Vascular development of the grapevine (Vitis vinifera L.) inflorescence rachis in response to flower number, plant growth regulators and defoliation.

The grapevine inflorescence is a determinate panicle and as buds emerge, shoot, flower and rachis development occur simultaneously. The growth and architecture of the rachis is determined by genetic and environmental factors but here we examined the role of flower and leaf number as well as hormones on its elongation and vascular development. The consequences of rachis morphology and vascular area on berry size and composition were also assessed. One week prior to anthesis, Merlot and Cabernet Sauvignon field vines were exposed to manual flower removal, exogenous plant growth regulators or pre-bloom leaf removal. Manual removal of half the flowers along the vertical axis of the inflorescence resulted in a shorter rachis in both cultivars. Conversely, inflorescences treated with gibberellic acid (GA3) and the synthetic cytokinin, 6-benzylaminopurine (BAP) resulted in a longer rachis while pre-bloom removal of all leaves on the inflorescence-bearing shoot did not alter rachis length relative to untreated inflorescences. Across the treatments, the cross-sectional areas of the conducting xylem and phloem in the rachis were positively correlated to rachis girth, flower number at anthesis, bunch berry number, bunch berry fresh mass and bunch sugar content at harvest. Conversely, average berry size and sugar content were not linked to rachis vascular area. These data indicate that the morphological and vascular development of the rachis was more responsive to flower number and plant growth regulators than to leaf removal.

Flowers regulate the growth and vascular development of the inflorescence rachis in Vitis vinifera L.

The rachis, the structural framework of the grapevine (Vitis vinifera L.) inflorescence (and subsequent bunch), consists of a main axis and one or more orders of lateral branches with the flower-bearing pedicels at their fine tips. The rachis is crucial both for support, and transport from the shoot. Earlier suggestions that the flowers per se affect normal rachis development are investigated further in this study. Different percentages (0, 25, 50, 75 or 100) of flowers were removed manually one week before anthesis on field-grown vines. Treatment effects on subsequent rachis development (curvature, vitality, anatomy, starch deposit) were assessed. Sections, both fixed and embedded, and fresh hand-cut were observed by fluorescence and bright-field optics after appropriate staining. Emphasis was on measurement of changes in cross-sectional area of secondary xylem and phloem, and on maturation of fibres and periderm. Specific defects in rachis development were dependent on the percent and location of flower removal one week prior to anthesis. The rachises curved inwards where most of the flowers were removed. When fully de-flowered, they became progressively necrotic from the laterals back to the primary axes and from the distal to the proximal end of those axes, with a concurrent disorganisation of their anatomy. A few remaining groups of flowers prevented desiccation and abscission of the rachis axes proximal to the group, but not distally. Flower removal (50%) reduced rachis elongation, while 75% removal reduced xylem and phloem area and delayed phloem fibre and periderm development. 75% flower removal did not affect starch present in the rachis during berry development. Developing flowers affect the growth and vitality of the rachis and the development of its vascular and support structures. The extent of these effects depends on the cultivar and the number and position of flowers remaining after some are removed one week before anthesis.

The amino acid distribution in rachis xylem sap and phloem exudate of Vitis vinifera 'Cabernet Sauvignon' bunches.

Amino acids are essential to grape berry and seed development and they are transferred to the reproductive structures through the phloem and xylem from various locations within the plant. The diurnal and seasonal dynamics of xylem and phloem amino acid composition in the leaf petiole and bunch rachis of field-grown Cabernet Sauvignon are described to better understand the critical periods for amino acid import into the berry. Xylem sap was extracted by the centrifugation of excised leaf petioles and rachises, while phloem exudate was collected by immersing these structures in an ethylenediaminetetraacetic acid (EDTA) buffer. Glutamine and glutamic acid were the predominant amino acids in the xylem sap of both grapevine rachises and petioles, while arginine and glycine were the principal amino acids of the phloem exudate. The amino acid concentrations within the xylem sap and phloem exudate derived from these structures were greatest during anthesis and fruit set, and a second peak occurred within the rachis phloem at the onset of ripening. The concentrations of the amino acids within the phloem and xylem sap of the rachis were highest just prior to or after midnight while the flow of sugar through the rachis phloem was greatest during the early afternoon. Sugar exudation rates from the rachis was greater than that of the petiole phloem between anthesis and berry maturity. In summary, amino acid and sugar delivery through the vasculature to grape berries fluctuates over the course of the day as well as through the season and is not necessarily related to levels near the source.

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus.

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.

Some properties of the walls of metaxylem vessels of maize roots, including tests of the wettability of their lumenal wall surfaces.

BACKGROUND AND AIMS: Since the proposal of the cohesion theory there has been a paradox that the lumenal surface of vessels is rich in hydrophobic lignin, while tension in the rising sap requires adhesion to a hydrophilic surface. This study sought to characterize the strength of that adhesion in maize (Zea mays), the wettability of the vessel surface, and to reconcile this with its histochemical and physical nature. METHODS: Wettability was assessed by emptying the maize root vessels of sap, perfusing them with either water or oil, and examining the adhesion (as revealed by contact angles) of the two liquids to vessel walls by cryo-scanning electron microscopy. The phobicity of the lumenal surface was also assessed histochemically with hydrophilic and hydrophobic probes. KEY RESULTS: Pit borders in the lumen-facing vessel wall surface were wetted by both sap/water and oil. The attraction for oil was weaker: water could replace oil but not vice versa. Pit apertures repelled oil and were strongly stained by hydrophilic probes. Pit chambers were probably hydrophilic. Oil never entered the pits. When vessels were emptied and cryo-fixed immediately, pit chambers facing away from the vessels were always sap-filled. Pit chambers facing vessel lumens were either sap- or gas-filled. Sap from adjoining tracheary elements entering empty vessels accumulated on the lumenal surface in hemispherical drops, which spread out with decreasing contact angles to fill the lumen. CONCLUSIONS: The vessel lumenal surface has a dual nature, namely a mosaic of hydrophilic and hydrophobic patches at the micrometre scale, with hydrophilic predominating. A key role is shown, for the first time, of overarching borders of pits in determining the dual nature of the surface. In gas-filled (embolized) vessels they are hydrophobic. When wetted by sap (vessels refilling or full) they are hydrophilic. A hypothesis is proposed to explain the switch between the two states.

Quantitative cryo-analytical scanning electron microscopy (CEDX): an important technique useful for cell-specific localization of salt.

Advances in the techniques required for the X-ray microanalysis of cryo-fixed, naturally hydrated plant tissues in the cryo-scanning electron microscope have reached the stage that accurate, cell-specific localization and quantification of the nutrient and toxic elements can be achieved. Advances are described in the successive processes of cryo-fixation, cryo-planing to produce flat surfaces, monitored minimal etching to reveal cell outlines, coating with aluminum, spectrum collection, and quantification by comparison with comparable frozen standard solutions of the elements.

Development and persistence of sandsheaths of Lyginia barbata (Restionaceae): relation to root structural development and longevity.

BACKGROUND AND AIMS: Strongly coherent sandsheaths that envelop perennial roots of many monocotyledonous species of arid environments have been described for over a century. This study, for the first time, details the roles played by the structural development of the subtending roots in the formation and persistence of the sheaths. METHODS: The structural development of root tissues associated with persistent sandsheaths was studied in Lyginia barbata, native to the Western Australian sand plains. Cryo-scanning electron microscopy CSEM, optical microscopy and specific staining methods were applied to fresh, field material. The role of root hairs was clarified by monitoring sheath development in roots separated from the sand profile by fine mesh. KEY RESULTS AND CONCLUSIONS: The formation of the sheaths depends entirely on the numerous living root hairs which extend into the sand and track closely around individual grains enmeshing, by approx. 12 cm from the root tip, a volume of sand more than 14 times that of the subtending root. The longevity of the perennial sheaths depends on the subsequent development of the root hairs and of the epidermis and cortex. Before dying, the root hairs develop cellulosic walls approx. 3 µm thick, incrusted with ferulic acid and lignin, which persist for the life of the sheath. The dead hairs remain in place fused to a persistent platform of sclerified epidermis and outer cortex. The mature cortex comprises this platform, a wide, sclerified inner rim and a lysigenous central region - all dead tissue. We propose that the sandsheath/root hair/epidermis/cortex complex is a structural unit facilitating water and nutrient uptake while the tissues are alive, recycling scarce phosphorus during senescence, and forming, when dead, a persistent essential structure for maintenance of a functional stele in the perennial Lyginia roots.

