The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana.

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the "phospho-dawn." Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants.

Levanase from Bacillus subtilis hydrolyses ß-2,6 fructosyl bonds in bacterial levans and in grass fructans.

A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3 U mg(-1) using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (ß-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.

The interplay between P uptake pathways in mycorrhizal peas: a combined physiological and gene-silencing approach.

Arbuscular mycorrhizal fungi (AMF) have a key role in plant phosphate (Pi) uptake by their efficient capture of soil phosphorus (P) that is transferred to the plant via Pi transporters in the root cortical cells. The activity of this mycorrhizal Pi uptake pathway is often associated with downregulation of Pi transporter genes in the direct Pi uptake pathway. As the total Pi taken up by the plant is determined by the combined activity of mycorrhizal and direct pathways, it is important to understand the interplay between these, in particular the actual activity of the pathways. To study this interplay we modulated the delivery of Pi via the mycorrhizal pathway in Pisum sativum by two means: (1) Partial downregulation by virus-induced gene silencing of PsPT4, a putative Pi transporter gene in the mycorrhizal pathway. This resulted in decreased fungal development in roots and soil and led to reduced plant Pi uptake. (2) Changing the percentage of AMF-colonized root length by using non-, half-mycorrhizal or full-mycorrhizal split-root systems. The combination of split roots, use of ³²P and ³³P isotopes and partial silencing of PsPT4 enabled us to show that the expression of PsPT1, a putative Pi transporter gene in the direct pathway, was negatively correlated with increasing mycorrhizal uptake capacity of the plant, both locally and systemically. However, transcript changes in PsPT1 were not translated into corresponding, systemic changes in actual direct Pi uptake. Our results suggest that AMF have a limited long-distance impact on the direct pathway.

The Arabidopsis transcription factor PHR1 is essential for adaptation to high light and retaining functional photosynthesis during phosphate starvation.

The transcription factor PHR1 (PHOSPHATE STARVATION RESPONSE 1; encoded by gene At4g28610) is central for adaptation to phosphate deficiency in Arabidopsis (Arabidopsis thaliana). A rapid turnover of phosphate pools in the leaves is essential for energy transfer and metabolism within photosynthesis, and consequently, we hypothesized that PHR1 is needed for adaptation to high-light stress during P deficiency. We analyzed three Arabidopsis plant lines: wild-type, a transgenic PHR1 overexpressor line and a knockout mutant, phr1. The plants were grown under phosphate-limiting and sufficient conditions and exposed to different light conditions. Photosynthetic activity and light stress of the leaves were characterized by analyzing accumulation of carbohydrates, chlorophyll fluorescence, immunoblot detection of photosystem subunits and anthocyanin accumulation. Compared to the wild-type and the overexpressor line, the phr1 mutant has decreased levels of phosphate, anthocyanins and carbohydrates during combined P deficiency and light stress. The stressed mutant also has strongly decreased photosystem II (PSII) quantum efficiency, and shows degradation of the core units of PSII demonstrating extensive irreversible photodamage. We conclude that PHR1 is needed for the metabolic balance, for retaining P(i) levels and for inducing anthocyanin production, and during P deficiency PHR1 is vital for adaptations to avoid permanent damage to photosystems during high-light conditions.

Overexpression of the MYB-related transcription factor GCC7 in Arabidopsis thaliana leads to increased levels of Pi and changed P-dependent gene regulation.

A proper concentration and turnover of inorganic phosphate (Pi) is essential to maintain cellular processes. Consequently, plants have mechanisms to control Pi homeostasis and to alleviate Pi limitation. The MYB-related transcription factor, PHR1, is important for gene induction during Pi starvation. PHR1 belongs to a family, characterised by the presence of a GARP- and a coiled coil domain. We propose that this family, with 15 members in Arabidopsis thaliana (L.) Heynh., be termed the GCC-family. In this study, transgenic plants overexpressing one member, GCC7, and a T-DNA knockout mutant, gcc7, are characterised. We find overexpressor plants to accumulate more Pi in shoots, irrespective of the Pi supply. Therefore, GCC7 was characterised in relation to Pi starvation. We conclude that GCC7 is not strictly required for a P-starvation response since the gcc7 mutant responds to Pi limitation. However, overexpression of GCC7 strongly interferes with the P-starvation response, abolishing induction of several P-responsive genes including AT4, IPS1 and several P-transporter genes, whereas GCC7 does not directly interfere with the PHR1 (GCC1) dependent regulation of miR399d. Thus GCC7 influences P-accumulation and P-dependent gene regulation, but GCC7 has a function entirely different from PHR1.

Effects of an onion by-product on bioactivity and safety markers in healthy rats.

Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, gamma-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota.

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase.

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) in regulation of carbon metabolism was investigated in transgenic potato plants (Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P(2) was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic (14)CO(2) metabolism, showed that in plant lines with reduced Fru-2,6-P(2) level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P(2) only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-(14)C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-(14)C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P(2) is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P(2) on tuber metabolism was limited.

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.

Starch phosphorylation: a new front line in starch research.

Starch is the primary energy reserve in higher plants and is, after cellulose, the second most abundant carbohydrate in the biosphere. It is also the most important energy source in the human diet and, being a biodegradable polymer with well-defined chemical properties, has an enormous potential as a versatile renewable resource. The only naturally occurring covalent modification of starch is phosphorylation. Starch phosphate esters were discovered a century ago but were long regarded as a curiosity, receiving little attention. Indeed, the mechanism for starch phosphorylation remained completely unknown until recently. The starch-phosphorylating enzyme is an alpha-glucan water dikinase. It is now known that starch phosphorylation plays a central role in starch metabolism.

Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach.

The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998) Plant Cell 10, 1699-1711). A new approach to the study of starch biosynthesis was developed based on "very short pulse" labeling of leaf starch through photosynthetic fixation of (14)CO(2). This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for the dbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.