Plant Abiotic Stress, Second Edition. Edited by Matthew A. Jenks and Paul M. Hasegawa. © 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

Figure 1.1 SUB1A-mediated submergence tolerance responses revealed by integrating omics tools (Jung et al., 2010). Orange boxes indicate events

upregulated in M202 (Sub1) after submergence, and blue boxes indicate events downregulated in M202 (Sub1) after submergence. Several of the AP2/ERF

TFs are associated with submergence tolerance response.

ScB8

ScB2

ScB10

SUB1C SUB1A

ScB7

ScB7SaB8

SaB20

SaB12 SaB4 SaB17SaB15

SaB19SaB16

SaB6

SaB1

SaB24

SaB14

SaB10

SaB2

SaB22

SaB21SaB9

SaB23

SaB18

SaB7

SaB3SaB6

SaB4

Figure 1.2 The rice SUB1A/SUB1C interactome. The interactome map represents 28 proteins identified

from high-throughput Y2H screening using SUB1A and SUB1C as baits. Proteins in blue represent interactors

with both SUB1A and SUB1C (Seo et al., 2011).

Growth regulationcell wall properties

Ubq

Posttranslational

Translationalcontrol

Alternative splicingand RNA processing

Water / Turgor loss

Stress perception

KinasesKinases

PPases

PPases

PPaseregulators

ActivityLocalization

Stability

Stressperception

Amino acidmetabolism

Metabolicregulation andcycling to bufferredox status

Alt OX, uncoupling, catabolism

SoluteAccumulation

Vacuole

NADP+

NADPH

SUMO

ROS

Cell deathsenescence

e–

e–e–

e–

e–

CO2

CHOO2H2O

Solutes

Protectivesolutes

Figure 2.1 Summary diagram of regulatory mechanisms and drought tolerance related cellular changes

discussed in this chapter. Sensing and signaling of drought stress (top half of diagram) begins with an initial

perception of water loss or loss of turgor that occurs via unknown mechanisms but may involve plasma

membrane or organelle localized sensors and cytoskeleton changes. Downstream signaling involves the action

of kinases and phosphatases (PPases), which can have opposing effects on a range of targets including trans-

porters, transcription factors, and other proteins. Alternative splicing, selective translation, and protein modifi-

cation by attachment of ubiquitin (Ubq) or small ubiquitin-like modifier proteins (SUMO) pathways generate

additional changes in protein content and activity important for drought response. The regulator events lead to

specific cellular changes related to drought tolerance (bottom half of diagram) including solute uptake and

synthesis of protective solutes, changes in chloroplast and mitochondrial metabolism to buffer cellular redox

status, and control of solute synthesis, as well as changes in cell wall properties. Adjustment to photosynthetic

metabolism as well as mitochondrial alternative oxidases and uncoupling proteins act to dissipate reducing

potential when necessary and prevent reactive oxygen species (ROS) formation. Even with this, the mitochon-

dria and chloroplast are also sources of ROS, which, along with other specific signals, determine the time and

extent of cell death and senescence during drought.

0

Col

aba2

-1 Col

aba2

-1

Col

aba2

-1 Col

aba2

-1

4

8

12

0

4

8

12

Roo

t elo

ngat

ion

(mm

)

8

20

40

60

80

10020

15

10

5

0

1.0

0.5

0.0

1.0

1.5

0.5

0.0

1.0

1.5

2.0

0.5

0.0

1.0

1.5

2.0

0.5

0.0

0.2

0.3

0.4

0.5

0.6

0.20

0.25

0.30

0.35

0.40

0.45

Dry

wei

ght (

mg

seed

ling–1

)

Fre

sh w

eigh

t (m

g se

edlin

g–1)

0.20

0.25

0.30

0.35

0.40

0.45

Control(–0.25 MPa)

