J. A. Cardoso, J. C. Jiménez, K. Odokonyero, L.M. Pineda, Hannah Vos, Fernando Vergara, Daniela Chamorro, I. M. Rao

Collaborators:CIAT: J. Polanía, J. Arango, J. Nuñez, Plant nutrition lab, Nutrition quality lab, Birthe Paul

Beca-ILRI HubKARI-KenyaRAB-RwandaAgResearch-New Zealand

Corpoica-ColombiaINTA-NicaraguaIDIAP-Panamá

Mechanisms of adaptation to drought and waterlogging in Brachiaria grasses

Brachiaria spp.: Important forage grasses in the tropics

• African origin

• Estimated 100 million hectares in Brazil alone.

• Breeding program started in the late 1980s.

• Wide range of adaptation to climatic and edaphic factors.

• Carbon accumulation in soil, reduction of greenhouse effect gases from soil (N20) and methane from livestock

Why do we need to know mechanisms of abiotic stress adaptation?

• Identifying plant attributes that contribute to resistance/tolerance to major abiotic stresses (e.g., drought and waterlogging)

• Developing rapid, reliable and high throughput screening methods

Phenotyping of Brachiaria genotypes developed by the

breeding program

Drought resistance(avoidance/tolerance)

Assessment methods

• Leaf gas exchange/porometry • Infrared thermometry• Carbon isotope

discrimination???

• Chlorophyll content (SPAD)

• Chlorophyll fluorescence

• Relative water content in leaves

• Weighing each container on a regular basis

• Vertical distribution of roots in soil cylinders (120 cm height x 22 cm width; 80 cm height x 7.5 cm width)

• Micrographs from root cross sections

• High stomatal conductance

• Delayed leaf senescence

• High quantum yield

• High osmotic adjustment

• High transpiration efficiency

• Deep root systems

• Increased root length density in medium and deep soil layers

• Decreased resistance to water movement from soil by increasing root hair growth and xylem diameters

Brachiaria hybridcv. Cayman

Drought

Shoot growth and biomass partitioning after 5 weeks of drought

Experiment 135 kg of soil

Experiment 262 kg of soil

Based on hue, chlorophyll contents (SPAD or analytical) can be estimated (r2 > 0.85)

Binary images are used to estimated shoot areas (difference of dark and white pixels) (r2 > 0.7)

Skeletonize to determine leaf apparition rates

Non-destructive assessment of shoot growth over time (semi-automated)

scripts at Github, juan_cardosinho1

2

3

4

5

0 days 7 days 14 days 21 days

Pro

ject

ed s

hoot

are

a (c

m2 ) 0 days 10 days 21 days

Assessment of shoot growth under progressive drought

• Faster growth rates for Napier grass, Rhodes grass and hybrid Cayman• Greater shoot area represents greater demands for water

Wilting plants

Root growth after 5 weeks of growth

Root growth after 5 weeks of growth

Root growth after 5 weeks of growth

Estimation of root length using digital images

Pho

tosy

nthe

tic ra

te (µ

mol

CO

2 m

2 s-1) Leaf gas exchange and chlorophyll fluorescence

Tran

spira

tion

rate

(mm

ol H

2O m

2 s-1)

Pho

tosy

nthe

tic e

ffici

ency

• Preliminary results indicate that reduction of growth under drought is mainly due to restriction of leaf gas exchange

• Photosynthetic efficiency (Fv/Fm) is relatively insensitive to drought stress indicating stomatal limitation of photosynthesis

0.0.0.0.0.0.0.0.0.

Brachiaria grasses

32.7º C

Growth of Napier grass under drought conditions for 21 daysGrowth of Mulato II under drought conditions for 21 days

Wilting plant

Growing plant

Mulato II shows superior resistance to terminal drought conditions than Napier grass

• Regulated use of water allows Mulato II to sustain growth under drought conditions.

• Mulato II responds to drought by early closing of stomata which reduces transpiration.

• Leaf and canopy temperature values are measured using infrared thermography.

Thermal infrared images

CTD

dro

ught Napier

RhodesCayman

Marandu

Toledo

LlaneroMulato II

BasiliskPiata

Mulato

Tully

Napier

RhodesCayman

Marandu

Toledo

Mulato II

Piata

Mulato

TullyTupi

Llanero

Basilisk

Canopy and leaf temperature as proxies for deep rooting

• Canopy temperature depression (CTD) = Air temperature - Canopy temperature

• A cooler canopy (higher CTD) indicates transpiring leaf and indicates access to water by roots

Observable root length (m plant-1)

Root depth(m plant-1)

Observable root length 80-120 cm root depth

(m plant-1)

CaymanMulato II

Mul

ato

IITime course of growth and water uptake under drought

conditions

Water uptake and growth under drought conditions

• Currently, a simultaneous analysis of shoot and root growth and water content across the soil profile is possible using digital image analysis and TDR.

7 days 14 days

Mulato II Napier Mulato II Napier

Root traits that influence water transport

XV

• More and greater xylem vessels (XV) conduct more water (decreased axial resistance)

Increased xylem vessel (XV) under drought conditions for Napier grass

• Increased root diameter facilitates penetration in drying soils

Greater root diameter in Napier grass

• Roots hairs for decreasing radial resistance

Longer and denser root hairs in Mulato II

Irrigated Drought

Nap

ier g

rass

Mul

ato

II

Mul

ato

II

N

apie

r gra

ssIrrigated Drought (10 days)

0 h 1 h 2 h 3 h

1cm

Root elongation rate

• Faster root elongation rates for Napier• Higher inhibition of root growth under drought conditions for Mulato

II (65%) than Napier (35%)

Irrigated Drought

Irrigated Drought

Nap

ier g

rass

Mul

ato

IIB

. hum

idic

ola

Aerenchyma development

AER

• Aerenchyma (AER) can improve the acquisition of water and nutrients by reducing the metabolic costs of soil exploration.

