Sodium Stress in the Halophyte Thellungiellahalophila and by mcu14908

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									Journal of Integrative Plant Biology 2007, 49 (10): 1484–1496



      Sodium Stress in the Halophyte Thellungiella halophila
          and Transcriptional Changes in a thsos1-RNA
                        Interference Line
              Dong-Ha Oh1 , Qingqiu Gong2 , Alex Ulanov2 , Quan Zhang3 , Youzhi Li4 , Wenying Ma3 ,
                                                                                 ∗
                             Dae-Jin Yun1 , Ray A. Bressan5 and Hans J. Bohnert2
        (1 Division of Applied Life Science (BK21 Program) and Environmental Biotechnology National Core Research Center, Graduate School of
                                                          Gyeongsang National University, Jinju 660-701, Korea;
                   2
                       Departments of Plant Biology and of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;
                       3
                           Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Jinan 250014, China;
          4
              Key Laboratory of the Ministry of Education for Microbial and Plant Genetic Engineering, Guangxi University, Nanning 530005, China;
                              5
                                  Department of Horticulture and Landscape Architecture, Purdue University, West-Lafayette, IN 47907, USA)




Abstract

The plasma membrane Na+ /H+ -antiporter salt overly sensitive1 (SOS1) from the halophytic Arabidopsis-relative Thel-
lungiella halophila (ThSOS1) shows conserved sequence and domain structure with the orthologous genes from
Arabidopsis thaliana and other plants. When expression of ThSOS1 was reduced by RNA interference (RNAi), pronounced
characteristics of salt-sensitivity were observed. We were interested in monitoring altered transcriptional responses
between Thellungiella wild type and thsos1-4, a representative RNAi line with particular emphasis on root responses to salt
stress at 350 mmol/L NaCl, a concentration that is only moderately stressful for mature wild type plants. Transcript profiling
revealed several functional categories of genes that were differently affected in wild-type and RNAi plants. Down-regulation
of SOS1 resulted in different gene expression even in the absence of stress. The pattern of gene induction in the RNAi plant
under salt stress was similar to that of glycophytic Arabidopsis rather than that of wild type Thellungiella. The RNAi plants
failed to down-regulate functions that are normally reduced in wild type Thellungiella upon stress and did not up-regulate
functions that characterize the Thellungiella salt stress response. Metabolite changes observed in wild type Thellungiella
after salt stress were less pronounced or absent in RNAi plants. Transcript and metabolite behavior suggested SOS1
functions including but also extending its established function as a sodium transporter. The down-regulation of ThSOS1
converted the halophyte Thellungiella into a salt-sensitive plant.

Key words: salt stress; salt overly sensitive1; RNA interference; Thellungiella halophila; transcript profiling.

Oh DH, Gong Q, Ulanov A, Zhang Q, Li Y, Ma W, Yun DJ, Bressan RA, Bohnert HJ (2007). Sodium stress in the halophyte Thellungiella halophila
and transcriptional changes in a thsos1-RNA interference line. J. Integr. Plant Biol. 49(10), 1484–1496.

Available online at www.blackwell-synergy.com/links/toc/jipb, www.jipb.net


                                                                                           An essential pathway for the exclusion of sodium ions from
                                                                                        entering plants has been described in Arabidopsis thaliana. This
Received 10 Mar. 2007 Accepted 10 May 2007                                              pathway, termed SOS (salt overly sensitive), consists of three
The work was supported by grants from the Biogreen 21 project of the                    proteins, which regulate not only the entry of Na+ but also dis-
Rural Development Administration (Korea), the Environmental Biotechnology               tribution within cell-lineages of the root and transport to shoots
National Core Research Center Project of KOSEF (R15-2003-012-01002-                     (Shi et al. 2000; Shi et al. 2002; Zhu 2003). The three component
00), Basic Science Project of KOSEF (RO1-2006-000-10123-0), NSF DBI-                    proteins form a functional unit: SOS3, a calcium-binding protein,
0223905 and by UIUC and Purdue University institutional funds.                          associates with the S/T protein kinase SOS2 (Guo et al. 2004),
∗
    Author for correspondence.                                                          and the SOS3/SOS2 complex activates the plasma membrane
Tel: 217 265 5475;                                                                      sodium/proton antiporter SOS1 (Halfter et al. 2000; Qiu et al.
Fax: 217 333 5574;                                                                      2002). In Arabidopsis, the SOS1 gene has been studied in
E-mail: <bohnerth@life.uiuc.edu>.                                                       detail. Lines in which the genes, individually or in combinations,
C    2007 Institute of Botany, the Chinese Academy of Sciences                          were eliminated (sos1, 2, 3), in comparison with wild type,
doi: 10.1111/j.1672-9072.2007.00548.x                                                   indicated that a main function of the sodium/proton-antiporter
                                                                      Thellungiella SOS1-RNAi Salt-stress Transcript Profiling     1485



