Characterization of growth and arachidonic acid production of Parietochloris incisa comb. nov (Trebouxiophyceae, Chlorophyta)

Arachidonic acid (AA) is a precursor of biologically activeprostaglandines and leukotrienes. The commercial source for AA at present is afungus, but the recently discovered coccoid green alga,Parietochlorisincisa comb. nov., in which over 90% of total AA is deposited intriacylglycerols, makes this species a potential candidate for commercialproduction of AA. We investigated the effect of the light-regime on cell-AAcontent and on culture productivity, by manipulating the intensity of the lightsource, the length of the light-path (LPL), and the population density ofcultures grown in flat plate glass reactors under both controlled laboratoryconditions (continuously illuminated) as well as outdoors. The effect ofnitrogen deprivation on culture content of AA and its productivity was alsostudied.In all experiments, the longer light-path reactors with the highest arealvolumes (L m^–2) yielded the highest culture-AA or the highestamount of AA harvested per illuminated reactor surface. Highest culture contentof AA was obtained in cultures exposed to strong light andnitrogen-deprivation.In contrast, highest cell-AA content was obtained in cultures receiving thelowest light-dose. Maximum culture content of AA obtained in the laboratory was2667 mg L^–1, reached after a 38-day growth period(of which the final 17 days took place in nitrogen-free medium), undercontinuous exposure to 2000 mol photon m^–2s^–1. Maximal culture content of AA outdoors wassignificantly lower compared with the maximum obtained in the laboratory.     

光强和氮源及其浓度对缺刻缘绿藻生长、油脂和花生四烯酸积累的影响

对缺刻缘绿藻（Parietochloris incisa（Reisigl） S.Watanabe）在不同光强和氮源及其浓度条件下的生长状况及油脂和花生四烯酸（AA）的积累规律进行了研究。结果显示,缺刻缘绿藻在3种氮源条件下均能较好地生长。在高氮浓度条件下,增大光强能显著提高缺刻缘绿藻的生物量并促进油脂和AA的积累。缺刻缘绿藻在300 μmol·m^-2·s^-1光强、8.8 mmol·L^-1NaNO₃条件下生物量达到最大（4.17 g·L^-1）。油脂含量在100 μmol·m^-2·s^-1光强、1.0 mmol·L^-1氮浓度下达到最高,分别为41.17%（NaNO₃）、42.04%（NH₄HCO₃）和39.96%（CO（NH₂）₂）。AA绝对含量在300 μmol·m^-2·s^-1光强、2.9 mmol·L^-1 NaNO₃条件下达到最高,占细胞干重的16.44%。油脂和AA产率,在300 μmol·m^-2·s^-1光强、以NaNO₃为氮源的条件下达到最大,分别为134.6 mg·L^-1·d^-1（1.0 mmol·L^-1）和35.85 mg·L^-1·d^-1（2.9 mmol·L^-1）。综合考虑成本等因素,选择NH₄HCO₃（5.9 mmol·L^-1）和CO（NH₂）₂（2.9 mmol·L^-1）为氮源、在300 μmol·m^-2·s^-1高光强下培养缺刻缘绿藻进行AA的生产为最优方案。     

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.     

氮、磷对缺刻缘绿藻生长、总脂及花生四烯酸积累的影响

本文以缺刻缘绿藻(Parietochloris incisa)为实验材料,研究了其在四种不同氮、磷浓度下的生长、总脂(TL)及花生四烯酸(AA)含量的变化,分析测定了低氮和低磷浓度下脂肪酸的组成。结果表明:在BG-11培养基基础上,氮浓度的改变对缺刻缘绿藻的生物量影响不明显,不同氮浓度下的最终生物量均在2.1 g.L-1左右。在低氮浓度下TL和AA达到最大,分别为33.37%(%干重)和36.63%(%总脂肪酸,TFA),其中AA占藻体干重的11.56%。不同磷浓度对缺刻缘绿藻的生长有显著影响,最终生物量在0.90～2.07 g.L-1之间。TL在8～20 mg.L-1的磷浓度范围内没有明显变化,为28%左右。TL与氮、磷浓度呈显著负相关关系,且低氮有利于AA的积累。     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Lipid and fatty acid composition of muscle and internal organs from Ruvettus pretiosus

The lipid composition of different muscular areas (dorsal, ventral, subcutaneous, periosteum) and organs (liver, gall bladder, testes) of a gempylid fish Ruvettus pretiosus was examined. Waxes were the main lipid fraction in muscular areas, while the liver and testes were quite enriched in triglycerides and phospholipids. Oleic acid and fatty acids of n-3 family were the most frequently found in total lipids.     

Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa

The relationships between pigment (carotenoid and chlorophyll) content with accumulation of total fatty acids (TFA) and arachidonic acid (AA) were studied in the green microalga Parietochloris incisa (Trebouxiophyceae, Chlorophyta) grown under different PFDs (35, 200, and 400 μmol photons m⁻² s⁻¹) and nitrogen availabilities. The growth of P. incisa under higher light and nitrogen deficiency was accompanied by accumulation of FA, an increase in carotenoid and a decline in chlorophyll content. It was found that the carotenoid-to-chlorophyll ratio (but not the individual pigment content) correlates closely with the volumetric content of both TFA and AA. Analysis of scattering-compensated absorption spectra of P. incisa suspensions revealed their tight relationship in the blue-green range of the spectrum with the carotenoid-to-chlorophyll ratio, TFA, and AA content. These findings allowed the development of algorithms for the non-destructive assay of TFA and AA in cell suspensions in the ranges of 0.09–3.04 and 0.04–1.7 μg mL⁻¹, with accuracy of 0.06 and 0.01 μg mL⁻¹, respectively, via analytically measured carotenoid-to-chlorophyll ratio and using the ratio of absorption coefficients at 510 and 678 nm, with accuracy of 0.07 and 0.02 μg mL⁻¹, respectively. The feasibility of obtaining essential spectral information concerning the physiological condition of P. incisa using a standard spectrophotometer is also shown.     

Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa

The effects of light and nitrogen deficiency on biomass, fatty acid content and composition were studied in Parietochloris incisa, the unicellular freshwater chlorophyte accumulating very high amounts of arachidonic-acid-rich triacylglycerols. P. incisa cultures grown on complete nutrient medium and under high light (400 μmol photons m^{− 2} s⁻¹) showed the highest rate of growth in comparison to medium (200 μmol photons m⁻² s⁻¹) and low (35 μmol photons m⁻² s⁻¹) light intensity. Cultures grown under high light (on complete BG-11 medium) attained higher volumetric contents of total fatty acids and arachidonic acid due to greater increase in biomass. Nitrogen starvation brought about a strong increase in the arachidonic acid proportion of total fatty acids. Thus, adjustments to cultivation conditions could serve as an efficient tool for manipulation of yield and relative content of arachidonic acid in P. incisa. The significance of the changes in lipid metabolism for adaptation of P. incisa to high-light stress and nitrogen deficiency is also discussed.     

Lipids and fatty acid composition of developing winged bean seeds

The moisture, lipids and fatty acid composition of developing winged bean (Psophocarpus tetragonolobus) seeds were studied. The moisture content decreased steadily as the seeds matured. The lipid content increased gradually and reached a maximum ca 6 weeks after flowering (WAF). In the early stage (2 WAF) of the developing seeds there were more polar lipids (glycolipids and phospholipids) than neutral lipids but, as the seeds developed, neutral lipids gradually accumulated while the polar lipids decreased until 6 WAF. Thereafter, both the neutral lipid and polar lipid levels remained little changed. The amounts of palmitic and stearic acids decreased, but the level of behenic acid increased as the seeds matured. On the other hand, the oleic acid content increased while that of linolenic acid decreased rapidly as the seeds matured. The concentration of linoleic acid, however, fluctuated during the development of the seeds.     

