Physiological and Anatomical Responses of Grapevine Roots to Drought Stress and Recovery ...

121 Pages·2017·3.76 MB·English

by  Italo Cuneo

Physiological and Anatomical Responses of Grapevine Roots to Drought Stress and
Recovery after Re-watering
By
ITALO F. CUNEO ARRATIA
B.S. (Pontificia Universidad Católica de Valparaíso) 2010
M.S. (Pontificia Universidad Católica de Valparaíso) 2013
Dissertation
Submitted in partial satisfaction of the requirements of the degree of
DOCTOR OF PHILOSOPHY
in
Horticulture and Agronomy
in the
OFFICE OF GRADUATE STUDIES
of the
UNIVERSITY OF CALIFORNIA
DAVIS

Approved:

Andrew J. McElrone, Chair

M. Andrew Walker

Astrid Volder

Committee in Charge
2017
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ABSTRACT
In this dissertation, root systems of different grapevine rootstocks have been
examined in their response to drought and re-watering and in their ability to take up water
through woody roots. Fine roots are commonly thought to be a weak link in the soil-plant-
atmosphere continuum, capable of breaking the water transport pathway in conditions of
drought stress. However, the exact site and the sequence of this dysfunction is not known.
In the first chapter, we utilized x-ray microtomography (microCT) to explore in vivo
changes in structure and embolism formation, and we complemented these studies with
hydraulic experiments to test how the hydraulic properties of the radial pathway were
affected by drought. We found that lacunae formation in fine root cortical cells, and not
embolism formation, is the initial and primary driver of reduced fine root hydraulic
conductivity (Lp). After re-watering, we found no recovery of Lp, even though the stem
r r
water potentials ( ) and stomatal conductance did recover. In the third chapter, we
stem
hypothesized that when root systems have limited amounts of fine roots, some water
uptake must happen through suberized woody roots. Using microCT and hydraulic
measurements, we found that when water is delivered to suberized woody roots,
hydration of the bark occurs first and is then followed by water absorption in the xylem
and embolism removal (~20% of vessels refilling completely within 15 h). Hydraulic
experiments showed that while Lp of suberized woody roots is 0.7% of the Lp found in
r r
fine roots, woody roots are still conductive and water uptake can occur when fine roots
are absent. For the fourth chapter, two commonly used grapevine rootstocks were tested
for their physiological performance under mild to severe drought stress and subsequent
recovery. We used neutron radiography (NR), microCT, fluorescent microcopy and
ii
hydraulic measurements to get a detailed picture of the physiological mechanisms that
might contribute to drought tolerance/susceptibility in grapevine rootstocks. We found that
the drought resistant rootstock examined (i.e. 110R) showed a faster decline in Lp during
r
mild drought stress and a rapid recovery of root elongation and Lp after re-watering. The
r
observed changes in hydraulic properties coincide with lacunae formation during mild
drought stress and with the recovery in root elongation after re-watering. The integration
of response to drought and hydraulic recovery after re-watering is important for
understanding drought tolerance in grapevine rootstocks.

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TABLE OF CONTENTS
LIST OF FIGURES .......................................................................................................... vi
LIST OF TABLES ............................................................................................................ ix
ACKNOWLEDGEMENTS ............................................................................................... x
Preface ............................................................................................................................ xi

Chapter 1 General Introduction ....................................................................................... 1
General overview .................................................................................................. 1
Woody root system morphology, classification and environmental importance .... 2
Water uptake and transport in roots ..................................................................... 4
Physiological responses to drought in roots ......................................................... 6
New visualization technologies for studying stress root physiology ...................... 8

Chapter 2 Mechanical failure of fine root cortical cells initiates plant hydraulic decline
during drought ............................................................................................................... 10
Abstract .............................................................................................................. 10
Introduction ......................................................................................................... 11
Material and Methods ......................................................................................... 14
Plant Material ........................................................................................... 14
Measurements of plant water status ........................................................ 15
X-ray microtomography (microCT) ........................................................... 15
MicroCT image analysis ........................................................................... 17
Root hydraulic conductivity (Lp) .............................................................. 18
r
Fluorescent light microscopy ................................................................... 19
Leaf gas exchange .................................................................................. 20
Results ............................................................................................................... 20
In-vivo observation of changes in root structure and function .................. 20
Changes in hydraulic properties .............................................................. 26
Discussion .......................................................................................................... 28
Acknowledgements ............................................................................................ 36
Supplemental data .............................................................................................. 37

