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Physiological and molecular basis of salt and waterlogging

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Physiological and molecular basis of salt and waterlogging ...

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									 Comparative molecular physiology of salt and
  waterlogging tolerance in Lotus tenuis and L.
corniculatus: towards a perennial pasture legume
                 for saline land

                   Natasha Lea Teakle




          This thesis is presented for the degree of
                    Doctor of Philosophy

            The University of Western Australia

                  School of Plant Biology

        Faculty of Natural and Agricultural Sciences

                         June 2008
ABSTRACT


Salinity and waterlogging interact to reduce the growth of most crop and pasture
species. Species that are productive on saline-waterlogging land are needed for
Australian farming systems. One option is Lotus tenuis, a perennial legume widely
grown for pasture in the flood-prone and salt-affected Pampa region of Argentina. To
identify mechanisms responsible for the adverse interaction between salinity and
waterlogging, Lotus tenuis with a reputation for tolerance was compared with L.
corniculatus, the most widely cultivated Lotus species.


The physiology of salt and waterlogging tolerance in L. tenuis (4 cultivars) was
evaluated, and compared with L. corniculatus (3 cultivars). Overall, L. tenuis cultivars
accumulated less Na+ and Cl-, and more K+ in shoots than L. corniculatus cultivars,
when exposed to 200 mM NaCl for 28 d in aerated or in anoxic (stagnant agar)
solutions. In a NaCl dose response experiment (0 to 400 mM NaCl in aerated solution),
Lotus tenuis (cv. Chaja) accumulated half as much Cl- in its shoots than L. corniculatus
(cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na+ in
treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants
at 200 mM NaCl; L. tenuis (cv. Chaja) accumulated about half as much Cl- in old
leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv.
San Gabriel). There were not, however, significant differences between the two species
for Na+ concentrations in the various shoot tissues under aerated NaCl treatment.


To determine the cause of the differences in shoot concentrations between the Lotus
species, xylem concentrations of Na+ and Cl- were measured in sap collected using
spittlebugs (Philaenus spumarius) from plants in saline (200 mM NaCl) and/or stagnant
treatments over 4 weeks. In aerated-NaCl solution (200 mM), L. corniculatus had 50%
higher Cl- concentrations in the xylem and shoot compared with L. tenuis, whereas
concentrations of neither Na+ nor K+ differed between the species after 28 d treatment.
In stagnant-plus-NaCl solution, xylem Cl- and Na+ concentrations of L. corniculatus
increased to twice those of L. tenuis. These differences in xylem ion concentrations,
which were not caused by variation in transpiration between the two species,
contributed to lower net accumulation of Na+ and Cl- in shoots of L. tenuis, indicating
ion transport mechanisms in roots of L. tenuis were contributing to better ‘exclusion’ of
Cl- and Na+ from shoots, compared with L. corniculatus. Thus, Cl- ‘exclusion’ is a key
trait contributing to salt tolerance of L. tenuis; and ‘exclusion’ of both Cl- and Na+ from
the xylem enables L. tenuis to better tolerate the interactive stresses of salinity and
waterlogging.


Enhanced root aeration would contribute to maintaining Na+ and Cl- transport processes
in roots of plants exposed to stagnant-plus-NaCl treatments. Measurements of radial O2
loss (ROL) under stagnant conditions indicated that L. tenuis roots exhibit a partial
barrier to ROL and had higher O2 concentrations at root tips, compared with L.
corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma.
Therefore, enhanced root aeration might have contributed to the maintenance of Na+
and Cl- ‘exclusion’ in roots of L. tenuis exposed to the stagnant-plus-NaCl treatments.
Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or
stagnant-plus-NaCl treatment, demonstrating greater tolerance to these stresses over
time.


To further understand the mechanisms of Cl- transport under salt stress,       36
                                                                                    Cl tracer
experiments were conducted at steady-state Cl- uptake after 4 d treatment at 200 mM
NaCl. The two species did not differ in unidirectional uptake of Cl-, but rather transport
of Cl- from roots to shoots started sooner in L. corniculatus and was twice the rate of L.
tenuis under saline treatment. In addition, L. tenuis has greater efflux of Cl-, and thus
maintains lower total Cl- concentrations over time. A possible candidate gene involved
in regulation of Cl- transport in L. tenuis could be a cation-chloride cotransporter
(CCC). CCCs are predicted to play a role in salt tolerance of plants, particularly when
Na+ and Cl- are co-transported across membranes. A CCC gene was cloned from L.
tenuis (LtCCC); protein sequence analysis showed LtCCC had 80% homology to an
Arabidopsis CCC, and 91% with a putative Medicago truncatula CCC. Results from
real-time qPCR showed expression of LtCCC increased under salt stress for L. tenuis
roots, but not in L. corniculatus. Therefore, LtCCC may contribute to the control of
root-to-shoot Cl- transport, and therefore differences in salt tolerance between L. tenuis
and L. corniculatus.


