Biochemical changes across a carbon saturation gradient: Lignin,cutin, and suberin decomposition and stabilization in fractionated carbon pools

Soils that exhibit soil organic carbon (SOC) saturation provide an opportunity to examine mechanisms of C storage in soils with increasingly limited C-stabilization potential. A manure rate experiment in Lethbridge, Alberta, in which SOC responded asymptotically to long-term manure C additions, allowed us to assess changes in SOC biochemical composition in response to soil C saturation. By quantifying the cupric oxide oxidation products of lignin, cutin, and suberin in fractionated SOC pools that are characterized by chemical (i.e., mineral-associated), physical (i.e., microaggregate-associated), or no protection (i.e., free particulate organic matter), we evaluated the interaction between C saturation and the biochemical characteristics of SOC.We tested the specific responses of soil fraction lignin, cutin, and suberin to C saturation level by using the bulk soil to approximate C-input composition across manure input treatments. Carbon-normalized lignin (lignin-VSC/OC) in the chemically protected fractions did not differ, while in the non-protected and physically protected soil fractions, it decreased with C saturation level. Neither the stabilization of cutin and suberin, nor the lignin:cutin + suberin ratio, differed in any of the measured soil fractions in response to C saturation level.These results indicate that with C saturation and decreased C stabilization potential, lignin, cutin, or suberin were not preferentially stabilized or depleted in mineral protected soil C pools. The lack of evidence for biochemical preference in mineral associations with C saturation supports the existence of an outer kinetic zone of organomineral associations, in which partitioning of organic compounds, rather than sorption, controls mineral SOC accumulation at high SOC loadings. Furthermore, despite theories of inherent lignin recalcitrance, depleted lignin concentrations with C saturation in the non-protected and aggregate protected fractions indicate that lignin was, in this study, preferentially decomposed when not protected by association with mineral phases in the soil. In conclusion, C-input quantity, and not quality, combined with physical and chemical protection mechanisms that govern long-term C storage, appeared to control C saturation and stabilization at this site.     

分层破胚剥皮玉米不同部位营养成分富集特征

为明确玉米籽粒营养成分的分布差异及不同部位富集特征,应用快速缓苏、微量着水半湿法分层破胚剥皮技术,结合靶向代谢组学方法,对郑单958玉米不同部位的营养成分及基础代谢物质进行分析与比较。结果表明玉米籽粒不同部位的淀粉、脂肪、矿物元素和膳食纤维等营养物质含量存在显著差异（P<0.05）。该研究中的玉米内皮层可能主要由种皮、糊粉层及部分外胚乳构成,该部位营养成分的种类及含量均较为丰富,其中水溶性膳食纤维含量显著高于其他部位（P<0.05）,可作为玉米水溶性膳食纤维的提取分离来源。K、P和Mg元素是玉米中含量最高的矿物元素,主要存在于胚芽中,Fe、Zn、Mn和Cu元素在胚芽和玉米皮层中均有较多分布,精制加工会导致这些矿物元素的损失。玉米胚芽中水解氨基酸种类较其他部位丰富且含量较高（P<0.05）,甜味氨基酸占总游离氨基酸含量的24.49%,高于玉米皮层部位、显著高于胚乳部位。研究结果为玉米营养健康食品的创制、玉米精深加工及相关专用装备的研发提供参考。     

Accuracy of Klason lignin and acid detergent lignin methods as assessed by bomb calorimetry.

An accurate method for estimation of lignin concentration is important for prediction of the digestible energy content of livestock feeds. The accuracy of lignin concentration estimates based on the Klason lignin and acid detergent lignin methods was compared. Ten diverse forage samples were analyzed for protein, carbohydrates, lipids, organic acids, ash, lignin (by both methods), and gross energy. The accuracy of the two lignin concentration estimates was examined by comparing the measured forage gross energy to a gross energy value calculated from the compositional analysis. Use of the acid detergent lignin estimate in this gross energy calculation accounted for 68-84% of the forage gross energy compared to 85-97% of the gross energy using Klason lignin. These results indicate that while Klason lignin estimates are substantially higher than acid detergent lignin estimates, Klason lignin is the more accurate lignin method and does not overestimate lignin because gross energy recoveries were less than 100%.     

高湿度贮藏环境保持香梨表皮蜡质延缓衰老进程

为了探讨相对湿度对库尔勒香梨表皮蜡质的影响及与耐贮性的关系,该文设置3个不同相对湿度(25℃,40%~45%、60%~65%和80%~85%),研究不同湿度贮藏下香梨表皮蜡质含量和化学组分的变化及与贮藏品质的相关性。通过气相色谱-质谱联用技术(gas chromatography-mass spectrometer,GC-MS)对不同湿度贮藏香梨表皮蜡质的化学组分进行比较分析,同时测定好果率、失重率、表观色泽、硬度、可溶性固形物含量、可滴定酸含量、乙烯释放速率和呼吸强度等贮藏品质参数,最后对蜡质含量、化学组分及贮藏参数做相关性分析。结果表明:对照组(相对湿度40%~45%)和处理组的蜡质均由烷烃、烯烃、脂肪酸、醇类、醛类、酯类和萜类组成,但所含具体物质种类和含量略有差异。不同湿度贮藏条件对蜡质含量和各化学组分均有影响,与对照组相比,相对湿度80%~85%贮藏可显著保持蜡质含量、烷烃和醛类物质,延缓脂肪酸和醇类物质增加(P<0.05)。蜡质化学组分及采后衰老之间存在密切相关性,各蜡质组分中,酯类和萜类物质对贮藏品质影响最大,其次是烷烃和醇类物质,其中:酯类物质与失重率、硬度和好果率极显著相关(P<0.01);萜类物质与失重率和好果率极显著相关(P<0.01);烷烃物质与失重率极显著相关(P<0.01)。总体来看,在贮藏过程中,高湿度贮藏环境通过保持表皮蜡质含量和化学组分延缓香梨衰老进程,维持稳定的蜡质化学组成对采后香梨贮藏品质的保持具有正向作用。     

Chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses by dogs to select corn fibers

The objective was to examine the chemical composition, in vitro fermentation characteristics, and in vivo digestibility responses of fiber-rich corn coproducts resulting from corn wet milling. Native corn fibers, native corn fibers with fines, hydrolyzed corn fibers, and hydrolyzed extracted corn fibers were analyzed chemically and their capacity to produce short-chain fatty acids determined. Ash content was low (<1.2%), crude protein content varied little, but fat and fiber concentrations varied widely. Most fiber was in the insoluble form, with glucose being predominant followed by xylose. Total short-chain fatty acid production ranged from 211.6 to 699.52 micromol/g of dry matter, whereas branched-chain fatty acid production was low. Four corn fibers (native and processed) were included in a canine diet matrix at the 7% inclusion level. Nutrient digestibility, food intake, and fecal characteristics were not affected by corn fiber inclusion in canine diets, suggesting that they should be considered as potential dietary fiber sources in dog foods.     

Characterization of Miscanthus giganteus Lignin Isolated by Ethanol Organosolv Process under Reflux Condition

Miscanthus giganteus lignin was extracted by an organosolv process under reflux conditions (4 h) with varying concentrations of ethanol (65%, 75%, 85%, 95%) and 0.2 M hydrochloric acid as catalyst. The resulting lignin was extensively characterized by size exclusion chromatography (SEC), Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and chemical analysis (residual sugars, Klason lignin, ash). The predominant linkage units present were β-O-4' (82-84%), resinol (6-7%), and phenylcoumaran (10-11%). The 65% ethanol solvent system gave the lowest lignin yield (14% of starting biomass) compared to 29-32% of the other systems. Increasing ethanol concentration resulted in decreasing carbohydrate content of the lignins (3.6-1.1%), a higher solubility in tetrahydrofuran (THF), a slight reduction of the molecular weight (M(w) 2.72-2.25 KDa), an increasing α-ethoxylation, and an increase in ethoxylated phenylpropenoic compounds (p-coumaric and ferulic acid), but the S/G ratio of the monolignols (0.63, GC/MS) and Klason lignin content (86-88%) were unaffected. An extraction method for these ethyl-esterified phenylpropenoids and smaller molecular weight lignin compounds was developed. The effect of reaction time (2, 4, and 8 h) was investigated for the 95% ethanol solvent system. Besides increased lignin yield (13-43%), a slight increase in M(w) (2.21-2.38 kDa) and S/G ratio (0.53-0.68, GC-MS) was observed. Consecutive extractions suggested that these changes were not from lignin modifications (e.g., condensations) but rather from extraction of lignin of different composition. The results were compared to similar solvent systems with 95% acetone and 95% dioxane.     

Multivariate characterization of the fatty acid profile of spanish cookies and bakery products

The fatty acid compositions of 39 brands of cookies and bakery products were analyzed with special attention to the their trans-fatty acid content. The average contents (percent w/w of the total fatty acids detected) for the different nutritional fractions were as follows: saturated fatty acids (SFA), 49.43% (SD = 17.04); monounsaturated fatty acids (MUFA), 32.87% (SD = 10.94); polyunsaturated fatty acids (PUFA), 12.48% (SD = 11.29); and trans-fatty acids (TFA), 5.20% (SD = 9.30), the mean fat content being 22.7% (SD = 7.4). C18:1t was the predominant trans isomer found in all of the analyzed samples (mean = 4.04, SD = 8.46). Factor analysis performed on the fatty acid profile allowed a meaningful classification of the samples according to the main source of fat employed in their elaboration. Four factors that explained 75% of the total variance were retained. According to the results obtained, the fat used to elaborate the samples was mainly composed of vegetable and animal fat, and only in a few samples were partially hydrogenated vegetable oils used as the main source of fat.     

Characterization of dietary fiber lignins from fruits and vegetables using the DFRC method

Insoluble fiber fractions from 11 fruits and vegetables were investigated for their lignin composition using the derivatization followed by reductive cleavage (DFRC) methodology. To enrich lignin contents and to minimize polysaccharide excess that led to nonanalyzable DFRC chromatograms, the insoluble fibers were degraded by a carbohydrolases mixture. The residues that were found to be representative for the insoluble fiber lignins were analyzed. The investigated fibers differ considerably in their lignin contents and also in their lignin compositions. With the exception of radish fiber, only trace amounts (or none) of the products resulting from p-hydroxyphenyl units were detected. Lignins noticeably differed in the ratio of the DFRC products resulting from syringyl units (S) and guaiacyl (G) units (G/S ratios ranged from approximately 39 to 0.2). The insoluble fiber lignins were classified as G-rich lignins (G/S ratio > 3; carrot, spinach, kiwi, curly kale, radish, and asparagus), S-rich lignins (S/G ratio > 3; rhubarb), or balanced lignins (0.3 < G/S ratio < 3; pear, apple, small radish, and kohlrabi). Information about further structural characteristics, for example, cinnamyl endgroups, was obtained from the analysis of DFRC minor products.     

潮土中残留小麦和玉米秸秆养分含量差异及与微生物群落组成的关系

黄淮海平原典型潮土上小麦和玉米收获后的秸秆往往直接还田,但驱动它们在不同质地潮土(砂质、壤质、黏质)中分解的微生物是否与残留秸秆养分含量有关尚不清楚.本研究基于尼龙网袋法,通过10个月的田间培育试验,监测秸秆分解率、残留秸秆养分含量及微生物群落组成,评估各指标在秸秆类型和土壤质地之间的差异,探究残留秸秆养分与微生物群落...     

