A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.     

Dynamic protein composition of Arabidopsis thaliana cytosolic ribosomes in response to sucrose feeding as revealed by label free MSE proteomics

Cytosolic ribosomes are among the largest multisubunit cellular complexes. Arabidopsis thaliana ribosomes consist of 79 different ribosomal proteins (r-proteins) that each are encoded by two to six (paralogous) genes. It is unknown whether the paralogs are incorporated into the ribosome and whether the relative incorporation of r-protein paralogs varies in response to environmental cues. Immunopurified ribosomes were isolated from A. thaliana rosette leaves fed with sucrose. Trypsin digested samples were analyzed by qTOF-LC-MS using both MS(E) and classical MS/MS. Peptide features obtained by using these two methods were identified using MASCOT and Proteinlynx Global Server searching the theoretical sequences of A. thaliana proteins. The A. thaliana genome encodes 237 r-proteins and 69% of these were identified with proteotypic peptides for most of the identified proteins. These r-proteins were identified with average protein sequence coverage of 32% observed by MS(E) . Interestingly, the analysis shows that the abundance of r-protein paralogs in the ribosome changes in response to sucrose feeding. This is particularly evident for paralogous RPS3aA, RPS5A, RPL8B, and RACK1 proteins. These results show that protein synthesis in the A. thaliana cytosol involves a heterogeneous ribosomal population. The implications of these findings in the regulation of translation are discussed.     

Cytosolic proline is required for basal freezing tolerance in Arabidopsis

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze–thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2–1 (p5cs2–1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1–1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2–1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2–1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.     

Soluble starch synthase I: a major determinant for the synthesis of amylopectin in Arabidopsis thaliana leaves

A minimum of four soluble starch synthase families have been documented in all starch-storing green plants. These activities are involved in amylopectin synthesis and are extremely well conserved throughout the plant kingdom. Mutants or transgenic plants defective for SSII and SSIII isoforms have been previously shown to have a large and specific impact on the synthesis of amylopectin while the function of the SSI type of enzymes has remained elusive. We report here that Arabidopsis mutants, lacking a plastidial starch synthase isoform belonging to the SSI family, display a major and novel type of structural alteration within their amylopectin. Comparative analysis of beta-limit dextrins for both wild type and mutant amylopectins suggests a specific and crucial function of SSI during the synthesis of transient starch in Arabidopsis leaves. Considering our own characterization of SSI activity and the previously described kinetic properties of maize SSI, our results suggest that the function of SSI is mainly involved in the synthesis of small outer chains during amylopectin cluster synthesis.     

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana

The mutants irt1-1 and irt1-2 of Arabidopsis thaliana were identified among a collection of T-DNA-tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutations responsible interfere with expression of IRT1, a nuclear gene that encodes the metal ion transporter IRT1. In irt1 mutants, photosensitivity and chlorophyll fluorescence parameters, as well as abundance and composition of the photosynthetic apparatus, are significantly altered. Additional effects of the mutation under greenhouse conditions, including chlorosis and a drastic reduction in growth rate and fertility, are compatible with a deficiency in iron transport. Propagation of irt1 plants on media supplemented with additional quantities of iron salts restores almost all aspects of wild-type behaviour. The irt2-1 mutant, which carries an En insertion in the highly homologous IRT2 gene of Arabidopsis thaliana, was identified by reverse genetics and shows no symptoms of iron deficiency. This, together with the finding that irt1-1 can be complemented by 35S::IRT1 but not by 35S::IRT2, demonstrates that, although the products of the two genes are closely related, only AtIRT1 is required for iron homeostasis under physiological conditions.     

The plant-specific VIMAG domain of Glutamine Dumper1 is necessary for the function of the protein in Arabidopsis

The over-expression of the arabidopsis GLUTAMINE DUMPER1 gene (GDU1) leads to increased amino acid content and transport. In a screening for mutations suppressing this phenotype, a mutant was isolated. The mutation leads to a glycine to arginine substitution in one of the two conserved domains of the protein, the VIMAG domain. More detailed structure function relationship analyses showed that the presence of this domain and the membrane localisation are both necessary for the function of the GDU1 protein. These results shed light on the function of the GDU1 protein whose family is specific to plants.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Tissue layer specific regulation of leaf length and width in Arabidopsis as revealed by the cell autonomous action of ANGUSTIFOLIA

In vascular plants the shoot apical meristem consists of three tissue layers, L1, L2 and the L3, that are kept separate during organ formation and give rise to the epidermis (L1) and the subepidermal tissues (L2, L3). For proper organ development these different tissue layers must interact with each other, though their relative contributions are a matter of debate. Here we use ANGUSTIFOLIA (AN), which controls cell polarity and leaf shape, to study its morphogenetic function in the epidermis and the subepidermis of Arabidopsis thaliana. We show that ANGUSTIFOLIA expression in the subepidermis cannot rescue epidermal cell polarity defects, indicating a cell‐autonomous molecular function. We demonstrate that leaf width is only rescued by subepidermal AN expression, whereas leaf length is also rescued by epidermal expression. Strikingly, subepidermal rescue of leaf width is accompanied by increased cell number in the epidermis, indicating that AN can trigger cell divisions in a non‐autonomous manner.     

Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonization by necrotrophic pathogens

The ability of bacterial or fungal necrotrophs to produce enzymes capable of degrading pectin is often related to a successful initiation of the infective process. Pectin is synthesized in a highly methylesterified form and is subsequently de-esterified in muro by pectin methylesterase. De-esterification makes pectin more susceptible to the degradation by pectic enzymes such as endopolygalacturonases (endoPG) and pectate lyases secreted by necrotrophic pathogens during the first stages of infection. We show that, upon infection, Pectobacterium carotovorum and Botrytis cinerea induce in Arabidopsis a rapid expression of AtPME3 that acts as a susceptibility factor and is required for the initial colonization of the host tissue.     

Aberrant temporal growth pattern and morphology of root and shoot caused by a defective circadian clock in Arabidopsis thaliana

Circadian clocks synchronized with the environment allow plants to anticipate recurring daily changes and give a fitness advantage. Here, we mapped the dynamic growth phenotype of leaves and roots in two lines of Arabidopsis thaliana with a disrupted circadian clock: the CCA1 over‐expressing line (CCA1ox) and the prr9 prr7 prr5 (prr975) mutant. We demonstrate leaf growth defects due to a disrupted circadian clock over a 24 h time scale. Both lines showed enhanced leaf growth compared with the wild‐type during the diurnal period, suggesting increased partitioning of photosynthates for leaf growth. Nocturnal leaf growth was reduced and growth inhibition occurred by dawn, which may be explained by ineffective starch degradation in the leaves of the mutants. However, this growth inhibition was not caused by starch exhaustion. Overall, these results are consistent with the notion that the defective clock affects carbon and energy allocation, thereby reducing growth capacity during the night. Furthermore, rosette morphology and size as well as root architecture were strikingly altered by the defective clock control. Separate analysis of the primary root and lateral roots revealed strong suppression of lateral root formation in both CCA1ox and prr975, accompanied by unusual changes in lateral root growth direction under light–dark cycles and increased lateral extension of the root system. We conclude that growth of the whole plant is severely affected by improper clock regulation in A. thaliana, resulting not only in altered timing and capacity for growth but also aberrant development of shoot and root architecture.     

MOL1 is required for cambium homeostasis in Arabidopsis

Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine‐rich repeat receptor‐like kinase (LRR‐RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR‐RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR‐RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis‐expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress‐related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.     

N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.