Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice.. Similar to results obtained from our previous Os
Trang 1phosphatases supports distinctive functions of these plant enzymes
Shuhei Shima1,2, Hirokazu Matsui2, Satoshi Tahara2and Ryozo Imai1
1 Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, NARO, Toyohira-ku, Sapporo, Japan
2 Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan
Trehalose is a nonreducing disaccharide in which two
glucose units are linked by an a,a-1,1-glycosidic
link-age The prevalent pathway for trehalose synthesis
includes two enzymatic reactions Trehalose
6-phos-phate (Tre6P) is generated from UDP-glucose and
glu-cose 6-phosphate (Glc6P) in a reaction catalyzed by
trehalose-6-phosphate synthase (TPS) Tre6P is then
dephosphorylated to form trehalose via
trehalose-6-phosphate phosphatase (TPP) [1] In yeast, trehalose
synthesis is carried out by a large enzyme complex that
is composed of four subunits, including TPS1, TPS2, and regulatory subunits TSL1 and TPS3 [2]
Trehalose is widely distributed in nature In bacteria, fungi, and insects, trehalose functions as a storage car-bohydrate or a blood sugar In addition, trehalose can protect cellular integrity against a variety of environ-mental stresses associated with desiccation, heat, and cold [3] In plants, the presence of trehalose has been
Keywords
functional analysis; kinetic analysis; Oryza
sativa; recombinant protein; trehalose
Correspondence
R Imai, Crop Cold Tolerance Research
Team, National Agricultural Research Center
for Hokkaido Region, National Agriculture
and Food Research Organization,
Hitsujigaoka 1, Toyohira-ku, Sapporo
0628555, Japan
Fax ⁄ Tel: +81 11 857 9382
E-mail: rzi@affrc.go.jp
(Received 8 November 2006, revised 14
December 2006, accepted 19 December
2006)
doi:10.1111/j.1742-4658.2007.05658.x
Substantial levels of trehalose accumulate in bacteria, fungi, and inverte-brates, where it serves as a storage carbohydrate or as a protectant against environmental stresses In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement
of the recombinant protein demonstrated that OsTPP2 encodes a func-tional trehalose-6-phosphate phosphatase enzyme OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses Enzy-matic characterization of recombinant OsTPP1 and OsTPP2 revealed strin-gent substrate specificity for trehalose 6-phosphate and about 10 times lower Km values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50C for 4 min These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular sub-strate concentration and tightly regulated gene expression These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses
Abbreviations
ABA, abcisic acid; Glc1P, glucose 1-phosphate; Glc6P, glucose 6-phosphate; GST, glutathione S-transferase; TPP, trehalose-6-phosphate phosphatase; TPS, trehalose-6-phosphate synthase; Tre6P, trehalose 6-phosphate.
Trang 2documented in a limited number of species, including
Myrothamnus flabellifolia, a desiccation-tolerant desert
plant [4,5], and Selaginella lepidophylla, a
desiccation-tolerant moss [6]; its occurrence in many other plant
species is uncertain
TPS and TPP genes were functionally identified in
Arabidopsis thaliana by complementation of
Saccharo-myces cerevisiae mutants [7,8] Homologous TPS and
TPP genes have now been identified in many other
plant species These results suggest that trehalose
synthesis may in fact be ubiquitous among
angio-sperms, although the levels to which it accumulates
are generally low [1,9] Attempts to increase trehalose
content in plants by overexpressing microbial TPS
and TPP genes resulted in transgenic tobacco and
potato plants with increased stress tolerance at the
tissue level [10–12] However, these transformants
exhibited pleiotropic phenotypes, such as stunted
growth and lancet-shaped leaves [11,12] On the
other hand, expression of an Escherichia coli TPS–
TPP fusion enzyme in transgenic rice resulted in
accumulation of 3–10 times more trehalose compared
to nontransgenic rice plants, imparting abiotic stress
tolerance without altering morphology [13,14]
There-fore, these findings suggested that accumulation of
Tre6P may result in the observed morphologic
alter-ations in the tobacco and potato studies
Although trehalose biosynthesis in higher plants has
