Molecular cloning and characterization of a nonsymbiotichemoglobin gene (GLB1) from Malus hupehensis Rehd.with heterologous expression in tomato

Xingzheng Shi • Xinliang Wang • Futian Peng •

Yu Zhao

Received: 18 September 2011 / Accepted: 16 April 2012 / Published online: 25 April 2012

� Springer Science+Business Media B.V. 2012

Abstract Nonsymbiotic hemoglobins (nsHbs) are

involved in a variety of cellular processes in plants. Previous

studies indicate that nsHb expression improves plant toler-

ance during waterlogging and hypoxia. In the present work,

the nsHb class-1 coding sequence was cloned from Malus

hupehensis Rehd. var. pinyiensis Jiang and subsequently

named MhGLB1. The results elucidated the expressed

characteristics and physiological effects of MhGLB1. The

full-length cDNA contained a 477 bp open reading frame

encoding a protein with a molecular mass of 17.8 KDa with

158 amino acids. Quantitative real-time PCR analysis

showed that MhGLB1 expresses in roots, stems and leaves

growing under normal and nitrate-induced conditions.

Hypoxic stress induced accumulation of MhGLB1 within

12 h, and abscisic acid significantly induced expression of

MhGLB1 in roots. The photosynthetic, transpiration and

stomatal conductance rates of transgenic MhGLB1 tomato

plants decreased more slowly than that of wild-type plants

under waterlogging treatment. These results indicated that

the MhGLB1 gene has an important role in hypoxia.

Keywords Hypoxic stress � Malus hupehensis Rehd �MhGLB1 � Nonsymbiotic hemoglobin

Introduction

Hemoglobins (Hbs) were originally described in animals as

cells that facilitate oxygen transport in blood. Plant Hbs

were first isolated from root nodules of symbiotic nitrogen

(N) -fixing plant species, where they were expected to play

a role in binding and transport of molecular oxygen.

However, since the discovery of expressed hemoglobin

genes in non-nodulating plant species, the research for non-

symbiotic functions of plant Hbs has been ongoing. Three

distinct types of Hbs have been characterized in plants:

symbiotic, nonsymbiotic and truncated. Symbiotic hemo-

globins (sHbs) are found primarily in plant nodules and

their function is to regulate the oxygen supply to N-fixing

bacteria for symbiotic N fixation [1]. Nonsymbiotic

hemoglobin (NsHbs) are not involved in symbiotic N fix-

ation, but have an affinity for oxygen and may serve a

physiological function. These forms of hemoglobin are

believed to exist throughout the plant kingdom. The role of

truncated Hbs remains unclear. However, the most recent

research suggested that truncated Hbs share some charac-

teristics with nsHbs [2].

The nsHbs are divided into class-1 and class-2 Hbs.

Class-1 (nsHb-1s) has a very high affinity for oxygen and is

most likely an evolutionary precursor to symbiotic Hbs [3].

NsHb-1s proteins are relatively conserved across the plant

kingdom, suggesting that members of this class might have

important physiological functions. Class-2 Hbs (nsHb-2s)

have a lower affinity for oxygen and are similar to the sHbs

[4]. Previous studies indicate that nsHb-1s gene expression

is induced by nitrate (NO3), nitrite (NO2), nitric oxide (NO)

[5] and hypoxia [4].

Recently, class-1 nsHb genes have been isolated from

monocots and dicots, including Arabidopsis, Trema,

Parasponia, Solanum, barley, rice, tomato, cotton and

maize [6]. In Arabidopsis, non-symbiotic hemoglobin

controls bolting by scavenging NO, which serves as a floral

transition signal molecule [7]. In barley, only one nsHb

gene exists with strong expression in plant roots grown

X. Shi � X. Wang � F. Peng (&) � Y. Zhao

State Key Laboratory of Corp Biology/College of Horticulture

Science and Engineering, Shandong Agricultural University,

Tai’an 271018, Shandong, People’s Republic of China

e-mail: pft@sdau.edu.cn

123

Mol Biol Rep (2012) 39:8075–8082

DOI 10.1007/s11033-012-1654-4

under hypoxic stress [8]. ZmHb mRNA levels in maize

seedlings are induced by high-salt and osmotic stress in

addition to hypoxic stress [9]. Similarly, two copies of

class-1 nsHb genes, OsGLB1a and OsGLB1b, were detec-

ted in rice with 94 % identity at the nucleotide level. Both

genes were strongly induced in roots by the addition of

NO3, NO2 or NO [5].

