High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of L. maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in CGA biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Comparing anatomy, chemical composition, and water permeability of suberized organs in five plant species: wax makes the difference.

MAIN CONCLUSION: The efficiency of suberized plant/environment interfaces as transpiration barriers is not established by the suberin polymer but by the wax molecules sorbed to the suberin polymer. Suberized cell walls formed as barriers at the plant/soil or plant/atmosphere interface in various plant organs (soil-grown roots, aerial roots, tubers, and bark) were enzymatically isolated from five different plant species (Clivia miniata, Monstera deliciosa, Solanum tuberosum, Manihot esculenta, and Malus domestica). Anatomy, chemical composition and efficiency as transpiration barriers (water loss in m s-1) of the different suberized cell wall samples were quantified. Results clearly indicated that there was no correlation between barrier properties of the suberized interfaces and the number of suberized cell layers, the amount of soluble wax and the amounts of suberin. Suberized interfaces of C. miniata roots, M. esculenta roots, and M. domestica bark periderms formed poor or hardly any transpiration barrier. Permeances varying between 1.1 and 5.1 × 10-8 m s-1 were very close to the permeance of water (7.4 × 10-8 m s-1) evaporating from a water/atmosphere interface. Suberized interfaces of aerial roots of M. deliciosa and tubers of S. tuberosum formed reasonable transpiration barriers with permeances varying between 7.4 × 10-10 and 4.2 × 10-9 m s-1, which were similar to the upper range of permeances measured with isolated cuticles (about 10-9 m s-1). Upon wax extraction, permeances of M. deliciosa and S. tuberosum increased nearly tenfold, which proves the importance of wax establishing a transpiration barrier. Finally, highly opposite results obtained with M. esculenta and S. tuberosum periderms are discussed in relation to their agronomical importance for postharvest losses and tuber storage.

Identifying new compounds with potential pharmaceutical and physiological activity in Areca catechu and Areca triandra via a non-targeted metabolomic approach.

INTRODUCTION: The fruits of Areca catechu, also called areca nuts, are widely used as popular masticatory and traditional herbal medicine in Asia. Besides arecoline and related alkaloids, limited information is available about further primary and secondary metabolites and their potential biological activities. OBJECTIVE: Here we aimed to further enhance our knowledge on phytochemical profiles of A. catechu and Areca triandra fruits. We intended to comprehensively identify metabolites in A. catechu and A. triandra fruits. METHODOLOGY: Metabolites were identified by ultra-performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS/MS). The occurrence of 12 selected bioactive compounds in 4 different developmental stages of A. catechu and A. triandra was quantified by LC-MS/MS. RESULTS: A total of 791 metabolites was identified. Of these, 115 metabolites could successfully be mapped to 44 Kyoto Encyclopedia of Genes and Genomes metabolic pathways, and 154 metabolites occurred at significantly different levels in A. catechu compared to A. triandra. Several components with known biological activities were identified for the first time in A. catechu and A. triandra. The abundance of many of these new components was similar in A. catechu and A. triandra, but significantly different between the pericarp and the seeds of A. catechu fruits. CONCLUSIONS: Metabolic profiles indicate that fruits of the Areca species compared here have similar primary and secondary metabolites. Our findings provide new insights into A. catechu and A. triandra as valuable sources for traditional medicine and they pave the way for further studies to potentially improve the underlying pharmaceutical and physiological effects.

Suberized transport barriers in plant roots: the effect of silicon.

Plant roots are the major organs that take up water and dissolved nutrients. It has been widely shown that apoplastic barriers such as Casparian bands and suberin lamellae in the endo- and exodermis of roots have an important effect on regulating radial water and nutrient transport. Furthermore, it has been described that silicon can promote plant growth and survival under different conditions. However, the potential effects of silicon on the formation and structure of apoplastic barriers are controversial. A delayed as well as an enhanced suberization of root apoplastic barriers with silicon has been described in the literature. Here we review the effects of silicon on the formation of suberized apoplastic barriers in roots, and present results of the effect of silicon treatment on the formation of endodermal suberized barriers on barley seminal roots under control conditions and when exposed to osmotic stress. Chemical analysis confirmed that osmotic stress enhanced barley root suberization. While a supplementation with silicon in both, control conditions and osmotic stress, did not enhanced barley root suberization. These results suggest that enhanced stress tolerance of plants after silicon treatment is due to other responses.

DEGENERATED PANICLE AND PARTIAL STERILITY 1 (DPS1) encodes a cystathionine ß-synthase domain containing protein required for anther cuticle and panicle development in rice.