Seasonal water relations of Lyginia barbata (Southern rush) in relation to root xylem development and summer dormancy of root apices.

*Periods of dormancy in shallow roots allow perennial monocotyledons to establish deep root systems, but we know little about patterns of xylem maturation, water-transport capacities and associated economies in water use of growing and dormant roots. *Xylem development, anatomy, conductance and in situ cellular [K] and [Cl] were investigated in roots of field-grown Lyginia barbata (Restionaceae) in Mediterranean southwestern Australia. Parallel studies of gas exchange, culm relative water loss and soil water content were conducted. *Stomatal conductance and photosynthesis decreased during summer drought as soil profiles dried, but rates recovered when dormant roots became active with the onset of wetter conditions. Anatomical studies identified sites of close juxtaposition of phloem and xylem in dormant and growing roots. Ion data and dye tracing showed mature late metaxylem of growing roots was located >or= 100 mm from the tip, but at only

Summer dormancy and winter growth: root survival strategy in a perennial monocotyledon.

Here, we tested the alternation of root summer dormancy and winter growth as a critical survival strategy for a long-lived monocotyledon (Restionaceae) adapted to harsh seasonal extremes of Mediterranean southwest Western Australia. Measurements of growth and the results of comparative studies of the physiology, water content, metabolites, osmotic adjustments, and proteomics of the dormant and growing perennial roots of Lyginia barbata (Restionaceae) were assessed in field-grown plants. Formation of dormant roots occurred before the onset of summer extremes. They resumed growth (average 2.3 mm d(-1)) the following winter to eventually reach depths of 2-4 m. Compared with winter-growing roots, summer dormant roots had decreased respiration and protein concentration and c. 70% water content, sustained by sand-sheaths, osmotic adjustment and presumably hydraulic redistribution. Concentrations of compatible solutes (e.g. sucrose and proline) were significantly greater during dormancy, presumably mitigating the effects of heat and drought. Fifteen root proteins showed differential abundance and were correlated with either winter growth or summer dormancy. None matched currently available libraries. The specific features of the root dormancy strategy of L. barbata revealed in this study are likely to be important to understanding similar behaviour in roots of many long-lived monocotyledons, including overwintering and oversummering crop species.

Invited Review: Cryo-scanning electron microscopy (CSEM) in the advancement of functional plant biology. Morphological and anatomical applications.

Cryo-scanning electron microscopy (CSEM) is reviewed by exploring how the images obtained have changed paradigms of plant functions and interactions with their environment. Its power to arrest and stabilise plant parts in milliseconds, and to preserve them at full hydration for examination at micrometre resolution has changed many views of plant function. For example, it provides the only feasible way of accurately measuring stomatal aperture during active transpiration, and volume and shape changes in guard cells, or examining the contents of laticifers. It has revealed that many xylem conduits contain gas, not liquid, during the day, and that they can be refilled with sap and resume water transport. It has elucidated the management of ice to prevent cell damage in frost tolerant plants and has revealed for the first time inherent biological and physical features of root/soil interactions in the field. CSEM is increasingly used to reveal complementary structural information in studies of metabolism, fungal infection and symbiosis, molecular and genetic analysis.

Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion.

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.

Distribution of glucosinolates and sulphur-rich cells in roots of field-grown canola (Brassica napus).

To investigate the role played by the distribution pattern of glucosinolates (GSLs) in root systems in the release of biocides to the rhizosphere, GSLs have been localized, for the first time, to specific regions and cells in field-grown roots. GSL concentrations in separated tissues of canola (Brassica napus) were determined by chemical analysis, and cell-specific concentrations by extrapolation from sulphur concentrations obtained by quantitative cryo-analytical scanning electron microscopy (SEM). In roots with secondary growth, GSL concentrations in the outer secondary tissues were up to 5x those of the inner core. The highest GSL concentrations (from sulphur measurements) were in two cell layers just under the outermost periderm layer, with up to 100x published concentrations for whole roots. Primary tissues had negligible GSL. Release and renewal of the peripheral GSLs is probably a normal developmental process as secondary thickening continues and surface cells senesce, accounting for published observations that intact roots release GSLs and their biocide hydrolosates to the rhizosphere. Absence of myrosin idioblasts close to the root surface suggests that GSLs released developmentally are hydrolysed by myrosinase in the rhizosphere, ensuring a continuous localized source of biotoxic hydrolysates which can deter soil-borne pests, and influence microbial populations associated with long-lived components of the root system.

