INTRODUCTION

Several thermal methods have been used for measuring sap flow in the trunk of trees: heat pulse methods (Marshall, 1958; Swanson & Whitfield, 1981) and those that depend on the temperature of a linear heat source inserted in the xylem (Granier, 1985). These methods give the sap flux density (kg s−1 m−2). The estimation of the sap flow rate (kg s−1) involves the measurement of the cross-sectional area of the functional xylem. An improvement of the heat pulse method (Cohen, Fuchs & Green, 1981) avoids this additional measurement by estimating the radial profile of sap velocity, but assembling the sensor is very difficult. Heat balance methods were applied also on sectors of tree trunks by Daum (1967), Cermák, n, Kucera & Penka (1976). With all these methods, there is a question of how representative the measurements are of the overall flow rate in the trunk.

Here we propose a direct measurement of the sap flow rate in the whole section of the stems or trunks as an improvement of the heat balance method (Sakuratani, 1981; Valancogne & Nasr, 1989a, 1989b).

INTRODUCTION

Some fruits show reversible shrinkage on a daily basis directly linked to their water balance. The recognized interpretation of this process based on a reverse flux of water from the fruit to the plant in response to the transpirational pull of the foliage (Kozlowski, 1972, 1982) is similar to that used to explain diurnal trunk shrinkage (Landsberg, Blanchard & Warrit, 1976; Powell & Thorpe, 1977). This interpretation based on a network of hydraulic resistances and capacitances, and a single water flux between “bark” and xylem (Fig. 5A) gives a good explanation of the time lag between xylem water potential and trunk shrinkage. Our results show that for fruits, the model of reverse flux between fruit and xylem stem is not valid particulary because it cannot explain the delay between fruit shrinkage and changes in xylem water potential.

MATERIALS AND METHODS

Experimental site and plant material

The experiment was carried out in a five-year-old peach orchard (Prunus persica L. cv. Fire-Red on GF 305 rootstock). This old variety was chosen because the growing curve of the fruit presents 3 stages (stage I, until end of May: high growth rate; stage II, from beginning of June to mid-July: slow growth rate; stage HI, from mid-July to mid-August: high growth rate).

INTRODUCTION

Maritime pine stands extend over more than four million hectares in southern Europe. In the natural area of this species, which includes the western Mediterranean coasts, and Atlantic coasts of Portugal, Spain and France up to the Loire estuary, summer drought lasts from one to three months. Even so, environmental and physiological control of water consumption and transpiration of Maritime pine {Pinus pinaster Ait.) are still poorly documented (Gash et al, 1989; Granier et al, 1990; Diawara, Loustau & Berbigier, 1991).

INTRODUCTION

The formation and spreading of gaseous emboli through the xylem are recognized as common events in water stressed plants (Milburn, 1979; Tyree & Sperry, 1989a). In particular, it is widely held that the increase of xylematic tension, which is caused by the drop of water potential between the soil and the atmosphere, frequently induces cavitation, i.e. the breakage of water columns and the formation of gas bubbles in the lumina of xylem conduits.

The mechanism of cavitation is still being debated, although the hypothesis that cavitation is caused by the aspiration of air bubbles through the intervessel pit membranes (the so-called air seeding hypothesis) is widely supported by experimental evidence (Sperry & Tyree, 1988; Sperry, Tyree & Donnelly, 1988).

INTRODUCTION

Transport properties of roots have been characterized by certain transport coefficients such as the hydraulic conductivity (LPr), the permeability (Psr), and reflection coefficient (σsr). So far, these coefficients have been measured only at positive root pressures. However, according to the cohesion theory of the ascent of sap, negative pressures exist in the xylem of transpiring plants. Therefore, it is of some interest whether or not the absolute values of the transport coefficients would change when pressures in the xylem drop to negative values, i.e. below vacuum (= 0 MPa).

In the present work, LPr, Psr σsr and asr of excised roots of Zea mays L. have been measured at positive and negative root pressures with the aid of the root pressure probe. Furthermore, responses of xylem pressures of intact maize plants to changes in the rate of transpiration have been followed directly.

MATERIALS AND METHODS

The hydrostatic hydraulic conductivity (LPrh) of root endsegments was determined with the aid of a root pressure probe. In ‘hydrostatic experiments’ a metal rod was moved into (out of) the probe and hence, root pressure was changed. LPrh was calculated from the following relaxation of root pressure (Steudle, Oren & Schulze, 1987). Alternatively, in ‘osmotic experiments’ the concentration of a solute in the medium was changed and LPr, Psr, and σsr were determined from the pressure responses (Steudle et al., 1987). The addition of a nonpermeating solute (mannitol) caused the root pressure to drop to a stationary negative pressure. Now, a permeating solute (Ethanol, NaNO3) was added or removed from the medium and again, LPr, Psr and σsr were calculated from root pressure responses.

INTRODUCTION

The cohesion theory as originally developed by Bohm in the 1880s (Bohm, 1893) and subsequently expanded by Askenasy (1895) and Dixon & Joly (1895) to explain the rise of sap in tall plants had to assume a metastable state of water that was difficult to reproduce in laboratory experiments. It was, therefore, not surprising that the theory was criticized, in particular by those scientists who had a thorough understanding of physics, and it was even insinuated that Bohm might be suffering from hallucinations.

An important International Workshop was held at the Vallombrosa Abbey, in the Forest of Vallombrosa, near Firenze, Italy, 29–31 May 1990. Eighty scientists participated in a discussion of water transport in plants. There have been many international workshops and conferences on plant-water relations but this was the first to focus on the failure of the hydraulic pathway within the xylem. It was possible to assemble practically all those scientists, worldwide, who have worked on the cavitation of water in the transport system. This phenomenon of cavitation, which was discovered only in the 1960s, is now being recognized as being widespread. It occurs in all the species of vascular plant so far examined, and can usually be detected on any summer's day. Its ecological significance is a matter for further research, but many consider that embolism in the xylem predisposes plants to further water stress, so that cavitation and refilling may hold the key to vegetational response to climatic warming and drying.

At the meeting it was resolved to prepare a manuscript for publication and this process (with peer review and revision) took place during 1991. Papers presented fall naturally into several subject groupings:

1. (i) analysis of the mechanism and pathway of water flow in the plant,

2. (ii) the natural repair of the hydraulic continuum, whereby emboli are redissolved in water,

3. (iii) survey of methodologies including acoustic detection of cavitation, thermoelectric techniques and nuclear magnetic resonance, and

4. (iv) case studies, examples of current work, mainly in the hot dry climates of the southern Mediterranean.

INTRODUCTION

The recent habitats occupied by the genus Gunnera L. are characterised by high rainfall, high humidity conditions in which low temperatures are rare (Osborne et al., 1991; Palkovic, 1974). The global distribution of the genus is also thought to have been more extensive in the past but has decreased dramatically due to a reduction in annual levels of precipitation and an increase in seasonal temperature variation (Osborne et al., 1991; Jarzen, 1980).

Gunnera tinctoria (Molina) Mirbel was introduced into Ireland about 100 years ago as an ornamental shrub and is now naturalised in westernmost regions of the country (Campbell & Osborne, 1990) in areas of high annual rainfall (> 1600 mm) and high humidity not unlike its tropical and sub-tropical habitats where rainfall approaches 2000 mm per year (Müller, 1982; Osborne, 1989).

INTRODUCTION

Modifications of climate may create more favourable conditions for the development and spread of fungal pathogens. On the other hand, plants may become more susceptible to pathogens if stress conditions (for instance water stress) occur as a consequence of climate changes (Palti, 1981).

Recently, two fungi species (Phomopsis occulta Trav. and Diplodia pinea (Desm.) Kickx) were found to infect the cambium and the xylem of Douglas-fir (Pseudotsuga menziesü (Mirb.) Franco) trees grown in Southern Italy (Basilicata); this observation represents a new record for this region.

Phomopsis occulta causes a definite die-back of young branches and shoots, a typical stem girdling where chromatic alterations are evident with a definite canker of limited growth on the trunk (Wilson & Hahn, 1929). Diplodia pinea, chiefly important as a cause of die-back in pines, is recognized as causing a “blight” of Douglas-fir seedlings (Peace, 1962).

As microorganisms which probably infect both the parenchyma and the xylem, these fungi may determine a reduction of hydraulic conductivity of stem and branches, either by physically blocking the xylem conduits or by promoting the formation and spread of gaseous emboli within the conduits (Zimmermann, 1983; Tyree & Sperry, 1989).

The papers collected in this volume are the contributions of invited speakers to a two-day meeting organised by the Plant Development and Plant Metabolism groups of the SEB and held at Lancaster in April 1992. One of the ideas of the meeting was to try and get away from the stereotyped divisions of ‘hormones and receptors’, ‘signal transduction’, ‘Golgi transport’, ‘protein targeting’, and so on, and look instead at things from the point of view of the proteins involved in these processes, and the effects of post-translational modifications on their activity. The result is a selection of papers on phosphorylation, glycosylation, acylation, ubiquitination, and protein processing, which illustrate the ways in which these modifications are brought about and their effects on cellular processes.