been demonstrated, details of both the physiologic
functions and regulation of this pathway remain
lar-gely unknown Genome sequencing of Arabidopsis and
rice has revealed complex genomic organization of
plant trehalose biosynthesis genes Eleven putative TPS
and 10 putative TPP genes were identified within the
Arabidopsis genome, and nine putative TPS and nine
putative TPP genes were found within the rice genome
Genetic studies have revealed that trehalose
biosyn-thesis genes function specifically in regulating plant
growth and development An Arabidopsis knockout
mutant of AtTPS1 exhibited impaired embryo
matur-ation [15] Further characterizmatur-ation of the mutant
dem-onstrated that AtTPS1 is also required for vegetative
growth and flowering [16] A recent study established
that a maize TPP gene is involved in inflorescence
development [17] A more specific function of trehalose
biosynthesis in the regulation of starch biosynthesis
has recently been revealed Trehalose feeding was
found to induce expression of ApL3, encoding a large
subunit of ADP-glucose pyrophosphorylase in
Arabid-opsis [18,19] It was demonstrated recently that Tre6P
directly regulated starch synthesis via
post-transla-tional redox activation of ADP-glucose
pyrophospho-rylase [20,21]
In our previous study, we demonstrated that expres-sion of the rice TPP gene OsTPP1 is rapidly and tran-siently induced by chilling stress and abcisic acid (ABA) treatment Induction of OsTPP1 was followed
by transient increases in total TPP activity and treha-lose content in rice root [22] Eight other members of the rice TPP gene family have not yet been character-ized, so it is not known if these members have diver-gent functions in rice In addition, the enzymatic properties of plant TPPs are largely unknown
In this article, we report the isolation of a second TPP gene from rice, OsTPP2, and its relative tran-scription in response to abiotic stresses, as well as the
in vivo and in vitro functionality of its translated prod-uct We also describe unique kinetic and biophysical properties of the plant TPPs
Results
Isolation of rice OsTPP2 Completion of the rice genome sequence revealed nine putative TPP genes To determine which of these TPP genes are expressed in rice seedlings, RT-PCR was car-ried out using specific primer sets designed to amplify transcripts from all of these OsTPP genes (OsTPP1– OsTPP9) [22] Only mRNA for OsTPP2 was detected
in addition to that of the previously characterized OsTPP1 after 28 cycles of PCR amplification (Fig 1A) OsTPP3–OsTPP9 mRNAs were not detec-ted in root and shoot tissues after up to 35 cycles of amplification (data not shown) These results suggested that OsTPP1 and OsTPP2 were the major TPP genes expressed in rice seedlings A full-length OsTPP2 cDNA was then isolated from root tissue by RT-PCR The OsTPP2 gene contained an ORF encoding a 42.6 kDa protein with 382 amino acid residues Overall amino acid sequence homology between OsTPP2 and OsTPP1 was 53% (Fig 1B) Greater similarity was observed between OsTPP2 and Arabidopsis AtTPPA (57%) OsTPP2 contains two motifs shared by all TPP enzymes (Fig 1B), known as phosphatase boxes: (FIL- MAVT)-D-(ILFRMVY)-D-(GSNDE)-(TV)-(ILVAM)-(ATSVILMC)-X-(YFWHKR)-X-(YFWHNQ) (domain A), and (KRHNQ)-G-D-(FYWHILVMC)-(QNH)-(FWYGP)-D-(PSNQYW) (domain B) [23]
Responses of OsTPP2 to chilling and other abiotic stresses
In our previous study, we demonstrated that OsTPP1 expression is transiently induced by multiple abiotic stresses [22] We therefore determined whether
Trang 3OsTPP2 expression is also responsive to abiotic
stres-ses RNA gel blot analysis was performed on total
RNA extracted from rice seedlings subjected to low
temperature (12C), drought, and salt stresses
(Fig 2) OsTPP2 mRNA levels were detectable prior
to stress treatments, and transiently increased in
response to low temperature, peaking at 10 h after
the initiation of treatment in both shoot and root
tis-sues This expression pattern contrasted with the
observed rapid induction of OsTPP1 and gradual
induction of OsMEK1 in response to low-temperature
treatment [22,24] Drought stress transiently induced
OsTPP2 expression, which peaked at 6 h in shoots
and 2 h in roots (Fig 2) The induction of OsTPP2
