Plant Phloem Research Articles

Silencing of δ‐aminolevulinic acid dehydratase via virus induced gene silencing promotes callose deposition in plant phloem

ABSTRACT The δ-aminolevulinic acid dehydratase (ALAD) enzyme is an intermediate in the biosynthetic pathway of tetrapyrroles. It combines two δ‐aminolevulinic acid (δ‐ALA) molecules to form the pyrrole, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, light-sensing, and nutrient uptake. Our recent efforts showed that, in citrus, silencing of ALAD gene via Citrus tristeza virus-induced gene silencing, caused yellow spots and necrosis in leaves and in developing new shoots. Silencing of ALAD gene reduced leaf pigments and altered leaf metabolites. Moreover, total phenolic content, H2O2, and reactive oxygen species (ROS) increased, indicating that silencing of ALAD induced severe stress. Herein, we hypothesized that conditions including lower sucrose, elevated ROS, alteration of microRNA involved in RNAi regulatory protein Argonaute 1 (AGO1) and ROS lead to higher deposition of callose in phloem tissues. Using aniline blue staining and gene expression analysis of callose synthases, we showed significant deposition of callose in ALAD-silenced citrus.

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.

Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element–specific proteins reveal differentiation of the endomembrane system

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Purification and Proteomics Analysis of Phloem Tissues from Virus-Infected Plants.

The plant phloem vasculature is crucial for plant growth and development, and is essential for the systemic movement (SM) of plant viruses. Recent transcriptomic studies of the phloem during virus infection have shown the importance of this tissue, yet transcript levels do not provide definitive answers how virus-host interactions favour successful viral SM. Proteomic analyses have been used to identify host-virus protein interactions, uncovering a variety of ways by which viruses utilize host cellular machinery for completion of the viral infection cycle. Despite this new evidence through proteomics, very few phloem centric studies during viral infection have been performed. Here, we describe a protocol for the isolation of phloem tissues and proteins and the subsequent label-free quantitation (LFQ), for identification of proteomic alterations caused by viral infection.

Impact of Huanglongbing Pathogen Infection on the Amino Acid Composition in Both Citrus Plants and the Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) Diaphorina citri is the main vector of the pathogen Candidatus Liberibacter asiaticus (CLas), which is the causal agent of citrus Huanglongbing disease. Feeding by both ACP nymphs and adults on host plants allows them to obtain nutrition. Therefore, the nutritional content within the plant phloem is of much importance for the development and reproduction of ACP. The infection by pathogenic microbiomes may affect the amino acid contents of their host plants and then indirectly affect the biology of sap-feeding insects. In this study, we investigated the amino acid contents and their proportions in both CLas-infected and CLas-free citrus plants, ACP adults, and also in honeydew produced by ACP nymphs. Results showed that infection by CLas had a large impact on the amino acid species and proportion in all the tested target plants, ACP adults, and in the honeydew of ACP nymphs. The content of total amino acids in CLas-infected citrus was much higher than that of CLas-free citrus. However, CLas infection significantly reduced the proportion of essential amino acids (EAAs) in these plants. When feeding on CLas-infected citrus plants, ACP adults absorbed less total amino acids than those adults feeding on healthy plants, but the proportion of EAAs was significantly higher when they fed on CLas-infected citrus plants. The proportion of EAAs also significantly increased in the honeydew secreted by ACP nymphs that fed on CLas-infected citrus plants. However, EAA detection in the honeydew of ACP nymphs indicated that the utilization rate of EAAs by CLas positive ACP nymphs was reduced. Our study has revealed that CLas infection significantly affects the contents, proportion, and utilization efficiency of different amino acids in citrus plants, ACP adults, and nymphs, leading to a developmental pattern of ACP that is more conducive to CLas transmission.

Effects of Arbuscular Mycorrhiza on Primary Metabolites in Phloem Exudates of Plantago major and Poa annua and on a Generalist Aphid.

