Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography (SEC) coupled online to sector-field ICP-MS. Our data identified known TM binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10-15 kD containing Cu, Fe, S and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

Whether in ant-aphid mutualism the ants exert evolutionary selection pressure on aphid morphology has not yet been fully tested. Here, we tested whether the long proboscises of Stomaphis yanonis (Aphididae Lachninae) aphids confer an advantage in preventing predation by the tending ants. Specifically, we tested the hypothesis that aphids with a shorter proboscis would excrete less honeydew, making them more likely to be preyed upon by ants. Our results showed that aphid individuals with a shorter proboscis took up less phloem sap and excreted less honeydew than individuals with a longer proboscis. In addition, among aphids with a similar body size, those with a shorter proboscis were more susceptible to predation by ants than those with a longer proboscis. These results suggest that predation by tending ants, by exerting selection pressure on aphid proboscis morphology, has caused the aphids to evolve longer proboscises.

OBJECTIVE: Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes.
METHODS: We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined.
RESULTS: Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region.
CONCLUSIONS: Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion.

Extracellular vesicles (EVs) are nanoparticles that are released by cells and participate in the transfer of information. It is now known that EVs from mammalian cells are involved in different physiological and pathophysiological processes (antigen presentation, tissue regeneration, cancer, inflammation, diabetes, etc.). In the past few years, several studies on plants have demonstrated that EVs are also key tools for plant intercellular and cross-kingdom communications, suggesting that these nanostructures may contribute to distinct aspects of plant physiology such as development, defense, reproduction, symbiotic relationships, etc. These findings are challenging the traditional view of signaling in plants. EVs are probably involved in the phloem\'s transport system, since this vascular tissue plays a crucial role in translocating nutrients, defensive compounds, and informational signals throughout the plant. The collection of phloem is experimentally challenging because sap is under high turgor pressure inside the sieve elements, which have a small diameter and are hidden within the plant organs. The goals of this work are to develop new protocols that allow us to detect EVs for the first time in the phloem of the plants, and to isolate these nanovesicles for in-depth analysis and characterization. Our protocols describe two distinct methods to collect the phloem sap from rice and melon. The first method (Basic Protocol 1) involves \'Aphid stylectomy by radiofrequency microcautery\' using rice plants and the aphid Sitobion avenae. This is considered the least invasive method for collecting phloem sap. The second method, \'Stem incision\', involves cutting the stem of melon plants for collecting the exuded sap. Phloem sap EVs are then isolated by size exclusion chromatography. The results obtained in this study represent the first report on typical EVs isolated from in vivo-collected phloem sap. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from phloem sap: Aphid stylectomy by radiofrequency microcautery Basic Protocol 2: Isolation of EVs from phloem sap: Stem incision method.

Banana is a perennial crop and typically consists of a mother plant and one or more suckers that will serve as the next generation. Suckers are photosynthetically active, but also receive photo-assimilates from the mother plant. While drought stress is the most important abiotic constraint to banana cultivation, its effect on suckers or banana mats as a whole remains unknown. To investigate whether parental support to suckers is altered under drought stress and to determine the photosynthetic cost to the parental plant, we conducted a 13C labeling experiment. We labeled banana mother plants with 13CO2 and traced the label up to two weeks after labeling. This was done under optimal and drought-stressed conditions in plants with and without suckers. We retrieved label in the phloem sap of the corm and sucker as soon as 24 hours after labeling. Overall, 3.1 ± 0.7% of label assimilated by the mother plant ended up in the sucker. Allocation to the sucker seemed to be reduced under drought stress. The absence of a sucker did not enhance the growth of the mother plant; instead, plants without suckers had higher respiratory losses. Furthermore, 5.8 ± 0.4% of the label was allocated to the corm. Sucker presence and drought stress each led to an increase in starch accumulation in the corm, but when both stress and a sucker were present, the amount was severely reduced. Furthermore, the second to fifth fully open leaves were the most important source of photo-assimilates in the plant, but the two younger developing leaves assimilated the same amount of carbon as the four active leaves combined. They exported and imported photo-assimilates simultaneously, hence acting as both source and sink. 13C labeling has allowed us to quantify source and sink strengths of different plant parts, as well as the carbon fluxes between them. We conclude that drought stress and sucker presence, respectively causing a reduction in supply and an increase in carbon demand, both increased the relative amount of carbon allocated to storage tissues. Their combination, however, led to insufficient availability of assimilates and hence a reduced investment in long-term storage and sucker growth.

