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The phloem performs essential systemic functions in tracheophytes, yet little is known about its molecular genetic specification. Here we show that application of the peptide ligand CLAVATA3/EMBRYO SURROUNDING REGION 45 (CLE45) specifically inhibits specification of protophloem in Arabidopsis roots by locking the sieve element precursor cell in its preceding developmental state. CLE45 treatment, as well as viable transgenic expression of a weak CLE45G6T variant, interferes not only with commitment to sieve element fate but also with the formative sieve element precursor cell division that creates protophloem and metaphloem cell files. However, the absence of this division appears to be a secondary effect of discontinuous sieve element files and subsequent systemically reduced auxin signaling in the root meristem. In the absence of the formative sieve element precursor cell division, metaphloem identity is seemingly adopted by the normally procambial cell file instead, pointing to possibly independent positional cues for metaphloem formation. The protophloem formation and differentiation defects in brevis radix (brx) and octopus (ops) mutants are similar to those observed in transgenic seedlings with increased CLE45 activity and can be rescued by loss of function of a putative CLE45 receptor, BARELY ANY MERISTEM 3 (BAM3). Conversely, a dominant gain-of-function ops allele or mild OPS dosage increase suppresses brx defects and confers CLE45 resistance. Thus, our data suggest that delicate quantitative interplay between the opposing activities of BAM3-mediated CLE45 signals and OPS-dependent signals determines cellular commitment to protophloem sieve element fate, with OPS acting as a positive, quantitative master regulator of phloem fate.

Significance Contamination of water and foods with arsenic (As) poses a threat to millions of people worldwide. Because the rice grain is the major source of As intake, reducing the transfer of As from soil to the grain is a pressing public health issue. We found that a member of the Oryza sativa C-type ATP-binding cassette transporter (OsABCC) family, OsABCC1, detoxifies As and reduces the amount of As in the rice grain. OsABCC1 in the upper nodes of rice plants restricts the distribution of As to the grain by sequestering it in the vacuoles of the phloem companion cells of diffuse vascular bundles directly connected to the grain. Our work suggests a strategy for limiting As accumulation in rice grains and thereby reducing human As exposure. Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice ( Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.

Currently, phloem transport in plants under field conditions is not well understood. This is largely the result of the lack of techniques suitable for the measurement of the physiological properties of phloem. We present a model that interprets the changes in xylem diameter and live bark thickness and separates the components responsible for such changes. We test the predictions from this model on data from three mature Scots pine trees in Finland. The model separates the live bark thickness variations caused by bark water capacitance from a residual signal interpreted to indicate the turgor changes in the bark. The predictions from the model are consistent with processes related to phloem transport. At the diurnal scale, this signal is related to patterns of photosynthetic activity and phloem loading. At the seasonal scale, bark turgor showed rapid changes during two droughts and after two rainfall events, consistent with physiological predictions. Daily cumulative totals of this turgor term were related to daily cumulative totals of canopy photosynthesis. Finally, the model parameter representing radial hydraulic conductance between phloem and xylem showed a temperature dependence consistent with the temperature-driven changes in water viscosity. We propose that this model has potential for the continuous field monitoring of tree phloem function.

Key message Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing. Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth ( Antheraea pernyi ), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus ( Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8 , these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed usingDissociatortransposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes,Bx1,Bx2, andBx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthasesTPS2andTPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.

Phloem sap quality can differ between and within plants, and affect the performance of aphids. In turn, aphid infestation may change the chemical composition and nutritional value of phloem sap. However, the effects of different aphid species on the overall phloem sap composition of distinct parts within plant individuals in relation to aphid performance remain unclear. To test the specificity of plant responses to aphids, we used two chemotypes of Tanacetum vulgare plants and placed the monophagous aphids Macrosiphoniella tanacetaria and Uroleucon tanaceti on different plant parts (stems close to the inflorescence, young and old leaves). Aphid population growth was determined and sugars, organic acids, amino acids and metabolic fingerprints of phloem exudates were analysed. Macrosiphoniella tanacetaria performed best on stems, whereas U. tanaceti performed best on old leaves, indicating differences in niche conformance. Aphid infestation led to distinct changes in the phloem exudate composition of distinct metabolite classes, differing particularly between plant parts but less between chemotypes. In summary, plant responses to aphids are highly specific for the chemotype, plant part, metabolite class and aphid species. These changes may indicate that aphids construct their own niche, optimizing the food quality on the plant parts they prefer.

