Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Evolutionary and Structural Analysis of PP16 in Viridiplantae.

Members of the phloem protein 16 (PP16) gene family are induced by elicitors in rice and the corresponding proteins from cucurbits, which display RNA binding and intercellular transport activities, are accumulated in phloem sap. These proteins facilitate the movement of protein complexes through the phloem translocation flow and may be involved in the response to water deficit, among other functions. However, there is scant information regarding their function in other plants, including the identification of paralog genes in non-vascular plants and chlorophytes. In the present work, an evolutionary and structural analysis of the PP16 family in green plants (Viridiplantae) was carried out. Data mining in different databases indicated that PP16 likely originated from a larger gene present in an ancestral lineage that gave rise to chlorophytes and multicellular plants. This gene encodes a protein related to synaptotagmin, which is involved in vesicular transport in animal systems, although other members of this family play a role in lipid turnover in endomembranes and organelles. These proteins contain a membrane-binding C2 domain shared with PP16 proteins in vascular plants. In silico analysis of the predicted structure of the PP16 protein family identified several ß-sheets, one α-helix, and intrinsically disordered regions. PP16 may have been originally involved in vesicular trafficking and/or membrane maintenance but specialized in long-distance signaling during the emergence of the plant vascular system.

Study of Mineral Composition and Quality of Fruit Using Vascular Restrictions in Branches of Sweet Cherry.

Calcium (Ca) and carbohydrate (CHO) supply in sweet cherry have been associated with fruit quality at harvest and during storage. There is little published information integrating CHO and Ca availability and distribution in sweet cherry and their effects on fruit quality. Accordingly, in the 2019-20 season, vascular restrictions were imposed on the phloem (girdling, G, stopping phloem flow) and xylem (transverse incision, S, cutting 50% of xylem cross-section area) of individual vertical branches of the sweet cherry combination 'Lapins'/Colt trained as Kym Green Bush system to modify mineral and CHO composition in fruit and associate such changes with quality at harvest and storage. The girdling to the phloem was used to induce changes in CHO distribution. The transverse incision to the xylem was a tool to modify Ca distribution. Five treatments (TR) were implemented: TR1-CTL = Control (without vascular restriction), TR2-G, at its base, TR3-G + G: at its base, and G further up at the change of year between the second and the third years of growth TR4--S and TR5-S + G. The vegetative (i.e., shoot and leaf growth), reproductive (i.e., fruit set and yield) development and stomatal conductance were monitored. Each branch was divided into the upper (1-and 2-year-old wood) and the lower (3-and 4-year-old wood) segments of the restriction applied. The quality and mineral composition (Ca, Mg, K, and N) of fruit borne on each segment were measured at harvest. The upper segment of TR3-G + G branches were harvested 10 d before the lower segment. The fruit from the upper segment of TR3-G + G was the largest, the sweetest, and had the higher titratable acidity concentration. However, fruits of this segment were the softest, had the lowest Ca concentrations, and had the highest ratios of N:Ca and K:Ca, compared with the other TRs. TR3-G + G branches developed the highest number of lateral current season shoots including shoots below the second girdling in the lower segment of the branch. This vegetative flow of growth would explain the mineral unbalance produced in the fruit from the upper segment of the branch. TR2-G did not register changes in fruit quality and mineral concentration compared with TR1-CTL. Surprisingly, the fruit from the branches with xylem restriction did not show changes in Ca concentration, suggesting that the xylem stream was enough to supply the fruit in branches without lateral shoot development. Fruit firmness was positively related to fruit Ca concentration and negatively related to the ratios of K:Ca and N:Ca.

Foliar Calcium Absorption by Tomato Plants: Comparing the Effects of Calcium Sources and Adjuvant Usage.