SOS1 is in excluding the ion from root cells, although accumu-        Gao et al. 2006; Volkov and Amtmann 2006). Significantly,
lation also is observed in vacuoles. The activity of SOS1 further     Thellungiella is also characterized by its small genome size.
controls the import of Na+ into the vasculature, and its eventual     Expressed sequence tags (ESTs) and bacterial artificial chro-
distribution throughout the plant body (Shi et al. 2002; Qiu et al.   mosome (BAC) sequences that are now becoming available
2003). Its main protective function for the entire plant seems to     indicate high conservation of gene sequences with Arabidopsis,
be in assuring controlled, slow Na+ influx, rather than completely     and the plant can be transformed by flower dipping (Inan et al.
abolishing uptake, which appears to then activate other defense       2004). In essence, Thellungiella shows advantages that char-
reactions in the plants that lead to acclimation (Oh et al. unpubl.   acterized the ascent of Arabidopsis to become the premier plant
data, 2007).                                                          genetic and molecular model, even including sequencing of the
   Genes for SOS1 have been detected in a number of                   genome that is being considered at present. Relevant for our
species including the moss Physcomitrella patens (Benito              objectives, the eu-halophytic character of Thellungiella makes
and Rodriguez-Navarro 2003) and several monocot and dicot             possible a comparison of the genetic components that operate
species (Garciadeblas et al. 2006). The rice SOS1 protein,            in the species and to compare them with their counterparts
for example, functions in a complex with the Arabidopsis              in Arabidopsis (Taji et al. 2004; Wang et al. 2006). Moreover,
SOS2/3 components, indicating a high functional conservation          the high sequence identity of coding regions between the two
of the pathway (Martinez-Atienza et al. 2007). SOS1 shares            species makes it feasible to apply Arabidopsis oligonucleotide
evolutionary origin with the prokaryotic transporter NhaP, with       microarray hybridizations (Inan et al. 2004) to Thellungiella
other Na+ /H+ -antiporters in cyanobacteria (Hamada et al. 2001;      for analyzing and comparing gene expression characteristics
Waditee et al. 2001), yeast (Banuelos et al. 1998) and animals        between both species. Significantly, comparison of the ex-
(Brett et al. 2005). The long hydrophilic and cytoplasmic C-          pression profiles under salt stress revealed that Thellungiella
terminal tail, a characteristic of SOS1-type antiporter proteins,     has a more determined reaction specific to salt stress, while
is also present in yeast Nha1 and animal Na+ /H+ -exchanger           Arabidopsis shows what may be termed a ‘panic’ response,
(NHE) transporters. In animals, the C-terminal tail of NHE1           activating pathways not specifically related to the salt stress
functions as a plasma membrane scaffold for the assembly              tolerance (Gong et al. 2005). While the unidirectional sodium
of various signaling molecules (Baumgartner et al. 2004), and         influx is slower and more controlled than in Arabidopsis (Wang
the protein functions in the regulation of intracellular pH, cell     et al. 2006), Thellungiella also harbors various metabolites that
volume, differentiation, migration and control over cell death        may act as osmolytes in the absence of stress, which further
(Brett et al. 2005; Willoughby et al. 2005; Zachos et al. 2005; De    accumulate under salt stress, while Arabidopsis accumulates
Vito 2006). However, the proteins that interact with SOS1 or the      less or fails to accumulate such compounds (Gong et al. 2005).
signal pathways regulated by either SOS1 or the components            Hence, Thellungiella survives more extreme stress conditions
of the SOS pathway complex are as yet only incompletely               and completes its life cycle in the presence of salt concentration
understood (Katiyar-Agarwal et al. 2006). Models that sum-            that cannot be tolerated by Arabidopsis (Inan et al. 2004).
marize our present knowledge assume cross-talk that affects              To characterize the role and significance of SOS1 in regu-
the activity of plasma membrane proton-ATPases (Shabala               lating salt tolerance in a halophyte, we developed transgenic
et al. 2005), tonoplast Na+ /H+ -antiporters (Qiu et al. 2004;        Thellungiella plants in which the SOS1 transcript was reduced
Yamaguchi et al. 2005), Ca2+ -transporters (Pittman et al. 2005),     by RNA interference (RNAi). Down-regulation of SOS1 resulted
potassium uptake proteins (Wu et al. 1996; Rus et al. 2004), and      in the complete loss of the halophytic character of Thellungiella,
possibly additional functions in ion homeostasis and exclusion        indicating the essentiality of the SOS pathway. We compare
and compartmentalization of sodium ions. To date, there have          gene expression profiles in the root of wild type and a rep-
been few studies on the targets of SOS1 or other components of        resentative RNAi line, thsos1-4, under normal and salt-stress
the SOS pathway, especially in aspects of transcriptional regu-       conditions. Similarly, metabolite contents were compared in
lation. Significantly, differential subtraction screening identified    mature plants. The results reveal differential regulation of genes
several genes regulated by SOS1 in Arabidopsis (Gong et al.           and limited accumulation of metabolites in the RNAi plants under
2001). However, the information on the global transcription pro-      salt stress, and document the essentiality of SOS1 for salinity
file and especially the regulation in a naturally halophytic plant     stress tolerance.
are limited. Studies on halophytic plants emerge as an important
additional aspect because Arabidopsis, a glycophytic model
plant, has only limited salt tolerance capacity (Gong et al. 2005).   Results
   Thellungiella halophila (salt cress), a close relative of Ara-
bidopsis thaliana, has recently emerged as a model species
                                                                      The domain structure of SOS1
for the analysis of abiotic stress responses in plants because
the plant exhibits extreme freezing, cold and salinity tolerance      Thellungiella halophila SOS1 (ThSOS1) was compared with
(Bressan et al. 2001; Inan et al. 2004; Vera-Estrella et al. 2005;    counterpart genes of the glycophyte Arabidopsis thaliana
1486   Journal of Integrative Plant Biology    Vol. 49   No. 10       2007