–1.2 MPa

–1.2 MPa +2 μm ABA

–1.2 MPa +10 mM Pro

*

*

*

**

*

*6

4

2

0

Figure 2.2 Regulated changes in growth at low water potential: stimulation of aba2-1 by proline. Seedlings

of Arabidopsis Columbia wild type or the ABA-deficient mutant aba2-1 were transferred from control media

to low water potential (−1.2 MPa) on polyethylene glycol-infused agar plates. Measurements of seedling

growth (root elongation, fresh weight and dry weight) were performed 10 days after transfer. Transpiration is

low in this experimental system, thus dehydration avoidance through stomatal closure has a relatively minor

role. Because of this, aba2-1 differed little from wild type in the high water potential control (top panels). At

low water potential aba2-1 was inhibited in root elongation, consistent with previous observations that ABA is

required to promote root elongation at low water potential. Fresh weight and dry weight, which indicate shoot

growth as well as root growth, were slightly decreased or unchanged. Adding a low level of ABA comple-

mented the reduced growth aba2-1. However, adding the compatible solute proline had a more dramatic effect

of increasing fresh weight and dry weight by 1.5 to nearly 2-fold (bottom panels). Thus, when the growth

restraining effect of ABA was removed, added proline could greatly stimulate shoot growth at low water poten-

tial. Asterisks (*) indicate significant differences between wild type and aba2-1. Pictures show representative

seedlings from a series of replicated experiments. Figure is modified from Sharma et al., 2011.

NA

DP

/NA

DP

H

(A)

(B)

6

5

4

3

2

1

0 0

Cont.

*

* *

–1.2 MPa

Pro source (photosynthetic tissue)

Pentose phosphate pathwayphotosynthesis

NADP+ NADP+

NADPH NADPH

P5CRP5C

Osmotic adjustment, other pro functions

PDH

Mitochondrial electron transport

e–

P5CDH

NAD(P)H

Glu

Pro sink (root, meristematic tissue)

P5C

?

P5CS1Glu

NA

D/N

AD

H

6

5

4

3

2

1

Cont. –1.2 MPa

colp5cs1-4pdh1-2

Pro

Pro

Pro

Figure 2.3 NADP/NADPH ratio as an indicator of redox status of plants at low water potential and the role of

proline metabolism in buffering NADP/NADPH. (A) Seven-day-old Arabidopsis seedlings of Columbia wild

type, p5cs1-4 (lacking expression of D1-pyrroline-carboxylate synthetase1, which encodes a stress-induced

enzyme of proline synthesis) and pdh1-2 (lacking expression of the proline catabolism enzyme proline dehydro-genase1), were transferred to control (−0.25 MPa) or low water potential (−1.2 MPa) media and pyridine nucleo-

tide levels measured 96 hours later. Low water potential caused a decline in NADP/NADPH, indicating that a

reduced supply of NADP in the chloroplast may increase the potential for reactive oxygen production or inhibi-

tion of photosynthesis. In contrast, NAD/NADH was less affected by low water potential. p5cs1-4 and pdh1-2

both had a greater decline in NADP/NADPH than wild type at low water potential. pdh1-2 also had decreased

NADP/NADPH in the unstressed control treatment. These data indicated a role of proline metabolism in control-

ling NADP/NADPH in addition to other protective roles of proline as a compatible solute that accumulates during

drought. (B) Model of proline metabolism and its roles in regenerating NADP in photosynthetic tissue and sup-

plying energy and reductant to meristematic and growing tissue to support continued growth during low water

potential. In photosynthetic shoot tissue, synthesis of proline is relatively high, indicated by induced expression

of P5CS1 and repressed expression of PDH1. Because of its probable location in the chloroplast (reviewed in

Verslues and Sharma, 2010; Szabados and Savoure, 2010), proline synthesis can serve to regenerate NADP as an

electron acceptor to avoid reactive oxygen production and inhibition of photosynthesis. In growing tissue, espe-

cially the root apex, PDH1 expression is induced rather than repressed by low water potential, and proline serves

as an alternative respiratory substrate to sustain growth. Additional investigation of the localization and regulation

of P5CS1 and PDH1 and mechanisms of proline transport are needed for further understanding of how proline

metabolism promotes drought tolerance. Both (A) and (B) are modified from Sharma et al., 2011.