NsHb-1s genes have been identified that exhibit physi-

ological functions. For example, it has been shown that

nsHb proteins modulate cellular nitric oxide (NO) levels in

plants and play an important role in abiotic tolerance and

other cellular processes [4]. In the current study, we cloned

a pingyitiancha (Malus hupehensis Rehd. var. pinyiensis

Jiang PYTC) nsHb-1s gene MhGLB1. Quantitative real-

time PCR assays revealed the expression of MhGLB1 was

induced by NO3, hypoxic stress and abscisic acid (ABA).

Furthermore, over-expression of MhGLB1 in transgenic

tomato plants improved plant tolerance to waterlogging.

Materials and methods

Plant material and growth conditions

PYTC seeds were germinated on vermiculite. Seedlings

were grown in vermiculite irrigated with distilled water for

4 weeks in a green house at 24 �C under fluorescent light

and a photoperiod of 16 h (day)/8 h (night). The 4 weeks

old seedlings were transferred to a growth chamber pro-

viding the same conditions as a greenhouse. However the

seedlings were grown hydroponically in aerated Hoa-

gland’s nutrient solution containing 0.8 mM NO3 for

1 week to generate N-deprived seedlings. Following

treatment, the seedlings were removed from the treatment

solution and dried with paper towels. Whole leaves, stems

and roots were excised, poured into liquid N and stored at

-80 �C.

Search for candidate MhGLB1 expressed sequence tags

(ESTs)

The amino acid sequences of each GLB1 in model plants

were used to search the apple (Malus) EST database with

tBLASTn (http://www.ncbi.nlm.nih.gov/BLAST/).

RNA isolation and cDNA synthesis

Total RNA was extracted from 0.1 g of tissue from

2 weeks old seedlings with TRIzol (Invitrogen Inc., USA)

reagent then treated with RNase-free DNaseI at room

temperature for 15 min in reaction buffer containing

20 mM Tris–HCl (pH 8.4), 2 mM MgCl2, and 50 mM

KCl. DNaseI was inactivated by adding EDTA (2.5 mM

final concentration) and heated to 65 �C for 10 min. The

quality and quantity of total RNA were measured using

both electrophoresis and optical absorbency. Complemen-

tary DNAs were synthesized with the SuperScriptIII Kit

(Invitrogen Inc, USA) following the manufacturer’s

instructions.

Polymerase chain reaction (PCR) amplification, plant

expression vector construction and tomato

transformation

Primers were designed for PCR to amplify the PYTC nsHb

gene using sequences at the start and stop codons of Ma-

lus 9 domestica nsHb cDNA (GenBank accession number

AY224132). The primer sequences were sp1 (sense 50-GC

GGATCCATGGAAGGCAAAGTTTTC-30) and sp2 (anti-

sense 50-GCGAGCTCCTAATTAAGGGGAGGCTTCAT-

30). The restriction sites for BamHI and SacI are under-

lined, respectively. Total PYTC cDNA (*0.1 lg) was

used as a template for PCR amplification. PCR reactions

were conducted in the following reaction mixture: 0.4 lM

of each sense and antisense primer, 100 lM of 10 9 PCR

buffer and 2 mM MgCl2 in a final volume of 25 ll. PCR

amplification was carried out for 30 cycles at an annealing

temperature of 55.2 �C using a Mycycler thermacycler

(Bio-Rad, CA, USA). PCR products were detected in a 1 %

agarose gel after staining with ethidium bromide, isolated

from the gel using the GeneClean kit (Sangon, Shanghai,

China), and cloned into the pMD18-T cloning vector

(TaKaRa) following the manufacturer’s instructions,

sequenced to confirm identity with known GLB genes.

Following excision with the restriction enzymes BamHI

and SacI, the products were inserted into the plant

expression vector PBI121 (Clontech, Palo Alto, CA)

behind the 35S cauliflower mosaic virus (CaMV 35S)

promoter. The binary vector containing the GLB1 cDNA

construct was introduced into Agrobacterium tumefaciens

LBA4404 [10], which was subsequently used for tomato

transformation (Lycopersicon esculentum wild type cv.

Sy12f) [11]. Regenerated shoots with kanamycin resistance

were selected in MS medium [12] supplemented with

50 mg L-1 kanamycin, 3 mg L-1 6-benzyladenine (6-BA)