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Chapter 3 Water uptake can occur through woody roots and facilitates embolism repair
in grapevine ................................................................................................................... 43
Abstract .............................................................................................................. 43
Introduction ......................................................................................................... 44
Material and Methods ......................................................................................... 47
Plant Material ........................................................................................... 47
X-ray microCT .......................................................................................... 48
Hydraulic properties of woody roots ......................................................... 51
FDA-PI staining ........................................................................................ 52
Wettability of the bark .............................................................................. 53
Results ............................................................................................................... 53
Discussion .......................................................................................................... 62
Acknowledgements ............................................................................................ 67
Supplemental data .............................................................................................. 69

Chapter 4 Performance of fine roots for contrasting grapevine rootstocks subjected to
drought and recovery using neutron radiography and microCT .................................... 73
Abstract .............................................................................................................. 73
Introduction ......................................................................................................... 74
Material and Methods ......................................................................................... 76
Plant Material ........................................................................................... 76
Measurements of plant water status ........................................................ 77
Neutron Radriography.............................................................................. 78
X-ray microCT .......................................................................................... 79
Hydraulic properties of fine roots ............................................................. 80
Fluorol yellow 088 staining ....................................................................... 81
Results ............................................................................................................... 82
Discussion .......................................................................................................... 91
Acknowledgements ............................................................................................ 95
Supplemental data .............................................................................................. 96

References .................................................................................................................... 99
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LIST OF FIGURES
FIGURE 2.1. Transverse microCT images through fine and coarse roots of grapevine
showing embolism and cortical lacunae at different stem ............................................. 22

FIGURE 2.2. Cortical lacunae formation over time induced using sucrose solutions with
osmotic potential (π) of 0, -0.5, -1.0 and -1.5 MPa ...................................................... 23

FIGURE 2.3. Typical examples of the spatial structure of drought-induced cortical lacunae
in grapevine fine roots ................................................................................................... 24

FIGURE 2.4. Theoretical percent loss of conductivity for grapevine fine and coarse roots
...................................................................................................................................... 25

FIGURE 2.5. Drought-induced changes in root hydraulic conductivity (Lp) and
r
percentage area covered by cortical lacunae (of total cortical area) in grapevine fine roots
as a function of stem water potential ............................................................................. 27

FIGURE 2.6. Schematic illustration of cortical lacunae progression and its implications on
Lpr ................................................................................................................................. 30

FIGURE S2.1. Cartoon illustrating how plants were grown in order to get access to coarse
and fine roots ................................................................................................................ 37

FIGURE S2.2. Representative relationship between osmotic pressure and volumetric flow
rate ................................................................................................................................ 38

FIGURE S2.3. Mean diameter of cells located in the outer, middle and inner layers of the
cortex ............................................................................................................................ 39

FIGURE S2.4. Frequency distribution of all vessel diameters in fine and coarse roots.
...................................................................................................................................... 40

FIGURE S2.5. Pre-dawn and midday stem water potentials ......................................... 41

FIGURE S2.6. Free-hand cross sections of fine roots obtained from grapevine plants
subjected to well-watered (Control, -0.4 MPa  ) and drought-stress (3 days of water
stem
deprivation -1.2 MPa stem) conditions .......................................................................... 42
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FIGURE 3.1. Cartoon illustrating the preparation procedure of excised root samples for
microCT imaging ........................................................................................................... 49

FIGURE 3.2. Representative time series of bark hydration dynamics. .......................... 54

FIGURE 3.3. Temporal changes in percentage of air-filled pores of the bark of woody
roots as a consequence of bark hydration. ................................................................... 55

FIGURE 3.4. Radial root hydraulic conductivity (Lp) of intact root, soaked root (i.e. intact
r
root soaked in water for 24 hours) and root without bark ............................................. 56

