Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Floral transition and nitric oxide emission during flower development in Arabidopsis thaliana is affected in nitrate reductase-deficient plants

The nitrate reductase (NR)-defective double mutant of Arabidopsis thaliana (nia1 nia2) has previously been shown to present a low endogenous content of NO in its leaves compared with the wild-type plants. In the present study, we analyzed the effect of NR mutation on floral induction and development of A. thaliana, as NO was recently described as one of the signals involved in the flowering process. The NO fluorescent probes diaminofluorescein-2 diacetate (DAF-2DA) and 1,2-diaminoanthraquinone (1,2-DAA) were used to localize NO production in situ by fluorescence microscopy in the floral structures of A. thaliana during floral development. Data were validated by incubating the intact tissues with DAF-2 and quantifying the DAF-2 triazole by fluorescence spectrometry. The results showed that NO is synthesized in specific cells and tissues in the floral structure and its production increases with floral development until anthesis. In the gynoecium, NO synthesis occurs only in differentiated stigmatic papillae of the floral bud, and, in the stamen, only anthers that are producing pollen grains synthesize NO. Sepals and petals do not show NO production. NR-deficient plants emitted less NO, although they showed the same pattern of NO emission in their floral organs. This mutant blossomed precociously when compared with wild-type plants, as measured by the increased caulinar/rosette leaf number and the decrease in the number of days to bolting and anthesis, and this phenotype seems to result from the markedly reduced NO levels in roots and leaves during vegetative growth. Overall, the results reveal a role for NR in the flowering process.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Effect of decreased irradiance on N and C metabolism in leaves and roots of maize

The effects of decreased irradiance on fresh and dry weight, root respiration, levels of carbohydrates and N-compounds, and extractable activities of enzymes involved in C and N metabolism were evaluated in maize ( Zea mays L. cv. Plauto) seedlings during the 7 days following transfer from 450 to 200 μmol m⁻² s⁻¹ PAR. The fresh weight of roots and stems, the initiation of new leaves, root respiration rate, and the accumulation of dry matter, soluble sugars, starch, malate and amino acids in both leaves and roots were strongly reduced at low irradiance. In contrast, the level of nitrate was increased in leaves and only marginally affected in roots. Leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity started to decrease after 24–34 h, whereas ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity and chlorophyll content were unaffected or only slightly reduced. In both leaves and roots, the adjustment of N metabolism to low irradiance occurred through a relatively rapid (30% after 10 h) and large (60% after 3 days) decrease of nitrate reductase (NR; EC 1.6.6.1) activity, followed by slower and smaller changes in the activity of nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). We suggest that the preferential decrease of NR activity relative to other N-assimilating enzymes may be important for preventing the accumulation of toxic N-compounds like ammonia in both leaf and root tissues.     

Enhanced photosynthetic capacity increases nitrogen metabolism through the coordinated regulation of carbon and nitrogen assimilation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Plant growth and productivity depend on interactions between the metabolism of carbon and nitrogen. The sensing ability of internal carbon and nitrogen metabolites (the C/N balance) enables plants to regulate metabolism and development. In order to investigate the effects of an enhanced photosynthetic capacity on the metabolism of carbon and nitrogen in photosynthetically active tissus (source leaves), we herein generated transgenic Arabidopsis thaliana plants (ApFS) that expressed cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in their chloroplasts. The phenotype of ApFS plants was indistinguishable from that of wild-type plants at the immature stage. However, as plants matured, the growth of ApFS plants was superior to that of wild-type plants. Starch levels were higher in ApFS plants than in wild-type plants at 2 and 5 weeks. Sucrose levels were also higher in ApFS plants than in wild-type plants, but only at 5 weeks. On the other hand, the contents of various free amino acids were lower in ApFS plants than in wild-type plants at 2 weeks, but were similar at 5 weeks. The total C/N ratio was the same in ApFS plants and wild-type plants, whereas nitrite levels increased in parallel with elevations in nitrate reductase activity at 5 weeks in ApFS plants. These results suggest that increases in the contents of photosynthetic intermediates at the early growth stage caused a temporary imbalance in the free-C/free-N ratio and, thus, the feedback inhibition of the expression of genes involved in the Calvin cycle and induction of the expression of those involved in nitrogen metabolism due to supply deficient free amino acids for maintenance of the C/N balance in source leaves of ApFS plants.     