Pest control in agriculture is mainly based on the application of insecticides, which may impact nontarget beneficial organisms leading to undesirable ecological effects. Neonicotinoids are among the most widely used insecticides. However, they have important negative side effects, especially for pollinators and other beneficial insects feeding on nectar. Here, we identify a more accessible exposure route: Neonicotinoids reach and kill beneficial insects that feed on the most abundant carbohydrate source for insects in agroecosystems, honeydew. Honeydew is the excretion product of phloem-feeding hemipteran insects such as aphids, mealybugs, whiteflies, and psyllids. We allowed parasitic wasps and pollinating hoverflies to feed on honeydew from hemipterans feeding on trees treated with thiamethoxam or imidacloprid, the most commonly used neonicotinoids. LC-MS/MS analyses demonstrated that both neonicotinoids were present in honeydew. Honeydew with thiamethoxam was highly toxic to both species of beneficial insects, and honeydew with imidacloprid was moderately toxic to hoverflies. Collectively, our data provide strong evidence for honeydew as a route of insecticide exposure that may cause acute or chronic deleterious effects on nontarget organisms. This route should be considered in future environmental risk assessments of neonicotinoid applications.

Plant roots rely on inorganic orthophosphate (Pi) transporters to acquire soluble Pi from soil solutions that exists at micromolar levels in natural ecosystems. Here, we functionally characterized a rice (Oryza sativa) Pi transporter, Os Phosphate Transporter-1;3 (OsPHT1;3), that mediates Pi uptake, translocation, and remobilization. OsPHT1;3 was directly regulated by Os Phosphate Starvation Response-2 and, in response to Pi starvation, showed enhanced expression in young leaf blades and shoot basal regions and even more so in roots and old leaf blades. OsPHT1;3 was able to complement a yeast mutant strain defective in five Pi transporters and mediate Pi influx in Xenopus laevis oocytes. Overexpression of OsPHT1;3 led to increased Pi concentration both in roots and shoots. However, unlike that reported for other known OsPHT1 members that facilitate Pi uptake at relatively higher Pi levels, mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only when external Pi concentration was below 5 μM. Moreover, in basal nodes, the expression of OsPHT1;3 was restricted to the phloem of regular vascular bundles and enlarged vascular bundles. An isotope labeling experiment with ³²P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from source to sink leaves. Furthermore, overexpression and mutation of OsPHT1;3 led to reciprocal alteration in the expression of OsPHT1;2 and several other OsPHT1 genes. Yeast-two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays all demonstrated a physical interaction between OsPHT1;3 and OsPHT1;2. Taken together, our results indicate that OsPHT1;3 acts as a crucial factor for Pi acquisition, root-to-shoot Pi translocation, and redistribution of phosphorus in plants growing in environments with extremely low Pi levels.

Huanglongbing (HLB) is an extremely destructive and lethal disease of citrus worldwide, presumably caused by phloem-limited bacteria, Liberibacter asiaticus ( Las). The widespread invasiveness of the HLB pathogen and lack of natural HLB-resistant citrus cultivars have underscored the need for identifying tolerant citrus genotypes to support the current citrus industry's survival and potentially to lead to future natural HLB resistance. In this study, transverse sections of leaf lamina and midribs were examined with light and epifluorescence microscopy to determine anatomical characteristics that underlie HLB-tolerant mechanisms operating among \"Bearss\" lemon, \"LB8-9\" Sugar Belle mandarin, and its sibling trees compared with HLB-sensitive \"Valencia\" sweet orange. The common anatomical aberrations observed in all Las-infected varieties are as follows: phloem necrosis, hypertrophic phloem parenchyma cells, phloem plugging with abundant callose depositions, phloem collapse with cell wall distortion and thickening, excessive starch accumulation, and sometimes even cambium degeneration. Anatomical distribution of starch accumulation even extended to tracheid elements. Although there were physical, morphological, and pathological similarities in the examined foliage, internal structural preservation in \"Bearss\" lemon and \"LB8-9\" Sugar Belle mandarin was superior compared with HLB-sensitive \"Valencia\" sweet orange and siblings of \"LB8-9\" Sugar Belle mandarin. Intriguingly, there was substantial phloem regeneration in the tolerant types that may compensate for the dysfunctional phloem, in comparison with the sensitive selections. The lower levels of phloem disruption, together with greater phloem regeneration, are two key elements that contribute to HLB tolerance in diverse citrus cultivars.