced to 4–10% of that present in wild typeleaves (Lin et al. 1988a). To determine whether the amount ofthe SSU protein in the apl1 mutant of rice was affected, westernblots of extracts of wild type and apl1 mutant leaves were probedwith an antiserum to the SSU of AGPase from maize endosperm(Brittle-2; from Curt Hannah, Gainesville, FL, USA) (Fig. 3).The most abundant SSU transcript in rice leaves is encoded byOs08 g25730 (Ohdan et al. 2005). In barley, the orthologue ofthis gene is required for >90% of the AGPase activity in leaves(Rosti et al. 2006). The predicted mass of the mature plastidialSSU in rice leaves is 50 kDa. A protein of approximately thismass was recognised by the Brittle-2 antiserum in extracts ofwild type leaves but not in extracts of apl1 mutant leaves. Thisshows that apl1 mutant rice leaves lacking the normal LSUencoded by OsAPL1 also lack the SSU protein and suggeststhat accumulation of the SSU in leaves requires the presence ofa functional LSU protein.
In barley, the gene that encodes the major leaf AGPaseSSU, the homologue of Os08 g25730, also encodes the majorendosperm SSU by a second transcript (Rosti et al. 2006). It islikely that in rice, as in barley, a single gene encodes the majorSSU proteins in both the leaf and the endosperm. In addition tothe transcript encoding the leaf SSU, Os08 g25730 encodes atranscript that is abundant in endosperm and predicts a cytosolicSSU with a mass of 52.9 kDa (Ohdan et al. 2005). The Brittle-2antiserum recognised a protein of approximately this size inendosperms from both the wild type and apl1 mutant (data notshown). Thus, the AGPase SSU protein is absent in the leaves
484 Functional Plant Biology S. Rosti et al.
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Fig. 3. The AGPase SSU protein was detected in leaf extracts byimmunoblotting. Proteins from leaves of wild type (WT) and apl1 mutant(MUT) plants were extracted, fractionated by SDS–PAGE, transferred toa nitrocellulose membrane, and probed with an antiserum to the maizeendosperm AGPase SSU Brittle-2. Each track contains the equivalent of2 mg of leaf. Molecular weights in kD of marker proteins (track M) areindicated.
of the apl1 mutant, which lacks a functional LSU but the majorendosperm SSU that is encoded by the same gene is not affectedin the apl1 mutant.
The effect of the mutation in OsAPL1 on AGPase activity
We investigated whether AGPase activity was altered in apl1mutant leaves and assessed the contribution of OsAPL1 tothe AGPase activity in the endosperm, the major starch-synthesising organ of the rice plant. Two assays for AGPaseactivity are commonly used: a spectrophotometric assaythat measures AGPase activity in the direction of ADP-glucose pyrophosphorolysis and a radioactive assay thatmeasures AGPase activity in the direction of ADP-glucosesynthesis (Smith 1990). Preliminary experiments indicatedthat the spectrophotometric assay could be used to reliablymeasure AGPase activity in endosperm extracts, but in leafextracts, the assay was non-linear with respect to time andextract concentration. The radioactive assay gave reliablemeasurements of AGPase activity in leaf extracts. Therefore,
the spectrophotometric assay was used for endosperm extractsand the radioactive assay for leaves. Both assays were optimisedwith respect to the concentrations of components of the assayand the pH, and were linear with respect to time and extractconcentration over the ranges used in our experiments.
The AGPase activity in apl1 mutant endosperm was notsignificantly different from that in the wild type endosperm(Table 2). However, the AGPase activity in apl1 mutant leaveswas very different from that in wild type leaves. AGPase activitycould not be detected in extracts of apl1 mutant leaves, evenafter increasing the assay time to 40 min instead of 10 min anddoubling the amount of extract in the assay. To determine theminimum AGPase activity that would be detectable in our assays,we measured AGPase activity in serial dilutions of wild typeleaf extracts (data not shown). AGPase activity was linear withrespect to extract concentration down to a 10-fold dilution ofthe wild type extract and was still detectable in extract diluted100-fold. This suggests that the AGPase activity in apl1 mutantleaves is less than 1% of that in wild type leaves.
To investigate whether inhibitors of AGPase activity presentin leaf extracts of the apl1 mutant but not the wild type mightaccount for the observed difference in AGPase activity, weextracted mixtures of apl1 mutant and wild type leaves. In twoindependent experiments, the activities in the mixed extractswere 83 and 99% of those expected from measurements madeof separate extracts of replicate apl1 mutant and wild type leafsamples. This suggests that the absence of detectable AGPaseactivity in the apl1 mutant leaves was not because of inhibition.