The first chapter, by the volume editors, is an introductory overview of the subject of post-translational modification in plants, in which key advances in the last few years are highlighted. The following chapters by Dixon and Hardie place the work on plant systems in a wider context by reviewing the roles of protein phosphorylation in bacteria and animals; Fallon & Trewavas provide a general discussion of phosphorylation in plant cells and some details on Ca2+/calmodulin regulated and tyrosine kinases in plant systems. In the chapters by Mattoo et al., Soll, and White et al., phosphorylation is discussed with particular reference to chloroplast function; the uses of peptides in the study of G proteins and chloroplast protein kinase are outlined.

Introduction

Many plant tissues produce ribosome-inactivating proteins (RIPs) which act as N-glycosidases removing a specific adenine residue from a highly conserved surface loop present in 23S, 26S and 28S ribosomal RNA (Lord, Hartley & Roberts, 1991). The adenine residue in question (adenine 4324 in rat liver 28S rRNA) (Endo et al., 1987), plays a necessary role in the binding of elongation factors, and ribosomes that have been depurinated by RIPs can no longer function in protein synthesis. Typically, a single molecule of RIP can depurinate 1500–2000 susceptible ribosomes per minute. Plant RIPs usually occur as monomeric proteins with molecular masses of around 30 kDa and are frequently but not always N-glycosylated. Although these RIPs can potently and irreversibly inactivate mammalian ribosomes they are not cytotoxic to mammalian cells since they are unable to enter such cells and reach the cytosol where their ribosome substrates are located. It has recently been found that these single-chain RIPs are also active against prokaryotic ribosomes (Hartley et al., 1991). In some instances, however, the RIP is joined via a disulphide bond to a second polypeptide which, in all cases described to date, is a galactose-binding lectin whose molecular mass is also around 30 kDa. These heterodimeric plant toxins are able to bind opportunistically to eukaryotic cells by interacting with galactose residues present on cellsurface glycoproteins and glycolipids. Such cytotoxic lectins are amongst the most potent cytotoxins in Nature.

Biochemical signal transduction is a term used for chains, or rather for networks, of reactions induced by chemical or physical signals. Chemicals as signals in plants may originate internally, for instance hormones or other compounds acting at low concentrations, or externally, for instance by cell–cell interactions (Scherer, 1990a). Typical physiological signals (besides hormones) in plants are gravity, light or touch. Usually, signal transduction reactions are triggered by a conformational change induced by binding of the signalling molecule or by other induced physical changes to a receptor structure, which are in turn transduced by conformational changes in proteins interacting with the receptor structure. A typical reaction chain in eukaryotic signal transduction leads from a membrane-bound receptor to interactions with G proteins, which may activate or inhibit second-messenger-generating enzymes (Gilman, 1984). The enzymes known to generate second messengers in animal cells are phospholipase C, phospholipase A2 and phospholipase D, all of which generate lipid breakdown products as second messengers, and adenylate cyclase and guanylate cyclase, which generate cAMP and cGMP, respectively. Cytosolic Ca2+ ions also function as second messengers, the concentration of which is regulated by several processes, including the action of other second messengers. Hence, it is more correct to envisage signal transduction in plants as a network rather than as a linear chain of reactions.

Second messengers often activate protein kinases specifically, and these in turn can regulate enzymatic activities by regulatory phosphorylation, leading eventually to cellular responses (Ranjeva & Boudet, 1987).

Our efforts are centred around the regulatory mechanisms controlling the assembly, function and turnover of the photosystem II (PSII) reaction centre. The PSII reaction centre consists of D1, D2, the α and β subunits of cytochrome b559 and the psbl gene product. Of these proteins, the metabolism of the D1 protein is relatively well understood. Because D1 is synthesised and rapidly degraded in the light, it is well suited for in vivo pulse–chase analysis. The D1 protein undergoes at least five post-translational modifications during its life cycle: C-terminal processing, removal of the initiating methionine residue, N-acetylation of the resulting N-terminal threonine residue, covalent palmitoylation, and O-phosphorylation of the N-terminal threonine of the mature protein. Processing of D1 occurs on stroma-exposed membranes while palmitoylation and phosphorylation occur in spatially distinct grana membranes. The palmitoylation is light-stimulated, inhibited by the herbicides atrazine and DCMU, and is apparently transient in nature. This modification might be important in the assembly of the protein in the PSII reaction centre and/or in its translocation from stroma membranes to grana. D1 phosphorylation increases with increasing light intensity, is inhibited by atrazine and DCMU and is redox regulated in vitro. In vivo, phosphorylated D1 undergoes light-dependent turnover as a function of light intensity; this process occurs under blue, green, and red illuminations and is inhibited by sodium fluoride but not by the PSII herbicides. We are investigating the role of phosphorylation–dephosphorylation in the function and dynamics of PSII.