CPMAS 13C NMR and IR spectra of spruce and pine litter and of the Klason lignin fraction at different stages of decomposition

Fresh and decomposed spruce and pine litter and the Klason lignin fraction of spruce needles at different stages of decomposition were studied by CPMAS ¹³C NMR and IR spectroscopy as well as by chemical methods. It was shown that decomposition of needles is accompanied by an increase in aliphatic substances and carboxyl group content; the amount of polysaccharides is reduced. It is assumed that stable aliphatic compounds like cutin and lipids of microbial origin will accumulate during litter decomposition and humification. Aromaticity is low and does not alter drastically. The NMR spectra of the Klason lignin fraction show pronounced peaks at 30, 55, 115, 130, 150 and 175 ppm. Obviously, this fraction contains appreciable amounts of aliphatic and carboxyl carbon besides the typical aromatic units of lignin. During decomposition aromaticity decreases whereas the relative amounts of aliphatic substances and carboxyl groups increase. This is probably due to splitting of aromatic ring structures and side chains. The findings agree with the results from chemical analyses.     

Effects of konjac glucomannan on putative risk factors for colon carcinogenesis in rats fed a high-fat diet

The aim of this study was to determine effects of konjac glucomannan (KGM) in a high fat corn oil diet on risk factors of colon carcinogenesis, that is, fecal β-glucuronidase, mucinase, and bile acids, and on preventive factors, that is, fecal microflora and cecal short-chain fatty acids (SCFAs). Sprague-Dawley rats (n = 8 animals per group) were fed a normal-fat fiber-free (5% corn oil, w/w) or high-fat (25% corn oil, w/w) diet containing no fiber, KGM (5%, w/w), or inulin (5%, w/w, as a prebiotic control) for 4 weeks. Results indicated that the high-fat fiber-free diet significantly elevated the fecal β-glucuronidase and mucinase activities and total bile acid concentration and decreased cecal SCFA contents, as compared with its normal-fat counterpart. The incorporation of KGM, as well as inulin, into the high-fat fiber-free diet beneficially reduced the fecal β-glucuronidase and mucinase activities and lithocholic acid (secondary bile acid) concentration. Although KGM elevated the daily fecal total bile acid excretion, the change was due to the primary, instead of the secondary, bile acids. In addition, KGM beneficially promoted the daily fecal excretion of bifidobacteria and lactobacilli and cecal SCFA contents, as compared with the high-fat fiber-free diet. Therefore, the present study suggests that KGM potentially attenuated the high fat-induced risk in colon carcinogenesis.     

Impact of plant cell wall network on biodegradation in soil: Role of lignin composition and phenolic acids in roots from 16 maize genotypes

Residue quality is a key factor governing biodegradation and the fate of C in soil. Most investigations of relationships existing between crop residue quality and soil decomposition have been based on determining the relative proportions of soluble, cellulose, hemicellulose and lignin components. However, cell wall cohesion is increased by tight interconnections between polysaccharides and lignin that involve cross-linking agents (phenolic acids). The aim of this study was to determine the role of lignin composition and phenolic acids on short- to medium-term decomposition of maize roots in soil. Sixteen maize genotypes, presenting a range of chemical characteristics related to root lignin and phenolic acids, were used. The main components were characterized by Van Soest (VS) extraction and cell wall acid hydrolysis, and the non-condensed Syringyl and Guaicyl lignin monomers, esterified phenolic acids and etherified phenolic acids were determined. Maize roots were then incubated in soil under controlled conditions (15 °C, −80 kPa moisture) for 796 days. Results showed that VS extraction over-estimated the structural hemicellulose content and that VS lignin was more recalcitrant than Klason lignin. The tremendous effect of cell wall chemical characteristics was shown by marked variations (almost two-fold differences in C mineralization), between the 16 maize roots. Decomposition was controlled by soluble residue components in the short term whereas lignin and the interconnections between cell wall polymers were important in the long-term. Notably the cell wall domain rich in non-condensed lignin and esterified phenolic acids was prone to decomposition whereas the presence of etherified ferulic acids seemed to hamper cell wall decomposition.     

NMR characterization of lignins isolated from fruit and vegetable insoluble dietary fiber

Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.     

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.     

