2

Moss as a Model System for Plant Stress Responses

Andrew C. Cuming

2.1

Introduction

We live on a green planet, yet it was not always so. An observer from space sees the

blue of the ocean and the green of the land, but this is a comparatively recent

development in our planet’s history. Only in the last 450 million years has the

planet’s land surface become colonized by plants. Until this time, the land was

bare: rock, sand, and mud, devoid of organic matter and unable to support life. It is

difficult to determine precisely when the first eukaryotes gained a secure foothold

in the terrestrial environment. The most direct evidence derives from early fossil

spores indicating colonization by plants, dating from the mid-Ordovician period

(about 440–490 million years ago) [1, 2], although the earliest surviving megafossils

indicating the anatomical features of early plants occur about 50 million years later

in the fossil record [3]. What were these plants like and where did they come from?

It is now generally accepted that today’s land plants evolved from aquatic green

algae, and molecular systematic analysis has identified the charophytes [4] and

more specifically the Charales [5] as the likely ancestral taxon to all modern land

plants. The extant members of the Charales (e.g., Chara) remain aquatic and grow

as branched filaments. They have a haplontic life cycle in which the product of

sexual fusion immediately undergoes meiosis to generate haploid tissue. This is by

contrast with the land plants, which exhibit a diplobiontic life cycle, with an

alteration of haploid gametophyte and diploid sporophyte generations.

Among today’s land plants, it is the bryophytes that represent the first group to

diverge in the land plant lineage. The bryophytes comprise three distinct sub-

groupings: the mosses, liverworts, and hornworts. All display characters that

might be considered ‘‘primitive,’’ such as branched filamentous protonemal tis-

sues (in the mosses) and cells containing a single, algal-like pyrenoid-containing

chloroplast (in the hornworts). The earliest fossil sporangia have been suggested as

most similar to those found in extant liverworts [2]. All have a dominant haploid

gametophyte generation, in which the sporophyte is dependent on the gameto-

phyte for its development and nourishment. While the relationship between these

Plant Stress Biology. Edited by H. HirtCopyright r 2009 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32290-9

| 17

groups, in terms of their order of origin, remains disputed, it is agreed that, among

extant plant species, it is the bryophytes that most closely resemble the likely first

common ancestor of the land plants.

The ancestors of land plants were most likely found in the marginal areas of

bodies of water and were exposed periodically as the water receded. The successful

colonization of the land would have required a number of adaptations to permit

the survival of such plants. Terrestrial environments are necessarily more variable

in nature than aquatic environments. There are greater fluctuations of tempera-

ture, over both short and long timescales. The availability of water – a necessity for

life – is uncertain and there are greatly enhanced levels of radiation: a factor

contributing to both temperature and the availability of water, and also directly

damaging through the mutagenic effects of ultraviolet-induced DNA damage.

Modern plants have acquired multiple anatomical and developmental adaptations

to enable them to survive environmental extremes. The vascular plants that

dominate today’s planetary surface have extensive, branching root systems that

ramify throughout the soil, enabling them to scavenge water from the substratum.

Evaporative water loss is reduced through the presence of protective surfaces: waxy

cuticles, suberin, and lignin all act to retain water within the plant, whilst lignified

vascular systems both mechanically support the development of massive struc-

tures, and enable the distribution of water scavenged from the soil to all parts of

the plant. Evaporative water loss and gas exchange is facilitated through the pre-

sence of stomatal apertures on the leaf surfaces. The effects of incident radiation

are ameliorated through the accumulation of pigments that serve as sunscreens

(e.g., anthocyanins). Sexual reproduction no longer requires a layer of water for the

dissemination of swimming gametes.

The first land plants lacked these adaptations. Their ability to survive and

prosper therefore must necessarily have depended, at first, on biochemical adap-

tations to withstand such environmental variability. Such adaptations would be

expressed as metabolic responses, rather than as developmental in nature, and

such adaptations can still be recognized in today’s land plants. Although such

traits are commonly characterized as ‘‘primitive,’’ it should be emphasized that

this term reflects their ancient origins, rather than their efficacy. These ‘‘primi-

tive’’ traits have been instrumental in the conquest of the land by plants and in

their subsequent shaping of the terrestrial environment. Their importance is

highlighted by the diversity of the bryophytes, specifically the mosses, in modern

ecosystems.

The terrestrial flora is today dominated by the angiosperms, of which there are

thought to be about 250 000 species. This diversity originated from a massive

radiation in the mid-Cretaceous period (about 100 million years ago) and occurred

in concert with a similarly extensive radiation among insect species that act as

pollinators. Nevertheless, the bryophytes remain a large, diverse and successful

group: it is estimated that there are approximately 10 000 moss species, which is

second only in number to the angiosperms. Clearly, primitive traits retain their

value, even after 450 million years. Mosses retain many of the properties we

ascribe to the first successful colonists of the land. Most significantly, they are

18 | 2 Moss as a Model System for Plant Stress Responses

habitat-forming organisms – they are capable of colonizing bare surfaces, such as

bare rock and mud, and growing to envelop the surface. Their death and decay

generates the organic matter that distinguishes a true soil from a simple mineral

sludge, and provides the basis for colonization by other plants, more demanding in

their substrate requirements. Whilst many mosses thrive best under conditions of

shade and moisture, others are able to colonize and exploit exposed bare habitats

apparently inimical to plant growth. Even in urban environments, the sight of

mosses growing on bare roofs and walls is commonplace.

The study of mosses, therefore, provides insights into the conquest of land by

living organisms, and the origins of terrestrial life. Mosses provide a starting point

for unraveling the evolution of plant gene function through comparative, ‘‘evo-

devo’’ genomic strategies and for the identification of molecular strategies for

adaptations to abiotic stress.

Can a ‘‘systems biology’’ approach be applied to the study of these processes in

mosses? Until recently, this might have appeared an unlikely prospect. However,

in recent years, considerable progress has been made in developing genomic

resources for one moss species, Physcomitrella patens, whose biology makes it

particularly amenable for such an analysis. This species has now become a pow-

erful model for deploying systems-level approaches for comparative analyses of

plant stress responses.

2.2

Model Systems

Model systems provide us with tools to investigate processes common to entire

classes of living organism. They are chosen for their experimental amenity, and

vary according to their purpose and the particular expertise of the experimentalist,

but certain features are more desirable than others. They should be representative

of their class; thus, mice are good experimental models for the study of mam-

malian development, but zebrafish may be more tractable for wider applications

including all vertebrates. Arabidopsis thaliana is pre-eminent as a model plant,

having a small sequenced genome, a genetic linkage map densely marked by

molecular markers, a rapid life cycle, small size, and straightforward procedures

for genetic manipulation by transgenesis (http://www.Arabidopsis.org). It is an

outstanding workhorse for identifying how cellular organization and development

are programmed within the flowering plants, and also for how many fundamental

plant processes are regulated, common to all taxa of green plants. However, in

order to explore how these processes have evolved, we need to look beyond a single

species. Other model plants include rice – as an example of the monocotyledonous

plants and a representative cereal (the most important group of food crops on the

planet) – and several other species of angiosperm, whose genomes have recently

been deciphered (soybean, poplar, grape). Nevertheless, to undertake a wider

comparative analysis, models are also required from outside the flowering plants.

To date, these are fewer in number, but include the diatom Ostreococcus [6], the

2.2 Model Systems | 19

unicellular green alga, Chlamydomonas rheinhardtii [7], the moss P. patens [8], andthe lycophyte Selaginella moellendorfii (http://selaginella.genomics.purdue.edu/;

http://genome.jgi-psf.org/Selmo1/Selmo1.home.html).

