Download LEA proteins in higher plants: Structure, function, gene expression

Colloids and Surfaces B: Biointerfaces 45 (2005) 131–135
LEA proteins in higher plants: Structure, function,
gene expression and regulation
Shao Hong-Bo a,b,c,∗ , Liang Zong-Suo a,b,d , Shao Ming-An a,e
a
b
State Key Laboratory of Soil Erosion and Dryland Farming, The Centre of Soil and Water Conservation & Ecoenvironmental Research,
Chinese Academy of Sciences and Northwestern Sci-tech University of Agriculture and Forestry, Yangling 712100, China
Molecular Biology Laboratory, Bioinformatics College, Chongqing University of Posts & Telecommunications, Chongqing 400065, China
c Biological Laboratory, Chemical Engineering College, Qingdao University of Science and Technology, Qingdao 266042, China
d Life Science College, Northwestern Sci-tech University of Agriculture and Forestry, Yangling 712100, China
e Institute of Graphical Sciences and Resource, Chinese Academy of Sciences, Beijing 100101, China
Received 9 May 2005; accepted 28 July 2005
Abstract
Late embryogenesis abundant (LEA) proteins are mainly low molecular weight (10–30 kDa) proteins, which are involved in protecting
higher plants from damage caused by environmental stresses, especially drought (dehydration). These findings and the fact that the breeding
of drought tolerant varieties would be of great value in agriculture, form the basis of search for anti-drought inducible genes and their
characterization. LEA proteins are generally classified into six groups (families) according to their amino acid sequence and corresponding
mRNA homology, which are basically localized in cytoplasm and nuclear region. LEA protein synthesis, expression and biological activities
are regulated by many factors (e.g. developmental stages, hormones, ion change and dehydration), signal transduction pathways and lea genes.
No tissue-specific lea gene expression has been considered as one main regulatory mechanism on the basis of extensive studies with the model
plant, Arabidopsis thaliana. The study of the regulatory mechanism of lea gene expression is an important feature of modern plant molecular
biology.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Seed plants; LEA proteins; Gene structure; Function and expression; Drought resistance
1. Introduction
Higher plants dominate terrestrial ecosystems and play
most important economic and social roles in human life
[1,2], which contribute to their strong adaptive ability owing
to a long period of evolution under the pressure of natural
and human selection [3,4]. During such time span, higher
plants have developed multi-pathway, multi-level and multiscale survival strategies for continual changes in the environment, which include anatomical, physiological, biophysical,
biochemical, genetic, developmental and reproductive biological changes in response to adverse conditions [5]. The
∗
Corresponding author. Tel.: +86 23 62477654; fax: +86 29 87012210.
E-mail addresses: [email protected],
[email protected] (S. Hong-Bo).
0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2005.07.017
evolution of LEA proteins is one of these changes, which
plays an important role in resistance to drought. This implies
that studies on tolerant proteins, and the isolation, identification and functional analysis of their genes will be of great
benefit to the breeding of drought-resistant crops [6,7,47–52].
It is common knowledge that drought related food shortages
is a worldwide problem. Especially in sub-Saharan Africa
[8,9,47–52]. Drought is one of the main factors limiting crop
production [10–16]. Research on the biology and genetics of
drought resistance and breeding of drought-resistant crops
have, thus, become important field of contemporary research
in plant molecular biology and molecular breeding. These
efforts have been greatly aided by the sequencing of the Arabidopsis thaliana genome [6,17,18]. Here, we discuss the role
of LEA protein structure, function and gene expression in the
development of drought-resistant crops.
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S. Hong-Bo et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 131–135
2. LEA protein distribution and types in higher
plants
LEA proteins are formed during the late period of seed
development accompanied by dehydration. They are proteins with small molecular weight ranging mainly from 10
to 30 kDa and above 30 kDa [19]. Dure and Croud [17] first
studied LEA proteins in developing cotton seeds. Subsequently, researchers detected their existence in wheat, barley,
maize, rice, sunflower, potato, grape, apple, bean, Arabidopsis, tomato, rye, soybean, carrot and so on [19–28]. LEA
proteins exist mainly in higher plant seeds, but have also been
found in seedlings, roots and other organs [29]. LEA proteins are mainly localized in cytoplasm and nuclear regions
[26,29].
