Wim Van den Ende

Sugar signalling and antioxidant network connections in plant cells

Sugars play important roles as both nutrients and regulatory molecules throughout plant life. Sugar metabolism and signalling function in an intricate network with numerous hormones and reactive oxygen species (ROS) production, signalling and scavenging systems. Although hexokinase is well known to fulfil a crucial role in glucose sensing processes, a scenario is emerging in which the catalytic activity of mitochondria‐associated hexokinase regulates glucose‐6‐phosphate and ROS levels, stimulating antioxidant defence mechanisms and the synthesis of phenolic compounds. As a new concept, it can be hypothesized that the synergistic interaction of sugars (or sugar‐like compounds) and phenolic compounds forms part of an integrated redox system, quenching ROS and contributing to stress tolerance, especially in tissues or organelles with high soluble sugar concentrations.

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept

Plants suffering from abiotic stress are commonly facing an enhanced accumulation of reactive oxygen species (ROS) with damaging as well as signalling effects at organellar and cellular levels. The outcome of an environmental challenge highly depends on the delicate balance between ROS production and scavenging by both enzymatic and metabolic antioxidants. However, this traditional classification is in need of renewal and reform, as it is becoming increasingly clear that soluble sugars such as disaccharides, raffinose family oligosaccharides and fructans--next to their associated metabolic enzymes--are strongly related to stress-induced ROS accumulation in plants. Therefore, this review aims at extending the current concept of antioxidants functioning during abiotic stress, with special focus on the emanate role of sugars as true ROS scavengers. Examples are given based on their cellular location, as different organelles seem to exploit distinct mechanisms. Moreover, the vacuole comes into the picture as important player in the ROS signalling network of plants. Elucidating the interplay between the mechanisms controlling ROS signalling during abiotic stress will facilitate the development of strategies to enhance crop tolerance to stressful environmental conditions.

Sucrose, sucrosyl oligosaccharides, and oxidative stress: scavenging and salvaging?

In nature, no single plant completes its life cycle without encountering environmental stress. When plant cells surpass stress threshold stimuli, chemically reactive oxygen species (ROS) are generated that can cause oxidative damage or act as signals. Plants have developed numerous ROS-scavenging systems to minimize the cytotoxic effects of ROS. The role of sucrosyl oligosaccharides (SOS), including fructans and the raffinose family oligosaccharides (RFOs), is well established during stress physiology. They are believed to act as important membrane protectors in planta. So far a putative role for sucrose and SOS during oxidative stress has largely been neglected, as has the contribution of the vacuolar compartment. Recent studies suggest a link between SOS and oxidative defence and/or scavenging. SOS might be involved in stabilizing membrane-associated peroxidases and NADPH oxidases, and SOS-derived radicals might fulfil an intermediate role in oxido-reduction reactions taking place in the vicinity of membranes. Here, these emerging features are discussed and perspectives for future research are provided.

Sugars and plant innate immunity

Sugars are involved in many metabolic and signalling pathways in plants. Sugar signals may also contribute to immune responses against pathogens and probably function as priming molecules leading to pathogen-associated molecular patterns (PAMP)-triggered immunity and effector-triggered immunity in plants. These putative roles also depend greatly on coordinated relationships with hormones and the light status in an intricate network. Although evidence in favour of sugar-mediated plant immunity is accumulating, more in-depth fundamental research is required to unravel the sugar signalling pathways involved. This might pave the way for the use of biodegradable sugar-(like) compounds to counteract plant diseases as cheaper and safer alternatives for toxic agrochemicals.

Myo-inositol and beyond--emerging networks under stress.

Myo-inositol is a versatile compound that generates diversified derivatives upon phosphorylation by lipid-dependent and -independent pathways. Phosphatidylinositols form one such group of myo-inositol derivatives that act both as membrane structural lipid molecules and as signals. The significance of these compounds lies in their dual functions as signals as well as key metabolites under stress. Several stress- and non-stress related pathways regulated by phosphatidylinositol isoforms and associated enzymes, kinases and phosphatases, appear to function in parallel to coordinatively adapt growth and stress responses in plants. Recent evidence also postulates their crucial roles in nuclear functions as they interact with the key players of chromatin structure, yet other nuclear functions remain largely unknown. Phosphatidylinositol monophosphate 5-kinase interacts with and represses a cytosolic neutral invertase, a key enzyme of sugar metabolism suggesting a crosstalk between lipid and sugar signaling. Besides phosphatidylinositol, myo-inositol derived galactinol and associated raffinose-family oligosaccharides are emerging as antioxidants and putative signaling compounds too. Importantly, myo-inositol polyphosphate 5-phosphatase (5PTase) acts, depending on sugar status, as a positive or negative regulator of a global energy sensor, SnRK1. This implies that both myo-inositol- and sugar-derived (e.g. trehalose 6-phosphate) molecules form part of a broad regulatory network with SnRK1 as the central regulator. Recently, it was shown that the transcription factor bZIP11 also takes part in this network. Moreover, a functional coordination between neutral invertase and hexokinase is emerging as a sweet network that contributes to oxidative stress homeostasis in plants. In this review, we focus on myo-inositol, its direct and more downstream derivatives (galactinol, raffinose), and the contribution of their associated networks to plant stress tolerance.