Segregation analysis
To establish whether the Tos17 insertion in OsAPL1 wasresponsible for the absence of AGPase activity in the leaf,a population of plants that was segregating for the insertionwas analysed. Heterozygous Apl1/apl1 plants were allowedto self-fertilise and the resulting seeds were germinated andgrown for 3 weeks. Each seedling was genotyped as in Fig. 1from DNA extracted from a section of leaf. This showed 9alp1/apl1 homozygotes, 27 Apl1/apl1 heterozygotes and 16 wildtype plants. These numbers do not differ significantly froma 1 : 2 : 1 segregation ratio (χ2 = 1.9615 < test value = 5.99)at a 95% confidence level. AGPase activity was measuredin extracts of leaves from each seedling. There was acomplete correlation between the homozygous apl1 mutantgenotype and the absence of detectable AGPase activity inthe leaf (Fig. 4A). AGPase activity measured in Apl1/apl1heterozygous seedlings (404 ± 9 nmol min−1 g−1 fresh weight)
Table 2. AGPase activity in wild type and apl1 mutant leaves and endospermLeaves were harvested after 8 h of light during a 12 h light period from plants that were starting to flower.Endosperm was harvested at 10 to 12 days after flowering, when the individual endosperm weights were 9 to12 mg. The radioactive AGPase assay was used for extracts of leaves and the spectrophotometric assay for extractsof endosperm. Values are means ± s.e. determined from four samples, each from a separate plant. Assays were
Characterisation of a rice mutant lacking leaf starch Functional Plant Biology 485
was approximately half of that measured in wild type seedlings(839 ± 24 nmol min−1 g−1 fresh weight). These results show thatthe Apl1 allele is semi-dominant with respect to AGPase activityand that the lack of AGPase activity co-segregates with the Tos17insertion in the OsAPL1 gene.
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Fig. 4. Rice seedlings segregating for the Tos17 mutation in the OsAPL1gene were grown in petri dishes in a growth cabinet and genotyped byPCR. The leaves were assayed for AGPase activity (A) and tested forthe presence of starch (B). (A) A population of 52 T3 seeds from T2heterozygous plants were germinated in petri dishes on moist filter paper,grown in continuous light (100 µmol m−2 s−1) at 25◦C and genotyped after3 weeks, when the root and shoot were established. The AGPase activityfor each genotype is represented as a boxplot. The boxes represent the 1stand 3rd quartiles, the median is shown as a cross and the whiskers indicatethe maximum and minimum values for each distribution. MUT, apl1/apl1mutant; HET, Apl1/apl1 heterozygote; WT, wild type. (B) A population of100 T3 seeds from T2 heterozygous plants was germinated in 1% agar,grown in continuous light and genotyped. Four weeks after germination, aleaf was removed from each seedling and stained with iodine to check forthe presence of starch. Typical leaves are shown.
The effect of the mutation in OsAPL1 on starch synthesis
The amounts of starch in leaves at the end of the day and ina range of non-photosynthetic tissues/organs were measuredto determine the overall effect of the mutation in OsAPL1on starch synthesis in the rice plant. Leaves, root tips, pollengrains and embryos from apl1 mutant and wild type plantswere stained with iodine solution and observed under a lightmicroscope. Black-staining starch granules were observed in allof the non-photosynthetic tissues/organs in both genotypes, andin wild type leaves, but not in apl1 mutant leaves (Fig. 5A, B,and data not shown). These data suggest that the LSU encodedby OsAPL1 is necessary for starch synthesis in leaves but notfor starch synthesis in many of the non-photosynthetic partsof the plant.
The starch content of leaves from plants that were headingwas quantified (Table 3). Blades from the leaf subtending thepanicle (flag leaf) and the leaf immediately below the flag leaf(–1 leaf) were harvested at the end of the light period,immediately frozen in liquid N2 and extracted by grinding inperchloric acid. The starch contents in both types of leaves fromthe apl1 mutant were more than 10-fold lower than those in leavesfrom the wild type.
