# A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

Cécile Vriet, Tracey Welham, Andreas Brachmann, Marilyn Pike, Jodie Pike, Jillian Perry, Martin Parniske, Shusei Sato, Satoshi Tabata, Alison Smith, et al.

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## To cite this version:

Cécile Vriet, Tracey Welham, Andreas Brachmann, Marilyn Pike, Jodie Pike, et al., A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism. Plant Physiology, 2010, 154 (2), pp.643-655. 10.1104/pp.110.161844. hal-04062009

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# A Suite of *Lotus japonicus* Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism<sup>1[W][OA]</sup>

Cécile Vriet, Tracey Welham, Andreas Brachmann, Marilyn Pike, Jodie Pike, Jillian Perry, Martin Parniske, Shusei Sato, Satoshi Tabata, Alison M. Smith, and Trevor L. Wang*

Metabolic Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (C.V., T.W., M. Pike, J. Perry, A.M.S., T.L.W.); Faculty of Biology, Genetics, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany (A.B., M. Parniske); The Sainsbury Laboratory, Colney, Norwich NR4 7UH, United Kingdom (J. Pike); and Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan (S.S., S.T.)

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (*Arabidopsis thaliana*) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in *Lotus japonicus* and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in *L. japonicus* and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of *L. japonicus* grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in *L. japonicus* than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

Recent studies in Arabidopsis (*Arabidopsis thaliana*) have greatly enhanced our knowledge about pathways of transitory starch metabolism (Zeeman et al., 2007; Keeling and Myers, 2010; Kötting et al., 2010; Zeeman et al., 2010). The pathway of synthesis is well established for several species, but the degradative pathway is understood only in Arabidopsis. During synthesis, the plastidial isoforms of phosphogluco-isomerase (PGI1) and phosphoglucomutase (PGM1), together with ADP-Glc pyrophosphorylase (AGPase), catalyze the conversion of the Calvin cycle intermediate Fru 6-P to ADPGlc, the substrate for starch syntheses (Supplemental Fig. S1). Leaves of mutants

lacking any of these three enzymes either have strongly reduced starch contents or lack starch almost completely (Caspar et al., 1985; Hanson and McHale, 1988; Lin et al., 1988a, 1988b; Kruckeberg et al., 1989; Harrison et al., 1998; Yu et al., 2000; Streb et al., 2009). In contrast, the phenotypes of mutants lacking individual enzymes that convert ADPGlc into starch vary between species and are often much less pronounced (starch syntheses [Delvallé et al., 2005; Zhang et al., 2005] and starch-branching enzymes [Tomlinson et al., 1997; Blauth et al., 2001; Dumez et al., 2006]).

The degradation of the starch granule in Arabidopsis leaves is catalyzed primarily by β-amylases and isoamylase 3 (Wattebled et al., 2005; Delatte et al., 2006; Fulton et al., 2008). Normal rates of degradation require phosphorylation of the starch polymers by two glucan, water dikinases, GWD1 (Ritte et al., 2002) and GWD3 (or PWD, for phosphoglucan water, dikinase; Baunsgaard et al., 2005; Kötting et al., 2005), followed by dephosphorylation by a phosphoglucan phosphatase, STARCH EXCESS4 (SEX4; Kötting et al., 2009). Maltose produced by starch degradation is exported from the chloroplast by a maltose transporter and further metabolized to hexose phosphates in the cytosol (Zeeman et al., 2007; Supplemental Fig. S1). Mutations in numerous components of this pathway result in a starch-excess phenotype, in which the starch

<footnote n="1">¹ This work was supported by a Marie Curie studentship (EU Early Stage Training program no. 019727) to C.V. and by a Core Strategic Grant to the John Innes Centre from the Biotechnology and Biological Sciences Research Council.</footnote>

<footnote>* Corresponding author; e-mail trevor.wang@bbsrc.ac.uk.</footnote>

<footnote>The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Trevor L. Wang (trevor.wang@bbsrc.ac.uk).</footnote>

<footnote>[W] The online version of this article contains Web-only data.</footnote>

<footnote>[OA] Open Access articles can be viewed online without a subscription.</footnote>

<footnote>www.plantphysiol.org/cgi/doi/10.1104/pp.110.161844</footnote>
content of leaves at the end of the night is higher than that of wild-type plants.

These studies have also revealed the importance of starch turnover for the productivity of the plant. Mutants of Arabidopsis that are essentially unable to synthesize transitory starch, or with reduced rates of starch degradation at night, have a reduced rate of growth and delayed flowering time relative to wild-type plants under most conditions (Caspar et al., 1985, 1991; Eimert et al., 1995; Corbesier et al., 1998; Smith and Stitt, 2007). However, it is not known whether information about the nature and importance of starch turnover in Arabidopsis is widely applicable. Plant species differ considerably in the extent to which starch is stored in leaves at night as well as in diurnal patterns of growth and metabolic demand. The function and regulation of starch metabolism in heterotrophic organs and its importance in major physiological and developmental processes such as perenniality, vegetative regrowth, symbiotic nitrogen fixation, and the accumulation of seed storage reserves cannot be studied easily in Arabidopsis and remain largely unknown. These processes represent traits of agronomic value in legumes (Fabaceae), a family that includes some of the most agriculturally important forage (e.g. alfalfa [Medicago sativa] and clover [Trifolium spp.]), grain (e.g. pea [Pisum sativum] and common bean [Phaseolus vulgaris]), and oilseed (e.g. soybean [Glycine max]) crops.

Some information is already available about starch metabolism in pea and other legume crops (Martin and Smith, 1995; Wang et al., 1998b, and refs. therein). However, characteristics including large genome sizes and recalcitrant transformation and regeneration have limited progress on these species. There is insufficient information to allow either an overview of the nature and importance of starch metabolism in legumes or a meaningful comparison with the detailed picture emerging for Arabidopsis. The development of both *Lotus japonicus* and *Medicago truncatula* as legume model systems, and the wide range of genetic and genomic resources generated for them, offer the opportunity for a systematic analysis.

To elucidate the pathway of starch synthesis and degradation in legumes and provide resources for future experimentation, we screened an ethyl methanesulfonate (EMS)-mutagenized population of *L. japonicus* (Perry et al., 2003) for mutants altered in transitory starch metabolism and carried out genetic mapping to identify the mutation responsible for their phenotype. We also used TILLING (for Targeting Induced Local Lesions IN Genomes; McCallum et al., 2000) to confirm that the mutations identified were indeed responsible for the mutant phenotype and to obtain additional mutations in genes known to affect leaf starch content in other species. We present the results of molecular and phenotypic analyses on the mutants that provide novel insights into the structure-function relationship of the AGPase and GWD1 enzymes. In addition, our analyses reveal new information on the nature and importance of starch metabolism for

plant growth and development in *L. japonicus*. The importance of starch accumulation and degradation and a comparison with pathways in other plant species are also discussed.

