Iwona Ciereszko

Phosphate status affects the gene expression, protein content and enzymatic activity of UDP-glucose pyrophosphorylase in wild-type and pho mutants of Arabidopsis.

Abstract. The effects of inorganic phosphate (Pi) deficiency on the expression of the UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), involved in sucrose synthesis/metabolism, and on carbohydrate status were investigated in different tissues of Arabidopsis thaliana (L.) Heynh. For leaves, a decrease in internal Pi status caused by growth of plants on a medium lacking Pi (−P conditions) led to an increase in the overall content of glucose and starch, but had little effect on sucrose content. The Pi deficiency also led to an increased carbohydrate content in stems/flowers, but not in roots. The expression of Ugp was upregulated in both leaves and roots, but not in stems/flowers. The effects of Pi status on Ugp expression were confirmed using leaves of both pho1-2 and pho2-1 mutants of Arabidopsis (Pi-deficient and Pi-accumulating, respectively) and by feeding the leaves with d-mannose, which acts as a sink for Pi. The Pi-status-dependent changes in Ugp expression followed the same patterns as those of ApS, a gene encoding the small subunit of ADP-glucose pyrophosphorylase, a key enzyme of starch synthesis. The changes in Ugp mRNA levels, depending on internal Pi status, were generally correlated with changes in UGPase protein content and enzymatic activity. This was demonstrated both for wild-type plants grown under Pi-deficiency and for Pi mutants. The data suggest that, under Pi-deficiency, UGPase represents a transcriptionally regulated step in sucrose synthesis/metabolism, involved in homeostatic mechanisms readjusting the nutritional status of a plant under Pi-stress conditions.

Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme producing UDP-glucose, which is involved in an array of metabolic pathways concerned with, among other functions, the synthesis of sucrose and cellulose. An Arabidopsis thaliana UGPase-encoding gene, Ugp, was profoundly up-regulated by feeding sucrose to the excised leaves and by an exposure of plants to low temperature (5 degrees C). The UGPase activity and its protein content also increased under conditions of sucrose feeding and exposure to cold. The sucrose effect on Ugp was apparently specific and was mimicked by exposure of dark-adapted leaves to light. Drought and O2 deficiency had some down-regulating effects on expression of Ugp. The sugar-signalling pathway for Ugp regulation was independent of hexokinase, as was found by using transgenic plants with increased and decreased expression of the corresponding gene. Subjecting mutants deficient in abscisic acid (ABA) to cold stress conditions had no effect on Ugp expression profiles. Okadaic acid was a powerful inhibitor of Ugp expression, whereas it up-regulated the gene encoding sucrose synthase (Sus1), indicating distinct transduction pathways in transmitting the sugar signal for the two genes in A. thaliana. We suggest that Ugp gene expression is mediated via a hexokinase-independent and ABA-insensitive pathway that involves an okadaic acid-responsive protein phosphatase. The data point towards Ugp as a possible regulatory entity that is closely involved in the homoeostatic readjustment of plant responses to environmental signals.

Sucrose Metabolism in Leaves and Roots of Bean (Phaseolus vulgaris L.) during Phosphate Deficiency

Summary The influence of phosphate deficiency on the accumulation and metabolism of sugars in the source and sink leaves and roots of bean ( Phaseolus vulgaris L.) was studied after 16 days of plant culture. Glucose, sucrose and starch contents increased in all tissues of phosphate-deficient plants as compared with control plants. Phosphate deficiency increased activities of enzymes involved in sucrose synthesis in the leaves and root; sucrose phosphate synthase (EC 2.4.1.14) activity increased about twice, while sucrose synthase (EC 2.4.1.13) activity increased about 3-fold in the mature leaves and 50% and 90% in the young leaves and roots, respectively. Phosphate deficiency also increased the activities of enzymes hydrolyzing sucrose in the leaves and roots; neutral invertase (EC 3.2.1.26) activity increased twice in the source leaves and about 50% in sink leaves and roots. Acid invertases (EC 3.2.1.26) were divided into soluble and insoluble forms; insoluble invertases activities were several times lower than those of soluble invertases, both in the leaves and roots. Pi starvation increased mainly the activities of insoluble forms of acid invertases in all tissues in bean plants.