expression occurs earlier during stress treatment com-pared with expression of another drought-induced gene (salT) [25] Treatment with 150 mm NaCl also induced OsTPP2 expression (Fig 2) in roots, suggest-ing that stresses associated with water deficit similarly affect expression of this gene However, in contrast to chilling and drought stress treatments, clear induction
of OsTPP2 was not observed in shoots, whereas the salt treatment effectively induced salT in both roots and shoots Slight and transient induction of OsTPP2 was observed in roots and shoots in response to exo-genous ABA Together, these expression analyses indi-cated involvement of OsTPP2 in multiple stress responses
A
B
A
B
Fig 1 Expression of putative OsTPP genes in young vegetative tissues, and alignment of TPP sequences (A) Expression analysis of puta-tive TPP genes with RT-PCR, using RNAs extracted from root and shoot tissues of rice seedlings (O sativa L cv Yukihikari) (B) Alignment
of the amino acid sequences of OsTPP2, OsTPP1 (O sativa [22]), AtTPPA and AtTPPB (A thaliana [8]), TPS2 (Sa cerevisae [34]) and OtsB (E coli [35]) Database accession numbers are: OsTPP1, BAD12596; OsTPP2, BAF34519; AtTPPA, AAC39369; AtTPPB, AAC39370; TPS2, CAA98893; and OtsB, CAA48912 Shading reflects the degree of amino acid conservation Black shading indicates amino acid identity The bars represent highly conserved domains.
Trang 4OsTPP2 complements a yeast Dtps2 mutant
To detect OsTPP2 enzyme function in vivo, a Sa
cere-visiae (YPH499) tps2 (TPP) deletion mutant [22] was
transformed with plasmid constructs based on the
pAUR123 vector (Takara) Whereas wild-type cells
grow at both 30C and 36 C, growth of the Dtps2
mutant at 36C was inhibited because of its inability
to synthesize trehalose (Fig 3) The same mutant yeast
strain transformed with OsTPP2 recovered wild-type
levels of growth at 36C, suggesting that OsTPP2 is a
functional TPP enzyme in yeast cells
TPP activity of recombinant OsTPP2
To determine whether OsTPP2 exhibits TPP activity
in vitro, it was purified as a recombinant protein To
accomplish this, the ORF of OsTPP2 was inserted into
a pGEX-6P-3 vector to produce a glutathione S-trans-ferase (GST)–OsTPP2 fusion protein After affinity column purifications and protease digestion, OsTPP2 proteins were purified to near homogeneity The size
of the purified recombinant enzyme was estimated to
be 45 kDa on SDS⁄ PAGE, in accordance with the size deduced from the nucleotide sequence (42.6 kDa) (Fig 4A) TPP activity was then measured using this purified recombinant enzyme Aliquots of purified enzyme were added to the reaction mixtures, and conversion of Tre6P into trehalose was detected as a measure of enzyme activity (Fig 4B) Under these same conditions, purified GST or NaCl⁄ Pi solution without enzyme did not result in this conversion (Fig 4B) We therefore concluded that OsTPP2 encodes a functional TPP enzyme
Enzymatic properties of OsTPP1 and OsTPP2 Although genes encoding plant TPPs have been identi-fied in several plant species, their enzymatic character-istics have not been explored To further characterize the enzymatic properties of plant TPP enzymes, we also purified recombinant OsTPP1 using the same methods Then, the kinetic parameters of these recom-binant OsTPP1 and OsTPP2 enzymes were determined The Km values for Tre6P of OsTPP1 and OsTPP2 were determined to be 0.0921 and 0.186 mm, respect-ively, using Hanes–Woolf plots (Table 1) The kcat val-ues of OsTPP1 and OsTPP2 were 6.52 and 13.4 s)1, respectively Therefore, the kcat⁄ Km values of OsTPP1 and OsTPP2 were approximately the same These
Fig 3 Complementation of the heat-sensitive phenotype of a
Sa cerevisiae tps2 deletion mutant by introduction of OsTPP2 A YPH499 tps2 deletion mutant was transformed with the pAUR123 vector (Dtps2) and pAUR123-OsTPP2 (Dtps2 ⁄ OsTPP2) As a posit-ive control, YPH499 wild-type cells were transformed with the empty pAUR123 vector (wt) These transformants were grown overnight in YPD liquid medium supplemented with 0.5 lgÆmL)1 aureobasidin A The cultures were then diluted 1–1000 times Five microliters of each dilution was then spotted onto an YPD agar plate supplemented with 0.5 lgÆmL)1aureobasidin A These plates were incubated at 30 C or 36 C for 2 days.
