Margaret E. McCully

Flavonoids Synthesized in Cortical Cells During Nodule Initiation Are Early Developmental Markers in White Clover

We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated βglucuronidase (GUS) transgene (CHS3:gusA) was upregulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4′-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a nodulating Rhizobium strain and by clover-specific lipo-chitinoligosaccharides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule organogenesis in clover and may have direct roles in nodule formation.

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.

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.

Niches for bacterial endophytes in crop plants: a plant biologist's view

Currently proposed means of entry of bacterial endophytes into roots of field-grown crop plants, as well as niches available for their successful colonization of living tissues, are evaluated from a plant biology perspective and the following conclusions drawn. (1) Opportunities for passive crack entry into healthy, undisturbed roots in the field may not be as available as studies with laboratory-grown plants have suggested. Consistent entry of endophytes into living root tissues in the field probably requires bacterial capability to hydrolyse the hydrophobic incrustations of the walls of epidermal, hypodermal, endodermal and other cortical cells. (2) Xylem lumen apoplast is an unsuitable niche for endophytes, especially in the grasses, because of the reduced fitness such colonization imposes on plants subjected to field stresses. (3) The intercellular space apoplast is the most suitable niche for endophytes. More data about the environment of this apoplast are urgently needed so that plant varieties can be tailored to provide an optimal environment in these spaces for particular endophytes. It is suggested that many bacterial endophytes may not have colonized living tissues, but are living in protective niches in dead surface tissues or closely adhering soil of rhizosheaths. Selection of strains of beneficial bacteria adapted for colonizing these external niches may be desirable.

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.

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.

Cryo-scanning electron microscopy (CSEM) in the advancement of functional plant biology: energy dispersive X-ray microanalysis (CEDX) applications.

The capacity to make measurements of elemental concentrations at the level of single cells by energy dispersive X-ray microanalysis of cryo-fixed, inherently-hydrated plant parts (CEDX) is changing or extending our understanding of many plant functions. We include in this review a wide-ranging catalogue of studies that have used CEDX which provides access to the literature on elements measured, plants and tissues studied, techniques used, level of quantitation and the significant findings. These findings include new perspectives on the following areas: salt tolerance; xylem maturation and solute content, root pressure and embolism refilling; the contents of intercellular spaces; sequestration of toxic elements; biomineralisation with silicon; movement of tracer homologues of native cations; indirect localisation of molecules with a distinctive element component; transfer of nutrients from vesicular-arbuscular (VA) mycorrhizas; the role of mucilages in protection and in generating mechanical force. In an Appendix we discuss the procedures involved in CEDX: cryo-fixation, specimen planing, etching, elemental quantitation and mapping. Limitations on sample numbers, elements measurable, spatial resolution, sensitivity and threshold concentrations quantifiable are outlined. A brief discussion of the potential of emerging technologies for cell-specific analysis of cryo-fixed, hydrated specimens is included. In the Accessory Publication we list our standard protocol for CEDX.

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 1u2009:u20092u2009:u20094, further evidence that this relationship may be universal for AMF, and that other P-balancing cations are present but undetectable by the analytical technique.

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 ~38u2009cm-1 at the base to ~10u2009cm-1 beyond 10u2009cm. Branch roots in the first ~8u2009cm were produced before assimilate was available. Branch length decreased from ~26u2009mm at 1u2009cm along the primary root to ~8u2009mm at 10u2009cm from the base. Without the cotyledon, branch root density in the basal region was ~10u2009cm-1 and roots were short (~5u2009mm). Beyond 8-10u2009cm both measurements matched those of intact seedlings. Dark-grown seedlings had basal branch root densities higher than those without cotyledons but none beyond 10u2009cm. 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.