Easy Come,Easy Go: Capillary Forces Enable Rapid Refilling of Embolized Primary Xylem Vessels

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.There is a pressing need to understand how plants manage the maintenance of water transport from roots through leaves under changing environmental conditions (Allen et al., 2010; Choat et al., 2012). The problem arises because water is transported through the xylem under tension (i.e. under negative absolute pressure). As tension increases, conduits become increasingly vulnerable to cavitation, which causes the conduits to lose their ability to transport water. Conduits can become embolized during normal diurnal function as a result of tensions induced by transpiration and in response to environmental conditions such as drought or freezing stress (Zimmermann and Tyree, 2002). Vulnerability to cavitation and embolism formation suggests that plants have mechanisms to regain lost hydraulic capacity, either through the formation of new conduits or by refilling embolized ones.The vulnerability of conduits to embolisms and the capacity for repair are related to the structural diversity of xylem tissue (Zwieniecki and Holbrook, 2009; Lens et al., 2011; Cai et al., 2014). In vascular plants, the classification of xylem tissues depends on the meristem that produced them (Evert and Eichhorn, 2006). Primary xylem is produced by apical meristems and includes both protoxylem and metaxylem conduits, which are distinguished by their wall structure and the timing of their development. Protoxylem matures during organ elongation, which results in loss of function due to stretching in some tissues and species, while in many others, functionality is maintained throughout the life of the organ. In contrast, metaxylem matures in elongated tissue. In herbaceous plants, primary xylem is the major hydraulic system of the roots, stems, and leaves. In woody plants, the primary xylem remains the main hydraulic system of the leaves, while the radial growth of stems occurs through the activity of a vascular cambium, which produces secondary xylem with only metaxylem conduits. As a woody plant grows, the secondary xylem (and hence the metaxylem) thus becomes of increasing importance to stem hydraulic function. However, protoxylem remains an integral component of the plant hydraulic system due to its function in leaves and elongating stems and roots.As discussed in a recent review (Brodersen and McElrone, 2013), refilling of embolized vessels has been shown to depend on the generation of positive pressure by roots in many monocots, herbaceous plants, and a few woody species. However, many species lack root pressure; thus, attention has focused on so-called novel refilling, which involves adjacent living cells in the repair of embolized metaxylem or secondary xylem in stems of mature plants. Novel refilling has been studied with a variety of methods to visualize temporal variation in the presence and subsequent absence of embolized vessels, including cryo-scanning electron microscopy (Cryo-SEM; Canny, 1997; McCully et al., 2014), double staining (Zwieniecki and Holbrook, 1998; Zwieniecki et al., 2000), NMR imaging (Holbrook et al., 2001; Zwieniecki et al., 2013), and high-resolution x-ray computed tomography (Lee and Kim, 2008; Brodersen et al., 2010; Kim and Lee, 2010; Lee et al., 2013; Suuronen et al., 2013). These observations, in combination with other measurements, led to a working hypothesis of an osmotically driven repair mechanism in which sugars pumped into embolized vessels by adjacent paratracheal parenchyma provide the osmotic pressure difference that refills the vessel (Nardini et al., 2011).Little is known about embolism and its repair in protoxylem, which has structural features that make it potentially more vulnerable to embolism than metaxylem in the same plant or tissue (Choat et al., 2005). These include a greater exposed area of the primary cell wall with annular or helical thickenings instead of secondary walls. This could enhance stretching of the primary wall when large pressure differences develop between functional and embolized vessels, thereby decreasing the pressure required for air seeding of bubbles (Choat et al., 2004). Choat et al. (2005) suggested that greater vulnerability of protoxylem to embolism might underpin the roles of petioles, leaves, and small stems in the hydraulic segmentation hypothesis of Zimmermann (1983), in which sacrifice of the most easily replaceable tissues protects the function of the main structure of a plant during water stress. If ease of protoxylem embolism were to contribute to the function of hydraulic fuses during mild water stress, then ease of refilling would be required to rapidly reset the system.This study focuses on embolism repair in two distantly related, vascular species, Azorella macquariensis (Apiaceae) and Colobanthus muscoides (Caryophyllaceae), that depend exclusively on protoxylem for vascular water transport. Both species form cushions, with the former being an endemic, keystone species in the alpine zone of subantarctic Macquarie Island and the latter being a regional endemic that plays a major role in rocky coastal areas often within the supralittoral zone (Selkirk et al., 1990; Orchard, 1993). Both species are of ecological interest, because the subantarctic region is under increasing threat from climate change (Adams, 2009). Specifically, the climate on Macquarie Island is progressively changing from one that is perpetually wet and misty to one with increased exposure to periodic drying (Bergstrom et al., 2015). Dieback of alpine vegetation was first observed in 2008, and by 2010, extensive and unprecedented decline of A. macquariensis led to its listing as critically endangered (Bricher et al., 2013).In this study, protoxylem structure was studied in relation to embolism repair. Refilling of gas-filled vessels was compared between excised tissue and that in intact, detached shoots. The results showed that the physical properties of the protoxylem facilitated refilling by capillary forces and that rapid refilling in detached shoots supplied with water occurred without root pressure.     