A
B
C
D
Fig 2 Expression of OsTPP2 in rice seedlings in response to
abiotic stress and exogenous ABA treatment Total RNAs were
iso-lated from rice seedlings subjected to chilling stress (A), drought
stress (B), 150 m M NaCl stress (C), and exogenous ABA (50 l M )
solution (D) The RNA blots were hybridized with an OsTPP2 probe.
The expression of OsTPP1 is shown for comparison of expression
patterns, and those of OsMEK1 [24] and salT [25] are shown as
positive controls for these treatments Ethidium bromide-stained
total RNA (10 lg) is presented as a loading control.
Trang 5results indicated that both enzymes exhibit similar cat-alytic activities It is interesting to note here that the
Kmvalues for rice TPPs are more than 10 times lower than those of bacterial TPP enzymes reported thus far For instance, others reported that the Km values for
E coli and Mycobacterium smegmatis TPPs were 2.5 mm and 1.5 mm, respectively [26,27]
To determine the substrate specificity of these recombinant proteins, phosphatase activities were measured using various sugar phosphate substrates [glucose 1-phosphate (Glc1P), Glc6P, galactose 6-phosphate, mannose 1-6-phosphate, mannose 6-phos-phate, fructose 1-phos6-phos-phate, fructose 6-phos6-phos-phate, sucrose 6-phosphate, lactose 1-phosphate, and ribose 5-phosphate] Both OsTPP1 and OsTPP2 exhibited strong phosphatase activity upon Tre6P, but almost
no activity (less than 1% relative to Tre6P) was detec-ted with any of the other sugar phosphates tesdetec-ted (data not shown)
The pH dependences of OsTPP1 and OsTPP2 enzyme activities were determined within a pH range
of 5.5–9.0, using two different buffers (Mes⁄ NaOH,
pH 5.5–7.5; Tris⁄ HCl, pH 7.0–9.0) The pH optima of OsTPP1 and OsTPP2 were approximately 7.0 and 6.5, respectively, whereas the enzymes had almost no activ-ity at pH 5.5 or 9 (Fig 5)
The heat stabilities of the recombinant OsTPP1 and OsTPP2 were determined by measuring residual activities after heat treatments (40–80C) (Fig 6)
A
B
Fig 4 Purification of recombinant OsTPP1 and OsTPP2 and deter-mination of their activities Recombinant OsTPP1 and OsTPP2 were purified according to the experimental procedure described previ-ously (A) SDS ⁄ PAGE (12%) was run with protein standards (lane M), crude extracts of the recombinant bacterial strains induced without (lane 2) or with (lane 3) isopropyl thio-b- D -galacto-side, and purified recombinant OsTPP1 or OsTPP2 (lane 4) (B) Chromatograms detailing TPP activities of recombinant OsTPP1, OsTPP2, and GST These proteins (0.5 lg) were used for assays in
100 lL reaction mixtures (2 m M Tre6P, 2 m M MgCl2, 50 m M
Tris ⁄ HCl, pH 7.0) T; trehalose; T6P, trehalose 6-phosphatase.
Table 1 Enzymatic properties of recombinant OsTPP1 and OsTPP2.
Protein
K ma (m M )
K cat (s)1)
K cat ⁄ K m (m M )1Æs)1) Reference
a Kmfor Tre6P.
Trang 6Heat treatment at 50C or higher for 4 min nearly
eliminated both OsTPP1 and OsTPP2 activity,
indica-ting that both enzymes are heat-labile
Discussion
Identification and functional characterization of
treha-lose biosynthesis genes have established trehatreha-lose
biosynthesis in higher plants However, low-level
accu-mulation of trehalose in plants suggests a distinctive
function of this substance compared with its role in
other organisms Organization of these trehalose
bio-synthesis genes is also quite unique in higher plants
Only one or two copies of TPS and TPP genes exist in
most bacteria, fungi, and insects, whereas these genes
constitute a large gene family in higher plants For
example, in Arabidopsis, 11 TPS and 10 TPP genes
have been identified from genomic information [28,29],
and nine TPS and nine TPP homologs are found in the rice genome Therefore, researchers have specula-ted that trehalose biosynthesis is tightly regulaspecula-ted dur-ing plant growth and development, and that each TPS and TPP gene is under specific regulation In this study, we identified a novel TPP gene (OsTPP2) from rice, and demonstrated that OsTPP2 and the previ-ously identified OsTPP1 are predominantly expressed
in young vegetative tissues of rice According to the results of yeast complementation analysis and enzyme assays with recombinant protein, OsTPP2 encodes a functional TPP
Expression of OsTPP2 was regulated by multiple stress factors, such as chilling, drought, and salt stresses,
as well as ABA treatment (Fig 2) It is interesting that OsTPP1is also regulated by the same stress factors but its induction kinetics are quite different in comparison
to those of OsTPP2 [22] OsTPP2 is transiently induced after 10 h of chilling stress (12C), whereas transient induction of OsTPP1 occurs much earlier) within 2 h
of the chilling stress [22] Similarly, the patterns of OsT-PP2 induction in response to drought and salt stresses
A
B
Fig 5 Optimal pH for recombinant OsTPP1 (A) and OsTPP2 (B)
activity The reaction buffer systems tested were Mes ⁄ NaOH
(pH 5.5–7.5) and Tris ⁄ HCl (pH 7.0–9.0) The enzyme assay was
car-ried out as described in Experimental procedures This activity was
determined by measuring inorganic phosphate released from
Tre6P.
A
B
Fig 6 Heat stability of recombinant OsTPP1 (A) and OsTPP2 (B) The purified recombinant OsTPP1 and OsTPP2 were incubated for various time periods at temperatures ranging from 40 C to 80 C The residual activity after treatment is expressed as a percentage
of the original activity.
Trang 7differ from those of OsTPP1 under similar conditions.
OsTPP2is induced by exogenous ABA during 1–2 h of
treatment, whereas ABA induction of OsTPP1 occurs
more rapidly (within 1 h) and pronouncedly [22] These
data clearly show that OsTPP1 and OsTPP2 are under
distinctive regulation Our preliminary results with
green fluorescent protein fusion proteins suggested that
both OsTPP1 and OsTPP2 are cytosolic proteins It was
therefore suggested that trehalose biosynthesis is tightly
regulated in response to multiple abiotic stress factors in
rice, the process involving two differentially regulated
TPP enzymes
Using recombinant enzymes, we conducted the first
detailed functional characterization of plant TPPs
These rice TPPs displayed three distinct properties
com-pared with the previously characterized microbial TPPs
First, the Km values for the recombinant OsTPP1 and
OsTPP2 enzymes are lower than values published for
the microbial enzymes Others have reported that the
Tre6P concentration in Arabidopsis is relatively very low
(10.1 ± 1.3 lgÆg)1 fresh weight) [30] Therefore, these
low Kmvalues for OsTPP1 and OsTPP2 correlate with
low concentrations of this substrate in plant cells
Second, these rice TPPs were overall less stable than
bacterial enzymes For example, others reported that a
TPP from Mycobacterium did not lose activity after heat
treatment at 60C for 6 min [31] In contrast, the results
of this study indicate that OsTPP1 and OsTPP2 are
completely inactivated after incubation at 50C for 3 or