The deficiency of calcium (Ca) reduces the quality and shelf life of fruits. In this scenario, although foliar spraying of Ca2+ has been used, altogether with soil fertilization, as an alternative to prevent deficiencies, little is known regarding its absorption dynamics by plant leaves. Herein, in vivo microprobe X-ray fluorescence was employed aiming to monitor the foliar absorption of CaCl2, Ca-citrate complex, and Ca3(PO4)2 nanoparticles with and without using adjuvant. We also investigated whether Sr2+ can be employed as Ca2+ proxy in foliar absorption studies. Moreover, the impact of treatments on the cuticle structure was evaluated by scanning electron microscopy. For this study, 45-day-old tomato (Solanum lycopersicum L., cv. Micro-Tom) plants were used as a model species. After 100 h, the leaves absorbed 90, 18, and 4% of aqueous CaCl2, Ca-citrate, and Ca3(PO4)2 nanoparticles, respectively. The addition of adjuvant increased the absorption of Ca-citrate to 28%, decreased that of CaCl2 to 77%, and did not affect Ca3(PO4)2. CaCl2 displayed an exponential decay absorption profile with half-lives of 15 h and 5 h without and with adjuvant, respectively. Ca-citrate and Ca3(PO4)2 exhibited absorption profiles that were closer to a linear behavior. Sr2+ was a suitable Ca2+ tracer because of its similar absorption profiles. Furthermore, the use of adjuvant affected the epicuticular crystal structure. Our findings reveal that CaCl2 was the most efficient Ca2+ source. The effects caused by adjuvant suggest that CaCl2 and Ca-citrate were absorbed mostly through hydrophilic and lipophilic pathways.

Inner bark vs sapwood is the main driver of nitrogen and phosphorus allocation in stems and roots across three tropical woody plant communities.

Nutrient allocation is central to understanding plant ecological strategies and forest roles in biogeochemical cycles. Thought to be mainly driven by environmental conditions, nutrient allocation to woody organs, especially to living tissues, is poorly understood. To examine the role of differences in living tissues (sapwood, SW, vs inner bark, IB), organs, ecological strategies, and environmental conditions in driving nutrient allocation and scaling in woody plants, we quantified nitrogen and phosphorus in main stems and coarse roots of 45 species from three tropical ecosystems with contrasting precipitation, fire regime, and soil nutrients. Nutrient concentration variation was mostly explained by differences between IB and SW, followed by differences between species and, in the case of phosphorus, soil nutrient availability. IB nutrient concentrations were four times those of SW, with root tissues having slightly higher concentrations than stem tissues. Scaling between IB and SW, and between stems and roots, was generally isometric. In cross-sections, IB contributed half of total nutrients in roots and a third in stems. Our results highlight the important role of IB and SW for nutrient storage, the coordination in nutrient allocation across tissues and organs, and the need to differentiate between IB and SW to understand plant nutrient allocation.

Anastomosing laticifer in the primary and secondary structures of Calotropis procera (Aiton) W.T.Aiton (Apocynaceae) stems.

An in-depth understanding of the development and distribution of laticifer (latex secretory structure) will be important for the production of both rubber and medicines and will support studies on plant adaptations to their environments. We characterize here and describe the ontogenesis of the laticifer sytem in Calotropis procera (Apocynaceae), an invasive subshrub species in arid landscapes. Anatomical and histochemical evaluations of the primary and secondary structures of the stem were carried out on a monthly basis during a full year, with ultrastructural evaluations of laticifer on the stem apex during the rainy season. In the primary structure, laticifer differentiate early from procambium and ground meristem cells of the cortex and medulla and become concentrated adjacent to the external and internal phloem of the bicollateral bundles. In the secondary structure, laticifer differentiates from fusiform derivative cells of the phloem close to the sieve-tube elements. The laticifer is of the articulated, anastomosing, branched type, and it originates from precursor cells that loose the transversal and longitudinal walls by dissolution. Latex is a mixture of terpenes, alkaloids, flavonoids, mucilage, and proteins. The apical meristem and vascular cambium where the laticifer system begins its development are active throughout the year, including during the dry season. The vascular cambium produces phloem with laticifer precursor cells during the rainy season, with high temperatures and long days. The ability of C. procera to grow under water deficit conditions and produce laticifer throughout the year contribute to its wide distribution in arid environments.

