A feruloyl transferase involved in the biosynthesis of suberin and suberin‐associated wax is required for maturation and sealing properties of potato periderm

Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the tuber periderm of potato (Solanum tuberosum). Suberin is a cell‐wall biopolymer with aliphatic and aromatic domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to play an important role in cross‐linking to the aromatic domain. In potato, ferulic acid esters are also the main components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains unknown. Here we report on a potato gene encoding a fatty ω‐hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid with ω‐hydroxyacid and fatty alcohols. FHT down‐regulation did not affect the typical suberin lamellar ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm. The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented. FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular architecture and periderm impermeability.     

CYP86A33-Targeted Gene Silencing in Potato Tuber Alters Suberin Composition,Distorts Suberin Lamellae,and Impairs the Periderm's Water Barrier Function

Suberin is a cell wall lipid polyester found in the cork cells of the periderm offering protection against dehydration and pathogens. Its biosynthesis and assembly, as well as its contribution to the sealing properties of the periderm, are still poorly understood. Here, we report on the isolation of the coding sequence CYP86A33 and the molecular and physiological function of this gene in potato (Solanum tuberosum) tuber periderm. CYP86A33 was down-regulated in potato plants by RNA interference-mediated silencing. Periderm from CYP86A33-silenced plants revealed a 60% decrease in its aliphatic suberin load and greatly reduced levels of C18:1 ω-hydroxyacid (approximately 70%) and α,ω-diacid (approximately 90%) monomers in comparison with wild type. Moreover, the glycerol esterified to suberin was reduced by 60% in the silenced plants. The typical regular ultrastructure of suberin, consisting of dark and light lamellae, disappeared and the thickness of the suberin layer was clearly reduced. In addition, the water permeability of the periderm isolated from CYP86A33-silenced lines was 3.5 times higher than that of the wild type. Thus, our data provide convincing evidence for the involvement of ω-functional fatty acids in establishing suberin structure and function.Periderm, the boundary tissue that replaces the epidermis in the secondary organs of plants, provides efficient protection against dehydration, UV radiation, and pathogens (Esau, 1965). Periderm is regularly found in the bark of woody plants, but herbaceous plants may also form a well-developed periderm in roots, tubers, and the oldest portions of stem. The periderm has been widely studied in potato (Solanum tuberosum) tubers because of the latter''s great agronomic significance (Schmidt and Schönherr, 1982; Vogt et al., 1983; Lulai and Freeman, 2001; Sabba and Lulai, 2002). Shrinkage and flaccidity occur in tubers if the protection afforded by the periderm against water loss is compromised (Lulai et al., 2006). Suberin and waxes embedded into the suberin matrix are considered essential for periderm imperviousness (Franke and Schreiber, 2007). Chemically, suberin is a complex lipid polymer consisting of a fatty acid-derived domain (aliphatic suberin) cross-linked by ester bonds to a polyaromatic lignin-like domain (aromatic suberin; Kolattukudy, 2001; Bernards, 2002; Franke and Schreiber, 2007). Aliphatic suberin has been widely analyzed in potato periderm (Kolattukudy and Agrawal, 1974; Graça and Pereira, 2000; Schreiber et al., 2005). The main monomers released from potato aliphatic suberin are a mixture of ω-hydroxyacids and α,ω-diacids with chain lengths ranging from C16 to C28 (mainly C18), together with glycerol. Small amounts of monofunctional fatty acids, alcohols, and ferulic acid are also released. Waxes are complex mixtures of lipids extractable with chloroform that in potato periderm consist mostly of linear very-long-chain aliphatics up to C32 (Schreiber et al., 2005). Suberin is deposited in the cell wall as a continuous deposit or secondary cell wall that overlays the polysaccharide primary cell wall from the inside (Esau, 1965). These suberin deposits appear under the transmission electron microscope (TEM) as regularly alternating opaque and translucent lamellae (Schmidt and Schönherr, 1982). Although several molecular models for suberin have been proposed (Kolattukudy, 1980; Bernards, 2002; Graça and Santos, 2007), how the suberin and wax components are organized in the lamellated suberin secondary cell wall is a matter of debate (Graça and Santos, 2007). Moreover, to what extent suberin and wax deposition and composition determine sealing properties of periderm still remains unclear (Schreiber et al., 2005). Several studies confirm the importance of waxes for the diffusion barrier (Soliday et al., 1979; Vogt et al., 1983; Schreiber et al., 2005), but the significance of aliphatic suberin has hardly been investigated at all. Interestingly, an Arabidopsis (Arabidopsis thaliana) knockout mutant for the GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE5 gene (GPAT5) with altered suberin showed higher tetrazolium salt permeability in the seed coat (Beisson et al., 2007).ω-Hydroxylation of fatty acids, a reaction carried out in plants by cytochrome P450 monooxygenases, is a crucial step in the biosynthesis of plant biopolymers (Kolattukudy, 1980; Nawrath, 2002). The Arabidopsis mutant lacerata, which shows phenotype defects compatible with a cutin deficiency, is defective in CYP86A8 encoding a fatty acid ω-hydroxylase (Wellesen et al., 2001). The aberrant induction of type three genes1 (att1) mutant, showing an altered cuticle ultrastructure and a higher transpiration rate than wild type, is defective in CYP86A2 and contains reduced amounts of ω-functionalized cutin monomers (Xiao et al., 2004). Moreover, a genome-wide study of cork oak (Quercus suber) bark highlighted a member of the cytochrome P450 of the CYP86A subfamily as a strong candidate gene for aliphatic suberin biosynthesis (Soler et al., 2007); and a role for CYP86A1 in the biosynthesis of suberin has recently been confirmed in the primary root of Arabidopsis knockout mutants (Li et al., 2007; Hofer et al., 2008). However, how the lack of fatty acid ω-hydroxylase activity may affect the structural features and sealing properties of suberin in periderm cell walls has not been documented.To provide experimental evidence of the role of CYP86A genes in periderm fine structure and water transpiration properties, especially quantitative permeability studies so far unexplored in Arabidopsis, we followed a strategy based on the potato (cv Desirée). The potato is a model of choice for such studies because transgenic plants can be produced in reasonable time and sufficient amounts of periderm can easily be obtained from tubers for chemical and physiological studies (Vogt et al., 1983; Schreiber et al., 2005). Based on the CYP86A gene that was highlighted in cork oak periderm as a strong suberin candidate for aliphatic suberin biosynthesis, we isolated the putative ortholog in potato and used a reverse genetic approach to analyze the effects of down-regulation on the chemical and ultrastructural features of suberin and on the physiological properties of the tuber periderm. Our findings indicate that down-regulation of CYP86A33, as this gene was designated, results in a strong decrease of the ω-functionalized monomers in aliphatic suberin, which are necessary for the suberin typical lamellar organization and for the periderm resistance to water loss.     