Effects of phosphate deficiency and sugars on expression of rab18 in Arabidopsis : hexokinase-dependent and okadaic acid-sensitive transduction of the sugar signal

The lack of phosphorus in the nutrient medium increased the expression of rab18, an abscisic acid (ABA)-responsive gene, in leaves of Arabidopsis thaliana. The expression of this gene was also upregulated after feeding the excised leaves with D-mannose and sucrose for both wild-type (wt) and aba1 (ABA-deficient) mutant plants. For aba1 mutants, both the phosphate deficiency and sugar effects on rab18 were weaker than in wt plants, suggesting possible involvement of both ABA-dependent and ABA-independent components in signalling. Transgenic Arabidopsis plants with increased hexokinase (HXK) expression had a much higher sucrose-dependent level of rab18 mRNA, implying the HXK involvement in sensing/transmitting the sugar signal. Sucrose-related induction of rab18 was completely inhibited by okadaic acid (OKA), suggesting the involvement of specific protein phosphatase(s) in transduction of the sugar signal. The results suggest that rab18 is regulated via interaction of a plethora of signals, including ABA, sugar and phosphate deficiency, and that the sugar effect is transmitted via a HXK-pathway, involving OKA-sensitive component(s). The findings prompt caution in linking the expression of rab18 solely to ABA signalling.

Sucrose hydrolysis in bean roots (Phaseolus vulgaris L.) under phosphate deficiency

Abstract Bean plants (Phaseolus vulgaris L.) were cultured for 16 days on phosphate-sufficient (+P) and phosphate deficient (−P) nutrient medium. Phosphate deficiency in the roots increased glucose and sucrose content more in the root tips than in the mature parts of roots and altered the sucrose/glucose ratio. Changes in the sucrose/glucose ratio in roots of −P plants compared with the control plants indicated differences in sucrose metabolism in these roots. Phosphate deficiency increased activities of cytosolic enzymes hydrolysing sucrose in the root tips; sucrose synthase (EC 2.4.1.13) activity increased more than twice, neutral invertase (EC 3.2.1.26) activity about 30%. However the activities of acid invertases (EC 3.2.1.26) in the roots of +P, −P plants were very similar. It is concluded that sucrose synthase have an increased role in sucrose hydrolysis in the growing parts of root under phosphate deficiency conditions. Intensification of sucrose hydrolysis in the cytoplasm may indicate that sucrose unloading from the phloem in bean roots occurs by symplastic route.

GROWTH AND METABOLISM OF CUCUMBER IN PHOSPHATE-DEFICIENT CONDITIONS

The influence of phosphate deficiency on the growth, phosphatase activity, CO2 exchange, and sugar accumulation in the leaves and roots of cucumber (Cucumis sativus L.) was studied during 4 weeks of plant culture. The growth on nutrient medium without phosphate decreased Pi (inorganic orthophosphate) content in leaves and roots of plants to about 10% of control. Phosphate starvation significantly decreased growth of shoots and mass of roots, whereas root elongation growth was enhanced (diameter of root decreased). Intensity of root elongation was most pronounced at the beginning of culture on P-deficient medium and remained similar even after transfer to full nutrient medium. Phosphate-deficient cucumber plants had higher acid phosphatase activity both in extracts from roots and leaves and in root exudates when compared to samples from phosphate-sufficient plants. Photosynthesis rate was not affected at the beginning of culture but decreased significantly in leaves of 36-day-old −P (phosphate-deficient) plants. Reducing sugars and sucrose content increased in all tissues of phosphate-deficient plants as compared to control plants but only at a moderate stage of phosphate deficiency (24-day-old plants). Carbohydrate content declined during the subsequent week of growth, possibly because of inhibitory effects of advanced phosphate deficiency on photosynthesis and sugar production.

Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis

The effects of inorganic phosphate (Pi) deficiency on expression of genes encoding ADP-glucose pyrophosphorylase small and large subunits (ApS and ApL1, ApL2, ApL3 genes), UDP-glucose pyrophosphorylase (Ugp gene), sucrose synthase (Sus1), soluble and insoluble acid invertases (Inv and Invcw) and hexokinase (Hxk1 gene), all involved in carbohydrate metabolism, were investigated in Arabidopsis thaliana (L.) Heynh. We used soil-grown pho mutants affected in Pi status, as well as wild-type (wt) plants grown under Pi deficiency conditions in liquid medium, and leaves of wt plants fed with D-mannose. Generally, ApS, ApL1, Ugp and Inv genes were upregulated, although to a varied degree, under conditions of Pi-stress. The applied conditions had differential effects on expression of other genes studied. For instance, Sus1 was downregulated in pho1 (Pi-deficient) mutant, but was unaffected in wt plants grown in liquid medium under P-deficiency. Mannose had distinct concentration-dependent effects on expression of genes under study, possibly reflecting a dual role of mannose as a sink for Pi and as glucose analog. Feeding Pi (at up to 200 mM) to the detached leaves of wt plants strongly affected the expression of ApL1, ApL2, Sus1 and Inv genes, possibly due to an osmotic effect exerted by Pi. The data suggest that ADP-glucose and UDP-glucose pyrophosphorylases (enzymes indirectly involved in Pi recycling) as well as invertases (sucrose hydrolysis) are transcriptionally regulated by Pi-deficiency, which may play a role in homeostatic mechanisms that acclimate the plant to the Pi-stress conditions.