Laticifer ontogenesis and the chemical constituents of Marsdenia zehntneri (Apocynaceae) latex in a semiarid environment.

MAIN CONCLUSION: Anastomosed laticifers with intrusive growth produce latex containing methyl comate and betulin with economic and ecological value in arid environments. Climatic factors influence laticifer development in the apical meristem and vascular cambium. Latex is a complex emulsion with high medicinal as well as ecological value related to plant survival. Marsdenia zehntneri is a shrubby plant that grows on limestone outcrops in the semiarid regions of Brazil. We sought to characterize the ontogenesis of the laticifers of this species and to relate that process to climatic seasonality and phenology through anatomical, ultrastructural, and micro-morphometric evaluations of the apical meristem and vascular cambium. The histochemistry of the secretory structure was investigated and the chemical composition of the latex was analyzed. Phenological assessments were performed by monitoring phenological events for 1 year. The laticifers network of M. zehntneri permeates the entire primary and secondary body of the plant, providing a wide distribution system of defensive compounds. Its laticifers, of a distinct mixed type (anastomosed, with intrusive growth), are numerous and voluminous in the apical meristem but scarce and minute in the secondary phloem. Latex secretion involves the participation of oleoplasts, polysomes, and dictyosomes. Methyl 2,3-dihydroxy-ursan-23-oate, methyl 3-hydroxy-ursan-23-oate, and betulin are encountered in high proportions in the latex and have ecological and medicinal functions. The development of primary laticifers is related to the resumption of apical meristem activity with increasing day length at the end of the austral winter. The development of secondary laticifers is related to high summer temperatures and rainfall that favor vascular cambium activity. The wide distribution of laticifers, their seasonal pattern of secretion, and their latex composition contribute to the adaptation of M. zehntneri to its natural environment.

Metabolomic Profiling of Phloem Sap from Different Pine Species and Implications on Black Capuchin.

In most commercial pine farms in southern Brazil, black capuchin causes damage to wood and financial losses when it removes bark from some pine species to feed upon underlying vascular tissues. Therefore, this study aimed to evaluate the variability of the primary metabolites of phloem saps from 10 different species of pine by NMR spectroscopy, as well as the aroma compounds using SPME-GC-MS. Each technique provided a different set of metabolites that we can correlate to monkey predilection. The PCA showed monosaccharide (detected by NMR) and α-pinene (pine-like and resinous flavor descriptors) as attractive compounds for monkeys. On the other hand, the low content of monosaccharide and the high content of ß-phellandrene (citrus odor descriptor) was observed in less attacked pine species (P. patula). The data fusion on primary metabolites and aroma compounds corroborated the individual analyses, complementing the comprehension of the monkey predilection. Thus, P. elliottii was an avoided tree even with high content of sugars possibly due to its high content of ß-phellandrene (citrus odor). The results are useful for further behavioral studies to determine the role that each highlighted metabolite plays in chemically mediated animal-plant interactions.

Phloem wedges in Malpighiaceae: origin, structure, diversification, and systematic relevance.

BACKGROUND: Phloem wedges furrowing the wood are one of the most notorious, widespread types of cambial variants in Angiosperms. Many lianas in Malpighiaceae show these variations in the arrangement of the secondary tissues. Here we explore their ontogeny, structure, and evolution in Malpighiaceae, where phloem wedges appeared multiple times, showing how they have contributed to the anatomical diversification of the family. Using a broad sampling with 143 species from 50 genera, covering all major lineages in Malpighiaceae, we crossed data from ontogeny, stem anatomy, and phylogenetic comparative methods to determine ontogenetic trajectories, final anatomical architectures, and evolution within the most recent phylogeny for the family. RESULTS: Phloem wedges appeared exclusively in lianas and disappeared in shrub lineages nested within liana lineages. At the onset of development, the vascular cambium is regular, producing secondary tissues homogeneously across its girth, but soon, portions of the cambium in between the leaf insertions switch their activity producing less wood and more phloem, initially generating phloem arcs, which progress into phloem wedges. In the formation of these wedges, two ontogenetic trajectories were found, one that maintains the continuity of the cambium, and another where the cambium gets dissected. Phloem wedges frequently remain as the main cambial variant in several lineages, while in others there are additional steps toward more complex cambial variants, such as fissured stems, or included phloem wedges, the latter a novel type of interxylary phloem first described for the family. CONCLUSIONS: Phloem wedges evolved exclusively in lianas, with two different ontogenies explaining the 10 independent origins of phloem wedges in Malpighiaceae. The presence of phloem wedges has favored the evolution of even more complex cambial variants such as fissured stems and interxylary phloem.