Wax and suberin development of native and wound periderm of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) and its relation to peridermal transpiration

Native and wound periderm was isolated enzymatically from potato (Solanum tuberosum L. cv. Desirée) tubers at different time intervals between 0 days up to 4 weeks after harvesting. Wound periderm formation was induced by carefully removing native periderm from freshly harvested tubers before storage. The chemical composition of lipids (waxes) obtained by chloroform extraction, as well as the monomeric composition of native and wound suberin polymer after transesterification by boron trifluoride/methanol, was analyzed using gas chromatography and mass spectrometry. Both types of periderm contained up to 20% extractable lipids. Besides linear long-chain aliphatic wax compounds, alkyl ferulates were detected as significant constituents. In wound periderm they amounted to more than 60% of the total extracts. Within 1 month of storage, suberin amounts in the polymer increased 2-fold in native periderm (180 g cm^–2), whereas in wound periderm about 75.0 g cm^–2 suberin polymer was newly synthesized. Native potato tuber periderm developed a very efficient transport barrier for water with permeances decreasing from 6.4×10^–10 m s^–1 to 5.5×10^–11 m s^–1 within 1 month of storage. However, the water permeability of wound periderm was on average 100 times higher with permeances decreasing from 4.7×10^–8 m s^–1 after 3 days to only 5.4×10^–9 m s^–1 after 1 month of storage, although suberin and wax amounts in wound periderm amounted to about 60% of native periderm. From this result it must be concluded that the occurrence of suberin with wax depositions in cell walls does not necessarily allow us to conclude that these cell walls must be nearly perfect barriers to water transport. In addition to the occurrence of the lipophilic biopolymer suberin and associated waxes, the still unknown molecular arrangement and precisely localized deposition of suberin within the cell wall must contribute to the efficiency of suberin as a barrier to water transport.     

Suberin-Associated Fatty Alcohols in Arabidopsis: Distributions in Roots and Contributions to Seed Coat Barrier Properties

