The Australian Plant Nutriomics Network

The Groups > Dr Sergey Shabala

Dr Sergey Shabala
Senior Lecturer
Crop Physiology and Plant Nutrition
University of Tasmania
Personal Website Address

Email: Sergey.Shabala@utas.edu.au

Research Aims

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The Big Question(s)

The group's research focus is on plant adaptive responses to environment (salinity, extreme temperatures, soil acidity, drought, waterlogging, nutritional disorders, biotic stresses). The "cornerstone" of all projects is the crucial role of cell membranes (and, therefore, membrane-transport processes) in plant-environment interaction. Research projects range from molecular (patch-clamp studies of membrane transport proteins) to the whole plant level. Most of projects use the MIFE technique, a non-invasive microelectrode technique for measurements on net ion fluxes from plant cells and tissues. During the last decade, the MIFE technique has become a powerful tool for in vivo studies of membrane-transport processes in living organisms. Pioneered at the University of Tasmania , this unique technology has been adapted by many leading laboratories across the world. Initially developed for plant physiology research, the MIFE technique is now used in other areas of life science such as microbiology, marine biology, medical biophysics etc. In the era of genomic research, the MIFE technique provides a unique possibility to link genetic / genomic data to cellular physiological behaviour thus providing a valuable contribution to functional genome/phenome research. In this context, we consider ourselves as being a "bridging element" between molecular biologists and whole plant physiologists and/or agronomists. As a result of that, there are several ongoing projects in the lab focusing on various aspects of plant-environment interaction. The following big questions are asked:

Q1. Salinity: what are ion transporters regulating plant adaptive responses to salinity, and how are they controlled?

The ability of plants to maintain a high cytosolic K + to Na + ratio is central to plant salt tolerance. Over millions of years, plants have evolved a sophisticated network of K + transport systems. In this project, a combination of biophysical techniques (non-invasive ion flux measurements, patch- and voltage-clamp, and a pressure-probe technique) are used to characterise the involvement of K + outward rectifying channels (KOR) into plant responses to salinity. Experiments on Arabidopsis and pea ( Pisum sativa L) plants have shown that KOR are abundant in both root (epidermis) and leaf (mesophyll) tissues. These channels showed a steep voltage-dependence and were blocked by elevated external [Ca 2+ ] (Mg 2+ and Ba 2+ caused similar effects). Divalent cations both shifted the activation curve of KOR to more depolarised voltages and decreased current amplitude. NaCl concentrations inducing salinity symptoms in crop plants caused significant net K + efflux from roots and leaf. By using Arabidopsis mutants (AKT1, SOS1, SOS2, and SOS3) it was demonstrated that salinity-induced K + efflux is mediated by KOR, rather than due to reduced K + uptake via K + inward rectifiers (KIR) or non-selective cation channels (NSCCs). This activation of K + efflux was not related to osmotic stress. Whole-plant assays, as well as experiments on excised leaves, have shown that NaCl-induced inhibition of photosynthesis can partially be prevented by elevated [Ca 2+ ] in growth media (Mg 2+ and Ba 2+ caused similar effect. Our data suggests a new mechanism of Ca 2+ action on salt toxicity - the prevention of excessive K + loss from the cell by blocking KOR. This mechanism and Ca 2+ -induced inhibition of Na + -transporting NSCCs could act in concert to enhance ameliorative effects of Ca 2+ on crop plants damaged by salt. This project has received funding from AusIndustry and includes collaboration with Drs Vadim Demidchik and Julia Davies ( University of Cambridge ), Prof Liz Van Volkenburgh ( University of Washington ) and Prof Theo Elzenga ( University of Groningen ).

The second part of this big question is related to control modes of ion transporters mediating plant adaptive responses to salinity. While recent progress in molecular cloning has identified a large number of genes mediating salt stress-induced elevation in the level of compatible solutes in plant cells, very little is known about the integration of the stress-responsive signalling cascades with membrane effectors. Of particular interest is the role of compatible solutes in this process. When confronted with a saline environment, plants respond with a significant elevation in the level of compatible solutes in the cytosol. Recent work shows that it is unlikely that these solutes are directly involved in conventional osmoprotection in plant cells. Rather, a regulatory role was identified. This project will investigate the control that compatible solutes exert on the activity of major plasma membrane transporters that are involved in plant adaptive responses to salinity. The modes of this control will be thoroughly addressed by application of a range of state-of-the-art biophysical and molecular techniques. This is a recently funded ARC Discovery project (2004-2006) to Dr S Shabala. It also involves collaboration with three leading physiological labs led by Prof Hans Bohnert ( University of Illinois , USA ), Prof Theo Elzenga ( University of Groningen , NL) and Dr Julia Davies ( University of Cambridge , UK ) who are Partner Investigators on this project.

