There are array of ways of studying plant response to drought or any kind of stress, ranging from physiological, morphological, cellular level, biochemical, anatomical or even at molecular level. This presentation deals or shows how plant tissues can respond under stress at anatomical level and hence contribution to tolerance.

1. Can changes in root anatomical traits
during stress enhance drought &
Salinity tolerance ?
KABEYA Jerry Muamba
Department of Crop Physiology, University of
Agricultural Sciences, Bangalore, India

2. Roots
 Usually underground individual
functions:
 anchor plant and hold upright
 absorb water and minerals form soil and conduct
to stem
 store food & propagation

3. A better understanding of the basic mechanisms of root
growth and development is necessary

4. Causes of plant’s action and reaction in her ever changing environment
Drought Temperature
Temperature
High Salinity Soil compaction
Others

5.  Plant’s rapid resilience to these unfriendly environments is thus crucial and matter
of urgency to keep surviving.
Resilience strategies
Physiological
e.g. stomata and non- Anatomical. Biochemical.
stomata water loss e.g. root diameter, etc. e.g. OA, proline level,
check. etc.
Morphological Molecular.
e.g. leaf angle, root e.g. Genes expression ,
length, etc. etc.

6.  Plant roots are crucial for the
absorption and translocation of water
and nutrients through xylem to above
ground for various cellular processes.
 Anatomical root traits such as;
 cortex thickness
 suberized exo and endodermis,
 Casparian bands,
 aerenchymatous tissues, etc.
play great role in root functioning in term
of hydraulic conductance, oxygenation,
protective measures etc.

7. Root Cortex size
 The size of cortex play important role in
water conductivity and hence plant
tolerance to stress
 Thus, smaller cortex width results in
higher conductivity because the
distance of water to reach vascular
cylinder is shorten, and better capacity
to tolerance to stress

8. Casparian Bands
The main role of this structure is to block non selective apoplastic
bypass flow of ions and water through the cortex into vascular
cylinder (no free water & ion movement through apoplast).

9. Suberin lamellae structure
 Suberin structure consists of two
domains,
 Polyaromatic
 Polyaliphatic
• Permeability of apoplastic
barriers to water or solute
transport strongly to the amount
and chemical composition of
aliphatic and aromatic domains
Polyaromatic Polyaliphatic
Roles:
a. It reduces ROL to the rizosphere and increase longitudinal diffusion in the
aerenchyma
b. It avoid partial absorption of phytotoxins from rhizosphere
c. It diminishes the release of excess gases such as ethylene and methane in soil
d. it decreases the intake of water and nutrients

10. Aerenchyma structure
Submerged roots
Aerenchyma and air spaces
Irrigated condition
 Aerenchyma, gas or air-spaces, are formed either as part of normal development or in
response to stress ( hypoxia or nutrient deficiency) by two main mechanisms
I. Lysigeny : gas- spaces are formed via cell lysis
II. Schizogeny : gas–spaces are formed during cell separation during tissue
development
 The importance is to provide an internal pathway for oxygen transport between roots and
the aerial environment. Along this pathway, O2 is supplied to the roots and rhizosphere,
while CO2, ethylene, etc. move from the soil to the shoot and the atmosphere

11. Morphological Response of root of Fraxinus angustifolia seedling under compaction
treatments
(A) No compaction of loam soil, (B) high compaction of loam soil, (C) no compaction of
sandy-loam soil and (D) high compaction of sandy-loam soil. For each root, the value of
soil bulk density is shown.

12. Pictures of root histology of Fraxinus angustifolia seedling under compaction
treatments: no compaction treatment of loam soil is shown in the left; high compaction
treatment of sandy-loam soil on the right.
X, xylem; P, phloem; S, suber; R, rhizodermis; XV, xylem vessels
D. Alameda and R. Villar, 2012

13. Causal relationships of the effects of soil compaction from root to whole plant functioning.

14. (January 2012)
Drought-Induced Root Aerenchyma Formation Restricts Water
Uptake in Rice Seedlings Supplied with Nitrate
Xiuxia Yang, Yong Li, Binbin Ren, Lei Ding, Cuimin Gao, Qirong Shen and Shiwei Guo,
The aims of the study were to investigate;
1. Whether the root aerenchyma and water uptake of water-stressed rice are related to
both nitrogen and water stress.
2. Also analyze the relationship between root aerenchyma and root hydraulic
conductivity in rice seedlings supplied with different forms of nitrogen under water
stress.