## RESULTS AND DISCUSSION

### Metabolism of Leaf Starch in *L. japonicus*

To examine the extent of diurnal starch turnover in leaves of *L. japonicus*, plants of the two wild-type ecotypes commonly used in research on this species, Miyakojima MG-20 and Gifu B-129 (referred to as MG-20 and Gifu, respectively), were grown in a 12-h-light/ 12-h-dark cycle in a growth chamber. Their leaf starch contents were measured at the end of the day and the end of the night at 2-week intervals during their first 10 weeks of growth. For the first 6 weeks, the two ecotypes showed similar, strong diurnal changes in starch content. The starch content was in excess of 7.5 mg g⁻¹ fresh weight at the end of the day and less than 0.6 mg g⁻¹ fresh weight at the end of the night (Fig. 1; Supplemental Table S1). This pattern is very similar to that in Arabidopsis plants under the same growth conditions (Yu et al., 2001, 2005; Baunsgaard et al., 2005; Fulton et al., 2008). In contrast to the situation in Arabidopsis, starch levels in older *L. japonicus* plants increased at the end of both the day and the night. At 8 weeks, the diurnal amplitude of change in starch content was unchanged, but leaves had about 14 mg starch g⁻¹ fresh weight at the end of the day and retained 5 mg starch g⁻¹ fresh weight at the end of the night. Between 8 and 10 weeks, leaf starch content increased dramatically. In Gifu, a diurnal

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Diurnal changes in leaf starch content through the development of *L. japonicus* plants. Plants were grown in 12 h of light and 12 h of dark. Mature leaves were harvested at the end of the day (EOD) and the end of the night (EON) and assayed for starch content at the indicated times after germination. Values are means ± SE of measurements on six samples, each of which consisted of three fully expanded leaves taken from the top of two plants.</figcaption></figure>
turnover of about 10 mg starch g⁻¹ fresh weight was maintained, but starch content at the end of the night was 20 mg g⁻¹ fresh weight. In MG-20, starch content was over 50 mg g⁻¹ fresh weight throughout the diurnal cycle. Increases in leaf starch content with leaf and/or plant age have been observed in several other species, including tobacco (*Nicotiana tabacum*; Matheson and Wheatley, 1962; Ölçer et al., 2001) and soybean (Ainsworth et al., 2006). It should be mentioned, however, that both the timing and magnitude of the rise in starch content with plant age in *L. japonicus* were highly dependent on the growth conditions (data not shown).

## In Silico Identification of Genes Involved in Starch Metabolism in *L. japonicus*

We first listed the Arabidopsis genes involved in the metabolism of leaf starch (Supplemental Table S2). This included genes encoding enzymes involved in the partitioning of photoassimilates into starch, in the synthesis and degradation of starch, and in the subsequent metabolism of maltose (Zeeman et al., 2007, 2010; Fulton et al., 2008; Kötting et al., 2009). Recent progress in EST and genome sequencing projects in *L. japonicus* allowed us to identify the orthologs of the Arabidopsis genes in this species (Supplemental Table S2). Whenever possible, these genes were mapped on the genetic linkage map of *L. japonicus* (Fig. 2) using

the marker information provided by the Miyakogusa genome database available at http://www.kazusa.jp/lotus/index.html (Sato et al., 2008).

We were able to identify *L. japonicus* genes encoding all of the classes of enzymes involved in starch metabolism in Arabidopsis, but there were several differences in isoform representation. For instance, we could not find *L. japonicus* sequences orthologous to Arabidopsis genes encoding the glucan, water dikinase GWD2 and the β-amylases BAM2 and BAM4 (Supplemental Table S2). These genes may be present but in an unsequenced region of the genome (9% of the gene space has not been sequenced; Sato et al., 2008) or expressed at a low level and so not represented in the EST collection. Alternatively, there may be differences in the composition of some gene families between the two species. We also found duplications of several starch metabolism genes in *L. japonicus*, including AGPase large subunit isoform 2 (APL2), starch synthase 2 (SS2), granule-bound starch synthase (GBSS), α-amylase 3 (AMY3), β-amylase 3 (BAM3), and cytosolic glucan phosphorylase PHS2 (also known as Pho2 in some species). Duplication of genes encoding SS2 and GBSS exist in numerous species, including other legumes and cereals (Pan et al., 2009). Interestingly, the duplications appear to have independent origins in different groups of plants. The legume duplication resulted from a whole-genome duplication within

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Chromosomal locations of starch metabolism genes of *L. japonicus*, and map positions of mutations discovered in the forward genetic screen. The genetic linkage map of *L. japonicus* is derived from a cross between the ecotypes Gifu and MG-20 (maternal and paternal parents, respectively). For each chromosome, the number is given at the top and the size (in cM) is given at the bottom (data from http://www.kazusa.or.jp/lotus/index.html). Genes involved in the synthesis and degradation of starch (lettered in white and black, respectively) are shown on the left of each chromosome. Mutations from the forward genetic screen are shown on the right of each chromosome. Identified mutations are labeled with allele numbers; mapped intervals of the mutations yet to be characterized are labeled with the SL number of the mutant lines. The starch metabolism genes are listed in Supplemental Table S2. Full details of the alleles are given in Supplemental Tables S4 and S6.</figcaption></figure>
Rosid clade I, whereas the cereal duplication resulted from the whole-genome duplication at the base of the grass lineages, well after the divergence of the monocots and dicots (Pan et al., 2009).

## Forward Genetic Screens to Identify Mutants Affected in Starch Metabolism

To identify mutants, we used a collection of lines impaired in starch metabolism from a screen on 1,428 M2 families (17,100 plants) from seeds of Gifu mutagenized with EMS (Perry et al., 2003). Our screen utilized the fact that decolorized leaves of wild-type plants stain strongly with iodine solution at the end of the day, when starch content is high, and much less strongly at the end of the night, when starch content is low (Fig. 3). We used this to isolate mutants that had either lower levels of leaf starch than wild-type plants at the end of the day ("synthesis mutants") or higher levels of leaf starch than wild-type plants at the end of a dark period ("degradation mutants"). Because starch content at the end of a normal night varied from batch to batch and with glasshouse conditions, screening for

degradation mutants was usually performed on plants subjected to an extended night of up to 44 h. The clearest results were obtained on young plants (about 4 weeks old) in which starch content was generally low at the end of the night (Fig. 1).

The screens led to the selection of 10 synthesis and six degradation mutants (Fig. 3). Allelism tests suggested that the mutations defined at least five loci for the synthesis mutants and four loci for the degradation mutants. The selected mutant lines were outcrossed at least once to MG-20 to reduce numbers of background mutations introduced by EMS mutagenesis and to establish a mapping population. Segregation ratios of the F2 progeny (data not shown) indicated a recessive monogenic mode of inheritance for all the starch mutant phenotypes, consistent with the nature of such mutations in other species.