Identification of unusual fatty acids of four alpine plant species from the Pamirs

Fatty acid composition and structure in total lipids from the green above-ground parts of four alpine plants, Oxygraphis glacialis, Primula macrophylla, Rhodiola pamiroalaica, and Swertia marginata, were established by GC and GC-MS. A total of 55 fatty acids was detected, and 48 of them were identified. Ubiquitous palmitate, linoleate, and linolenate predominated in the lipids accounting for about 72-90% of the total fatty acids. At the same time, the latter contained numerous species, which were unusual for higher plants and included saturated odd-numbered n-acids (six C15-C25 species, 0.26-1.40%), saturated even-numbered very-long-chain n-acids (six C20-C30 species, 1.00-2.49%), iso-acids (nine C15-C26 species, 0.64-1.53%), anteiso-acids (four C15-C20 species, 0.08-1.57%), certain uncommon mono- and dienoic acids, as well as 16:3omega3, 18:3omega6, and 18:4omega3 acids that are absent from the most higher plants. Nine fatty acids were found here for the first time in higher plants and two may be new to science. The evidence on the unusual fatty acids is discussed with respect to their distribution in living organisms, pathways of biosynthesis, and chemotaxonomic role.     

Influence of nitrogen deficiency on biochemical composition of the green alga <Emphasis Type="Italic">Botryococcus</Emphasis>

A study has been carried out to investigate the influence of nitrogen deficiency on intracellular lipid composition, including total fatty acid composition of lipids, polar lipids, and triacylglycerols, of the alga Botryococcus braunii Kütz IPPAS H-252 in batch culture. Under nitrogen limitation, the alga accumulates lipids as triacylglycerols and the total fatty acid (FA) composition changes: trienoic acids decrease (from 52.8–57.2 to 19.5–24.7% of the total FAs) and the oleic acid increases (from 1.1–1.2 to 17.1–24.4%) as does the saturated acids (from 23.7–26 to 32.9–46.1%). A similar rearrangement in the FA spectrum occurs at later times in the control culture, but it is less pronounced. Under nitrogen limitation, considerable changes in the polar lipid FAs are registered at day 13: saturated acids increase (from 28.6–35.5 to 76.8%) and all polyenoic acids markedly decrease (from 56.9–64.1 to 6.8%). Changes in the triacylglycerol fatty acid spectrum are seen on day 7: the oleic acid increases (from 14.7 to 34.2%) and remains at a high level till the end of the culture. In the control, triacylglycerols with large contents of oleic acid are detected at day 13, the total lipids and triacylglycerols still remaining unchanged.     

氮源类型和水平对3株球状绿藻生长、油脂和花生四烯酸积累的影响

【背景】缺刻叶球藻(Lobosphaera incisa Reisigl)是一种单细胞球状绿藻,是已知花生四烯酸(Arachidonic acid,AA)含量最丰富的植物资源之一。然而目前其分类和命名仍然较为混乱。【目的】明确3株球状绿藻(SAG2468、SAG2043、H4301)的分类地位及在不同氮源(硝酸钠、尿素、碳酸氢铵、碳酸铵、硝酸铵、氯化铵和硫酸铵)和氮浓度(18mmol/L,3mmol/L)条件下油脂和AA积累的特性。【方法】通过分子系统学和形态观察的方法对3株球状绿藻进行分类界定;采用干重法、重量法和气相色谱分析的方法分别对其生物量、油脂含量、脂肪酸组成及AA含量进行测定。【结果】3株球状绿藻均隶属于叶球藻属(Lobosphaera),SAG2468原定名为缺刻缘绿藻(Parietochloris incisa),现修订为缺刻叶球藻(Lobosphaera incisa),与H4301为缺刻叶球藻的不同地理株系。SAG2043原定名为双隔蚁形藻(Myrmecia bisecta),现修订为双隔叶球藻(Lobosphaera bisecta)。3株微藻在高氮(18 mmol/L)和低氮(3 mmol/L)浓度的硝酸钠和尿素培养条件下均可良好生长,铵盐对藻细胞生长普遍有抑制作用,且浓度越高抑制作用越显著。低氮胁迫能显著促进油脂和AA的积累(P0.05),SAG2043在3 mmol/L硝酸钠条件下油脂和AA产率最高,分别为142.15mg/(L·d)和35.51mg/(L·d),明显高于另外2株微藻(P0.05)。此时SAG2043对应获得的生物量为4.9 g/L,油脂含量为43.49%,AA含量高达干重的10.86%,占总脂肪酸含量的31.75%。【结论】SAG2043是一株更具AA开发潜力的微藻。     

Influence of culture temperature on the growth,biochemical composition and fatty acid profiles of six Antarctic microalgae