(AtSOS1) and a second halophytic species, Mesembryan-                        intensity columns were swapped and normalized as described in
themum crystallinum (McSOS1) (Figure 1). They shared                         the methods section, to also compare the data for wild type and
74.5% (307/412) identity and 88.6% (365/412) similarity in the               thsos1-4 under either normal or stressed conditions (Figure 4;
transmembrane domain and 51.6% (366/709) identity and                        C[i4/Wt] and S[i4/Wt]). The salt stress included incubation in the
70.1% (497/709) similarity in their cytosolically located C-                 presence of 350 mmol/L NaCl for 24 h, a concentration and time
termini. More than 64 phosphorylation sites (54 serine, 11 thre-             chosen to enhance differences between the wild type and RNAi
onine) were predicted by the NetPhos 2.0 program (http://www.                lines. After background filtering and normalization by TIGR-
cbs.dtu.dk/services/NetPhos/). Overall, 81.5% of the phospho-                TM4-MIDAS (Gong et al. 2005), 15858 out of a total of 29551
rylation sites (42 out of 54 serine residues and all 11 threo-               probes remained in all slide hybridizations. These were included
nine residues) were in the C-terminal region of Thellungiella                in further analyses. One-way ANOVA analysis (P = 0.01) iden-
SOS1 (Figure 1). Pfam-directed (http://www.sanger.ac.uk/                     tified 1464 genes as regulated significantly, which were further
Software/Pfam/) sequence analysis identified two different con-               classified by fuzzy k-means clustering (Table 1). The program
served domains in the SOS1 proteins; first a Na+ /H+ -exchanger               placed these genes into seven clusters (C0–C6), where each
domain covering the transmembrane domains (amino acids 31                    gene was assigned to a cluster for which its ‘membership value’
to 444), and second a cyclic nucleotide binding domain (amino                was highest.
acids 753 to 843) that is located centrally in the long C-terminal              Each cluster revealed different regulation of genes by salt
tail (Figure 2A). This C-terminal tail, located on the cytoplasmic           stress, and, as well, by decreasing SOS1 expression based on
side of the plasma membrane, comprises approximately 60%                     the RNAi effect. Cluster C0 described up-regulated genes, and
of the entire protein of 1146 amino acids (molecular mass:                   clusters C1 and C6 included genes that were down-regulated by
126 kDa). The cyclic nucleotide-binding domain was present in                decreasing SOS1, even under non-stressed conditions. Genes
the three SOS1 sequences with the consensus glycine residues                 in cluster C1 were also down-regulated by the stress in wild type,
conserved in all SOS1 sequences (Figure 2B).                                 while cluster C6 showed less significant regulation by the stress.
                                                                             Clusters C2 and C3 included genes induced by the stress, with
                                                                             the level of induction higher in either wild type (C2) or the thsos1-
Generation of ThSOS1 RNAi plants and their stress                            4 line (C3). The C4 cluster showed a slight up-regulation in wild
phenotype                                                                    type and down-regulation in thsos1-4 by salt stress. The C5
                                                                             cluster included genes that were down-regulated only in the
To characterize the role of SOS1 in a halophyte, we devel-
                                                                             wild type, while they were less down-regulated or not changed
oped transgenic plants expressing an ThSOS1 RNAi construct
                                                                             in thsos1-4 by salt stress (Figure 4).
(Figure 3A). A segment of the C-terminal region of ThSOS1
was inserted in an inverted orientation on either side of a β-
glucuronidase (GUS) reporter sequence that allowed for moni-
toring of the expression strength of the RNAi construct. Among               Regulation affected by the presence of SOS1 (C0, C1 and
approximately 20 homozygous lines in the T3 generation, line                 C6 clusters)
thsos1-4 showed approximately 70% reduction in ThSOS1
                                                                             Cluster C0 contained 381 genes whose expression level
mRNA abundance in seedlings and more than 50% reduction
                                                                             was higher in thsos1-4 under normal conditions. Most of the
in mature plants under normal and salt-stress conditions (not
                                                                             genes in C0 were not regulated in wild type and slightly
shown). Mature thsos1-4 plants did not show a difference in
                                                                             down-regulated in thsos1-4, by salt stress treatment. When
growth and biomass compared to the wild type under non-stress
                                                                             compared with the expression profiles in Genevestigator
conditions. However, the RNAi plants showed decreased salt
                                                                             (https://www.genevestigator.ethz.ch/at/), a collection of gene
tolerance compared to wild type plants (Figure 3B), and the
                                                                             expression data under various treatment conditions that have
deleterious effect was strictly correlated with the strength of the
                                                                             been reported for Arabidopsis, the C0 cluster genes did not
stress treatment.
                                                                             show an apparent similarity with any single stress or hor-
                                                                             mone response (Figure 5 and Supplement). In C0, the Ca2+ -
                                                                             transporter CAX9, a protein similar to CAX2 and a Ca2+ -ATPase
Expression profile analyses by oligonucleotide-based
                                                                             were found. In addition, transcripts for the Ca2+ -binding protein
microarrays
                                                                             RD20, a vacuolar Ca2+ -binding protein and a putative caltactin
Using oligonucleotide microarray slides that covered ap-                     were 2 to 4-fold more abundant in thsos1-4 under normal, no-
proximately 90% of the known Arabidopsis transcriptome                       stress conditions. A group of sugar transporters, such as STP1,
(http://www.ag.arizona.edu/microarray/), the gene expression                 SFP1 and 2, ERD6 and a putative mannitol transporter were
profile was analyzed comparing stressed (S) and non-stressed                  found in C0. The complete list of genes in C0 cluster and
(C) roots from 6-week-old plants of wild type (Wt) and thsos1-               the comparison with Genevestigator data are included in the
4 (i4) (Figure 4; Wt[S/C] and i4[S/C]). After hybridization, the             supplementary materials.
                                                                             Thellungiella SOS1-RNAi Salt-stress Transcript Profiling            1487




Figure 1. Comparison of salt overly sensitive1 (SOS1) amino acid sequences of Thellungiella halophila (ThSOS1), Arabidopsis thaliana (AtSOS1)
and Mesembryanthemum crystallinum (McSOS1).

Transmembrane domains (boxed) were predicted by AraMemNon consensus prediction (http://aramemnon.botanik.uni-koeln.de/). Putative phospho-
rylation (circled) sites were predicted by NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) with conserved positions indicated by triangles.
1488      Journal of Integrative Plant Biology         Vol. 49     No. 10     2007




                                Na+\H+ Exchanger                              cNMP binding domain




                          Conserved cNMP binding domains in SOS1


                          Consensus             lrsfk-kGevifreGdpadslYivlsGkvkvykdedgreqilg-ilgpGdffGelallgg
                          AtSOS1            759 -EPMKLRGVTLYKEGSKPTGVWLIFDGIVKWKSKILSNNHSLHPTFSHGSTLGLYEVLTG
                          ThSOS1            753 -ETMKLRGVTLYKEGSKPTGVWLIFDGIVKWKSKGLSNNHSLHPTFSHGSTLGLYEVLTG
                          McSOS1            765 -EVVKVRGLTLYKEGSRPNGIWLISNGVVKWGSKSRVIKHAFHPTFTHGSTLGLYEVLTG
                                                 . .* :* .:::**. . .:::: .* ** ..     :: :   : *. :*    :* *

                          Consensus                endspSrhaprsatvvAltdsellvipredflelleedpe-
                          AtSOS1                   K--------PYLCDLITDSMVLCFFIDSEKILS-LQSDSTI 849
                          ThSOS1                   K--------PYMCDVITDSVVLCFFINSERILSYVQSDSTI 843
                          McSOS1                   K--------PYMCDVITDSVVLCFFINSERILSYVQSDFEM 855
                                                   : ... .* . ::: :       :.* * :*. ::.*


Figure 2. Schematic representation of conserved domains in Thellungiella halophila (ThSOS1) and sequence alignment of the cyclic nucleotide-
binding domain with the domain consensus sequences.