Q2. The mystery of biological clocks: membrane transporters as an essential component of cellular clock mechanisms

This "open-ended" project elucidates physiological implications and underlying ionic mechanisms of fast (ultradian) oscillations in plants. Although oscillatory processes are ubiquitous in the Plant Kingdom , many plant physiologists ignored these as physiologically unimportant, unwanted "noise". This project addresses various issues such as functional expression of ultradian membrane oscillators; diversity and hierarchy; advantages of oscillatory control; role in plant evolution and adaptation (e.g. osmoregulation; plant movements; root nutrient acquisition; cell growth and elongation; tropisms); ultradian rhythms in timekeeping (link with circadian systems); ionic and molecular mechanisms of membrane pacemakers; and oscillations as a part of encoding mechanisms. We showed that each plant cell possesses its own, feedback-controlled, oscillators, and that the proton pump is a key component of plant membrane oscillators. Experimental evidence suggests that ultradian membrane oscillators provide a very efficient way of plant adaptation to extreme environmental conditions, including temperature, salt and osmotic stress and soil pH. An ensemble of ultradian membrane oscillators may also interact in a way of producing circadian rhythms. The project has received an official ARC funding (1998-2000) and continues in extensive collaboration with Prof Steve Tyerman ( University of Adelaide ), Dr Mary Beilby (UNSW) and Prof Dietrich Gradmann ( University of Goettingen ), specifically on modelling of membrane-transport oscillations.

Q3. Light-induced bioelectrogenesis in plant cells: receptors, effectors and signalling cascades

This is another open-ended project, which received the formal ARC funding in 2000-2004. Under its "umbrella" interaction between specific photoreceptors (phytochromes and cryptochromes) and their contribution to plant growth and developmental processes, as well as to regulation of circadian clock mechanisms, is investigated. Of particular interest are blue light perception mechanisms. Blue light is a key factor controlling a large number of physiological processes in plants, including plant growth and morphogenesis. Of the special interest to this project are phototropism, cotyledon expansion and inhibition of hypocotyl elongation. These reactions are preceded or accompanied by significant changes in electrochemical properties of cells and tissues including changes in membrane potential and ion transport across membranes. The project aims to elucidate the ionic mechanisms of blue light perception and signalling in plants. The experimental approach includes in planta characterisation of BL-induced ion flux kinetics from WT and several Arabidopsis ( nph and cry ) and pea ( pcd2 ) mutants from mesophyll, epidermal and parenchyma tissues. These measurements are complemented by measurements of light-induced changes in cytosolic free calcium using fluorescent dyes (in collaboration with Prof Theo Elzenga, University of Groningen ). Our results provide a strong evidence that some of the above mentioned mutations affected either functional expression of plasma ( nph ) or internal ( pcd2 ) membrane Ca 2+ transporters or some component of the downstream signalling cascade of the "bending" or "extension" signal, which includes changes in cytosolic Ca 2+ concentrations. This is consistent with the ubiquitous role of Ca 2+ as a second messenger in plant cells. Two postdoctoral Research Fellows, Dr Olga Babourina (currently at UWA) and Dr Branka Zhivanovic, have been involved.

Q4. Plant screening for environmental fitness

See a list of the students' projects below.

The Short-term goals

Immediate goals arising from the above projects are as follows:

Verify the hypothesis that the key function of compatible solutes in plant cells is to maintain an optimal K + /Na + ratio in the cytosol and that the activity of plasma membrane ion transporters is directly modulated by changes in the levels of compatible solutes in the cytosol ( Q1 );
Assess the solute sensitivities of the different types of membrane transporters mediating plant adaptive responses to salinity, both at the cellular and the tissue levels ( Q2 );
Elucidate signalling pathways that mediate control by these compatible solutes over transporter activity and to reveal the tissue specificity of this control ( Q3 );
Establish the role that oscillatory processes have in plant life and to support the hypothesis of frequency encoding mechanism operating in plant cells ( Q2 );
Elucidate the role of ultradian rhythms in biological timekeeping (link with cellular circadian systems) ( Q2 );
Distinguish between plasma membrane transporters involved in blue light control of plant elongation and of phototropic bending and to elucidate the ionic components of the two signal transduction pathways ( Q3 );
Develop efficient methods of screening plants for environmental fitness (salinity, waterlogging, drought, temperature extremes, Al toxicity, nutrient deficiencies) ( Q4 )