Suberin is found in a variety of tissues, such as root endoderms and periderms, storage tuber periderms, tree cork layer, and seed coats. It acts as a hydrophobic barrier to control the movement of water, gases, and solutes as well as an antimicrobial barrier. Suberin consists of polymerized phenolics, glycerol, and a variety of fatty acid derivatives, including primary fatty alcohols. We have conducted an in-depth analysis of the distribution of the C18:0 to C22:0 fatty alcohols in Arabidopsis (Arabidopsis thaliana) roots and found that only 20% are part of the root suberin polymer, together representing about 5% of its aliphatic monomer composition, while the remaining 80% are found in the nonpolymeric (soluble) fraction. Down-regulation of Arabidopsis FATTY ACYL REDUCTASE1 (FAR1), FAR4, and FAR5, which collectively produce the fatty alcohols found in suberin, reduced their levels by 70% to 80% in (1) the polymeric and nonpolymeric fractions from roots of tissue culture-grown plants, (2) the suberin-associated root waxes from 7-week-old soil-grown plants, and (3) the seed coat suberin polymer. By contrast, the other main monomers of suberin were not altered, indicating that reduced levels of fatty alcohols did not influence the suberin polymerization process. Nevertheless, the 75% reduction in total fatty alcohol and diol loads in the seed coat resulted in increased permeability to tetrazolium salts and a higher sensitivity to abscisic acid. These results suggest that fatty alcohols and diols play an important role in determining the functional properties of the seed coat suberin barrier.Suberin is a cell wall-linked polymeric barrier that plays a critical role in the survival of plants by protecting them against various biotic and abiotic stresses. It primarily acts as a hydrophobic barrier to control the movement of water, gases, and solutes, but also contributes to the strength of the cell wall (Ranathunge et al., 2011). Suberin is deposited at the inner face of primary cell walls next to the plasma membrane (Kolattukudy, 1980; Franke and Schreiber, 2007). It is typically found as lamellae (alternating dark and light bands when viewed by transmission electron microscopy) in the endodermis, exodermis, and peridermis of roots, as well as in the peridermis of underground storage tubers (Bernards, 2002). Suberin is also found in shoot periderms of trees (i.e. cork layer) and in seed coats (Molina et al., 2006, 2008) and is deposited in response to wounding (Kolattukudy, 2001).Suberin is a polymer consisting of aliphatics (fatty acid derivatives), phenolics, and glycerol. The predominant aliphatic components of suberin are ω-hydroxy fatty acids, α,ω-dicarboxylic acids, very-long-chain fatty acids, and primary fatty alcohols, while the major phenolic components are p-hydroxycinnamic acids, especially ferulic acid (Kolatukudy, 1980; Bernards et al., 1995; Pollard et al., 2008; Ranathunge et al., 2011). In the periderm of underground storage organs, suberin is found in association with waxes, which are isolated either by extensive extraction in solvent (Soliday et al., 1979; Serra et al., 2009) or by brief immersion of tubers in chloroform (Espelie et al., 1980). These suberin-associated waxes consist of linear aliphatics (e.g. alkanes, fatty acids, and fatty alcohols), which are similar to cuticular wax components of aerial tissues but generally of shorter chain lengths (Espelie et al., 1980). In waxes extracted from 3-week-old wounded potato (Solanum tuberosum) periderms, alkyl ferulates (i.e. ferulic acid linked by an ester bond to a C16:0–C32:0 fatty alcohol) represent up to 60% of the total wax load (Schreiber et al., 2005). Root waxes are also found in 6- to 7-week-old mature taproots of Arabidopsis (Arabidopsis thaliana) with a fully developed periderm (Li et al., 2007; Kosma et al., 2012). They are enriched in alkyl hydroxycinnamates (AHCs) made of C18:0 to C22:0 fatty alcohols esterified with coumaric, caffeic, or ferulic acids (Kosma et al., 2012). The