With the advent of new detection techniques [30], it has
become possible to classify LEA proteins based on molecular
hybridization (Northern hybridization, Western blotting) and
immunological methods. Based on their amino acid sequence
and mRNA homology, LEA proteins are basically divided
into five groups, Group 1–5 (Dure, 1993; Ingram and Bartels
[40]; He and Fu [19]; Zhang and Zhao [20]).
3. LEA protein structure
LEA proteins in higher plants are mainly composed of
hydrophilic amino acids ordered in repeated sequence (e.g.
Gly and Lys), forming hyper-hydrophilicness and thermal
stability. Advanced structure of such protein contains nonperiodic linear and ␣-helixed structure without thermal dominative state and corresponding dehydrated proteins exist in
a natural form of dimers.
Group 1 proteins (such as D19) have a conservative
sequence made up of 20 amino acid residues in repeated
copies, which can absorb a large amount of bound water [24].
Group 1 protein genes play a role in endosperm development
and osmotic protection of vegetative organs in higher plants
(Clark and Marcotle, 1999). Group 2 proteins (e.g. D11)
include a 15-amino acid conservative structure (EKKGIMDKIKEKLPG) at the C-terminus, and play an important role in
metabolism as molecular chaperones and defending protein
structure with a close connection to higher plant resistance to
drought [25,31,32]. Group 3 proteins (e.g. D7) are made up of
11 amino acids (TAQAAKEKAGE) in 13 repeats, resulting in
amphypathic ␣-helixed structure, and are involved in enriching ions during dehydration of higher plants (Zhang and Zhao
[20]; Yu [21]). Barley PMA1949, carrot Dc8, and soybean
pGmPM2 proteins belong to this family [22,29,33]. Group
4 proteins include cotton LEA14, D113 and Craterostigma
PGC27-45, which are devoid of repeated motif sequences and
contain a conservative region at N-end to form amphypathic
␣-helixed structure. The above proteins can further form a
subsidiary structure adaptive to conformational changes of
other proteins and function in protecting membrane stability and integration during drying and dehydration [34,35]. A.
thaliana AtECP31, carrot DcECP31 and cotton LEA D34,
D29 are involved in Group 5 proteins, in which there is little high-amino acid-residue specificity. On the basis of this
structure, it is predicted that Group 5 proteins play roles in
seed maturation, dehydration and combining concentrated
ions [23,29,30].
4. LEA protein functions
LEA proteins mainly play functions in dehydration tolerance and storage of seeds and in whole-plant stress resistance
to drought, salt, and cold. Farrant et al. (1992) experimented
on the recalcitrant seeds of Avicennia marina and Podocarpus henkelii and concluded that one of the important reasons
was the absence of LEA proteins in dehydration sensitive
seeds. Further study showed that LEA proteins are expressed
through all the developmental stages with different expression levels and no tissue specificity. For instance, Em, RAB21
and dehydrins in seeds can be found in the root, stem,
leaf, callus and suspension cultures of higher plants under
ABA or/and NaCl induction [21,28,36]. In cotton seedlings,
in vitro treatment could lead to the accumulation of LEA
protein mRNAs [22,29,31,34,37]. Almonguera et al. (1992)
reported that LEA mRNAs and heat shock mRNAs accumulated in a concerted way in sunflowers. Zhang et al.
(2000) applied yeast expression systems to investigate the
effect of LEA proteins on cellular metabolism under different
water stresses and found that transfer of two genes, tomato
le4 (Group 2) and barley HVA1 (Group 3) into such systems could promote earlier expression of HVA1 gene in the
medium containing 1.2 mol L−1 NaCl under GAL1 promoter,
increase the expression of both genes in 1.2 mol L−1 KCl
medium, resulting in growth reduction. These results imply
that LEA proteins play a special role in protecting cytoplasm
from dehydration. Functions related to other aspects remains
unknown.
5. Gene expression and regulation of LEA proteins in
higher plants
LEA protein gene expression in terms of time course starts
from the late period of maturation and initiation period of drying reaches its peak in progressive dehydration and sharply
decreases after some hours of germination [19,23,27,38].
Many reports show that LEA protein gene expression has no
tissue-specificity at the levels of tissues and organs as the gene
can express in cotyledons, panicles of seeds [26] and also
in stems, leaves and roots (vegetative tissues) [16,18,28,36].