Lab members and areas of research

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There are currently 9 members in the lab, including one post-doctoral Research Fellow (to be appointed soon), 6 PhD students, one visiting researcher and myself.

Name	Position	Current research focus
Sergey Shabala	Group Research Leader	Potassium transporters and salinity; oscillations in roots and leaves; light-induced signalling
to be appointed	Post-Doctoral Fellow	Ion transporters regulating plant adaptive responses to salinity and the modes of their control by compatible solutes in plant cells
Andrew Knowles).	PhD student	Basic cation uptake in plantation eucalyptus and pines
Yuda Hariadi	PhD student	Physiological assessment of magnesium deficiency in beans
Tim Wherrett	PhD student	Nutrient uptake and acid soil related disorders in crops
Christiane Smethurst	PhD student	Lucerne screening for salt and waterlogging tolerance
Jiayin Pang	PhD student	Physiological mechanisms of waterlogging tolerance in barley
Robert Tegg	PhD student	Thaxtomin toxicity in plant cells
Zhonghua Chen	Visiting Scholar	Ionic mechanisms of salt tolerance in barley

The brief summary of students' projects is given below.

" Basic cation uptake in plantation eucalyptus and pines " (Andrew Knowles). This project is aimed to find the optimal ratios between K, Mg and Ca supply to eucalyptus and pine seedlings. Hydroponics experiments in the glasshouse are complemented by MIFE measurements of net fluxes of ions of interest from different part of the root (meristematic, elongation, mature and root hair zones). For the first time, Mg flux kinetics were measured and quantified using the ion-selective microelectrode technique. The project is funded by CRC for Temperate Wood Production and CSIRO Forestry (co-supervised by Dr Philip Smethurst).

" Physiological assessment of magnesium deficiency in beans " (Yuda Hariadi). Despite the wide recognition of Mg deficiency in several soil types, there has been little documentation of the relationship between Mg availability, growth and yield responses, and kinetics of development of deficiency symptoms in crops. The aim of this work is to characterize the development of magnesium deficiency symptoms in Vicia faba L. plants and make a comparative evaluation of the suitability of various physiological characteristics as prospective tools for early diagnostics of Mg deficiency in broad beans. Whole-plant studies (hydroponics experiments) are complemented by measuring light-induced changes in ion flux kinetics from the mesophyll tissue of bean leaves grown at various levels of available Mg. The ultimate aim of this project is to understand the association between Mg deficiency and changes in membrane-transport activity in deficient leaves and to find out the early way of diagnostics of Mg deficiency symptoms in plants.

" Nutrient uptake and acid soil related disorders in crops " (Tim Wherrett). Using a unique combination of the MIFE and patch-clamp technique, this project is aimed to understand the specific ionic mechanisms mediating plant adaptive responses to acid soil related disorders, including tolerance to Al. The major focus is on comparison of "electrophysiological behaviour" of two contrasting in Al-tolerance wheat cultivars, ET8 and ES8. This data will then be compared with results from the Signal grass, an Al-tolerant species that doesn't use organic acid extrusion to tolerate high Al level in soil solution. Possible mechanisms of amelioration of Al toxicity by Si and Ca will also be investigated. Dr Peter Ryan (CSIRO Plant Industry, Canberra ) co-supervises this project.

" Lucerne screening for salt and waterlogging tolerance " (Christiane Smethurst). This ACIAR-funded project aims at improving the adaptation of lucerne in China and Australia through the development of germplasm and novel screening techniques. The project (commenced in April 2001) is undertaken in collaboration with SARDI ( Adelaide ), University of WA ( Perth ) and seven research institutes from Inner Mongolia , Gansu and Shandong provinces ( China ). The core activities of the project are genetic resources acquisition and screening, development of screening techniques, plant screening, selection and breeding, and extension of project findings and training of scientists. The University of Tasmania contribution to this project is in developing new screening tools for salt and waterlogging tolerance.