We investigated the reliability of our measurements of starchcontent as follows. We assessed the recovery of starch from wildtype leaves by adding a known amount of starch purified fromArabidopsis leaves to one of two replicate samples of wild typeleaf blades (i.e. the two halves of a single leaf) before extraction.The amount of starch added was the same as that in wild type riceleaves (Table 3). The recoveries of starch in two independentexperiments were 100% and 92% of the amount added. Toinvestigate the accuracy of our measurements of the very lowlevels of starch found in apl1 mutant leaves, we prepared serialdilutions of extracts of wild type leaves and assayed thesefor starch. This showed that our measurements were accuratedown to 5% of the wild type levels of starch. To determinewhether very small starch granules that might be present inthe apl1 mutant leaves could have been lost during extraction,we increased the speed of centrifugation of the extract from2000g to 12 000g. This did not yield a higher recovery ofstarch from apl1 mutant leaf samples. These experiments showthat the data in Table 3 accurately reflect the starch content ofthe wild type leaves and suggest that the amount of starch inapl1 mutant leaves is less than or equal to 5% of that in wildtype leaves.
To establish whether the Tos17 insertion in OsAPL1 wasresponsible for the absence of starch in the leaf, we genotyped100 seedlings that were segregating for the insertion and scoredtheir leaf starch phenotypes by iodine staining (Fig. 4B). Eachplant was genotyped as in Fig. 1 with DNA extracted froma section of leaf. This showed 24 alp1/apl1 homozygotes,51 Apl1/apl1 heterozygotes and 25 wild type plants. Thesenumbers do not differ significantly from a 1 : 2 : 1 segregationratio (χ2 = 0.060 < test value = 5.99) at a 95% confidence level.There was a complete correlation between the homozygousapl1 mutant genotype and the absence of starch in the leaf. Allof the heterozygous plants had normal leaf-starch phenotypes.These results show that the low-starch phenotype is recessiveand that it co-segregates with the Tos17 insertion in theOsAPL1 gene.
486 Functional Plant Biology S. Rosti et al.
A
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Fig. 5. Leaves from mature wild type (A) and apl1 (B) plants were stainedwith iodine to show their starch content and viewed under a microscope.The bar is 10 mm. Despite the lack of starch in their leaves, the apl1 mutantplants [on the right (C, D)] grew normally and were indistinguishable fromwild type plants [on the left (C, D)] at both the vegetative (C) and floral (D)stages of growth.
Table 3. Starch content in wild type and apl1 mutant leavesSamples were taken from plants less than 1 week after the emergenceof the first panicle. Leaves were harvested at the end of the light periodand immediately frozen in liquid N2. Starch was extracted by grinding inperchloric acid and separated from soluble sugars by centrifugation. Glucosereleased from starch by digestion with amylolytic enzymes was quantifiedby a spectrophotometric assay. Values are means ± s.e. determined from five
samples, each from a separate plant. Assays were performed in triplicate
Starch content (mg starch g−1 FW)Wild type Apl1 mutant
The effect of the mutation in OsAPL1 on the starchand sugar contents of leaves and stems
To investigate whether the apl1 mutant rice plant stores starchtransiently during the day in cells other than those of the leafblade or whether it stores carbohydrate transiently in the leavesin another form, such as sugar, we measured the starch, sucrose,glucose and fructose contents of the blade and sheath of the flagleaf and in the stem supporting the panicle. These organs wereharvested at the end of the day and at the end of the night fromplants that were starting to flower (Fig. 6). The starch and sucrosecontents in wild type leaf blades and sheaths were comparableto previous estimates for rice plants grown in similar growthconditions (for example, Makino et al. 1997) but were lowerthan those for rice grown in high light levels in controlled growthconditions (Makino et al. 1997) or in the field (Watanabe et al.1997; Hirose et al. 1999).
The data in Fig. 6 show that in the wild type, less starchaccumulated in the leaf sheaths and stems than in the leaf blades.The starch in the wild type leaf blades and sheaths was almostcompletely turned over during the night but there was no turnoverof starch in the stem. In the mutant, the leaf blades and sheathslacked starch at both sampling times. There was some starch inthe mutant stems, and iodine staining (data not shown) showedthat, as in the wild type, this was located mainly at the base.The sucrose content of all three organs was greater at the endof the day than at the end of the night. The apl1 mutationhad no statistically significant effect on the sucrose contents ateither time of day. The glucose and fructose contents showed nostatistically significant variations between the two time pointsand like the sucrose contents, they were not affected by the apl1mutation. In the leaf blade and sheath, the glucose and fructosecontents were less than the sucrose contents. In the stem, theglucose and fructose contents were approximately similar to thesucrose contents at the end of the day.