The combination of salinity and anoxic stress leads to differences in Na + transport, in
addition to Cl-, between L. tenuis and L. corniculatus. At the root level, L. tenuis had
17% higher root and 25% lower shoot Na+ concentration than L. corniculatus after 9 d
stagnant-plus-NaCl treatment. Therefore, during early stages of exposure to salinity, L.
tenuis accumulated a higher proportion of total Na+ in the roots under combined
stagnant-plus-NaCl treatment (55% versus 39% for L. corniculatus). Na+ transporters,
particularly those relying on H+ gradients across membranes, which in turn require
adequate ATP levels, could be impaired under O2 deficits that inhibit respiration. To
study the effect of O2 deficiency on a Na+ transporter, an NHX1-like gene was cloned
from L. tenuis and identity established via sequencing and yeast complementation
studies. Real-time qPCR showed expression of NHX1 in L. tenuis roots increased under
stagnant-plus-NaCl treatment, whereas it was reduced in L. corniculatus. Thus,
maintaining O2 transport to roots, together with up-regulation of an NHX1-like gene for
Na+ accumulation in vacuoles, contributes to tolerance of L. tenuis to combined salinity
and waterlogging stresses.


This study highlights the importance of minimising Cl- transport to shoots as a
mechanism of salt tolerance and has identified a CCC-like gene in L. tenuis as a
candidate for mediating root-to-shoot Cl- transport. Under combined stagnant-plus-NaCl
treatment, control of Na+ transport is another mechanism contributing to tolerance in
these Lotus species. Enhanced root aeration in L. tenuis maintains root Na+ transport
processes, such as accumulation in vacuoles via NHX1-like genes, to diminish xylem
loading to the shoot. Overall, this thesis has contributed new knowledge on the potential
of Lotus tenuis as a saltland pasture and has significantly enhanced current
understanding on the mechanisms of salinity and waterlogging tolerance in plants.
ACKNOWLEDGEMENTS

Firstly, I would like to thank my supervisors Tim Colmer and Daniel Real. Thank you
for encouraging and motivating me during the last few years. No matter how busy, you
always found time to discuss protocols, read drafts, listen to my complaints about failed
experiments and ‘de-stress’ me! You have also assisted greatly in my career
development by helping create new contacts, encouraging me to go overseas for
extended research trips and assisting with applications. The enthusiasm, attention to
detail and work ethic you both display inspires me to strive for excellence in my own
work.

I am grateful to the Grains Research and Development Corporation for my PhD
Scholarship and the AW Howard fund for a top-up stipend. Special thanks also to the
Salinity CRC for extra research funds and professional development. The overseas
research would not have been possible without the financial support of the AW Howard
travel awards, Mary Janet Lindsay of Yanchep Memorial fund and the UWA travel
award.

Thanks to Anna Amtmann for letting me complete 12 months research towards my
thesis at Glasgow University, UK. I loved my time in Scotland and the Amtmann/Blatt
group was fantastic to work with. Special thanks to Annegret, for assisting with toad
operations, and surviving with me the frustrating months of dying oocytes! Thanks also
to Bernie, Patrick, Richard and Bo for all your advice.

Special thanks to Tim Flowers for letting me complete 3 months research at Sussex
University, UK, and most importantly for introducing me to the wonderful spittlebugs! I
am very appreciative of how welcoming you and your family were to me during my
time in Sussex.

Thanks to the members of the Plant Ecophysiology group at UWA (‘Colmerites’!), both
past (Jeremy, Imran, Kirsten) and present members (there are now too many to name!)
who assisted me greatly in preparing for experiments and harvests. Thanks to Hans and
the office staff for assisting with the administrative requirements of my PhD. I also
received fantastic assistance from Gary, Elizabeth and Hai (student labs), Leon and staff
(glasshouses) and Alan and Sean (computing).

Special thanks to my family for their support during the last few years and putting up
with me being a ‘poor student’ for so long. Having a family of farmers helped me
always keep the science in perspective.