Types, structure and potential for axial water flow in the deepest roots of field-grown cereals.

Deep root systems that extend into moist soil can significantly increase plant productivity. Here, the components of soil-grown root systems of wheat, barley and triticale are characterized, and types and water conducting potential of deep roots in the field are assessed. Root system components were characterized in plants grown in soil in PVC tubes, based on their origin and number and the arrangement of xylem tracheary elements (XTE) viewed using fluorescence microscopy. A new nomenclature is proposed. Deep roots were harvested in the field, and root types of the current crop and remnant roots from previous crops were identified by fluorescence and cryo-scanning electron microscopy. Four types of axile (framework) and five types of branch root were distinguished in the three cereals. Six per cent of deep roots were axile roots that originated from the base of the embryo; 94% were branch roots, of which 48% had only two XTE (10 microm diameter), and thus potentially low axial flow. Only 30% of roots in the cores were from the current crop, the remainder being remnants. Selection for more deep-penetrating axile roots and increased vascular capacity of deep branches is of potential benefit. Conventional root-length density measurements should be interpreted and applied cautiously.

Electrolyte distribution and yolk sac morphology in frozen hydrated equine conceptuses during the second week of pregnancy.

To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concept of osmotic intake of fluid from the uterine environment as far as the compartments. However, electrolyte concentrations in the compartments consistently exceeded those found in the yolk sac, making it likely that 'uphill' water transport, rather than a purely osmotic uptake, is involved in yolk sac fluid accumulation. We also speculate that capsule formation could actively contribute to conceptus expansion and thereby to fluid intake.

Relative amounts of soluble and insoluble forms of phosphorus and other elements in intraradical hyphae and arbuscules of arbuscular mycorrhizas.

Transport of phosphorus (P) into host plants and its release to root cells is an important function of arbuscular mycorrhizal fungi (AMF). However, relatively little is known about the forms and water solubilities of P compounds in specific locations in the intraradical fungal structures. We determined concentrations and solubility of P components in these structures in white clover (Trifolium repens L.). Plants were grown in the field (colonised by indigenous AMF) or in the glasshouse (inoculated with Glomus intraradices). Mycorrhizas were cryo-fixed in liquid nitrogen immediately (control) or after treatments designed to destroy cell membranes and extract solubles. Thirty to 70% of total P in hyphae and 100% in arbuscules was not extracted. The unextracted proportion of P was higher in the inoculated plants suggesting an environmental effect. It is proposed that the large component of non-extractable P in the arbuscules is involved in the tight regulation of inorganic P release to the host cells. In control roots magnesium, potassium and P were present in hyphae in molar ratios 1 : 2 : 4, further evidence that this relationship may be universal for AMF, and that other P-balancing cations are present but undetectable by the analytical technique.

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions.

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.

Branch roots of young maize seedlings, their production, growth, and phloem supply from the primary root.

Branch root development on the primary root of maize (Zea mays L.) seedlings was followed for 9 d after planting. This period includes the shift from seedling heterotrophy to autotrophy. Linear density of branches in the basal region ranged from ~38 cm-1 at the base to ~10 cm-1 beyond 10 cm. Branch roots in the first ~8 cm were produced before assimilate was available. Branch length decreased from ~26 mm at 1 cm along the primary root to ~8 mm at 10 cm from the base. Without the cotyledon, branch root density in the basal region was ~10 cm-1 and roots were short (~5 mm). Beyond 8-10 cm both measurements matched those of intact seedlings. Dark-grown seedlings had basal branch root densities higher than those without cotyledons but none beyond 10 cm. There were more and smaller diameter sieve tubes in the basal region of the primary root. These decreased distally in number but had larger diameters where branches formed after assimilate was available. Proliferation of basal branch roots in very young seedlings can have major advantages for successful seedling establishment in the field and could be screened for without difficulty.