What are the general features of mosses, how do they differ from the flowering

plants, and how can we use them experimentally? Anatomically, mosses are

simpler than the flowering plants [9]. Many of their structures are only a single cell

in thickness (a particularly attractive feature for cell biologists, since the processes

occurring within a single cell can be conveniently observed using modern imaging

techniques). Genetically, the mosses (like all the bryophytes) are also distinctively

different. All plants exhibit an alteration of generations, with a haploid gameto-

phyte generation and a diploid sporophyte generation. In the so-called ‘‘higher’’

plants (pteridophytes, lycopods, gymnosperms, and angiosperms), the diploid

sporophyte represents the dominant generation. The pollen grain and embryo sac

(the microspore and megaspore) represent the gametophyte generation that derive

from meiotic cell division and (in the case of the embryo sac) that depend on the

sporophyte for their transitory existence. By contrast, the bryophytes have a

dominant, haploid gametophyte generation, which produces gametes by mitotic

division. Upon fusion of gametes, a diploid sporophyte develops that is entirely

dependent on the gametophyte for its nourishment and growth. Within the

sporophyte, meiocytes are formed that generate haploid spores by meiotic division.

These spores are dispersed to initiate a new gametophyte. Stages in moss devel-

opment are illustrated in Figure 2.1.

When a moss spore germinates, it does so by extending a filamentous cell

known as a protonema. This cell divides, generating a uniseriate protonemal

filament in which the apical cell undergoes continuous mitotic divisions to extend

the filament. Whilst the apical cell remains continuously active in cell division – in

effect it is a unicellular meristem – the subapical cells are less active. Typically they

may only undergo one more mitotic division to generate a side-branch initial. This

initial can itself then divide to generate another uniseriate filament or it may

initiate the formation of a three-dimensional ‘‘bud’’ containing a more-or-less

tetrahedral meristematic apical cell that will proliferate the leafy shoots that are

characteristic of mature moss colonies.

There are thus a very limited number of cell types in a moss. The protonemal

tissue may comprise either slower growing, chloroplast-rich chloronemal cells or

rapidly growing caulonemal cells (the caulonemata enable the moss to spread

over the substrate). The cells of the leafy shoot may include specialized midrib

cells, in addition to those comprising the lamina, and in some mosses, such as

Sphagnum spp. specialized water storage cells (hyaline cells). The leafy shoots

represent the sexual organs of mosses, in that they bear the specialized male and

female reproductive structures – gametangia – at their apices. The female

gametangia are flask-shaped archegonia, each containing a single egg cell, and

the male gametangia – the antheridia – produce large numbers of motile fla-

gellate spermatozoids.

Although there are some rudimentary conducting tissues in some mosses, they

generally lack a vascular system and the analysis of the Physcomitrella genome

20 | 2 Moss as a Model System for Plant Stress Responses

Figure 2.1 Stages in the development of the moss, P. patens. (a) The

mature sporophyte is borne at the tip of the leafy shoot (the

gametophore). (b) A mature spore. Each sporophyte contains between

2000 and 5000 haploid spores. The mature spores are enclosed in a

sporopollenin wall. (c) Spores germinate to produce chloronemal

filaments. This panel shows a germinated sporeling and an

ungerminated spore (top left). (d) Protonemal filaments grow by

repeated division and elongation of the apical cell. The subapical cells

divide to generate side-branch initials or buds. (e) A bud initial at an

early stage of development. Buds develop to form the leafy shoots. (f)

Gametophores, with rhizoids just discernable at the base.

(g) A mature gametophyte comprises many gametophores. This plant

was initiated on agar medium inoculated with a ‘‘spot inoculum’’ of

chloronemal tissue about 4 weeks earlier. (h) Following incubation at

low temperature, the gametangia develop at the gametophore apex.

The leaves have been stripped from the gametophore to reveal the

flask-shaped archegonium (the female structure containing the egg

cell) and the small ovoid antheridia that produce flagellate sperm. (i)

Following fertilization of the egg cell by a sperm, the sporophyte

develops within the archegonium. (j and k) Protoplasts are easily

obtained by digestion of chloronemal filaments with the cell-wall

degrading enzyme mixture ‘‘Driselase.’’ Each protoplast is totipotent

and capable of regenerating to produce a new plant. Protoplasts are

also readily transformed by uptake of exogenous DNA.

sequence indicates that this moss, at least, lacks the genes necessary to synthesize

lignin of the type found in the tracheophytes [8]. Neither do mosses produce

extensive ramifying root systems that are able to scavenge water from the sub-

strate, although the protonemal network may penetrate soils to a shallow depth.

Gametophores are often characterized by a proliferation of empty cells – rhizoids –

at their base and these have been postulated to have some root-like functions [10],

possibly in nutrient assimilation or support, but the gametophyte in general lacks

many of the adaptations used by the tracheophytes to restrict water loss, such as

cuticular wax and somatal apertures. Nevertheless, these adaptations are not

absent: they are restricted to the sporophyte phase of the life cycle, indicating their

early evolution among the land plants.

2.3

Physcomitrella as a Model System

Very few mosses have been studied intensively at the molecular or biochemical

level. However, one moss species, P. patens, has emerged as an excellent model

system for undertaking genome-level analyses. Physcomitrella has been studied

since the early years of the twentieth century and at the genetic level since 1968,

when the first mutants were isolated [11]. First, the cells of a fully differentiated

plant retain their totipotency. Mosses are easily grown in axenic tissue culture by

the simple expedient of fragmenting plants in water with a laboratory blender and

dispersing the suspension over the surface of an agar plate [12]. The resultant

suspension rapidly regenerates as a uniform mat of protonemal filaments, thus

providing an excellent source of homogeneous tissue for biochemical or molecular

analysis. Alternatively, small explants of filamentous tissue can be subcultured as

‘‘spot inocula’’ and they will recapitulate the entire developmental pathway to

generate new, clonal plants.

The haploid nature of the dominant gametophyte generation clearly facilitates

the recognition of mutant phenotypes immediately following mutagenic treatment

and this was exploited by Cove et al. in the isolation of auxotrophic mutants [13], as

well as mutants affecting a number of processes relating to cellular differentiation

[14], hormone responses [15], and the polar growth responses of single cells: the

protonemal apical cell is the site of perception of, transduction and response to

environmental stimuli such as light and gravity [16–19]. This provides the oppor-

tunity to elucidate how such processes operate within a single, easily observed cell,

rather than in a multicellular organ containing a number of different cell types, as

occurs in anatomically more complex plants. Furthermore, cellular differentiation

processes are easily studied and manipulated: the transitions between chloronemal

and caulonemal cells can be regulated by auxin [9, 20] and the carbohydrate

nutritional status of the cell [21] whilst bud formation leading to gametophore

development is triggered by the action of cytokinins [9, 22, 23, 24]. Owing to

the simple anatomy of the moss, allowing accurate and quantitative analysis, and

the predictable nature of the developmental transitions that occur, Physcomitrella

22 | 2 Moss as a Model System for Plant Stress Responses

developmental progression is highly amenable to a detailed computational analysis

that allows predictions to be made and tested [25]. Consequently, Physcomitrellaprovides a highly suitable organism for systems-level analysis – especially when the

genetic and genomic resources now available are considered.

Whilst the utility of Physcomitrella as a subject for cellular level investigations

was clear, its potential for comparative genomic analysis of the evolution of gene

function was not immediately apparent. Only after the establishment of routine

genetic transformation did one of the more remarkable properties of this organ-

ism emerge. Physcomitrella is routinely transformed by the polyethylene glycol-

mediated uptake of naked DNA by protoplasts [26]. This is not a particularly

efficient means of DNA delivery in any plant species, but development of the

technique was aided by the ability of Physcomitrella protoplasts to regenerate with

very high efficiency. During the early development of the protoplast transforma-

tion procedure, it was discovered that retransformation of a transgenic line with a

second DNA construct containing vector sequences homologous with the first

transgene resulted (i) in an increased frequency of transformation by the second

construct and (ii) genetic cosegregation of the two transgenes [27]. It was postu-

lated that this arose from a propensity for the second transgene to become inte-

grated at the first transgenic locus by homologous recombination – a form of gene

targeting. This was subsequently confirmed by a landmark molecular analysis [28],

which showed that transformation with a DNA construct containing sequence

homology with an endogenous genomic sequence resulted in targeting of the

transgene to the endogenous locus. This occurred at very high frequency (up to

100%) – an efficiency of gene targeting hitherto only observed in yeast and cer-

tainly very much higher than occurs in flowering plants.

Gene targeting technology thus allows ‘‘reverse genetic’’ analysis of gene

function to be undertaken with great facility in Physcomitrella – a concept proven

by the first predetermined targeted knockout phenotype to be described in any

plant, that of the Physcomitrella FTSZ gene. This gene encodes a plastid tubulin

required for chloroplast division, the cells of ftsZ mutants generated by targeted

disruption containing single giant chloroplasts [29].

Deployment of such a powerful tool for genetic manipulation can only be

effective if the sequences of the genes to be targeted are known. This requirement

stimulated a series of gene discovery programmes. Initially these comprised the

accumulation of expressed sequence tag (EST) collections [30, 31] and culminated

in the acceptance of the Physcomitrella genome for complete sequence determi-

nation by an international consortium based on the United States Department of

Energy’s Joint Genome Institute Community Sequencing Program. The first-draft

sequence assembly of the Physcomitrella genome was released in 2007 [8].

Currently, the use of Physcomitrella as a model is supported by the genome

sequence assembly – representing about 486 Mbp and encoding approximately

25 000 genes – complemented by a recently developed, molecular-marker-based

linkage map which is anchored to the genome sequence. This will facilitate

mutagenesis-based ‘‘forward genetic’’ screening to undertake the map-based

cloning of genes responsible for selected phenotypic traits [32]. The EST resource

2.3 Physcomitrella as a Model System | 23

comprises about 300 000 sequences, and microarray chips are available based on

the EST resource and on the genome sequence. The facility with which gene

targeting may be undertaken allows the ‘‘reverse genetic’’ analysis of gene func-

tion by gene disruption to generate knockout mutants. It is also possible to carry

out more sophisticated manipulations that include the ability to construct

‘‘knock-in’’ fusions – for example, with reporter genes (b-glucuronidase, Green

Fluorescent Protein, etc.) that allow gene expression to be visualized in lines where

the reporter is introduced into the correct genetic locus, rather than at an ectopic

site elsewhere in the genome, or with molecular tags to facilitate the isolation of

molecular complexes (e.g., by generating epitope-tagged or affinity-tagged pro-

teins). Additionally, it is a relatively simple matter to undertake site-directed

mutagenesis of specific genes, containing as little as a single base alteration. Such

surgical precision is not available in any other plant model.

2.4

Water Stress and Abscisic Acid

As has been indicated above, the mosses retain many features that must have been

characteristic of the first land plants. These features include relatively high levels

of tolerance to abiotic stresses. Most strikingly, many mosses exhibit a char-

acteristically high degree of dehydration tolerance [33]. At its most extreme, this

occurs in the form of desiccation tolerance – the ability to withstand dehydration to

about 5–10% of the plant’s original water content. At this point, it is important to

be clear about how this property is defined. In plain English, the term ‘‘desiccation

tolerance’’ simply refers to the ability to survive and recover from the desiccated

state. However, this expression provides insufficient precision, in that it does not

specify the timescale over which a tolerant state is achieved. Consequently, a

distinction must be made between plants that are able to achieve a viable, desic-

cated state only following a period of adaptation through relatively prolonged

exposure to conditions that cause dehydration to occur over a period of time, and

plants that can equilibrate rapidly with a dry atmosphere and reach the dehydrated

state without a need for prior physiological adaptation [34]. The term ‘‘poikilo-

hydric’’ is used to describe the latter class of plant [35]. Poikilohydry is a generally

rare phenomenon that remains relatively common among the bryophytes,

whereas the ability of plants to achieve a state of desiccation tolerance following a

longer adaptive period is more generally widespread. However, poikilohydry is

relatively rare among the tracheophytes, implying that it is a characteristic that has

been lost during the evolution of anatomical complexity [35, 36]. Nevertheless, a

small number of taxa among the tracheophytes still display desiccation tolerance.

These so-called ‘‘resurrection plants’’ are able to undergo complete dehydration of

the vegetative tissues and recover normal metabolic function rapidly following

rehydration. From the distribution of this character within the land plant phylo-

geny, it is apparent that it is has independently re-evolved several times [36],

24 | 2 Moss as a Model System for Plant Stress Responses

implying that only a relatively small number of genes are required to mutate to

result in the gain or loss of tolerance.

Among poikilohydric mosses, the best-characterized is Tortula ruralis, the

characteristic features of which will be discussed in Section 2.5. Many mosses that

are not poikilohydric nevertheless still display a high tolerance of extreme dehy-

dration, culminating in the ability to tolerate the desiccated state so long as they

have undergone prior adaptation as a consequence of relatively slow drying [35].

Physcomitrella is in this second class of moss and in its responses to the application

of water stress, it exhibits many features more commonly associated with the

responses observed in flowering plants. Most angiosperms are not thought of as

desiccation-tolerant plants. However, this is misleading, because most angios-

perms retain the property of surviving desiccation – but only during specific parts

of their life cycle; in particular, during the development of reproductive propagules

such as seeds and pollen grains. Owing to its economic importance, the acquisi-

tion of desiccation tolerance by seeds has been most extensively characterized. The

ability to retain dry seeds in a viable form from one growing season to the next

underpins all agricultural practice and the acquisition of desiccation tolerance is

therefore intimately associated with the most significant development in human

history – the transition from hunter-gatherer populations with a limited resource

base to agricultural societies capable of acquiring surpluses, undergoing popula-

tion growth and initiating economic and cultural development.

Desiccation tolerance in seeds is acquired as a result of controlled, progressive

dehydration exerting biochemical and molecular changes within cells through the

agency of the plant growth regulator, abscisic acid (ABA). ABA imposes growth

inhibition in developing seeds (dormancy) to prevent precocious germination of

embryos that would be otherwise unsupported by storage reserves and it also

stimulates the accumulation of cellular components that are required for the

survival of the seed tissues in the dry state. These components include sugars and

protective proteins – the ‘‘late embryogenesis abundant’’ (LEA) proteins – whose

requirement for the acquisition of desiccation tolerance is well established, but

whose protective mechanisms remain enigmatic [37]. ABA also acts in the

responses of vegetative tissues to water stress. The de novo synthesis and accu-

mulation of ABA is an immediate response to water deficit, and the processes

stimulated by ABA include physiological changes, such as the closure of stomata,

senescence, and abscission of leaves, as a further measure to restrict evaporative

water loss, and the accumulation, in vegetative tissues of osmoregulatory com-

ponents (compatible osmolytes that include amino acids (proline, glycine betaine),

polyhydric alcohols, and disaccharides (principally sucrose) [38–41] as well as a

subset of the LEA proteins that accumulate in dehydrating seeds. However, very

few angiosperms have vegetative tissues that will survive desiccation, no matter

how gentle or prolonged the prior period of adaptive stress. This is by contrast with

nonpoikilohydric mosses, many of which undergo physiological adaptation to

enable their vegetative tissues to survive desiccation.

In Physcomitrella, the acquisition of vegetative stress tolerance is an ABA-

mediated process. Treatment with exogenous ABA, as well as slow dehydration

2.4 Water Stress and Abscisic Acid | 25

imposed over a period of several days, results in the acquisition of desiccation

tolerance [42, 43]. Figure 2.2 shows some of the structural and anatomical

consequences of stress and ABA treatment. These experimental conditions likely

reflect the ecological situation of P. patens; typically, this species occupies the

Figure 2.2 Effects of stress and ABA treatment of chloronemal tissue.