15. Methodology
Rice seeds (O. sativa L., cv. ‘Shanyou 63’ hybrid Indica China, and cv. ‘Yangdao 6’
Indica China) were disinfected in 10% H2O2 for 30 min and then immersed in
saturated CaSO4 solution for 2 hrs.
When the seedlings had developed an average of 2.5 visible leaves in distilled water,
they were transplanted to 3 liter buckets and into a full-strength mixture of
ammonium and nitrate nutrition (ANN)
One week later, the seedlings were supplied with either ammonium (AN) or nitrate
(NN) nutrition alone
Two weeks after, water stress was conducted by adding 10% PEG (10%, w/v; mol.
wt 6,000 Da) to the nutrient solutions ( -0.15MPa) for 1 week.
Four treatments were applied: AN, NN, ANP and NNP (ANP and NNP represent the
ammonium and nitrate nutrition with 10% added PEG6000, respectively).

16. Nutrient solutions were changed every 3 days and pH was adjusted to 5.50 ± 0.05
every day with 0.1 mol l-1 HCl or 0.1 mol l-1 NaOH.
Observations and measurements
Xylem sap flow rate measurements
plants were de-topped approximately 2 cm above the interface of the roots
and shoots, and the exudation was immediately cleaned with filter paper to
avoid contamination.
Absorbent cotton was placed on the top of each piece of de-topped xylem
and covered with plastic film to avoid evaporation.
Xylem sap was collected from 20:0 h to 06:00 h, and the exudation rate was
calculated from the differences in cotton weight.

17. Determination of water uptake rate and aquaporin activity
Water uptake of intact plants, with or without addition of 0.1mM HgCl2 (aquaporin inhibitor) to
the hydroponic solutions, was determined by the depletion of nutrient solution (by weighing).
The contribution of aquaporin to the water uptake rate was calculated from the difference in
the water uptake rate between the control and HgCl2-treated plants.
Root aerenchyma measurements
4mm to 5mm root pieces were cut from the middle of 7–8 cm long newly formed adventitious
roots using a razor blade.
Root tissues were then fixed with 3% glutaraldehyde in 0.1M phosphate buffer (pH 7.0) for 3–5 h.
The tissues were thereafter rinsed in with phosphate buffer and sections were dehydrated in a
graded ethanol series and dried using a vacuum freeze-dryer.
The dried root sections were coated with gold-palladium and glued onto specimen stubs.
Finally, root samples were viewed and photographed with a scanning electron microscope (model
3000N; Hitachi).
Aerenchyma formation was calculated from the observed electron microscope images with Motic
Images Plus 2.0 (China Group Co., Ltd.)

18. Results
Effects of nitrogen form and water stress on shoot and root DW (g plant1), the
root/shoot ratio and tillers of rice plants (No. plant1) (cv. Shanyou 63 and cv.
Yangdao 6)
AN -ammonium , NN- nitrate (NN) under either non-water stress or water stress simulated
by adding 10% PEG6000 (NH4+ + PEG as ANP; NO3- + PEG as NNP).
NH+4 nutrition improves water holding in rice seedlings and subsequently enhances
their resistance to drought

19. Effect of nitrogen form and water stress on root aerenchyma formation in rice plants
AN AN
Aerenchyma formation (%)
NN NN
ANP ANP
 NNP NNP
Aerenchyma formation during stress (either hypoxia or anoxia) promotes radial oxygen
release from roots, leading to rhizosphere oxygenation
 Also facilitate methane diffusion from water-loggedShanyou 63 to the atmosphere.
sediments Yamgdao 6
Shanyou 63 Yamgdao 6
 The formation of root aerenchyma may be an adaptation to low P availability by reducing
Rice plants were supplied with ammoniumP (AN) or nitrate (NN) under either 2010).
the metabolic costs of soil exploration and recycling (Fan et al. 2003, Zhu et al. non-water
stress or water stress simulated by adding 10% PEG6000 (NH+4 + PEG as ANP; NO 3 + PEG as
NNP). ae, aerenchyma. of water stress on root aerenchyma development the electron
Nevertheless, the effects Aerenchyma formation was determined from depend not
micrographs in Fig. above. or the environment but also on the plant species.
only on the nitrogen form

20. Effects of nitrogen form and water stress on root hydraulic conductance in rice plants (cv.
Shanyou 63 and cv. Yangdao 6)
Aerenchyma formation (%)
Shanyou 63 Yamgdao 6 Shanyou 63 (26%) Yamgdao 6 (18%)
 Aerenchyma formation restrict water uptake ability in nitrate nutrition
Root aerenchyma increased significantly in water-stressed plants fed with nitrate. Root
 Water stress can enhance lignificationplants fed with of cell walls in in those fed with
hydraulic conductivity was lower in and suberization nitrate than the endodermis and
ammonium, probably caused by the hydraulic water transport pathways in plants fed with
exodermis where major apoplastic different resistance exists.
nitrate and those fed with ammonium.

