RICERCA ITALIANA     

VEGETATIVE GROWTH CONTROL MECHANISMS IN OLIVE (OLEA EUROPAEA L.)
GENOTYPES CHARACTERIZED BY DIFFERENT VIGOUR

Università degli Studi di Palermo

Abstract

Aim of the research project is the study of morphologic, hysto-anatomical, ecophysiological and molecular of processes involved in growth control in olive. Object of research will be two clones of cv. "Leccino" olive characterized by contrasting vegetative vigour: "Leccino Minerva", with standard vigour, and "Leccino dwarf", characterized by low vigour. Furthermore, to verify the level of control exerted by the environment on growth processes, the study will be performed under two different radiative regimes.
The structure of the research proposed finds its rationale in the importance, recognized in a number of species, of the mechanisms of regulation of plant water relations in the control of vegetative growth and dry matter partitioning processes. To this aim, a central role has been given to the integration of the information that will be gathered in studying plant hydraulic architecture and its regulation at a molecular level, with data on primary production and assimilate and nutrients partitioning within tree. This integration will be possible also due to the use of innovative methodologies that allow to evaluate "in vivo" important physiological parameters of plant water relations with high accuracy and on different scale levels.
In the research, particular emphasis will be dedicated to the processes contributing to the realization of primary production, such as water transport, photosynthesis, accumulation and partitioning of dry matter and mineral elements. These aspects will be studied both under the ecophysiological and a molecular approach. One of the main targets is to obtain an assessment ofhydraulic limitations to water dynamics in the tree in relation to the interaction between the above-ground and th below-ground parts of the tree. Form the molecular point of view, the level of expression aquaporine genes will be related to hydraulic architecture parameters and to growth potential of the genotypes under study. To isolate the hydraulic resistance components in the tree, ecophysiological observations will be carried out "in situ" by a high-pressure flowmeter (HPFM). Tree hydraulic architecture and growth processess, will be integrated with hysto-anatomical observations on vascular system characters and cambial activity. Observations on canopy gas-exchange, in relation to net assimilation and within-tree dry matter and carbohydrates partitioning will give information on photosynthetic and hydraulic efficiency of the two genotypes, also in response to the two radiative regimes imposed. These measurements, together with observations on seasonal growth dynamics and the specific respiration rates measured on the different vegetative organs of the tree, will lead to a detailed carbon balance of the tree These parameters will be integrated in a specific dynamic growth simulation model which will allow the evaluation of the importance of the processess involved in growth control and an appraisal of the level of control imposed by the environmental variables on the expression of growwth potential of the two genotypes. <<<

Principal Investigator

Tiziano CARUSO Università degli Studi di PALERMO

Research Objectives

In olive culture, the necessity to reduce costs of cultural practices and to obtain a fast return of investment costs, urges the integral mechanization of the cultivation techniques, namely harvest and pruning. The availability of small-sized trees is the fundamental prerequisite for the intensification of planting systems and the adoption of machinery with high operative efficiency. While in many fruit tree species, control of tree size has been obtained through the use of rootstocks, in olive, to date, the only way to control tree size is the use of low vigour genotypes. In recent years, some examples of high density hedge-row olive orchards have been setup, with the use of low-vigour genotypes, in which the integral mechanization of olive picking operations have been achieved through the use of the same overhead harvesting machines developed for the grapevine industry.
To date, within the world olive germplasm, only few genotypes have been recognized as suitable for high-density hedge-row systems. This could lead to an impoverishment of the diversity of olive cultivars which contribute to world oil production and, for this reason, a more extensive study of olive germplasm keeping into consideration the phenotypic characters related to tree growth and vegetative vigour is highly desirable. To this purpose, it becomes crucial a deeper knowledge on mechanisms of growth limitation.
Aim of the research project is the study of morphologic, hysto-anatomical, ecophysiological and molecular of processes involved in growth control in olive. Object of research will be two clones of cv. "Leccino" olive characterized by contrasting vegetative vigour: "Leccino Minerva", with standard vigour, and "Leccino dwarf", characterized by low vigour. Furthermore, to verify the level of control exerted by the environment on growth processes, the study will be performed under two different radiative regimes.
The structure of the research proposed finds its motivations in the importance, recognized in a number of species, of the mechanisms of regulation of plant water relations in the control of vegetative growth and dry matter partitioning processes. To this aim, a central role has been given to the integration of the information that will be gathered in studying plant hydraulic architecture and its regulation at a molecular level, with data on primary production and assimilate and nutrients partitioning within tree. This integration will be possible also due to the use of innovative methodologies that allow to evaluate "in vivo" important physiological parameters of plant water relations with high accuracy and on different scale levels.
In the research, particular emphasis will be dedicated to the processes contributing to the realization of primary production, such as water transport, photosynthesis, accumulation and partitioning of dry matter and mineral elements. These aspects will be studied both under the ecophysiological and a molecular approach. One of the main targets is to obtain an assessment ofhydraulic limitations to water dynamics in the tree in relation to the interaction between the above-ground and th below-ground parts of the tree. Form the molecular point of view, the level of expression aquaporine genes will be related to hydraulic architecture parameters and to growth potential of the genotypes under study. To isolate the hydraulic resistance components in the tree, ecophysiological observations will be carried out "in situ" by a high-pressure flowmeter (HPFM). Tree hydraulic architecture and growth processess, will be integrated with hysto-anatomical observations on vascular system characters and cambial activity. Observations on canopy gas-exchange, in relation to net assimilation and within-tree dry matter and carbohydrates partitioning will give information on photosynthetic and hydraulic efficiency of the two genotypes, also in response to the two radiative regimes imposed. These measurements, together with observations on seasonal growth dynamics and the specific respiration rates measured on the different vegetative organs of the tree, will lead to a detailed carbon balance of the tree These parameters will be integrated in a specific dynamic growth simulation model which will allow the evaluation of the importance of the processess involved in growth control and an appraisal of the level of control imposed by the environmental variables on the expression of growwth potential of the two genotypes. <<<