The effect of the mutation in OsAPL1 on plantdevelopment and yield
Wild type and apl1 mutant plants were grown together ina controlled environment with a light regime of 12 h lightand 12 h dark. In these growth conditions, there was nodifference between the wild type and apl1 mutant plants in size,morphology or development (Fig. 5C, D). At plant maturity,various components of yield were measured (Table 4). Therewere no detectable differences between the wild type and
Characterisation of a rice mutant lacking leaf starch Functional Plant Biology 487
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Fig. 6. The blade and sheath of the flag leaf and the stem supporting thepanicle were harvested from wild type (WT) and apl1 mutant (MUT) plantsthat were starting to flower either at the end of the light period or at the end ofthe dark period. The plant material was immediately frozen in liquid nitrogen,extracted with perchloric acid and assayed for starch, sucrose, glucose andfructose. Values are means ± s.e. determined from 4 to 10 samples, eachfrom a separate plant. Each assay was replicated four (sucrose, glucose andfructose) or eight (starch) times.
Table 4. Analysis of yield componentsPlants were grown in a controlled environment with a photoperiod of 12 hlight and 12 h dark. When completely mature, the plants were allowedto dry to ambient humidity for 2 weeks before harvest. Values are meanweights ± s.e. determined from seven plants. Weights are in grams except
mean individual seed weight, which is in milligrams
apl1 mutant plants for any of these yield components. Thissuggests that in the growth conditions used in these experiments,the OsAPL1 gene is not required for normal growth anddevelopment.
Discussion
Our data show that a Tos17 insertion in the OsAPL1 gene of riceencoding a LSU of AGPase disrupts its function by resulting inthe exclusion of exon 4 from the mature transcript. No normalOsAPL1 transcript is present in the mutant plant. The leavesof the mutant also lack AGPase activity and starch. Strongevidence that the mutation in OsAPL1 is directly responsiblefor these phenotypes is provided by segregation analysis. Boththe absence of detectable AGPase activity and the reductionin starch content co-segregated with the insertion in OsAPL1.There are on average, 10 insertions per mutant line in the plantsgenerated by the Rice Genome Project at National Instituteof Agrobiological Sciences (Miyao et al. 2003). The line weused in this study, NG7525, is known currently to contain threeinsertions in addition to that in the OsAPL1 gene on chromosome3. Two of these additional insertions are on chromosome 9 andone is on chromosome 8. None are insertions in the codingregions of genes. The co-segregation of the starch and AGPasephenotypes with the insertion in OsAPL1 makes it very unlikelythat these additional Tos17 insertions, or mutations in othergenes induced by tissue culture, are responsible for the observedphenotypes. Other genes in which mutations might give riseto low AGPase and/or low starch contents are those encodingisoforms of the SSU of AGPase, plastidial phosphoglucomutaseand plastidial phosphoglucose isomerase. None of these is tightlylinked to OsAPL1 on chromosome 3.
The effects of the mutation show that OsAPL1 is required for>99% of the AGPase activity and >95% of the starch contentin the leaf blades. This shows that, in the apl1 mutant, OsAPL2,OsAPL3 and OsAPL4 together account for less than 1% of theAGPase activity in leaves. This is consistent with an analysis oftranscript abundance, which showed that in leaves, OsAPL1 isexpressed much more strongly than the other three LSU genes[Ohdan et al. (2005); Table 1]. The mutation also affects thestarch content of the leaf sheath but does not significantly affectAGPase activity or starch synthesis in other parts of the plant
488 Functional Plant Biology S. Rosti et al.
that were examined. Thus, OsAPL1 is responsible for the LSUof AGPase in the leaf but does not contribute significantly to theLSU protein in many other plant organs. We assume that one ormore of the other three LSUs genes in rice is/are responsible forthe AGPase activity in these other organs.