monomer composition (in terms of major chemical species and chain length) of both suberin and suberin-associated waxes varies considerably between plant species, tissues, and developmental stages. Aliphatic suberin and suberin-associated waxes are considered the major contributors to the diffusion resistance of suberized cell walls to radial transport of water and solutes (Soliday et al., 1979; Espelie et al., 1980; Zimmermann et al., 2000; Ranathunge and Schreiber, 2011). The organization of suberin components in the lamellated structure as well as how waxes may be associated with the polymer is a matter of debate (Graça and Santos, 2007).Primary fatty alcohols are long-chain hydrocarbons containing a single hydroxyl group at the terminal position. They are ubiquitously detected as components of the suberin polymer, representing 1% to 10% of the total monomer mass recovered after transesterification (Holloway, 1983; Bernards, 2002; Pollard et al., 2008). Primary fatty alcohols are also typical components of suberin-associated waxes, where they can be found either in free form or linked by an ester bond with a hydroxycinnamic acid (i.e. as AHCs; Soliday et al., 1979; Espelie et al., 1980; Bernards and Lewis 1992; Li et al., 2007; Kosma et al., 2012). In mechanically isolated endodermis of soybean (Glycine max) roots, fatty alcohols represent about 1.5% and 0.2% of the total aliphatics found in suberin-associated waxes and suberin polymer, respectively (Thomas et al., 2007). In onion (Allium cepa) root exodermis, fatty alcohols (C14:0–C28:0) account for 7% to 12% of the soluble fraction, while the suberin fraction contains only C22:0 fatty alcohol, which makes up 3% of the suberin fraction across all exodermal maturation zones (Meyer et al., 2011). In suberizing potato periderms 7 d post wounding, C16:0 to C28:0 fatty alcohols represent about 10% and 18% of the total aliphatics in the insoluble poly(aliphatic) domain (suberin polymer) and in the soluble (nonpolymeric) fraction, respectively (Yang and Bernards, 2006). A similar study on native periderms from 21-d-stored potato (Serra et al., 2009) reported that fatty alcohols represent about 20% of the total aliphatic components found in the suberin polyester, while unlinked fatty alcohols and alkyl ferulates accounted for about 23% and 44% of the total aliphatics in the soluble waxes.In Arabidopsis, C18:0, C20:0, and C22:0 fatty alcohols account for slightly less than 3% of the polymerized aliphatics in roots of soil-grown plants (Domergue et al., 2010), but as much as 36% [w/w] of the soluble wax load (Li et al., 2007). Arabidopsis fatty acyl reductases FAR1 (At5g22500), FAR4 (At3g44540), and FAR5 (At3g44550) generate, respectively, the C22:0, C20:0, and C18:0 fatty alcohol present in the suberin of root, seed coat, and wounded leaf tissues (Domergue et al., 2010). These three enzymes also generate the C18:0 to C22:0 fatty alcohol components that make up AHCs of root waxes (Kosma et al., 2012). Although one particular chain length of primary alcohol was reduced in each far single mutant line (C18:0-OH, C20:0-OH, and C22:0-OH in far5, far4, and far1, respectively), the total fatty alcohol load of the suberin polymer and its composition were only slightly affected and mutant plants had no obvious developmental or physiological defects (Domergue et al., 2010). In this study, we report on the distribution of primary fatty alcohols in the soluble (nonpolymeric) and insoluble (suberin polymer) fractions from mature roots of Arabidopsis. We report that far double and triple mutants have highly reduced fatty alcohol levels, in a chain length-specific manner, in both fractions as well as in the seed coat suberin polymer. The significant reductions in total fatty alcohol and diol levels in the seed coat of these mutants lead to increased permeability and higher sensitivity to abscisic acid (ABA), bringing to light insights on the roles of fatty alcohols and diols in determining functional properties of suberin.     