Arbuscular mycorrhiza (AM), i.e., the interaction of plants with arbuscular mycorrhizal fungi (AMF), often influences plant growth, physiology, and metabolism. Effects of AM on the metabolic composition of plant phloem sap may affect aphids. We investigated the impacts of AM on primary metabolites in phloem exudates of the plant species Plantago major and Poa annua and on the aphid Myzus persicae. Plants were grown without or with a generalist AMF species, leaf phloem exudates were collected, and primary metabolites were measured. Additionally, the performance of M. persicae on control and mycorrhizal plants of both species was assessed. While the plant species differed largely in the relative proportions of primary metabolites in their phloem exudates, metabolic effects of AM were less pronounced. Slightly higher proportions of sucrose and shifts in proportions of some amino acids in mycorrhizal plants indicated changes in phloem upload and resource allocation patterns within the plants. Aphids showed a higher performance on P. annua than on P. major. AM negatively affected the survival of aphids on P. major, whereas positive effects of AM were found on P. annua in a subsequent generation. Next to other factors, the metabolic composition of the phloem exudates may partly explain these findings.

Metal tolerance protein MTP6 is involved in Mn and Co distribution in poplar

With the booming demand of the electric vehicle industry, the concentration of manganese (Mn) and cobalt (Co) flowing into land ecosystems has also increased significantly. While these transition metals can promote the growth and development of plants, they may become toxic under high concentrations. It is thus important to understand how Mn and Co are distributed in plants to develop novel germplasms for the remediation of these heavy metals in contaminated soils. Here, an MTP gene that encodes the CDF (cation diffusion facilitator) protein in Populus trichocarpa, PtrMTP6, was screened as the key gene involved in the distribution of both Mn and Co in poplar. The PtrMTP6-GFP fusion protein was co-localized with the mRFP-VSR2, showing that PtrMTP6 proteins are present at the pre-vacuolar compartment (PVC). Yeast mutant complementation assays further identified that PtrMTP6 serves as a Mn and Co transporter, reducing yeast cell toxicity after exposure to excessive Mn or Co. Histochemical analyses showed that PtrMTP6 was mainly expressed in phloem, suggesting that PtrMTP6 probably involved in the Mn and Co transport via phloem in plants. Under excess Co, PtrMTP6 overexpressing poplar lines were more severely damaged than the control due to higher Co accumulations in young tissue. PtrMTP6 overexpressing lines showed little change in their tolerance to excess Mn, although young tissues also accumulated more Mn. PtrMTP6 play important roles in Mn and Co distribution in poplar and further research on its regulation will be important to increase bioremediation in Mn and Co polluted ecosystems.

Host plant preference of Trioza erytreae on lemon and bitter orange plants

Trioza erytreae, the African citrus psyllid, is a vector of Candidatus Liberibacter spp., the causal agent of the citrus greening disease or Huanglongbing (HLB). The spread of the vector throughout the Iberian Peninsula has been continuous since its introduction in mainland Spain in 2014. The patterns of host preference and feeding behaviour largely depend on olfactory cues. Understanding these patterns is crucial to prevent further dispersion and develop management measures against the pest. In this work, a series of settlement, olfactometric, probing, and feeding experiments were conducted to assess the host preference of T. erytreae for lemon or bitter orange plants. The settlement experiment provided evidence on the preference of both sexes of T. erytreae for lemon plants, whereas males did not show any significant choice pattern in the case of the olfactometric assays. Forty EPG variables were analysed to describe and compare the probing and feeding behaviour of T. erytreae on lemon and bitter orange plants. The EPG variables indicated that T. erytreae has some difficulties in accepting the phloem of bitter orange plants. This suggests that lemon plants would be a better source for the acquisition of Candidatus Liberibacter asiaticus (CLas) by T. erytreae since the psyllid spends much longer periods feeding from the phloem on lemon than on bitter orange.

Detection of Oxytetracycline in Citrus Phloem and Xylem Saps Using Europium-Based Method.

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited ‘Candidatus Liberibacter asiaticus’. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the ‘Ca. L. asiaticus’ pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The ‘Ca. L. asiaticus’-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in ‘Ca. L. asiaticus’-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE.