Timescale

24 months

National and international background

As for large part of temperate zone fruit trees species, the olive, a species able to live in dry environmental conditions, the aspects related to the control of the vegetative growth of the tree, in the last thirty years, has become object of research activity at the more important scientific institution of the Mediterranean Countries (De Rio et al., 2000). The availability of rootstocks that reduce tree size represents, in fact, an important instrument, especially in the Mediterranean European Countries, in order to encourage a productive activity that today evidences signs of weakness. Similarly to what happened in fruit growing, also for olive it becomes necessary to renew the systems, intensifying planting densities to reduce the unproductive period, to increase yield and, in relation to the destination of the product, to mechanize integrally or, at least, to facilitate harvest (Tous et al., 1999). To give a new impulse to olive growing, it becomes therefore important to get new genotypes characterized by weak vegetative growth and early fructification.
Recently, the researchers attention on cultivar choices, moved towards on above mentioned characteristics instead to productivity and oil yield. In fact, the introduction of olive trees in new countries was done with cultivars like "Arbequina", "Koroneiki" e "Arbosana", in the past considered cultivars of minor importance.
The tendency to prefer cultivars of modest vigour and early fructification, is investing all the olive countries that, in the within of the respective genetic resources, watch with greater attention to increase the genetic pool privileging the genotypes of modest vigour. Also within the breeding activity, the olive growth control objective has been assuming a key role until researchers are cheeking again their germplasm collections for this new character, neglected in the past.
In this way, are emerging new genotypes with vegetative interesting characteristics. In particular, from the breeding programs for induced mutagenesis, was obtained a mutation of cultivar "Leccino", named "Leccino dwarf". It is characterized by low vegetative growth with size reduction up to 50 % of the standard cultivar and it has been assessing as rootstock and self-rooted cultivar (Pannelli et al., 2000).
In spite of several years of field observations, still few are the information on the physiological mechanisms that are involved in vigour control in such type of genotype. In particular, in olive, is still unclear if the size reductions are due to a source limitations, for a reduced of leaf photosynthetic activity or due to a sink limitations in relation with low vegetative apex growth potential.
Other factors involved in growth potential reduction could be linked to a poor functionality of the organs for resources absorption and transport (Tyree e Zimmerman, 2002) or in the different sensibility to abiotic stresses (Raveh et al., 2003).
In other temperate fruit tree species, in witch have been already found a low vigour genotypes, the mechanism that control growth is partially unknown and, generally, it takes into account aspects of sink-source limitations (Ho et al., 1989). For instance, in brachytic peach genotypes has been observed a drastic reduction of canopy net assimilation respect standard trees (Corelli et al., 1995).
This reductions has been related to a smaller photosynthetic activity for a different canopy architecture instead of a smaller specific source activity.
Is well known that, modification in vegetative growth model can be achieved in the nursery by using shading nets (Camilo et al, 2002). This effects have been related ether to a photoinhibition reduction or smaller respiration ratio due to e lower thermal regime of the leaf (Sorrentino et al., 1997). In olive the use of shading nets have the effects to induce an higher vegetative growth and, as a consequence, obtain in the nursery a reduction of plant production cycle.
For the reasons mentioned above, therefore, to analyse vegetative growth control in fruit trees is necessary take into consideration the interactions between source and sink organs of the tree. For this purpose, very useful could be the system dynamic analysis techniques that are able to summarise in a unique growth simulation model whole physiologic relationships involved in the process.
Several eco-physiological model have been developed to simulate the vegetative and reproductive growth in plants (Grossman e DeJong, 1995). In such kind of models, growth is expressed as the result of the interaction between physiologic process like assimilation, respiration, translocation and carbon accumulation that could be observed at different organisation levels: cellular, organ and plant. According to a mechanistic approach that characterise the theoretical base of the models, trees are collections of semi-autonomous but interacting organs and that carbon partitioning is driven by competition among organs (Grossman e DeJong, 1995). In particular the carbohydrate flow and partitioning are driven by sink competition for the available resources. In carbon balance, hence, the assimilated carbon represents the "supply" part of the model and the carbon pool available for respiration and growth is the "demand" part.
Growth simulation and carbon balance models have been setting up successfully for several species both in fruit trees and in forest trees. Furthermore, carbon balance and growth simulation models have been adopted to analyse physiologic phenomena like fruit response in citrus and peach to crop load reduction (Goldschmidt et al., 1992; Grossman e DeJong, 1995).
In the literature is reported that plants living in the shade show a higher allocation of biomass in the leaves, a higher leaf area for unit leaf mass and longer stems (Popma & Bongers, 1988). Shade leaves, in fact, have a slow respiration rate as well as a reduced metabolic activity. This reduces costs of maintenance of the orchards, makes smaller the carbon losses and enhances the potential relative growth (Letho & Grace, 1994). On the contrary, plants living at high light intensity show a more important root mass to get a better compensation of water losses due to high transpiration rates. A number of studies show that plant growth doesn't increase at increasing irradiance values, in particular during water and nutrient stresses (Conhan et al., 1996). Moreover, Poorter (1999) pointed out that most of the genotypes under study reached their optimum relative growth rate at intermediate light conditions.
At the best of our knowledge, no studies are available on changes of cambial activity of individuals growing in different irradiances; however, studies on growth in such plants have been carried out (Poorter,1999).