Susceptibility to the severe Citrus tristeza virus (CTV), T36, is higher for Citrus macrophylla (CM) than for C. aurantium (CA). How host-virus interactions are reflected in host physiology is largely unknown. In this study, the profile of metabolites and the antioxidant activity in the phloem sap of healthy and infected CA and CM plants were evaluated. The phloem sap of quick decline (T36) and stem pitting (T318A) infected citrus, and control plants was collected by centrifugation, and the enzymes and metabolites analyzed. The activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in infected plants increased significantly in CM and decreased in CA, compared to the healthy controls. Using LC-HRMS2 a metabolic profile rich in secondary metabolites was assigned to healthy CA, compared to healthy CM. CTV infection of CA caused a drastic reduction in secondary metabolites, but not in CM. In conclusion, CA and CM have a different response to severe CTV isolates and we propose that the low susceptibility of CA to T36 may be related to the interaction of the virus with the host\'s metabolism, which reduces significantly the synthesis of flavonoids and antioxidant enzyme activity.

Plant sap is a nutritionally unbalanced diet that constitutes a challenge for insects that feed exclusively on it. Sap-sucking hemipteran insects generally overcome this challenge by harboring beneficial microorganisms in their specialized symbiotic organ, either intracellularly or extracellularly. Genomic information of these bacterial symbionts suggests that their primary role is to supply essential amino acids, but empirical evidence has been virtually limited to the intracellular symbiosis between aphids and Buchnera. Here we investigated the amino acid complementation by the extracellular symbiotic bacterium Ishikawaella harbored in the midgut symbiotic organ of the stinkbug Megacopta punctatissima. We evaluated amino acid compositions of the phloem sap of plants on which the insect feeds, as well as those of its hemolymph, whole body hydrolysate, and excreta. The results highlighted that the essential amino acids in the diet are apparently insufficient for the stinkbug development. Experimental symbiont removal caused severe shortfalls of some essential amino acids, including branched-chain and aromatic amino acids. In vitro culturing of the isolated symbiotic organ demonstrated that hemolymph-circulating metabolites, glutamine and trehalose, efficiently fuel the production of essential amino acids. Branched-chain amino acids and aromatic amino acids are the ones preferentially synthesized despite the symbiont\'s synthetic capability of all essential amino acids. These results indicate that the symbiont-mediated amino acid compensation is quantitatively optimized in the stinkbug-Ishikawaella gut symbiotic association as in the aphid-Buchnera intracellular symbiotic association. The convergence of symbiont functions across distinct nutritional symbiotic systems provides insight into how host-symbiont interactions have been shaped over evolutionary time.

Aphids feed on plant phloem sap that contains massive amounts of sucrose; this not only provides vital nutrition for the aphids but also produces high osmotic pressure. To utilize this carbon source and overcome the osmotic pressure, sucrose is hydrolyzed into the monosaccharides, glucose and fructose. In the green peach aphid (Myzus persicae), we show that this process is facilitated by a key α-glucosidase (MpAgC2-2), which is abundant in the aphid salivary gland and is secreted into leaves during feeding. MpAgC2-2 has a pH optimum of 8.0 in vitro, suggesting it has adapted to the environment of plant cells. Silencing MpAgC2-2 (but not the gut-specific MpAgC3-4) significantly increased the amount of sucrose ingested and hindered aphid feeding on the phloem of tobacco seedlings, resulting in a smaller body size, as well as lower α-glucosidase activity and glucose levels. These effects could be rescued by feeding aphids on tobacco plants transiently expressing MpAgC2-2. The transient expression of MpAgC2-2 also led to the hydrolysis of sucrose in tobacco leaves. Taken together, these results demonstrate that MpAgC2-2 is a salivary protein that facilitates extra-intestinal feeding via sucrose hydrolysis. Our findings provide insight into the ability of aphids to digest the high concentration of sucrose in phloem, and the underlying mechanism of extra-intestinal digestion.

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 β-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 β-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.

Changes in the substances in phloem sap can effectively reflect the nutritional status of cucumber plants during their growth. Because of the limitations of the time-consuming and complex operations of existing phloem sap extraction methods, the authors proposed a new extraction method based on the capillary-air pressure principle and designed a new sap sampling device. To examine the feasibility of the new sampling device, sap sampled from the same plant with the new method and the common EDTA method was analyzed by gas-phase mass spectrometry. The data showed that the number of substances in the sap sampled using capillary-air pressure was higher than that observed using the EDTA method. The concentration of substances sampled using capillary-air pressure was much higher than that observed using EDTA.