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Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of H. vulgare (susceptible, mildew locus o (mlo) and Mildew locus a (Mla) resistant). Multispectral imaging of barley plants was daily conducted 0–8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with mlo and Mla based resistances 0–120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of Blumeria graminis f.sp. hordei (Bgh) conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The Mla resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening. [ABSTRACT FROM AUTHOR]

There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response. [ABSTRACT FROM PUBLISHER]

Expression of BZI-1ΔN, a dominant-negative form of the tobacco ( Nicotiana tabacum) basic leucine zipper (bZIP) transcription factor BZI-1 leads to severe defects in pollen development which coincides with reduced transcript abundance of the stamen specific invertase gene NIN88 and decreased extracellular invertase enzymatic activity. This finding suggests a function of BZI-1 in regulating carbohydrate supply of the developing pollen. BZI-1 heterodimerises with the bZIP factors BZI-2, BZI-3 and BZI-4 in vitro and in planta. Whereas BZI-1 exhibits only weak activation properties, BZI-1/BZI-2 heterodimers strongly activate transcription. Consistently, approaches leading to reduced levels of functional BZI-1 or BZI-2 both significantly interfere with pollen development, auxin responsiveness and carbohydrate partitioning. In situ hybridisation studies for BZI-1 and BZI-2 confirmed temporal and spatial overlapping expression patterns in tapetum and pollen supporting functional cooperation of these factors during pollen development. Plants over-expressing BZI-4 produce significantly reduced amounts of intact pollen and are also impaired in NIN88 transcription and enzymatic activity. BZI-4 homodimer efficiently binds to a G-box located in the NIN88 promoter but exhibits almost no transcriptional activation capacity. As BZI-4 does not actively repress transcription, we propose that its homodimer blocks G-box mediated transcription. In summary, these data support a regulatory model in which BZI-4 homodimers and BZI-1/BZI-2 heterodimers perform opposing functions as negative or positive transcriptional regulators during pollen development. [ABSTRACT FROM AUTHOR]

The expression pattern of the genes coding for vacuolar and extracellular invertase activity was analyzed in sugar beet ( Beta vulgaris) and compared with the expression of sucrose synthase in this important sucrose-storing crop. Northern blot analysis revealed that sucrose synthase is the predominant sucrose-cleaving enzyme in tap roots, whereas vacuolar invertase was specifically expressed in petioles. Extracellular invertase transcripts showed low abundance in all the sugar beet organs and were not detected in northern blots. Relative RT-PCR analysis revealed differential expression of the two extracellular invertase genes: BVInv-CW1 was almost exclusively expressed in tap roots and BVInv-CW2 was widely expressed in all the organs analyzed. A remarkable result of this analysis was the high expression of vacuolar invertase (BVInv-V3) in petioles. Two factors had a clear influence on vacuolar invertase gene expression in petioles: light and the developmental stage, so that expression was higher in petioles from juvenile plants. BVInv-V3 transcripts showed circadian oscillation in petioles, with maximal accumulation during the light period. A similar pattern of diurnal oscillation was also observed for the vacuolar invertase activity, showing a delay with respect to the level of transcripts. The analysis of sugars in petioles revealed oscillation of the hexoses, with a remarkably higher content of glucose than fructose. In contrast, the level of sucrose in petioles was very low. This pattern of expression suggests an important role of petiole vacuolar invertase in plant development and photoassimilate partitioning. [ABSTRACT FROM AUTHOR]

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the... [ABSTRACT FROM AUTHOR]

Summary Brassinosteroids (BRs) induce various growth responses when applied exogenously to plant tissues, and the analysis of biosynthetic mutants reveals an essential role for plant growth and development. Only a few BR-regulated genes have been identified so far, and the corresponding gene products are assumed to be involved in cell elongation. The present study shows that BR growth responses are linked to the regulation of carbohydrate metabolism by induction of the mRNA for the key enzyme of an apoplastic phloem-unloading pathway. Addition of BRs to autotrophic tomato suspension culture cells specifically elevates the activity of cell-wall-bound invertase, whereas the intracellular invertase activities were not affected. This enhanced enzyme activity was shown to correlate with the induction of the mRNA of extracellular invertase Lin6, whereas the mRNA levels of the other three extracellular invertase isoenzymes were not affected. The induction level induced by different BRs correlates with their growth-promoting activity. The physiological significance of this regulation is further supported by the low concentrations and short incubation times required to induce Lin6 mRNA. This regulatory mechanism results in an elevated uptake of sucrose via the hexose monomers, and thus an increased supply of carbohydrates to the BR-treated cells. Experiments with tomato seedlings showed that the localized BR-dependent growth response of the hypocotyl elongation zone was accompanied by a specific induction of Lin6 mRNA that is restricted to the corresponding tissues. This study demonstrates a role of BRs in tissue-specific source/sink regulation. [ABSTRACT FROM AUTHOR]

Summary: Different plant invertase isoenzymes are characterized by a single amino-acid difference in a conserved sequence, the WEC-P/V-D box. A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences. The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed muta- genesis. The mutated gene was heterologously expressed in an invertase-deficient Saccharomyces cerevisiae strain. The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity. A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position. [ABSTRACT FROM AUTHOR]

I n an effort to understand factors that control glycosylation of proteins and processing of carbohydrate chains, invertase from Saccharomyces cerevisiae was expressed in a heterologous system. Microinjection of invertasespecific in vitro transcripts into oocytes from Xenopus laevis resulted in synthesis, glycosylation and secretion of enzymatically active invertase. It was found that although the number of carbohydrate chains acquired is the same as in yeast, the carbohydrate processing is different. This is consistent with the notion that the usage of a glycosylation site is determined by the protein part, whereas subsequent processing occurs in a host-dependent manner. Both, high-mannose and complex type glycans, most likely tri- and tetra-antennary structures, were synthesized in oocytes. The data obtained suggests that in this system the core chains of yeast invertase remain high-mannose type, whereas the more extensively processed polymannose chains are modified to complex oligosaccharides. In the presence of the glycosylation inhibitor, tunicamycin, and the glucosidase processing inhibitor, methyldeoxynojirimycin, secretion of invertase is significantly decreased, whereas in the presence of the mannosidase inhibitor, deoxymannojirimycin, no influence of secretion is seen. This may suggest that glycosylation of invertase is important for early secretion events. Expression of invertase lacking the leader sequence results in loss of glycosylation and secretion in oocytes. This indicates that yeast signals for secretion are functional in this higher eukaryote. [ABSTRACT FROM AUTHOR]