## Genetic Linkage Mapping and TILLING as Complementary Approaches to Identify Mutations Affecting Starch Metabolism

To discover genes necessary for starch metabolism in *L. japonicus*, we took two approaches: mapping, to

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Iodine staining of starch synthesis and degradation mutants. A to C, Iodine staining of leaves of mutants obtained from the forward genetic screen and from TILLING. All mutant lines shown here except *gwd1-3* and *gwd3-4* were derived from outcrosses of the original mutant with MG-20. Each leaflet is from a different plant. Plants were grown in 16 h of light and 8 h of dark and were approximately 4 weeks old at the time of harvest. The name of the mutant allele is given where this is known (Supplemental Table S5). A, Starch synthesis mutants from the forward genetic screen and TILLING. Leaflets were harvested at the end of the day. B and C, Mutants from the forward genetic screen (B) and TILLING (C). Leaf phenotypes of the *gwd1-3* and *gwd3-4* TILLING mutants are shown in comparison with those of their segregating wild types (WT). Leaflets were harvested at the end of an extended night of up to 44 h. D, Starch contents of roots and embryos of wild-type and starch synthesis mutant plants. Plants were harvested when approximately 3 months old. Roots and embryos were decolorized prior to staining with iodine solution. Note that roots and embryos of wild-type plants have high starch contents, those of *pgm1-4* have no detectable starch, those of the *aps1-1* mutant have drastically reduced starch contents, and those of *pgi1-2* (SL4308-12) appear very similar to the wild type.</figcaption></figure>
identify the mutations responsible for the phenotypes identified above, and TILLING, to identify mutations in selected genes encoding enzymes known to be necessary for starch metabolism in other plant species (Supplemental Table S2). TILLING was also used to identify additional mutant alleles of genes for which only one mutant allele was isolated from the forward genetic screen.

We initially carried out rough mapping on the forward screen mutant lines by bulk segregant analysis, using genomic DNA from mutants isolated from F2 populations from crosses with MG-20. Microsatellite simple sequence repeat (SSR) markers evenly distributed over the arms of each of the six chromosomes of *L. japonicus* (three to four SSR markers per chromosome) were selected from the markers developed by the Kazusa DNA Research Institute (http://www.kazusa.jp/lotus/index.html; Supplemental Table S3). The mapping interval thus identified (Supplemental Table S4) was confirmed by further mapping on the mutant individuals.

Using information from the genome sequence of *L. japonicus* (Sato et al., 2008), we searched within the confirmed interval for candidate genes (Fig. 2; Supplemental Table S2). Synteny with the genomes of the legume species soybean (http://www.phytozome.net/ soybean) and *M. truncatula* (http://www.medicago. org/genome/) were also used in some cases where the *L. japonicus* genome sequence was not available. Where sequencing of candidate genes (for primers, see Supplemental Table S5) revealed mutations, biochemical and genetic approaches were used to check rigorously that the mutations were indeed responsible for the starch phenotype. As described below, we were able to identify the mutated genes in six out of the 10 synthesis mutants and three out of the six degradation mutants.

TILLING was carried out using two DNA populations of *L. japonicus* (GENPOP and STARPOP; Perry et al., 2009). Most of the mutants isolated were heterozygous (Perry et al., 2009), so homozygous mutant, heterozygous, and wild-type segregants in the M3 generation were identified by sequencing (for primers, see Supplemental Table S5).

## Mutations in the Starch Synthesis Genes *LjPGI1*, *LjPGM1*, *LjAPL1*, *LjAPL2*, and *LjAPS1*

Based on knowledge gained from Arabidopsis (see introduction and Supplemental Fig. S1), we examined whether the almost starchless and low-starch plants (synthesis mutants) selected in the forward screen lacked pPGM, pPGI, or AGPase. As described below, mutations affecting these three enzymes accounted for the phenotypes of six of the 10 synthesis mutants.

Three of the synthesis mutants from the forward screen (SL4308-12, SL4715-2, and SL5069-2) had leaf starch contents that were only 10% of wild-type values (Table I), but their embryo and root starch contents were indistinguishable by iodine staining from those

<caption>**Table I.** Starch content at the end of the day in leaves of mutants with starchless or low-starch phenotypes</caption>

Values are means ± SE of measurements on six samples, each consisting of fully expanded leaves from the upper part of the shoot of a single plant. n.d., Not detected.

<table><thead><tr><th rowspan="2">Genotype<sup>a</sup></th><th colspan="2">Starch Content</th></tr><tr><th>Wild Type</th><th>Mutant</th></tr><tr><td colspan="3">mg Glc equivalents g<sup>-1</sup> fresh wt</td></tr></thead><tbody><tr><td>PGM1</td><td></td><td></td></tr><tr><td>pgm1-3</td><td>25.3 ± 6.4</td><td>n.d.</td></tr><tr><td>pgm1-4</td><td>20.1 ± 4.8</td><td>n.d.</td></tr><tr><td>pgm1-5</td><td>29.7 ± 6.2</td><td>6.1 ± 1.3</td></tr><tr><td>PGI1</td><td></td><td></td></tr><tr><td>pgi1-1</td><td>17.2 ± 5.9</td><td>1.83 ± 0.35</td></tr><tr><td>pgi1-2</td><td>50.6 ± 4.7</td><td>4.56 ± 1.90</td></tr><tr><td>APS1</td><td></td><td></td></tr><tr><td>aps1-1</td><td>47.0 ± 4.1</td><td>3.73 ± 1.29</td></tr><tr><td>APL1</td><td></td><td></td></tr><tr><td>apl1-1</td><td>59.6 ± 10.5</td><td>12.7 ± 2.0</td></tr></tbody></table>

of wild-type plants (Fig. 3 for SL4308-12; data not shown). This pattern of starch distribution is also seen in the Arabidopsis *pgi1* mutant (Yu et al., 2000). The three mutants were shown by crossing to be allelic, and the mutations in all three mapped to the same interval at the top of chromosome I. The *LjPGI1* gene is located in this interval (Fig. 2; Supplemental Tables S2 and S4). Sequencing of the *PGI1* gene in the three mutants revealed two mutations that create stop codons (*pgi1-1*, SL4715-2; *pgi1-2*, SL4308-12) and one (*pgi1-3*, SL5069-2) at a splice-site junction (Fig. 4; Supplemental Table S6). Native gel electrophoresis followed by activity staining on protein extracts from leaves revealed two bands of PGI activity in wild-type extracts, attributable to the cytosolic and plastidial isoforms of the enzyme (Shaw and Prasad, 1970). The band attributable to the plastidial isoform was missing in extracts of the mutants (Supplemental Fig. S2), consistent with the specific loss of the plastidial isoform of PGI in the *pgi1* mutants.

One of the synthesis mutants, SL4725-4, appeared from iodine staining and starch quantification to lack starch in leaves, stems, roots, and embryos, suggesting that it might be a *pgm1* mutant (Fig. 3; Table I). Arabidopsis, tobacco, and pea mutants lacking pPGM are starchless in all plant parts examined (Caspar et al., 1985; Hanson and McHale, 1988; Harrison et al., 1998). In a second mutant (SL4867-11) with a strongly reduced starch content relative to wild-type plants in all organs examined, the mutation mapped to an interval of about 10 centimorgan (cM) on chromosome V, in which the *PGM1* gene is located (Fig. 2; Supplemental Tables S2 and S4). Sequencing revealed that the *PGM1* gene in SL4725-4 contained a mutation affecting a splice-site

<footnote><sup>a</sup>For each genotype, F3 homozygous mutant and wild-type plants for analysis were selected by genotyping individual plants in a single F2 population derived from a cross between the original mutant in Gifu and MG-20. Plants were grown for 7 weeks, as described for starch quantification in "Materials and Methods."</footnote>
**Figure 4.** Structures of the starch metabolism genes and positions of the nonsilent mutations discovered in the forward genetic screen and by TILLING. Boxes and lines indicate exons and introns, respectively. Only the TILLING mutations leading to a starch phenotype and/or predicted by the PARSESNP program to have position-specific scoring matrix values greater than 0 are shown. The length of the gene sequence (in bp) is given on the right. The symbols above the gene specify the mutation type and position: octagons, stop codons; diamonds, splice site junction mutations; triangles, amino acid changes. Details of the nature and position of the mutations are given in Supplemental Table S6.