Molecular biological study on the survival strategy of phytoplasma

Phytoplasmas (genus ‘Candidatus Phytoplasma’) are plant pathogenic bacteria that reside intracellularly within the plant phloem. They infect hundreds of plant species worldwide and induce a wide range of symptoms, including stunting, yellowing, witches’ broom, phyllody, virescence, proliferation, and purple top. The phytoplasma genome has unique features that overturn commonly known wisdom. For example, ATP synthase, which is thought to be essential for life, is not encoded, which provides opportunities for reconsidering the question what is life? Phytoplasmas cause dramatic changes in plant development and parasitizes not only plants but also vector insects by host switching. Therefore, the molecular mechanisms of how phytoplasmas manipulate host plants and how they infect different hosts are of particular interest. This review summarizes the history of phytoplasma discovery, unique survival strategies revealed by genome sequencing, and the molecular mechanisms by which phytoplasmas manipulate host plants.

Biochemical Origin of Raman-Based Diagnostics of Huanglongbing in Grapefruit Trees.

Biotic and abiotic stresses cause substantial changes in plant biochemistry. These changes are typically revealed by high-performance liquid chromatography (HPLC) and mass spectroscopy-coupled HPLC (HPLC-MS). This information can be used to determine underlying molecular mechanisms of biotic and abiotic stresses in plants. A growing body of evidence suggests that changes in plant biochemistry can be probed by Raman spectroscopy, an emerging analytical technique that is based on inelastic light scattering. Non-invasive and non-destructive detection and identification of these changes allow for the use of Raman spectroscopy for confirmatory diagnostics of plant biotic and abiotic stresses. In this study, we couple HPLC and HPLC-MS findings on biochemical changes caused by Candidatus Liberibacter spp. (Ca. L. asiaticus) in citrus trees to the spectroscopic signatures of plant leaves derived by Raman spectroscopy. Our results show that Ca. L. asiaticus cause an increase in hydroxycinnamates, the precursors of lignins, and flavones, as well as a decrease in the concentration of lutein that are detected by Raman spectroscopy. These findings suggest that Ca. L. asiaticus induce a strong plant defense response that aims to exterminate bacteria present in the plant phloem. This work also suggests that Raman spectroscopy can be used to resolve stress-induced changes in plant biochemistry on the molecular level.

Metabolomic Analysis Provides New Insight Into Tolerance of Huanglongbing in Citrus.

There have been efforts to develop citrus cultivars that are tolerant of Huanglongbing (HLB), a catastrophic phloem-limited disease. Previous studies demonstrated that continuous plant growth with phloem regeneration is one of the major characteristics of HLB tolerance. In this study, the metabolic mechanisms of HLB tolerance in citrus were elucidated using a multiple pathway-targeted metabolomic approach. Comparative analysis of healthy and infected HLB-tolerant and HLB-sensitive mandarin cultivars (Citrus reticulata) revealed differentially expressed metabolic responses among different groups. Pathway enrichment analysis indicated aspartate and glutamate metabolism, purine metabolism, and biosynthesis of plant hormones were upregulated in the tolerant group, except salicylic acid signaling. Catabolic pathways linked to energy-yielding metabolism were also upregulated in the tolerant group. These metabolisms and pathways were interconnected with each other, unveiling a pivotal metabolic network associated with HLB tolerance. In the network, auxins and cytokinins, the plant hormones responsible for plant growth and phloem regeneration, were accumulated. In addition, purine metabolites serving as energy carriers and nitrogen sources of plants were increased. Only salicylic acid-related metabolites for plant defense responses were decreased in the tolerant group. Our findings may evidence the strategy of HLB-tolerant cultivars that sustain plant growth and phloem formation rather than displaying direct plant defense to overcome the disease.

Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem.