Figure 2.4 Intron sequence polymorphisms lead to varying rates of P5CS1 alternative splicing and varying capacity for proline accumulation among Arabidopsis

accessions. The Arabidopsis P5CS1 transcript can be alternatively spliced into two transcripts: a full-length transcript that encodes P5CS1 protein and a transcript

missing exon 3 (exon 3-skip P5CS1) that cannot be translated to P5CS1 protein. Most accessions produce only a low level of exon 3-skip P5CS1 and can accumu-

late high levels of proline in response to low water potential stress. However, in some accessions up to half of the P5CS1 transcript is the non-functional exon 3-skip

P5CS1. These accessions have reduced levels of proline accumulation. Accessions having high levels of exon 3-skip P5CS1 have extra TA repeats in intron 2 and

a specific G to T transversion in intron 3; these are sufficient to drive high levels of alternative splicing. The percentage of exon 3-skip P5CS1 is correlated with

temperature and rainfall conditions from the accessions sites of origin, indicating the P5CS1 and proline synthesis are under selection as part of local adaptation of

Arabidopsis accession to climates differing in rainfall and temperature patterns. Figure is modified from Kesari et al., 2012.

OsmoprotectantsProline

Glycine betaine

SOS1 SOS2 SOS2

V-PPase

PPi

V-ATPaseNHXs

Vacuole

Cytosol

CDPKs

CBLs

CIPKs

HKTsCNGCsNSCCs GLRs

Na+

H +

2×Pi

ATPADP + P

i

Na+

H+

H+

Na+Na+Na+Na+ K+/ Na+

Ca2+

H+

Figure 6.3 A selection of well-characterized cellular processes involved in salt tolerance. Na+ can passively enter

into plant cells through ion channels, such as non-selective cation channels (NSCCs), glutamate receptor-like chan-

nels (GLRs), cyclic-nucleotide gated channels (CNGCs), and the Na+ or K+/Na+ HKT transporters. Na+/H+ antiport-

ers like SOS1 or NHXs transport Na+ out of the cell or into the vacuole and require the establishment of an H+

gradient by membrane bound PPases and ATPases. SOS2 and SOS3 regulate the activity of SOS1, ensuring the

tranporter is active during salt stress—SOS3 and SOS2 are members of the CBL/CIPK calcium signalling pathway.

Both CDPKs and CBLs/CIPKs are involved in the Ca2+ dependent salt stress signalling pathways and regulate the

cell response to salt stress by post-translationally modifying a variety of proteins, such as transporters. Finally, salt

stressed cells accumulating high concentrations of Na+ produce proline and glycine betaine as osmoprotectants.

Zn2+

Zn

Zn

Zn

Zn

To shoot

Xylem

To shoot

Xylem

NcZNT1

ZIP? ZIP? ZIP?

MTP1Vacuole

NcZNT1

Zn transport into and across the root

Nocceae caerulescens root

“Non-hyperaccumulator” root

NcZNT1

HMA4

HMA4

Xylem parenchymaEndodermis / PericycleEpidermis / Cortex

MTP1Vacuole

Xylem parenchymaEndodermis / PericycleEpidermis / Cortex

Zn2+

Figure 7.1 Model of Zn transport into and across the root of the Zn/Cd hyperaccumulator, Nocceae caerulescens. This

is a speculative model for Zn2+ transport from the soil into the root, radial Zn transport to the center of the root, and