Glycerol is a suberin monomer. New experimental evidence for an old hypothesis

The monomer composition of the esterified part of suberin can be determined using gas chromatography-mass spectroscopy technology and is accordingly believed to be well known. However, evidence was presented recently indicating that the suberin of green cotton (Gossypium hirsutum cv Green Lint) fibers contains substantial amounts of esterified glycerol. This observation is confirmed in the present report by a sodium dodecyl sulfate extraction of membrane lipids and by a developmental study, demonstrating the correlated accumulation of glycerol and established suberin monomers. Corresponding amounts of glycerol also occur in the suberin of the periderm of cotton stems and potato (Solanum tuberosum) tubers. A periderm preparation of wound-healing potato tuber storage parenchyma was further purified by different treatments. As the purification proceeded, the concentration of glycerol increased at about the same rate as that of α,ω-alkanedioic acids, the most diagnostic suberin monomers. Therefore, it is proposed that glycerol is a monomer of suberins in general and can cross-link aliphatic and aromatic suberin domains, corresponding to the electron-translucent and electron-opaque suberin lamellae, respectively. This proposal is consistent with the reported dimensions of the electron-translucent suberin lamellae.     

Natural Variation in Small Molecule–Induced TIR-NB-LRR Signaling Induces Root Growth Arrest via EDS1- and PAD4-Complexed R Protein VICTR in Arabidopsis

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest.     

Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.Potato (Solanum tuberosum) is the world’s fourth most important food crop after maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). Potato steroidal glycoalkaloids (SGAs) are abundant poisons in tuber sprouts and green tubers and are described as bitter tasting, burning, scratchy, or acrid (Friedman, 2006; Ginzberg et al., 2009; Taylor et al., 2007). SGAs in the tuber are induced by exposure to light, low temperature, and mechanical injury (Valkonen et al., 1996). Producers and consumers have called for the removal of SGAs from potatoes. Only potato with SGAs of major food crops has such broad industry consensus on the need to solve this important worldwide problem. Controlling the SGA content is also important for potato breeding. Wild germplasm has been used in potato breeding programs as a source of pest and disease resistance. Since high SGA concentrations are found in most wild tuber-bearing species, introgression of wild germplasm may increase the risk for high SGA levels. Although the initial concentrations are still low in new breeding lines, dangerous levels of SGAs can accumulate due to environmental factors, pathogen infections, and postharvest treatments (Valkonen et al., 1996).SGAs are biosynthesized from a common precursor, cholesterol (CHR; Sawai et al., 2014), but little is known about intermediates and enzymes in the SGA biosynthetic pathway. To change a biosynthetic flow to CHR and decrease SGA contents, transgenic potatoes overexpressing a heterologous soybean sterol methyltransferase gene were produced (Arnqvist et al., 2003). Three genes responsible for glycosylating potato SGA have been identified (McCue et al., 2005, 2006, 2007). However, changing the expression of the sterol methyltransferase or glycosyltransferase genes does not effectively decrease SGA levels. To control the SGA content of potato, we focused on the biosynthetic steps from CHR to the aglycone, solanidine. Few details about the biosynthetic pathway are verified; however, the pathway is hypothesized to require at least three oxidization steps at positions C16, C22, and C26 of CHR structure and the addition of one nitrogen atom at the position C26 (Fig. 1; Kaneko et al., 1977; Erich, 1983; Eich, 2008). The later step was shown to be another oxidation and an amination reaction at the position C26 (Ohyama et al., 2013). Here, we identified two cytochrome P450 monooxygenase (CYPs) genes, POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2 that encode enzymes mediating two oxidation steps. Silencing PGA1 and PGA2 resulted in a significant reduction in SGA composition and the creation of novel phenotypes, including the suppression of flower development and tuber sprouting. Sprouting reduces the quality and yield of potato tubers in storage. Suppression of tuber sprouting is of significant benefit to the industry for the long-term storage of tubers. Thus, controlling tuber sprouting is another important objective in potato breeding (Sonnewald and Sonnewald, 2014).Open in a separate windowFigure 1.Biosynthetic pathway for SGAs in potato. The structures of CHR and solanidine, two biosynthetic intermediates of potato SGAs, are shown with the structures of the SGA products. Circles indicate putative carbon positions that are oxidized in the hypothesized pathway.     