欧洲黑杨MARs的分离克隆及序列分析

为获得林木植物核基质附着区(MARs)序列,诱导欧洲黑杨(Populus nigra)愈伤组织并制备悬浮细胞,裂解释放细胞核,除去非MARs的游离DNA片段,得到核基质。碱性缓冲液分离残留DNA与结合蛋白,回收并克隆残留DNA,测序后得到两个具有MARs序列特征的DNA片段A7和A23。序列特征分析结果表明它们都含有MARs的特征基元,通过与其他已发表的MARs序列比较,认为是从杨树中分离到的两个MARs片段,并预测了其功能。该研究是首次从木本植物获得MARs序列。     

Xylem Sap from Actinidia chinensis: Apparent Differences in Sap Composition Arising from the Method of Collection

Large differences in composition were found between xylem sapcollected from Actinidia chinensis (Chinese gooseberry or kiwifruit) as bleeding sap and sap collected by vacuum extraction.A comparison of saps collected by the two methods has littlemeaning, however, unless the position on the plant from whichsap was collected and the prior treatment of the plant are specified.Furthermore the composition of bleeding sap changes rapidlywith time, probably because of marked gradients in concentrationof individual solutes in the xylem sap from the base to thetop of the plant. Contamination of vacuum-extracted sap by cellularcontents was shown to be insignificant. Sap collected as bleeding sap and by vacuum extraction are ofsomewhat different origins. It would be difficult to predictthe composition of bleeding sap simply from a knowledge of vacuum-extractedsap: it may be similarly unwise to predict the composition ofthe transpiration stream from that of vacuum extracted sap. Actinidia chinensis, kiwi fruit, Chinese gooseberry, xylem sap, bleeding sap, vacuum-extraction     

Pit Membrane Degradation and Air-Embolism Formation in Ageing Xylem Vessels of Populus tremuloides Michx

Air-embolism formation in xylem vessels of Populus tremuloidesMichx. was quantified by its reduction of hydraulic conductivityin branch segments. Embolism was induced by increasing xylemtension in drying stems, or by inserting one end of a hydratedstem in a pressure bomb and increasing air pressure in the bomb.Both treatments produced the same response suggesting that embolismby water stress was caused by air entering water-filled vessels,presumably through inter-vessel pits. In rapidly-growing P.tremuloides branches, the vessels of the outer growth ring werefunctional whereas vessels in older xylem were mostly embolized.This selective embolizing of older vessels was associated witha marked increase in permeability of their inter-vessel pitsto air, relative to pits of younger vessels. Air-injection pressuresless than 1·0 MPa completely embolized older vesselsthat had been re-filled in the laboratory, whereas pressuresover 4·0 MPa were required to embolize young vessels.Greater permeability of old vessels was due to degradation oftheir pit membranes as seen in the scanning electron microscope;large openings were present that were not seen in pit membranesof young vessels. These holes would allow air to penetrate vesselends at low pressure differences causing embolism. Degradationof pit membranes causing the selective dysfunction of oldersapwood may be a general phenomenon initiating heartwood formationin many species. Key words: Xylem embolism, hydraulic conductivity, heartwood formation, cavitation, Populus tremuloides, Michx     

转Bt 基因抗虫棉(Gossyposium)伤流中Bt 毒蛋白的运输

本文以常规棉品种‘石远321’和抗虫棉品种‘SGK321’与‘99B’为材料, 通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量。结果发现, 转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉, 并在抗虫棉伤流中检测到了Bt毒蛋白, 而常规棉伤流中没有Bt毒蛋白的存在。通过嫁接实验研究发现, 将常规棉嫁接到转Bt基因抗虫棉砧木上, 叶片中也有Bt毒蛋白的积累。这些结果说明, Bt毒蛋白可以通过木质部伤流液向地上部运输, 并在叶片中积累, Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的。     