It is reported in the literature that vessel width and length influences the plant water transport (according to the Poiseuille law) and that hydraulic architecture of the vascular system is a central point in the regulation of stomatal conductance and photosynthetic rate (Sperry, 2000). At this regard, in the past decade a number of studies appeared on the relationships between xylem cavitation, leaf water potential and stomatal closure (e.g. Trifilò et al., 2003) and between leaf gas exchange, hydraulic properties of the leaf and productivity (Lo Gullo et al., 2000). Vein embolism seems to be at least co-responsible for initiating stomatal closure (Trifilò et al., 2003), thus triggering a plant response to prevent runaway embolism in a negative feedback mechanism.
It is known that water stress induces modifications of the hydraulic properties of roots (Lo Gullo et al. 1998), stems (Sperry & Tyree 1988) and leaves (Nardini et al. 2001). In some cases, changes of plant hydraulic properties are caused by morpho-anatomical changes i.e. changes in the xylem conduits diameter. This is the case when the plant is exposed to a given environmental factor over the long-term which affects plant developmental pattern. However, short-term variations of plant hydraulic efficiency can also occur as a consequence, for example, of cycles of xylem embolism and refilling (Salleo et al. 2004) or as the result of new expression and/or regulation of aquaporins which are known to be transport proteins involved in the up-regulation of water permeability of plant cell membranes (Maurel & Chrispeels 2001). Recent studies have shown that light is an environmental factor that regulates the hydraulic properties of roots and leaves both over the long- (Tyree et al. 1998) and the short-term (Nardini et al. 2005; Tyree et al. 2005). In this last case, a role of aquaporins has been suggested and, in some cases, demonstrated, possibly involving changes in the patterns of expression and or post-transcriptional regulation.
To the best of our knowledge, very few studies have attempted at measuring the effect of different irradiance levels on whole plant hydraulic properties and in particular on the partitioning of hydraulic resistances between roots, stems and leaves. Such a knowledge would be especially important for Olive breeding. In fact, it is quite common practice in Olive plantations to use shading nets to protect young plants from excessive irradiance during summer. Moreover, it is known that Olive plants in southern Italy are generally pruned in such a way that round-shaped canopies are maintained in order to induce a large degree of self-shading of the leaves, while in northern Italy plants are generally pruned to maximize the exposure of leaves to direct solar irradiance. However, the effect of these different practices on plant hydraulic architecture and, hence, on plant gas exchange, is unknown. The aim of this research is to quantify the impact of different irradiance levels on the hydraulic architecture of two genotypes of olive, one of which (LD) is characterized by reduced vegetative growth.
Even though light is the source of energy for photosynthesis, it can also be harmful to plants. Strong light often causes the reduction of photosynthetic efficiency through photoinhibition (Long et al. 1994). The susceptibility of plants to photoinhibition depends on the species and growth light-environments. In general, shade plants or low-light grown plants are more susceptible to photoinhibition than sun plants or high-light grown plants (Osmond 1994). Since photoinhibition has a potential to lower productivity and plant growth, especially in former stages of development, avoidance of photoinhibition is critical for the fitness and survival of plants in natural habitats (Takahashi et al. 2002).
It is now widely accepted that Reactive Oxygen Species (ROS) produced upon illumination are involved in the mechanism of photoinhibition (Asada 1999). ROS refer to chemicals produced during water photolysis in PSII: hydroxyl radical (•OH), hydrogen peroxide (H2O2), superoxide radical (O2-), singlet-excited oxygen (O2). These ROS can oxidize molecules in chloroplasts to inhibit partial reactions of photosynthesis and they have been shown to also bring about photoinhibition through the inhibition of de novo synthesis of D1 protein of PSII that is essential to recover from photoinhibition (Nishiyama et al. 2001).
Olive tree is generally a sun plant (or high-light grown plant), defending against high light shock, thanks to enzymatic antioxidant systems, such as superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, indoleacetate oxidase and polyphenol oxidase, and a higher activity of the enzymatic antioxidant system in full-light conditions with respect to semi-shade conditions is required for a better protection against a more pronounced oxidative stress. Interestingly, water stress and rewatering can modulate the activity of enzymatic antioxidant system of olive trees, showing that the ability of olive trees to up-regulate the enzymatic antioxidant system might be an attribute linked to drought tolerance (Sofo et al. 2005).
There is strong evidence that aquaporins (= MIPs, Major Intrinsic Proteins) are central components in plant water relations, especially during water stress (Siefritz et al. 2002). Recently Secchi et al. (2004) cloned two different genes encoding for aquaporins of Olea europea plasma membrane, OePIP1 belonging to the PIP1 subfamily, and OePIP2 belonging to the PIP2 subfamily.
Light-dependent expression of aquaporin genes and, more specifically, diurnal fluctuations of specific aquaporin transcripts have been reported in the roots of several plant species, including radish, arabidopsis (Harmer et al. 2000) and Nicotiana excelsior. A striking parallel between aquaporin gene expression levels and regulation of water transport at the tissue and cell level has been often observed under conditions of varying light. For instance, diurnal variation of root hydraulic conductivity in Lotus japonicus was well correlated to changes in abundance of mRNAs coding PIP1 homologues (Henzler et al. 1999). These fluctuations may allow coupling of root Lp to stomatal functions (Javot & Maurel 2002), and thus reduce xylem tension and prevent cavitation of root vessels at high transpiration rates (Jackson et al. 2000).
However, a few information is available about aquaporin expression and activity upon high light levels, and/or in presence of stress induced by ROS (Luu & Maurel 2005). Reactive oxygen species were also found to inhibit the activity of water channels in Chara corallina internodal cells (Henzler et al. 2004). With an induced decrease in cell hydraulic conductivity by 90%, these compounds proved to be even more efficient than mercurials. It was proposed that hydroxyl radicals act through a so-called oxidative gating mechanism either by direct oxidation of the aquaporins or indirectly through lipid membrane oxidation and formation of secondary radicals (Henzler et al. 2004). <<<