and 0.2 mg l-1 indoleacetic acid (IAA). Shoots were roo-

ted in 1/2MS medium supplemented with 0.3 mg l-1

indoleacetic acid (IAA). The REDExtract-N-Amp plant

PCR Kit (Sigma-Aldrich, Poole, Dorset, UK) was used to

confirm the kanamycin-resistant plantlets (T0) with sp1 and

sp2 for the transgenic tomato. The PCR-positive plantlets

were transplanted into soil. After 4 weeks of growth, the

plants were collected for quantitative real-time PCR anal-

ysis. The transgenic lines were selected by T1 seedling

segregation analysis following germination on kanamycin

medium. The kanamycin-resistant T1 seedlings were grown

8076 Mol Biol Rep (2012) 39:8075–8082

123

to maturity to collect T2 seeds. The transgenic lines were

selected by T2 seedling segregation analysis following

germination on kanamycin medium. Total RNA was pre-

pared from leaves of 4 week old T2 plants for quantitative

real-time PCR analysis. The methods of RNA isolation and

cDNA synthesis have been described in the previous par-

agraph. T1 plants were also self-pollinated to obtain

homozygous plants, and T2 transgenic plants from T1

independent lines were analyzed.

Wild-type (WT) and transgenic tomato plants were

grown in the greenhouse under natural light supplemented

by sodium vapor lamps with a photoperiod of 14/10 h

(light/dark). Average day/night temperatures were

approximately 28/18 �C. The transgenic tomato plants

were transplanted into plastic pots and placed on plastic

trays in the greenhouse at 25 �C and a 14/10 h light/dark

cycle. Simulated flooding treatments were applied by

adding tap water to the plastic trays. The tap water for

waterlogging was equilibrated in the growing chamber at

25 �C for 24 h and the water level was maintained at the

pot soil surface. The waterlogging treatments were con-

ducted in both the transgenic lines and the controls. The

portable photosynthesis system CI-340 was used to mea-

sure the photosynthetic rate, stomatal conductance and

transpiration rate of the transgenic and control plants.

Quantitative real-time PCR

We examined the response of MhGLB1 in the presence of

NO3, hypoxia and ABA. Plants were N-deprived for 48 h

for the NO3 experiments, and subsequently transferred to

fresh medium supplemented with 10 mM KNO3 and

allowed to grow under the same conditions for 2, 6 and 8 h.

Seedlings grown in Hoagland’s nutrient solution containing

10 mM KCl served as controls. For ABA experiments,

seedlings were pretreated with 200 lM the NO scavenger

2-(4-carboxypheny)-4, 4, 5, 5,-tetramethilimidazoline-1-

oxyl-3-oxide (c-PTIO) and distilled water for 4 h, then

treated with 100 lM ABA for 4 h. The NO Detection Kit

(nitrate reductase) from Nanjing Jiancheng Bioengineering

Institute measured NO content. Seedlings were grown in

distilled water as controls. Seedlings were grown under

anaerobic conditions for 12 and 24 h for the hypoxia

experiments. Aerated seedlings served as controls. At

harvest, plants were separated into roots, stems and leaves.

Specific gene primers were designed from the MhGLB1

cDNA sequence and analyzed using the Primer 5.0 soft-

ware (PE Applied Biosystems) following the manufac-

turer’s guidelines. The r18s gene was used as the internal

constitutively expressed control (house-keeping gene).

Primers (reverse-R and forward-F) and [50] 6-FAM, [30]TAMRA-labeled probes (-P) listed in Table 1 were syn-

thesized by Invitrogen and used at a 200 nM final

concentration. PCR reactions were performed in a 25 ll

final volume according to the manufacturer’s protocol and

three PCR replicates were assayed for each sample. Real-

time quantitative PCR was conducted on a FTC2000

machine. PCR cycles were as follows: 1 cycle of 2 min at

50 �C; 10 min at 95 �C; followed by 40 cycles each of 15 s

at 95 �C, and 1 min at 60 �C. Control reactions were per-

formed without a template. Relative gene expression was

calculated according to the relative method (DCT) using

r18s as a constitutively expressed gene. Mean values of

2-DCT (DCT = CT(gene of interest) - CT(r18s)) were calcu-

lated from three independent experiments. PCR efficiencies

of MhGLB1, LeGLB1, Mhr18S and Ler18S were 98.12,

99.01, 98.30 and 98.57 %, respectively. In order to ensure

the accuracy of the experiment, intraassay variation was

\1.20 % and interassay variation \1.10 %.

