Update on Cuticular Wax Biosynthesis and Its Roles in Plant Disease Resistance.

The aerial surface of higher plants is covered by a hydrophobic layer of cuticular waxes to protect plant tissues against enormous environmental challenges including the infection of various pathogens. As the first contact site between plants and pathogens, the layer of cuticular waxes could function as a plant physical barrier that limits the entry of pathogens, acts as a reservoir of signals to trigger plant defense responses, and even gives cues exploited by pathogens to initiate their infection processes. Past decades have seen unprecedented proceedings in understanding the molecular mechanisms underlying the biosynthesis of plant cuticular waxes and their functions regulating plant-pathogen interactions. In this review, we summarized the recent progress in the molecular biology of cuticular wax biosynthesis and highlighted its multiple roles in plant disease resistance against bacterial, fungal, and insect pathogens.

Cytological cycles and fates in Psidium myrtoides are altered towards new cell metabolism and functionalities by the galling activity of Nothotrioza myrtoidis.

The morphogenesis of galls occurs by the redifferentiation of cells that assume new functions in the modified host plant organs. The redifferentiated cells in the galls of Nothotrioza myrtoidis on Psidium myrtoides have low complexity metabolism and are photosynthesis-deficient. These galls were studied in search for evidences of the establishment of new cell cycles and fates and cytological gradients that corroborate their metabolic profile. Young and mature leaves of P. myrtoides and leaf galls induced by N. myrtoidis at different developmental stages were collected along 24 months and analyzed under light and transmission electron microscopy. The leaves of P. myrtoides are long-lasting and did not senesce within the analyzed period, while the galls have a shorter cycle, and senesce within 1 year. A homogenous parenchyma is established by a "standby-redifferentiation" of the chlorophyllous tissues, and sclerenchyma cells redifferentiate from parenchyma cells in the outer cortex of the mature galls. The lack of organelles, the underdeveloped lamellation of chloroplasts, and the occurrence of few plastoglobules are related to the photosynthetic deficiency of the galls. No cytological gradients were observed, but the organelle-rich cells of the vascular and perivascular parenchymas are similar to those of the nutritive tissues of galls induced by other insect taxa. These cells nearest to the feeding sites of N. myrtoidis present higher metabolism and well-developed apparatus for the prevention of oxidative stress. The features herein described corroborate the low metabolic profile of the galls as the cell cycles and fates of P. myrtoides are manipulated for completely new functionalities.

[Micromorphology of leaf epidermis of some Venezuelan rice cultivars (Poaceae) associated with the mechanical damage of Sogata T. orizicolus (Homoptera: Delphacidae)]. / Micromorfología de la epidermis foliar de cultivares de arroz Venezolano (Poaceae) asociado con el daño mecánico de sogata Tagosodes orizicolus (Homoptera: Delphacidae).

Rice cultivars are affected directly and indirectly by the insect sogata. The mechanical damage or direct loss, is produced after feeding and ovoposition on the young leaves tissues, while the indirect damage is produced after the transmission of the Rice hoja blanca virus. We studied the morpho-anatomic structures associated with the resistance of the mechanical damage produced by the insect, in six rice cultivars, including controls for resistance and susceptibility (Makalioka and Bluebonnet 50), during August 2011, in Fundacion Danac, Venezuela. Samples were taken from leaf 3, where cuticle thickness, presence of macrohair, microhair and silica bodies in the second third of the leaf was evaluated. A significant difference to thickness of the cuticle, the presence of microhair in the leaves, and presence of silica bodies was observed among cultivars, determining a significant correlation among the number of macrohair and microhair in the adaxial leaf blade with the presence of silica bodies, and thickness of the cuticle and number of posed insects. Thickness of the cuticle and presence of silica bodies in the intercostals space of microhair and macrohair showed to be the variables most related to mechanical damage and resistance mechanism.

Micromorfología de la epidermis foliar de cultivares de arroz Venezolano (Poaceae) asociado con el daño mecánico de sogata Tagosodes orizicolus (Homoptera: Delphacidae) / Micromorphology of leaf epidermis of some Venezuelan rice cultivars (Poaceae) associated with the mechanical damage of Sogata T. orizicolus (Homoptera: Delphacidae)

Rice cultivars are affected directly and indirectly by the insect sogata. The mechanical damage or direct loss, is produced after feeding and ovoposition on the young leaves tissues, while the indirect damage is produced after the transmission of the Rice hoja blanca virus. We studied the morpho-anatomic structures associated with the resistance of the mechanical damage produced by the insect, in six rice cultivars, including controls for resistance and susceptibility (Makalioka and Bluebonnet 50), during August 2011, in Fundacion Danac, Venezuela. Samples were taken from leaf 3, where cuticle thickness, presence of macrohair, microhair and silica bodies in the second third of the leaf was evaluated. A significant difference to thickness of the cuticle, the presence of microhair in the leaves, and presence of silica bodies was observed among cultivars, determining a significant correlation among the number of macrohair and microhair in the adaxial leaf blade with the presence of silica bodies, and thickness of the cuticle and number of posed insects. Thickness of the cuticle and presence of silica bodies in the intercostals space of microhair and macrohair showed to be the variables most related to mechanical damage and resistance mechanism.