(a) Chloronemal filaments incubated on normal growth medium. (b)

Chloronemal filaments incubated in medium containing 10% mannitol

as an osmotic stress treatment, for 2 h. Note the commencement of

plasmolysis, as the cellular constituents shrink away from the cross-

walls of each filament. (c) Chloronemal filaments subjected to drought

by equilibration with an atmosphere of 75% relative humidity for 48 h.

The tissue has undergone about 90% fresh weight loss and the

cellular constituents have shrunk to form strands within each cell.

(d) The same tissue shown in (c), seconds following rehydration. The

cellular structure is rapidly becoming reorganized. The majority of the

rehydrated cells will regain viability. (e) The structure of chloronemal

cells grown on unsupplemented growth medium. (f) Chloronemal

tissue incubated on medium supplemented with 10�5 M ABA for 2

weeks. Colonies grown on ABA appear smaller, as a consequence of

the reduced length of the individual cells in each filament. The cells

are generally shorter and fatter. (g) Cells in a colony grown on 10�4 M

ABA for 14 days. The majority of the cells are smaller, rounded,

densely cytoplasmic and thick-walled, and are differentiating into

‘‘brood cells.’’ A few empty cells (‘‘tmema,’’ arrowed and labeled ‘‘t’’)

can be discerned.

26 | 2 Moss as a Model System for Plant Stress Responses

muddy margins of reservoirs, lakes, and ponds, and when exposed is probably

subjected to relatively slow dehydration due to its close association with the

underlying, generally water-retentive substratum (by contrast with species that

colonize bare rock surfaces and that likely undergo much more rapid drying/

wetting cycles). The acquisition of desiccation tolerance is associated with

membrane stabilization (cellular membranes do not undergo a phase transition

that would result in leakiness) and the physical transition of the cytosol from a

liquid to a glassy state [43]. Such changes are characteristic of organisms that

exhibit anhydrobiotic survival.

ABA induces a number of additional changes in Physcomitrella. Most striking is

the growth arrest and differentiation of the chloronemal cells to form rounded,

thick-walled ‘‘brood cells’’ (brachycytes). These cells become interspersed by

empty, fragile cells (‘‘tmema cells’’) that are easily fractured, thus releasing the

brachycytes that act, in effect, as vegetative spores, each able to initiate the for-

mation of a new colony following dispersal and rehydration [44, 20].

Analysis of the biochemical and metabolic changes that occur following ABA or

stress treatment highlights the similarities between the responses of Physcomitrellaand of the developing seeds of angiosperm species. Transcriptional profiling of

protonemal tissue subjected to such treatments has revealed a number of char-

acteristic changes in gene expression that are initiated very rapidly. Within min-

utes of the application of ABA, transcripts encoding a number of LEA proteins can

be observed to accumulate and these transcripts very rapidly become highly

abundant [45]. Many of the genes expressed in response both to application of ABA

and of the imposition of drought stress have homologs that are similarly regulated

in angiosperms: they include a substantial component encoding LEA proteins, as

well as a number of genes encoding proteins that have been identified as stress-

associated in flowering plants.

Additionally, the underlying mechanisms by which these genes are regulated in

response to ABA and to drought stress appear to have been conserved during the

evolution of the land plants. In flowering plants, drought-stress and ABA-induced

gene expression is regulated through two principal classes of transcription factor.

These are the abscisic-responsive element binding (AREB) factors [46, 47] and

dehydration-responsive element binding (DREB) factors [48, 49] The AREB factors

are members of the large and diverse basic domain/leucine zipper (bZIP) tran-

scription factor family that interacts with cis-acting sequences that include a core

ACGT motif. The DREB factors are members of the equally large APETALA2/

ethylene-responsive element binding (AP2/EREB) transcription factor family that

recognizes cis-acting sequences containing the CCGAC motif. These two classes

of factor interact in a number of the signal transduction pathways that lead to

stress-induced gene expression in the vegetative tissues of flowering plants [50,

51], and factors of both classes have also been identified as mediating both

ABA- and osmotic stress-induced expression of LEA genes during seed develop-

ment [52, 47, 48].

Many of the genes upregulated by ABA and drought stress in Physcomitrellacontain promoter motifs characteristic of these recognition sites [45], and the

2.4 Water Stress and Abscisic Acid | 27

function of the ACGT-core ABRE motif has been demonstrated to be required

both for the expression of a cereal Group 1 LEA gene in moss cells [53] and for the

homologous expression of its Physcomitrella ortholog [54]. Interestingly, the Group

1 LEA genes in flowering plants are highly seed-specific in their pattern of

expression. This is a consequence of their requirement for transcriptional activa-

tion by the ABI3 (ABA-INSENSITIVE3) transcription factor (encoded, in Arabi-dopsis, by the ABI3 gene) which is highly seed-specific in its pattern of expression,

and which is required both for the onset of embryonic dormancy and for the

acquisition of desiccation tolerance. Whereas in those flowering plant genomes

analyzed to date, there resides only a single copy of this key developmental reg-

ulatory gene, the Physcomitrella genome contains at least three ABI3 paralogs [8,

55], suggesting that the evolutionary origins of this gene were related to a primary

role in mediating the drought stress-tolerance pathway, and that recruitment for a

more specialized role in coordinating the seed developmental pathway was

accompanied by loss of the additional members of this gene family.

2.5

T. ruralis: A Model for Poikilohydry

The moss T. ruralis is probably the best-characterized poikilohydric moss. It is able

to survive rapid dehydration and recover full metabolic activity within minutes of

rehydration [56]. Unlike Physcomitrella, which stands at the polar opposite end of

the dehydration tolerance spectrum, Tortula does not show any striking alterations

in the pattern of gene expression during the period of prior dehydration [57].

Instead, this species appears to be constitutively prepared for dehydration. This

likely reflects its ecological distribution, being common in sand dunes and open

grassland [35]. Significant changes in gene expression associated with the desic-

cated state are apparent only following rehydration, when a number of novel

transcripts appear in the polysomal mRNA pool. Designated ‘‘rehydrins’’ [58],

these gene products were suggested to be involved in the repair of dehydration-

associated damage, early in the recovery phase of rehydration. However, it is

interesting that an EST-based analysis of the rehydration-associated transcriptome

of T. ruralis identified a significant number of transcripts encoding LEA proteins

among the most abundant class [59]. One rehydration associated gene product – a

protein designated ‘‘Tr288’’ – is a member of the Group 2 LEA proteins [60]: a class

of polypeptide initially defined as ‘‘dehydrins’’ following their identification as

ABA- and dehydration-induced gene products in cereals [61]. In Physcomitrella – asin angiosperms – the orthologous gene is expressed during drought stress, salt

stress, and in response to ABA treatment, [62] and the mutant strain derived by

gene knockout exhibited hypersensitivity to osmotic and salt stress.

Whilst the protective functions of LEA proteins are far from clearly understood,

at the molecular level, it is generally agreed that they serve to protect macro-

molecular constituents of the cell from the consequences of dehydration, which

include irreversible denaturation and the formation of inactive macromolecular

28 | 2 Moss as a Model System for Plant Stress Responses

aggregates. Dehydrins, like most LEA proteins, are highly hydrophilic, and retain a

high potential for sequestering water molecules [63]. It has been suggested that

they might retain a minimal level of hydration within the cell, offer their hydro-

philic side-chains as participants in hydrogen-bonding interactions with other

macromolecules as ‘‘replacement water’’ [37, 64] or associate to form macro-

molecular fibrillar structures that reinforce the structure of the cell as the compo-

nents undergo the vitrification that is characteristic of dry cells [65, 66]. In

considering why supposedly protective proteins might accumulate during the

recovery from dehydration, an alternative possibility is that such highly hydrophilic

proteins might act as moderators of dehydration/rehydration rates, restricting too-

rapid water loss during dehydration and acting to sequester incoming water upon

rehydration. In this model, water would be retained in an osmotically unresponsive

form: cells accumulating a significant quantity of such proteins would become

osmotically relatively stable – a property akin to that of camel erythrocytes (essential

for survival in an animal that can undergo prolonged periods of exercise in a desert

environment, punctuated by occasional, but substantial, intake of water), which has

been ascribed to the increased water-binding capacity of the unusually hydrophilic

form of hemoglobin found in this species [67].

