Long-wavelength absorbing and fluorescent chameleon labels for proteins, peptides, and amines

Long-wavelength absorbing labels that change their color and fluorescence upon conjugation to proteins and other biomolecules provide two critical advantages over the wealth of conventional amine-reactive labels. At first, the progress of the labeling reaction can be monitored continuously either visually or by spectrometry without prior purification. Then, the labeled biomolecule can be investigated with red or near-infrared light, which minimizes background interference in biological samples. These unique characteristics are met by a group of long-wavelength absorbing cyanine dyes carrying a reactive chloro substituent for nucleophilic substitution with primary amines, which is accompanied by a color change from green to blue. In addition to this so-called chameleon effect, the dyes display an increase in fluorescence during the labeling reaction. Despite their structural similarity, the reactivity of the dyes differs strongly. The fastest labeling kinetics is observed with dye S 0378 as its five-membered ring affords a stabilizing effect on the intermediate carbocation during an S(N)1-type of nucleophilic substitution. The reaction mechanism of the amine-reactive cyanine dyes provides a blueprint for the design of future long-wavelength absorbing chameleon dyes.     

Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

Tolerance of crop plants to oxygen deficiency stress: fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia

The study investigates the reactions of rice, wheat and maize to anoxia (plants without access to oxygen) and hypoxia (roots with very limited access to oxygen). We studied the adaptations of these intact crop plants because they are known to differ widely in their tolerance to oxygen deficiency. In hypoxia, there was an accumulation of sugars, especially in wheat and maize, although both flood-sensitive species significantly increased the activities of fermentative and glycolytic enzymes, clearly more than in rice. In rice, avoiding an oxygen limitation due to the effective aeration system (30% of root cross-sectional area) may have accounted for only a minor metabolic reaction to hypoxia. In anoxia, maize and wheat quickly lost viability and nearly all photosynthetic capacity, while most rice leaves stayed turgid and green, losing only 50% of the photosynthetic capacity. A strong metabolic arrest under anoxia was obvious for the sucrolytic, glycolytic and fermentative enzymes in all tested species, but was most pronounced in rice. Of the 14 enzymes studied, rice showed the lowest activity increase in hypoxia for 11 enzymes, and the strongest activity decrease in anoxia for 8 enzymes. However, rice was able even under anoxia to keep a 1/4 of the ATP level of the aerated control, while it was at the detection limit in maize and wheat. It appears that in anoxic rice, the switch to metabolic dormancy and maintenance of basic shoot meristems diminishes the needs for energy and substrate. Additionally, rice already has lower sugar demand under hypoxia, and sugar supply appears to be sustained under anoxia by a functioning anaerobic amylase and by the photosynthetically active shoot.     

Localization of sucrose synthase in wheat roots: increased in situ activity of sucrose synthase correlates with cell wall thickening by cellulose deposition under hypoxia

Sucrose synthase (SuSy; EC 2.4.1.13) plays a prominent role in O(2) deficiency and functions at a branch point, partitioning sucrose between cell wall biosynthesis and glycolysis. The cleavage of sucrose by SuSy was localized in wheat ( Triticum aestivum L. cv. Alcedo) roots subjected to 4 days of hypoxia. Increased SuSy activity was observed by in situ activity staining in the tip region and in the stele of root axes. The pattern of cellulose deposition correlated with regions of high SuSy activity. Cellulose accounted for more than 30% of root dry weight and the cellulose content increased substantially under hypoxia. The strongest accumulation of cellulose occurred in the base and mid-regions of the roots where the content rose to 163% and 182% of controls, respectively. In the root axis, cellulose deposition occurred in the endodermis and walls of pith cells. In root tips, cellulose was primarily deposited in developing xylem and phloem. The marker enzyme for O(2) shortage, pyruvate decarboxylase (EC 4.1.1.17), exhibited a 14-fold increase in the root apex, whereas in basal root tissues, which contained more aerenchyma, pyruvate decarboxylase activity was only doubled. The root apex also contained the highest concentration of sucrose and hexoses. The elevated sugar content in all root zones was partially used to synthesize cellulose for secondary wall thickening.     

Characterisation of the ATP-dependent phosphofructokinase gene family from Arabidopsis thaliana

Plants possess two different types of phosphofructokinases, an ATP-dependent (PFK) and a pyrophosphate-dependent form (PFP). While plant PFPs have been investigated in detail, cDNA clones coding for PFK have not been identified in Arabidopsis thaliana. Searching the A. thaliana genome revealed 11 putative members of a phosphofructokinase gene family. Among those, four sequences showed high homology to the alpha- or beta-subunits of plant PFPs. Seven cDNAs resulted in elevated PFK, but not PFP activity after transient expression in tobacco leaves suggesting that they encode Arabidopsis PFKs. RT-PCR revealed different tissue-specific expression of the individual forms. Furthermore, analysis of GFP fusion proteins indicated their presence in different sub-cellular compartments.