21. Effects of nitrogen form and water stress on water uptake rate (with or without HgCl2)
based on per unit root FW for 2 h [gH2O (g root FW)-1] and decreased percentage after
addition of 0.1 mmol-1 HgCl2 to rice plants (cv. Shanyou 63 and cv. Yangdao 6)
 This indicates that water transport occurs mainly through Hg-sensitive aquaporins
(symplastic) in roots under ammonium-fed conditions,
 Whereas it occurs mainly through an apoplastic pathway in water-stressed plants fed with
nitrate (aerenchyma formation is often accompanied by formation of apoplastic barrier)

22. Inference of the study
 The outcome of the study showed that rice growth, root aerenchyma formation,
root hydraulic conductivity and root xylem flow rate in two water-stressed rice
cultivars were affected by the different nitrogen forms
 Under the water-stressed condition, ammonium nutrition increased the tolerance of
rice plants to water stress and resulted in higher water uptake rates and xylem sap
flow rates than found in plants fed with nitrate
 This phenomenon was caused by lower root aerenchyma formation and relatively
higher aquaporin activity
 Thus, water transport from the medium to the xylem varied between water-stressed
plants fed with the two nitrogen forms and impacted the drought tolerance of rice
seedlings.

23.  Under unfavorable situations, root anatomical and Physiological traits have
played immense role to plant resistance to salinity stress.
 Rice is a very salt-sensitive crop species and damage from salinity on rice
seedlings may result in an excess transport of NaCl through the root system
to the leaves

24. The aim of this study was;
 To assess net Na+ fluxes and shoot Na+ content by means of SIET
analysis and atomic absorption spectrometry to elucidate the
contribution of net sodium fluxes at anatomically distinct root
zones of rice seedlings
 To provide data for further interpretation of roles of Casparian
bands in sodium fluxes.
With regard to rice and this study, the assessment of the Na+ fluxes into plant
system was carried out at three anatomically distinct regions in terms of
development of Casparian bands in a developing roots.
SIET –Scanning ion-selective electrode techinique

25. Root distinct zonation
1. Close to root apex, - where no exo- or endo-dermal casparian bands are
formed- Zone I
2. Further from the apex,- where casparian bands of either the endo –or exo-
dermal or both, are mature- Zone II
3. More further from apex, - where root primodia are initiated and casparian
bands are interrupted by developing lateral roots- Zone III
Methods:
 Seeds of rice (Oryza sativa L. cv.Nipponbare ) were germinated for 5 days on wet
filter paper in the light at 27±2 ◦C.
 Seedlings were transferred to an aerated hydroponic culture system and grown in
the same growth chamber in which all of the following experiments were
conducted
 The solution contained the macronutrients 0.09mM (NH4)2SO4, 0.05mM KH2PO4,
0.05mM KNO3, 0.03mM K2SO4, 0.06mM Ca(NO3)2, 0.07mM MgSO4, 0.11mM Fe-EDTA
and the micronutrients 4.6 M H3BO3, 1.8 M MnSO4, 0.3 M ZnSO4 and 0.3 M CuSO4,
pH 5.5–6.0.

26. Root anatomy and microscopy measurements
To examine Casparian bands in root exodermal and endodermal cells, sections were
made at different distances from the root tip (3, 6, 10, 12, 15, 18, 21, 25, 30, 35, 40, 50,
60, 80, 90, 100, 120, 150, 180, 200, 250 mm) of 15 days-old rice.
a. To localize suberin deposition, the sections were stained with Sudan III dissolved
in ethanol/water (1:1; v/v).
b. To localize casparian bands , the sections were stained with 0.1% berberine
hemisulphate for 1 h and with 0.5% (w/v) aniline blue for another 1 h
 The stained sections were observed under an epifluorescence microscope (Q 500
IW, Carl Zeiss, Göttingen, Germany)

27. Freehand cross-sections of rice roots
At 15mm : no At 25mm : casparian
casparian detection detection in radial
or morphological cell wall
change
At 30mm :
casparian detection At 35mm :
was observed in endodermal suberin
exodermis lamellae first
appearance
At 60mm : At 25mm : lysis of
endodermal suberin cortical cells into
lamellae full aerenchyma
development formation
(A) Part of a cross-section at 15mm from the root tip where the endodermis was immature, stained with aniline
blue. (B) Endodermal cell walls with Casparian bands were shown in root zone II, 25mm behind the root tip.
(C)Exodermal cell walls with Casparian bands were shown in root zone II, 30mm behind the root
tip.(D)Endodermal suberin lamellae were well-developed at 50mm (E) Exodermal suberin lamellae were well
developed at about 60mm. (F) Cortical cells started to collapse and aerenchyma gradually formed at a distance of
25mm from the root tip, stained with Sudan III.

