Detection and Characterization of Protein Kinases in Rice Phloem Sap

Calcium-dependent protein kinases were detected and characterizedin the phloem sap of rice plants. Protein phosphorylation wasactivated in the presence of micromolar levels of free Ca²⁺ions but was not activated by a polyamine in vitro. Mg²⁺ ionswere essential for protein phosphorylation and K⁺ ions inhibitedthe protein phosphorylation. Analysis by two-dimensional polyacrylamideelectrophoresis revealed that 17-kDa protein with a pI of 5.0was the most highly phosphorylated protein in the phloem sapof rice plants. A protein of 65 kDa, which was autophosphorylated,had a Ca²⁺-dependent protein kinase activity and the mobilityof this band in SDS gel was changed in the presence of calcium.These results suggest that a signal-transport system may existin the sieve tubes of rice plants that operates via the phosphorylationof proteins by calcium dependent protein kinases. (Received December 20, 1993; Accepted October 8, 1994)     

Detection of Several mRNA Species in Rice Phloem Sap

Evidence is reported for the presence of the mRNAs of thioredoxinh, oryzacystatin-I, and actin in the rice phloem sap collectedby the insect laser method. As the sieve element, the core componentof the phloem, is enucleated, these macromolecules are probablytransported from the companion cells. ¹Current address: Ajinomoto Co., Inc., Fermentation & BiotechnologyLaboratories, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, 210-8581Japan.     

Sugar, Amino Acid and Inorganic Contents in Rice Phloem Sap

Rice phloem sap was obtained through severed stylets of brownplanthoppers and its chemical composition was determined. Sucrose,the only sugar detected, was present at 17–25% (w/v) inthe sap. Amino acids were present at a total of 3–8% (w/v)and were found to be mostly in a free form. The amount of boundamino acids was estimated to be small, if any were present.Among the free amino acids, asparagine (17–33% on a molarbasis), glutamate (6–14%), serine (10–13%), glutamine(7–15%), threonine (5–6%) and valine (6–7%)were dominant, while cystine and methionine (0–0.2%) werepresent in minor amounts and -aminobutyric acid was not detected.The sap had a slightly alkaline pH (ca. 8.0). The inorganicconstituents detected by electron probe x-ray microanalyzerwere Na, S, P and K, with the K content being the highest. Theosmotic pressure was estimated to be 13–15 atm. The aminoacid composition of the plant parts was determined and the differencesin the ease of phloem loading among amino acids were compared. (Received October 5, 1981; Accepted December 17, 1981)     

Chemical Composition of Phloem Sap from the Uppermost Internode of the Rice Plant

The chemical composition of phloem sap from the uppermost internodeof rice plants (Oryza sativa L., var. Kantou), one week afteranthesis, was compared with that of phloem sap from the leafsheath of a young seedling. The pure phloem sap from rice plantswas collected by an insect laser technique. The phloem sap from the uppermost internode contained a highlevel of sucrose (573.8 mM) which was the only sugar detected.The concentrations of total amino acids, potassium and ATP were124.8 mM, 40.4 mM and 1.76 mM, respectively. The concentrationof sucrose was three times higher and the potassium level wasone third as high in the internode sap as in the phloem sapfrom the leaf sheath. The total concentration of amino acidswas almost the same, but the relative amount of each amino acidwas quite different. The ratios of levels of Glu to Gln andof levels of Asp to Asn in the phloem sap from the uppermostinternode were smaller than those in the phloem sap from theleaf sheath. The adenylate energy charge was 0.92–0.93in both types of phloem sap. The amino acid composition of the phloem sap from the uppermostinternode was compared with that of the phloem sap of the flagleaf and the endosperm sap of the same plant, one week afteranthesis. The differences in composition along the phloem pathwaysuggest the selective translocation of amino acid. (Received July 21, 1989; Accepted December 11, 1989)     

Water Deficit-Induced Changes in Concentrations in Proline and Some Other Amino Acids in the Phloem Sap of Alfalfa