A final consideration is that the resilience of the desiccated state in poikilohydric

mosses appears to be a feature only of the gametophyte generation. A study of the

desert moss, Tortula inermis, revealed that whereas the gametophyte was highly

tolerant of extreme desiccation, the sporophyte was far more sensitive [68]. Since

sporophytes display a number of anatomical features that are more characteristic

of the vascular plants (including the differentiation of conducting tissues, and the

presence of stomata and cuticles) their desiccation sensitivity may be correlated

with this additional anatomical complexity.

2.6

Cold Stress and Abscisic Acid

Many abiotic stresses elicit similar responses at the molecular and biochemical

level. This reflects the extent of cross-talk that exists between the various stress-

associated perception and signal transduction pathways (reviewed in Chapter 4),

but also some of the common cellular consequences of individual stresses and the

mechanisms that must subsequently be activated to bring about the amelioration

of these stresses or the repair of stress-induced damage. Thus, water-deficit stress

responses have much in common with low-temperature and freezing stress. Both

drought and freezing have the effect of reducing the available water in the cell.

Both require the accumulation of solutes that can act as either compatible

osmolytes or as antifreeze compounds, and both require an alteration of mem-

brane lipids (typically the desaturation of fatty acids) to alter membrane fluidity

and reduce the likelihood of membrane phase transitions. As with the drought

stress response, the freezing stress response and low-temperature acclimation

have been studied in some detail in Physcomitrella.

2.6 Cold Stress and Abscisic Acid | 29

Physcomitrella is highly susceptible to temperatures below freezing. However,

pretreatment with ABA for as little as 24 h substantially enhances freezing tol-

erance, with the temperature at which 50% of cells surviving freezing falling from

about �2 to �8 1C as a consequence of this treatment [43, 69]. Analysis of gene

expression by differential display, to identify novel genes, and of the expression of

candidate genes selected by their similarity with genes known to mitigate freezing

stress in flowering plants indicated a significant upregulation of these genes was

required for the acquisition of tolerance. As well as genes encoding LEA proteins,

these included genes for enzymes associated with sugar metabolism and the

detoxification of reactive oxygen species – the generation of which is a common

consequence of abiotic stress treatments. ABA-treated tissue additionally exhibited

a significant degree of membrane stabilization following freezing [69] suggesting

that modification of membrane lipids comprised a substantial component of the

mechanism of induced tolerance. Treatment with osmotically challenging con-

centrations of mannitol and NaCl also induces the expression of such genes [45,

69]. Oldenhof et al. [43] also demonstrated that ABA treatment increased tolerance

to freezing, and that this was associated with an increase in endogenous sucrose

levels. This is likely derived from the rapid breakdown of chloroplast-localized

starch following the administration of ABA [70] and is accompanied by the

accumulation of additional novel sugars such as the trisaccharide, theanderose

[71]. However, it is an open question as to whether such treatments reflect the true

mechanism by which freezing tolerance is mediated in Physcomitrella or simply

highlight the extensive overlap in the responses of a stress-induced gene set to

multiple agents. Thus, acclimation of Physcomitrella protonemal tissue by

incubation at low temperatures for prolonged periods (up to 1 week) will also

substantially enhance freezing tolerance, and induce the expression of many of the

same genes upregulated by ABA treatments. Although these changes are essen-

tially similar to those rapidly and massively induced by ABA treatment and

although mutants that are insensitive to ABA exhibit a reduced level of freezing

tolerance [72], the changes occurring during low-temperature acclimation do so

in the apparent absence of any measurable increase in the endogenous con-

centrations of ABA present in the moss tissue [73], thus implicating multiple,

parallel signal transduction pathways in the regulation of these stress responses.

2.7

Future Perspectives

The development of genomic resources for Physcomitrella has resulted in a con-

comitant burgeoning of interest among plant scientists for using this species for

comparative studies of plant processes. In the near future, we can expect to see a

more comprehensive accumulation of large transcriptomic, proteomic, and

metabolomic datasets of the type that have been established for other model

organisms. Proteomic approaches have already been applied to identify the poly-

peptide content of protonemal cells [74, 75] and to begin to decipher the

30 | 2 Moss as a Model System for Plant Stress Responses

Physcomitrella phosphoproteome – an important prerequisite for the dissection of

signal transduction pathways associated with cellular differentiation and stress

responses [76, 77].

Whilst Physcomitrella provides the most easily manipulated species among the

mosses, we should not focus on this species to the exclusion of others, whose study

may return insights into processes not evident in Physcomitrella. Thus, T. ruraliswill remain an exemplar for the analysis of poikilohydric desiccation tolerance and

we can expect analysis of other mosses adapted to specialized environments to be

of value in determining the basis of tolerance to these stresses. One such example

is adaptation to heavy metal ions deposited at industrially polluted sites. Physco-mitrella is generally susceptible to the toxic effects of heavy metal ions [78], unlike

other mosses such as Fontinalis antipyretica [79], Scopelophila cataractae [80, 81], orTaxithelium nepalense [82]. As well as being of academic interest, an insight into the

way in which nonmodel bryophytes adapt to such adverse conditions may provide

novel approaches to engineering stress tolerance in crop species, through the

identification of novel genes, and for strategies for the phytoremediation of pol-

luted environments [83]. The rapid advances in massively parallel DNA sequen-

cing technology can be expected to make transcriptomic analysis possible for

organisms that have previously not enjoyed the benefits of extensive research

funding. The use of technology such as the Roche 454 GS-FLX DNA sequencing

procedure enables the acquisition of about 400 000 sequence traces of about 500

bases each, in a single day. This technology has already been used to sample the

Physcomitrella epigenome [84]. When used to sequence ESTs derived from specific

tissues or treatments, quantitative analysis of the clustered sequences generates

mRNA abundance profiles over a wide dynamic range [85]. Whilst complete

genome sequencing for many organisms remains a relatively distant prospect,

transcriptome sampling by new-generation sequencing promises a much more

immediate return, that can generate a sequence database that will interface with

proteomic and metabolomic profiling for nonmodel species.

References

1 Strother, P.K., Al-Hajri, S., and Traverse,

A. (1996) New evidence for land plants

from the lower Middle Ordovician of

Saudi Arabia. Geology, 24, 55–58.2 Wellman, C.H., Osterloff, P.L., and

Mohiuddin, U. (2003) Fragments of the

earliest land plants. Nature, 425, 282–285.

3 Edwards, D. and Feehan, J. (1980)

Records of Cooksonia-type sporangia

from late Wenlock strata in Ireland.

Nature, 287, 41–42.4 Manhart, J.R. and Palmer, J.D. (1990)

The gain of two chloroplast introns

marks the green algal ancestors of land

plants. Nature, 345, 268–270.5 Karol, K.G., McCourt, R.M., Cimino,

M.T., and Delwiche, C.F. (2001) The

closest living relatives of land plants.