Magnesium deficiency results in accumulation of carbohydrates and amino acids in source and sink leaves of spinach

Accumulation of assimilates in source leaves of magnesium‐deficient plants is a well‐known feature. We had wished to determine whether metabolite concentrations in sink leaves and roots are affected by magnesium nutrition. Eight‐week‐old spinach plants were supplied either with a complete nutrient solution (control plants) or with one lacking Mg (deficient plants) for 12 days. Shoot and root fresh weights and dry weights were lower in deficient than in control plants. Mg concentrations in deficient plants were 11% of controls in source leaves, 12% in sink leaves and 26% in roots, respectively. As compared with controls, increases were found in starch and amino acids in source leaves and in sucrose, hexoses, starch and amino acids in sink leaves, whereas they were only slightly enhanced in roots. In phloem sap of magnesium‐deficient and control plants no differences in sucrose and amino acid concentrations were found. To prove that sink leaves were the importing organs they were shaded, which did not alter the response to magnesium deficiency as compared with that without shading. Since in the shaded sink leaves the photosynthetic production of metabolites could be excluded, those carbohydrates and amino acids that accumulated in the sink leaves of the deficient plants must have been imported from the source leaves. It is concluded that in magnesium‐deficient spinach plants the growth of sink leaves and roots was not limited by carbohydrate or amino acid supply. It is proposed that the accumulation of assimilates in the source leaves of Mg‐deficient plants results from a lack of utilization of assimilates in the sink leaves.     

The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar.     

Floral determination in axillary buds of Nicotiana silvestris

At anthesis of the terminal flower the developmental fates of axillary buds of the long-day plant Nicotiana silvestris were assessed in situ and in isolation. The in situ developmental fate was assessed by decapitating the plant above the bud in question and letting the bud mature. The developmental fate of isolated buds was assessed by removing the bud from the main axis, rooting it, and letting it mature. The number of nodes below the terminal flower of the mature shoot was indicative of the developmental fate of the bud. Terminal meristems of rooted axillary buds exhibited two patterns of development: (1) Their developmental fate was the same as that of in situ buds at the same node or (2) their developmental fate was the same as that of seed-derived plants. For example, terminal meristems of rooted buds from the fourth node below the inflorescence produced either 15 to 19 nodes or 36 to 40 nodes. In situ fourth buds produced 12 to 14 nodes while seed-derived plants produced 33 to 39 nodes. Terminal meristems of rooted axillary buds that exhibited the same developmental fate as that of in situ buds were determined for floral development. Although determined buds produced a terminal flower, all but one had abnormal inflorescences. That is, in the place of floral branches determined buds produced vegetative branches. Four buds that were not determined for floral development had their shoot tips rooted each time the plant bolted. Only when the plants were allowed to grow without being rerooted did they flower. These results indicate that roots may prevent and/or destabilize floral determination in N. silvestris.     

Effect of Zn deficiency on net photosynthetic rate, 14C partitioning,and oil accumulation in leaves of peppermint

Changes in growth, CO₂ exchange rate, and distribution of photosynthetically fixed ¹⁴CO2 into the primary photosynthetic metabolic pool (sugars, amino acids and organic acids) and essential oil accumulation were determined in leaves (leaf positions 1-6 from apex) of developing peppermint grown in a solution culture at Zn concentrations of 0 and 0.05 g m-3. There was a significant decrease in ¹⁴C incorporation in total, ethanol-soluble and ethanol-insoluble fractions in Zn deficient plants at all leaf positions. 14C incorporated in essential oil and in sugars were significantly higher in leaf pairs 1 to 3 than in leaf pairs 4 to 6. ¹⁴C incorporation into amino acids and organic acids was higher in all leaf pairs in Zn deficient plants. Statistical analysis showed a positive significant association between Zn content of leaf and ¹⁴C incorporation into ethanol-soluble fraction and sugars and a negative correlation with ¹⁴C incorporation into amino acids and organic acids. Hence the content of sugars in leaves significantly influences essential oil accumulation under Zn stress. This revised version was published online in September 2006 with corrections to the Cover Date.     

The Influence of Nitrogen Nutrition on the Carbohydrate and Nitrogen Status of Emergent Macrophyte Acorus calamus L.

Carbon and nitrogen balance in Acorus calamus, a wetland species colonising littoral zones with a high trophic status, was studied under experimental conditions using water or sand culture with a defined composition of the nutrient solution. Influence of graded level of N (1.86, 7.5 and 18.6 mM) and/or forms of N (NH₄⁺ versus NO ₃^–) on the content of non-structural carbohydrates, free amino acids, total C, and total N was studied in Acorus rhizomes and roots to find possible connection with a reduced growth of Acorus plants under high N and NH₄⁺–N nutrition described in our previous study [Vojtíšková et al., 2004. Hydrobiologia 518: 9–22]. High N availability and pure NH₄⁺–N nutrition affected the C/N balance of rhizome and root systems of Acorus in a similar way. NH₄⁺–N was the only form of N elevated under the high N treatment. The major proportion of the total non-structural carbohydrates (TNC) was starch (91–93% and 51–64% in rhizomes and roots, respectively). The content of starch was significantly and and negatively affected by high N availability (P = 0.001), as well as by NH₄⁺–N nutrition (P=0.001). Amounts of simple soluble carbohydrates (sucrose, glucose, and fructose) were negligible in comparison to starch in rhizomes and branched roots (up to 5% of TNC), while roots without developed lateral roots (unbranched) contained up to 33% of TNC in the form of simple soluble sugars. Moreover, high hexoses/sucrose ratio, low starch/soluble sugars ratio, high content of N, and low C/N ratio support the notion that unbranched roots are metabolically active young roots with tissue differentiation in progress. A high content of free amino acids, typically with dominance of N-rich amino acids (Arg-46%, Gln-8%, Asn-7%), was found simultaneously with a low carbohydrate content under high N supply, which indicates that NH₄⁺ received is effectively incorporated into the organic form by this species. Since the decrease in carbohydrate content was not accompanied by luxurious growth, other possible carbon consuming processes were discussed in relation to NH₄⁺ nutrition. More dramatic changes in total N than C were found under high N availability resulting a shift in C/N ratio in favour of N. Although the shift towards N metabolism was obvious, no serious carbohydrate depletion occurred, which could explain the reduced growth of Acorus plants under high N and sole NH₄⁺–N nutrition described previously.     

Intrinsic morphology and spatial distribution of non-structural carbohydrates contribute to drought resistance of two mulberry cultivars

• Drought is one of the most adverse environmental stresses limiting plant growth and productivity. However, the underlying mechanisms regarding metabolism of non-structural carbohydrates (NSC) in source and sink organs are still not fully elucidated in woody trees.
• Saplings of mulberry cv Zhongshen1 and Wubu were subjected to a 15-day progressive drought stress. NSC levels and gene expression involved in NSC metabolism were investigated in roots and leaves. Growth performance and photosynthesis, leaf stomatal morphology, and other physiological parameters were also analysed.
• Under well-watered conditions, Wubu had a higher R/S, with higher NSC in leaves than in roots; Zhongshen1 had a lower R/S with higher NSC in roots than leaves. Under drought stress, Zhongshen1 showed decreased productivity and increased proline, abscisic acid, ROS content and activity of antioxidant enzymes, while Wubu sustained comparable productivity and photosynthesis. Interestingly, drought resulted in decreased starch and slightly increased soluble sugars in leaves of Wubu, accompanied by notable downregulation of starch-synthesizing genes and upregulation of starch-degrading genes. Similar patterns in NSC levels and relevant gene expression were also observed in roots of Zhongshen1. Concurrently, soluble sugars decreased and starch was unchanged in roots of Wubu and leaves of Zhongshen1. However, gene expression of starch metabolism in roots of Wubu was unaltered, but in leaves of Zhongshen1 starch metabolism was more activated.
• These findings revealed that intrinsic R/S and spatial distribution of NSC in roots and leaves concomitantly contribute to drought resistance in mulberry.