Phloem Tissue Research Articles

Isolating intact phytoplasma particles from plants for sequencing applications and for testing host responses to phytoplasmas

Candidatus Phytoplasma species are obligate plant parasites limited to the phloem tissue. The difficulties to grow phytoplasma in culture seriously impedes the study of these prokaryotes. In this study, a protocol to extract intact phytoplasmas from infected periwinkle plants using filtration, differential and density-gradient centrifugations was optimized. Confocal and transmission electron microscopy was used to visualize intact phytoplasmas in the preparation. Quantitative-PCR and DNA-seq analyses revealed that phytoplasma DNA was significantly enriched following the extraction procedure. Challenging periwinkle plants with phytoplasma preparation induced the expression of one defence-related gene. This study provides an optimized procedure for phytoplasma DNA enrichment and for extracting intact phytoplasmas that can be used to examine host responses to them.

Super-Resolution Microscopy of Phloem Proteins.

Super-resolution microscopy bridges the gap between light and electron microscopy and gives new opportunities for the study of proteins that contribute to phloem function. The established super-resolution techniques are derived from fluorescence microscopy and depend on fluorescent dyes, proteins tagged with GFP variants or fluorochrome-decorated antibodies. Compared with confocal microscopy they improve the resolution between 2.5 and 10 times and, thus, allow a much more precise (co-) localization of membranes, plasmodesmata, and structural proteins. However, they are limited to thin tissue slices rather than intact plant organs and can only show immobilized or only slowly moving targets. Accordingly, the first super-resolution micrographs of the phloem were recorded from fixed tissue which was sectioned using a vibratome or microtome. As with transmission electron microscopy, preparation of phloem tissue for super-resolution microscopy is challenged by the sudden pressures release when dissecting the functional tissue (see Chapter 2 ).This chapter describes a protocol for investigation of proteins in the plasma membranes of sieve elements and companion cells. It illustrates how high-resolution fluorescence imaging can provide information that could not be obtained with confocal or electron microscopy. Further, a brief introduction outlines the theoretical background of super-resolution techniques suitable for phloem imaging and summarizes the findings of the first available super-resolution studies on the phloem. The protocol focusses on the crucial steps for super-resolution microscopy of immunolocalized phloem proteins, adjusted to the use of three-dimensional structured illumination microscopy (3D-SIM).

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.

Evaluation of pomological traits of pear decline-resistant rootstocks

Twenty pear rootstock genotypes were examined for their pear decline resistance and their pomological traits in a six years field trial. All rootstock selections were grafted with the susceptible cultivar Williams followed by graft-inoculation with a severe strain of ‘Candidatus Phytoplasma pyri’ prior plantation. Seven years post inoculation all surviving trees were positive in PCR analyses. The concentration of the phytoplasmas was determined by quantitative real-time PCR and ranged from 3x106 to 6x107 phytoplasma cells per gram of phloem tissue. Among the grafted rootstock genotypes clear differences in mortality, symptom expression and pomological performance were observed. Pear decline-tolerant rootstocks with promising pomological traits were selected for further evaluation.

Agrilus mali Matsumara (Coleoptera: Buprestidae), a new invasive pest of wild apple in western China: DNA barcoding and life cycle.

Agrilus mali Matsumara (Coleoptera: Buprestidae) is a wood‐boring beetle distributed to eastern China that occasionally injures apple species. However, this wood‐boring beetle is new to the wild apple forests (Malus sieversii) of the Tianshan Mountains (western China) and has caused extensive tree mortality. The development of a biological control program for these wild apple forests is a high priority that requires exploration of the life cycle, DNA barcoding and taxonomic status of A. mali. In this study, to determine the diversity of invasive beetles, a fragment of the mitochondrial cytochrome oxidase gene was analyzed. Based on the results, beetles from Gongliu and Xinyuan counties of Xinjiang were identical but differed from those in the apple nursery of Gongliu by a single‐nucleotide substitution. We summarize the taxonomic status, relationships, and genetic distances of A. mali among other Agrilus species using the Tajima‐Nei model in maximum likelihood phylogeny. Analysis revealed that A. mali was closely related to Agrilus mendax and both belong to the Sinuatiagrulus subgenus. The life cycle of A. mali was investigated based on a monthly regular inspection in the wild apple forests of Tianshan. Similar to congeneric species, hosts are injured by larvae of A. mali feeding on phloem tissue, resulting in serpentine galleries constructed between bark and xylem that prevent nutrient transport and leading to tree mortality. Future studies will focus on plant physiological responses to the invasive beetles and include surveys of natural enemies for a potential classical biological control program.