4 min, so these enzymes are heat-labile This further
suggests that the turnover rates of OsTPP1 and OsTPP2
are relatively high Relatively rapid turnover of these
enzymes would better enable tight control of trehalose
or Tre6P levels in rice Third, the substrate specificity of
OsTPP1 and OsTPP2 is higher than for the
correspond-ing bacterial enzymes [31] For instance, the
mycobacte-rial TPP exhibited approximately 18% and 5% relative
activities against Glc1P and Glc6P, when compared
with Tre6P substrate In contrast, no phosphatase
activ-ity was detected when OsTPP1 and OsTPP2 were
incubated with various sugar phosphate substrates,
including Glc1P and Glc6P (data not shown)
The function of trehalose in stress tolerance has
been documented in several transgenic plants [13,32]
However, whether trehalose has a direct stress
protec-tion funcprotec-tion in wild-type plants (as in the case of
microorganisms) or a regulatory function remains
unclear Rice transgenic plants expressing an otsA–otsB
fusion gene exhibited improved stress tolerance;
how-ever, trehalose in these plants did not reach high
enough levels to function as an osmoprotectant
[14,33] Moreover, trehalose accumulated in wild-type
rice plants at very low levels, and changed minimally
and transiently in response to chilling stress [22] In this study, we discovered that stress-induced OsTPP2 expression was transient and distinct from the expres-sion pattern of salT (which encodes a protein with a putative stress protection function) under those same conditions [25] Moreover, others have shown recently that the trehalose biosynthesis pathway is intercon-nected with the glucose and ABA signaling pathways
in Arabidopsis [30] These current studies suggest that trehalose or Tre6P is involved in regulation of stress responses in higher plants
Although the trehalose biosynthesis pathways are conserved during evolution, a unique function of this substance in higher plants has yet to be elucidated Rather than overproduction of trehalose as a stress protectant or as a storage carbohydrate, fine-tuned biosynthesis is required to produce the putative signa-ling molecules trehalose or Tre6P in higher plants Our genetic and biochemical analyses presented here sup-port this hypothesis
Experimental procedures
Plant materials, growth conditions and stress treatments
Seeds of Japonica rice (Oryza sativa L cv Yukihikari) were surface-sterilized in 70% ethanol for 20 min, further steril-ized in 2.5% sodium hypochlorite solution for 25 min, and then washed several times with sterile water These steril-ized seeds were then soaked in distilled water for 4 days and set for germination in the dark at 20C Germinated seeds were uniformly distributed onto a plastic mesh grid that was supported by a plastic container filled with water
up to the base of the mesh grid Seeds were then grown under continuous illumination in a growth chamber at
25C After growth for 7 days, the seedlings were subjected
to various abiotic stress treatments Chilling treatment was imposed by transferring mesh grids containing seedlings into containers filled with prechilled water at 12C in a growth chamber set at the respective temperature Roots and shoots of the treated seedlings were collected after 0 (control), 1, 2, 4, 6, 10, 24 and 48 h of chilling treatment, immediately frozen in liquid nitrogen, and stored at)80 C for further analysis For NaCl and ABA treatments, 7-day-old rice seedlings were transferred, along with the mesh grid, and placed into solutions containing 150 mm NaCl and 50 lm ABA, respectively For ABA treatments, shoots were also sprayed with ABA solution Drought treatment was imposed by shifting the mesh grid with seedlings (immediately removing free water from roots by blotting on
a paper towel) into a container without water Roots and shoots of the treated seedlings were then collected and stored as mentioned previously
Trang 8RT-PCR and cDNA cloning
Expression analysis of all putative TPP genes was carried
out by RT-PCR Total RNA was extracted from root and
shoot tissues of rice seedlings (O sativa L cv Yukihikari)
