Xylem Parenchyma Cells Research Articles

Vascular bundle cell-specific expression of a phosphate transporter improves phosphate use efficiency of transgenic Arabidopsis without detrimental effects

Constitutive overexpression of phosphate (Pi) transporter family 1 often results in the accumulation of toxic levels of Pi, which causes growth retardation in plants. In contrast, we had previously reported that root epidermis-specific overexpression of the phosphate transporter TaPT2 in Arabidopsis leads to improved growth and Pi use efficiency. In the present study, we used promoters AtHKT1;1 and SKOR, which are predominantly expressed in the vascular bundle tissues, to overexpress TaPT2. Transgenic lines exhibited increased shoot growth compared to wild type plants under normal- and low-Pi conditions, along with elevated root Pi and total P content, and higher xylem sap Pi concentration, specifically under low-Pi conditions. This was attributed to moderate Pi accumulation in the xylem parenchyma cells, enhancing the Pi uploading capacity to the xylem. SKOR-TaPT2, however, did not complement pho1 mutant, which was defective in uploading Pi to the xylem. The transcriptional levels of VPT1 and VPT3, which are responsible for transporting excess Pi into a vacuole, were upregulated in SKOR promoter lines under normal-Pi conditions. Our results suggested that root vascular bundle-specific expression of TaPT2 is another promising strategy for increasing biomass production, Pi uptake, and Pi use efficiency while preventing growth retardation in transgenic plants.

Weight-induced radial growth in plant stems depends on PIN3

How multiple growth programs coordinate during development is a fundamental question in biology. During plant stem development, radial growth is continuously adjusted in response to longitudinal-growth-derived weight increase to guarantee stability.1,2,3 Here, we demonstrate that weight-stimulated stem radial growth depends on the auxin efflux carrier PIN3, which, upon weight increase, expands its cellular localization from the lower to the lateral sides of xylem parenchyma, phloem, procambium, and starch sheath cells, imposing a radial auxin flux that results in radial growth. Using the protein synthesis inhibitor cycloheximide (CHX) or the fluorescent endocytic tracer FM4-64, we reveal that this expansion of the PIN3 cellular localization domain occurs because weight increase breaks the balance between PIN3 biosynthesis and removal, favoring PIN3 biosynthesis. Experimentation using brefeldin A (BFA) treatments or arg1 and arl2 mutants further supports this conclusion. Analyses of CRISPR-Cas9 lines for Populus PIN3 orthologs reveals that PIN3 dependence of weight-induced radial growth is conserved at least in these woody species. Altogether, our work sheds new light on how longitudinal and radial growth coordinate during stem development.

STUDY ON ANATOMICAL CHARACTERIZATIONOF RHINACANTHUS NASUTUS (L.) KURZ

Rhinacanthus nasutus (L.) Kurz, belonging to the family Acanthaceae, is an herbaceous shrub. This plant holds significant value in traditional medicine. The leaves of R. nasutus are commercially available as tea bags, marketed as a natural remedy for various ailments. However, the conservation status of R. nasutus necessitates appropriate protective measures to ensure its sustainability. Anatomical studies provide various histological layers, including the epidermis, cortex, vascular bundles, and pith. The leaves feature numerous diacytic stomata and hypostomatic nature. The leaf midrib exhibits a narrow epidermis with numerous glandular hairs. The medulla contains arc-shaped vascular bundles and lateral accessory bundles, with the stele comprising linear xylem elements towards the inner side and phloem elements on the outer region. Transfusion tracheids are present between the palisade and spongy cells, with tissues containing large quantities of oil droplets. The petiole shows a narrow epidermis with numerous glandular hairs. The vascular region features a V-shaped stele. The stem reveals the stelar region is extensive and the inner region is rich in developed xylem elements with linear multiple vessels. The medullary ray cells are narrow, and the pith region displays intruded primary xylem with thick-walled parenchyma cells. Wood anatomical analysis shows xylem parenchyma cells that are paratracheal. The vessel elements possessing perforation plates and transverse walls with bordered pits. Tracheids have a narrow lumen, and the lateral walls contain simple pits. The wood demonstrates multiseriate ray parenchyma and the bark consists of phloem rays and narrow fibers. In conclusion, R. nasutus exhibits a variety of histological and anatomical characteristics that support its traditional medicinal uses. The detailed anatomical studies contribute to understanding the plants structural adaptations and potential environmental interactions, thereby facilitating its identification and utilization in herbal medicine. The conservation of this species is critical to preserving its medicinal value and ensuring its availability for future research and therapeutic applications.