A
                                           AscI    SwaI BamHI   SpeI

          pMAS   BAR   MAS 3’   pCaMv35S             GUS           OCS 3’

    LB                                                                       RB


                                   721bps ThSOS1 cDNA fragment (1326-2046)


B
         NaCl(mM)        Th WT                    thsos1-4


            0


           250


           350
                                           2 week treatment

                                                                                     Figure 4. Overview of centroids generated by fuzzy k-means clustering.
Figure 3. Development of transgenic plant expressing a Thellungiella
halophila (ThSOS1) RNA interference (RNAi) construct.                                Wt (S/C), wild type stressed/wild type control; i4 (S/C), thsos1-4 stressed/
                                                                                     thsos1-4 control; C(i4/Wt), thsos1-4 control/wild type control; S(i4/Wt),
(A) Schematic representation of the RNAi vector. BAR, BASTA (glu-                    thsos1-4 stressed/wild type stressed. The heatmap presents log
fosinate) resistance; GUS, β-glucuronidase; LB, left border; MAS,                    2 values.
mannopine synthase; OCS, octopine synthase; pMAS, promoter
mannopine synthase; RB, right border.
(B) Twelve-week-old plants were treated with the indicated concentration             resembled profiles observed under potassium deficiency in
of NaCl for 2 weeks.                                                                 Arabidopsis, also similar to low glucose and programmed cell
                                                                                     death (PCD), and opposite to transcript changes observed in
   Cluster C1 identified 216 genes down-regulated by decreas-                         high glucose and sucrose treatment (Figure 5 and Supplement).
ing SOS1 amount under normal condition. The genes in this                            A large group of heat-shock proteins and histones were down-
cluster were mostly down-regulated by salt stress in wild type.                      regulated in C1, which was again similar to observations of
When compared with Genevestigator expression profiles, C1                             plants that have undergone programmed cell death (PCD).
cluster genes showed expression pattern in thsos1-4 that                             Defense proteins, many of which are located in the apoplast, like
                                                                               Thellungiella SOS1-RNAi Salt-stress Transcript Profiling             1489



Table 1. Summary of fuzzy K-clusters
             WtS/WtC          i4S/i4C         i4C/WtC          i4S/WtS        Number of genes                          Explanation
C0          0.03 ± 0.08    −0.37 ± 0.09      0.82 ± 0.17      0.17 ± 0.40             381          Up-regulated by decreasing SOS1 (under normal
                                                                                                      conditions)
C1         −0.35 ± 0.06      0.13 ± 0.06   −0.63 ± 0.12     −0.13 ± 0.10              217          Down-regulated by stress and by decreasing SOS1
                                                                                                      (under normal conditions)
C2          0.73 ± 0.06      0.30 ± 0.06     0.13 ± 0.11    −0.34 ± 0.13              195          Up-regulated by stress, less in thsos1-4
C3          0.32 ± 0.06      0.85 ± 0.07   −0.05 ± 0.13       0.51 ± 0.12             102          Up-regulated by stress, more in thsos1-4
C4          0.12 ± 0.07    −0.23 ± 0.06    −0.21 ± 0.09     −0.58 ± 0.11              186          Down-regulated by decreasing SOS1 (under stress
                                                                                                      conditions)
C5         −0.52 ± 0.07    −0.01 ± 0.06    −0.05 ± 0.12       0.49 ± 0.15             234          Down-regulation by stress in Wt but not in i4
a
    C6      0.16 ± 0.06      0.42 ± 0.08   −0.50 ± 0.11     −0.23 ± 0.08              149          Down-regulated by decreasing SOS1 (under normal
                                                                                                      conditions)
Total       0.03 ± 0.07      0.03 ± 0.07     0.05 ± 0.13      0.05 ± 0.12            1 464         Total significantly regulated genes (ANOVA P < 0.01)
C, control (no stress); i4, thsos1-4; S, stressed (350 mmol/L NaCl, 24 h); Wt, wild type. Values are in Log 2
a
    Cluster 6 consisted of genes with membership lower than 0.5.




thaumatin-like proteins and a chitinase were two- to threefold                 bidopsis (Figure 5 and Supplement). Functional categoriza-
decreased, suggesting a down-regulation of secretory path-                     tion by the COG database (http://www.ncbi.nlm.nih.gov/COG/)
ways, which coincided with the down-regulation of, for example,                showed enrichment of genes involved in post-translational
a signal peptidase subunit gene. A large group of transcripts                  modifications and lipid and amino acid transport in C2 rel-
encoding Ras-GTPase, a PIP5-kinase and a VPS53-related                         ative to cluster C3. In contrast, cluster C3 contained higher
protein were included in cluster C1.                                           proportions of defense- and cell wall biogenesis-related genes
   The cluster C6 included 149 genes that were down-regulated                  (Table 3).
by decreasing SOS1 transcript amounts under normal condi-                         A group of MYB transcription factors, DREB2A, NAM fam-
tions, but was not regulated or slightly up-regulated by salt stress           ily proteins and an AP-domain transcription factor were up-
in the wild type. Comparison with other expression profiles by                  regulated in wild type in C2. CHX17 and a CNGC were also
the Genevestigator meta-analyzer showed similarity between                     included in cluster C2, as well as two CBL-interacting pro-
the C6 cluster and PCD, potassium deficiency, the cytokinin BA,                 tein kinases (CIPK1 and CIPK17). In cluster C3, a calcium-
cycloheximide and ozone treatment (Figure 5 and Supplement).                   binding ATPase and an EF-hand containing protein were signif-
A group of genes related to carbohydrate metabolism, such as                   icantly up-regulated only in thsos1-4 by salt stress. The BON1-
a starch synthase, a starch branching enzyme, a member of                      associated protein 1 (BAP1) and a BAP1 homolog were also
the ADP-glucose pyrophosphorylase family, a β-amylase and                      identified in C3. The complete list of genes in these clusters is
a component of the pyruvate dehydrogenase complex were                         included in the supplemental materials, including comparisons
included in C6. Also included were genes encoding PI3,4-                       with Genevestigator data.
kinases and a PLC1 protein.