Structure, evolution, and expression of the two invertase gene families of rice.

Invertases catalyze the irreversible hydrolysis of sucrose to glucose and fructose. Plants contain two unrelated families of these enzymes: acid forms that derive from periplasmic invertases of eubacteria and are found in cell wall and vacuole, and neutral/alkaline forms evolved from the cytosolic invertases of cyanobacteria. Genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliana) contain multiple genes encoding these two families. Here for rice we identify the member genes of a cell-wall group (designated OsCIN1–9), a vacuolar group (OsVIN1–2), and two ancient neutral/alkaline groups: α (OsNIN1–4) and β (OsNIN5–8). In Arabidopsis these groups contain six, two, four and five members, respectively. It is believed that the vacuolar group evolved from the cell-wall group. We provide evidence that the N-terminal signal peptide that directs cell-wall invertases co-translationally into the endoplasmic reticulum for secretion was replaced in the vacuolar group by a sequence similar to the complex N-terminal motif that targets alkaline phosphatase post-translationally to the vacuolar membrane of yeast. Since the last common ancestor of Arabidopsis and rice, the two invertase families evolved equally rapidly via gene duplication and gene loss, but the acid invertase family underwent ∼10 events of intron loss compared with a single event of intron gain in the neutral/alkaline invertase family. Transcripts were detected for all rice invertase genes except OsCIN9. The acid invertase genes showed greater spatial and temporal diversity of expression than the neutral/alkaline genes.

Multifunctional fructans and raffinose family oligosaccharides.

Fructans and Raffinose Family Oligosaccharides (RFOs) are the two most important classes of water soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure-function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called “defective invertases”. Both fructans and RFOs can act as reserve carbohydrates, membrane stabilizers and stress tolerance mediators. Fructan metabolism can also play a role in osmoregulation (e.g. flower opening) and source-sink relationships. Here, two novel emerging roles are highlighted. First, fructans and RFOs may contribute to overall cellular ROS homeostasis by specific ROS scavenging processes in the vicinity of organellar membranes (e.g. vacuole, chloroplasts). Second, it is hypothesized that small fructans and RFOs act as phloem-mobile signaling compounds under stress. It is speculated that such underlying antioxidant and oligosaccharide signaling mechanisms contribute to disease prevention in plants as well as in animals and in humans.

Fructan 1-Exohydrolases. β-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 andw2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [β-(2,1)] are much better substrates than levan-type fructans [β-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a β-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.

Extraction of high-quality genomic DNA from latex-containing plants.

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA. A cetyltrimethylammonium bromide protocol has been optimized for isolation of genomic DNA from latex-containing plants. Key steps in the modified protocol are the use of etiolated leaf tissue for extraction and an overnight 25 degrees C isopropanol precipitation step. The purified DNA has excellent spectral qualities, is efficiently digested by restriction endonucleases, and is suitable for long-fragment PCR amplification.

Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications

Glycoside hydrolases (GH) have been shown to play unique roles in various biological processes like the biosynthesis of glycans, cell wall metabolism, plant defence, signalling, and the mobilization of storage reserves. To date, GH are divided into more than 100 families based upon their overall structure. GH32 and GH68 are combined in clan GH-J, not only harbouring typical hydrolases but also non-Leloir type transferases (fructosyltransferases), involved in fructan biosynthesis. This review summarizes the recent structure-function research progress on plant GH32 enzymes, and highlights the similarities and differences compared with the microbial GH32 and GH68 enzymes. A profound analysis of ligand-bound structures and site-directed mutagenesis experiments identified key residues in substrate (or inhibitor) binding and recognition. In particular, sucrose can bind as inhibitor in Cichorium intybus 1-FEH IIa, whereas it binds as substrate in Bacillus subtilis levansucrase and Arabidopsis thaliana cell wall invertase (AtcwINV1). In plant GH32, a single residue, the equivalent of Asp239 in AtcwINV1, appears to be important for sucrose stabilization in the active site and essential in determining sucrose donor specificity.