The growth, biochemical composition and fatty acid profiles of six Antarctic microalgae cultured at different temperatures, ranging from 4, 6, 9, 14, 20 to 30 ^∘C, were compared. The algae were isolated from seawater, freshwater, soil and snow samples collected during our recent expeditions to Casey, Antarctica, and are currently deposited in the University of Malaya Algae Culture Collection (UMACC). The algae chosen for the study were Chlamydomonas UMACC 229, Chlorella UMACC 234, Chlorella UMACC 237, Klebsormidium UMACC 227, Navicula UMACC 231 and Stichococcus UMACC 238. All the isolates could grow at temperatures up to 20 ^∘C; three isolates, namely Navicula UMACC 231 and the two Chlorella isolates (UMACC 234 and UMACC 237) grew even at 30 ^∘C. Both Chlorella UMACC 234 and Stichococcus UMACC 238 had broad optimal temperatures for growth, ranging from 6 to 20 ^∘C (μ = 0.19 – 0.22 day^–1) and 4 to 14 ^∘C (μ = 0.13 – 0.16 day^–1), respectively. In contrast, optimal growth temperatures for NaviculaUMACC 231 and Chlamydomonas UMACC 229 were 4 ^∘C (μ = 0.34 day^–1) and 6–9 ^∘C (μ = 0.39 – 0.40 day^–1), respectively. The protein content of the Antarctic algae was markedly affected by culture temperature. All except Navicula UMACC 231 and Stichococcus UMACC 238 contained higher amount of proteins when grown at low temperatures (6–9 ^∘C). The percentage of PUFA, especially 20:5 in Navicula UMACC 231 decreased with increasing culture temperature. However, the percentages of unsaturated fatty acids did not show consistent trend with culture temperature for the other algae studied.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

Changes in glycerolipids and their fatty acid composition during maturation of tobacco seeds

Triacylglycerol, which was one of the minor lipid components in immature seeds of tobacco, accumulated dramatically between 7 and 27 days after flowering and, in mature seeds at 37 days, the fatty acid methyl esters of the triacylglycerols comprised 96.3% of those of the total lipids. Diacylglycerols and sterol ester also increased significantly during seed development. Phosphatidylcholine and phosphatidylethanolamine, which were major components in immature seeds, decreased constantly with increasing maturation as well as the quantities of phosphatidylinositol and phosphatidylglycerol. Monogalactosyldiacylglycerols, digalactosyldiacylglycerols and sulfoquinovosyldiacylglycerols also decreased and disappeared in mature seeds. In the triacylglycerols the percentages of palmitate, stearate and linolenate fell with increasing seed age, while that of linoleate increased up to 75.3% in mature seeds. A similar trend was observed in the fatty acid composition in the diacylglycerols and sterol ester. Generally, in the phospholipids the proportions of linoleate and linolenate decreased with concomitant increases of stearate and oleate.     

Lipid classes and fatty acid composition of rotifers (Brachionus plicatilis) fed two algal diets

Rotifers (Brachionus plicatilis), maintained on baker's yeast, were fed for 24h upon two algal diets, Isochrysis galbana (diet A) and Isochrysis galbana + Nannochloropsis gaditana (diet B). (These algal diets were selected for their potential use as essential fatty acid (EFA) boosters, taking into account the requirements of fish larvae). The effect of these algal diets on total lipid content, lipid classes and fatty acid composition was studied. The total lipid content increased after feeding upon both diets but no significant differences were found between the two types. Neutral lipid and polar lipid contents increased and a positive correlation was observed between the neutral lipids content of rotifers and that of the food supplied. However, the content of polar lipids in rotifers did not depend upon that of the diet. The increase in neutral lipid content was found to be higher in rotifers fed upon diet B, compared to diet A which increased the phospholipid content. Non-enriched rotifers contained only small amounts of polyenoic fatty acids, i.e. 18:3n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3, the contents of which increased significantly by feeding both diets. The EFA composition (20:4n-6, 20:5n-3 and 22:6n-3) of neutral lipids and phopholipids in rotifers reflected the EFA composition of each diet. Diet B-fed rotifers had the highest content in 20:4n-6 and 20:5n-3, whereas rotifers fed diet A and the highest 22:6n-3 content. The mixed diet I. galbana + N. gaditana enhanced substantially the composition of lipid classes i.e. neutral lipids and of n-3 PUFA of rotifers in comparison with Isochrysis or yeast diets.     