<figure><img src="image_6.png" /></figure>

junction, and the gene in SL4867-11 contained a mutation predicted to result in the amino acid change G95D. These mutants are referred to as *pgm1-4* and *pgm1-5*, respectively (Fig. 4; Supplemental Table S6). Native gel electrophoresis followed by activity staining on protein extracts from leaves revealed two bands of PGM activity in wild-type extracts, attributable to the cytosolic (PGM2) and plastidial (PGM1) isoforms of the enzyme (Harrison et al., 1998). The band attributable to the plastidial isoform was missing from extracts of the mutants, confirming that chloroplastic PGM activity is strongly reduced or absent in both cases (Supplemental Fig. S2). We used TILLING to select lines homozygous for three additional mutant alleles of the *PGM1* gene. Two of the alleles (*pgm1-2* from SL755-1 and *pgm1-3* from SL1837-1; Fig. 4; Supplemental Table S6) contained mutations creating stop codons; both of the mutant lines appeared from iodine staining to lack starch in all plant parts and were identical in phenotype to *pgm1-4* (Fig. 3; described in more detail below). The mutation in the third allele (*pgm1-1* from SL4490-1) was predicted to result in the amino acid change D436N. Activity of the plastidial isoform of PGM was reduced in *pgm1-1* (Supplemental Fig. S2), but no reduction in starch content of the leaves was apparent from iodine staining (Fig. 3).

We used similar approaches to identify the mutation accounting for the phenotype of another synthesis mutant, SL5127-5, in which leaf starch content at the end of the day is typically reduced by about 80% (Table I). The mutation mapped to an interval on

chromosome IV encompassing a gene encoding a large subunit of AGPase, APL1 (Fig. 2; Supplemental Tables S2 and S4). AGPase in higher plants is a heterotetramer composed of two small (APS) and two large (APL) subunits. Many species possess one or two *APS* genes and several *APL* genes that are differentially expressed between organs (Crevillén et al., 2003, 2005, and refs. therein). At least one *APS* gene and six *APL* genes are present in the *L. japonicus* genome (Supplemental Table S2). In Arabidopsis, loss of the small subunit (in the *adg1* mutant) almost eliminates AGPase activity and starch synthesis (Lin et al. 1988a; Wang et al., 1998a), and loss of the major leaf isoform of the large subunit (in the *adg2* mutant) reduces activity by 95% and starch synthesis by 60% (Lin et al., 1988b; Wang et al., 1997). Sequencing revealed a single mutation in the *APL1* gene of SL5127-5, predicted to result in the amino acid change S400L. To discover whether this mutation can account for the starch phenotype of the mutant, we assayed AGPase activity in extracts of wild-type and mutant leaves. Activity was 93% lower in mutant than in wild-type extracts (Table II). Activity in extracts made from mixtures of wild-type and mutant leaves was 98.5% of that predicted from separate extracts of the two genotypes; hence, the large difference in activity is likely to result from a loss of APL function in the mutant rather than from the presence of inhibitory substances in the mutant leaf.

To understand further the importance of AGPase subunits in *L. japonicus*, we used TILLING to identify
<table><caption>**Table II.** AGPase activities in leaves of the apl and aps synthesis mutants</caption><caption>Values are means ± SE of measurements on three plants.</caption><thead><tr><th>Genotype<sup>a</sup></th><th>AGPase Activity<br>nmol min<sup>-1</sup> g<sup>-1</sup> fresh wt</th></tr></thead><tbody><tr><td>MG-20</td><td>1,457 ± 41</td></tr><tr><td>Gifu</td><td>1,367 ± 140</td></tr><tr><td>apl1-1</td><td>105 ± 9</td></tr><tr><td>apl1-2</td><td>1,166 ± 166</td></tr><tr><td>apl2-1</td><td>1,844 ± 32</td></tr><tr><td>apl2-3</td><td>1,996 ± 63</td></tr><tr><td>apl2-4</td><td>1,364 ± 174</td></tr><tr><td>aps1-1</td><td>439 ± 10</td></tr><tr><td>aps1-2</td><td>1,376 ± 160</td></tr><tr><td>aps1-3</td><td>1,412 ± 168</td></tr></tbody></table>

<sup>a</sup>Plants were grown in a glasshouse as described in "Materials and Methods" and harvested 10 h into the 16-h light period when 7 to 12 weeks old.

mutations in a second large subunit gene, *APL2*, and in the single small subunit gene, *APS1*. For *APL2*, we isolated four alleles containing mutations predicted to have a deleterious effect on activity of the encoded protein. None of these had an effect on starch content (assessed by iodine staining) in any of the organs examined, including roots, leaves, and embryos (data not shown). In contrast, plants carrying one of the four mutant alleles identified for *APS1* (*aps1-1* from SL529-1), which had a mutation predicted to bring about the amino acid change A111T (Supplemental Fig. S4; Supplemental Table S6), displayed a strong reduction in starch content in leaves, roots, and embryos (Fig. 3). AGPase activity in leaves of this mutant was about 70% lower than in wild-type leaves (Table II), and leaf starch content was reduced by up to 90% (Table I).

## Mutations in the Starch Degradation Genes *LjGWD1* and *LjGWD3*

Following the same approach as for the starch synthesis mutants, we identified the mutations responsible for the starch-excess phenotype of three of the starch degradation mutants. The mutations in SL5215-2 and SL5358-3 mapped to the same interval on chromosome IV, and the mutation in line SL5104-12 mapped to the top of chromosome V (Fig. 2; Supplemental Table S4). These two intervals encompass genes encoding *GWD1* (Ritte et al., 2002) and *GWD3* (Baunsgaard et al., 2005; Kötting et al., 2005), respectively (Fig. 2; Supplemental Table S2). In Arabidopsis, the *gwd1* (or *sex1*) mutant has a severe starch-excess phenotype and reduced growth under short-day conditions (Caspar et al., 1991; Yu et al., 2001). The *gwd3* (*pwd*) mutant (Baunsgaard et al., 2005; Kötting et al., 2005) also has a starch-excess phenotype, although less severe than that of *gwd1*. Sequencing revealed that SL5215-2 and SL5358-3 both carry a mutation in *GWD1*, while SL5104-12 has a mutation in *GWD3*.