using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
The total RNA (1 lg) was reverse-transcribed using the
GeneAmp Gold RNA PCR Reagent Kit (Applied
Biosys-tems, Foster City, CA, USA) with an oligo-dT primer The
following PCR was carried out using gene-specific forward
and reverse primers (listed in Table 2) according to the
protocol supplied with the kit GeneAmp 9700 (Applied
Biosystems) was used for amplification with the following
program: 28 or 35 cycles of 94C for 45 s, 53 C for
45 min, and 72C for 1.5 min, with a final extension at
72C for 5 min The amplified bands were cloned into a
pGEM-T easy vector (Promega, Madison, WI, USA) and
subsequently sequenced
DNA sequencing and analysis
DNA sequencing was carried out using an ABI PRISM 310
Genetic Analyzer (PE Biosystems, Foster City, CA, USA)
The BigDye Terminator v1.1 Cycle Sequencing Kit
(Applied Biosystems) was used for the sequencing reaction
Sequence analysis was performed using genetyx software
(Software Development, Tokyo, Japan) Multiple amino
acid alignments were performed using the online clustal w
alignment program at a website maintained by DDBJ
(http://www.ddbj.nig.ac.jp/search/clustalw-e.html)
Northern blot analyses
Total RNA was isolated from plant tissues using TRIzol
reagent (Invitrogen) Ten micrograms of total RNA was
then denatured in formamide and formaldehyde, separated
on 0.8% agarose gels, and transferred onto Hybond-N+ membranes (GE Healthcare, Piscataway, NJ, USA) The blots were hybridized in Rapid-Hyb Buffer (GE Health-care) at 65C with a 32
P-labeled full-length OsTPP2 frag-ment as a probe The blots were washed twice with wash buffer (2· NaCl ⁄ Cit, 0.1% SDS) for 15 min at 65 C, and then washed twice with another wash buffer (0.2· NaCl ⁄ -Cit, 0.1% SDS) for 15 min at 65C The blots were then exposed to X-ray film for signal detection
Yeast complementation
A Sa cerevisiae tps2 deletion mutant of the YPH499 (MATa his3-D200 leu2-D1 lys2-801 trp1-D1 ade2-101 ura3-52) strain was used as a host cell population for complementation analysis [22] The OsTPP2 ORF region was cloned into the pAUR123 vector (Takara, Kyoto, Japan), which allows constitutive expression of the insert under control of the ADH1 promoter Transformation of
Sa cerevisiae was carried out with the S.c EasyComp Transformation Kit (Invitrogen) The plasmid vectors pAUR123-OsTPP2 and pAUR123 were transformed into both the wild-type and the mutant YPH499 strains These transformants were cultured in YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose) until a D600of 0.5 was reached The collected cells were resuspended in sterilized water, and a series of dilutions (10)1, 10)2, and
10)3) was made Five microliters of each dilution was then dropped onto YPD plates and cultured for 2 days at either
30C or 36 C
Recombinant protein production and purification The ORFs of OsTPP1 and OsTPP2 were PCR-amplified with BamHI and SalI linker sequences from the correspond-ing cDNA clones These PCR products were digested and ligated with a predigested pGEX-6P-3 vector (GE Health-care) E coli BL21 cells were then transformed with the resulting pGEX-OsTPP1 and pGEX-OsTPP2 vectors, respectively The transformant cells were grown overnight in
LB medium containing ampicillin (50 lgÆmL)1), inoculated into 2· YT medium containing ampicillin (50 lgÆmL)1), and cultured at 37C for 3 h Recombinant protein expres-sion was induced with 0.5 mm isopropyl thio-b-d-galacto-side and incubated for another 3 h Pelleted cells were resuspended in 10 mL of NaCl⁄ Pi (pH 7.4, 0.14 m NaCl,
3 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4) and disrup-ted with sonication Lysed samples were centrifuged in an
AR 0/5-24 rotor (MX-300; Tomy, Tokyo, Japan) at
14 000 g for 5 min at 4C Recombinant proteins were purified from the soluble fractions with a glutathione-seph-arose 4B affinity column (GE Healthcare), and then digested with precision protease at 4C The protein samples were separated by SDS⁄ PAGE (12%) and stained with
Coomas-Table 2 Oligonucleotide primers used for RT-PCR analysis.