Stem girdling uncouples soybean stomatal conductance from leaf water potential by enhancing leaf xylem ABA concentration

To understand the impact of shoot-to-root ABA transport on water potential of, and xylem ABA concentration in, different plant tissues during soil drying, soybean (Glycine max cv. Siverka) plants were subjected to drought and girdling in a factorial experiment. Girdling was achieved by surgically excising the phloem tissue from just above the cotyledonary node. After girdling and withholding water, ABA concentrations were determined in xylem saps extracted from individual leaves, detached shoots and de-topped roots, after measuring stomatal conductance (gs), tissue water potentials, and root ABA concentrations. Soil drying decreased water potential throughout the plant and approximately doubled xylem ABA concentrations, coinciding with stomatal closure. Girdling slightly enhanced water potential, especially in droughted plants. Girdling diminished the soil-drying induced increase in xylem sap ABA concentration, and completely prevented root tissue ABA accumulation. Furthermore, girdling decreased root ABA concentration and increased leaf xylem ABA concentration of well-watered (WW) plants. Stomatal conductance declined linearly with leaf water potential only in intact plants, while gs declined as leaf xylem ABA concentration increased, independently of girdling. Thus shoot to root ABA transport not only determines (soil-drying induced) root ABA accumulation, but also limits ABA accumulation in the shoot to maintain stomatal opening of WW plants.

Cloning and Functional Characterization of CsUGD2 in Cucumber (Cucumis sativus L.)

UGD gene encodes UDP-glucose dehydrogenase (UGD) which is a key enzyme in the biosynthesis of cell wall, and it catalyses the irreversible oxidation of UDP-glucose (UDP-Glc) into UDP-glucuronic acid (UDP-GlcA). In cucumber, the expression level of CsUGD2 genes was higher in phloem tissues of pedicel and fruit than that in stalk. This study investigated the function of CsUGD2 in cucumber by different methods. Structure analysis indicated that CsUGD2 gene only has an exon with a length of 1,443 bp. Protein alignment suggested that UGD protein was highly conservative in different species. Phylogenetic analysis showed that CsUGD2 protein and CmoUGD2 protein form a same clade which is far away from UGDs in Arabidopsis. Real-time fluorescence quantitative analysis of CsUGD2 in different tissues of cucumber in the same period showed that CsUGD2 expressed highest in the root of cucumber. When we transformed CsUGD2 into wild type Arabidopsis, the roots of transgenic plants were shorter and the flowering time was delayed. These results suggested that CsUGD2 may play an important role in the development of cucumber roots and only act on the development of cucumber fruit when compared with other plants fruits. However, the role of CsUGD2 in regulating the growth and development process of cucumber need to further study.

Scavenging effect of oxidized biochar against the phytotoxicity of lead ions on hydroponically grown chicory: An anatomical and ultrastructural investigation

