Lipid Composition of Latex and Rubber Particles in Hevea brasiliensis and Taraxacum kok-saghyz.

Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Pará rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85-99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (< 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.

Heterologous activation of the Hevea PEP16 promoter in the rubber-producing laticiferous tissues of Taraxacum kok-saghyz.

Hevea brasiliensis, the most abundant rubber crop, is used widely for the commercial production of natural rubber. To reduce the risk of a shortage in the supply of natural rubber that may arise from a single major rubber crop, rubber dandelion (Taraxacum kok-saghyz) has been developed as an alternative rubber-producing crop by using a transgenic approach. However, it is necessary to identify a suitable promoter for the transfer of rubber biosynthesis-related genes to the species. In this study, the promoter region of H. brasiliensis PEP16, which was isolated as a potentially important component in rubber biosynthesis, was sequenced and a pPEP16::GUS fusion construct was introduced into T. kok-saghyz. Histological and fluorometric studies using transgenic T. kok-saghyz plants indicated that the HbPEP16 promoter was highly activated in a laticiferous tissue-specific manner under normal growth conditions and that promoter activation was tightly regulated by various hormones and external signals. These findings suggested that the HbPEP16 promoter may be a useful molecular tool for the manipulation of gene expression in the laticiferous tissues of T. kok-saghyz.

Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects.

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR-enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)-the synthesis of the high molecular weight rubber polymer itself-is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.

SCYL2 Genes Are Involved in Clathrin-Mediated Vesicle Trafficking and Essential for Plant Growth.

Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a ß-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.

Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Translocation of phospholipase A2α to apoplasts is modulated by developmental stages and bacterial infection in Arabidopsis.

Phospholipase A(2) (PLA(2)) hydrolyzes phospholipids at the sn-2 position to yield lysophospholipids and free fatty acids. Of the four paralogs expressed in Arabidopsis, the cellular functions of PLA(2)α in planta are poorly understood. The present study shows that PLA(2)α possesses unique characteristics in terms of spatiotemporal subcellular localization, as compared with the other paralogs that remain in the ER and/or Golgi apparatus during secretory processes. Only PLA(2)α is secreted out to extracellular spaces, and its secretion to apoplasts is modulated according to the developmental stages of plant tissues. Observation of PLA(2)α-RFP transgenic plants suggests that PLA(2)α localizes mostly at the Golgi bodies in actively growing leaf tissues, but is gradually translocated to apoplasts as the leaves become mature. When Pseudomonas syringae pv.~tomato DC3000 carrying the avirulent factor avrRpm1 infects the apoplasts of host plants, PLA(2)α rapidly translocates to the apoplasts where bacteria attempt to become established. PLA(2)α promoter::GUS assays show that PLA(2)α gene expression is controlled in a developmental stage- and tissue-specific manner. It would be interesting to investigate if PLA(2)α functions in plant defense responses at apoplasts where secreted PLA(2)α confronts with invading pathogens.

Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination.

The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.

Phospholipase A(2) is required for PIN-FORMED protein trafficking to the plasma membrane in the Arabidopsis root.

Phospholipase A(2) (PLA(2)), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA(2)s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA(2)alpha, a PLA(2) isoform, colocalized with the Golgi marker. Impairments of PLA(2) function by PLA(2)alpha mutation, PLA(2)-RNA interference (RNAi), or PLA(2) inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA(2) hydrolytic product) restored the PM localization of PINs in the pla(2)alpha mutant and the ONO-RS-082-treated seedling. Suppression of PLA(2) activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair-specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA(2) inhibitor treatments, PLA(2)alpha mutation, or PLA(2)-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA(2), likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.

Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis.

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis.

Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A(1) activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.

Multiple forms of secretory phospholipase A2 in plants.

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.