Zn loading into the xylem. The model depicts differences between the Zn hyperaccumulator, Noccea caerulescens, compared with a “typical” non-accumulating plant species. Elevated root Zn2+ influx into the root from the soil is

depicted for N. caerulescens (larger green arrows compared with root Zn2+ influx in the non-hyperaccumulator). It

has been suggested that NcZNT1 may be the transporter facilitating this uptake, but recent localization of the

Arabidopsis homolog of the TcZNT1 gene suggests it may also be involved in metal loading into the stele. Hence we

also show NcZNT1 facilitating Zn influx from the apoplast into cells of the pericycle and other cells within the

stele. The model also indicates there is less vacuolar sequestration of Zn in roots of N. caerulescens. Thus in the

hyper accumulator, there would be a larger pool of mobile Zn in the root that can more readily move through the

endodermis and pericycle to the xylem parenchyma. We also show the elevated Zn loading into the xylem in

N. caerulescens, via the Zn/Cd ATPase, HMA4, for subsequent transport to the shoots.

Organic acid-Alcomplexes

Glycolysis

PEP

Oxaloacetate

Pyruvate

TCACYCLE

Organic acid-Alcomplexes

Organic acid-Alcomplexes

Al3+ Al3+

Activation

MalateCitrate

?

Al

Tonoplast

Internal Al-detoxification

Al exclusion

Als1

Nrat1SbMATE

ALMT1

Plasmamembrane

Mitochondria

Figure 7.2 Physiological mechanisms of aluminum (Al) tolerance. The model depicts the well-characterized

root tip Al exclusion and less well-studied internal Al detoxification mechanisms of Al tolerance. The Al exclu-

sion mechanism involves the transport of organic acids (OA) across the root-cell plasma membrane into the

rhizosphere via an Al-gated anion channel for malate (ALMT1) or an Al-activated citrate efflux transporter

(MATE). Activation of the ALMT channels appears to be due to direct activation of the transport protein by Al.

Al activation of the MATE may be more indirect and could involve Al interacting with a second membrane-

bound receptor protein that associates with the MATE protein, or by Al entering the cytosol and triggering

MATE activation. It is known that Al also triggers changes in expression of genes involved in Al tolerance. The

internal Al detoxification involves the entry of Al via an Al transporter that is known to be OsNrat1 in the rice

root but has not been identified in Al accumulators such as buckwheat and Hydrangea, where the Al is seques-

tered in the leaf vacuole. For these shoot Al accumulators, the tolerance mechanism involves chelation of

cytoplasmic Al by organic acids with the subsequent sequestration into the vacuole. Here we also suggest that

in the rice root, possibly Al transported into the root cell by OsNrat1 might be transported into the vacuole by

OsALS1 mediated by the transport of Al complexed with organic acids.

RDR2

dsRNA

24 nt dsRNA

Target Gene / Loci

POLIV

CLSY1

SHH1

ssRNA

DCL3

siRNA

°°

HEN1

AGO4

KTF1

DRM2

Pol V/Pol II

AGO4

IDN2

RdDM Complex

DRD1

= cytosine methylation

DMS4

Figure 8.2 RNA-directed DNA methylation in plants. The SNF2 chromatin remodeling protein CLASSY 1

(CLSY1) facilitates chromatin decondensation and access of RNA Pol IV to the target loci. Pol IV trasncripts

are converted into dsRNA by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). These dsRNAs serve as

substrate for DICER-LIKE 3 (DCL3) catalyzed production of 24-nt small RNA duplex. HUA ENHANCER-1

(HEN1), an RNA methyltransferase, catalyses the methylation of the 2′-OH group of three-terminal nucleo-

tides in the small-RNA duplex. SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) is required for Pol IV

dependent siRNA production. The 24nt siRNAs are loaded onto Argonaute 4 (AGO4) protein. DEFECTIVE

IN RNA-DIRECTED DNA METHYLATION 1 (DRD1, chromatin-remodelling factor) and DEFECTIVE IN