To evaluate the scavenging effect of functionalized biochar against the phytotoxicity of Pb2+, original biochar (O-B) was chemically oxidized with either HNO3 or KMnO4 to serve as biofilters (O-BF, HNO3-BF and KMnO4-BF) to hydroponically grown chicory (Cichorium intybus L. var. intybus). Plants subjected to Pb-stress showed various deteriorations in cell organelles including visible alterations in chloroplasts, malformations in plant cells, abnormalities in the mitochondrial system, inward invagination of cell walls, distortions in the plasma membrane, oversized vacuoles and irregular increase in plastoglobuli formation. In addition, disorganization in xylem and phloem tissues and numerous variations in the stomatal number, density and dimensions as well as stomata movement were noticeable in the abaxial leaf surface. Pb-stressed plants showed increments in root diameter, vascular cylinder and metaxylem vessels as well as an obvious increase in the thickness of cortex, intercellular aerenchyma and endodermis layer. Furthermore, a noticeable disturbance in macro-and micronutrient concentrations was recorded in Pb-stressed plants due to the defect in their water status. O-BF showed a limited scavenging effect against the phytotoxicity of Pb2+. However, oxidized biochar filters (particularly KMnO4-BF) recorded a noticeable safeguard effect due to their high affinity to Pb2+ ions. The higher sorption capacity of KMnO4-BF reduced the concentration of Pb in leaf tissues compared to the unequipped filtration treatment (117 vs. 19 µg g−1). In conclusion, data of this hydroponic study provides baseline information regarding the detoxification mechanisms of functionalized biochar against the phytotoxicity of trace elements.

Connection between the disease resistance of sour cherry genotypes and the carbohydrate content of the leaf and phloem tissues

The objective of the present study was to establish a possible connection between disease resistance and the carbohydrate content of plant tissues by examining sour cherry genotypes with different tolerance levels in homeostasis. Research on the sour cherry – Monilinia laxa interaction involved the comparison of two Hungarian cultivars (‘Érdi bőtermő’ and ‘Csengődi’) and their offsprings (8) by measuring the quantity of homeostatic carbohydrate fractions in their leaves and phloem tissues. The results demonstrated that the glucose quantity and the ratio of glucose and fructose to sucrose were correlated with the disease resistance of sour cherry cultivars and their hybrids. The glucose content was higher in susceptible genotypes and lower in tolerant genotypes. The hexose:sucrose ratios of susceptible genotypes were significantly higher than those of tolerant genotypes.

Transcriptome Analysis of Oleoresin-Producing Tree Sindora Glabra and Characterization of Sesquiterpene Synthases.

Terpenes serve important physiological and ecological functions in plants. Sindora glabra trees accumulate copious amounts of sesquiterpene-rich oleoresin in the stem. A transcriptome approach was used to determine the unique terpene biosynthesis pathway and to explore the different regulatory mechanisms responsible for the variation of terpene content among individuals. Analysis of de novo-assembled contigs revealed a complete set of genes for terpene biosynthesis. A total of 23,261 differentially expressed unigenes (DEGs) were discovered between high and low oil-yielding plants. DEG enrichment analysis suggested that the terpene biosynthesis process and the plant hormone signal transduction pathway may exert a major role in determining terpene variation in S. glabra. The expression patterns of candidate genes were further verified by quantitative RT-PCR experiments. Key genes involved in the terpene biosynthesis pathway were predominantly expressed in phloem and root tissues. Phylogenetic analysis and subcellular localization implied that S. glabra terpene synthases may evolve from a common ancestor. Furthermore, two sesquiterpene synthase genes, SgSTPS1 and SgSTPS2, were functionally characterized. SgSTPS1 mainly generated β-caryophyllene from farnesyl pyrophosphate. SgSTPS2 is a versatile enzyme that catalyzes the formation of 12 sequiterpenes from farnesyl pyrophosphate and synthesis of three monoterpenes using geranyl pyrophosphate. Together, these results provide large reservoir for elucidating the molecular mechanism of terpene biosynthesis and for exploring the ecological function of sesquiterpenes in S. glabra.

Laser Microdissection of Phytoplasma-Infected Grapevine Leaf Phloem Tissue for Gene Expression Study.

Phytoplasmas have been found confined mainly in leaf phloem sieve elements. In spite of this, few researches have been focused on the infected phloem tissue, whereas the plant response at the infection site could be quite different compared to distal parts and almost completely masked when whole organs are considered. Herein, we provide a protocol for the isolation of leaf phloem from paraffin-embedded samples by Laser Microdissection, followed by RNA purification and RNA amplification to generate cDNA libraries. Our protocol, which has been set up for phytoplasma-infected field-grown grapevine and successfully used for gene expression profiling, can be modified according to different plant species.