The NAC Transcription Factor ANAC087 Induces Aerial Rosette Development and Leaf Senescence in Arabidopsis.

CmNACP1 mRNA has been shown to move long distance through the phloem in Cucurbita maxima (pumpkin) and through a graft junction. Whereas the phloem transport of several different mRNAs has been documented in other systems as well, its function remains, for most of these RNAs, largely unknown. To gain insight into the possible role of these RNAs, we searched for the closest homologs of CmNACP1 in Arabidopsis, a model plant much more amenable for analysis. A phylogenetic approach using the predicted NAC domain indicated that ANAC059, ANAC092, ANAC079, ANAC100, ANAC046, and ANAC087 form a single clade with CmNACP1. In the present work, we analyzed the possible function of the ANAC087 gene in more detail. The promoter region of this gene directed expression in the vasculature, and also in trichomes, stem, apexes, and developing flowers which supports the notion that ANAC087 and CmNACP1 are orthologs. Overexpression of the ANAC087 gene induced increased branching in inflorescence stem, and also development of ectopic or aerial rosettes in T1 and T2 plants. Furthermore, overexpression of ANAC087 leads to accelerated leaf senescence in 44 days post-germination (dpg). Interestingly, a similar phenotype was observed in plants expressing the ANAC087 gene upstream region, also showing an increase in ANAC087 transcript levels. Finally, the results shown in this work indicate a role for ANAC087 in leaf senescence and also in rosette development.

Mobile forms of carbon in trees: metabolism and transport.

Plants constitute 80% of the biomass on earth, and almost two-thirds of this biomass is found in wood. Wood formation is a carbon (C)-demanding process and relies on C transport from photosynthetic tissues. Thus, understanding the transport process is of major interest for understanding terrestrial biomass formation. Here, we review the molecules and mechanisms used to transport and allocate C in trees. Sucrose is the major form in which C is transported in plants, and it is found in the phloem sap of all tree species investigated so far. However, in several tree species, sucrose is accompanied by other molecules, notably polyols and the raffinose family of oligosaccharides. We describe the molecules that constitute each of these transport groups, and their distribution across different tree species. Furthermore, we detail the metabolic reactions for their synthesis, the mechanisms by which trees load and unload these compounds in and out of the vascular system, and how they are radially transported in the trunk and finally catabolized during wood formation. We also address a particular C recirculation process between phloem and xylem that occurs in trees during the annual cycle of growth and dormancy. A search of possible evolutionary drivers behind the diversity of C-carrying molecules in trees reveals no consistent differences in C transport mechanisms between angiosperm and gymnosperm trees. Furthermore, the distribution of C forms across species suggests that climate-related environmental factors will not explain the diversity of C transport forms. However, the consideration of C-transport mechanisms in relation to tree-rhizosphere coevolution deserves further attention. To conclude the review, we identify possible future lines of research in this field.

Phytoplasma of 16SrVII-B subgroup associated to shoot proliferation in Physalis peruviana plants

Physalis is an herbaceous species native to the Andes region. Currently, it is cultivated in various Brazilian states due to the economic interest of growers for this new fruit. Physalis plants grown in the field showed symptoms of shoot proliferation, leaf malformation, and chlorosis. Since these symptoms are commonly induced by phytoplasmas, this study investigated to confirm the presence of these prokaryotes in symptomatic plants. After DNA extraction from symptomatic and asymptomatic plants, phytoplasmas were found in all affected plants through the nested PCR. Examination by transmission electron microscopy (TEM) using appropriately prepared segments of leaf veins allowed the visualization of typical pleomorphic cells of phytoplasmas in the phloem of symptomatic plants. The computer-simulated RFLP patterns and the phylogenetic analysis allowed identifying the detected phytoplasmas as a 'Candidatus Phytoplasma fraxini'-related strain belonging to the 16SrVII-B subgroup. Moreover, physalis was identified as an additional host species for phytoplasmas in the 16SrVII group, expanding the current knowledge on the host range of phytoplasmas in this group.