Changes in amino acid composition of alfalfa (Medicago sativa L.) phloem sap were studies in response to a water deficit. Sap was collected by stylectomy. As the leaf water potential ([psi]) decreased from -0.4 to -2.0 MPa, there was significant increase of the total amino acid concentration, due to that of some amino acids: proline, valine, isoleucine, leucine, glutamic acid, aspartic acid, and threonine. Asparagine concentration, which is the main amino acid assayed in the phloem sap of alfalfa (it accounts for 70% of the total content), did not vary with the plant water status. The other amino acid concentrations remained stable as [psi] varied; in particular, [gamma]-amino butyric acid concentration remained unchanged, whereas it varied in response to wounding. The more striking change in the sieve tubes was the accumulation of proline, which was observed below a [psi] threshold value of about -0.9 MPa (concentration x60 for a decrease of [psi] from -0.9 to -2.0 MPa). The role of such changes in phloem sap amino acid concentration in osmotic adjustment of growing tissues is discussed.     

Effect of Potassium Supply on the Rate of Phloem Sap Exudation and the Composition of Phloem Sap of Ricinus communis

The composition of phloem sap has been investigated in Ricinus communis var. gibsonii, grown for 2 weeks on nutrient solution of low and high potassium content (K(1) and K(2)). Diagonal cuts were made in the bark of the stem resulting in the exudation of clear droplets which mainly consisted of phloem sap. Although the plants at low K (0.4 mm) and high K (1 mm) did not differ in growth, leaf area, height, or stem circumference, the rate of exudation of the high K plants was about twice as high as that of the plants with the lower K supply. This promoting effect of K on exudation did not result in a dilution of organic (sucrose, UDP-glucose, ATP, UTP) and inorganic constituents of the phloem sap. For the following compounds, even significantly higher concentrations in the exudate were observed in the K(2) plants: potassium, raffinose, glucose 6-phosphate, and fructose 6-phosphate. Also, the osmotic pressure of the phloem sap was substantially increased in the higher K treatment. Experiments in which labeled (14)CO(2) was applied to one leaf showed that K had a favorable effect on the assimilation of CO(2), and in particular promoted the export of photosynthates from the leaf. It is suggested that the higher rate of phloem-loading in the plants with the better K supply is due to the higher CO(2) assimilation rate and especially to a better provision of ATP required for phloem loading. Higher phloem-loading rates result in higher osmotic pressure in the sieve tubes which probably gave rise to the higher flow rates observed in the plants with improved K supply.     

水稻响应缺铁的韧皮部汁液蛋白质组学分析

为鉴定水稻(Oryza sativa)响应缺铁的根冠长距离信号转导物质, 采用TMT标记技术分析了不同浓度铁处理下水稻韧皮部汁液的蛋白质组学变化, 共鉴定出206个差异蛋白, 其中54个蛋白表达丰度上调, 152个蛋白表达丰度下调。差异蛋白的KEGG通路分类主要包括激素信号代谢、谷胱甘肽代谢、碳代谢以及mRNA转运等代谢途径。此外, 对差异蛋白对应的生理指标进行测定, 发现激素、蔗糖、谷胱甘肽和转运蛋白等在缺铁条件下变化显著, 后续对这些差异蛋白的功能研究有助于揭示水稻响应铁素营养的长距离信号途径。     

Collection of Pure Phloem Sap from Wheat and its Chemical Composition

Wheat Phloem sap was collected without contamination from thestylets of small brown planthopper severed by a YAG laser beam.The sugar, amino acid and inorganic ion composition was determinedusing only one µl of the sap. The sap had a high sucrose level (251 mM), and also a high K⁺level (299 mM). Total amino acid compounds in the sap reached262 mM. The dominant amino acids were glutamic acid, asparticacid and serine, while r-amino butylic acid was absent. Themajor anion in the sap was Cl^– and its concentration was25.1 mM. Nitrate was also present at a concentration of 8.1mM. These results suggested that the sap obtained from the cut endof the stylets of the small brown planthopper was a phloem originof wheat. (Received May 21, 1986; Accepted August 7, 1986)     

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C₁₈ reversed-phase high performance liquid chromatography → NH₂ normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.     