" Physiological mechanisms of waterlogging tolerance in barley " ( Jiayin Pang). This is a part of GRDC-funded project to Tasmanian Institute of Agricultural Research (Drs Neville Mendham and Meixue Zhou) to create waterlogged-tolerant barley genotypes using QTL markers. Several contrasting barley genotypes will be studied in an attempt to find out the specific details of difference in waterlogging tolerance between these genotypes. The project includes studies of whole-plant responses to waterlogging (chlorophyll fluorescence, photosynthetic parameters, transpiration etc), anatomical and biochemical studies on plant roots, and measuring net ion flux and O 2 profiles near the root surface.

" Thaxtomin toxicity in plant cells " (Robert Tegg). This is a first attempt to use the MIFE system to characterise the effect of pathogen toxins on root membrane transport. The major focus of this project is on the interplay between thaxtomin, auxin content in roots, and Ca 2+ signalling. A range of Arabidopsis mutants will be used to functionally characterise the kinetics of this interaction. The project is led by TIAR phytopathologist Dr Calum Wilson.

Techniques in the Laboratory

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Currently active

Our main expertise is in non-invasive ion flux measuring (the MIFE) technique. Currently, there are 3 working MIFE stations available in the lab. There is also one MIFE setup in the Physics department available for research visitors in the lab. Other techniques available in the lab include:

Patch-clamp electrophysiology
Voltage-clamp
Membrane potential measurements
Cell pressure-probe
Microelectrode O 2 flux measurements
A range of techniques for plant ecophysiological studies (infra-red gas analyser for photosynthetic measurements, stomatal conductance, tisse elemental analysis etc)
Chlorophyll fluorescence

In development

We are currently working on developing the invasive microelectrode technique to measure intracellular ionic concentrations (to be linked with the MIFE non-invasive ion flux measurements).

Main areas of expertise

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Top three areas of knowledge

Ion transport across plant membranes
Salinity tolerance in plants
Circadian and ultradian oscillations in plant systems

Top three technical skills

Non-invasive ion flux measurements
Whole-plant/ecophysiology
Chlorophyll fluorescence technique

Funding

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Current

Year	Funding body	Title	Amount
2001-2004	ACIAR	Lucerne Adapted to Adverse Environments in China & Australia	67,642
2004	DEST	A Novel Approach to Study Ion Transport in Leaf Chloroplasts under Saline Conditions	36,000
2004 - 2006	ARC	Ion Transporters Regulating Plant Adaptive Responses to Salinity and the Modes of their Control by Compatible Solutes in Plant Cells	210,000

