Suberin is a lipid and phenolic cell wall heteropolymer found in the roots and other organs of all vascular plants. Suberin plays a critical role in plant water relations and in protecting plants from biotic and abiotic stresses. Here we describe a transcription factor, AtMYB41 (At4g28110), that can activate the steps necessary for aliphatic suberin synthesis and deposition of cell wall-associated suberin-like lamellae in both Arabidopsis thaliana and Nicotiana benthamiana. Overexpression of AtMYB41 increased the abundance of suberin biosynthetic gene transcripts by orders of magnitude and resulted in the accumulation of up to 22 times more suberin-type than cutin-type aliphatic monomers in leaves. Overexpression of AtMYB41 also resulted in elevated amounts of monolignols in leaves and an increase in the accumulation of phenylpropanoid and lignin biosynthetic gene transcripts. Surprisingly, ultrastructural data indicated that overexpression led to the formation of suberin-like lamellae in both epidermal and mesophyll cells of leaves. We further implicate AtMYB41 in the production of aliphatic suberin under abiotic stress conditions. These results provide insight into the molecular-genetic mechanisms of the biosynthesis and deposition of a ubiquitous cell wall-associated plant structure and will serve as a basis for discovering the transcriptional network behind one of the most abundant lipid-based polymers in nature.

Background and Aims Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. Methods We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. Key Results The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. Conclusions Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

The three-dimensional crystal structure of tomato (Lycopersicon esculentum) ␤-mannanase 4a (LeMAN4a) has been determined to 1.5 Å resolution. The enzyme adopts the (␤/␣) 8 fold common to the members of glycohydrolase family GH5. The structure is comparable with those of the homologous Trichoderma reesei and Thermomonospora fusca ␤-mannanases: There is a conserved three-stranded ␤-sheet located near the N terminus that stacks against the central ␤-barrel at the end opposite the active site. Three noncanonical ␣-helices surround the active site. Similar helices are found in T. reesei but not T. fusca ␤-mannanase. By analogy with other ␤-mannanases, the catalytic acid/base residue is E204 and the nucleophile residue is E318. The active site cleft of L. esculentum ␤-mannanase most closely resembles that of the T. reesei isozyme. A model of substrate binding in LeMAN4a is proposed in which the mannosyl residue occupying the −1 subsite of the enzyme adopts the 1 S 5 skew-boat conformation.

SummaryThe benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3¢-hydroxy-N-methylcoclaurine-4¢-O-methyltransferase (4¢OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H þ -ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4¢OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3¢-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4¢OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.

Activity of endo-beta-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. beta-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-beta-mannanase. The increase in endo-beta-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-beta-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-beta-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-beta-mannanase activity in the fruit.

Endo-␤-mannanase cDNAs were cloned and characterized from ripening tomato (Lycopersicon esculentum Mill. cv Trust) fruit, which produces an active enzyme, and from the tomato cv Walter, which produces an inactive enzyme. There is a two-nucleotide deletion in the gene from tomato cv Walter, which results in a frame shift and the deletion of four amino acids at the C terminus of the full-length protein. Other cultivars that produce either active or inactive enzyme show the same absence or presence of the two-nucleotide deletion. The endo-␤-mannanase enzyme protein was purified and characterized from ripe fruit to ensure that cDNA codes for the enzyme from fruit. Immunoblot analysis demonstrated that non-ripening mutants, which also fail to exhibit endo-␤-mannanase activity, do so because they fail to express the protein.In a two-way genetic cross between tomato cvs Walter and Trust, all F 1 progeny from both crosses produced fruit with active enzyme, suggesting that this form is dominant and homozygous in tomato cv Trust. Self-pollination of a plant from the heterozygous F 1 generation yielded F 2 plants that bear fruit with and without active enzyme at a ratio appropriate to Mendelian genetic segregation of alleles. Heterologous expression of the two endo-␤-mannanase genes in Escherichia coli resulted in active enzyme being produced from cultures containing the tomato cv Trust gene and inactive enzyme being produced from those containing the tomato cv Walter gene. Site-directed mutagenesis was used to establish key elements in the C terminus of the endo-␤-mannanase protein that are essential for full enzyme activity.In addition to helping produce a fruit suitable for consumption, ripening-associated softening in tomato (Lycopersicon esculentum Mill.) fruit leads to an increased susceptibility to physical damage during harvest and/or pathogen attack during shipping and storage. This results in crop losses that are costly to producers and consumers alike. Hence, there has been a great deal of research conducted to elucidate the mechanisms involved in ripening-associated softening. Softening in fleshy fruits is caused by the dissolution of pectin in the middle lamella, which reduces cell adhesion (Wakabayashi, 2000), and also by the breakdown of the cell walls themselves. Cell wall breakdown is caused by the concerted action of a number of proteins/enzymes including, but not limited to, expansins, which dissociate the xyloglucan/cellulose network (Brummell et al., 1999); pectin methylesterase, which cleaves methylester groups from pectic polysaccharides; polygalacturonase, which hydrolyzes pectin; and ␤-galactosidase, which removes the galactan side-chains from rhamnogalacturonan I (Brummell and Harpster, 2001).Glucomannans, galactoglucomannans, and galactomannans are polysaccharides present in type I cell walls, which are thought to cross-link cellulose microfibrils in the same manner as xyloglucans, although to a lesser degree (Carpita and Gibeaut, 1993). Of these three types of mannans, the cell walls of tomato a...

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Fruits of the tomato cultivar Walter undergo normal development to the red-ripe stage but, unlike those of the cultivar Trust, they do not produce any active endo-beta-mannanase. Reasons for this failure to produce the enzyme were sought. The cv. Walter contains genes for endo-beta-mannanase, as shown by Southern blot analysis, and transcripts for the enzyme are present in ripening fruits, as revealed using Northern hybridization. Moreover, the enzyme protein is detectable by Western blots using an endo-beta-mannanase-specific antibody from tomato. In addition, the inactive enzyme is localized appropriately in the wall regions of the outer layers of the fruit (skin and outer pericarp). Mixing inactive fruit extracts of cv. Walter, in excess, with extracts from cv. Trust fruits, which contain active enzyme, leads to an increase rather than a reduction in enzyme activity, showing that there are no inhibitors of endo-beta-mannanase in cv. Walter fruits. Similar results were obtained with fruits of the tomato cv. Heinz 1439. In contrast to the situation in fruits, the seeds of both cvs Walter and Heinz 1439 produce active enzyme, especially following germination.