Combined effects of irradiance level and carbon source on fatty acid and lipid class composition in the microalga Pavlova lutheri commonly used in mariculture

Pavlova lutheri, a marine Pavlovophyceae, has been well documented as it is commonly used as a food source in mariculture. In this study, we investigated the combined effects of carbon sources and irradiance levels on the growth, lipid classes and fatty acid profiles of this microalga. The microalga was cultured at 15 °C with a 14 h photoperiod in artificial seawater containing bicarbonate or acetate as carbon source.The growth and lipid composition of P. lutheri were more sensitive to variations in light intensity than in carbon source. However, P. lutheri seems to be able to use acetate to growth cell and lipid metabolism. With the both carbon source, the lowest cellular lipid contents were obtained under low light intensity. The proportions of PUFAs, especially EPA, were significantly higher under low light, and saturating fatty acids and DHA levels were significantly higher under high light. In P. lutheri, galactolipids, a major component of chloroplast lipid membranes, made up approximately 54-66% of total lipids. The highest PUFA levels, such as those of EPA, were predominantly found in the galactolipid fraction when the cells were grown at low light, regardless of the carbon source. The consequent accumulation of n-3 fatty acids in the galactolipids could facilitate thylakoid membrane fluidity, and therefore the velocity of electron flow involved in photosynthesis during light acclimatization. These results could be used to optimize the culture conditions and the nutritional value of this microalga, which is used to feed marine invertebrates and fish larvae in mariculture hatcheries, and to produce n-3 fatty acids for human health care and nutrition.     

脂肪酸对中华哲水蚤摄食两种海洋微藻的指示作用

在室内以饥饿培养为对照,以海洋原甲藻(Prorocentrum micans)和中肋骨条藻(Skeletonema costatum)培养中华哲水蚤(Calanus sinicus),研究了各脂肪酸标记对中华哲水蚤摄食不同饵料的指示作用。结果显示,海洋原甲藻中18 ∶ 4ω3、22 ∶ 6ω3含量较高,中肋骨条藻中16 ∶ 1ω7、20 ∶ 5ω3的含量较高。二者分别表现出典型的甲藻门和硅藻门的脂肪酸组成特征。中华哲水蚤的脂肪酸组成有两个特点:(1)20 ∶ 5ω3和22 ∶ 6ω3的含量均较高;(2)其体内表征桡足类浮游植物食性、由桡足类自身合成的20 ∶ 1和22 ∶ 1脂肪酸占有相当的比例。虽然中华哲水蚤对不同脂肪酸的吸收和转化效率不同,但以脂肪酸作为标记还是成功的指示了中华哲水蚤对微藻的摄食。在饥饿培养中,首先消耗的是那些浮游动物自身不能合成的多不饱和脂肪酸,而结构脂肪酸都表现出了较高的保守性。结合各脂肪酸标记变化趋势和Pearson相关性分析的结果认为,18 ∶ 4ω3、18 ∶ 4ω3/16 ∶ 1ω7、∑18/∑16能较好的指示中华哲水蚤对海洋原甲藻的摄食,仅16 ∶ 1ω7/18 ∶ 4ω3能指示中华哲水蚤对中肋骨条藻的摄食。     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Chemotaxonomy of the Rubiaceae family based on leaf fatty acid composition

With 10,700 species distributed in 637 genera, the Rubiaceae family is one of the largest of the angiosperms. Since it was previously evidenced that the fatty acid composition of photosynthetic tissues can be a tool for chemotaxonomic studies, the fatty acid composition of leaves from 107 Rubiaceae species highly representative of the diversity of the family was determined. Principal component analysis allowed a clear-cut separation of Coffeae, Psychotrieae and Rubieae. The occurrence of C16:3 fatty acid, a marker of the prokaryotic plastidial lipid biosynthetic pathway, concerned at least two branches: Theligoneae/Rubieae and Anthospermeae-Anthosperminae which appeared to be in close relationship. Additional experiments were carried out to ensure the correlation between the presence of C16:3 fatty acid and the prokaryotic biosynthetic pathway.