Science, 294, 2351–2353.6 Palenik, B., Grimwood, J., Aerts, A.,

Rouze, P., Salamov, A., Putnam, N.,

Dupont, C., Jorgensen, R., Derelle, E.,

Rombauts, S., Zhou, K., Otillar, R.,

Merchant, S.S., Podell, S., Gaasterland,

T., Napoli, C., Gendler, K., Manuell, A.,

Tai, V., Vallon, O., Piganeau, G., Jancek,

S., Heijde, M., Jabbari, K., Bowler, C.,

References | 31

Lohr, M., Robbens, S., Werner, G.,

Dubchak, I., Pazour, G.J., Ren, Q.,

Paulsen, I., Delwiche, C., Schmutz, J.,

Rokhsar, D., Van de Peer, Y., Moreau,

H., and Grigoriev, I.V. (2007) The tiny

eukaryote Ostreococcus provides genomic

insights into the paradox of plankton

speciation. Proc. Natl. Acad. Sci. USA,104, 7705–7710.

7 Merchant, S.S., Prochnik, S.E., Vallon,

O., Harris, E.H., Karpowicz, S.J.,

Witman, G.B., Terry, A., Salamov, A.,

Fritz-Laylin, L.K., Marechal-Drouard, L.,

Marshall, W.F., Qu, L.-H., Nelson, D.R.,

Sanderfoot, A.A., Spalding, M.H.,

Kapitonov, V.V., Ren, Q., Ferris, P.,

Lindquist, E., Shapiro, H., Lucas, S.M.,

Grimwood, J., Schmutz, J., Cardol, P.,

Cerutti, H., Chanfreau, G., Chen, C.-L.,

Cognat, V., Croft, M.T., Dent, R.,

Dutcher, S., Fernandez, E., Fukuzawa,

H., Gonzalez-Ballester, D., Gonzalez-

Halphen, D., Hallmann, A., Hanikenne,

M., Hippler, M., Inwood, W., Jabbari,

K., Kalanon, M., Kuras, R., Lefebvre,

P.A., Lemaire, S.D., Lobanov, A.V., Lohr,

M., Manuell, A., Meier, I., Mets, L.,

Mittag, M., Mittelmeier, T., Moroney,

J.V., Moseley, J., Napoli, C., Nedelcu,

A.M., Niyogi, K., Novoselov, S.V.,

Paulsen, I.T., Pazour, G., Purton, S.,

Ral, J.-P., Riano-Pachon, D.M., Riekhof,

W., Rymarquis, L., Schroda, M.,

Stern, D., Umen, J., Willows, R.,

Wilson, N., Zimmer, S.L., Allmer, J.,

Balk, J., Bisova, K., Chen, C.-J.,

Elias, M., Gendler, K., Hauser, C.,

Lamb, M.R., Ledford, L., Long, J.C.,

Minagawa, J., Page, M.D., Pan, J.,

Pootakham, W., Roje, S., Rose, A.,

Stahlberg, E., Terauchi, A.M., Yang, P.,

Ball, S., Bowler, C., Dieckmann, C.L.,

Gladyshev, V.N., Green, P.,

Jorgensen, R., Mayfield, S.M., Mueller-

Roeber, B., Rajamani, S., Sayre, R.T.,

Brokstein, P., Dubchak, I., Goodstein,

D., Hornick, L., Huang, Y.W., Jhaveri,

J., Luo, Y., Martınez, D., Ngau, W.C.A.,

Otillar, B., Poliakov, A., Porter, A.,

Szajkowski, L., Werner, G., Zhou, K.,

Grigoriev, I.V., Rokhsar, D.S., and

Grossman, A.R. (2007) The

Chlamydomonas genome reveals the

evolution of key animal and plant

functions. Science, 318, 245–250.8 Rensing, S.A., Lang, D., Zimmer, A.,

Terry, A., Salamov, A., Shapiro, H.,

Nishiyama, T., Perroud, P.-F., Lindquist,

E., Kamisugi, Y., Tanahashi, T.,

Sakakibara, K., Fujita, T., Oishi, K.,

Shin-I, T., Kuroki, Y., Toyoda, A.,

Suzuki, Y., Hashimoto, S., Yamaguchi,

K., Sugano, S., Kohara, Y., Fujiyama, A.,

Anterola, A., Aoki, S., Ashton, N.,

Barbazuk, W.B., Barker, E., Bennetzen,

J., Blankenship, R., Cho, S.H., Dutcher,

S., Estelle, M., Fawcett, J.A., Gundlach,

H., Hanada, K., Heyl, A., Hicks, K.A.,

Hughes, J., Lohr, M., Mayer, K.,

Melkozernov, A., Murata, T., Nelson, D.,

Pils, P., Prigge, M., Reiss, B., Renner,

T., Rombauts, S., Rushton, P.,

Sanderfoot, A., Schween, G., Shiu, S.-

H., Stueber, K., Theodoulou, F.L., Tu,

H., Van de Peer, Y., Verrier, P.J.,

Waters, E., Wood, A., Yang, L., Cove,

D.J., Cuming, A.C., Hasebe, M., Lucas,

S., Mishler, B.D., Reski, R., Grigoriev, I.,

Quatrano, R.S., and Boore, J.L. (2008)

The Physcomitrella genome reveals

insights into the conquest of land by

plants. Science, 319, 64–69.9 Reski, R. (1998) Development, genetics

and molecular biology of mosses.

Botanica Acta, 111, 1–15.10 Menand, B., Yi, K.K., Jouannic, S.,

Hoffman, L., Ryan, E., Linstead, P.,

Schaefer, D.G., and Dolan, L. (2007) An

ancient mechanism controls the

development of cells with a rooting

function in land plants. Science, 316,1477–1480.

11 Engel, P.P. (1968) Induction of

biochemical and morphological mutants

in moss Physcomitrella patens. Am. J.Bot., 55, 438–446.

12 Knight, C.D., Cove, D.J., Cuming, A.C.,

and Quatrano, R.S. (2002) Moss gene

technology, in Molecular Plant Biology,Vol. 2 (eds P.M. Gilmartin and C.

Bowler), Oxford University Press,

Oxford, pp. 285–299.

13 Ashton, N.W. and Cove, D.J. (1977) The

isolation and preliminary

characterisation of auxotrophic and

analogue resistant mutants of the moss.

32 | 2 Moss as a Model System for Plant Stress Responses

Physcomitrella patens. Mol. Genet.Genomics, 154, 87–95.

14 Ashton, N.W., Cove, D.J., and

Featherstone, D.R. (1979) The isolation

and physiological analysis of mutants of

the moss, Physcomitrella patens, whichover-produce gametophores. Planta, 144,437–442.

15 Ashton, N.W., Grimsley, N.H., and

Cove, D.J. (1979) Analysis of

gametophytic development in the moss,

Physcomitrella patens, using auxin and

cytokinin resistant mutants. Planta, 144,427–435.

16 Jenkins, G.I. and Cove, D.J. (1983)

Phototropism and polarotropism of

primary chloronemata of the moss

Physcomitrella patens: responses of thewild-type. Planta, 158, 357–364.

17 Jenkins, G.I. and Cove, D.J. (1983)

Phototropism and polarotropism of

primary chloronemata of the

moss Physcomitrella patens:responses of mutant strains.

Planta, 158, 432–438.18 Jenkins, G.I., Courtice, G.R.M., and

Cove, D.J. (1986) Gravitropic responses

of wild-type and mutant strains of the

moss Physcomitrella patens. Plant CellEnviron., 9, 637–644.

19 Knight, C.D., Futers, T.S., and Cove,

D.J. (1991) Genetic analysis of a mutant

class of Physcomitrella patens in which

the polarity of gravitropism is reversed.

Mol. Genet. Genomics, 230, 12–16.20 Decker, E.L., Frank, W., Sarnighausen,

E., and Reski, R. (2006) Moss systems

biology en route: phytohormones in

Physcomitrella development. Plant Biol.,8, 397–406.