Single-cell RNA sequencing reveals a high-resolution cell atlas of petals in Prunus mume at different flowering development stages.

Prunus mume (mei), a traditional ornamental plant in China, is renowned for its fragrant flowers, primarily emitted by its petals. However, the cell types of mei petals and where floral volatile synthesis occurs are rarely reported. The study used single-cell RNA sequencing to characterize the gene expression landscape in petals of P. mume 'Fenhong Zhusha' at budding stage (BS) and full-blooming stage (FS). Six major cell types of petals were identified: epidermal cells (ECs), parenchyma cells (PCs), xylem parenchyma cells, phloem parenchyma cells, xylem vessels and fibers, and sieve elements and companion cells complex. Cell-specific marker genes in each cell type were provided. Floral volatiles from mei petals were measured at four flowering development stages, and their emissions increased from BS to FS, and decreased at the withering stage. Fifty-eight differentially expressed genes (DEGs) in benzenoid/phenylpropanoid pathway were screened using bulk RNA-seq data. Twenty-eight DEGs expression increased from BS to FS, indicating that they might play roles in floral volatile synthesis in P. mume, among which PmBAHD3 would participate in benzyl acetate synthesis. ScRNA-seq data showed that 27 DEGs mentioned above were expressed variously in different cell types. In situ hybridization confirmed that PmPAL2, PmCAD1, PmBAHD3,5, and PmEGS1 involved in floral volatile synthesis in mei petals are mainly expressed in EC, PC, and most vascular tissues, consistent with scRNA-seq data. The result indicates that benzyl acetate and eugenol, the characteristic volatiles in mei, are mostly synthesized in these cell types. The first petal single-cell atlas was constructed, offering new insights into the molecular mechanism of floral volatile synthesis.

The OsZIP2 transporter is involved in root-to-shoot translocation and intervascular transfer of cadmium in rice.

Cadmium (Cd) is a toxic metal that poses serious threats to human health. Rice is a major source of dietary Cd but how rice plants transport Cd to the grain is not fully understood. Here, we characterize the function of the ZIP (ZRT, IRT-like protein) family protein, OsZIP2, in the root-to-shoot translocation of Cd and intervascular transfer of Cd in nodes. OsZIP2 is localized at the plasma membrane and exhibited Cd2+ transport activity when heterologously expressed in yeast. OsZIP2 is strongly expressed in xylem parenchyma cells in roots and in enlarged vascular bundles in nodes. Knockout of OsZIP2 significantly enhanced root-to-shoot translocation of Cd and alleviated the inhibition of root elongation by excess Cd stress; whereas overexpression of OsZIP2 decreased Cd translocation to shoots and resulted in Cd sensitivity. Knockout of OsZIP2 increased Cd allocation to the flag leaf but decreased Cd allocation to the panicle and grain. We further reveal that the variation of OsZIP2 expression level contributes to grain Cd concentration among rice germplasms. Our results demonstrate that OsZIP2 functions in root-to-shoot translocation of Cd in roots and intervascular transfer of Cd in nodes, which can be used for breeding low Cd rice varieties.

An oligo peptide transporter family member, OsOPT7, mediates xylem unloading of Fe for its preferential distribution in rice.

Iron (Fe) needs to be delivered to different organs and tissues of above-ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe-deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long-distance distribution to the developing organs and local distribution within the leaf in rice.

Visualizing plant intracellular inorganic orthophosphate distribution.

Intracellular inorganic orthophosphate (Pi) distribution and homeostasis profoundly affect plant growth and development. However, its distribution patterns remain elusive owing to the lack of efficient cellular Pi imaging methods. Here we develop a rapid colorimetric Pi imaging method, inorganic orthophosphate staining assay (IOSA), that can semi-quantitatively image intracellular Pi with high resolution. We used IOSA to reveal the alteration of cellular Pi distribution caused by Pi starvation or mutations that alter Pi homeostasis in two model plants, rice and Arabidopsis, and found that xylem parenchyma cells and basal node sieve tube element cells play a critical role in Pi homeostasis in rice. We also used IOSA to screen for mutants altered in cellular Pi homeostasis. From this, we have identified a novel cellular Pi distribution regulator, HPA1/PHO1;1, specifically expressed in the companion and xylem parenchyma cells regulating phloem Pi translocation from the leaf tip to the leaf base in rice. Taken together, IOSA provides a powerful method for visualizing cellular Pi distribution and facilitates the analysis of Pi signalling and homeostasis from the level of the cell to the whole plant.