Gene Orientation Oligonucleotide sequence (5¢- to 3¢)
OsTPP1 (AB120515) Forward TCAGTCATGCCCGGTGGC
Reverse ACACTGAGTGCTTCTTCC OsTPP2 (AB277360) Forward ATGGATTTGAAGACAAGCAAC
Reverse TTAAGTGGATTCCTCCTTCCA OsTPP3 (AP004341) Forward ATGACGAACCACGCCGGC
Reverse CTACTTGCCAATCAGCCCTTT OsTPP4 (AP004119) Forward CTGTTCGTCTCGACGAGT
Reverse TCTTACGGCCTCTACACC OsTPP5 (AL606633) Forward CACGCACCTACACCAAGA
Reverse TGATGGGCCTCTCAGCAT OsTPP6 (AP004658) Forward TCAACGGATGGGTGGAGT
Reverse ACTTGGACACGAGGATGC OsTPP7 (AP005580) Forward CACGACGCTGTTCCCGTA
Reverse TCAACCGTGTCCTGGACA OsTPP8 (AP004727) Forward AGTACGACGCGTGGACGA
Reverse GTGTGCTGCGAAGTCATG OsTPP9 (AC103551) Forward TGCTCTCTCGCTCTCGTT
Reverse AGTGTCACTGTGGTCAGG
Trang 9sie Brilliant Blue The protein concentrations were measured
with a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA,
USA) using IgG as a standard
Assay of TPP activity
Assays for TPP enzyme activity were carried out in reaction
mixtures (100 lL) containing the following components:
2 mm Tre6P, 2 mm MgCl2, 50 mm Tris⁄ HCl buffer
(pH 7.0), and an appropriate amount of enzyme (0.5 lg)
After incubation at 37C for 30 min, these mixtures were
boiled for 4 min to stop the reaction The amount of
treha-lose produced was determined using a Dionex (DX-500)
gradient chromatography system coupled with pulse
amper-ometric detection (Dionex Corporation, Sunnyvale, CA,
USA) The samples were applied to a CarboPac PA1 colum
(Dionex) equilibrated with 0.1 m NaOH, using a flow rate
of 1 mLÆmin)1 A 0.2 m sodium acetate gradient buffered in
0.1 m NaOH was applied over 3–8 min; the sodium acetate
concentration was then increased to 1 m for 2 min before
equilibrating the column again with 0.1 m NaOH Under
these conditions, the retention times of trehalose and Tre6P
were 2.8 min and 12.0 min, respectively
For the analysis of optimum pH, substrate specificity,
and heat tolerance, TPP activity was assayed by
determin-ing released inorganic phosphate levels with BIOMOL
GREEN Reagent (Biomol Research Laboratories,
Plymouth Meeting, PA, USA) Two volumes of this reagent
were added to each terminated enzyme reaction, and then
incubated for 20 min at room temperature The absorbance
of each mixture was determined at 620 nm with a Beckman
DU-65 spectrophotometer (Beckman Instrument, Inc.,
Full-erton, CA, USA) and compared with that of a standard
solution
Substrate specificity
TPP enzymes (0.5 lg) were added to a reaction mixture
(100 lL) containing 50 mm Tris⁄ HCl (pH 7.0), 2 mm
MgCl2, and 2 mm sugar phosphate, and incubated at 37C
for 30 min The sugar phosphate substrates tested were
Glc1P, Glc6P, galactose 6-phosphate, mannose
1-phos-phate, mannose 6-phos1-phos-phate, fructose 1-phos1-phos-phate, fructose
6-phosphate, sucrose 6-phosphate, lactose 1-phosphate and
ribose 5-phosphate All sugar phosphates were purchased
from Sigma Chemical Co (St Louis, MO, USA)
PH optimum
The pH optimum of TPP was determined using two
differ-ent buffer systems, Mes⁄ NaOH (pH 5.5–7.5) and Tris ⁄ HCl
(pH 7.0–9.0) The conditions for the enzyme reaction and
determination of inorganic phosphate levels were as
des-cribed above
Heat stability of TPP The purified proteins were heat-treated at different temper-atures (40–80C) for 0, 1, 2, 3 or 4 min, and then cooled immediately on ice After centrifugation at 20 000 g for
5 min by using MX-300 (Tomy), the supernatants were used to determine residual activity Enzyme reactions and determination of inorganic phosphate levels were carried out as described above
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