Finally, and most importantly, my heart-felt thanks to ‘Sir’ James, the world’s greatest
pot lid alfoiler! You put up with the crazy hours I worked and tolerated coming second
to Lotus plants. I dedicate this thesis to you, as you understood how important it was to
me, and without your endless love, support and encouragement, I would not have
survived my PhD.
TABLE OF CONTENTS

     Content                                                              Start page #


     Cover page…………………………………………….……………………….. i
     Thesis abstract ………………………………………………………….…….. iii
     Acknowledgements……..……………………………………………………. vii
     Table of contents……………………………………………………………… ix
     Thesis declaration ……………………………………………………………..xi




     Chapter 1: General Introduction…………………………………………… 1
     1.1 Salinity and waterlogging in agriculture                                   2
     1.2 Perennial pastures for saline agriculture                                  3
     1.3 Lotus tenuis: a potential new perennial pasture option for saline land     3
     1.4 Thesis outline and objectives                                              4


     Chapter 2: Review of the Literature………………………………………... 7
     2.1 Summary                                                                    8
     2.2 Introduction                                                               8
     2.3 Root aeration traits and tissue tolerance                                 10
           2.3.1 Adventitious roots                                                11
            2.3.2 Aerenchyma                                                       13
           2.3.3 Barrier to radial oxygen loss                                     15
            2.3.4 Cellular anoxia tolerance                                        16
     2.4 Regulation of ion transport under salt and waterlogging stress            18
            2.4.1 Influx of Na+ and Cl- into roots                                 19
            2.4.2 Efflux of Na+ and Cl- from roots                                 22
            2.4.3 Accumulation in root vacuoles                                    23
           2.4.4 Xylem net loading                                                 24
     2.5 Conclusions                                                               29
Content                                                            Start page #


Chapter 3: Growth and ion relations in response to combined salinity
and waterlogging in the perennial forage legumes Lotus corniculatus
and Lotus tenuis……………………………………………………………… 31
Abstract                                                                     32
Introduction                                                                 32
Materials and Methods                                                        34
Results                                                                      37
Discussion                                                                   49
Acknowledgements                                                             51


Chapter 4: Lotus tenuis tolerates the interactive effects of salinity and
waterlogging by ‘excluding’ Na+ and Cl- from the xylem…………………. 53
Abstract                                                                     54
Introduction                                                                 54
Materials and Methods                                                        56
Results                                                                      60
Discussion                                                                   72
Acknowledgements                                                             76


Chapter 5: Minimising root-to-shoot transport of Cl- contributes to salt
tolerance of the perennial legume Lotus tenuis and is correlated with
expression of a CCC-like gene………………………..…………………….…77
Abstract                                                                     78
Introduction                                                                 78
Materials and Methods                                                        80
Results                                                                      89
Discussion                                                                  102
Acknowledgements                                                            107
Content                                                                  Start page #


Chapter 6: Lotus tenuis tolerates combined salinity and waterlogging:
maintaining O2 transport to roots and expression of an NHX1-like gene
contribute to regulation of Na+ transport ………………………………………….109
Abstract                                                                         110
Introduction                                                                     110
Materials and Methods                                                            113
Results                                                                          120
Discussion                                                                       133
Acknowledgements                                                                 138


Chapter 7: General Discussion……………………………………………………. 139
7.1 Summary of key findings                                                      140
7.2 Mechanisms for tolerance to combined salinity and waterlogging               142
7.3. Implications of results for breeding Lotus tenuis for saline land           146
7.4 Limitations of the thesis and future research priorities                     147
7.5 Conclusions and outlook                                                      149


Bibliography…………………………………………………………………………151
THESIS DECLARATION


This thesis contains published work which has been co-authored. The bibliographical
details of the work and where it appears in the thesis are outlined below. Work carried
out for these papers and the rest of the thesis is entirely my own with one exception:
xylem Cl- readings (Chapter 4), which were taken by T. Flowers on samples I had
collected, but analysed by T. Flowers after I left the UK due to equipment problems
during my visit and subsequent time constraints. The co-authors provided technical
advice on experiments when required and editorial input into drafts.


Chapter 3: Teakle NL, Real D, Colmer TD (2006). Growth and ion relations in
response to combined salinity and waterlogging in the perennial forage legumes Lotus
corniculatus and Lotus tenuis. Plant and Soil 289: 369-383.


Chapter 4: Teakle NL, Flowers TJ, Real D, Colmer TD (2007). Lotus tenuis tolerates
the interactive effects of salinity and waterlogging by ‘excluding’ Na+ and Cl- from the
xylem. Journal of Experimental Botany 58: 2169-2180.

								
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