Chemical Composition and Ultrastructure of Suberin from Hollow Heart Tissue of Potato Tubers (Solanum tuberosum)

The disorder of potato tubers (Solanum tuberosum var. Russet Burbank) called “hollow heart” is manifested by the occurrence of hollow regions in internal parts of the tuber. The structure and composition of the suberin from the tissue lining of these internal cavities were determined by gas chromatography and mass spectrometry of the LiAlH₄-hydrogenolysis products. Identification of octadecene-1,18-diol as the major component and the presence of hexadecane-1,16-diol and very long chain (>C₁₈) alcohols in the hydrogenolysate showed that the suberin lining the internal cavities is quite similar to that found in the periderm of external wounds and the natural skin. Electron microscopic examination showed similar lamellar structure for the suberin of hollow heart, external wound periderm, and the natural skin of potato tubers. The results show that suberin can develop in a tissue which is not exposed to the external environment.     

Cadmium induces hypodermal periderm formation in the roots of the monocotyledonous medicinal plant Merwilla plumbea

Background and Aims

Merwilla plumbea is an important African medicinal plant. As the plants grow in soils contaminated with metals from mining activities, the danger of human intoxication exists. An experiment with plants exposed to cadmium (Cd) was performed to investigate the response of M. plumbea to this heavy metal, its uptake and translocation to plant organs and reaction of root tissues.

Methods

Plants grown from seeds were cultivated in controlled conditions. Hydroponic cultivation is not suitable for this species as roots do not tolerate aquatic conditions, and additional stress by Cd treatment results in total root growth inhibition and death. After cultivation in perlite the plants exposed to 1 and 5 mg Cd L⁻¹ in half-strength Hoagland''s solution were compared with control plants. Growth parameters were evaluated, Cd content was determined by inductively coupled plasma mass spectroscopy (ICP-MS) and root structure was investigated using various staining procedures, including the fluorescent stain Fluorol yellow 088 to detect suberin deposition in cell walls.

Key Results

The plants exposed to Cd were significantly reduced in growth. Most of the Cd taken up by plants after 4 weeks cultivation was retained in roots, and only a small amount was translocated to bulbs and leaves. In reaction to higher Cd concentrations, roots developed a hypodermal periderm close to the root tip. Cells produced by cork cambium impregnate their cell walls by suberin.

Conclusions

It is suggested that the hypodermal periderm is developed in young root parts in reaction to Cd toxicity to protect the root from radial uptake of Cd ions. Secondary meristems are usually not present in monocotyledonous species. Another interpretation explaining formation of protective suberized layers as a result of periclinal divisions of the hypodermis is discussed. This process may represent an as yet unknown defence reaction of roots when exposed to elemental stress.     