DAPI and Confocal Laser-Scanning Microscopy for In Vivo Imaging of Phytoplasmas.

As phytoplasmas are located inside the phloem tissue, always surrounded by numerous layers of other cells, they can result difficult candidates for microscopical investigations. Moreover, the necessity to kill the plant tissues for microscopy observations causes instantaneous and irreversible modifications in the sieve elements, leading to misleading information and erroneous interpretations. Phytoplasmas were here investigated in intact Vicia faba host plants using DAPI as fluorescent probe and confocal laser scanning microscopy. The described nondestructive technique may be applied for the imaging of phytoplasmas and of different pathogen-related responses in planta.

Phytoplasmas: An Introduction.

Phytoplasmas are among the most recently discovered plant pathogens. They are wall-less prokaryotes restricted to phloem tissue, associated with diseases affecting several hundred plant species. The impact of phytoplasma diseases on agriculture is impressive and, at the present day, no effective curative strategy has been developed. The availability of rapid and sensitive techniques for phytoplasma detection as well as the possibility to study their relationship with the host plants is a prerequisite for the management of phytoplasma-associated diseases.

Real-Time PCR Protocol for Phytoplasma Detection and Quantification.

Phytoplasmas are mollicutes restricted to plant phloem tissue and are normally present at very low concentrations. Real-time polymerase chain reaction (qPCR) offers several advantages over conventional PCR. It is a fast, sensitive, and reliable detection technique amenable to high throughput. Two fluorescent chemistries are available, intercalating dyes or hybridization probes. Intercalating dyes are relatively less expensive than TaqMan® hybridization probes but the latter chemistry is the most commonly used for phytoplasma detection. qPCR may be designed for universal detection of phytoplasma, group or subgroup specific detection, or for simultaneous detection of up to three or four phytoplasmas (multiplexing). qPCR may be used for relative or absolute quantification in host plants and in insect vectors. Therefore, qPCR plays an important role in phytoplasma detection as well as in host-pathogen interaction and in epidemiological studies. This chapter outlines the protocols followed in qPCR assay for phytoplasma detection and quantification, focusing mainly on the use of TaqMan® probes.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N.benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N.benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N.benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Effect of Cold Stress on Photosynthetic Traits, Carbohydrates, Morphology, and Anatomy in Nine Cultivars of Stevia rebaudiana.

Stevia rebaudiana Bertoni is a sweet medicinal herb that is cultivated worldwide. This study aimed to identify the genotypic responses and function of nine cultivars of S. rebaudiana (accession numbers 1–9 from the EUSTAS Stevia Gene Bank) to low temperature. Plants were grown in vitro and incubated under controlled conditions at 5° or 25°C for 1 month. Cold stress significantly decreased the maximum quantum yield of photosystem II (Fv/Fm) in all cultivars, which was more pronounced in cultivars 5, 6, 8, and 9. The efficiency of photosystems I and II (PIABS) also declined in cold-stressed plants and was accompanied by reductions in net photosynthesis (PN), intercellular CO2 (Ci), water use efficiency (WUE), and chlorophyll a, chlorophyll b and carotenoid contents, more so in cultivars 5, 6, 8, and 9. Regardless of the downregulation of photosynthetic capacity, the cold stress increased water-soluble carbohydrates in all cultivars, which was accompanied by an increase in fresh leaf mass and area, more so in cultivars 5, 6, 8, and 9. Furthermore, cold stress increased the stomatal index and density, epidermal cell density, stem diameter, xylem vessel width, phloem tissue width, and number of sclerenchyma in all cultivars. Even though the nine cultivars of S. rebaudiana had lower PSII efficiencies at low temperatures, the increase in carbohydrates and leaf mass suggests that damage to PSII is not responsible for the reduction in its efficiency.