Solid-substrate fermentation of corn fiber by Phanerochaete chrysosporium and subsequent fermentation of hydrolysate into ethanol

The goal of this study was to develop a fungal process for ethanol production from corn fiber. Laboratory-scale solid-substrate fermentation was performed using the white-rot fungus Phanerochaete chrysosporium in 1 L polypropylene bottles as reactors via incubation at 37 degrees C for up to 3 days. Extracellular enzymes produced in situ by P. chrysosporium degraded lignin and enhanced saccharification of polysaccharides in corn fiber. The percentage biomass weight loss and Klason lignin reduction were 34 and 41%, respectively. Anaerobic incubation at 37 degrees C following 2 day incubation reduced the fungal sugar consumption and enhanced the in situ cellulolytic enzyme activities. Two days of aerobic solid-substrate fermentation of corn fiber with P. chrysosporium, followed by anaerobic static submerged-culture fermentation resulted in 1.7 g of ethanol/100 g of corn fiber in 6 days, whereas yeast ( Saccharomyces cerevisiae) cocultured with P. chrysosporium demonstrated enhanced ethanol production of 3 g of ethanol/100 g of corn fiber. Specific enzyme activity assays suggested starch and hemi/cellulose contribution of fermentable sugar.     

Policosanol Contents and Compositions in Wax‐Like Materials Extracted from Selected Cereals of Korean Origin

Policosanols, long‐chained alcohols, have been reported to have beneficial physiological activities. Content and composition of policosanols in wax‐like materials extracted from selected cereals of Korean origin were determined. Wax‐like materials were extracted using hot hexane. Yields of wax‐like materials from unpolished grain sorghum, polished grain sorghum, brown rice, purple rice, wheat, and maize were 223, 37, 33, 61, 10, and 10 mg/100 g of dry kernels, respectively. Policosanol contents, as determined using HPLC, in the wax‐like materials from the cereals were 33, 29, 6, 0, and 2% (w/w, db), respectively. Major alcohols in the policosanols from grain sorghum were octacosanol and triacontanol. Docosanol was the major alcohol in the policosanols from brown rice, purple rice, wheat, and maize.     

Relationship between fatty acid profile and vitamin E content in maize hybrids (Zea mays L.)

The balance between the vitamin E (tocochromanols) and polyunsaturated fatty acid (PUFA) contents mainly determines the susceptibility to lipid peroxidation and the storage stability of corn oil. In 1997, field experiments were conducted at two different locations to evaluate a collection of 30 corn hybrids for fatty acid profiles and tocochromanol contents. Hybrids differed significantly (p < 0.01) for major fatty acids, as well as for tocochromanol contents and composition. The major fatty acids were palmitic, oleic, and linoleic acids, whose contents were in the ranges 9.2-12.1%, 19.5-30.5%, and 53.0-65.3%, respectively. The tocopherol contents ranged as follows: alpha-tocopherol, 67-276 mg (kg of oil)(-1); beta-tocopherol, 0-20 mg (kg of oil)(-1); gamma-tocopherol, 583-1048 mg (kg of oil)(-1); delta-tocopherol, 12-71 mg (kg of oil)(-1); total tocopherol, 767-1344 mg (kg of oil)(-1). gamma-Tocopherol was the predominant derivative among all tocopherols. The tocotrienol contents were in the ranges 46-89, 53-164, and 99-230 mg (kg of oil)(-1) for alpha-, gamma-, and total tocotrienol contents, respectively. The tocotrienol profile was not characterized by the predominance of any tocotrienol homologue. alpha-Tocopherol was positively correlated with PUFA (r = 0.41) and with the vitamin E equivalent (vit E equiv) (r = 0.84), and it was not correlated with gamma-tocopherol. gamma-Tocopherol was highly correlated with total tocopherol and tocochromanol contents (r = 0.93 and r = 0.90, respectively), indicating that the contribution of this vitamer to the total tocochromanol content is the most important among all tocochromanols. The high positive correlation found between the vit E/PUFA ratio and the vit E equiv, as well as the absence of correlation between this ratio and PUFA indicates that a higher vit E/PUFA ratio can be easier achieved be increasing the vitamin E content than by modifying fatty acid profile in corn oil.     