El arroz es afectado directa e indirectamente por el insecto sogata. El daño directo o mecánico es debido a la postura de huevos y alimentación del tejido foliar por parte del insecto y el daño indirecto que ocurre es por la trasmisión del virus de la hoja blanca del arroz. El ensayo fue conducido en la Fundación DANAC en agosto 2011, con la finalidad de evaluar las estructuras morfoanatómicas asociadas con la resistencia al daño mecánico en seis cultivares de arroz que incluyen a los testigos de resistencia y susceptibilidad (Bluebonnet 50 y Makalioka). En el tercio medio de la tercera hoja, se evaluaron las variables grosor de la cutícula, presencia de macropelos, micropelos y cuerpos silíceos. Se observaron diferencias significativas entre los cultivares para el grosor de cutícula, número de micropelos y presencia de cuerpos silíceos, se determinó una correlación significativa entre el número de macro-pelos y micropelos en la cara adaxial con la presencia de cuerpos silíceos y el grosor de la cutícula y el número de insectos posados a las 96h. El grosor de la cutícula y la presencia de cuerpos silíceos en los espacios intercostales de los micropelos y macropelos mostraron ser los caracteres más relacionados con la resistencia al daño mecánico y los mecanismos de resistencia al insecto.

Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita.

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode's life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest.

The mechanism of pollinator specificity between two sympatric fig varieties: a combination of olfactory signals and contact cues.

BACKGROUND AND AIMS: Pollinator specificity facilitates reproductive isolation among plants, and mechanisms that generate specificity influence species boundaries. Long-range volatile attractants, in combination with morphological co-adaptations, are generally regarded as being responsible for maintaining extreme host specificity among the fig wasps that pollinate fig trees, but increasing evidence for breakdowns in specificity is accumulating. The basis of host specificity was examined among two host-specific Ceratosolen fig wasps that pollinate two sympatric varieties of Ficus semicordata, together with the consequences for the plants when pollinators entered the alternative host variety. METHODS: The compositions of floral scents from receptive figs of the two varieties and responses of their pollinators to these volatiles were compared. The behaviour of the wasps once on the surface of the figs was also recorded, together with the reproductive success of figs entered by the two Ceratosolen species. KEY RESULTS: The receptive-phase floral scents of the two varieties had different chemical compositions, but only one Ceratosolen species displayed a preference between them in Y-tube trials. Specificity was reinforced at a later stage, once pollinators were walking on the figs, because both species preferred to enter figs of their normal hosts. Both pollinators could enter figs of both varieties and pollinate them, but figs with extra-varietal pollen were more likely to abort and contained fewer seeds. Hybrid seeds germinated at normal rates. CONCLUSIONS: Contact cues on the surface of figs have been largely ignored in previous studies of fig wasp host preferences, but together with floral scents they maintain host specificity among the pollinators of sympatric F. semicordata varieties. When pollinators enter atypical hosts, post-zygotic factors reduce but do not prevent the production of hybrid offspring, suggesting there may be gene flow between these varieties.

Structural evidence in Plectroniella armata (Rubiaceae) for possible material exchange between domatia and mites.

Domatia are small structures on the lower surface of a leaf, usually taking the form of cavities, pouches, domes with an opening, or hairs (or a combination of these), and located in the axils between the main veins. They are found in many dicotyledons including certain members of the Rubiaceae. As part of an ongoing study of selected southern African members of the tribe Vanguerieae of this family, their structure in transverse section was investigated. In some taxa, such as Plectroniella armata, light microscopic (LM) observations revealed large numbers of stomata in the domatia as well as a number of channel-like structures extending across the cuticle toward the cavity of the domatia. The cuticle of the epidermis lining the domatia also appeared thicker than in other parts of the leaves. The epidermis in P. armata was also examined using transmission electron microscopy (TEM). Domatia have been shown to house mainly mites, many of which are predatory or fungivorous, in a symbiotic (mutualistic) relationship with the plant. To date, much research has focussed on the role of domatia in providing shelter for various organisms, their eggs and their young. However, the TEM study revealed the apparent "channels" and thick cuticle seen under LM to be electron dense non-cellulosic branching fibrils within pronounced, often closely spaced cuticular folds. The functional significance of these fibrils and folds requires further investigation. Folding of cell walls and membranes at ultrastructural level is usually functionally associated with an increased surface area to facilitate active exchange of compounds/metabolites. This may indicate that translocation of substances and/or other forms of communication is possible between the domatium and its inhabitants. This therefore suggests a far more active role for the leaf in the symbiotic relationship than was previously thought. More work is required to test such a possibility.