21 Thelander, M., Olsson, T., and Ronne,

H. (2005) Effect of the energy supply on

filamentous growth and development in

Physcomitrella patens. J. Exp. Bot., 56,653–662.

22 Wang, T.L., Horgan, R., and Cove, D.J.

(1981) Cytokinins from the moss

Physcomitrella patens. Plant Physiol., 68,735–738.

23 Reski, R., and Abel, W.O. (1985)

Induction of budding on chloronemata

and caulonemata of the moss,

Physcomitrella patens, using

isopentenyladenine. Planta 165,354–358.

24 Reutter, K., Atzorn, R., Hadeler, B.,

Schmulling, T., and Reski, R. (1998)

Expression of the bacterial ipt gene in

Physcomitrella rescues mutations in

budding and in plastid division. Planta,206, 196–203.

25 Fracchia, F.D. and Ashton, N.W. (1995)

A visualization tool for studying the

development of the moss Physcomitrellapatens, in Proceedings of the IEEEConference on Visualization (eds G.M.

Nielson and D. Silver). IEEE, New York,

pp. 364–367.

26 Schaefer, D., Zryd, J.P., Knight, C.D.,

and Cove, D.J. (1991) Stable

transformation of the moss

Physcomitrella patens. Mol. Genet.Genomics, 226, 418–424.

27 Kammerer, W. and Cove, D.J. (1996)

Genetic analysis of the effects of re-

transformationof transgenic lines of the

moss Physcomitrella patens. Mol. Genet.Genomics, 250, 380–382.

28 Schaefer, D.G. and Zryd, J.P. (1997)

Efficient gene targeting in the moss

Physcomitrella patens. Plant J., 11,1195–1206.

29 Strepp, R., Scholz, S., Kruse, S.,

Speth, V., and Reski, R. (1998) Plant

nuclear gene knockout reveals a role in

plastid division for the homolog of the

bacterial cell division protein FtsZ, an

ancestral tubulin. Proc. Natl. Acad. Sci.USA, 95, 4368–4373.

30 Nishiyama, T., Fujita, T., Shin-I, T., Seki,

M., Nishide, H., Uchiyama, I., Kamiya,

A., Carninci, P., Hayashizaki, Y.,

Shinozaki, K., Kohara, Y., and Hasebe,

M. (2003) Comparative genomics

of Physcomitrella patens gametophytic

transcriptome and Arabidopsisthaliana: implication for land plant

evolution. Proc. Natl. Acad. Sci. USA, 100,8007–8012.

31 Lang, D., Eisinger, J., Reski, R., and

Rensing, S.A. (2005) Representation and

high-quality annotation of the

Physcomitrella patens transcriptome

demonstrates a high proportion of

proteins involved in metabolism in

moses. Plant Biol., 7, 238–250.

References | 33

32 Kamisugi, Y., von Stackelberg, M.,

Lang, D., Care, M., Reski, R., Rensing,

S.A., and Cuming, A.C. (2008) A

sequence-anchored genetic linkage map

for the moss Physcomitrella patens. PlantJ., 56, 855–866.

33 Dilks, T.J.K. and Proctor, M.C.F. (1974)

The pattern of recovery of bryophytes

after desiccation. J. Bryol., 8, 97–115.34 Alpert, P. (2006) Constrains of tolerance:

why are desiccation-tolerant organisms

so small or rare? J. Exp. Biol., 209,1575–1584.

35 Proctor, M.C.F. and Tuba, Z. (2002)

Poikilohydry and homoihydry: antithesis

or spectrum of possibilities? New Phytol.,156, 327–349.

36 Oliver, M.J., Tuba, Z., and Mishler, B.

(2000) The evolution of vegetative

desiccation tolerance in land plants.

Plant Ecol., 151, 85–100.37 Cuming, A.C. (1999) LEA proteins, in

Seed Proteins (eds P.R. Shewry and

R. Casey), Kluwer, Dordrecht,

pp. 753–780.

38 Yoshiba, Y., Kiyosue, T., Nakashima, K.,

Yamaguchi-Shinozaki, K., and

Shinozaki, K. (1997) Regulation of levels

of proline as an osmolyte in plants

under water stress. Plant Cell Physiol.,38, 1095–1102.

39 McKue, K.F. and Hanson, A.D. (1996)

Drought and salt tolerance: towards

understanding and application. TrendsBiotechnol., 8, 358–362.

40 Popp, M. and Smirnoff, N. (1995) Polyol

accumulation and metabolism during

water deficit, in Environment and PlantMetabolism (ed. N. Smirnoff), BIOS,

Oxford, pp. 199–215.

41 Bianchi, G., Gamba, A., Murelli, C.,

Salamini, F., and Bartels, D. (1991)

Novel carbohydrate metabolism in the

resurrection plant Craterostigmaplantagineum. Plant J., 1, 35–39.

42 Frank, W., Ratnadewi, D., and Reski, R.

(2005) Physcomitrella patens is highlytolerant against drought, salt and

osmotic stress. Planta, 220, 384–394.43 Oldenhof, H., Wolkers, W.F.,

Bowman, J.L., Tablin, F., and Crowe,

J.H. (2006) Freezing and desiccation

tolerance in the moss Physcomitrella

patens: an in situ Fourier transform

infrared spectroscopic study. Biochim.Biophys. Acta, 1760, 1226–1234.

44 Schnepf, E. and Reinhard, C. (1997)

Brachycytes in Funaria protonemata:induction by abscisic acid and fine

structure. J. Plant Physiol., 151, 166–175.45 Cuming, A.C., Cho, S.H., Kamisugi, Y.,

Graham, H., and Quatrano, R.S. (2007)

Microarray analysis of transcriptional

responses to abscisic acid and osmotic-

salt- and drought-stress in the moss,

Physcomitrella patens. New Phytol., 176,275–287.

46 Marcotte, W.R., Russell, S.H., and

Quatrano, R.S. (1989) Abscisic acid-

responsive sequences from the Em gene

of wheat. Plant Cell, 1, 969–976.47 Finkelstein, R.R. and Lynch, T.J. (2000)

The Arabidopsis abscisic acid response

gene ABI5 encodes a basic leucine

zipper transcription factor. Plant Cell,12, 599–609.

48 Shen, Q. and Ho, T.-H.D. (1995)

Functional dissection of an abscisic acid

(ABA)-inducible gene reveals two

independent ABA-responsive complexes

each containing a G-box and a novel cis-acting element. Plant Cell, 7, 295–307.

49 Sakuma, Y., Liu, Q., Dubouzet, J.G.,

Abe, H., Shinozaki, K., and Yamaguchi-

Shinozaki, K. (2002) DNA-binding

specificity of the ERF/AP2 domain of

Arabidopsis DREBs, transcription factors

involved in dehydration- and cold-

inducible gene expression. Biochem.Biophys. Res. Commun., 290, 998–1009.

50 Shinozaki, K. and Yamaguchi-Shinozaki,

K. (1997) Gene expression and signal

transduction in water-stress response.

Plant Physiol., 115, 327–334.51 Shinozaki, K., Yamaguchi-Shinozaki, K.,

and Seki, M. (2003) Regulatory network

of gene expression in the drought and

cold-stress response. Curr. Opin. PlantBiol., 6, 410–417.

52 Guiltinan, M.J., Marcotte, W.R., and

Quatrano, R.S. (1990) A plant leucine

zipper protein that recognizes an

abscisic acid response element. Science,250, 267–271.

53 Knight, C.D., Seghal, A., Atwal, K.,

Wallace, J.C., Cove, D.J., Coates, D.,

34 | 2 Moss as a Model System for Plant Stress Responses

Quatrano, R.S., Bahadur, S.,

Stockley, P.G., and Cuming, A.C. (1995)

Molecular responses to abscisic acid and

stress are conserved between moss and

cereals. Plant Cell, 7, 499–506.54 Kamisugi, Y. and Cuming, A.C. (2005)

The evolution of the abscisic acid-

response in land plants: comparative

analysis of group 1 LEA gene expression

in moss and cereals. Plant Mol. Biol., 59,723–737.