The role of the secondary phloem during the development of the grapevine Berry Shrivel ripening disorder

Berry Shrivel (BS) is a post-veraison physiological ripening disorder of grapevine berries. Its symptoms encompass low pH, reduced content of sugars and anthocyanins, and loss of turgor leading to berries shriveling. Evidence for the primary causes of BS is still speculative and anatomical studies are scarce. So far, anatomical studies have determined necrotic cells, degraded primary phloem cells and hardening of secondary phloem cells in the rachis of BS affected grapes. The picture is far from being complete. Herein we report in-depth analyses of the ultrastructure, anatomy and spatial elementary analysis of rachis and pedicel tissues of BS symptomatic grape clusters with different symptom severity. We hypothesize that structural changes in the vascular system of BS affected grape clusters could alter transport functions of the phloem tissue and contribute to the appearance of BS symptoms. By applying different microscopic techniques (LM, SEM, TEM and EDS) we found a number of anatomical differences in both, rachis and pedicels, between H and BS symptomatic grapes, which include: (i) extended areas of collapsed cells and cell wall thickenings in the secondary phloem in BS samples; (ii) reduced number of cell layers in the cambium in BS samples; (iii) higher rate of callose deposition on sieve plates that are additionally covered with a carbohydrate-like material in BS samples; and (iv) reduced (up to 60%) estimated sieve tube conductivity in BS samples.

Biochemical, histopathological and genetic analysis associated with leaf rust infection in wheat plants (Triticum aestivum L.)

The response of Egyptian wheat cultivars (Gemmeiza-7, Gemmeiza-10, Gemmeiza-11 and Gemmeiza-12) against leaf rust was studied at adult stage at two locations during two seasons. The results differentiated cultivars to susceptible and resistant depend upon the epidemiological parameters; FRS (%), AUDPC and r-value. The resistant cultivars, (Gemmeiza-10 and Gemmeiza-12) have the resistant genes; Lr10 and Lr19. However, Gemmeiza-7 and Gemmeiza-11 haven't any resistant genes. The discoloration of superoxide and hydrogen peroxide was decreased in moderately resistant and resistant cultivars compared to the susceptible cultivars. Also, electrolyte leakage increased in susceptible cultivars. Catalase and peroxidase activities were increased in resistant cultivars. Our results were confirmed with the anatomical studies which proved that epidermis thickness, mesophyll and phloem tissues were decreased in susceptible cultivars. Therefore, this explains why these cultivars still resistant till now.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 μm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

RNA sequencing analysis provides new insights into dynamic molecular responses to Valsa mali pathogenicity in apple ‘Changfu No. 2’

Valsa canker caused by the necrotrophic pathogen Valsa mali (Vm) severely affects apple production in Eastern Asia. The molecular basis underlying the apple response to Vm infection is poorly understood. Hence, we performed RNA sequencing (RNA-seq) to investigate the dynamic gene expression profiles of a major apple cultivar, ‘Changfu No.2’, during Vm infection. Compared with the control (C), 104, 313, and 1059 differentially expressed genes (DEGs) were detected from the phloem tissue within the range of 0.9–1.3 cm (T1), 0.5–0.9 cm (T2), and 0.1–0.5 cm (T3) beyond the lesion periphery, respectively. Gene ontology (GO) enrichment analysis revealed that the DEGs associated with plant growth and development were down-regulated, whereas those related to defense responses were up-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that hormonal and Ca2+ signaling and phenylpropanoid biosynthesis were involved in the defense responses. In conclusion, multiple defense responses associated with ABA, JA, ET, Ca2+, and cell wall signals contributed to the defense against Vm infection in ‘Changfu No.2’. In contrast, the DEGs with inhibited expression were involved in plant growth and development; auxin signaling and several resistance genes might weaken the resistance of ‘Changfu No.2’ to pathogens. Our results offer a new insight into plant responses against necrotrophs and could benefit programs aimed at breeding for Vm resistance.