Rapid mobilization of membrane lipids in wheat leaf sheaths during incompatible interactions with Hessian fly.

Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time polymerase chain reaction analyses of 168 lipid-metabolism-related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.

Chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation.

Artificial aphid infestation experiments on the three chrysanthemum cultivars 'Keiun', 'Han6' and 'Jinba' showed that the three cultivars vary markedly in their resistance. Of the three cultivars, the most resistant ('Keiun') produced the longest, highest and densest trichomes, the largest and fullest gland cells, and the most wax on the lower leaf epidermis. Superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), polyphenol oxidase activity (EC 1.14.18.1) and phenylalanine ammonia lyase (EC 4.3.1.5) were enhanced by aphid herbivory. In the two more resistant cultivars ('Keiun' and 'Han6'), the activity of superoxide dismutase and ascorbate peroxidase enzymes rapidly increased following infestation, and their levels remained high from seventy-two to one hundred and sixty-eight hours after inoculation. We suggest that these two antioxidant enzymes contribute to aphid resistance of these chrysanthemum cultivars. All three cultivars showed quick responses to aphid infestation by increasing polyphenol oxidase and phenylalanine ammonia lyase activities during the early period after inoculation. Activities of these two defense enzymes were higher in the two resistant cultivars after 72h after inoculation, suggesting involvement of these two enzymes in aphid resistance.

Induction of phenolic glycosides by quaking aspen (Populus tremuloides) leaves in relation to extrafloral nectaries and epidermal leaf mining.

We studied the effect of epidermal leaf mining on the leaf chemistry of quaking aspen, Populus tremuloides, during an outbreak of the aspen leaf miner, Phyllocnistis populiella, in the boreal forest of interior Alaska. Phyllocnistis populiella feeds on the epidermal cells of P. tremuloides leaves. Eleven days after the onset of leaf mining, concentrations of the phenolic glycosides tremulacin and salicortin were significantly higher in aspen leaves that had received natural levels of leaf mining than in leaves sprayed with insecticide to reduce mining damage. In a second experiment, we examined the time course of induction in more detail. The levels of foliar phenolic glycosides in naturally mined ramets increased relative to the levels in insecticide-treated ramets on the ninth day following the onset of leaf mining. Induction occurred while some leaf miner larvae were still feeding and when leaves had sustained mining over 5% of the leaf surface. Leaves with extrafloral nectaries (EFNs) had significantly higher constitutive and induced levels of phenolic glycosides than leaves lacking EFNs, but there was no difference in the ability of leaves with and without EFNs to induce phenolic glycosides in response to mining. Previous work showed that the extent of leaf mining damage was negatively related to the total foliar phenolic glycoside concentration, suggesting that phenolic glycosides deter or reduce mining damage. The results presented here demonstrate that induction of phenolic glycosides can be triggered by relatively small amounts of mining damage confined to the epidermal tissue, and that these changes in leaf chemistry occur while a subset of leaf miners are still feeding within the leaf.

Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry.

Trichomes are specialized epidermal structures that function as physical and chemical deterrents against arthropod herbivores. Aerial tissues of cultivated tomato (Solanum lycopersicum) are populated by several morphologically distinct trichome types, the most abundant of which is the type VI glandular trichome that produces various specialized metabolites. Here, the effect of the hairless (hl) mutation on trichome density and morphology, chemical composition, and resistance to a natural insect herbivore of tomato was investigated. The results show that the major effect of hl on pubescence results from structural distortion (bending and swelling) of all trichome types in aerial tissues. Leaf surface extracts and isolated type VI glands from hl plants contained wild-type levels of monoterpenes, glycoalkaloids, and acyl sugars, but were deficient in sesquiterpene and polyphenolic compounds implicated in anti-insect defence. No-choice bioassays showed that hl plants are compromised in resistance to the specialist herbivore Manduca sexta. These results establish a link between the morphology and chemical composition of glandular trichomes in cultivated tomato, and show that hl-mediated changes in these leaf surface traits correlate with decreased resistance to insect herbivory.

Food bodies in Cissus verticillata (Vitaceae): ontogenesis, structure and functional aspects.