                                                                               Genes down-regulated by salt stress (C4 and C5)
Induction of genes by salt stress (C2 and C3 clusters)
                                                                               Clusters C4 and C5 identified genes differently down-regulated
The C2 and C3 clusters indicated salt stress up-regulated                      by salt stress in the comparison between wild type and thsos1-
genes in a slightly different way between wild type and thsos1-                4. In C4, 186 genes, thsos1-4 down-regulated genes are con-
4. Cluster C2 contained 191 genes, which showed higher                         trasted by genes that are not regulated or show a trend to up-
induction by salt stress in wild type than in thsos1-4. Many                   regulation in wild type by salt stress. Significantly, SOS1 was
of these genes in C2 were similarly induced by abscisic acid                   included in the C4 cluster showing up-regulation in wild type
(ABA) but have been reported as being down-regulated by                        by salt stress, but was low in thsos1-4 both under normal and
cycloheximide treatment. In contrast, C3 cluster genes (102)                   stress conditions, which was confirmed by quantitative reverse
showed higher induction in thsos1-4 than in the wild type.                     transcription-polymerase chain reaction (RT-PCR) results (not
These genes were less related to the ABA response than                         shown). Cluster C5 with 234 genes showed down-regulation
those in cluster C2. Genes showing the highest up-regulation in                in wild type under salt stress, but no regulation in thsos1-4.
C3 also showed higher up-regulation when treated by ozone,                     According to Genevestigator data for Arabidopsis, the genes in
hydrogen peroxide, salt or cycloheximide in wild type Ara-                     C5 were down-regulated by many different stress treatments,
1490    Journal of Integrative Plant Biology       Vol. 49   No. 10     2007




Figure 5. Comparison of salt stress regulated genes in wild type and RNA interference (RNAi) Thellungiella lines with stress responses in Arabidopsis.

Expression pattern of genes with membership values higher than 0.7 in each cluster were compared with the regulation of corresponding Arabidopsis
genes by Genevestigator (https://www.genevestigator.ethz.ch/at/). Included are regulatory changes in Arabidopsis by programmed cell death (PCD),
osmotic stress, salinity, ozone, hydrogen peroxide and abscisic acid (ABA) treatment.


similar to the expression pattern observed under conditions that               treated with 0 and 350 mmol/L NaCl, respectively, for 1 week.
promoted PCD (Figure 5). Functional categorization revealed,                   The concentration of each compound was determined by com-
when compared with the total population, a higher proportion                   parisons with known amounts of the identified metabolite with
of genes related to translation and cell division in cluster C5                internal standards (Table 2). Among the organic acids, citric
(Table 3).                                                                     acid and malic acid were most abundant and accumulated in
                                                                               the wild type in response to salt stress, both in the taproot and
                                                                               mature leaves. These and other acids in the tricarboxylic acid
                                                                               (TCA) cycle accumulated to a much lower degree or decreased
Metabolite analyses
                                                                               in thsos1-4. The most significant accumulation appeared in
To analyze a possible effect of SOS1 down-regulation on                        proline among the amino acid species, but thsos1-4 failed to
metabolites, the taproot (Vera-Estrella et al. 2005) and mature                accumulate proline to the same degree as the wild type both
leaves were isolated from 12-week-old plants, which were                       in mature leaves and taproots. Only aspartic acid accumulated
                                                                           Thellungiella SOS1-RNAi Salt-stress Transcript Profiling    1491



Table 2. Functional categorization of genes in each cluster
Category                                                          C0         C1        C2       C3        C4        C5        C6       Total

INFORMATION STORAGE AND PROCESSING
Translation, ribosomal structure and biogenesis                   2.0         2.9      0.6       0.0       2.6       4.7      0.7       2.2
RNA processing and modification                                    2.0         2.4      1.7       0.9       2.6       0.9      0.7       1.7
Transcription                                                     6.5         7.7      7.8       6.6      11.8       4.3      3.6       6.8
Replication, recombination and repair                             0.6         1.9      0.6       0.0       1.3       3.0      2.9       1.5
Chromatin structure and dynamics                                  0.8         2.4      0.6       0.0       0.7       2.2      0.7       1.2
CELLULAR PROCESSES AND SIGNALING
Cell cycle control, cell division, chromosome partitioning        1.1         1.0      0.0       0.9       0.0      2.6       0.7       1.0
Nuclear structure                                                 0.0         0.0      0.0       0.0       0.0       0.0      0.0       0.0
Defense mechanisms                                                2.8         2.4      0.6       4.7       2.0       2.2      2.9       2.4
Signal transduction mechanisms                                    8.8         9.2     10.6      12.3       9.9       9.9     12.2      10.0
Cell wall/membrane/envelope biogenesis                            3.4         4.3      0.6       6.6       0.0      4.3       2.9       3.1
Cell motility                                                     0.0         0.0      0.0       0.0       0.0       0.0      0.7       0.1
Cytoskeleton                                                      1.4         1.4      0.6       2.8       1.3       1.7      0.0       1.3
Extracellular structures                                          0.6         0.5      1.1       0.0       0.7       0.9      0.7       0.7
Intracellular trafficking, secretion, and vesicular transport      2.0         3.9      1.1       0.9       3.3       3.0      2.9       2.5
Posttranslational modification, protein turnover, chaperones       8.2         8.7      8.4       1.9       5.3       3.0     10.1       6.8
METABOLISM
Energy production and conversion                                  1.7         1.9      1.1       1.9       0.7       2.6      1.4       1.7
Carbohydrate transport and metabolism                             4.5         5.8      4.5       7.5       2.6       5.2      5.0       4.9
Amino acid transport and metabolism                               2.5         1.4      6.1       2.8       2.6       2.2      2.9       2.8
Nucleotide transport and metabolism                               1.1         1.0      0.6       0.9       0.7      1.3       1.4       1.0
Coenzyme transport and metabolism                                 0.0         0.5      0.6       0.0       0.7       1.3      0.0       0.4
Lipid transport and metabolism                                    4.0         4.8      5.6       1.9       2.0       3.9      4.3       3.9
Inorganic ion transport and metabolism                            3.1         1.0      3.4       1.9       4.6       1.7      2.2       2.6
Secondary metabolites biosynthesis, transport and catabolism      2.8         1.4      6.1       4.7       2.6      5.6       5.0       3.9
POORLY CHARACTERIZED                                             40.1        33.3     38.0      40.6      42.1     33.6      36.0      37.5
TOTAL                                                            100 (%)




