Marilyn J. Pike

Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

Callose Synthase GSL7 Is Necessary for Normal Phloem Transport and Inflorescence Growth in Arabidopsis

One isoform of callose synthase, Glucan Synthase-Like7 (GSL7), is tightly coexpressed with two isoforms of sucrose synthase (SUS5 and SUS6) known to be confined to phloem sieve elements in Arabidopsis (Arabidopsis thaliana). Investigation of the phenotype of gsl7 mutants of Arabidopsis revealed that the sieve plate pores of stems and roots lack the callose lining seen in wild-type plants. Callose synthesis in other tissues of the plant appears to be unaffected. Although gsl7 plants show only minor phenotypic alterations during vegetative growth, flowering stems are reduced in height and all floral parts are smaller than those of wild-type plants. Several lines of evidence suggest that the reduced growth of the inflorescence is a result of carbohydrate starvation. Levels of sucrose, hexoses, and starch are lower in the terminal bud clusters of gsl7 than in those of wild-type plants. Transcript levels of “starvation” genes expressed in response to low sugars are elevated in the terminal bud clusters of gsl7 plants, at the end of the night, and during an extended night. Pulse-chase experiments with 14CO2 show that transport of assimilate in the flowering stem is much slower in gsl7 mutants than in wild-type plants. We suggest that the callose lining of sieve plate pores is essential for normal phloem transport because it confers favorable flow characteristics on the pores.

Plastidial glycolysis in developing Arabidopsis embryos

During oilseed embryo development, carbon from sucrose is utilized for fatty acid synthesis in the plastid. The role of plastidial glycolysis in Arabidopsis embryo oil accumulation was investigated. Genes encoding enolases (ENO) and phosphoglyceromutases (PGlyM) were identified, and activities and subcellular locations were established by expression of recombinant and green fluorescent protein (GFP)-fusion proteins. Mutant Arabidopsis plants lacking putative plastidial isoforms were characterized with respect to isoform composition and embryo oil content. In the developing embryo, ENO1 and ENO2 account for most or all of the plastidial and cytosolic ENO activity, respectively, and PGLYM1 accounts for most or all of the plastidial PGlyM activity. The eno1 and pglym1 mutants, in which plastidic ENO and PGlyM activities were undetectable, had wild-type amounts of seed oil at maturity. It is concluded that although plastids of developing Arabidopsis embryos have the capacity to carry out the lower part of the glycolytic pathway, the cytosolic glycolytic pathway alone is sufficient to support the flux from 3-phosphoglycerate to phosphoenolpyruvate required for oil production. The results highlight the importance for oil production of translocators that facilitate interchange of glycolytic intermediates between the cytosol and the plastid stroma.

The Transport of Sugars to Developing Embryos Is Not via the Bulk Endosperm in Oilseed Rape Seeds

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [14C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [14C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [14C]Suc converted to hexose falls dramatically. Instead, most of the [14C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.

Starch turnover in developing oilseed embryos

*Starch accumulates early during embryo development in Arabidopsis and oilseed rape, then disappears during oil accumulation. Little is known about the nature and importance of starch metabolism in oilseed embryos. *Histochemical and quantitative measures of starch location and content were made on developing seeds and embryos from wild-type Arabidopsis plants, and from mutants lacking enzymes of starch synthesis and degradation with established roles in leaf starch turnover. Feeding experiments with [(14)C]sucrose were used to measure the rate of starch synthesis in oilseed rape embryos within intact siliques. *The patterns of starch turnover in the developing embryo are spatially and temporally complex. Accumulation is associated with zones of cell division. Study of mutant plants reveals a major role in starch turnover for glucan, water dikinase (absent from the sex1 mutant) and isoforms of beta-amylase (absent from various bam mutants). Starch is synthesized throughout the period of its accumulation and loss in embryos within intact siliques of oilseed rape. *We suggest that starch turnover is functionally linked to cell division and differentiation rather than to developmental or storage functions specific to embryos. The pathways of embryo starch metabolism are similar in several respects to those in Arabidopsis leaves.

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

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.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

Altered Starch Turnover in the Maternal Plant Has Major Effects on Arabidopsis Fruit Growth and Seed Composition

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night

Background: Maltose metabolism during leaf starch degradation requires a multidomain glucanotransferase and a complex polysaccharide. Results: A conventional bacterial glucanotransferase rescues an Arabidopsis mutant lacking the multidomain glucanotransferase. Conclusion: Both the plant glucanotransferase-polysaccharide couple and the bacterial enzyme provide a glucosyl buffer in the starch degradation pathway. Significance: New light is shed on the regulation and evolution of maltose metabolism. Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a “glucosyl buffer” to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.

Isolation of Amyloplasts

Two different methods for the preparation of starch‐rich plastids are described together with protocols for the determination of plastid yield, purity, and intactness. The preparation of amyloplasts from maize endosperm and oilseed rape embryos are given as examples, but the protocols could be adapted for the isolation of starch‐rich plastids from other plant organs. A method for the determination of the quantitative distribution of an enzyme between the plastids and cytosol is given. Typical results and references for marker enzymes for a range of subcellular compartments are listed. Curr. Protoc. Cell Biol. 38:3.28.1‐3.28.15.