BACKGROUND AND AIMS: The distinction between pearl bodies (or pearl glands) and food bodies (FBs) is not clear; neither is our understanding of what these structures really represent. The present work examined the ontogenesis, structure, ultrastructure and histochemical aspects of the protuberances in Cissus verticillata, which have been described since the beginning of the 19th century as pearl glands or pearl bodies, in order to establish a relationship between their structure and function. METHODS: Segments of stems and leaves in different stages of development were collected and fixed for study under light microscopy as well as electron transmission and scanning microscopy. Samples of FBs were subjected to chemical analysis using thin-layer chromatography. KEY RESULTS: The FBs in C. verticillata are globose and attached to the plant by a short peduncle. These structures are present along the entire stem during primary growth, and on the inflorescence axis and the abaxial face of the leaves. The FBs were observed to be of mixed origin, with the participation of both the epidermis and the underlying parenchymatic cells. The epidermis is uniseriate with a thin cuticle, and the cells have dense cytoplasm and a large nucleus. The internal parenchymatic cells have thin walls; in the young structures these cells have dense cytoplasm with a predominance of mitochondria and plastids. In the mature FBs, the parenchymatic cells accumulate oils and soluble sugars; dictyosomes and rough endoplasmic reticulum predominate in the cytoplasm; the vacuoles are ample. Removal of the FBs appears to stimulate the formation of new ones, at the same place. CONCLUSIONS: The vegetative vigour of the plant seems to influence the number of FBs produced, with more vigorous branches having greater densities of FBs. The results allow the conclusion that the structures traditionally designated pearl glands or pearl bodies in C. verticillata constitute FBs that can recruit large numbers of ants.

Anti-herbivore structures of Paulownia tomentosa: morphology, distribution, chemical constituents and changes during shoot and leaf development.

BACKGROUND AND AIMS: Recent studies have shown that small structures on plant surfaces serve ecological functions such as resistance against herbivores. The morphology, distribution, chemical composition and changes during shoot and leaf development of such small structures were examined on Paulownia tomentosa. METHODS: The morphology and distribution of the structures were studied under light microscopy, and their chemical composition was analysed using thin-layer chromatography and high-performance liquid chromatography. To further investigate the function of these structures, several simple field experiments and observations were also conducted. KEY RESULTS: Three types of small structures on P. tomentosa were investigated: bowl-shaped organs, glandular hairs and dendritic trichomes. The bowl-shaped organs were densely aggregated on the leaves near flower buds and were determined to be extrafloral nectarines (EFNs) that secrete sugar and attract ants. Nectar production of these organs was increased by artificial damage to the leaves, suggesting an anti-herbivore function through symbiosis with ants. Glandular hairs were found on the surfaces of young and/or reproductive organs. Glandular hairs on leaves, stems and flowers secreted mucilage containing glycerides and trapped small insects. Secretions from glandular hairs on flowers and immature fruits contained flavonoids, which may provide protection against some herbivores. Yellow dendritic trichomes on the adaxial side of leaves also contained flavonoids identical to those secreted by the glandular hairs on fruits and flowers. Three special types of leaves, which differed from the standard leaves in shape, size and identity of small structures, developed near young shoot tips or young flower buds. The density of small structures on these leaf types was higher than on standard leaves, suggesting that these leaf types may be specialized to protect young leaves or reproductive organs. Changes in the small structures during leaf development suggested that leaves of P. tomentosa are primarily protected by glandular hairs and dendritic trichomes at young stages and by the EFNs at mature stages. CONCLUSIONS: The results indicate that P. tomentosa protects young and/or reproductive organs from herbivores through the distribution and allocation of small structures, the nature of which depends on the developmental stage of leaves and shoots.

The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.

Isoflavonoids are a group of secondary metabolites common to leguminous plants that play roles in nodulation and defense responses. Isoflavone synthase (IFS) catalyzes the key entry point step of isoflavone biosynthesis from the general phenylpropanoid pathway. We have cloned the 5' upstream regions of the genes encoding the two isoflavone synthase isoforms from soybean. We characterized the tissue-specific expression patterns of IFS1 and IFS2 genes of soybean by quantitative RT-PCR. Isoflavone synthase transcripts were detected primarily in the roots and seeds. In transgenic soybean plants in which the IFS1 promoter drove expression of the bacterial uidA (GUS) gene, the expression was localized to the root epidermis and root hairs. We detected differential tissue-specific expression of IFS1:GUS in response to nodulation and defense signals. Bradyrhizobium japonicum, a diazotropic symbiont of soybean, induced the expression of IFS1:GUS in root hairs and xylem poles in the young differentiation zone of the root. This observation suggests novel roles for isoflavonoids in nodulation. The defense related hormone salicylic acid induced the expression of IFS1:GUS in all cell types of the root. We also noticed corresponding alterations in the transcript levels of IFS1 and IFS2 in response to B. japonicum or salicylic acid as identified by quantitative RT-PCR. Using hairy root transformed soybean composite plants, we have identified regions of the IFS1 and IFS2 promoters mediating response to B. japonicum. We have also shown conserved root-specific expression of IFS1 in rice and Arabidopsis. The expression pattern of IFS1 in soybean is consistent with the physiological roles of isoflavonoids as defense compounds against pathogens and signal molecules to symbiotic bacteria in soybean.

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy.

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.