Sap Concentrations in Halophytes and Some Other Plants

Freezing point depression in xylem sap of mangroves was found to range from 0.05 to 0.5 degrees , in desert plants from 0.01 to 0.16 degrees . In crush juices from leaves of Batis and Salicornia, 90% or more of the freezing point depression was made up of sodium and chlorine ions; in mangroves they constituted 50 to 70%, the rest probably being organic solutes. Plants growing in seawater have -30 to -60 atmospheres pressure in the xylem sap. As shown earlier, at zero turgor pressure the intracellular freezing point of the parenchyma cells matches closely the negative pressure in the xylem sap. This agrees with the present data, that the fluid which exudes from the xylem by applying gas pressure on the leaves is practically pure water; freezing point is rarely above 0.01 to 0.02 degrees . To perform this ultrafiltration, the plasma membrane is subjected to a hydrostatic pressure gradient which in some cases may exceed 100 atmospheres.     

The Arabidopsis Nitrate Transporter NRT1.8 Functions in Nitrate Removal from the Xylem Sap and Mediates Cadmium Tolerance

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd²⁺) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd²⁺-sensitive phenotype. Further analyses showed that Cd²⁺ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd²⁺ tolerance.     

Collection and Chemical Composition of Pure Phloem Sap from Zea mays L.

Pure phloem sap was collected from leaf sheaths of Zea maysL. by the insect laser technique, and its chemical compositionwas analyzed. Sucrose was the only sugar detected. The predominantinorganic ions were K⁺ and Cl^–. The adenylate energy chargeof phloem sap was between 0.72 and 0.86. (Received October 18, 1989; Accepted May 11, 1990)     

Free Amino Acid Composition of Phloem Sap and Growing Fruit of Lycopersicon esculentum

Pure phloem sap of tomato leaves was collected by stylectomy.Glutamine and glutamate were the predominant free amino acidstranslocated by the phloem stream. In developing fruits glutaminecontent increased significantly, reaching 35% of the total freeamino acids. Comparison in the amino acid composition betweenthe two tissues are discussed. (Received October 6, 1997; Accepted January 27, 1998)     

Sugar Selectivity and Other Characteristics of Phloem Loading in Beta vulgaris L

The rate of phloem loading, its selectivity, and the disposition of labeled carbon were studied following application of (14)C-labeled sugars to the free space of source leaves of sugar beet (Beta vulgaris L.). Buffered 10 mm solutions of (14)C-labeled sucrose, fructose, stachyose, mannitol, 3-0-methyl glucose or l-glucose were applied to the abraded epidermis of source leaves held in the dark. Distribution of the labeled carbon from sugar taken up from the free space was studied by micro-densitometry of autoradiographs. Uptake of labeled sugar from the free space, partition between mesophyll and minor veins, metabolic conversions, export and respiration were followed during the 3-hr time course studies. Rates of sugar uptake into the minor veins, flux rates through the sieve element-companion cell complex membrane and concentration ratios between free space and the interior of the minor vein phloem cells were compared for the six sugars studied for evidence of active uptake. The composition of the free space solution in leaves photosynthesizing in (14)CO(2) was studied by vacuum infiltration of the source leaf air spaces and removal of the solution by centrifugation. Labeled compounds in this solution were compared to those in an aqueous ethanol extract of the same leaf pieces.The results in sugar beet source leaves support the concept of direct, active uptake of sucrose from free space into minor veins. This is not the case for fructose, 3-0-methyl glucose, mannitol, or stachyose. The latter two sugars, which are translocated in some plants, are not loaded into the minor veins at a rate sufficient to make them a significant component of the material translocated. The rate of phloem loading is controlled in part by mesophyll metabolism, especially as it affects the availability of sucrose to the free space. Both the rate and selectivity of export are controlled by uptake from the free space into the sieve element-companion cell complex of the minor veins.     