Tissue and cellular phosphorus storage during development of phosphorus toxicity in Hakea prostrata (Proteaceae).

Storage of phosphorus (P) in stem tissue is important in Mediterranean Proteaceae, because proteoid root growth and P uptake is greatest during winter, whereas shoot growth occurs mostly in summer. This has prompted the present investigation of the P distribution amongst roots, stems, and leaves of Hakea prostrata R.Br. (Proteaceae) when grown in nutrient solutions at ten P-supply rates. Glasshouse experiments were carried out during both winter and summer months. For plants grown in the low-P range (0, 0.3, 1.2, 3.0, or 6.0 micromol d(-1)) the root [P] was > stem and leaf [P]. In contrast, leaf [P] > stem and root [P] for plants grown in the high-P range (6.0, 30, 60, 150, or 300 micromol P d(-1)). At the highest P-supply rates, the capacity for P storage in stems and roots appears to have been exceeded, and leaf [P] thereafter increased dramatically to approximately 10 mg P g(-1) dry mass. This high leaf [P] was coincident with foliar symptoms of P toxicity which were similar to those described for many other species, including non-Proteaceae. The published values (tissue [P]) at which P toxicity occurs in a range of species are summarized. X-ray microanalysis of frozen, full-hydrated leaves revealed that the [P] in vacuoles of epidermal, palisade and bundle-sheath cells were in the mM range when plants were grown at low P-supply, even though very low leaf [P] was measured in bulk leaf samples. At higher P-supply rates, P accumulated in vacuoles of palisade cells which were associated with decreased photosynthetic rates.

Soil strength and rate of root elongation alter the accumulation of Pseudomonas spp. and other bacteria in the rhizosphere of wheat.

Results from a controlled environment system and the field showed that slow root elongation rate was associated with accumulation of Pseudomonas spp. in the rhizosphere; fast root elongation avoided accumulation. In the controlled environment system, total bacteria and bacteria belonging to the genus Pseudomonas were quantified along wheat (Triticum aestivum L. cv. Janz) seminal roots elongating at rates of 2.4 or 0.8 cm d-1 in loose and compacted field soil, respectively. Although total numbers of bacteria were similar for both rates of elongation, more Pseudomonas spp. accumulated on the slow-growing roots and their numbers were greatest 0.5-1 cm from the root tips. A reduced rate of root elongation in compacted soil accelerated the differentiation of root hairs, branch roots and adhesion of rhizosheath soil. Elongation rate and distance between the root tip and the zone of root hair development were positively correlated (r=0.9), providing a morphological indicator of root elongation rate in the field. Slow-growing roots from the field had 20 times more Pseudomonas spp. per unit root length than fast-growing field roots, while total bacteria were 8-fold higher; differences were greatest 0-1 cm from the tips. These results may explain how soil structure and Pseudomonas spp. interact in conservation farming. Rapid root elongation is identified as a desirable trait for avoiding accumulations of bacteria.

Mucilage production by wounded xylem tissue of maize roots - time course and stimulus.

As a reaction to invasion by pathogens, plants block their xylem conduits with mucilage, restricting pathogen advance. Wounding soil-grown roots of maize revealed that pectinaceous mucilage could be found in the vessels after 6 h, and abundantly filled most vessels up to 3 cm proximal to the wound after 1 d. Phenolics increased in the mucilage at later times. The same reactions occurred in vessels following mechanical wounding of axenically-grown roots, showing that the presence of microbes is not necessary for the response. The xylem mucilage is similar to root-cap mucilage in mode of extrusion from the periplasmic space of living cells through primary wall, apparent phase transition, and staining indicative of acidic polysaccharides. Whether other known properties of root-cap mucilage which might alter vessel functioning, such as reduction of surface tension and increased viscosity produced by dissolved solutes, are also common to xylem mucilage requires further investigation. However, our results indicate that possible influence of wounding-induced mucilage in xylem vessels should be considered in all experimental investigations of xylem function.