Sean E. Weise

A Putative Phosphatase, LSF1, Is Required for Normal Starch Turnover in Arabidopsis Leaves

A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.

Gravitropism of inflorescence stems in starch-deficient mutants of Arabidopsis

Previous studies have assayed the gravitropic response of roots and hypocotyls of wild type Arabidopsis thaliana, two reduced‐starch strains, and a starchless strain. Because there have been few reports on inflorescence gravitropism, in this article, we use microscopic analyses and time‐course studies of these mutants and their wild type to study gravitropism in these stems. Sedimentation of plastids was observed in endodermal cells of the wild type and reduced‐starch mutants but not in the starchless mutant. In all of these strains, the short inflorescence stems (1.0–2.9 cm) were less responsive to the gravistimulus compared with the long stems (3.0–6.0 cm). In both long and short inflorescence stems, the wild type initially had the greatest response; the starchless mutant had the least response; and the reduced starch mutants exhibited an intermediate response. Furthermore, growth rates among all four strains were approximately equal. At about 6 h after reorientation, inflorescences of all strains returned to a position parallel to the gravity vector. Thus, in inflorescence stems, sedimentation of plastids may act as an accelerator but is not required to elicit a gravitropic response. Furthermore, the site of perception appears to be diffuse throughout the inflorescence stem. These results are consistent with both a plastid‐based statolith model and the protoplast pressure hypothesis, and it is possible that multiple systems for gravity perception occur in plant cells.

The role of transitory starch in C3, CAM, and C4 metabolism and opportunities for engineering leaf starch accumulation

Essentially all plants store starch in their leaves during the day and break it down the following night. This transitory starch accumulation acts as an overflow mechanism when the sucrose synthesis capacity is limiting, and transitory starch also acts as a carbon store to provide sugar at night. Transitory starch breakdown can occur by either of two pathways; significant progress has been made in understanding these pathways in C(3) plants. The hydrolytic (amylolytic) pathway generating maltose appears to be the primary source of sugar for export from C(3) chloroplasts at night, whereas the phosphorolytic pathway supplies carbon for chloroplast reactions, in particular in the light. In crassulacean acid metabolism (CAM) plants, the hydrolytic pathway predominates when plants operate in C(3) mode, but the phosphorolytic pathway predominates when they operate in CAM mode. Information on transitory starch metabolism in C(4) plants has now become available as a result of combined microscopy and proteome studies. Starch accumulates in all cell types in immature maize leaf tissue, but in mature leaf tissues starch accumulation ceases in mesophyll cells except when sugar export from leaves is blocked. Proper regulation of the amount of carbon that goes into starch, the pathway of starch breakdown, and the location of starch accumulation could help ensure that engineering of C(4) metabolism is coordinated with the downstream reactions required for efficient photosynthesis.

Carbon Balance and Circadian Regulation of Hydrolytic and Phosphorolytic Breakdown of Transitory Starch

Transitory starch is formed in chloroplasts during the day and broken down at night. Transitory starch degradation could be regulated by light, circadian rhythms, or carbon balance. To test the role of these potential regulators, starch breakdown rates and metabolites were measured in bean (Phaseolus vulgaris) and Arabidopsis (Arabidopsis thaliana) plants. In continuous light, starch and maltose levels oscillated in a circadian manner. Under photorespiratory conditions, transitory starch breakdown occurred in the light faster than at night and glucose-6-P (G6P) was elevated. Nonaqueous fractionation showed that the increase in G6P occurred in the chloroplast. When Arabidopsis plants lacking the plastidic starch phosphorylase enzyme were placed under photorespiratory conditions, G6P levels remained constant, indicating that the increased chloroplastic G6P resulted from phosphorolytic starch degradation. Maltose was increased under photorespiratory conditions in both wild type and plants lacking starch phosphorylase, indicating that regulation of starch breakdown may occur at a point preceding the division of the hydrolytic and phosphorolytic pathways. When bean leaves were held in N2 to suppress photosynthesis and Suc synthesis without increasing photorespiration, starch breakdown did not occur and maltose and G6P levels remained constant. The redox status of the chloroplasts was found to be oxidized under conditions favoring starch degradation.

The interactions between the circadian clock and primary metabolism.

Primary metabolism in plants is tightly regulated by environmental factors such as light and nutrient availability at multiple levels. The circadian clock is a self-sustained endogenous oscillator that enables organisms to predict daily and seasonal changes. The regulation of primary metabolism by the circadian clock has been proposed to explain the importance of circadian rhythms in plant growth and survival. Recent transcriptomic and metabolomic analyses indicate a wide spread circadian regulation of different metabolic processes. We review evidence of circadian regulation of pathways in primary metabolism, discuss the challenges faced for discerning the mechanisms regulating circadian metabolic oscillations and present recent evidence of regulation of the circadian clock by metabolites.

β-Maltose Is the Metabolically Active Anomer of Maltose during Transitory Starch Degradation

Maltose is the major form of carbon exported from the chloroplast at night as a result of transitory starch breakdown. Maltose exists as an α- or β-anomer. We developed an enzymatic technique for distinguishing between the two anomers of maltose and tested the accuracy and specificity of this technique using β-maltose liberated from maltoheptose by β-amylase. This technique was used to investigate which form of maltose is present during transitory starch degradation in bean (Phaseolus vulgaris), wild-type Arabidopsis (Arabidopsis thaliana), two starch deficient Arabidopsis lines, and one starch-excess mutant of Arabidopsis. In Phaseolus and wild-type Arabidopsis, β-maltose levels were low during the day but were much higher at night. In Arabidopsis plants unable to metabolize maltose due to a T-DNA insertion in the gene for the cytosolic amylomaltase, (Y. Lu, T.D. Sharkey [2004] Planta 218: 466–473) levels of α- and β-maltose were high during both the day and night. In starchless mutants of Arabidopsis, total maltose levels were low and almost completely in the α-form. We also found that the subcellular concentration of β-maltose at night was greater in the chloroplast than in the cytosol by 278 μm. We conclude that β-maltose is the metabolically active anomer of maltose and that a sufficient gradient of β-maltose exists between the chloroplast and cytosol to allow for passive transport of maltose out of chloroplasts at night.