Because of different classification methods for developmental stages in diverse plants, there exist some controversial
results (Galau et al., 1991). Results from our lab are in support of Huges and Galau (1989) who used barley seeds and
in vitro seedlings as experimental materials at four defined
periods of development (cotyledon, seedling, maturation and
mature termination) [38,39,51].
S. Hong-Bo et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 131–135
Higher plant adaptation to changing environment has
many mechanisms involved at different levels, one of which
is LEA protein gene expression and regulation formed during long evolutionary history of natural selection and artificial selection. The characteristics, which are perceived in
anatomy, physiology, biochemistry, biophysics and developmental biology are mainly the responses to related geneexpression and de-regulation at the molecular level. Up to
now, it is difficult to describe a detailed clear picture concerning this aspect [25,33,38–52]. In fact, various factors
and conditions and processes influence LEA protein gene
expression, among which ABA is considered the most important, especially in reducing the harm caused by drought
and is connected directly or indirectly with other regulatory circuits (Shao et al. [12,46]; Somerville and Dangl [1];
Chaves et al. [35]). Information from the model plant A.
thaliana has shown that there is a universal signal transduction network system in higher plants, on the basis of
and through which other factors produce different effects,
such as ABA concentration changes and drought as signal
stimulus [12,34,38,39,47–50]. On the other hand, there is
a stress-threshold problem (stress intensity), over-maximum
stress degree will lead to initiation and cascade reactions
of other gene expression pathways like related senescence
and death gene expression reactions (Zhu, 2001; Xiong et
al. [45]; Bray [47]; Shao et al. [12,46]). So, four steps
at least are involved in LEA protein gene expression and
regulation induced by drought: signal recognition, signal
transduction, signal amplification and integration, LEA protein gene expression responses and its product formation.
We will take ABA, which has recently been shown to be
involved clearly in inducing LEA protein gene expression,
as an example for further review. Concerning this aspect,
there are at least three different induced pathways: ABAdependent type; ABA-induced type; ABA-irresponsive type
[27,40–42,52].
ABA-dependent type gene expression is determined by
endogenous ABA accumulation or exogenous ABA levels.
Under drought stress, the endogenous ABA level increases.
In barley seedlings and beans, drought can result in high
levels of ABA by 57–160 times compared with those of
controls [36,40]. ABA physiological functions are realized
by corresponding gene expression. Many genes induced by
ABA have been isolated, cloned, identified, and subjected
to functional analysis (Bray [47]; Chaves et al. [35]). It
has been proved that cis-acting and trans-acting factors are
related to ABA-responsive gene transcriptional regulation.
There is some difference in the DNA sequence containing ABRE, in which there exists a G-box made of ACGT
sequence, and it can bind the transcriptional factor, bZIP.
Shen et al. (1995) reported that CE1-binding elements needed
ABA-responsiveness under the promoter of barley HVA22.
In A. thaliana, there are at least 58 genes coding bZIP
[36,40,41].
Many of LEA protein genes can be induced by ABA. A.
thaliana drought-induced gene RD22 is regulated by ABA
133
[42]. Under severe stress, two DNA elements (MYC-like element and MYB-like element) were identified, which were
related to ABA-induced-gene expression. The DNA-binding
protein gene rd22 BP1, coding MYC-like elements, can be
induced by drought and ABA [43]. Flores et al. [44] studied Phaseolus vulgaris by treatments of ABA and water
stress, induced 6 cDNA clones, in which there were two
types of LEA protein genes [44]. Under the water pressure of
0.35 MPa (16 h), these genes reached their expression peak
with a decrease in the progressive stress and after recovery of
water; these genes could obtain transient expression with the
above level. These results showed that short-term and rapid
treatment cannot lead to changes in the expression of these
genes.
In higher plant adaptation to drought, mRNA accumulation and activities can respond to gene expression to large
extent. ABRE is related to water stresses, which contains
CACGTG and G-box. Wheat Emla and rice rab16A gene promotion elements are ABREs, combined with 35S promoter
gene in many copies and regulated by ABA [42,44]. Iuchi
et al. [43] induced 10 cDNA clones from a high-tolerantdrought cowpea. Northern hybridization implied that higher
salt stress could result in the expression of three genes,
cPRD8, cPRD14, cPRD22, but not cold or heat stress. In
a defined range of ABA applied exogenously, cPRD and
cPRD22 could be induced, but not cPRD14, which suggested that there were two signal transduction pathways
between water stress and the expression of cPRD genes in
cowpea.