Local distribution of manganese to leaf sheath is mediated by OsNramp5 in rice.

To play essential roles of manganese (Mn) in plant growth and development, it needs to be transported to different organs and tissues after uptake. However, the molecular mechanisms underlying Mn distribution between different tissues are poorly understood. We functionally characterized a member of rice natural resistance-associated macrophage protein (NRAMP) family, OsNramp5 in terms of its tissue specificity of gene expression, cell-specificity of protein localization, phenotypic analysis of leaf growth and response to Mn fluctuations. OsNramp5 is highly expressed in the leaf sheath. Immunostaining revealed that OsNramp5 is polarly localized at the proximal side of xylem parenchyma cells of the leaf sheath. Both the gene expression and protein abundance of OsNramp5 are unaffected by different Mn concentrations. Knockout of OsNramp5 decreased the distribution of Mn to the leaf sheath, but increased the distribution to the leaf blade at both low and high Mn supplies, resulting in reduced growth of leaf sheath. Furthermore, expression of OsNramp5 under the control of root-specific promoter in osnramp5 mutant complemented Mn uptake, but could not complement Mn distribution to the leaf sheath. These results indicate that OsNramp5 expressed in the leaf sheath plays an important role in unloading Mn from the xylem for the local distribution in rice.

Changes in the physiological activity of parenchyma cells in Dalbergia odorifera xylem and its relationship with heartwood formation

BackgroundThe formation of a tree’s heartwood gives the wood properties such as natural decay resistance and aesthetic color, and often directly determines the value of wood products. Regulating the quantity and quality of heartwood is of great importance to the use of wood. However, the mechanism of heartwood formation has been poorly understood.ResultsUsing Dalbergia odorifera as the study species, the number of starch grains, the morphology of the nuclei, the changes in the content of water and secondary metabolites were observed continuously in the radial direction of the xylem. The results show that from the outer toward inner sapwood, the starch grains are abundant, the length to diameter ratio of the nuclei is decreasing, and the morphology changes from elongated elliptical and then to round. In the outer transition zone, the starch grains begin to decrease abruptly and the nuclei shrink at a slower rate, with a radial width of approximately 2 mm. In the inner transition zone, the heartwood color begins to appear, the starch grains disappear and a few nuclei with reduced fluorescence are present, with a radial width of approximately 1 mm. Heartwood formation after complete disappearance of the nuclei. The moisture content of the heartwood is higher than that of the sapwood, and the inner transition zone is where the content rises. The secondary metabolites of the heartwood begin to accumulate in large quantities in the inner transition zone.ConclusionBased on the physiological changes of parenchyma cells in the xylem, the radial width of the transition zone of Dalbergia odorifera is clearly defined as approximately 3 mm. Both the water and secondary metabolite abrupt changes occur at the final stage of programmed cell death, and neither is a direct cause of programmed cell death in parenchyma cells.

Differential gene expression patterns and physiological responses improve adaptation to high salinity concentration in pepper accessions.

High salinity decreases the productivity of crops worldwide. Pepper is particularly sensitive to high salt concentrations. Herein, we subjected three tolerant pepper accessions (C12, B14 and A25) to high sodium chloride concentration (70 mM NaCl). The aerial and root biomass, leaf and root osmotic potential (Ψπ ), Na+ , Cl- , K+ and proline concentrations and the relative expression of the putative genes CaSOS1, CaHKT1, three CaNHXs and CaP5CS were measured. Different salinity tolerance strategies depending on the pepper accession were identified. In C12, tolerance was attributed to the accumulation of Na+ in vacuoles and endosomes by the activation of vacuolar CaNHXs genes and the reduction in Ψπ ; additionally, the activation of CaHKT1 and CaSOS1 in leaves and roots moved and accumulated Na+ ions in the xylem and xylem parenchyma cells (XPC) as well as expulsed it out of the root cells. A25 accession, on the contrary, was specialized in compartmentalizing Na+ ions in root and leaf vacuoles and root XPC by the up-regulation of CaNHXs and CaHKT1, respectively, avoiding a toxic accumulation in leaves. Finally, B14 accession moved and accumulated Na+ in xylem and XPC, reducing its concentration in roots by the activation of CaSOS1 and CaHKT1. This study shade light on different tolerance mechanisms of pepper plants to overcome salt stress.

