Rhamnogalacturonan-I is a determinant of cell-cell adhesion in poplar wood.

The molecular basis of cell-cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717-1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan-I (RG-I) using either dilute alkali or a combination of xylanase and RG-lyase. Acidic chlorite followed by dilute alkali treatment enables cell-cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell-cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG-lyase (AtRGIL6) showed enhanced cell-cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG-I, and also for xylan, as determinants of cell-cell adhesion in poplar wood cell walls. Genetic control of RG-I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.

Migration and proliferation of cancer cells in culture are differentially affected by molecular size of modified citrus pectin.

While chemically and thermally modified citrus pectin (MCP) has already been studied for health benefits, it is unknown how size-fractionated oligo- and polysaccharides differentially affect cancer cell behavior. We produced thermally MCP and fractionated it by molecular size to evaluate the effect these polymers have on cancer cells. MCP30/10 (between 30 and 10 kDa) had more esterified homogalacturonans (HG) and fewer rhamnogalacturonans (RG-I) than MCP and MCP30 (higher than 30 kDa), while MCP10/3 (between 10 and 3 kDa) showed higher amounts of type I arabinogalactans (AGI) and lower amounts of RG-I. MCP3 (smaller than 3 kDa) presented less esterified HG and the lowest amount of AGI and RG-I. Our data indicate that the enrichment of de-esterified HG oligomers and the AGI and RG-I depletions in MCP3, or the increase of AGI and loss of RGI in MCP30/10, enhance the anticancer behaviors by inhibiting migration, aggregation, and proliferation of cancer cells.

Ripening-induced chemical modifications of papaya pectin inhibit cancer cell proliferation.

Papaya (Carica papaya L.) is a fleshy fruit with a rapid pulp softening during ripening. Ripening events are accompanied by gradual depolymerization of pectic polysaccharides, including homogalacturonans, rhamnogalacturonans, arabinogalactans, and their modified forms. During intermediate phases of papaya ripening, partial depolymerization of pectin to small size with decreased branching had enhanced pectin anti-cancer properties. These properties were lost with continued decomposition at later phases of ripening. Pectin extracted from intermediate phases of papaya ripening markedly decreased cell viability, induced necroptosis, and delayed culture wound closing in three types of immortalized cancer cell lines. The possible explanation for these observations is that papaya pectins extracted from the third day after harvesting have disrupted interaction between cancer cells and the extracellular matrix proteins, enhancing cell detachment and promoting apoptosis/necroptosis. The anticancer activity of papaya pectin is dependent on the presence and the branch of arabinogalactan type II (AGII) structure. These are first reports of AGII in papaya pulp and the first reports of an in vitro biological activity of papaya pectins that were modified by natural action of ripening-induced pectinolytic enzymes. Identification of the specific pectin branching structures presents a biological route to enhancing anti-cancer properties in papaya and other climacteric fruits.

Rhamnose-Containing Cell Wall Polymers Suppress Helical Plant Growth Independently of Microtubule Orientation.

Although specific organs in some plant species exhibit helical growth patterns of fixed or variable handedness, most plant organs are not helical. Here we report that mutations in Arabidopsis RHAMNOSE BIOSYNTHESIS 1 (RHM1) cause dramatic left-handed helical growth of petal epidermal cells, leading to left-handed twisted petals. rhm1 mutant roots also display left-handed growth. Furthermore, we find that RHM1 is required to promote epidermal cell expansion. RHM1 encodes a UDP-L-rhamnose synthase, and rhm1 mutations affect synthesis of the pectic polysaccharide rhamnogalacturonan-I. Unlike other mutants that exhibit helical growth of fixed handedness, the orientation of cortical microtubule arrays is unaltered in rhm1 mutants. Our findings reveal a novel source of left-handed plant growth caused by changes in cell wall composition that is independent of microtubule orientation. We propose that an important function of rhamnose-containing cell wall polymers is to suppress helical twisting of expanding plant cells.

Structural alteration of cell wall pectins accompanies pea development in response to cold.

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype 'Terese' and a frost-tolerant genotype 'Champagne'. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-ß-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-ß-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

Novel rhamnogalacturonan I and arabinoxylan polysaccharides of flax seed mucilage.

The viscous seed mucilage of flax (Linum usitatissimum) is a mixture of rhamnogalacturonan I and arabinoxylan with novel side group substitutions. The rhamnogalacturonan I has numerous single nonreducing terminal residues of the rare sugar l-galactose attached at the O-3 position of the rhamnosyl residues instead of the typical O-4 position. The arabinoxylan is highly branched, primarily with double branches of nonreducing terminal l-arabinosyl units at the O-2 and O-3 positions along the xylan backbone. While a portion of each polysaccharide can be purified by anion-exchange chromatography, the side group structures of both polysaccharides are modified further in about one-third of the mucilage to form composites with enhanced viscosity. Our finding of the unusual side group structures for two well-known cell wall polysaccharides supports a hypothesis that plants make a selected few ubiquitous backbone polymers onto which a broad spectrum of side group substitutions are added to engender many possible functions. To this end, modification of one polymer may be accompanied by complementary modifications of others to impart functions to heterocomposites not present in either polymer alone.

The Arabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydration properties.

Seed coat development in Arabidopsis thaliana involves a complex pathway where cells of the outer integument differentiate into a highly specialized cell type after fertilization. One aspect of this developmental process involves the secretion of a large amount of pectinaceous mucilage into the apoplast. When the mature seed coat is exposed to water, this mucilage expands to break the primary cell wall and encapsulate the seed. The mucilage-modified2 (mum2) mutant is characterized by a failure to extrude mucilage on hydration, although mucilage is produced as normal during development. The defect in mum2 appears to reside in the mucilage itself, as mucilage fails to expand even when the barrier of the primary cell wall is removed. We have cloned the MUM2 gene and expressed recombinant MUM2 protein, which has beta-galactosidase activity. Biochemical analysis of the mum2 mucilage reveals alterations in pectins that are consistent with a defect in beta-galactosidase activity, and we have demonstrated that MUM2 is localized to the cell wall. We propose that MUM2 is involved in modifying mucilage to allow it to expand upon hydration, establishing a link between the galactosyl side-chain structure of pectin and its physical properties.

Phosphate starvation responses are mediated by sugar signaling in Arabidopsis.

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be down-stream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.

Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged storage of apple.

Growth and maturation of the edible cortical cells of apples (Malus domestica Borkh) are accompanied by a selective loss of pectin-associated (1-->4)-beta-D-galactan from the cell walls, whereas a selective loss of highly branched (1-->5)-alpha-L-arabinans occurs after ripening and in advance of the loss of firm texture. The selective loss of highly branched arabinans occurs during the overripening of apples of four cultivars (Gala, Red Delicious, Firm Gold, and Gold Rush) that varied markedly in storage life, but, in all instances, the loss prestages the loss of firm texture, measured by both breaking strength and compression resistance. The unbranched (1-->5)-linked arabinans remain associated with the major pectic polymer, rhamnogalacturonan I, and their content remains essentially unchanged during overripening. However, the degree of rhamnogalacturonan I branching at the rhamnosyl residues also decreases, but only after extensive loss of the highly branched arabinans. In contrast to the decrease in arabinan content, the loss of the rhamnogalacturonan I branching is tightly correlated with loss of firm texture in all cultivars, regardless of storage time. In vitro cell separation assays show that structural proteins, perhaps via their phenolic residues, and homogalacturonans also contribute to cell adhesion. Implications of these cell wall modifications in the mechanisms of apple cortex textural changes and cell separation are discussed.