转Bt基因抗虫棉(Gossyposium)伤流中Bt毒蛋白的运输

本文以常规棉品种‘石远321'和抗虫棉品种‘SGK321'与‘99B'为材料,通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量.结果发现,转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉,并在抗虫棉伤流中检测到了Bt毒蛋白,而常规棉伤流中没有Bt毒蛋白的存在.通过嫁接实验研究发现,将常规棉嫁接到转Bt基因抗虫棉砧木上,叶片中也有Bt毒蛋白的积累.这些结果说明,Bt毒蛋白可以通过木质部伤流液向地上部运输,并在叶片中积累,Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的.     

Assimilate Transport in the Xylem Sap of Pedunculate Oak (Quercus robur) Saplings

Abstract: The rates of photosynthesis and transpiration, as well as the concentrations of organic compounds (total soluble non-protein N compounds [TSNN], soluble carbohydrates), in the xylem sap were determined during two growth seasons in one-year-old Quercus robur saplings. From the data, the total C gain of the leaves, by both photosynthesis and the transpiration stream, was calculated. Large amounts of C were allocated to the leaves by the transpiration stream; depending on the time of day and the environmental conditions the portion of C originating from xylem transport amounted to 8 to 91% of total C delivery to the leaves. Particularly under conditions of reduced photosynthesis, e.g., during midday depression of photosynthesis, a high percentage of the total C delivery was provided to the leaves by the transpiration stream (83 to 91 %). Apparently, attack by phloem-feeding aphids lowered the assimilate transport from roots to shoots; as a consequence the portion of C available to the leaves from xylem transport amounted to only 12 to 16 %. The most abundant organic compounds transported in the xylem sap were sugars (sucrose, glucose, fructose) with concentrations of ca. 50 to 500 μmol C ml^-1, whereas C from N compounds was of minor significance (3 to 20 μmol ml^-1 C). The results indicate a significant cycling of C in the plants because the daily transport of C with the transpiration stream exceeded the daily photosynthetic CO₂ fixation in several cases. This cycling pool of C may sustain delivery of photosynthate to heterotrophic tissues, independent of short time fluctuations in photosynthetic CO₂ fixation.     

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     

GC-MS analysis of compounds extracted from buds of Populus balsamifera and Populus nigra

The composition of hexane and ether extracts from buds of two poplar species (Populus balsamifera and P. nigra) was investigated by GC-MS method. In hexane extracts, 54 "neutral" compounds were recorded. The greatest amounts of them are sesquiterpenes and n-alkanes. Among 56 components of ether extracts, many aliphatic acids and hydroxyacids were detected. However, the main fraction consists of phenolcarboxylic acids, substituted cinnamic acids, and their esters. It was established that chemotaxonomic differences between Populus balsamifera and P. nigra are observed in the case of both hexane and ether bud extracts.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

欧美杨水分利用效率相关基因 PdEPF1的克隆及表达

提高植物水分利用效率(WUE)是未来解决我国甚至世界干旱缺水的最重要手段之一。在对植物WUE的众多研究方法中,大多集中在生理手段,但通过分子生物学手段研究其表达调控机制的较少。欧美杨(Populus deltoides×Populus nigra)是中纬度地区最适合种植的短轮伐期工业用材集约经营树种之一。近年来我国引进了许多优良的欧美杨无性系用于营造大面积的速生丰产林并取得很好的经济和社会效益。但高耗水量的缺点限制了其进一步的推广。通过基因芯片技术从欧美杨中找到一个可能调控WUE的基因-PdEPF1.荧光定量表达进一步验证了这一结果。荧光定量表达分析表明该基因受ABA、盐、冷、干旱等胁迫诱导表达。组织特异表达分析说明PdEPF1基因在顶端叶和根中有表达,成熟叶衰老叶中则无表达。克隆到启动子分析表明该启动子含有多种干旱响应元件(drought response elememt),赤霉素响应元件(GA response elememt),低温响应元件(coldresponse elememt),ABA响应元件(ABA response elememt)等逆境相关的作用元件。     

Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 °C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2±24.7 μl h^?1g^?1) and post-thermogenic (50.5±30.4 μl h^?1g^?1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Glutamine Transfer from Xylem to Phloem and Translocation to Developing Leaves of Populus deltoides