The Composition of Phloem Exudate and Xylem Sap from Tree Tobacco (Nicotiana glauca Grah.)

The composition of xylem sap and exudate from stem incisionsof Nicotiana glauca Grah. was compared in detail. Exudationfrom stem incisions occurred over a 5 min period in certainplants, enabling collection of 5–30 µl of sap. Therate of exudation showed an exponential decline. Exudate hada high dry matter content (170–196 mg ml^–1) andhigh sugar (sucrose) levels. Xylem sap had a low pH (5.8) andexudate a pH of 7.9. Glutamine dominated the amino compoundsin xylem sap and exudate, and K⁺ was the major cation. Totalamino compounds in stem exudate reached 10.8 mg ml^–1 whereasxylem sap contained much lower levels (0.28 mg ml^–1).All mineral elements and amino compounds with the exceptionof calcium were more concentrated in stem exudate than in xylemsap. Sucrose was labelled heavily in stem exudate following pulsingof an adjacent leaf with ¹⁴CO₂. A concentration gradient ofsugar (2.1 bar m^–1) was recorded for stems. Levels ofsucrose, amino compounds and K⁺ ions in stem exudate showeda diurnal periodicity. Each commodity reached maximum concentrationat or near noon and minimum concentration about dawn. The evidencesuggests that exudate from stem incisions of N. glauca is arepresentative sample of solutes translocated in the phloem. Nicotiana glauca Grah., phloem sap, xylem sap, sucrose, amino compounds, mineral ions     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

Collection and Chemical Composition of Phloem Sap from Citrus sinensis L. Osbeck (Sweet Orange)

Through utilizing the nutrient-rich phloem sap, sap feeding insects such as psyllids, leafhoppers, and aphids can transmit many phloem-restricted pathogens. On the other hand, multiplication of phloem-limited, uncultivated bacteria such as Candidatus Liberibacter asiaticus (CLas) inside the phloem of citrus indicates that the sap contains all the essential nutrients needed for the pathogen growth. The phloem sap composition of many plants has been studied; however, to our knowledge, there is no available data about citrus phloem sap. In this study, we identified and quantified the chemical components of phloem sap from pineapple sweet orange. Two approaches (EDTA enhanced exudation and centrifugation) were used to collect phloem sap. The collected sap was derivatized with methyl chloroformate (MCF), N-methyl-N- [tert-butyl dimethylsilyl]-trifluroacetamide (MTBSTFA), or trimethylsilyl (TMS) and analyzed with GC-MS revealing 20 amino acids and 8 sugars. Proline, the most abundant amino acid, composed more than 60% of the total amino acids. Tryptophan, tyrosine, leucine, isoleucine, and valine, which are considered essential for phloem sap-sucking insects, were also detected. Sucrose, glucose, fructose, and inositol were the most predominant sugars. In addition, seven organic acids including succinic, fumaric, malic, maleic, threonic, citric, and quinic were detected. All compounds detected in the EDTA-enhanced exudate were also detected in the pure phloem sap using centrifugation. The centrifugation technique allowed estimating the concentration of metabolites. This information expands our knowledge about the nutrition requirement for citrus phloem-limited bacterial pathogen and their vectors, and can help define suitable artificial media to culture them.     

水稻硝酸还原酶的RNA干涉及其酶活性分析

分析水稻硝酸还原酶（NR）基因生物信息学的结果显示：水稻基因纽中有2个NR基因成员：一个为NR[NADH]（NR1）：另一个为NR[NAD（P）H]（NR2）。两者的蛋白序列相似性为70％。用RT—PCR技术从水稻cDNA中获得了NR1和NR2的cDNA片段,其大小分别为1086bp和892bp。构建RNA干涉载体（称pRNAi—NR1和pRNAi-NR2）转化水稻愈伤组织后检测转基因后代酶活性的结果表明：两种干涉植株的根叶中的NR活性均大幅度下降,并且根叶中的活性变化呈线性正相关关系。表明2个基因可能均有调控根叶中NR活性的作用。