10Boron Is Mobile in Cowpea Plants.

Cowpea [Vigna unguiculata (L.) Walp] is cultivated in tropical and subtropical regions worldwide, but its production is usually limited by boron (B) deficiency, which can be mitigated by applying B via foliar spraying. In plants with nutrient mobility, the residual effect of foliar fertilization increases, which might improve its efficiency. An experiment was carried out to evaluate the concentration and mobility of the B isotopic tracer (10B) in different organs of cowpea plants, after the application of this micronutrient in the growing media and also to leaves. Treatments were designed based on B fertilization as follows: without B in the growth media, with 10B applied via foliar spraying (10B-L), with B in the growth media (substrate) and 10B via foliar spraying (10B-L + B-S), and with 10B in the growth media (substrate) without foliar spraying (10B-S), and a control without fertilization. A redistribution of 10B was observed in new leaves when the element was supplied via foliar spraying, resulting in greater leaf area, dry mass and dry matter production of aerial parts, and also the whole plant. 10Boron was redistributed when applied via foliar spraying in cowpea plants, regardless of the plant's nutritional status, which in turn might increase internal B cycling.

Genetic and Transcription Profile Analysis of Tissue-Specific Anthocyanin Pigmentation in Carrot Root Phloem.

In purple carrots, anthocyanin pigmentation can be expressed in the entire root, or it can display tissue specific-patterns. Within the phloem, purple pigmentation can be found in the outer phloem (OP) (also called the cortex) and inner phloem (IP), or it can be confined exclusively to the OP. In this work, the genetic control underlying tissue-specific anthocyanin pigmentation in the carrot root OP and IP tissues was investigated by means of linkage mapping and transcriptome (RNA-seq) and phylogenetic analyses; followed by gene expression (RT-qPCR) evaluations in two genetic backgrounds, an F2 population (3242) and the inbred B7262. Genetic mapping of 'root outer phloem anthocyanin pigmentation' (ROPAP) and inner phloem pigmentation (RIPAP) revealed colocalization of ROPAP with the P1 and P3 genomic regions previously known to condition pigmentation in different genetic stocks, whereas RIPAP co-localized with P3 only. Transcriptome analysis of purple OP (POP) vs. non-purple IP (NPIP) tissues, along with linkage and phylogenetic data, allowed an initial identification of 28 candidate genes, 19 of which were further evaluated by RT-qPCR in independent root samples of 3242 and B7262, revealing 15 genes consistently upregulated in the POP in both genetic backgrounds, and two genes upregulated in the POP in specific backgrounds. These include seven transcription factors, seven anthocyanin structural genes, and two genes involved in cellular transport. Altogether, our results point at DcMYB7, DcMYB113, and a MADS-box (DCAR_010757) as the main candidate genes conditioning ROPAP in 3242, whereas DcMYB7 and MADS-box condition RIPAP in this background. In 7262, DcMYB113 conditions ROPAP.

Inner bark as a crucial tissue for non-structural carbohydrate storage across three tropical woody plant communities.