ABCG Transporters Are Required for Suberin and Pollen Wall Extracellular Barriers in Arabidopsis

Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.     

Histochemical Probing of Potato Periderm with Neutral Red: A Sensitive Cytofluorochrome for the Hydrophobic Domain of Suberin

A technique is described which uses the lipid fluorochrome neutral red as a cytochemical probe to detect the hydrophobic domain of the lignosuberin matrix in native and wound periderm of potato tuber. Toluidine blue O is used as a counterstain to quench autofluorescence. The neutral red technique appears to be specific for the hydrophobic/lipid domain of suberin and is significantly more sensitive than Sudan III and IV. The fluorochrome was extensively used on paraffin-embedded tissue with excellent results but also worked on freehand sections of fresh periderm tissue. In tuber tissue undergoing wound-healing, the pattern of suberin fluorescence obtained with the neutral red probe was identical in specificity to the color pattern obtained with Sudan III/IV, but somewhat different than that observed when berberine was used. Results obtained with the neutral red probe and berberine probe visually demonstrated that during ligno-suberin biosynthesis, the depositions of hydrophobic/lipid and phenolic/lignin-like components in potato tuber periderm were separate processes. The deposition of these components does not necessarily require their simultaneous presence because the fluorescence from these probes showed that the components were not consistently present together on the cell walls.     

Unraveling ferulate role in suberin and periderm biology by reverse genetics

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.Key words: BAHD feruloyl acyltransferases, ferulate, periderm, potato tuber skin, suberin, suberized tissues, waxRecently published reverse genetic studies in Arabidopsis and potato identified two orthologous genes that encode a BAHD feruloyl transferase acting on aliphatics and showed that deficiency in these enzymes produces a decrease in suberin-associated ferulic acid. These results, here discussed, signify an important advance in suberin biochemistry and ultrastructure, providing a valuable new insight into the organization of the suberized tissues and their role in the control of water vapour loss.     

Fatty acid ω-hydroxylases from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1.

Abstract

Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

     

Water Uptake along the Length of Grapevine Fine Roots: Developmental Anatomy,Tissue-Specific Aquaporin Expression,and Pathways of Water Transport