Although SL5215-2 and SL5358-3 were independently selected, they carry the same mutation in *GWD1*, predicted to result in the amino acid change E566K (Fig. 4; Supplemental Table S6). Plants carrying this mutation are referred to as *gwd1-1* mutants. The mutation in *GWD3* affects a splice-site junction and results in the nucleotide change G7871A (Fig. 4; Supplemental Table S6).

To confirm that the mutations identified in these two genes were responsible for the starch-excess phenotype of the *gwd1-1* and *gwd3-1* mutants, we generated additional mutant alleles by TILLING. We targeted a region of the *LjGWD1* gene encompassing the sequence encoding the second of the two putative starch-binding domains of the protein (SBD, Carbohydrate-Binding Module [CBM45] family; Mikkelsen et al., 2006) and a second region encoding part of the phospho-His domain of the enzyme (Yu et al., 2001). Two mutant lines isolated by TILLING (from SL1833-1 and SL3001-1, *gwd1-2* and *gwd1-3*, respectively; Fig. 4; Supplemental Table S6) had point mutations in *GWD1* leading to stop codons. Leaves of both mutants showed very strong starch-excess phenotypes (Fig. 3), stronger than that of *gwd1-1*, and quantification confirmed very high levels of starch in the *gwd1-2* mutant (end-of-night values: wild type, 0.9 ± 0.3, *gwd1-2*, 43.1 ± 3.3 mg g⁻¹ fresh weight [mean ± SE of at least five replicates]; Supplemental Fig. S3A). Immunoblotting with an antiserum to *GWD1* from potato (*Solanum tuberosum*) revealed that an immunoreactive protein of the expected mass was present in extracts of wild-type plants but missing in extracts of the *gwd1-2* mutant (Supplemental Fig. S3B). Several other mutant alleles were identified by TILLING, but none were predicted by the PARSESNP (see "Materials and Methods") program to have a deleterious effect on enzyme function, and leaves of the homozygous mutants did not have a starch-excess phenotype (data not shown).

TILLING performed on *LjGWD3* targeted the region encoding the nucleotide-binding domain involved in the dikinase activity of the enzyme (Kötting et al., 2005). A suite of mutant alleles with missense change mutations was identified. Homozygous mutants for one of these alleles, *gwd3-4* (from SL639-1; Fig. 4; Supplemental Table S6) had a clear starch-excess phenotype in leaves (Fig. 3). The mutation in this line is predicted to affect a residue (amino acid change G980E) that is identical across all the predicted protein homologs of *LjGWD3* we analyzed and lies within a conserved motif (PWD_ARATH, Q6ZY51; PWD_ORYSA, NP_001066613; putative homologs in *Vitis vinifera*, XP_002265211; *Ricinus communis*, XP_002518612; and *Sorghum bicolor*, XP_002453659 [multiple sequence alignment not shown]). The starch-excess phenotypes of both *gwd3-1* and *gwd3-4* strongly suggest that *LjGWD3* plays an important role in the degradation of starch in leaves of *L. japonicus*. It seems likely that, as in Arabidopsis, *LjGWD3* acts in synergy with *LjGWD1* to phosphorylate the starch granule as a prerequisite for degradation (Baunsgaard et al., 2005; Kötting et al., 2005).
## Isolation of Mutants Affected in Novel Proteins Involved in Starch Metabolism in *L. japonicus*

We mapped the chromosomal locations of the mutations responsible for the remaining four synthesis mutants (SL5249-3, SL5143-3, SL5074-12, and SL4618-12) and three degradation mutants (SL4841-4, SL5035-7, and SL5272-11) from the forward screen. To date, four of the mutations have been mapped to intervals of less than 5 cM (synthesis mutants SL5249-3 and SL4618-12 and degradation mutants SL4841-4 and SL5035-7; Fig. 2; Supplemental Table S4). No obvious candidate genes are present in these intervals, such as those encoding pathway enzymes or established regulators. Hence, the genes responsible for the phenotypes are likely to encode previously undiscovered components of starch metabolism pathways.

Altogether, this comprehensive collection of starch metabolism mutants of *L. japonicus* is a valuable resource for further study of starch metabolism in general and its importance in legumes in particular. To illustrate this, we report below new information about the structure-function relationships of AGPase and GWD1 derived from study of the mutations affecting these enzymes in *L. japonicus* and provide new insights into the importance of starch metabolism in *L. japonicus* through the analysis of the phenotypes of the *pgm1* and *gwd1* mutants.

## Identification of Novel Amino Acid Residues Important for the Activity of the LjAPL1 and LjGWD1 Enzymes

The nature of the mutation in the *APL1* gene of *L. japonicus* (in line SL5127-5, from the forward screen) was unexpected in the light of our current understanding of the structure-function relationships of the enzyme. Although leaves of the *apl1* mutant have only 7% of the AGPase activity of wild-type leaves (Table II), the mutation changes an amino acid (S400L; Supplemental Fig. S4) not known previously to be important for enzyme activity. The crystal structure of the small subunit of AGPase from potato tubers (Jin et al., 2005) and mutagenesis studies (Ballicora et al., 1998; Greene et al., 1998; Kavakli et al., 2002) have enabled the identification of residues important for either substrate affinity or sensitivity to the allosteric effectors inorganic phosphate and 3-phosphoglycerate. The strong sequence similarities between the small and large subunits (48% identity at the amino acid level between LjAPL1 and StAPS) allowed us to use this three-dimensional structure to analyze the impact of the mutation in *apl1*. The mutated residue (Ser-400; equivalent to Ser-331 in the potato small subunit) is positioned at the interface between subunits (Supplemental Fig. S5). It is tempting to speculate that this residue is crucial for activity because it is involved in interaction between the subunits. Consistent with this idea, Cross et al. (2005) identified a 55-amino acid region of the potato small subunit including Ser-331 that influences both the catalytic and the allosteric

properties of the enzyme by modulating interaction between the subunits. However, preliminary in silico analysis indicates that the change from Ser to Leu at this position is unlikely to affect subunit interaction (Supplemental Fig. S5); the backbone, rather than the side chain, of the residue at this position is involved in subunit interaction. The side chain of Ser-400, however, interacts with other amino acids in the β-helix domain in which it is located. These interactions are lost when a Leu is substituted for this Ser residue. We suggest that Ser-400 may be important for the three-dimensional structure of the large subunit itself and hence for heterotetramer stability as a whole.

The mutation in *gwd1-1* also provides new information about the structure-function relationships of an enzyme essential for normal starch metabolism. The mutation (E566K) lies in the second of the two starch-binding domains (SBD-1 and SBD-2) located in the N-terminal region of the GWD1 protein (Mikkelsen et al., 2006). These domains have been classified into the newly defined CBM45 family in the CAZy database (http://www.cazy.org). Members of this family are also found in an extraplastidial glucan, water dikinase of unknown function (GWD2; Glaring et al., 2007) and in plastidial α-amylases. Expression of an N-terminally truncated form of the Arabidopsis plastidial α-amylase AMY3 has established that the CBM module is not required for the catalytic activity of this enzyme (Yu et al., 2005). Expression of a truncated form of Arabidopsis GWD1, lacking SBD-1, indicates that this module may be important in determining both the specific activity and the glucan substrate preference of the enzyme (Mikkelsen et al., 2006). However, nothing is known thus far about the role of SBD-2. None of the reported Arabidopsis *gwd1* mutants carry mutations in the SBD-2 module (Yu et al., 2001). Our results indicate that the SBD-2 module is crucial for the in vivo function of the enzyme. The Glu at position 566 is conserved at the equivalent position in GWD1 from other species and lies in a generally conserved region (Supplemental Fig. S6) predicted to be a coiled-coil domain.