MERISTEM SILENCING 3 (DMS3) facilitate Pol V/II access to the target loci. Transcription factors such

as RNA-DIRECTED DNA METHYLATION4 (RDM4)/DMS4 help recruit RNA polymerase V. Pol V transcribes

target loci to produce ssRNAs. These transcripts serve as scaffolding for recruitment of complementary siRNA

bound AGO4. The KOW DOMAIN-CONTAINING TRANCRIPTION FACTOR 1 (KTF1)/SUPPRESSOR

OF TY INSERTION 5-LIKE (SPT5L) binds to nascent scaffold transcript RNA and recruits AGO4-bound

siRNAs to the RdDM effector complex. INVOLVED IN DE NOVO 2 (IDN2)/RNA-dependent DNA methyla-

tion 12 (RDM12) binds to the hybrid siRNA-nascent scaffold transcripts and aids recruitment or retention of

de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2). DRM2

catalyzes cytosine methylation in DNA sequences complementary to siRNAs.

Active chromatin

Active chromatin

AbioticStresses

Changes in expression and/or activity of epigenetic effectors

Transcriptome changeAcclimation response

Str

ess

relie

f

Str

ess

relie

f

Within generation and Transgenerational stress memory

Stable epialleles with adaptive value

Repressed chromatin

Repressed chromatin

= DNA methylation

= Active histone marks

= Repressive histone marks

Figure 8.4 Epigenetic regulation of stress tolerance. Abiotic stresses alter epigenetic state, which determines

stress resposive gene expression. Transient chromatin modifications mediate acclimation response. Heritable

epigenetic modifications provide within generation and transgenerational stress memory.

Control 200 mM NaCl Control 200 mM NaCl

Arabidopsis thaliana Thellungiella parvula

Figure 9.1 Thellungiella parvula compared with its relative Arabidopsis thaliana. Seeds were germinated

and plants grown on root wash mix (http://pcf.aces.illinois.edu/services/soil.html) in a growth room with

14 hours light (120 μmol/m2sec) and 10 hours dark, for 4 weeks before treatment. Plants were irrigated with

1/20 Hoagland solution once a week. Treatment was done by adding of 200 mM NaCl to the irrigation solution.

0.05

MYB transcription factors in A. thaliana (Blue) and

T. parvula (Red)

(A)

Figure 9.2 Possible mechanisms underlying the divergence of T. parvula and A. thaliana genomes and

lifestyles. (A) Copy number variation of orthologs. Evolutionary relationships exemplified by a phylogenetic

tree including all MYB family genes in T. parvula (red) and A. thaliana (blue) for 126 Arabidopsis and 130

Thellungiella R2R3 MYB proteins. The phylogenetic tree was inferred by the Neighbour-Joining method,

using the pairwise deletion option and 1000 bootstraps (Mega5; http://www.megasoftware.net/).

(E)

(C)

(A)

(F)

(D)

(B)

1614

LOD

cM cM

0

14

cM

LD

cM

LD

X

121086420

Figure 10.2 Basis of linkage analysis and association study. Linkage analysis relies on biparental population

with low recombination (A) resulting in low resolution mapping of QTL but high power of detecting a signifi-

cant linkage between trait and loci (C and E). By contrast association study uses historical recombination

accumulated over time in a diversity panel (B), which results in high-resolution mapping (D and F).

QTLregion

Parentalchromosomes

MarkersRecombination events

Flankingrecombinants

Desiredoutcome

Figure 10.3 Introgression of favorable QTL allele from a donor line (pink) into a recipient line (blue) with

desirable genetic background. A recombinant population is screened with markers flanking the QTL region.

Lines showing a recombination event on each side of the QTL (boxed) are crossed to generate progeny contain-

ing the QTL allele of the donor line in the genetic background of the elite recipient line. The strategy is effec-

tive if there is no recombination between the actual QTL and its flanking markers, and therefore relies on

markers tightly linked to the QTL.