The distribution of ¹⁴C from xylem-borne [¹⁴C]glutamine, the major nitrogen compound moving in xylem sap of cottonwood (Populus deltoides Bartr. ex Marsh), was followed in rapidly growing shoots with a combination of autoradiographic, microautoradiographic, and radioassay techniques. Autoradiography and ¹⁴C analyses of tissues showed that xylem-borne glutamine did not move with the transpiration stream into mature leaves. Instead, most of it was transferred from xylem to phloem in the upper stem and then translocated to young developing tissues. Microautoradiography showed that metaxylem parenchyma, secondary xylem parenchyma, and rays were the major areas of uptake from xylem vessels in the stem. Accumulation in phloem (high ¹⁴C concentrations in sieve tubes) took place in internodes subtending recently mature leaves. Little ¹⁴C from xylem-borne glutamine was found in phloem of mature leaves, which indicates restricted retransport of glutamine that did enter the leaf. In the primary tissues of the upper stem, most ¹⁴C was found in the phloem. Cottonwood stems have an efficient uptake and transfer system that enhances glutamine movement to developing tissues of the upper stem.     

Chemical Composition of Hypodermal and Endodermal Cell Walls and Xylem Vessels Isolated from Clivia miniata (Identification of the Biopolymers Lignin and Suberin)

The occurrence of the biopolymers lignin and suberin was investigated with hypodermal (HCW) and endodermal cell walls (ECW) and xylem vessels (XV) isolated from Clivia miniata Reg. roots. Both biopolymers were detected in HCW and ECW, whereas in XV, typical aliphatic suberin monomers were missing and only representative lignin monomers such as guaiacyl (G) and syringyl (S) units could be detected. The absolute amounts of lignin were about one order of magnitude higher compared with suberin in both HCW and ECW. The ratios of the two aromatic lignin units (G/S) decreased from 39 in XV and 10 in HCW to about 1 in ECW, indicating significant differences in lignin structure and function between the three investigated samples. Additionally, compared with the detectable lignin-derived aromatic units G and S, significantly higher amounts of esterified p-coumaric acid-derived aromatic monomers were obtained with HCW, but not with ECW. This is interpreted as a functional adaption of HCW toward pathogen defense at the root/soil interface. The final aim of this study was to provide a thorough chemical characterization of the composition of HCW, ECW, and XV, which in turn will form the basis for a better understanding of the relevant barriers toward the passive, radial, and apoplastic diffusion of solutes from the soil across the root cortex into the root cylinder.     

Enzymatic digestibility and pretreatment degradation products of AFEX‐treated hardwoods (Populus nigra)

There is a growing need to find alternatives to crude oil as the primary feed stock for the chemicals and fuel industry and ethanol has been demonstrated to be a viable alternative. Among the various feed stocks for producing ethanol, poplar (Populus nigra × Populus maximowiczii) is considered to have great potential as a biorefinery feedstock in the United States, due to their widespread availability and good productivity in several parts of the country. We have optimized AFEX pretreatment conditions (180°C, 2:1 ammonia to biomass loading, 233% moisture, 30 minutes residence time) and by using various combinations of enzymes (commercical celluloses and xylanases) to achieve high glucan and xylan conversion (93 and 65%, respectively). We have also identified and quantified several important degradation products formed during AFEX using liquid chromatography followed by mass spectrometry (LC‐MS/MS). As a part of degradation product analysis, we have also quantified oligosaccharides in the AFEX water wash extracts by acid hydrolysis. It is interesting to note that corn stover (C4 grass) can be pretreated effectively using mild AFEX pretreatment conditions, while on the other hand hardwood poplar requires much harsher AFEX conditions to obtain equivalent sugar yields upon enzymatic hydrolysis. Comparing corn stover and poplar, we conclude that pretreatment severity and enzymatic hydrolysis efficiency are dictated to a large extent by lignin carbohydrate complexes and arabinoxylan cross‐linkages for AFEX. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Summary Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10⁶ protoplasts per g fresh weight using 2.0% Cellulase Onozuka R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH₄NO₃. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 M BAP and 9.0 M 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.Abbreviations CPW Cell and Potoplast Wash solution - LPM Liquid Plating Method - LDM Liquid Drop Method - HDM Hanging Drop Method - FSPM Fabric supported Semi-solid agar Plating Method - DTT Dithiothreitol - MES 2-(N-morpholino) ethane sulfonic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - MS Murashige and Skoog (1962)