PHOSPHATE DEFICIENCY AFFECTS ACID PHOSPHATASE ACTIVITY AND GROWTH OF TWO WHEAT VARIETIES

The effects of phosphate deficiency on the plant growth and acid phosphatase activity in two wheat cultivars (Triticum aestivum L. cv. ‘Bryza’ and ‘Opatka’) were studied. Pi content decreased significantly in the shoots and roots of all plants grown for one to three weeks in the nutrient medium without phosphate (−P). Phosphate starvation affected growth of both wheat cultivars in a similar way: significantly decreased shoot and root mass, especially after three weeks growth on −P medium, even up to 8–11% of control. However the ratio of root/shoot fresh weight of −P plants increased (2–3 times more than control). The shoot height of −P wheat plants decreased to about 50–60% of the control after two to three weeks of culture; less affected by Pi deficiency was root elongation—no significant changes were observed for both wheat cultivars during one to three weeks of culture. Phosphate deficiency increased the activity of extracellular and intracellular acid phosphatases in comparison to phosphate-sufficient plants. The increase of intracellular acid phosphatase activity in leaves and roots was generally more significant in ‘Bryza’ cultivar as compared to ‘Opatka’, especially after two to three weeks of growth on −P medium. The increase of root surface acid phosphatase activity was the highest for Pi-deficient wheat cv. ‘Bryza’, after three weeks of culture in the −P medium. Generally, the increase of activity of acid phosphatases secreted by the roots of −P wheat plants was higher than those of intracellular acid phosphatases. These results indicate an important role of acid phosphatases in the acclimation of both wheat cultivars to early Pi deficiency.

Compartmentation and Fluxes of Sugars in Roots of Phaseolus Vulgaris Under Phosphate Deficiency

The influence of phosphate deficiency on the sugar accumulation and sugar partitioning in the root cells of bean (Phaseolus vulgaris L.) was studied. Bean plants were cultured 17 - 19 d on a phosphate-sufficient and phosphate-deficient nutrient medium. Phosphate deficit in the growth medium resulted in increased sugar concentration for about 30 % in the apoplastic and cytoplasmic compartments as well as in the vacuoles of root cells. However, the distribution of sugars between apoplast and cytoplasm compartment and vacuole was not affected by decreased phosphate concentration. About 20 % of sugars were found in the apoplast and cytoplasm, about 80 % in the vacuole. Low phosphate concentration enhanced influx of exogenous 14C-sucrose into meristematic and elongation zones of root. The 14C-labelled sugar content in the root tips increased for about 60 % as compared to control plants. Phosphate deficiency increased also 14C-glucose uptake and content in the root tips. However, the amount of 14CO2 liberated during respiration of P-deficient roots (after feeding with uniformly labelled 14C-glucose) was lower than 14CO2 respired by control plants, thus a large part of accumulated sugars seems to be metabolically inactive.

In situ activities of hexokinase and fructokinase in relation to phosphorylation status of root meristem cells of Vicia faba during reactivation from sugar starvation.

The plant cell cycle is equipped with two principal control points: PCP1 in G1 and PCP2 in G2 phase. These checkpoints can arrest the cell cycle in response to carbohydrate starvation, while sugar presence can revive the replication and mitotic activity. The process of cell cycle revival is strongly repressed by okadaic acid (OA) or 6-dimethylaminopurine (6-DMAP), inhibitors of specific protein phosphatases 1 or 2A or kinases (cyclin-dependent kinases), respectively. In the present study, it was investigated whether inhibition of cell cycle revival is performed through interference of the above-mentioned inhibitors with the metabolic pathway of sucrose applied to the cells. Changes of hexokinase (HK) and fructokinase (FK) activities, key enzymes of hexose metabolism, were analyzed in Vicia faba root meristem cells arrested in G1 and G2 phase by carbohydrate starvation as well as in those recovered with glucose or sucrose in the presence of OA or 6-DMAP. It was shown that in the sugar-starved cells, the activity of both enzymes decreased significantly. During cell regeneration with carbohydrates, the activity of HK was induced more by sucrose than by glucose, while FK remained inactive after glucose addition. Moreover, in situ investigation of the activities of HK and FK showed that OA-induced and 6-DMAP-induced repression of the cell cycle revival is connected with the interference of these drugs in the metabolic pathway of sucrose. It was also indicated that stronger OA-induced and 6-DMAP-induced inhibition of the replication and mitosis revival, at the early stages of sucrose regeneration, was correlated with the stronger influence of these inhibitors on HK and FK activities.