AERt

% Inhibition root elongation303540455055606570

05

10152025

% A

er ro

ots

drou

ght

Napier B. humidicola

Mulato II

• A erenchyma (AER) can improve the acquisition of water and nutrients by reducing the metabolic costs of soil exploration.

• We plan to quantify respiration rates of aerenchymatyous roots vs. non aerenchmatous roots using an IRGA.

Aerenchyma development and root growth

Root anglesM

ulat

o II

Nap

ier g

rass

Irrigated Drought (10 days)

• Phenotypic plasticity for root angles• Straighter angles under drought conditions• Evaluation for root angles under irrigated and drought conditions underway

1m

1m

2.5 cm

Recovery after drought

• Short term drought restricts growth of Mulato II and is reflected in final biomass

Mec

hani

sms

Drought resistance in Brachiaria

Water spenders

Maintaining water uptake

Napier grassCayman

Water savers

Reducing water loss

• Piatá

• Deep roots• Increased root length

density at depth• Increased root growth at

expense of shoots

• Closing of stomata• Leaf senescence• Reduced leaf area

• Cayman

Both mechanisms

• Napier grass• Rhodes grass

Terminal drought Intermittent drought

• Mulato II• Basilisk

• Mulato• Marandu• Tupi

• Toledo• Tully• Llanero

Cul

tivar

s

Productivity

Effects of waterlogging on Brachiaria grasses

Tully

T

oled

o

R

uzi g

rass

Drained (D) Waterlogged (W) D W D W

Tully Toledo Ruzi grass Tolerant Mod. tolerant Sensitive

• Increased root death and smaller root system in non-tolerant genotypes

• Reduction in shoot growth in non-tolerant genotypes

• Increased leaf senescence, chlorophyll loss, stomatal closure, lower values of photosynthetic efficiency (fv’/fm’) in non-tolerant genotypes.

21 days of treatment

Effects of waterlogging on Brachiaria grasses

Traits associated with waterlogging tolerance in Brachiaria grasses

• Aerenchyma (air spaces) allows oxygen difussion from shoot to root to maintain root aerobic respiration.

• Brachiaria adapts to waterlogging by the development or increase or aerenchyma in root tissues. Constitutive formation of aerenchyma in B. humidicola allows immediate adaptation to waterlogging

• Better adapted genotypes show thicker roots, greater aerenchyma formation and smaller steles (conductive tissue)

Drained Waterlogged

B. humidicola Tully(tolerant)

B. ruziziensis Br 44-02(sensitive)

• Cross sections taken at 10 cm form the root tip

• Scale = 0.5mm

Aerenchyma

stel

e

Increased suberization of the outer part of the root (OPR)

B. h

umid

icol

aB

. ruz

izie

nsis

Drained Waterlogged Waterlogged

O2

O2

B. humidicola

B. ruziziensis

O2

O2

O2

O2

AE AE

• Roots with greater aerenchyma formation and increased suberization of OPR shows deeper penetration into waterlogged soil

Leaf

she

ath

Inte

rnod

eR

oot

• Aerenchyma (arrows) formation in shoots (internode and leaf sheath) and roots

• Continuum of ventilation form shoot to roots facilitates gas exchange between atmosphere and rhizosphere

B. humidicola after 21 days of growth under waterlogging

O2

CH4N2O? C2H4

Replacement rooting

Roots produced after waterlogging (white)

• Roots produced before waterlogging (rotten or decaying)

• Brachiaria genotypes without constitutive formation of aerenchyma in roots depend on the formation of new roots with aerenchyma for adaptation to waterlogging

Replacement rooting

Cayman Mulato II

Decaying or rotten roots

Decaying or rotten roots

Replacement roots

Cayman

• Most of damage (e.g. leaf chlorosis and leaf senescence) occurs between 7 and 14 days after waterlogging, period where new roots with aerenchyma started to develop.

• After this, aerenchymatous roots confer adaptation to waterlogging.

Screening for waterlogging tolerance in Brachiaria hybrids

1 day of waterlogging treatment 7 days of waterlogging treatment

14 days of waterlogging treatment 21 days of waterlogging treatment

Recovery after drought and waterlogging

• B. humidicola responds to accumulated ethylene in waterlogged soil by internode elongation and hyponastic growth of leaves

• This plastic response allows leaves of B. humidicola to escape form water and continue photosynthesis

Irrigated Drought Waterlogging Recovery period

Irrigated Drought Waterlogging

Conclusions so far…..

• Cayman seems to be a water spender, not a saver. It attempts to maximize carbon gain (growth) when water is available.

• Most Brachiaria grasses combine water saving mechanisms (by regulation of water loss by closing leaf stomata) with deep rooting ability to avoid drought stress.

• Digital images (e.g., RGB, Thermal IR) allows recordings of growth and responses to stresses.

• Leaf and canopy temperatures could be used as proxies for rooting depth in Brachiaria genotypes

• Aerenchyma might aid root elongation under drought conditions

Conclusions so far….• Brachiaria grasses adapt to waterlogging by

increasing or developing aerenchyma in roots to sustain root aerobic respiration

• Plants without constitutive formation of aerenchyma in roots depend on the formation of new roots with aerenchyma for adaptation to waterlogging

• Maximum rooting depth can be used as an indicator of root aeration efficiency and waterlogging tolerance

Challenges ahead

• Determine the role of endophytes in drought adaptation

• Determine the contribution of climate smart Brachiaria grasses to soil carbon accumulation and greenhouse balance

• Scale up for automated phenotyping in breeding populations

Thank you