Non-structural carbohydrates (NSC) are crucial for forest resilience, but little is known regarding the role of bark in NSC storage. However, bark's abundance in woody stems and its large living fraction make it potentially key for NSC storage. We quantified total NSC, soluble sugar (SS) and starch concentrations in the most living region of bark (inner bark, IB), and sapwood of twigs, trunks and roots of 45 woody species from three contrasting tropical climates spanning global extremes of bark diversity and wide phylogenetic diversity. NSC concentrations were similar (total NSC, starch) or higher (SS) in IB than wood, with concentrations co-varying strongly. NSC concentrations varied widely across organs and species within communities and were not significantly affected by climate, leaf habit or the presence of photosynthetic bark. Starch concentration tended to increase with density, but only in wood. IB contributed substantially to NSC storage, accounting for 17-36% of total NSC, 23-47% of SS and 15-33% of starch pools. Further examination of the drivers of variation in IB NSC concentration, and taking into account the substantial contribution of IB to NSC pools, will be crucial to understand the role of storage in plant environmental adaptation.

The retranslocation of boron is influenced by the nutritional status of cherry tomato plants / A retranslocação do boro é influenciada pelo estado nutricional do tomateiro cereja

Boron (B) retranslocation may confer greater tolerance to B deficiency or toxicity, resulting in plants and fruits with better nutritional quality. Most plants do not demonstrate phloem mobility for B; however, some plants have natural mobility and others express it under stress conditions, such as nutritional stress. Therefore, we investigated the phloem mobility of B in cherry tomato plants with different nutritional status. The plants were cultivated in nutrient solution with adequate and insufficient doses of Potassium (K) and B. Furthermore, the plants received foliar fertilization with B in a mature leaf (F) in a localized form. The B content and amount in leaves near F were compared with leaves of the same age and position on control plants. The experiment was carried out in a protected environment and conducted in a randomized complete block design in a subdivided plot scheme with four replicates. Re-translocation of B between neighboring leaves was observed when the plant was without K deficiency and with B deficiency. In this nutritional status, leaves next to fertilized leaf with B (F) showed higher B amount when compared to leaves of the same position of control plants. Evidence suggests that retranslocation of B over relatively short distances (between leaves) can be influenced by the nutritional status the cherry tomato plants.

A retranslocação de Boro (B) pode conferir maior tolerância à deficiência ou toxidez de B, resultando em plantas e frutos com melhor qualidade nutricional. A maioria das plantas não demonstra mobilidade floemática para o B, contudo, algumas plantas possuem mobilidade natural e outras a expressam sob condições de estresse, como o estresse nutricional. Nesse sentido, foi investigada a mobilidade floemática do B em plantas de tomate cereja com diferentes status nutricionais. As plantas foram cultivadas em solução nutritiva com doses adequada e insuficiente de Potássio (K) e B. Adicionalmente, as plantas receberam, de forma localizada, fertilização foliar com B em uma folha madura (F). Os teores e os conteúdos de B nas folhas vizinhas a F foram comparados com folhas de mesma idade e posição de plantas controle. O experimento foi realizado em ambiente protegido e conduzido no delineamento de blocos casualizados em esquema de parcelas subdivididas com quatro repetições. Evidenciou-se retranslocação de B entre folhas vizinhas quando a planta estava sem deficiência de K e com deficiência de B. Neste status nutricional, as folhas vizinhas à folha aplicada (F) apresentaram maiores teores e conteúdos de B quando comparadas às folhas de mesma posição das plantas controle. As evidências sugerem que a retranslocação de B em distâncias relativamente curtas (entre folhas) pode ser influenciada pelo status nutricional de plantas de tomate cereja.

Leaf and Stem Anatomy of Cherry Tomato Under Calcium and Magnesium Deficiencies

Abstract Studies evaluating the influence of nutrients on plant anatomy are very important because nutritional deficiencies can alter the thickness and shape of certain tissues, compromising their functionality what can explain the reduction of productivity. The aim of this study was to characterize the anatomical changes in cherry tomato plants subjected to calcium (Ca) and magnesium (Mg) deficiencies. The experiment was conducted in nutrient solution and the plants subjected to three treatments: complete solution (Control), Ca restriction (1 mmol L-1 of Ca) and Mg omission (without Mg). The experimental design was completely randomized, with three repetitions. Sixty days after seedling transplanting leaves and stem were collected and submitted to anatomical evaluations. Ca or Mg deficiency promotes most evident anatomical changes in chlorophyllous and vascular tissues of the leaves, rather than in the stems. Leaves of 'Sindy' tomato plants with a concentration of 1.7 g kg-1 of Mg and visual symptoms of Mg deficiency present hyperplasia of both tissues, phloem and xylem. This deficiency also promotes increases in the thickness of mesophyll, spongy parenchyma and palisade parenchyma, and consequently of leaf thickness. The midrib of the leaves with a concentration of 10 g kg-1 of Ca, without visual symptoms of deficiency presented phloem hypertrophy and hyperplasia.