in thsos1-4 more than in the wild type. Several hexoses and                sequences found in the moss Physcomitrella patens, and in a
sucrose, along with proline, malic acid and citric acid, constituted       number of higher plants (Benito and Rodriguez-Navarro 2003;
the major metabolites by mass in wild type under salt stress.              Garciadeblas et al. 2006). In sequences from diverse plant
The accumulation of these sugars was comparable to the wild                species, the high conservation of the SOS1 sequence, includ-
type in leaves, however, significantly lower in the taproots of             ing the positions of putative phosphorylation sites, is obvious
thsos1-4. Exceptions were fructose, glucose and galactose,                 (Figure 1). Also, pathway components have successfully been
which were higher in thsos1-4 than in wild type in the leaves              transferred between Arabidopsis and rice (Martinez-Atienza
under stress. Finally, in the thsos1-4 tap roots raffinose and              et al. 2007), and the transfer of the entire pathway, using
trehalose accumulated to a larger extent.                                  genes from Arabidopsis, confers salinity tolerance to yeast cells
                                                                           (Quintero et al. 2002). The highly conserved cyclic nucleotide-
                                                                           binding domain, found in the C-terminal region of SOS1 in plant
Discussion                                                                 species (Figure 2), appears to indicate that SOS1 activity or
                                                                           location may be regulated by other factors as well, or that
The sodium/proton antiporter SOS1 has emerged as a crucial                 SOS1 affects other components of the plant salinity stress
locus determining the exclusion and, over time, slow uptake                response.
of sodium ions, and hence the limited salt tolerance of the                   Cyclic nucleotides have recently emerged as important com-
glycophyte Arabidopsis thaliana. SOS1 is regulated via the                 ponents in stress signaling in plants (Gobert et al. 2006;
SOS2/3 complex by Ca2+ -dependent phosphorylation (Halfter                 Yoshioka et al. 2006). Cellular cyclic GMP concentrations rise
et al. 2000; Quintero et al. 2002; Cheng et al. 2004). This                following osmotic or salt stress treatment, and pre-treatment
pathway appears to be highly conserved in plants based on                  with a guanylyl cyclase inhibitor abolishes Ca2+ transients
                                                                                                                                                                                                                   1492


Table 3. Accumulation of metabolites under salt stress
                                                             Mature leaves                                                                                 Taproots

                                         Wild type                                     thsos1-4                                Wild type                                            thsos1-4

                           Control                   Stressed                Control              Stressed           Control               Stressed                   Control                   Stressed

Citric acid               2 589.7 (121.7)            2 471.6 (185.7)         777.1 (160.6)         339.1 (52.9)      315.6 (27.2)          1 548.4 (12.4)             382.5 (52.6)              704.9 (143.9)
Dehydroascorbate            12.7 (15.7)                 77.4 (18.3)           81.3 (18.4)            62.4 (9.1)        38.1 (5.4)             75.3 (12.3)              58.6 (9.7)                 88.2 (0.8)
Fumaric acid                39.8 (10.4)                 53.8 (12.2)           60.5 (18.4)            26.5 (2.2)        12.2 (3.2)             28.9 (8.6)               12.6 (0.0)                 22.1 (3.4)
Malic acid                1 381.5 (154.3)            5 209.6 (1 647.6)   1 987.9 (160.5)          2 306.4 (128.4)    851.5 (122.4)         1 345.9 (132.0)            817.2 (137.5)             995.6 (185.2)
                                                                                                                                                                                                                   Journal of Integrative Plant Biology




Quinic acid                112.0 (20.6)                 67.2 (14.3)           25.1 (3.0)             25.3 (3.6)       39.3 (0.6)              61.4 (0.8)               37.0 (5.7)                 67.7 (2.1)
Succinic acid               28.1 (43.4)               133.1 (47.6)            47.6 (7.7)            28.4 (4.2)        49.0 (6.0)             87.0 (18.0)               47.4 (6.1)                 67.8 (7.0)
Threonic acid               28.2 (26.4)               241.0 (44.4)            23.9 (3.1)            37.9 (3.7)        16.0 (2.6)             26.4 (10.2)               36.5 (9.1)                 34.3 (4.8)
                                                                                                                                                                                                                   Vol. 49




GABA                       366.2 (75.8)               362.5 (6.7)             50.7 (1.0)            51.5 (4.5)        96.6 (23.7)           234.8 (34.6)              113.3 (15.1)              208.3 (55.0)
Alanine                     11.5 (0.5)                 13.4 (2.5)              5.7 (0.4)              5.7 (1.0)         9.8 (1.9)            36.0 (3.2)                11.8 (0.8)                 42.9 (4.3)
Aspartic acid              411.0 (95.5)               399.4 (13.6)           292.8 (27.5)          202.0 (12.0)      138.9 (3.9)            149.7 (11.6)              277.3 (36.2)              436.4 (46.5)
                                                                                                                                                                                                                   No. 10




Glutamic acid              388.4 (112.2)              334.8 (69.8)           311.7 (90.1)          276.6 (54.7)       91.5 (10.8)           228.8 (48.4)              112.2 (3.6)               141.9 (13.6)
Glycine                     10.8 (0.0)                 18.8 (6.8)                n/d                  3.3 (0.5)       11.0 (0.5)             32.8 (1.3)                11.4 (0.9)                26.3 (1.2)
                                                                                                                                                                                                                   2007




