In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

Physiology and biochemistry of budburst in Vitis vinifera

Both the physiological and biochemical control of budburst in the grapevine, Vitis Vinifera L. were investigated. It was found that the accuracy of a predictive model for grapevine budburst based on ambient temperature was limited under the experimental conditions. There was a significant correlation of 4.7 ± 0.3 days between the days of maximal xylem exudation and budburst over the 3 years of investigation. The co-relationships between daily xylem exudate volume and a range of environmental parameters were considered. It was found that soil temperature was highly correlated against daily xylem exudation. Ambient temperature and soil moisture were significantly correlated with xylem exudation, however the coefficients of correlation were much lower than that of soil temperature. Rainfall showed only a very limited correlation with daily xylem exudate flow. Seasonal variations in the pH and the carbohydrate and inorganic nutrient concentrations of xylem exudate were investigated. Exudate carbohydrate concentrations fell from 660 µM before the day of maximal xylem exudation to zero levels within 4 weeks. Xylem exudate pH was found to consistently fall to a minimum at the time of maximal exudate flow. Exudate concentrations of the metallic cofactors Ca, K, Mg, Mn and Zn varied directly with daily exudate flow, suggesting some sort of flow-dependent mobilisation of these nutrients. A growth promontory oligosaccharide fraction was prepared by partial acid hydrolysis of grapevine primary cell wall material. This fraction significantly increased control growth of the Lemna minor L. bioassay over a limited ‘window’ of bioactivity. A growth inhibitory oligosaccharide fraction, similar in activity to abscisic acid was isolated from grapevine xylem exudate prior to budburst. The exudate concentration or efficacy of this substance declined after budburst such that there was no apparent growth inhibition. A model is proposed for grapevine budburst whereby an oligosaccharide growth inhibitor is gradually removed from the xylematic stream under the effects of soil temperature, allowing the surge of metabolic activity and vegetative growth that constitute budburst.

Use of phosphite as a control for Phytophthora cinnamomi in southeastern Victorian vegetation communities

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

Real-time measurement of phloem turgor pressure in Hevea brasiliensis with a modified cell pressure probe

Background: Although the pressure flow theory is widely accepted for the transport of photoassimilates in phloem sieve elements, it still requires strong experimental validation. One reason for that is the lack of a precise method for measuring the real-time phloem turgor pressure from the sink tissues, especially in tree trunks. Results: Taking the merits of Hevea brasiliensis, a novel phloem turgor pressure probe based on the state of the art cell pressure probe was developed. Our field measurements showed that the phloem turgor pressure probe can sensitively measure the real-time variation of phloem turgor pressure in H. brasiliensis but the calculation of phloem turgor pressure with xylem tension, xylem sap osmotic potential and phloem sap osmotic potential will under-estimate it. The measured phloem turgor pressure gradient in H. brasiliensis is contrary to the Münch theory. The phloem turgor pressure of H. brasiliensis varied from 8-12 bar as a consequence of water withdrawal from transpiration. Tapping could result in a sharp decrease of phloem turgor pressure followed by a recovery from 8-45 min after the tapping. The recovery of phloem turgor pressure after tapping and its change with xylem sap flow suggest the importance of phloem water relationship in the phloem turgor pressure regulation. Conclusion: The phloem turgor pressure probe is a reliable technique for measuring the real-time variation of phloem turgor pressures in H. brasiliensis. The technique could probably be extended to the accurate measurement of phloem turgor pressure in other woody plants which is essential to test the Münch theory and to investigate the phloem water relationship and turgor pressure regulation. © 2014 An et al.

First report of a phytogeographically mixed (transitional) Middle-Late Permian fossil wood assemblage from the Hami area, northwest China, and implications for Permian phytogeographical, paleogeographical and paleoclimatic evolution in central Asia

A diverse and well-preserved fossil wood assemblage is described, for the first time, from the Middle Permian Taerlang Formation and the Upper Permian Quanzijie Formation in the vicinity of the Tianshan Town, Hami City of northwestern China. On the basis of wood microstructure, the fossil woods are classified into three genera and five species, including one new genus: Prototianshanoxylon gen. nov. and two new species: Prototianshanoxylon erdaogouense sp. nov., Prototianshanoxylon hamiense sp. nov. The new genus is characterized by window-like cross-field pitting and mixed tracheid radial wall pitting that suggest a transitional type between araucarioid-type and protopinoid-type pittings.Phytogeographically, the fossil wood assemblage is characterized by an admixture of elements of both temperate Angaran (represented by wood specimens with moderately to well defined growth rings in their secondary xylem) and tropical-subtropical north subregion of the Cathaysian floras (with wood specimens lacking well-defined growth rings). Such a phytogeographically mixed fossil wood assemblage is interpreted to represent a transitional and complex climate condition between a cool temperate and tropical to subtropical zones, showing both seasonal variation and unstable climate conditions. Previously, similarly mixed floras have already been found to exist widely in northern China ranging in age from Early to Late Permian, but the mechanisms thought to be responsible for their formation were varied and remain controversial. In this study, the formation of these mixed Permian floras of North China is linked to the closure of the Tianshan-Hingan seaway coupled with the collision and amalgamation of Siberia with North China and the Tarim block, in a manner much like closing a pair of scissors with the closure of the seaway proceeding gradually and progressively from west to the east.

Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography

Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis.