High-amylose starch: Structure, functionality and applications.

Starch with a high amylose (AM) content (high AM starch, HAS) has attracted increasing research attention due to its industrial application potential, such as functional foods and biodegradable packaging. In the past two decades, HAS structure, functionality, and applications have been the research hotspots. However, a review that comprehensively summarizes these areas is lacking, making it difficult for interested readers to keep track of past and recent advances. In this review, we highlight studies that benefited from rapidly developing techniques, and systematically review the structure, functionality, and applications of HAS. We particularly emphasize the relationships between HAS molecular structure and physicochemical properties.

Precursor biosynthesis regulation of lignin, suberin and cutin.

The extracellular matrix of plants can contain the hydrophobic biopolymers lignin, suberin and/or cutin, which provide mechanical strength and limit water loss and pathogen invasion. Due to their remarkable chemical resistance, these polymers have a high potential in various biotechnological applications and can replace petrol-based resources, for example, in the packing industry. However, despite the importance of these polymers, the regulation of their precursor biosynthesis is far from being fully understood. This is particularly true for suberin and cutin, which hinders efforts to engineer their formation in plants and produce customised biopolymers. This review brings attention to knowledge gaps in the current research and highlights some of the most recent findings on transcription factors that regulate lignin, suberin and cutin precursor biosynthesis. Finally, we also briefly discuss how some of the remaining knowledge gaps can be closed.

Cutin:xyloglucan transacylase (CXT) activity covalently links cutin to a plant cell-wall polysaccharide.

The shoot epidermal cell wall in land-plants is associated with a polyester, cutin, which controls water loss and possibly organ expansion. Covalent bonds between cutin and its neighbouring cell-wall polysaccharides have long been proposed. However, the lack of biochemical evidence makes cutin-polysaccharide linkages largely conjectural. Here we optimised a portfolio of radiochemical assays to look for cutin-polysaccharide ester bonds in the epidermis of pea epicotyls, ice-plant leaves and tomato fruits, based on the hypothesis that a transacylase remodels cutin in a similar fashion to cutin synthase and cutin:cutin transacylase activities. Through in-situ enzyme assays and chemical degradations coupled with chromatographic analysis of the 3H-labelled products, we observed that among several wall-related oligosaccharides tested, only a xyloglucan oligosaccharide ([3H]XXXGol) could acquire ester-bonds from endogenous cutin, suggesting a cutin:xyloglucan transacylase (CXT). CXT activity was heat-labile, time-dependent, and maximal at near-neutral pH values. In-situ CXT activity peaked in nearly fully expanded tomato fruits and ice-plant leaves. CXT activity positively correlated with organ growth rate, suggesting that it contributes to epidermal integrity during rapid expansion. This study uncovers hitherto unappreciated re-structuring processes in the plant epidermis and provides a step towards the identification of CXT and its engineering for biotechnological applications.

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation.

Certain transglucanases can covalently graft cellulose and mixed-linkage ß-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-ß-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.

Mini Review: Transport of Hydrophobic Polymers Into the Plant Apoplast.

The plant apoplast contains the four hydrophobic polymer, lignin, suberin, cutin, and cutan, that are crucial for stress resistance, controlling solute diffusion, and strengthening the cell wall. Some of these polymers are widely used in industry and daily life products, such as all wood-containing goods (lignin) and wine cork (suberin). Despite the importance of these polymers, several aspects of their formation remain unknown. This mini review highlights technical bottlenecks in the current research and summarizes recent insights into the precursor transmembrane transport, an essential step in the polymer formation. We also briefly discuss how some of the remaining knowledge gaps can be closed and how a better understanding of these biopolymers will benefit other research fields.

Homogalacturonan Accumulation in Cell Walls of the Green Alga Zygnema sp. (Charophyta) Increases Desiccation Resistance.

Land plants inherited several traits from their green algal ancestors (Zygnematophyceae), including a polysaccharide-rich cell wall, which is a prerequisite for terrestrial survival. A major component of both land plant and Zygnematophyceaen cell walls is the pectin homogalacturonan (HG), and its high water holding capacity may have helped algae to colonize terrestrial habitats, characterized by water scarcity. To test this, HG was removed from the cell walls of Zygnema filaments by pectate lyase (PL), and their effective quantum yield of photosystem II (YII) as a proxy for photosynthetic performance was measured in response to desiccation stress by pulse amplitude modulation (PAM). Old filaments were found to contain more HG and are more resistant against desiccation stress but relatively lose more desiccation resistance after HG removal than young filaments. After rehydration, the photosynthetic performance recovered less efficiently in filaments with a HG content reduced by PL, independently of filament age. Immunolabeling showed that partial or un-methylesterified HG occurs throughout the longitudinal cell walls of both young and old filaments, while no labeling signal occurred when filaments were treated with PL prior labeling. This confirmed that most HG can be removed from the cell walls by PL. The initial labeling pattern was restored after ~3 days. A different form of methylesterified HG was restricted to cell poles and cross cell walls. In conclusion, it was shown that the accumulation of HG in Zygnema filaments increases their resistance against desiccation stress. This trait might have played an important role during the colonization of land by Zygnematophyceae, which founded the evolution of all land plants.