Morphoanatomical Responses and Stomatal Conductance in Shaded and Sun-Exposed Broussonetia papyrifera Seedlings

Investigating invasive species’ morphoanatomical characteristics, such as leaf area and thickness of vascular tissues and storage cells, can reveal information about their adaptive ability and competitive advantage. Thus, we analyzed the variation in leaf, stem, and root morphoanatomical structure and stomatal conductance between shaded and sun-exposed seedlings of Broussonetia papyrifera to provide insights into management strategies to control its invasive ability. The variations in leaf area (LA), Leaf Mass per Area (LMA), stomatal density, thickness of palisade and spongy mesophyll tissues, thickness of xylem parenchyma cells, diameter of all xylem vessels in stem and roots and stomatal conductance were analyzed between shaded and sun-exposed seedlings. Results revealed that the LA and LMA of B. papyrifera were significantly larger in the sun-exposed than in the shaded condition. Seedlings exposed to sunlight also had thicker palisade mesophyll and xylem parenchyma cells. The diameter of xylem vessels in roots was also significantly higher in sun-exposed seedlings. Contrarily, a reverse pattern was observed in the diameter of stem xylem vessels (i.e., shaded > sun-exposed seedlings). Physiologically, a higher stomatal conductance was also observed in sun-exposed seedlings. In conclusion, we found significant variations in leaf, stem, and root morphoanatomy and stomatal conductance between shaded and sun-exposed seedlings of B. papyrifera. The findings of the present study may provide valuable implications for managing or controlling its invasibility in the introduced range. Keywords: freehand technique, invasive species, Moraceae, paper mulberry, stomatal conductance

Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.

Structural Variation Patterns in Xylem Vessels and Parenchyma Cells and Their Association with Tree Evolution

Xylem vessels and parenchyma cells perform functions such as water transport and nutrient storage in trees. However, they are highly variable in different trees. Therefore, this study aimed to explore the structural change patterns in vessels and parenchyma cells in the sapwood, transition wood, and heartwood of tree species with different degrees of evolution. The structural characteristics of the two types of cells in the sapwood, transitional wood, and heartwood were measured in six species with different levels of evolution, namely, Michelia macclurei Dandy, Cinnamomum camphora (L.) presl, Erythrophleum fordii Oliv, Melaleuca leucadendron L., Parashorea chinensis Wang Hsie and Tectona grandis L.F. The results showed that the more evolved species had larger earlywood vessel lumen diameters, thicker walls, and wider hydraulic diameters, as well as smaller latewood vessel densities, reflecting better water transport effectiveness and higher safety. From the sapwood to the transition wood and heartwood, the earlywood vessel lumen diameter of the more primitive species tended to be stable and then decrease, while that of the more evolved species gradually decreased. The latewood vessel density of the more primitive species tended to be stable and then increase, while that of the more evolved species tended to be stable. Additionally, the starch grains of the more primitive species were mainly distributed in the axial or ray parenchyma cells of the sapwood, while those of the more evolved species were abundantly distributed in the axial and ray parenchyma cells of the sapwood, and the distribution of the starch grains was reduced in the transition wood and heartwood. From the sapwood to the heartwood, the ray parenchyma cell fraction tended to be stable in the more primitive species, and tended to be stable before decreasing in the more evolved species. The pit density in the horizontal wall of the ray parenchyma cells tended to be stable and decrease in the more primitive species, while in the more evolved species it tended to be stable or decrease before stabilizing. Overall, trees’ vessels have gradually undergone the optimal selection of vessels during evolution, and the structural variation in the parenchyma cells contributes to their nutrient storage and transport.

Interactive effect between tree ageing and trunk-boring pest reduces hydraulics and carbon metabolism in Hippophae rhamnoides.