Mutations affecting LSUs of leaf isoforms of AGPase areknown in only one other species, A. thaliana. The adg2 mutantin Arabidopsis has a single nucleotide substitution in one ofthe four genes encoding LSU of AGPase (Wang et al. 1997).The leaves of the mutant have only 5% of the normal AGPaseactivity and 40% of the normal starch content at the end of theday when grown in a 12 h light/12 h dark photoperiod (Lin et al.1988a). Thus, in both rice and Arabidopsis, one of the four LSUgenes is necessary for most of the AGPase activity in leaves.Another similarity between these species is the effect of thelack of the LSU on the presence of the SSU in the leaves. Inthe leaves of the rice apl1 mutant, the SSU of AGPase was notdetected (this study) and in the leaves of the Arabidopsis adg2mutant, the amount of the SSU protein is severely reduced (Linet al. 1988a). These data suggest that, in both species, the SSUtranscript and/or protein is rapidly turned over in the absence ofa functional AGPase LSU.
The lack of leaf starch in apl1 mutant rice plants hadno significant effect on the growth and development of theplant in a 12 h light/12 h dark photoperiod (Table 4, Fig. 5). Insome growth conditions, starch-less mutants of other speciescan also grow normally. The starch-less mutants of Nicotianasylvestris Speg. (NS 458; Hanson and McHale 1988; Huberand Hanson 1992) and Pisum sativum L. (rug-3; Harrison et al.1998) that are deficient in plastidial phosphoglucomutase areindistinguishable from wild type controls when grown in 16 hlight/8 h dark photoperiods. Similarly, starch-less Arabidopsisplants lacking plastidial phosphoglucomutase (pgmP-1; Casparet al. 1985) or the SSU of AGPase (adg1; Lin et al. 1988b)grow as vigorously as the wild type when grown in continuouslight. However, unlike the apl1 mutant of rice, the growth ofthe Nicotiana and Arabidopsis starch-less mutants is impaired(relative to the wild type) when grown in a 12 h light/12 h darkphotoperiod. This suggests that rice is much less dependent onleaf starch synthesis for normal growth than are Nicotiana andArabidopsis.
The lack of dependence of growth on starch accumulation andturnover in rice may be a reflection of the fact that rice partitionsmost of its photosynthate to sucrose rather than to starch, asdo some other cereals, such as wheat and barley (Huber 1980).Consequently, the ratio of starch to sucrose in rice leaves is low(Fig. 6 and for example, Ohashi et al. 2000) compared to thatin other plants, such as Arabidopsis (Gibon et al. 2004; Walterset al. 2004). In the starch-less apl1 mutant of rice, the levels ofsugars in the flag leaf were not statistically significantly differentfrom those in the wild type. Thus, maintenance of normal growthrates in apl1 plants was not accompanied by a compensatoryaccumulation of sugars during the day, such as was seen in thestarch-less mutant of Nicotiana. Greater than 2-fold higher thannormal levels of soluble sugars were observed in starch-lessNicotiana leaves at the end of the day, in conditions where growthwas not affected (Hanson and McHale 1988). Nor was there adecline in the levels of sugars in apl1 at the end of the nightsimilar to that seen in the starch-less mutants of Arabidopsis.
In pgmP-1, the sugar levels at the end of the night are less thanhalf those in the wild type and this decline in sugar levels isthought to trigger the inhibition of growth (Gibon et al. 2004).It is not clear how sugar levels are maintained in the mutant riceleaves throughout the night, neither is it known how plants likerice with low leaf starch content, maintain a supply of carbon tothe non-photosynthetic parts of the plant at night.
Although in the conditions used here, the lack of leafstarch in the apl1 mutant had no effect on yield, it is unlikelythat starch synthesis in rice leaves is unnecessary for normalyield in all growth conditions. Positive correlations betweenleaf starch content and yield have been observed for rice (forexample: Seneweera et al. 1994; Ishimaru 2003). Light levelsand nitrogen availability can greatly influence the amount ofstarch synthesised in rice leaves. A comparison of the growth ofrice under different irradiances (Makino et al. 1997) showed thatstarch and sucrose levels in the leaf blades and sheaths were 5- to10-fold greater at high irradiance (1000 µmol quanta m−2 s−1)than at levels of irradiance similar to those used in ourexperiments (350 µmol quanta m−2 s−1). Further experimentswill be required to compare the effect of apl1 on growth andyield in different environmental conditions.
Acknowledgements
We are grateful to Alison Smith (John Innes Centre) and Peter Keeling (BASFPlant Sciences) for constructive criticism of the manuscript. This work wassupported by a CASE studentship from the Biotechnology and BiologicalSciences Research Council UK (BBSRC). The industrial partner for theCASE studentship was Syngenta. The John Innes Centre is supported by acore strategic grant from the BBSRC.
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Manuscript received 13 October 2006, accepted 19 February 2007