First report of laticifers in lianas of Malpighiaceae and their phylogenetic implications.

PREMISE: Laticifers have evolved multiple times in angiosperms and have been interpreted as a key innovation involved in plant defense mechanisms. In Malpighiaceae, laticifers were previously known from a single lineage of trees and shrubs, the Galphimia clade, but with detailed anatomical analyses here, we show that their distribution is broader in the family, also encompassing large clades of lianas. METHODS: From 15 genera, 70 species of Malpighiaceae were surveyed through careful anatomical ontogenetic analysis of roots, stems, and leaves and detailed histochemical tests to elucidate the nature of laticifers and latex in the family. RESULTS: Articulated anastomosing laticifers were encountered in roots, stems, and leaves of two distantly related megadiverse genera of Malpighiaceae lianas: Stigmaphyllon (stigmaphylloid clade) and Tetrapterys s.s. (tetrapteroid clade). From the apex downward, in Stigmaphyllon the laticifers are derived from the procambium and from the cambium during its early activity and are present in the outermost part of the vascular cylinder of stems and leaves and in the pericycle of roots, whereas in Tetrapterys s.s. they are derived from the ground meristem, procambium, and cambium throughout the plant body and are present in the cortex and pith, either the pericycle in roots or the outermost part of the vascular system in stems and leaves, and the primary and secondary phloem. CONCLUSIONS: Laticifers seem to have evolved at least three times independently in Malpighiaceae, once in a lineage of trees and shrubs and twice in two distantly related megadiverse lianescent lineages. Laticifer evolution in Malpighiaceae is homoplastic and may be related to increases in species diversification.

Optimal Preparation of Tissue Sections for Light-Microscopic Analysis of Phloem Anatomy.

In order to successfully analyze and describe any plant tissue, the first challenge is preparation of good anatomical slides. The challenge is even greater when the target tissue has heterogeneous characteristics, such as the phloem where soft and stiff tissues occur side by side. The goal of this chapter is to present a detailed protocol containing various techniques for optimal preparation of phloem tissue samples for light microscopic analysis. The process typically involves the steps of fixation, softening, embedding, sectioning, staining, and mounting. The protocol can be applied to make samples of phloem and surrounding tissues of stems and roots, from woody to herbaceous plants.

Molecular characterization of a phytoplasma associated with a commercial variety of Momordica charantia

Momordica charantia (bitter melon) presents two distinct types or varieties, known as wild type and commercial type. Plants of the wild type are hosts of a phytoplasma of the group 16SrIII-J, which is associated with a disease known as witches broom. However, this disease has not yet been reported in commercial bitter melon. Thus, symptomatic plants of the commercial type were analyzed in order to demonstrate the association between phytoplasmas and disease. In further assays, strains found in symptomatic plants of the commercial type were subjected to analysis of sequences of the secY gene to determine the extent of genetic diversity. Amplification of DNA fragments from genes 16Sr rRNA (1.2Kb) and secY (1.6Kb) revealed association of phytoplasma with symptomatic plants of the commercial type. Virtual Restriction Fragment Length Polymorphism (RFLP) analysis identified this phytoplasma as a member of the subgroup 16SrIII-J. Phylogenetic analysis showed that the phytoplasma was closely related to the representative of the 16SrIII-J subgroup. Molecular analysis indicated that the secY gene, in spite of the greater genetic variation compared with 16S rRNA gene, did not separate strains of the phytoplasma of the subgroup 16SrIII-J among those strains present in M. charantia.(AU)