Sea-buckthorn (Hippophae rhamnoides) is widely distributed across the Eurasian continent. Recently sea-buckthorn has shown premature ageing and decline when confronted with water deficiency and Holcocerus hippophaecolus damage in northwest China and the Loess Plateau region. However, the physiological process of sea-buckthorn senescence in response to drought and pest damage is still unknown. In this study, 4-year-old (4y), 15-year-old normal growth (15yN) and 15-year-old seriously moth-damaged sea-buckthorn plants (15yH) were used as the research objects. The growth of branches and roots, branch water potential and percentage loss of conductivity (PLC), branch vulnerability to embolism (quantified by P50, xylem water potential at 50 % of PLC), branch xylem parenchyma cell viability, photosynthesis and the non-structural carbohydrate (NSC) content in branches and roots in dry and wet seasons were measured. The results showed that the length, basal diameter of 1-year-old branches and the leaf area of 4y trees were significantly larger than that of 15yN and 15yH trees, and the fine root density of 15yH trees was significantly lower than that of 15yN trees in all measured areas. The branch-specific hydraulic conductivity of 15yN and 15yH trees was only 50.2 % and 12.3 % of that of 4y trees, and the P50 of 4y, 15yH and 15yN trees was -3.69 MPa, -2.71 MPa and -1.15 MPa, respectively. The midday water potential and photosynthetic rate were highest in 4y trees, followed by 15yN and then 15yH trees in both the dry season and wet seasons, while branch PLC declined in the opposite direction (15yH trees highest, 4y trees lowest). The degree of PLC repair within a day was highest in 4y trees, followed by 15yN and then 15yH trees, and the viability of xylem cells was consistent with this pattern. The branch xylem starch and NSC content of 4y and 15yN trees were significantly higher than that of 15yH trees in the dry season, and the root starch and NSC content of 4y trees were significantly higher than that of 15yH trees in the two seasons. The above results suggest that the hydraulic properties of the normal elderly and seriously pest-damaged sea-buckthorn were significantly worse than in juvenile plants. Narrower early wood width and vessel density, high embolism vulnerability and weak embolism repair capacity led to the decline in water-conducting ability, and similarly further affected photosynthesis and the root NSC content. The decline in xylem parenchyma cell viability was the main reason for the limited embolism repair in the branches.

Tonoplast-localized transporter ZmNRAMP2 confers root-to-shoot translocation of manganese in maize.

Almost all living organisms require manganese (Mn) as an essential trace element for survival. To maintain an irreplaceable role in the oxygen-evolving complex of photosynthesis, plants require efficient Mn uptake in roots and delivery to above-ground tissues. However, the underlying mechanisms of root-to-shoot Mn translocation remain unclear. Here, we identified an Natural Resistance Associated Macrophage Protein (NRAMP) family member in maize (Zea mays), ZmNRAMP2, which localized to the tonoplast in maize protoplasts and mediated transport of Mn in yeast (Saccharomyces cerevisiae). Under Mn deficiency, two maize mutants defective in ZmNRAMP2 exhibited remarkable reduction of root-to-shoot Mn translocation along with lower shoot Mn contents, resulting in substantial decreases in Fv/Fm and plant growth inhibition compared to their corresponding wild-type (WT) plants. ZmNRAMP2 transcripts were highly expressed in xylem parenchyma cells of the root stele. Compared to the WT, the zmnramp2-1 mutant displayed lower Mn concentration in xylem sap accompanied with retention of Mn in root stele. Furthermore, the overexpression of ZmNRAMP2 in transgenic maize showed enhanced root-to-shoot translocation of Mn and improved tolerance to Mn deficiency. Taken together, our study reveals a crucial role of ZmNRAMP2 in root-to-shoot translocation of Mn via accelerating vacuolar Mn release in xylem parenchyma cells for adaption of maize plants to low Mn stress and provides a promising transgenic approach to develop low Mn-tolerant crop cultivars.

Variation of wood color and chemical composition in the stem cross-section of oak (Quercus spp.) trees, with special attention to the sapwood-heartwood transition zone