By studying A. thaliana ABA-insensitive mutants (aba
mutants), some of the genes can be induced by drought,
salt, and cold, so it is considered that the expression of
these genes do not need ABA under cold or drought [45].
Some of these genes, such as rd29A, lti78 and cor78 have
been analyzed under drought-inducing conditions [46–52].
During drying and cold stress, there are at least two independent systems, by which gene expression can be regulated,
i.e. ABA-dependent and non-ABA-dependent type. rd29A
is a non-ABA-dependent cis-acting element (YamaguchiShinozaki et al., 1993, 1994). By fusing rd29A promoter
with GUS reporter gene and transferring the combined construct into A. thaliana and tobacco plants, dehydration, low
temperature or higher salt conditions could produce obvious expression of the constructed gene under such promoter.
Further analysis demonstrated that the promoter region of
rd29A contained a conservative sequence of 9 bp (TACCGACAT, DRE), which is necessary for rd29A expression. It
is easy to know that dehydration, low temperature or higher
salt can each induce the genes with DRE cis-acting elements,
but cannot induce the genes with ABRE.
6. Summary and perspective
Over the past 10 years, many advances have taken place in
this challenging field. Although many molecular aspects are
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S. Hong-Bo et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 131–135
from the model plant A. thaliana, these have indeed deepened
our understanding of higher plant resistance to drought. This
anti-drought character is a quantitative character controlled
by many genes and impacted by a large number of environmental, anatomical, physiological, biophysical, biochemical and developmental factors. The characteristic result we
observe under a given condition is practically a comprehensive character. Many related genes (cDNA clones) have been
isolated and sequenced, but the functions and biochemical
properties of the proteins coded by these genes depend upon
predicting according to public database resources, and remain
unclear. Their space structure is also a challenge. Besides the
evolutionary relationship of different LEA protein groups in
diverse higher plant species with their adaptation to adverse
environment needs further effort, so the range of the tested
materials should be enlarged to explore the universal principle in functions, structure, gene expression, and evolution.
All these beg answers.
Information from LEA protein gene expression shows
that it is a multi-functional stress protein to maintain normal metabolism for higher plants under severe conditions,
and its gene can code RNA-regulatory-proteins, which can
regulate related events, such as gene expression and development. The mechanism is unknown (Wu et al., 2003). In
addition, what is the stress threshold (e.g. drought degree)?
How much drought intensity can prime LEA protein gene
expression? Which is the signal transduction pathway? What
is the cross-talk between this pathway and others? How many
components are involved in transcriptional and translational
level regulation, respectively? How to integrate the related
data into a clear picture and use it in practice for orientedbiotechnological anti-drought breeding is another big
challenge.
Up to date, there have been mainly two ways to study
LEA protein genes: select mutant genes from model plants
and analyze stress responses of crops. The extensive application of transgenic plants and microarrays is one developing direction. The related evidence directly from transgenic
plants to support anti-drought functions of LEA proteins is
not so much. By utilizing an individual-function gene, the
obtained transgenic plants can exhibit tolerant-stress characters, but the practical effect is not so good as that in
production, So, transferring transcription factor genes (like
CBF gene) into crops may be of great benefit to bringing
out fine functions of LEA proteins and genes and antidrought breeding with comprehensive and optimal traits,
which will be the second promising direction in this field.
Many results are gained under a given condition in laboratories and most of them at subcellular, cellular, tissue
or organ levels. Higher plants in natural field suffer from
many environmental stresses, which cannot be controlled
by human being. Therefore, the above results are inconsistent with, to some extent, those in nature and at wholeplant level, which needs further investigation in the third
direction from whole-plant level to community level in field
environment.
Acknowledgements
This work is jointly supported by State Key Basic
Research Development Plan of China (G2000018605), Major
Research Program of NSFC (90102012), Knowledge Innovation Engineering of the Chinese Academy of Sciences
(KZCX1-06-2-4), Shao MA-Innovation Group Program
Project of Northwestern Sci-tech University of Agriculture
and Forestry, Doctoral Initiation Foundation of Chongqing
University of Posts & Telecommunications and West-China
Key Research Project of National Natural Science Foundation of China (90302005). Many thanks are given Professor
Adam Wilkins in Cambridge University, UK and Professor
I.K. Vasil in University of Florida, USA, for their critical
reading of the manuscript and providing many instructive
suggestions.
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