Proline                    870.4 (172.8)             4 501.8 (752.8)         964.4 (81.0)         1 494.0 (122.0)    531.0 (72.0)          3 279.6 (426.8)            220.0 (71.0)             2 205.9 (97.2)
Pyroglutamic acid          271.2 (84.0)               432.2 (163.7)          241.5 (62.5)           78.8 (8.5)        95.4 (8.9)            231.3 (86.0)              108.6 (11.8)              288.6 (18.8)
Serine                     220.3 (107.3)              404.4 (95.5)           129.0 (14.4)          155.9 (12.7)       89.1 (11.9)           162.5 (37.8)              101.2 (2.9)               144.6 (1.5)
Threonine                  101.0 (13.0)               365.6 (85.3)           112.0 (7.2)           114.6 (37.2)      160.3 (11.3)           325.7 (65.8)              180.8 (19.6)              241.7 (27.0)
Valine                      22.7 (0.3)                 52.8 (14.0)             8.9 (0.6)            26.1 (6.3)        40.5 (5.9)             65.2 (18.7)               49.6 (7.3)                72.8 (6.9)
Fructose                 2 049.3 (467.6)             3 409.5 (781.0)     2 857.4 (92.9)           4 481.5 (73.8)    1 523.9 (275.0)        2 888.6 (574.7)       1 376.2 (131.8)               1 434.3 (217.1)
Galactose                1 286.2 (172.8)             2 622.6 (619.5)     1 226.2 (209.1)          3 184.6 (68.0)    1 182.5 (23.7)         1 894.1 (103.5)            862.9 (36.7)             1 410.1 (121.9)
Glucose                  2 193.0 (406.8)             3 943.8 (1 433.8)   1 622.7 (185.3)          4 941.2 (186.2)   1 445.1 (91.2)         2 199.5 (81.0)        1 471.4 (267.8)               1 694.2 (188.3)
Maltose                     38.5 (7.4)                 73.1 (15.6)            35.9 (6.8)            63.1 (11.9)       15.3 (3.0)             21.8 (3.3)                18.8 (0.7)                32.8 (3.1)
Raffinose                    23.0 (12.4)                47.1 (8.6)                n/d                94.6 (5.4)       109.9 (1.6)            112.2 (24.4)              235.2 (7.5)               206.0 (21.1)
Ribose                      33.5 (8.6)                 49.8 (16.4)            14.8 (5.8)            26.4 (4.5)        11.6 (0.9)             22.8 (9.1)                 5.7 (0.7)                  6.8 (0.7)
Sorbose                  1 089.7 (254.6)             1 040.9 (209.4)         343.0 (48.5)          394.6 (35.8)      855.8 (15.7)          1 026.5 (347.9)            695.9 (43.6)              856.9 (80.5)
Sucrose                    890.2 (157.5)             2 535.3 (250.8)         925.2 (150.6)        2 224.8 (231.3)   2 088.0 (350.4)        6 704.0 (977.6)       2 112.5 (348.6)               4 300.1 (1 076.8)
Trehalose                      n/a                        n/a                    n/a                   n/a            36.6 (4.1)             54.0 (11.3)              228.5 (15.6)              309.1 (8.6)
Values are ug/g fresh weight. Standard deviations (n = 5) in parentheses. GABA, γ - aminobutyric acids.
                                                                       Thellungiella SOS1-RNAi Salt-stress Transcript Profiling      1493



induced by salt stress, but not by osmotic stress, indicating              Second, interference with SOS1 expression resulted in the
that cGMP may act upstream of Ca2+ specifically in a ionic              activation of genes that are regulated differently by salt stress
stress response pathway (Donaldson et al. 2004). Treatment             in wild type plants. Stress-dependent up-regulated genes in
of Arabidopsis roots by a membrane-permeable cGMP ho-                  wild type (cluster C2) included genes with functions in post-
molog increased salt tolerance accompanied by reduced Na+ -            transcriptional modifications (Table 3), which appears to be
influx and enhanced K+ -uptake, and significantly induced the            another indication of the more determined salt-response of the
expression of SOS1 and SOS3 in concert with other cation               halophyte Thellungiella (Gong et al. 2005). In contrast, genes
transporters (Maathuis and Sanders 2001; Maathuis 2006). In            with higher induction in thsos1-4 by salt stress (cluster C3)
Arabidopsis, cyclic-nucleotide binding sites were found in more        were more similar to typical salt stress response genes in
than 30 proteins, including 20 CNGCs, potassium transporters           Arabidopsis. Functional categorization showed that C3 included
(AKT1, KAT1) and SOS1 (Talke et al. 2003; Bridges et al. 2005;         more genes involved in general defense (Table 3). Cluster C3
Maathuis 2006). It is possible that regulation of SOS1 by cyclic       genes were also more similar to transcription profiles of the
nucleotides, both in transcriptional and post-transcriptional in-      Arabidopsis wild type treated with ozone and hydrogen peroxide
teractions, plays a critical role in establishing ion homeostasis      (Figure 5), mirroring the role of SOS1 in inhibition of ROS
in plants in general, not simply restricted to episodes of salinity    production during salt stress (Katiyar-Agarwal et al. 2006).
stress. This function, shared by glycophytes and halophytes,               Finally, the thsos1-4 RNAi line failed to regulate genes that
may account for the high conservation of SOS1.                         are down-regulated in wild-type under salt stress. Gong et al.
   Reduction of SOS1 by RNAi changed the halophytic Thel-              (2005) had shown down-regulation of ribosomal protein genes
lungiella halophila into a salt-sensitive plant (Figure 3). The role   and genes involved in cell division in wild type Thellungiella, and
of SOS1 as the primary sodium excluder leading to protection           genes in these categories are included in cluster C5. The loss
under salt stress has been documented for glycophytes (Shi             of down-regulation in thsos1-4 appears to suggest impairment
et al. 2002), and a more general function for SOS1 in the              of stress sensing in the RNAi line, which may also explain the
regulation of ion uptake has been suggested based on the               reduced capacity to activate protective pathways, such as the
structure of its unusually long C-terminus (Zhu 2003). Our             incomplete accumulation of osmolytes, proline in particular, as
experiments now indicate that SOS1 appears to be critical for          it is obvious from the metabolite profiles (Table 3).
salt tolerance in a halophytic species as well. Experimental               In conclusion, down-regulation by more than 50% of the
evidence has also been provided suggesting the involvement of          SOS1 transcript in the halophyte Thellungiella generated a plant
Arabidopsis SOS1 in the regulation of pH (Shabala et al. 2005)         whose response to elevated NaCl was virtually indistinguishable
and the control of reactive oxygen species (ROS) during salt           from the behavior of a glycophytic plant. In different experi-
stress (Katiyar-Agarwal et al. 2006). Here, the down-regulation        ments, we have shown that SOS1 down-regulation leads to
of THSOS1 by RNAi expression in Thellungiella was used to              the entry of Na+ into the root vasculature that is much faster
probe the role of this protein in the regulation of salt tolerance     than in the wild type, along with accelerated death of root
by a true halophyte. The significantly lower expression of SOS1         cells originating from the region of the meristem (Oh et al.
in the thsos1-4 line converted Thellungiella into a glycophyte,        unpubl. data, 2007). While this result confirms the role that
which lead us to suggest that SOS1 expression and amount may           is traditionally assigned to SOS1, the profiling of transcripts
be the crucial difference between glycophytes and halophytes.          and metabolites seems to indicate a much broader role for
   An analysis of the Thellungiella root transcriptome and the         this sodium/proton antiport protein of the plasma membrane.
profile of major metabolites in wild type and thsos1-4 support          Absence or significant reduction of SOS1 affects plant behavior
this interpretation. We compared the transcript profiles of plants      not only during episodes of osmotic or ionic stress but SOS1
in the presence of 350 mmol/L NaCl (24 h), which in wild type          appears to have a function under normal growth as well. The
Thellungiella represents a strong but non-lethal stress. The           recognition, as reported here, of additional functions by the
results allowed for several conclusions.                               SOS1 gene and protein should inspire further studies.
   First, even in the absence of NaCl, lower SOS1 transcript
amounts affected gene expression and metabolite profiles. In
particular, lowering the amount of SOS1 seemed to interfere
with some functions regulated by Ca2+ and calcium-related              Materials and Methods
processes and, in addition, carbohydrate transport. Genes re-
lated to these functions showed higher expression in thsos1-4,         Plant growth
possibly indicating compensatory regulation (C0 cluster). Down-
regulated genes in thsos1-4 suggested processes similar to             Plants were grown on artificial Isolite soil (Sundine Enterprise,
PCD at least in parts of the root, a significant down-regulation        www.sundine.com) under 14-h day and 10-h night conditions
of secretory pathway functions and symptoms of potassium               and were irrigated with half strength Hoagland’s solution every
deficiency (C1 and C6).                                                 4 days.
1494   Journal of Integrative Plant Biology   Vol. 49   No. 10       2007