Numerous piercing-sucking insects can horizontally transmit viral pathogens together with saliva to plant phloem, but the mechanism remains elusive. Here, we report that an important rice reovirus has hijacked small vesicles, referred to as exosomes, to traverse the apical plasmalemma into saliva-stored cavities in the salivary glands of leafhopper vectors. Thus, virions were horizontally transmitted with exosomes into rice phloem to establish the initial plant infection during vector feeding. The purified exosomes secreted from cultured leafhopper cells were enriched with virions. Silencing the exosomal secretion-related small GTPase Rab27a or treatment with the exosomal biogenesis inhibitor GW4869 strongly prevented viral exosomal release in vivo and in vitro. Furthermore, the specific interaction of the 15-nm-long domain of the viral outer capsid protein with Rab5 induced the packaging of virions in exosomes, ultimately activating the Rab27a-dependent exosomal release pathway. We thus anticipate that exosome-mediated viral horizontal transmission is the conserved strategy hijacked by vector-borne viruses.

How Do Trees Grow in Girth? Controversy on the Role of Cellular Events in the Vascular Cambium

Radial growth has long been a subject of interest in tree biology research. Recent studies have brought a significant change in the understanding of some basic processes characteristic to the vascular cambium, a meristem that produces secondary vascular tissues (phloem and xylem) in woody plants. A new hypothesis regarding the mechanism of intrusive growth of the cambial initials, which has been ratified by studies of the arrangement of cambial cells, negates the influence of this apical cell growth on the expansion of the cambial circumference. Instead, it suggests that the tip of the elongating cambial initial intrudes between the tangential (periclinal) walls, rather than the radial (anticlinal) walls, of the initial(s) and its(their) derivative(s) lying ahead of the elongating cell tip. The new concept also explains the hitherto obscure mechanism of the cell event called ‘elimination of initials’. This article evaluates these new concepts of the cambial cell dynamics and offers a new interpretation for some curious events occurring in the cambial meristem in relation to the radial growth in woody plants.

Seasonal changes in temperate woody plant phloem anatomy and physiology: implications for long-distance transport.

Seasonal changes in climate are accompanied by shifts in carbon allocation and phenological changes in woody angiosperms, the timing of which can have broad implications for species distributions, interactions and ecosystem processes. During critical transitions from autumn to winter and winter to spring, physiological and anatomical changes within the phloem could impose a physical limit on the ability of woody angiosperms to transport carbon and signals. There is a paucity of the literature that addresses tree (floral or foliar) phenology, seasonal phloem anatomy and seasonal phloem physiology together, so our knowledge of how carbon transport could fluctuate seasonally, especially in temperate climates is limited. We review phloem phenology focussing on how sieve element anatomy and phloem sap flow could affect carbon availability throughout the year with a focus on winter. To investigate whether flow is possible in the winter, we construct a simple model of phloem sap flow and investigate how changes to the sap concentration, pressure gradient and sieve plate pores could influence flow during the winter. Our model suggests that phloem transport in some species could occur year-round, even in winter, but current methods for measuring all the parameters surrounding phloem sap flow make it difficult to test this hypothesis. We highlight outstanding questions that remain about phloem functionality in the winter and emphasize the need for new methods to address gaps in our knowledge about phloem function.

A Salivary Odorant-Binding Protein Mediates Nilaparvata lugens Feeding and Host Plant Phytohormone Suppression.

Odorant-binding proteins (OBPs) typically act as transporters of odor molecules and play an important role in insect host location. Here, we identified an OBP in brown planthopper (BPH) Nilaparvata lugens salivary glands via transcriptome sequencing. Real-time quantitative PCR and Western blotting analysis results showed that NlugOBP11 was highly expressed in salivary glands and secreted into rice plant during feeding, suggesting that it assists in BPH feeding on rice. Functional analysis in N. lugens saliva revealed that silencing this gene by RNA interference decreased the BPH stylet performance in the phloem of rice plants, reduced sap sucking, and ultimately led to insect death. Moreover, overexpression of NlugOBP11 in rice protoplasts or Nicotiana benthamiana leaves inhibited the production of defense-related signaling molecule salicylic acid in rice plant. The results demonstrate that NlugOBP11 is not only essential for BPH feeding, but also acts as an effector that inhibits plant defense.