Engineering starch accumulation by manipulation of phosphate metabolism of starch

A new understanding of leaf starch degradation has emerged in the last 10 years. It has been shown that starch phosphorylation and dephosphorylation are critical components of this process. Glucan, water dikinase (GWD) (and phosphoglucan, water dikinase) adds phosphate to starch, and phosphoglucan phosphatase (SEX4) removes these phosphates. To explore the use of this metabolism to manipulate starch accumulation, Arabidopsis (Arabidopsis thaliana) plants were engineered by introducing RNAi constructs designed to reduce expression of AtGWD and AtSEX4. The timing of starch build-up was altered with ethanol-inducible and senescence-induced gene promoters. Ethanol induction of RNAi lines reduced transcript for AtGWD and AtSEX4 by 50%. The transgenic lines had seven times more starch than wild type at the end of the dark period but similar growth rates and total biomass. Elevated leaf starch content in maize leaves was engineered by making an RNAi construct against a gene in maize that appeared to be homologous to AtGWD. The RNAi construct was expressed using the constitutive ubiquitin promoter. Leaf starch content at the end of a night period in engineered maize plants was 20-fold higher than in untransformed plants with no impact on total plant biomass. We conclude that plants can be engineered to accumulate starch in the leaves with little impact on vegetative biomass.

The relationship between leaf area growth and biomass accumulation in Arabidopsis thaliana

Leaf area growth determines the light interception capacity of a crop and is often used as a surrogate for plant growth in high-throughput phenotyping systems. The relationship between leaf area growth and growth in terms of mass will depend on how carbon is partitioned among new leaf area, leaf mass, root mass, reproduction, and respiration. A model of leaf area growth in terms of photosynthetic rate and carbon partitioning to different plant organs was developed and tested with Arabidopsis thaliana L. Heynh. ecotype Columbia (Col-0) and a mutant line, gigantea-2 (gi-2), which develops very large rosettes. Data obtained from growth analysis and gas exchange measurements was used to train a genetic programming algorithm to parameterize and test the above model. The relationship between leaf area and plant biomass was found to be non-linear and variable depending on carbon partitioning. The model output was sensitive to the rate of photosynthesis but more sensitive to the amount of carbon partitioned to growing thicker leaves. The large rosette size of gi-2 relative to that of Col-0 resulted from relatively small differences in partitioning to new leaf area vs. leaf thickness.

Chloroplast to Leaf

Photosynthesis is highly responsive to environmental changes. Even so, fundamental modifications of the photosynthetic processes during the evolution of plant life have been relatively limited in comparison to the enormous variations in climatic conditions that have occurred during this period. This is evidence of the remarkable plasticity within the photosynthetic process allowing plants to adapt to different life conditions and environmental changes. Currently, the earth is experiencing a series of rapidly developing environmental changes, often collectively referred to as global climate change, and predominantly caused by anthropogenic activities. These may positively or negatively affect photosynthesis as well as trigger yet further adaptive responses. The rapidity of these environmental changes is exemplified by the increases in CO2 and CH4 over the last century (Fig. 9.1). It is essential to know if plants have the capacity to adapt to such rapid environmental changes and thereby mitigate the impact on photosynthetic productivity of crops as well as natural plant communities. The effects of global change on photosynthesis can be extremely complex, reflecting not only natural plant biodiversity but also microclimate diversity. As an example, rising atmospheric CO2 rise per se can enhance photosynthetic carbon fixation and incremental plant growth, but this may be counteracted by the associated temperature increase. Higher temperatures might exceed the optimal temperature for photosynthesis as well as enhance photorespiration, an energetically wasteful process that competes with photosynthetic carbon fixation.

Cellular and organ level localization of maltose in maltose-excess Arabidopsis mutants

Maltose is the predominant form of carbon exported from the chloroplast at night. Plants that lack either the chloroplast maltose transporter or disproportionating enzyme 2 (DPE2, EC 2.4.1.25) have excess maltose in leaves. We confirmed that DPE2 is not associated with the chloroplast in Arabidopsis thaliana. Using non-aqueous fractionation methods, we found that essentially all the maltose in mex1-1 leaves is located inside chloroplasts but only 40% of maltose in dpe2-1 leaves is located inside chloroplasts. We found that maltose exists in a significant amount in the exudates collected from maltose-accumulating dpe2-1 Arabidopsis petioles. However, the amount of maltose in the exudates from mex1-1 petioles was not significantly different from that in wild-type phloem exudates. We found twice as much maltose in the roots of dpe2-1 plants relative to wild type but the maltose level in the roots of mex1-1 plants was not higher than wild type. We conclude that maltose accumulated in the cytosol of leaves can be carried from the shoots to the roots and that maltose accumulated in the chloroplast of mex1-1 leaves is not mobilized. By measuring the transcript levels and enzymatic activities, we show that maltose-metabolizing enzymes are active in wild-type roots. The amount of maltose moved from the shoots to the roots increased in dpe2-1 plants. The roots of dpe2-1 plants must have the capacity to metabolize the excess maltose.