Isolation and Analysis of Differential Expressed ESTs from Stem Trichomes of Lycopersicon esculentum (Solanaceae)

Glandular trichomes are special organs involved in plant defense response and synthesis of volatile secondary metabolites, analyzing trichome specific expressed sequence tags will help us further understand the specific function of plant trichomes. In this paper, suppression subtractive hybridization（SSH） based on magnetic beads technology was used to isolate differential expressed genes of the glandular trichomes in Lycopersicon esculentum. The differential expressing cDNA library was constructed using the glandular trichomes cDNA as tester and the cDNA from the stem without glandular trichomes as driver. After randomly sequencing 108 differential ESTs, Blast2go program was used to do blastx, functional annotation and metabolism analysis. The results show that most ESTs are related to substance metabolism, response to stress, biotic or abiotic stimulus, and have binding and catalytic function. These differential genes lay the foundation for further research on defense mechanism of the tomato trichomes.     

Glandular Trichomes on Alfalfa Impede Searching Behavior of the Potato Leafhopper Parasitoid

New cultivars of alfalfa, Medicago sativa L., have been released with glandular trichomes for resistance to potato leafhopper, Empoasca fabae (Harris). Yet, the impact of the glandular trichomes on the primary natural enemy of the leafhopper, Anagrus nigriventris Girault, is unknown. We compared the host searching behavior of the egg parasitoid on four alfalfa clones varying in trichome characters. Female wasps were videotaped on Ranger, a susceptible clone with relatively sparse trichomes, B14, a resistant clone with dense but nonglandular trichomes, and FG12 and FG18, two resistant clones with glandular trichomes. Although the number of leafhopper eggs per stem exposed to wasps did not significantly differ among the four clones, the frequency of foraging and total foraging time were less on the two clones with glandular trichomes than on the two clones with nonglandular trichomes. In addition, an analysis of covariance demonstrated that, although the number of ovipositional probes increased with egg density on a stem, the number of probes on stems with glandular trichomes was significantly less than that on stems without glandular trichomes. The allocation of time by wasps among drumming, probing, and grooming behaviors was similar among the clones. Wasps tended to fly off of clones with glandular trichomes more often than off of clones with nonglandular trichomes. This study suggests that cultivars with glandular trichomes may interfere with host searching by A. nigriventris.     

Role of the glandular trichomes in resistance of perennial alfalfa to the potato leafhopper (Homoptera: Cicadellidae)

Experiments were performed to elucidate resistance of glandular-haired alfalfa, Medicago sativa L., to the potato leafhopper, Empoasca fabae (Harris). The primary objective was to examine the potential role of the glandular trichomes and stem lignification for imparting resistance to this pest. During free-choice foliar discoloration experiments, the resistant alfalfa clone FGplh13 expressed lower levels of injury than the susceptible P5373 after 13 d. No-choice tests performed using nymphal potato leafhoppers showed higher levels of mortality associated with the resistant glandular-haired clone FGplh13, and no nymphs survived after 48 h. Additional no-choice experiments using FGplh13 and P5373 alfalfa with the glandular and nonglandular trichomes intact or removed show that mortality of nymphs and adults decreased after removal of the glandular trichomes from FGplh13. Nymphal and adult mortality and nymphal development time did not differ on FGplh13 with the trichomes removed and P5373 with the trichomes intact or removed. Firstinstar, and possibly second-instar, potato leafhoppers were entrapped by an exudate produced by the glandular trichomes on the FGplh13 alfalfa. Stem lignification and the number of vascular bundles did not differ between FGplh13 and P5373 alfalfas. The glandular trichomes on FGplh13 alfalfa appeared to provide the major host resistance factor, with resistance to adults being chemically based and resistance to nymphs being chemically and mechanically based. To maintain levels of potato leafhopper resistance, breeders would appear to benefit by continuing to select for the expression of the glandular trichome phenotype.     

Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562 aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.     

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.     

EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol

The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.     

龙葵腺毛发育的细胞学研究

利用光学显微镜、扫描电镜和透射电镜技术,观察了龙葵“四叶一心”期时叶片及茎表皮的腺毛的种类、分布,探究了不同类型腺毛的起源、生长、成熟、分泌、衰老等发育过程的细胞学特征;通过组织化学染色和荧光显微技术,观察了龙葵腺毛成分、分布,为龙葵的进一步开发利用提供参考。结果表明：（1）龙葵腺毛分为单细胞头腺毛和多细胞头腺毛两类,前者主要分布于茎表面和叶上下表皮,后者主要分布于茎表面的单细胞头腺毛之间、叶脉及叶边缘;（2）龙葵腺毛发育起始于表皮细胞突起,单细胞头腺毛行顶端生长,具1-4个柄细胞,四种类型;多细胞头腺毛可再分为一层、两层与三层多细胞头腺毛,另具三种特殊类型;（3）龙葵成熟腺毛具分泌能力,通过皮下空间的物质积累导致腺毛头细胞表面形成突起、包块、破口,最终释放分泌物;而头细胞与柄细胞随即皱缩、衰老。（4）超微结构显示,腺毛头细胞中内质网与高尔基体极为丰富,合成代谢及分泌活动活跃,产生大量包裹嗜锇物质的囊泡,囊泡与细胞壁融合,进而将嗜锇物质转移至细胞壁并积累,随后储存在角质层下的皮下空间直至分泌释放;（5）组织化学染色结果表明,腺毛含有萜类、生物碱、脂类、蛋白质、酚类和多糖。头细胞中主要含有萜类、生物碱、脂类、蛋白质、酚类和中性多糖;柄细胞中主要含有萜类、生物碱、脂类。     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.     

Immunolocalization of two ligninO-methyltransferases in stems of alfalfa (Medicago sativa L.)

Summary Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.