Comparison of values derived from selected flours by fat acidity methods of the American Association of Cereal Chemists and the International Organization for Standardization

Fat acidity values were determined on 8 commercially available flours by the American Association of Cereal Chemists method (AACC-02-01A) and by the International Organization for Standardization method (ISO-7305). Comparisons of means obtained by these 2 methods indicated that the ISO method produced results that were 1.4-2.5 times greater (P less than 0.001) than those for the AACC method. These data are consistent with a previous report which indicated that acidic materials (phosphates and amino acids) other than fatty acids are extracted by 95% alcohol (ISO method), whereas fatty acids alone are extracted by petroleum ether (AACC method). Fat acidity values obtained by AACC methodology employing Soxhlet and continuous extraction were also compared. No difference in fat acidity means existed with respect to extraction technique except for an aged whole wheat flour sample which produced a higher value (P = 0.05) by continuous extraction.     

Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products

The contents of free and total phenolic acids and alk(en)ylresorcinols were analyzed in commercial products of eight grains: oat (Avena sativa), wheat (Triticum spp.), rye (Secale cerale), barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), millet (Panicum miliaceum), rice (Oryza sativa), and corn (Zea mays). Avenanthramides were determined in three oat products. Free phenolic acids, alk(en)ylresorcinols, and avenanthramides were extracted with methanolic acetic acid, 100% methanol, and 80% methanol, respectively, and quantified by HPLC. The contents of total phenolic acids were quantified by HPLC analysis after alkaline and acid hydrolyses. The highest contents of total phenolic acids were in brans of wheat (4527 mg/kg) and rye (4190 mg/kg) and in whole-grain flours of these grains (1342 and 1366 mg/kg, respectively). In other products, the contents varied from 111 mg/kg (white wheat bread) to 765 mg/kg (whole-grain rye bread). Common phenolic acids found in the grain products were ferulic acid (most abundant), ferulic acid dehydrodimers, sinapic acid, and p-coumaric acid. The grain products were found to contain either none or only low amounts of free phenolic acids. The content of avenanthramides in oat flakes (26-27 mg/kg) was about double that found in oat bran (13 mg/kg). The highest contents of alk(en)ylresorcinols were observed in brans of rye (4108 mg/kg) and wheat (3225 mg/kg). In addition, whole-grain rye products (rye bread, rye flour, and whole-wheat flour) contained considerable levels of alk(en)ylresorcinols (524, 927, and 759 mg/kg, respectively).     

Biosynthesis, molecular structure, and domain architecture of potato suberin: a (13)C NMR study using isotopically labeled precursors

Although suberin in potato wound periderm is known to be a polyester containing long-chain fatty acids and phenolics embedded within the cell wall, many aspects of its molecular structure and polymer-polymer connectivities remain elusive. The present work combines biosynthetic incorporation of site-specifically (13)C-enriched acetates and phenylalanines with one- and two-dimensional solid-state (13)C NMR spectroscopic methods to monitor the developing suberin polymer. Exogenous acetate is found to be incorporated preferentially at the carboxyl end of the aliphatic carbon chains, suggesting addition during the later elongation steps of fatty acid synthesis. Carboxyl-labeled phenylalanine precursors provide evidence for the concurrent development of phenolic esters and of monolignols typical of lignin. Experiments with ring-labeled phenylalanine precursors demonstrate a predominance of sinapyl and guaiacyl structures among suberin's phenolic moieties. Finally, the analysis of spin-exchange (solid-state NOESY) NMR experiments in ring-labeled suberin indicates distances of no more than 0.5 nm between pairs of phenolic and oxymethine carbons, which are attributed to the aromatic-aliphatic polyester and the cell wall polysaccharide matrix, respectively. These results offer direct and detailed molecular information regarding the insoluble intermediates of suberin biosynthesis, indicate probable covalent linkages between moieties of its polyester and polysaccharide domains, and yield a clearer overall picture of this agriculturally important protective material.