55 Marella, H.H., Sakata, Y., and

Quatrano, R.S. (2006) Characterization

and functional analysis of ABSCISICACID INSENSITIVE 3-like genes from

Physcomitrella patens. Plant J., 46,1032–1044.

56 Bewley, J.D. (1979) Physiological aspects

of desiccation-tolerance. Annu. Rev. PlantPhysiol., 30, 195–238.

57 Oliver, M.J. (1991) Influence of

protoplasmic water loss on the control

of protein synthesis in the desiccation-

tolerant moss Tortula ruralis:ramifications for a repair-based

mechanism of desiccation-tolerance.

Plant Physiol., 97, 1501–1511.58 Scott, H.B.I.I. and Oliver, M.J. (1994)

Accumulation and polysomal

recruitment of transcripts in response to

desiccation and rehydration of the moss

Tortula ruralis. J. Exp. Bot., 45, 577–583.59 Oliver, M.J., Dowd, S.E., Zaragoza, J.,

Mauget, S.A., and Payton, P.R. (2004)

The rehydration transcriptome of the

desiccation-tolerant bryophyte Tortularuralis: transcript classification and

analysis. BMC Genomics, 5, 89.60 Velten, J. and Oliver, M.J. (2001) Tr288:

a rehydrin with a dehydrin twist. PlantMol. Biol., 45, 713–722.

61 Close, T.J. (1996) Dehydrins: emergence

of a biochemical role of a family of plant

dehydration proteins. Physiol. Planta.,97, 795–803.

62 Saavedra, L., Svensson, J., Carballo, V.,

Izmendi, D., and Vidal, S. (2006) A

dehydrin gene in Physcomitrella patens isrequired for salt and osmotic stress

tolerance. Plant J., 45, 237–249.63 Campbell, S.A. and Close, T.J. (1997)

Dehydrins: genes, proteins and

associations with phenotypic traits. NewPhytol., 137, 61–74.

64 Hoekstra, F.A., Golovina, E.A., and

Buitink, J. (2001) Mechanisms of plant

desiccation tolerance. Trends Plant Sci.,6, 431–440.

65 Wise, M.J. (2003) Leaping to

conclusions: a computational reanalysis

of late embryogenesis abundant proteins

and their possible roles. BMCBioinformatics, 4, 52.

66 Wise, M. and Tunnacliffe, A. (2004)

POPP the question: what do LEA

proteins do? Trends Plant Sci., 9, 13–17.67 Bogner, P., Csutora, P., Cameron, I.L.,

Wheatley, D.N., and Miseta, A. (1998)

Augmented water binding and low

cellular water content in erythrocytes

of camel and camelids. Biophys J., 75,3085–3091.

68 Stark, L.R., Oliver, M.J., Mishler, B.D.,

and McLetchie (2007) Generational

differences in response to desiccation

stress in the desert moss Tortula inermis.Ann. Bot., 99, 53–60.

69 Minami, A., Nagao, M., Arakawa, K.,

Fujikawa, S., and Takezawa, D. (2003)

Abscisic acid-induced freezing tolerance

is accompanied by increased expression

of stress-related genes. J. Plant Physiol.,160, 475–483.

70 Nagao, M., Minami, A., Arakawa, K.,

Fujikawa, S., and Takezawa, D. (2005)

Rapid degradation of starch in

chloroplasts and concomitant

accumulation of soluble sugars

associated with ABA-induced freezing

tolerance in the moss Physcomitrellapatens. J. Plant Physiol., 162, 169–180.

71 Nagao, M., Oku, K., Minami, A.,

Mizuno, K., Sakurai, M., Arakawa, K.,

Fujikawa, S., and Takezawa, D. (2006)

Accumulation of theanderose in

association with the development of

freezing tolerance in the moss

Physcomitrella patens. Phytochemistry, 67,702–709.

72 Takezawa, D. (2007) Reduced sensitivity

to freezing and dehydration in the

abscisic acid-insensitive mutant in

mosses. Plant Cell. Physiol. Suppl., 48,893.

References | 35

73 Minami, A., Nagao, M., Ikegama, K.,

Koshiba, T., Arakawa, K., Fujikawa, S.,

and Takezawa, D. (2005) Cold

acclimation in bryophytes: low-

temperature-induced freezing tolerance

in Physcomitrella patens is associatedwith increases in expression levels of

stress-related genes but not with

increase in level of endogenous abscisic

acid. Planta, 220, 414–423.74 Sarnighausen, E., Wurtz, V., Heintz, D.,

van Dorsselaer, A., and Reski, R. (2004)

Mapping of the Physcomitrella patensproteome. Phytochemistry, b65,1589–1607.

75 Cho, S.H., Hoang, Q.T., Kim, Y.Y.,

Shin, H.Y., Ok, S.H., Bae, J.M., and

Shin, J.S. (2006) Proteome analysis of

gametophores identified a

metallothionein involved in various

abiotic stress responses in

Physcomitrella patens. Plant Cell Rep., 25,475–488.

76 Heintz, D., Wurtz, V., High, A.A., Van

Dorsselaer, A., Reski, R., and

Sarnighausen, E. (2004) An efficient

protocol for the identification of protein

phosphorylation in a seedless plant,

sensitive enough to detect members of

signalling cascades. Electrophoresis, 25,1149–1159.

77 Heintz, D., Erxleben, A., High, A.A.,

Wurtz, V., Reski, R., Van Dorsselaer, A.,

and Sarnighausen, E. (2006) Rapid

alteration of the phosphoproteome in

the moss Physcomitrella patens aftercytokinin treatment. J. Proteome Res., 5,2283–2293.

78 Rother, M., Krauss, G.-J., Grass, G., and

Wesenberg, D. (2006) Sulphate

accumulation under Cd2þ stress in

Physcomitrella patens – combined

transcript, enzyme and metabolite

profiling. Plant Cell Environ., 29,1801–1811.

79 Rau, S., Miersch, J., Neumann, D.,

Weber, E., and Krauss, J.-G. (2007)

Biochemical responses of the aquatic

moss Fontinalis antipyretica to

Cd, Cu, Pb and Zn determined by

chlorophyll fluorescence and

protein levels. Environ. Exp. Bot., 59,299–306.

80 Shaw, J.A. and Beer, S.C. (1989)

Scopelophila cataractae (Mitt.) Broth. in

Pennsylvania. Bryologist, 92, 112–115.81 Shaw, J.A. (1995) Genetic biogeography

of the rare ‘‘copper moss’’, Scopelophilacataractae (Pottiaceae). Plant Systemat.Evol., 197, 43–58.

82 Choudhury, S. and Panda, S.K. (2005)

Toxic effects, oxidative stress and

ultrastructural changes in moss

Taxithelium nepalense (Schwaegr.) Broth.under chromium and lead toxicity.

Water Air Soil Pollut., 167, 73–90.83 Prasad, M.N.V. and De Oliveira

Freitas, H.M. (2003) Metal

hyperaccumulation in plants –

biodiversity prospecting for

phytoremediation technology. Electron. J.Biotechnol., 6, 285–321.

84 Axtell, M.J., Snyder, J.A., and Bartell,

D.P. (2007) Common functions for

diverse small RNAs of land plants. PlantCell, 19, 1750–1769.

85 Weber, A.P.M., Weber, K.L., Carr, K.,

Wilkerson, C., and Ohlrogge, J.B. (2007)

Sampling the Arabidopsis transcriptome

with massively parallel pyrosequencing.

Plant Physiol., 144, 32–42.

36 | 2 Moss as a Model System for Plant Stress Responses