RNA i lines                                                                 C6) were generated, and genes were grouped into the cluster in
                                                                            which they had the highest membership value. The expression
A partial cDNA fragment of 721 bp, nucleotides 1326 to
                                                                            profile of each cluster was compared with previous microarray
2033 of the open reading frame of ThSOS1 (Genbank
                                                                            data, by the Meta-analyzer provided by the Genevestigator
accession: EF207775), was cloned into the AscI-SwaI and
                                                                            (https://www.genevestigator.ethz.ch/at/). Functional categoriza-
BamHI-SpeI sites of vector pGSA1252 (Arabidopsis Biological
                                                                            tion was determined using the COG categorization program
Resource Center, Columbus, OH, USA) to generate the
                                                                            (http://www.ncbi.nlm.nih.gov/COG/).
vector for ThSOS1 RNAi expression, which was transferred
to Agrobacterium tumefaciens GV1101 and introduced into
Thellungiella halophila, ecotype Shandong, using conventional               Metabolites analysis
flower dipping. For analysis of SOS1 expression, quantitative                Mature leaves (ML) and taproots (TR) were harvested from
real-time PCR was carried out as described by Gong et al.                   12-week-old plants after treatment with 0, 250 or 350 mmol/L
(2005) with primers 5 -TGAACGAGCGATGCAACTTA-3                               NaCl for 1 week. Samples (100–150 mg FW) were extracted
and 5 -GTTATCTTGTGCCTTGTTATTGTTCA-3 for ThSOS1                              and derivatized according to (Roessner et al. 2000; Roessner
and 5 -TCTAAGCTCTCAAGAAAAAGGCTAA-3                  and 5 -                 et al. 2001). Sample volumes of 1–2 µL were injected with a
AAACAAACAAATGGAGAAGCAAAT-3 for an actin gene                                split ratio of 5:1. The gas chromatography–mass spectroscopy
(ThACT2).                                                                   (GC-MS) system consisted of a HP5890 gas chromatograph, a
                                                                            HP5973 mass selective detector and HP 7673A (Agilent, Palo
Microarray hybridizations                                                   Alto, CA, USA) autosampler. Gas chromatography was carried
                                                                            out on a 30 m SPB-50 column with 0.25 mm inner diameter and
Root samples were harvested from more than ten 6-
                                                                            0.25 µm film thickness (Supelco, Belfonte, CA, USA) with an
week-old plants of wild type and thsos1-4 plants treated
                                                                            injection temperature of 230 ◦ C, the interface set to 250 ◦ C, and
with 0 or 350 mmol/L NaCl for 24 h. The preparation of
                                                                            the ion source adjusted to 200 ◦ C. The helium carrier gas was
cDNA and hybridization was carried out essentially as
                                                                            set at a constant flow rate of 1 mL/min. The temperature program
described by Gong et al. (2005), hybridizing the cD-
                                                                            was 5min isothermal heating at 70 ◦ C, followed by an oven
NAs prepared from wild type control (Wt-C) and stressed
                                                                            temperature increase of 5 ◦ C/min to 310 ◦ C and a final 10 min at
(Wt-S) into a 70-mer oligonucleotide microarray slide
                                                                            310 ◦ C. Mass spectra were recorded in the m/z 50–600 scanning
(http://ag.arizona.edu/microarray), and the thsos1-4 control
                                                                            range. Spectra evaluation was carried out as described by
(i4-C) and stressed (i4-S) cDNA into another slide, after labeling
                                                                            Lozovaya et al. (2006). The experiment was carried out with
each cDNA with Cy3 and Cy5, respectively. The experiment was
                                                                            five independent biological replications for each variant. Data
repeated with a different set of RNA and cDNA preparation and
                                                                            were analyzed statistically using the algorithm incorporated
with the dye swapped, constituting total four slide hybridization.
                                                                            into Microsoft Excel 2002 (Microsoft Corporation, Seattle, WA,
                                                                            USA). Differences were determined to be statistically significant
Microarray evaluation
                                                                            at P < 0.05.
The results were analyzed by GenePix4000B (Axon Instru-
ments, Union City, CA, USA) and the resulting GPR files were
converted into MEV files (Gong et al. 2005). To compare the
                                                                            Acknowledgments
expression profiles of wild type and thsos1-4 under normal and
                                                                            We thank Drs Shisong Ma and Valeriy Poroyko for technical
stress conditions, another set of MEV files were generated by
                                                                            support (UIUC) and discussions.
positioning the integrated intensity columns for wild type control
(Wt-C) and thsos1-4 control (i4-C) together into one MEV file,
and wild type stressed (Wt-S) and thsos1-4 stressed (Wt-S)                  References
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