28. Lysigenous aerenchyma channels and lateral root primordium of rice roots.
At 120mm : fully Aerenchyma caused
developed main separation
Aerenchyma between outer and
inner root part
No observation of
At 60mm: Initiation either liginified nor
of lateral root suberised exodermis
primordia in direction of lateral
root primordia
formation
(A) Well-developed aerenchyma of roots was observed at about 120mm from the root tip. (B) Well-
defined OPR (the outer part of roots) comprised rhizodermis, exodermis, sclerenchyma and one central
cortical cell layer. (C and D) A lateral root primordium was formed from the stele of the parent root. C,
from a clearing material of roots, D, from an anatomical section of roots

29. Measurement of Na+ contents from different zones along roots
 For each 15-day-old rice seedling, five adventitious roots were selected for experiments
 Root were divided into three zones
I. Zone I ( 0-20mm from the apex), neither exodermis nor endodermis is matured
II. Zone II (40- 60mm)
III. Zone III, basal root part, where lateral root primordia were initiated and lateral
roots had emerged from parent root.
 In order to measure net fluxes of Na+ in different zones, the seedlings were allowed to
grow in a plastic box with three compartments.
 Alternatively, the root zones were allowed to take up Na+ in the root xylem through
the shoot, for 48hrs by subjecting the portion with NaCl.
 The whole shoot was harvested and Na+ content determined by atomic absorption
spectrometry

30. Frequency distribution of shoot sodium content in plants under different
treatment conditions and comparisons of Na+ transport between the three root
zones of rice seedlings.
A B  Considerable variation in individual Na+
content was observed in the plants
from different treatments
 The uptake of Na+ capacity in different
treatments ranged from; 0.02-0.16
µmol (zone I), 0.01-0.23 µmol (zone II)
and 0.06-3.10 µmol (zone III)
D respectively.
C
 The average shoot Na+ contents in the
plants from treatments I , II and III
were 0.092±0.048 µmol, 0.074 ± 0.067
µmol and 1.382 ± 0.911 µmol,
respectively (D)

31. Study summary
 The study revealed that Casparian bands in the endo-/exodermis started to form at a
position about 25–30mm from the root tip and suberin lamellae started to develop at
about 35mm from the root tip
 The anatomical data also showed that a region of the exodermis opposite to developing
lateral root primordia lacked impregnation with lignin and suberin
 Studies have showed that Casparian bands of the endo- and exo-dermis developed much
closer to the root tip in salt-stressed rice plants than in the controls
 Atomic absorption spectrometry revealed that the average Na+ contents entering the
root were dependent on
• Root zonation (lower in zone I & II than in III)
• Amount and chemical composition of apoplastic barriers in root zonation.

32. Study inference
 Casparian bands and suberin hampered with free water movement and ions through
the apoplast of roots into stele or further into root, thereby offering tolerance at the
root tip.
 But, the regions of discontinuity in endodermal Casparian bands created during the
development of lateral root primordia from the pericycle allowed significant
apoplastic leakage of ions from the external medium into root tissues.
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33. The nutrient composition of the solutions was as follows: macronutrients (mg l1): 40 N as
(NH4)2SO4 or Ca(NO3)2, 10 P as KH2PO4, 40 K as K2SO4 and KH2PO4, and 40 Mg as
MgSO4; micronutrients (mg l1): 2.0 Fe as Fe-EDTA, 0.5 Mn as MnCl24H2O, 0.05 Mo as
(NH4)6Mo7O244H2O, 0.2 B as H3BO3, 0.01 Zn as ZnSO47H2O, 0.01 Cu as CuSO45H2O,
and 2.8 Si as Na2SiO39H2O. The Ca content in AN was compensated for by adding CaCl2.
A nitrification inhibitor (dicyandiamide) was added to each nutrient solution to maintain
the identified condition.

34. Basis sets for the partial independence constraints implied by each of the three models shown in Fig. 5. The notation ‘(X,Y)|{A,B,…}’ means
that variables X and Y are hypothesized to be probabilistically independent, conditional on the set of variables {A,B,…} and the ‘φ’
represents the null (empty) set. Pearson’s partial correlation coefficient (r), and probability (P) are given for each conditional independence
statement. Values in bold have a probability below 0.05. The overall model is tested with Fisher’s C statistic and the probability is shown in
brackets (below each model). In those models where the C value was not significant (P > 0.05), the model was
accepted. Model 1 and 2 are rejected (P <0.05) and model 3 is accepted (P >0.05). Codes
The codes of the variables of the models are: 1, Photosynthetic rate; 2, Transpiration rate; 3, ψ, leaf water potential; 4, Soil compaction
(soil bulk density); 5, Plant height (not used in the models); 6, Plant area; 7, Xylem vessels proportion; 8, Xylem vessels diameter; 9, SRL,
specific root length; 10, Root tissue mass density (TMDR) (not used in the models); 11, Plant Biomass.