The transformation of sapwood (SW) into heartwood (HW) during ageing of wood tissues is the result of physiological and biochemical changes initiated in the transition zone (TZ). These changes contribute to the evolution of active (living) wood cells in SW into less/non-active (dead) wood cells in HW. Previous studies established that the biosynthesis of extractive contents is the most prominent process that occurs in the TZ. To improve our understanding of the extent and characteristics of the TZ in oak wood, the present study reports the results of color parameters (using CIELab color space) and molecular structure and composition of polysaccharide and lignin compounds (using FTIR-ATR and Py-GC–MS). For that purpose, six wood cores from individual living oak (Quercus spp.) trees were collected from two forests with similar environmental conditions, located in the Basque Country (Northern Spain). The color data indicated significant differences between SW, TZ and HW by showing that SW samples were characterized by higher hue (h°) and lower redness (a*) values than the HW, and intermediate values for the TZ. They also suggested that the variations of wood color from SW to HW occur gradually, along a wide TZ counting 4–10 measurement points in a row, depending on the tree. Furthermore, FTIR and Py-GC–MS data gave evidence of the variation trends of polysaccharide and lignin contents in the radial direction, through various FTIR ratios (1735/1325, 1590/1735, 1590/1230, and 1230/1325 cm−1) and one pyrolysis ratio (acetic acid/total polysaccharide: Ps01/Tot_Ps). The observed variations in this present study suggest that the cross-sectional transition patterns can be related to the continuous lignification process of xylem parenchyma cells, as well as the storage of polysaccharide compounds. These results contribute to our fundamental knowledge on the TZ, which may be valuable in research and industrial applications where a clear delimitation of sapwood and/or heartwood is required.

Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

Histological, Morpho-Physiological, and Biochemical Changes during Adventitious Rooting Induced by Exogenous Auxin in Magnolia wufengensis Cuttings

Magnolia wufengensis, a rare ornamental tree species, is now in a huge gap between market demand and actual supply of seedlings. As cutting propagation is one of the most important means to solve the shortage of seedling supply, this study developed an efficient cutting propagation procedure of M. wufengensis, revealed the morphological and histological changes of adventitious root formation, and explored the rhythm correlation between rooting process and physiological and biochemical changes. Cuttings pre-treated with NAA:IBA (2:1) exhibited the best rooting performance. Anatomical analysis demonstrated that adventitious root primordia of M. wufengensis were initiated from cambial and parenchyma cells of xylem, with no relationship to the callus formed on the epidermis. The rooting process of M. wufengenis can be divided into four periods: induction phase (0–8 dap) (dap means days after planting), initiation phase (8–13 dap), expression phase (13–18 dap), and extension phase (18–28 dap). NAA:IBA (2:1) induced the accumulations of 3-indoleacetic-acid and increased the contents of peroxidase and polyphenol-oxidase near the wounding at induction phase. The initiation phase, with the first histological modifications to the formation of meristemoids, correspond to the increase of peroxidase, polyphenol-oxidase, and soluble protein contents. The synergistic reaction of low 3-indoleacetic-acid and high levels of gibberellins and zeatin also stimulates the initiation phase. In the expression and extension phase, high activities of polyphenol-oxidase, IAA-oxidase, and increased contents of soluble protein co-stimulate the emergence and outgrowth of adventitious roots. The present study not only provides optimized protocol by application of auxin combination but also presents insights in the histological, morpho-physiological, and biochemical changes in stem cuttings of M. wufengensis.

Apoplastic sugar may be lost from grape berries and retrieved in pedicels.

In ripening grape (Vitis sp.) berries, the combination of rapid sugar import, apoplastic phloem unloading, and water discharge via the xylem creates a potential risk for apoplastic sugar to be lost from the berries. We investigated the likelihood of such sugar loss and a possible sugar retrieval mechanism in the pedicels of different Vitis genotypes. Infusion of D-glucose-1-13C or L-glucose-1-13C to the stylar end of attached berries demonstrated that both sugars can be leached from the berries, but only the nontransport sugar L-glucose moved beyond the pedicels. No 13C enrichment was found in peduncles and leaves. Genes encoding 10 sugar transporters were expressed in the pedicels throughout grape ripening. Using an immunofluorescence technique, we localized the sucrose transporter SUC27 to pedicel xylem parenchyma cells. These results indicate that pedicels possess the molecular machinery for sugar retrieval from the apoplast. Plasmodesmata were observed between vascular parenchyma cells in pedicels, and movement of the symplastically mobile dye carboxyfluorescein demonstrated that the symplastic connection is physiologically functional. Taken together, the chemical, molecular, and anatomical evidence gathered here supports the idea that some apoplastic sugar can be leached from grape berries and is effectively retrieved in a two-step process in the pedicels. First, sugar transporters may actively retrieve leached sugar from the xylem. Second, retrieved sugar may move symplastically to the pedicel parenchyma for local use or storage, or to the phloem for recycling back to the berry.

Visualization of phosphorus re‐translocation and phosphate transporter expression profiles in a shortened annual cycle system of poplar

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P are considered an important strategy to support the vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under 'a shortened annual cycle system', which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32 P and/or 33 P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.