## Importance of Starch Metabolism for Plant Growth, Development, and Nodule Function in *L. japonicus*

We used our synthesis and degradation mutants to examine whether loss of normal starch metabolism affected growth and development and especially the legume-specific capacity for nitrogen fixation in *Rhizobium*-containing root nodules. The essentially starchless phenotype of the *pgm1-4* mutant (Figs. 3 and 5) indicates that plastidial PGM1 is necessary for starch synthesis throughout the plant. Interestingly, this mutant grows normally even under relatively short days (12 h of light and 12 h of dark; Fig. 5). This is also true of the equivalent mutant in pea (*rug3*; Harrison, 1996). In contrast, although the almost starchless *pgm1* mutant of Arabidopsis grows at approximately the same rate as the wild type under
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Phenotypes of the *pgm1-4* and *gwd1-2/3* mutants. A, Plants grown in 16 h of light and 8 h of dark (top) and 12 h of light and 12 h of dark (bottom). B and C, Root systems and nodules of 3-month-old plants grown in 16 h of light and 8 h of dark and inoculated with *M. loti* at 6 weeks. Nodules are shown either whole (top left), cut open (bottom left) or cut open and stained with iodine (right). The cortical zone and noninfected cells in wild-type (WT) nodules contain starch. Mutant nodules contain no starch. D to F, Iodine staining of root systems and of the interior of a nodule of a mutant plant (F; compare with the wild type shown in B). Plants were grown in 16 h of light and 8 h of dark and were 6 months old. G and H, Pollen grains from plants stained with iodine (G) and acetocarmine (H). The mutation increases the starch content of pollen but does not affect its viability. I, Three-month-old wild-type,</figcaption></figure>

continuous light, it grows much more slowly under 12-h-light/12-h-dark conditions (Caspar et al., 1985).

Both the *gwd1-2* and *gwd1-3* mutants accumulated excessive amounts of starch in their leaves, roots, stems, nodules, and pollen (Figs. 3 and 5; Supplemental Fig. S3). These mutants showed severe growth defects, including chlorosis, premature leaf senescence, and very poor or no seed set. Only a small proportion of mutant plants in a given batch produced flowers, and they did so about 2 months later than wild-type plants. These characteristics were present regardless of the daylength under which the plants were grown. This is in contrast to the situation in Arabidopsis, in which *gwd1* (*sex1*) knockout mutants display almost no growth phenotype under long days but retain very high starch contents and can reproduce even under short days when growth is significantly retarded (Caspar et al., 1991). Thus, loss of GWD seems to have much more serious consequences for *L. japonicus* than for Arabidopsis.

It is not clear why growth rates are reduced in some mutant plants impaired in leaf starch synthesis or degradation or why mutations in homologous starch-metabolism genes have different consequences for growth in *L. japonicus* and Arabidopsis. In Arabidopsis, genetic and environmental factors that reduce carbohydrate availability at night can also cause temporary cessation of growth (Smith and Stitt, 2007). It may be that there is variation between genes and species in the extent to which mutations affecting starch turnover result in reduced carbohydrate availability and hence cessation of growth during the night. Alternatively, for starch-excess mutants, the severity of the phenotype may depend on the extent to which starch accumulation physically impairs normal chlo-roplast functions.

We explored further the causes of the loss of fertility in *gwd1-2* and *gwd1-3* mutants. In the progeny of plants heterozygous for either mutant allele, segregation did not follow the Mendelian ratio of 1:2:1 mutant: heterozygote: wild type. From 219 progeny of a *gwd1-2* heterozygote, 13 were mutants, 116 were heterozygotes, and 90 were wild type. For 202 progeny of a *gwd1-3* heterozygote, 15 were mutants, 98 were heterozygotes, and 89 were wild type. The low numbers (6%–7%) of homozygous mutant plants suggested that loss of GWD1 is partially lethal at the gametophyte stage, or that mutant embryos generally failed to mature, or that mutant seeds did not germinate. In tomato (*Solanum lycopersicum*), loss of GWD1 leads to pollen with an abnormally high starch content that fails to germinate (Nashilevitz et al., 2009). However, two lines of evidence suggested that pollen from *L. japonicus* *gwd1* mutants is at least partially viable despite its high starch content. First, heterozygous plants could be obtained in the F1 progeny of recip-

<footnote>heterozygous, and mutant segregants for *gwd1-3* (left three plants) and *gwd1-2* (right three plants).</footnote>
rocal crosses made between heterozygote and wild-
type plants. Second, staining the pollen with acetocar-
mine, a dye used to assess whether pollen grains
have matured, showed no differences between the
wild type, heterozygotes, and homozygous mutants
(Fig. 5).

Most *gwd1* flowers did not produce pods, and when they did, pods and embryos often aborted early in development. However, fully developed mutant pods and seeds could be obtained by detaching flowers and placing the peduncles in Murashige and Skoog liquid medium supplemented with 3% Suc (data not shown). This result suggested that the effect of the mutation on the production of viable seed was maternal rather than due to a defect in embryo development. As a further test, we analyzed embryos and seeds from 40 pods each of wild-type and heterozygous (GWD1-2/gwd1-2) plants from the same segregating population. Both the number of seeds per pod and the average seed weight were very similar for the two genotypes (an average of 9.1 and 10.2 seeds per pod for heterozygous and wild-type plants, respectively, and an average seed weight of 1.0 mg for both genotypes). Similar results were obtained for the *gwd1-3* mutation (data not shown). These data support the idea that reduced production of viable seeds on homozygous mutant plants is a maternal effect. However, genotyping of each individual mature embryo from heterozygous plants was not wholly consistent with a maternal effect. Out of 63 mature seeds, only seven contained homozygous mutant embryos (one in nine rather than the expected one in four). Thus, mutant embryos were disadvantaged whether in mutant or heterozygous pods. The effect of the *gwd1* mutations on fertility thus appears to be complex. Gametophytic, maternal, and embryo effects may all be involved. In general, our mutants show that the phosphorylation of starch via the glucan, water dikinases GWD1 and GWD3 is essential for normal starch degradation in most parts of the *L. japonicus* plant and that this, in turn, is crucial for normal plant growth and development.