Applications submitted or planned

2004 ARC Discovery on the use of the MIFE technique for a rapid assessment of bacterial viability and growth.

Publications for last 5 years

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Shabala S (2003) Regulation of potassium transport in leaves: from molecular to tissue level. Ann Bot 92: 627-634.
Smethurst CF, Shabala S (2003) Screening methods for waterlogging tolerance in lucerne: comparative analysis of waterlogging effects on chlorophyll fluorescence, photosynthesis, biomass and chlorophyll content. Funct Plant Biol 30 : 335-343.
Shabala S , Shabala L, Van Volkenburgh E (2003) Effect of calcium on root development and root ion fluxes in salinised barley seedlings. Funct Plant Biol 30 : 507-514.
Babourina OK, Newman IA , Shabala SN (2003) Electrophysiological localization of blue light sensory sites in etiolated dicotyledon seedlings. Plant Cell Environ 26 : 1505-1514.
Shabala S (2003) Physiological implications of ultradian oscillations in plant roots. Plant & Soil 255 : 217-226.
Demidchik V, Shabala SN , Coutts KB, Tester MA, Davies JM (2003) Free oxygen radicals regulate plasma membrane Ca 2+ and K + - permeable channels in plant root cells. J Cell Science 116 : 81-88.
Demidchik V, Bowen HC, Maathuis FJM, Shabala SN , Tester MA, White PJ, Davies JM (2002) Arabidopsis thaliana root non-selective cation channels mediate calcium uptake and are involved in growth. Plant J 32 : 799-808.
Levina NN, Dunina-Barkovskaya AY, Shabala S , Lew RR (2002) Blue light modulation of ion transport in the slime mutant of Neurospora crassa. J Membr Biol 188 : 213-226.
Shabala S , Shabala L (2002) Kinetics of net H + , Ca 2+ , K + , Na + , NH 4 + , and Cl - fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues. Physiologia Plantarum 114 : 47-56.
Babourina O, Newman I, Shabala S (2002) Blue light-induced kinetics of H + and Ca 2+ fluxes in etiolated wild-type and phototropin-mutant Arabidopsis seedlings. Proceed Natl Acad Sci USA 99 : 2433-2438.
Shabala S , Schimanski LJ, Koutoulis A (2002) Heterogeneity in bean leaf mesophyll tissue and ion flux profiles: Leaf electrophysiological characteristics correlate with the anatomical structure. Ann Bot 89 : 221-226.
Shabala S , Knowles A (2002) Rhythmic patterns of nutrient acquisition by wheat roots. Functional Plant Biol 29 : 595-605.
Shabala SN , Lew RR (2002) Turgor regulation in osmotically stressed Arabidopsis epidermal root cells. Direct support for the role of inorganic ion uptake as revealed by concurrent flux and cell turgor measurements. Plant Physiol 129 : 290-299.
Shabala L, Ross T, Newman I, McMeekin T, Shabala S (2001) Measurements of net fluxes and extracellular changes of H + , Ca 2+ , K + , and NH 4 + in Escherichia coli using ion-selective microelectrodes. J Microbiol Methods 46 : 119-129.
Babourina O, Hawkins B, Lew RR, Newman IA, Shabala S (2001) K+ transport by Arabidopsis root hairs at low pH. Australian J Plant Physiology 28: 635-641
Tyerman SD , Beilby M, Whittington J, Juswono U, Shabala S (2001) Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp. Austral J Plant Physiol 28 : 591-604.
Garnett TP, Shabala SN , Smethurst PJ, Newman IA (2001) Simultaneous measurement of ammonium, nitrate and proton fluxes along the length of eucalypt roots. Plant and Soil 236 : 55-62.
Shabala S , Wilson S (2001) Fluctuations in light intensity modulate ion fluxes from grape berry mesocarp: direct evidence from microelectrode ion flux estimations. Austral J Grape Wine Res 7 : 137-143.
Shabala L, Shabala S , Ross T, McMeekin T (2001) Membrane transport activity and ultradian ion flux oscillations associated with cell cycle of Thraustochytrium sp . Austral J Plant Physiol 28 : 87-99.
Mariani MM, Shabala S , Gehring CA (2000) Plant natriuretic peptide immunoreactants modulate plasma-membrane H + gradients in Solanum tuberosum L. leaf tissue vesicles. Arch Biochem Biophys 376 : 456-458 .
Babourina O, Shabala S , Newman I (2000) Verapamil-induced kinetics of ion fluxes in oat seedlings. Australian Journal of Plant Physiology 27 : 1031-1040.
Shabala S (2000) Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll. Plant Cell Environ 23 : 825-838
Shabala S , Babourina O, Newman IA (2000) Ion-specific mechanisms of osmoregulation in bean mesophyll cells. J Exp Bot 51: 1243-1253
Babourina O, Leonova TI, Shabala S , Newman IA (2000) Effect of sudden salt stress on ion fluxes in intact wheat suspension cells. Ann Bot 85 : 759-767.
Shabala S , Newman I (2000). Salinity effects on the activity of plasma membrane H + and Ca 2+ transporters in bean leaf mesophyll: masking role of the cell wall. Ann Bot 85 : 681-686
Shabala S , Newman I, Wilson S, Clark R (2000). Nutrient uptake patterns over the surface of germinating wheat seeds. Austral J Plant Physiol 27: 89-97.
Pharmawati M, Shabala SN , Newman IA, Gehring CA. (1999). Natriuretic peptides and cGMP modulate K + , Na + , and H + fluxes in Zea mays roots. Mol Cell Biol Res Com 2 : 53-57.
Shabala SN , Newman IA (1999) Light-induced transient changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis. Plant Physiol 119 : 1115-1124

Book chapters

Shabala S (2002) Screening plants for environmental fitness: chlorophyll fluorescence as a holy grail for plant breeders. In: Advances in Plant Physiology (ed. A.Hemantaranjan). Vol. 5. Chapter 10. pp. 287-340. Scientific Publishers: Jodhpur , India .