Degeneration of apical spikelets and reduced panicle fertility are common reasons for low seed-setting rate in rice (Oryza sativa). However, little is known about the underlying molecular mechanisms. Here, we report a novel degenerated panicle and partial sterility 1 (dps1) mutant that showed panicle apical degeneration and reduced fertility in middle spikelets. dps1 plants were characterized by small whitish anthers with altered cuticle morphology and absence of pollen grains. Amounts of cuticular wax and cutin were significantly reduced in dps1 anthers. Panicles of dps1 plants showed an accumulation of reactive oxygen species (ROS), lower antioxidant activity, and increased programmed cell death. Map-based cloning revealed that DPS1 encodes a mitochondrial-localized protein containing a cystathionine ß-synthase domain that showed the highest expression in panicles and anthers. DPS1 physically interacted with mitochondrial thioredoxin proteins Trx1 and Trx20, and it participated in ROS scavenging. Global gene expression analysis in dps1 revealed that biological processes related to fatty acid metabolism and ROS homeostasis were significantly affected, and the expression of key genes involved in wax and cutin biosynthesis were downregulated. These results suggest that DPS1 plays a vital role in regulating ROS homeostasis, anther cuticle formation, and panicle development in rice.

PERSISTENT TAPETAL CELL2 Is Required for Normal Tapetal Programmed Cell Death and Pollen Wall Patterning.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

Root cortical senescence decreases root respiration, nutrient content and radial water and nutrient transport in barley.

The functional implications of root cortical senescence (RCS) are poorly understood. We tested the hypotheses that RCS in barley (1) reduces the respiration and nutrient content of root tissue; (2) decreases radial water and nutrient transport; and (3) is accompanied by increased suberization to protect the stele. Genetic variation for RCS exists between modern germplasm and landraces. Nitrogen and phosphorus deficiency increased the rate of RCS. Maximal RCS, defined as the disappearance of the entire root cortex, reduced root nitrogen content by 66%, phosphorus content by 63% and respiration by 87% compared with root segments with no RCS. Roots with maximal RCS had 90, 92 and 84% less radial water, nitrate and phosphorus transport, respectively, compared with segments with no RCS. The onset of RCS coincided with 30% greater aliphatic suberin in the endodermis. These results support the hypothesis that RCS reduces root carbon and nutrient costs and may therefore have adaptive significance for soil resource acquisition. By reducing root respiration and nutrient content, RCS could permit greater root growth, soil resource acquisition and resource allocation to other plant processes. RCS merits investigation as a trait for improving the performance of barley, wheat, triticale and rye under edaphic stress.

Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Pollen Development.

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction.

Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development.

Silicon Promotes Exodermal Casparian Band Formation in Si-Accumulating and Si-Excluding Species by Forming Phenol Complexes.

We studied the effect of Silicon (Si) on Casparian band (CB) development, chemical composition of the exodermal CB and Si deposition across the root in the Si accumulators rice and maize and the Si non-accumulator onion. Plants were cultivated in nutrient solution with and without Si supply. The CB development was determined in stained root cross-sections. The outer part of the roots containing the exodermis was isolated after enzymatic treatment. The exodermal suberin was transesterified with MeOH/BF3 and the chemical composition was measured using gas chromatography-mass spectroscopy (GC-MS) and flame ionization detector (GC-FID). Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) was used to determine the Si deposition across root cross sections. Si promoted CB formation in the roots of Si-accumulator and Si non-accumulator species. The exodermal suberin was decreased in rice and maize due to decreased amounts of aromatic suberin fractions. Si did not affect the concentration of lignin and lignin-like polymers in the outer part of rice, maize and onion roots. The highest Si depositions were found in the tissues containing CB. These data along with literature were used to suggest a mechanism how Si promotes the CB development by forming complexes with phenols.

Partial depolymerization of genetically modified potato tuber periderm reveals intermolecular linkages in suberin polyester.

Suberin is a biopolyester found in specialized plant tissues, both internal and external, with key frontier physiological functions. The information gathered so far from its monomer and oligomer composition, and in situ studies made by solid state techniques, haven't solved the enigma of how the suberin polyester is assembled as a macromolecule. To investigate how monomers are linked in suberin, we analyzed oligomer fragments solubilized by the partial depolymerization of suberin from potato (Solanum tuberosum) tuber periderms. The structure of the suberin oligomers, namely which monomers they included, and the type and frequency of the inter-monomer ester linkages, was assessed by ESI-MS/MS and high resolution NMR analysis. The analyzed potato periderms included the one from wild type (cv. Desirée) and from plants where suberin-biosynthesis genes were downregulated in chain elongation (StKCS6), ω-hydroxylation (CYP86A33) and feruloylation (FHT). Two building blocks were identified as possible key structures in the macromolecular development of the potato periderm suberin: glycerol - α,ω-diacid - glycerol, as the core of a continuous suberin aliphatic polyester; and glycerol - ω-hydroxyacid - ferulic acid, anchoring this polyaliphatic matrix at its periphery to the vicinal polyaromatics, through linking to ferulic acid. The silencing of the StKCS6 gene led to non-significant alterations in suberin structure, showing the relatively minor role of the very-long chain (>C28) fatty acids in potato suberin composition. The silencing of CYP86A33 gene impaired significantly suberin production and disrupted the biosynthesis of acylglycerol structures, proving the relevance of the latter and thus of the glycerol - α,ω-diacid - glycerol unit for the typical suberin lamellar organization. The silencing of the FHT gene led to a lower frequency of ferulate linkages in suberin polyester but to more polyphenolic guaiacyl units as seen by FTIR analyses in the intact polymer.

Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30°C), MS2 exhibits a K(m) for 16:0-Acyl Carrier Protein of 23.3 ± 4.0 µm, a V(max) of 38.3 ± 4.5 nmol mg⁻¹ min⁻¹, and a catalytic efficiency/K(m) of 1,873 M⁻¹ s⁻¹. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.

SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs.

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions.

Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

A radioactive assay allowing the quantitative measurement of cuticular permeability of intact Arabidopsis thaliana leaves.

Cuticular penetration of five different ¹4C-labeled chemicals (benzoic acid, bitertanole, carbaryl, epoxiconazole and 4-nitrophenol) into Arabidopsis thaliana leaves was measured and permeances P (ms⁻¹) were calculated. Thus, cuticular barrier properties of A. thaliana leaves have been characterized quantitatively. Epoxiconazole permeance of A. thaliana was 2.79 × 10⁻8 ms⁻¹. When compared with cuticular permeances measured with intact stomatous and astomatous leaf sides of Prunus laurocerasus, frequently used in the past as a model species studying cuticular permeability, A. thaliana has a 48- to 66-fold higher permeance. When compared with epoxiconazole permeability of isolated cuticles of different species (Citrus aurantium, Hedera helix and P. laurocerasus) A. thaliana permeability is between 17- to 199-fold higher. Co-permeability experiments, simultaneously measuring ¹4C-epoxiconazole and ³H2O permeability of isolated cuticles of three species (C. aurantium, H. helix and P. laurocerasus) showed that ³H2O permeability was highly correlated with epoxiconazole permeability. The regression equation of this correlation can be used predicting cuticular transpiration of intact stomatous leaves of A. thaliana, where a direct measurement of cuticular permeation using ³H2O is impossible. Water permeance estimated for A. thaliana was 4.55 × 10⁻8 m⁻¹, which is between 12- and 91-fold higher than water permeances measured with isolated cuticles of C. aurantium, H. helix and P. laurocerasus. This indicates that cuticular water permeability of the intact stomatous leaves of the annual species A. thaliana is fairly high and in the upper range compared with most P values of perennial species published in the past.

Phosphoenolpyruvate provision to plastids is essential for gametophyte and sporophyte development in Arabidopsis thaliana.

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/-)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/-) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/-) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.

The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

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 omega-hydroxyacid (approximately 70%) and alpha,omega-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 omega-functional fatty acids in establishing suberin structure and function.

Silencing of StKCS6 in potato periderm leads to reduced chain lengths of suberin and wax compounds and increased peridermal transpiration.

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C(28) and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C(26) and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C(28) and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration.

Differences between water permeability of astomatous and stomatous cuticular membranes: effects of air humidity in two species of contrasting drought-resistance strategy.

Cuticular water permeabilities of adaxial and abaxial leaf surfaces and their dependence on relative air humidity (RH) applied in long-term and short-term regimes have been analysed for Hedera helix, native in a temperate climate, and Zamioculcas zamiifolia, native in subtropical regions. The water permeability of cuticular membranes (CM) isolated from the adaxial (astomatous) and abaxial (stomatous) leaf sides was measured using a method which allowed the separation of water diffusion through the remnants of the original stomatal pores from water diffusion through the solid cuticle. The long-term effects of low (20-40%) or high (60-80%) RH applied during plant growth and leaf ontogeny ('growth RH') and the short-term effects of applying 2% or 100% RH while measuring permeability ('measurement RH') were investigated. With both species, water permeability of the solid stomatous CM was significantly higher than the permeability of the astomatous CM. Adaxial cuticles of plants grown in humid air were more permeable to water than those from dry air. The adaxial CM of the drought-tolerant H. helix was more permeable and more sensitive to growth RH than the adaxial CM of Z. zamiifolia, a species avoiding water stress. However, permeability of the solid abaxial CM was similar in both species and independent of growth RH. The lack of a humidity response in the abaxial CM is attributed to a higher degree of cuticular hydration resulting from stomatal transpiration. The ecophysiological significance of higher permeability of the solid stomatous CM compared to the astomatous CM is discussed.