Legume nodules generally contain starch in cortical
cells and noninfected cells within the infected zone
(Gordon, 1992; Gordon and James, 1997; Szczyglowski
et al., 1998). In *pgm1-4* mutants inoculated with *Mes-
orhizobium loti*, nodules had a normal pink color, and
their size and number per root system were similar to
those of wild-type segregants (Fig. 5). In spite of the
apparent lack of starch in the nodules, assays for
nitrogen fixation (acetylene reduction) revealed no
statistically significant differences (Student's *t* test
*P* value of greater than 0.05) between mutant, hetero-
zygote, and wild-type plants 36 d after inoculation
(0.57 ± 0.06, 0.75 ± 0.11, and 0.67 ± 0.20 µmol ethylene
h⁻¹ root system⁻¹, respectively; mean ± SE of five root
systems). Thus, storage of carbon as starch in the
nodule is not necessary for normal nodule function
under our growth conditions. At two stages of nodule
development, nodules on *gwd1-2* plants contained
about 15 times more starch than nodules on wild-

type plants (Supplemental Fig. S3C). Nonetheless,
mutant nodules were capable of nitrogen fixation. In
one batch of plants, nitrogen fixation (acetylene re-
duction) was comparable in *gwd1* mutants and their
segregating wild-type and heterozygous lines. In an-
other batch, fixation was reduced in mutant relative to
wild-type and heterozygous plants. Both batches
showed that the *gwd1* mutants were capable of nitro-
gen fixation (Supplemental Fig. S3D). Taken together
with the results of measurements on the nodules of
*pgm1-4* mutant plants, these data indicate that starch
storage and normal starch metabolism are not essen-
tial for nodule function.

CONCLUSION

We have linked the identification of candidate genes
from rough mapping with TILLING reverse genetics
to assemble a comprehensive suite of starch mutants in
the model legume *L. japonicus*. This is a valuable new
resource with which to examine the partitioning of
carbon in relation to several crop traits of agronomic
importance, such as nitrogen fixation and perenniality.
Our research has additional significance. It shows that
the main components of the pathways of leaf starch
metabolism are conserved across different families
and life histories. In contrast, the control of flux
through these pathways and their importance in sus-
taining normal plant growth are very different in
Arabidopsis and *L. japonicus*. Whereas a severe defi-
ciency in starch synthesis has much less effect in *L.
japonicus* than in Arabidopsis, loss of the capacity for
starch degradation has much more profound conse-
quences for plant growth. Further investigation of
these differences and the genes affecting starch me-
tabolism that were previously unknown could lead to
a better understanding of the regulatory mechanisms
linking the metabolism of starch with plant growth.
Our analysis also revealed significant differences in
starch metabolism between plants of MG20 and Gifu,
two wild-type ecotypes of *L. japonicus* with large
differences in several other traits, including biomass
production. This finding is particularly interesting in
light of the recent demonstration that rates of starch
turnover are positively, causally linked to productivity
in Arabidopsis (Sulpice et al., 2009). We suggest that *L.
japonicus* is an excellent system with which to test the
wider applicability of this finding.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Initial screening for starch metabolism mutants was carried out as de-
scribed by Perry et al. (2003, 2009). Plants for starch quantification were grown
in a growth chamber on F2 compost (Levington; Scotts Professional) with 12 h
of light and 12 h of dark at a light intensity of 100 µmol m⁻² s⁻¹ and day/night
temperatures of 22°C/18°C. Plants for iodine staining, acetylene reduction
assay, enzyme assay, and native gel electrophoresis were grown in a glass-
house on compost with 16 h of light and 8 h of dark (with or without
supplementary lighting; minimum light intensity at 200 µmol m⁻² s⁻¹) or in
a controlled-environment room (light intensity at 200 µmol m⁻² s⁻¹; 75% humidity).

Plants for acetylene reduction assay were transferred from compost to perlite:vermiculite (1:1, v/v) at 7 weeks and fed twice a week with nutrient solution (Broughton and Dilworth, 1971) containing 0.1 mM KNO₃. Roots were truncated at the time of transfer to stimulate lateral root formation and hence nodule production upon inoculation with *Mesorhizobium loti* strain Tono.

For in vitro culture of pods, peduncles carrying mature flowers were cut and the ends were immediately immersed in distilled water. Peduncles were then surface sterilized with 10% (v/v) bleach (1% available chlorine) and rinsed repeatedly in sterile water. After recutting, peduncles were inserted through a hole in the lid of a 50-mL tube of sterile Murashige and Skoog medium (microelements and macroelements including vitamins, pH 5.7) with 3% (w/v) Suc and then kept for 4 weeks in a growth chamber with 16 h of light and 8 h of dark.

## In Silico Analysis of Gene and Protein Sequences

Genes encoding known enzymes of starch metabolism in *Lotus japonicus* were identified from the Miyakogusa genome database (Kazusa DNA Research Institute; http://www.kazusa.jp/lotus/index.html; Sato et al., 2008) using gene names as keywords and/or by performing BLASTp with the protein sequences of their Arabidopsis (*Arabidopsis thaliana*) orthologs. Hits that included clones and selected genome assembly sequences were selected for further analyses. These included use of the ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw2/index.html) for protein sequence alignment of homologs and other members of the same gene family from other species. Alignments allowed the identification of problems associated with incorrect annotations, incorrect predictions, or incomplete sequences for the *L. japonicus* genes and proteins.

Retrieved coding sequences that were incorrectly predicted or partial were used to search for the contig containing the corresponding genomic sequence. A predicted full-length coding sequence was then obtained using FGENESH (http://linux1.softberry.com/berry.phtml), Genscan (http://genes.mit.edu/GENSCAN.html; Burge and Karlin, 1997; Burge, 1998), and GenomeScan (http://genes.mit.edu/genomescan.html; Yeh et al., 2001).

When orthologs of known genes could not be identified, BLASTn or tBLASTn searches were performed against the *L. japonicus* EST database (Dana-Farber Cancer Institute Gene Index of *L. japonicus*; http://compbio.dfci.harvard.edu/tgi/plant.html). Where possible, a partial or full-length coding sequence was obtained by aligning several EST clones, and the resulting sequence was used to search the Miyakogusa genome database or to screen transformation-competent bacterial artificial chromosome vector and bacterial artificial chromosome libraries.

Gene structures were predicted using the CODDLE program (for Codons Optimized to Discover Deleterious Lesions; http://www.proweb.org/coddle/), and PARSESNP (for Project Aligned Related Sequences and Evaluate SNPs; http://www.proweb.org/parsesnp/) was used to determine potential effects on protein function.

## Isolation of Nucleic Acids

Genomic DNA was extracted with phenol:chloroform according to Heckmann et al. (2006). The DNA pellet was washed with 70% (v/v) aqueous ethanol, air dried, and resuspended in 50 to 100 µL of nuclease-free water. Samples of 1 µL were subjected to electrophoresis on 1% agarose gels to check integrity. Five- to 10-fold dilutions were used for further experiments.

## Genotyping and Sequencing

Genotyping of individual plants was carried out using gene-specific primers (Supplemental Table S5). PCR primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; Rozen and Skaletsky, 2000). PCR was performed using ExTaq polymerase (Takara) following the manufacturer's instructions, and the products were sequenced.