Delivery of Rice Gall Dwarf Virus Into Plant Phloem by Its Leafhopper Vectors Activates Callose Deposition to Enhance Viral Transmission.

Rice gall dwarf virus (RGDV) and its leafhopper vector Recilia dorsalis are plant phloem-inhabiting pests. Currently, how the delivery of plant viruses into plant phloem via piercing-sucking insects modulates callose deposition to promote viral transmission remains poorly understood. Here, we initially demonstrated that nonviruliferous R. dorsalis preferred feeding on RGDV-infected rice plants than viruliferous counterpart. Electrical penetration graph assay showed that viruliferous R. dorsalis encountered stronger physical barriers than nonviruliferous insects during feeding, finally prolonging salivary secretion and ingestion probing. Viruliferous R. dorsalis feeding induced more defense-associated callose deposition on sieve plates of rice phloem. Furthermore, RGDV infection significantly increased the cytosolic Ca2+ level in rice plants, triggering substantial callose deposition. Such a virus-mediated insect feeding behavior change potentially impedes insects from continuously ingesting phloem sap and promotes the secretion of more infectious virions from the salivary glands into rice phloem. This is the first study demonstrating that the delivery of a phloem-limited virus by piercing-sucking insects into the plant phloem activates the defense-associated callose deposition to enhance viral transmission.

Effect of Cardinium Infection on the Probing Behavior of Bemisia tabaci (Hemiptera: Aleyrodidae) MED.

Facultative endosymbionts can affect the growth, physiology, and behavior of their arthropod hosts. There are several endosymbionts in the invasive whitefly Bemisia tabaci Mediterranean (MED, Q biotype) that influence host fitness by altering stylet probing behavior. We investigated the probing behavior of B. tabaci MED infected with the facultative endosymbiont Candidatus Cardinium hertigii (Cardinium (Sphingobacteriales: Flexibacteraceae)). We generated genetically similar Cardinium-infected (C*+) and uninfected (C-) clonal sublines and analyzed the probing behavior of newly emerged adult on cotton (Malvales: Malvaceae), Gossypium hirsutum L., using electropenetrography (EPG). The C- subline demonstrated a longer duration of E2 (2.81-fold) and more events of E2 (2.22-fold) than the C*+ subline, indicating a greater level of sustained ingestion of plant phloem. These findings provide insight into the fitness costs (fitness of a particular genotype is lower than the average fitness of the population) of the Cardinium-infected B. tabaci.

New insight into iron biogeochemical cycling in soil-rice plant system using iron isotope fractionation

Iron (Fe) migration in soil-plants is a critical part of Fe biogeochemical cycling in the earth surface system. Fe isotope fractionation analysis in the soil-rice system is promising for quantitatively assessing various pathways and clarifying Fe transformation processes. However, the mechanisms of Fe isotope fractionation in the soil-rice system are not well understood. In this study, the Fe isotopic compositions (δ56Fe) of rhizosphere soils, pore water, Fe plaque, and rice plant tissues at the jointing and mature stages of the plants were determined. The rice plants were slightly enriched in heavier δ56Fe by ∼0.3‰ relative to the soil, and the stele and cortex showed similar δ56Fe values, indicating that the uptake of Fe by rice plants predominantly occurred via Fe(III)-phytosiderophores (Fe(III)-PS) chelation, but not Fe(III) reduction. Additionally, at both the jointing and mature stages, the rice plant tissues showed similar δ56Fe values. However, the Fe isotope fractionation between the roots and stems (Δ56Feroot−stem) was 1.39 ± 0.13‰, which is similar to the previously Ab initio-calculated values between Fe(III)-citrate and Fe(III)- 2-deoxymugineic acid (DMA), indicating that both the phloem and xylem have similar δ56Fe values, and the major Fe-chelating substances in the phloem of the rice plants are Fe(III)-DMA and Fe(II)- Nicotianamine (NA). Therefore, this study demonstrates that Fe isotope fractionation can be used as a signature for interpreting the Fe uptake and translocation mechanism in the soil-rice system.

RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants.

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.