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lp_r) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lp_r and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lp_r, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.In woody perennial root systems, the majority of water uptake is often attributed to unsuberized fine roots (Kramer and Boyer, 1995), even though woody portions can constitute the vast majority of root surface area for these plants at maturity (Nightingale, 1934; Kramer and Bullock, 1966). This assumption has likely been reinforced by the fact that most studies investigating root water uptake have been done with herbaceous species, whose roots function more like the tips of woody perennials. Although unsuberized fine roots typically have a greater ability to absorb water (i.e. they are more conductive per unit of surface area), it has been shown that older suberized portions of woody taproots can still contribute significantly to root system water uptake (Kramer and Bullock, 1966; Queen, 1967; Chung and Kramer, 1975; MacFall et al., 1990, 1991). Despite this knowledge and the fact that unsuberized roots of many woody perennials are scarce or absent during periods of the growing season when peak transpiration requires much water (MacFall et al., 1991), we still know little about how suberized portions of perennial rooting systems contribute to radial water absorption across species.The composite transport model (Steudle, 2001) is a conceptual framework describing water transport into plant roots. This model posits that water is able to flow into the root via multiple parallel pathways, traveling either in the cell walls (apoplastic) and/or from cell to cell (symplastic and/or transcellular). Transport across the cell-to-cell pathway can involve water crossing plasma membranes; thus, the rate of water uptake can be influenced by the abundance and activity of aquaporins (i.e. water channels). The contribution of aquaporins to root water uptake has been the focus of numerous studies, and the absolute magnitude of this contribution appears to be highly variable, ranging from 20% to 90% across species (for review, see Javot and Maurel, 2002). Steudle (2000) suggested that radial water flow would be dominated by aquaporin regulation in heavily suberized roots, as flow through the apoplast would be minimized. The localization of aquaporins should play a critical role in defining their impact on radial water uptake across suberized and unsuberized roots. For herbaceous species, peak aquaporin mRNA and/or protein levels have been found in root tips and the endodermis, pericycle, phloem, and xylem tissues (Schäffner, 1998; Otto and Kaldenhoff, 2000; Suga et al., 2003; Fraysse et al., 2005; Knipfer et al., 2011). Few aquaporin localization studies have been conducted in woody perennials (Vandeleur et al., 2009). Recent work from our laboratory revealed a precipitous drop in aquaporin expression between the grapevine (Vitis spp. rootstocks) root tips and older root portions (Gambetta et al., 2012). These observations led to this study, where we explore patterns of aquaporin localization in Vitis species fine roots and how they intersect with the structural anatomy and patterns of suberization to affect water uptake along the root length.Hydraulic conductivity (Lp_r) of the apoplastic pathway can be altered through changes in cell wall chemistry, especially through the deposition of suberin. Suberized apoplastic barriers in plant roots include the Casparian band of the endodermis and the suberin lamella of the endodermis, exodermis, and periderm in woody species (Esau, 1977). Casparian bands and suberin lamella are solute impermeable (for review, see Peterson and Enstone, 1996), but across studies, the extent to which they impede the flow of water is highly variable (Peterson et al., 1993; Steudle et al., 1993; Peterson and Enstone, 1996; Schreiber et al., 2005). Regardless, studies support the idea that in roots there is always some flow across the cell-to-cell pathway due to apoplastic barriers and/or an osmotic component to the driving gradient (Steudle et al., 1993; Miyamoto et al., 2001; Knipfer and Fricke, 2011). In the cell-to-cell pathway, Lp_r can be altered by intrinsic plasma membrane properties, plasmodesmata (Oparka and Prior, 1992; Roberts and Oparka, 2003), and/or the abundance and activity of aquaporins. Changes in aquaporin gene expression and protein activity remain potentially dynamic and can occur within hours, while alterations of suberized apoplastic barriers are permanent and would manifest over longer developmental time frames.The total water potential gradient across a fine root can be composed of both osmotic (ΔΨOs) and hydrostatic (ΔΨHy) pressure gradients. A purely ΔΨOs requires that some portion of the pathway be cell to cell. A purely ΔΨHy should drive flow through both pathways, and the proportion of flow through the two pathways will be determined by their Lp_r. Experimentally, Lp_r generated under ΔΨHy is typically greater than Lp_r generated under ΔΨOs, typically ranging from 2-fold to more than 100-fold greater (Steudle et al., 1987; Hallgren et al., 1994; Miyamoto et al., 2001; Knipfer and Fricke, 2011). In some cases, Lp_r is nearly equal under both types of gradients (Miyamoto et al., 2001; Knipfer and Fricke, 2011). These results suggest that if Lp_r through the apoplast were to be reduced by the presence of an apoplastic barrier, this would force flow across a cell-to-cell pathway regardless of the driving gradient (Steudle, 2000).In this study, we sought to provide a more detailed understanding of the localization of aquaporin expression and its contribution to radial water uptake in different zones of grapevine fine roots, from the unsuberized actively growing root tip to portions of the fine root undergoing secondary growth and containing a developed periderm. We characterized the developmental anatomy along the length of the fine root, including the localization of suberized structures, and quantified tissue-specific mRNA levels of plasma membrane aquaporin isogenes via a combination of laser-capture microdissection (LCM) and quantitative PCR. Finally, we determined the Lp_r of root tips and secondary growth root zones under both ΔΨOs and ΔΨHy while investigating the contribution of aquaporin activity to Lp_r via chemical inhibition.     

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.     

EXO70A1-Mediated Vesicle Trafficking Is Critical for Tracheary Element Development in Arabidopsis

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type– or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.