FIGURE 3.5 Representative time series of transverse microCT images through excised
woody roots of grapevine. ............................................................................................. 58

FIGURE 3.6. Temporal changes of refilled vessels (presented as % of initially embolized)
in excised woody roots obtained from grapevine plants ............................................... 59

FIGURE 3.7. Process of vessel refilling in woody roots documented using microCT
scanning in the same position at different times ............................................................ 60

FIGURE 3.8. Transverse woody root sections of grapevine stained with fluorescein-
diacetate/propidium-iodide (FDA/PI) and observed under fluorescent light (G/R filter
system for blue and green excitation). .......................................................................... 61

Figure S3.1. Representative masked microCT images before processing (A) and after
processing (B) for quantifying the volume occupied by empty porous spaces during
hydration. ..................................................................................................................... 69

Figure S3.2. Cartoon illustrating the set-up of hydraulic measurements. Every root (n =
6) was measured 3 times (i.e. Intact root, root without bark, axial) by tracking flow in a
glass capillary. ............................................................................................................... 70

Figure S3.3. Representative pressure – flow relationship of intact root, soaked root, and
root without bark treatments. Volume flow was induced using a hydrostatic pressure. 71

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Figure S3.4. Advancing contact angle – surface of contact relationship. ..................... 72

FIGURE 4.1. Neutron radiography was used to track root elongation dynamics during
drought .......................................................................................................................... 83

FIGURE 4.2. Representative transverse microCT images of 110R and 101-14MGT
through fine roots (i.e. maturation root zone, 5 cm back from tip) showing cortical lacunae
at different stem ........................................................................................................... 85

FIGURE 4.3. Percentage of lacunae in the cortex of fine roots of 110R and 101-14Mgt.
...................................................................................................................................... 86

FIGURE 4.4. Free-hand cross sections of fine roots of 110R and 101-14Mgt obtained
from plants under well-watered (i.e. ~0.3 MPa  ) and drought (i.e. ~1.0 MPa)
stem
conditions and from two different developmental root regions (i.e. secondary growth zone
and tip). ......................................................................................................................... 87

FIGURE 4.5. Fine root hydraulic conductivity (Lp) for 110R and 101-14Mgt rootstocks at
r
different stress levels. .................................................................................................... 88

FIGURE 4.6. Water-depletion slopes in the soil surrounding the root tip and maturation
zone for 110R and 101-14 Mgt. ..................................................................................... 90
Figure S4.1. Experimental set-up of neutron radiography at the McClellan Nuclear
Research Center. .......................................................................................................... 97

Figure S4.2. Representative plot profiles of neutron radiographs across root tips and
maturation root zones of 110R and 101-14 Mgt. .......................................................... 98

Figure S4.3. Representative relationship between osmotic pressure and volumetric flow
rate for 110R and 101-14Mgt fine roots. ....................................................................... 99

viii
LIST OF TABLES
Table 4.1. Stem water potential ( ), estimated root elongation, and number of laterals
stem
of 110R and 101-14MGT at different levels of stress.
………………………………………………………………………………………………….84

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ACKNOWLEDGEMENTS
This dissertation is dedicated to my wife, Claudia Airola, who embraced the idea
of leaving behind our beloved family and country, to help me accomplish my goals. Her
support and love during these almost 4 years were absolutely the key of my success.
I would like to extend a deep sense of respect and gratitude to my supervisor, Dr.
Andrew J. McElrone, for his expert guidance and advice during every step of my PhD.
His office door was always open for every kind of scientific discussion. Yet, more
important than this, he consistently motivated me to extract the very best of myself.
I am very grateful to Dr. Thorsten Knipfer for his guidance and genuine disposition
to critically read and assist my work. His technical comments and tips on root hydraulics
and anatomy were indispensable and all the philosophical discussions were invaluable.
I would like to thank Dr. M. Andrew Walker and Dr. Astrid Volder for fruitful and
thorough comments to this dissertation. I extend my thanks Dr. Felipe Barrios-Masias,
Caetano Albuquerque, Jake Uretsky and Clarissa Reyes for technical support.

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