## TILLING

Primers were designed using Primer3 in the region identified by CODDLE as having the maximum likelihood of producing deleterious alleles following EMS mutagenesis. TILLING on the genes *LjPGM1*, *LjAPS1*, *LjAPL2*, and *LjGWD1* was performed as described previously (Horst et al., 2007) using gene-specific primers. TILLING for *LjGWD3* was carried out by RevGenUK

(http://revgenuk.jic.ac.uk/) on an ABI3730 capillary sequencer following the method of Le Signor et al. (2009). Gene-specific primers used for TILLING are shown in Supplemental Table S4. The effect of the mutations was predicted using the PARSESNP program. Mutations resulting in the loss of a splicing site or a premature stop codon and those with a position-specific scoring matrix difference score greater than zero, or likely to have an effect according to our own analysis of a multiple sequence alignment, were selected for further analysis.

## Mapping

Mapping populations were created from crosses between mutants (in the Gifu background) and the wild-type ecotype *L. japonicus* MG-20. F1 plants were allowed to self, and mutant plants were selected in the F2 generation based on iodine staining of leaves. Mapping was carried out using microsatellite SSR markers designed at the Kazusa DNA Research Institute (from Sigma Genosys, Sigma-Aldrich). Rough mapping was carried out initially by bulk segregant analysis (markers shown in Supplemental Table S2) on at least two pools of eight genomic DNA samples of mutants from F2 segregating populations. The mapping interval thus identified was confirmed on the mutant individuals. For five of the mutant lines shown in Supplemental Table S6 (SL5074-12, SL5249-3, SL4618-12, SL4841-4, and SL5035-7), finer mapping was performed on a larger set of mutant individuals from F2 segregating populations from a cross with MG-20. Finer mapping used SSR markers designed either at the Kazusa DNA Research Institute or with the Simple Sequence Repeat Identification Tool (Temnykh et al., 2001). SSR markers within the target regions were PCR amplified and fluorescently labeled (Schuelke, 2000). The method used multiplex PCR amplification and automated capillary electrophoresis analysis on an ABI 3730 DNA Analyzer (M. Groth, Biocenter Ludwig-Maximilians-Universität Munich) and GeneMapper software (Applied Biosystems).

## Iodine Staining and Starch Quantification

Tissue was heated in 80% (v/v) aqueous ethanol to remove pigments before staining with iodine (Lugol's solution), except for embryos, which were incubated in chloroform:ethanol:water (5:5:1, v/v/v) prior to treatment with 80% ethanol.

For starch quantification, 50 to 200 mg of tissue was rapidly frozen in liquid nitrogen after harvest and then homogenized on dry ice using a pulverizing mill (MM300; Retsch). After extraction with perchloric acid (1 mL of 0.7 M), insoluble material was washed, heated to gelatinize starch, and incubated with amyloglucosidase and α-amylase prior to measurement of Glc on the solubilized material, according to Smith and Zeeman (2006).

## Enzyme Assays

For assays of AGPase, leaf material (200-300 mg) was harvested on ice, extracted in 3 mL of 50 mM HEPES (pH 7.4), 1 mM EDTA, 2 mM MgCl₂, and 1 mM dithiothreitol and clarified by centrifugation at 4°C. Each assay contained 10 µL of soluble extract in 190 µL of 50 mM HEPES (pH 7.4), 15 mM MgCl₂, 15 mM 3-phosphoglycerate, 1.5 mM ATP, 0.5 mg mL⁻¹ bovine serum albumin, 0.5 mM [U-¹⁴C]Glc 1-P at 1 MBq mmol⁻¹, and 12.5 units mL⁻¹ inorganic pyrophosphatase (from baker's yeast). After incubation for 10 min at 37°C, samples were processed by treatment with DEAE cellulose discs according to Ghosh and Preiss (1966) and the ADPGlc product was measured by liquid scintillation counting.

## Gel Electrophoresis and Immunoblotting

Native gel electrophoresis for enzyme activities was modified from the method of Shaw and Prasad (1970). Samples of 200 to 300 mg of leaves were homogenized in 1 mL of cold 100 mM MOPS, pH 7.2, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol. Following centrifugation (10,000g, 10 min at 4°C), samples (50-100 µg of protein) of supernatant were subjected to electrophoresis on native polyacrylamide gels (7.5%) containing 1% (w/v) potato (Solanum tuberosum) starch. Gels were then incubated for 1 h at 37°C in 100 mM Tricine (pH 8), 10 mM MgCl₂, 0.25 mM NADP, 1 mM thiazolyl blue tetrazolium bromide, a trace of Meldola's blue (Sigma-Aldrich), 1 unit mL⁻¹ Glc 6-P dehydrogenase from *Leuconostoc mesenteroides*, and either 7 mM Glc 1-P (PGM activity) or 8 mM Fru 6-P (PGI activity).

For immunoblotting, samples of frozen leaves were powdered and mixed with 2× Laemmli sample buffer. The homogenate was clarified by centrifu-
gation. Samples of 10 µg of protein were loaded onto SDS-polyacrylamide gels (4%-12% gradient). After electrophoresis, the gel was blotted onto a nitrocellulose membrane and probed with rabbit serum containing antibodies to potato GWD1 at a dilution of 1:1,000 (Yu et al., 2001).

## Acetylene Reduction Assay

The amount of ethylene produced by whole root systems incubated with acetylene was quantified as described by Horst et al. (2007).

Novel sequence data from this article have been deposited in the GenBank/EMBL/DDBJ databases under the following accession numbers: AP011538, LjT05B14 (TM2333; DPE2); AP011539, LjB22L24 (BM2254; 5' region of LjGWD1).

## Supplemental Data

The following materials are available in the online version of this article.

**Supplemental Figure S1.** Pathway of transitory starch metabolism in *Arabidopsis* leaves.

**Supplemental Figure S2.** Separation of PGM and PGI isoforms by native gel electrophoresis.

**Supplemental Figure S3.** Phenotypes of *gwd1* TILLING mutants.

**Supplemental Figure S4.** Comparison of the amino acid sequences of *L. japonicus* AGPase subunits with those of other species.

**Supplemental Figure S5.** Predicted effect on AGPase function of the S400L amino acid change identified in the *L. japonicus* mutant *apl1-1*.

**Supplemental Figure S6.** Protein domains of *L. japonicus* GWD1, and comparison of its amino acid sequence with those of other species.

**Supplemental Table S1.** Diurnal changes in leaf starch content through the development of *L. japonicus* plants.

**Supplemental Table S2.** Orthologs in *L. japonicus* of the core set of genes encoding enzymes of starch metabolism in *Arabidopsis*.

**Supplemental Table S3.** Microsatellite SSR markers used for rough mapping of loci with mutant phenotype from the forward genetic screen.

**Supplemental Table S4.** Mapped interval and flanking microsatellite markers of the loci with mutant phenotypes from the forward genetic screen.

**Supplemental Table S5.** Primers used for gene sequencing, TILLING, and genotyping.

**Supplemental Table S6.** Mutant alleles of genes encoding enzymes of starch metabolism in *L. japonicus*.

## ACKNOWLEDGMENTS

We thank Martin Groth (Biocenter Ludwig-Maximilians-Universität) for access to the ABI3730 mapping method and Dr. Gerhard Ritte (University of Potsdam) for the gift of GWD1 antibodies. We thank RevGenUK (http://revgenuk.jic.ac.uk/) for TILLING the *LjGWD3* gene.

Received June 28, 2010; accepted July 30, 2010; published August 10, 2010.

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