Results

Full length MhGLB1 isolation and sequence analysis

We designed PCR primers from Malus cDNA to charac-

terize nsHb named MhGLB1. The full-length 477 bp PYTC

cDNA template, encoding a protein of 158 amino acids and

a mass of 17.8 KDa was amplified. The deduced nsHb

amino acid sequence exhibited a respective 95.57, 80.12,

82.82 and 79.50 % identity to pear, alfalfa, cotton and

soybean, and lower sequence identities of 65.76, 67.65,

71.17 and 71.69 % to orange, rice, wheat and maize,

Table 1 Primer and [50] 6-FAM, [30] TAMRA-labelled probe

sequences used in real-time quantitative PCR assays of genes

MhGLB1 and r18S. Accession numbers are given in parenthesis

Primer Sequence

MhGLB1 (EF690362)

GLB1-F: 50-CGCATTGTTGGAAACCATAAAG-30

GLB1-R: 50-TCATAAGCTTCTCCCCATGCA-30

GLB1-P: 50-AGGCCTTACCGGAAATGTGGTCA-30

Mhr18S (DQ341382)

Mhr18 s-F: 50-AAACGGCTACCACATCCA-30

Mhr18 s-R: 50-CACCAGACTTGCCCTCCA-30

Mhr18 s-P: 50-AGCAGGCGCGCAAATTACC-30

LeGLB1(AY026343)

LeGLB1-F: 50-GGTGTGGTTGATGAGCACTTTGA-30

LeGLB1-R: 50-AGGCCTCTCCCCATGCATTCT-30

LeGLB1-P: 50-CAAAATATGCCTTGTTGGAGA-30

Ler18S (X51576)

Ler18s-F: 50-GCCCGGGTAATCTTTGAAAT-30

Ler18s-R: 50-AGTAAGCGCGAGTCATCAGC-30

Ler18s-P: 50-CGGATCATTCAATCGGTAGG-30

Mol Biol Rep (2012) 39:8075–8082 8077

123

respectively (Table 2). We performed a phylogenetic

reconstruction to elucidate the relationships of nsHb to

nsHbs of other plants. The phylogenetic tree (Fig. 1) shows

MhGLB1 forms a cluster with pear (Pyrus communis)

GLB1 (AY224133) and grape GLB1 (XM_002284648)

genes and appears to be more closely related to alfalfa

GLB1 (Q9FVL0) and alder GLB1 (AB221344) genes.

MhGLB1 spatial expression patterns

The levels of MhGLB1 expression in different tissues was

evaluated by preparing total RNA from various tissues of

2 week old seedlings. The r18s gene was used as an

internal standard [13]. Quantitative real-time PCR analysis

revealed that higher levels of MhGLB1 transcripts were

expressed in roots relative to stems and leaves (Fig. 2).

NO3 induced MhGLB1 expression in various tissues

The rapid transient accumulation of MhGLB1 transcripts in

response to NO3 was assessed by monitoring transcriptional

levels in different tissues by quantitative real-time PCR

analysis during induction by NO3. When NO3 was resup-

plied to N-deprived seedlings, MhGLB1 transcripts were

rapidly induced in roots and peaked after 2 h of treatment.

Subsequently, accumulation decreased. MhGLB1 induction

in stems and leaves occurred more slowly relative to roots.

Control plants treated with 10 mM KCl exhibited nearly

consistent MhGLB1 levels from 0 to 12 h (Fig. 3).

MhGLB1 expression under ABA treatment

ABA treatment increased MhGLB1 mRNA and NO con-

tent. The amount of MhGLB1 mRNA accumulated under

ABA treatment was 1.6-fold and 1.2-fold higher than that

under c-PTIO pretreatment and the control, respectively.

However, no significant difference was found between the

c-PTIO pretreatment and the control (Fig. 4). These results

indicated that NO might activate the effects of ABA on

MhGLB1.

Table 2 Comparison of the deduced amino acid sequences of nsHb

from Malus hupehensis with the known related protein sequences

Species Sequence

length

Sequence identity

(%)

Accession

numbers

Pingyitiancha 158 100 GQ423619

Grape 168 76.94 XM_002284648

Pear 158 95.57 AY224133

Orange 183 65.76 AY026338

Arabidosis 160 76.40 NM_127165

Tomato 152 75.32 AY026343

Potato 152 74.68 AY151389

Alfalfa 160 80.12 Q9FVL0

Barley 162 73.01 Q42831

Wheat 162 71.17 AY151390

Maize 152 71.69 NM_001111496

Rice 169 67.65 NM_001055972

Cotton 163 82.82 AY899302

Soybean 161 79.50 U47143

Fig. 1 A phylogenetic tree for the conserved region of non-symbiotic

hemoglobins. The tree was made with the ClustalW program.

(http://align.genome.jp/clustalw) Genbank accession numbers:

Malus hupehensis, GQ423619; Grape, XM_002284648; Pear,

AY224133; Orange, AY026338; Arabidosis, NM_127165; Tomato,

AY026343; Potato, AY151389; Alfalfa, Q9FVL0; Barley, Q42831;

Wheat, AY151390; Maize, NM_001111496; Rice, NM_001055972;

Cotton, AY899302; Soybean, U47143; Radish, AY286331; Crowtoe,

AB238220; Alder, AB221344

0

0.2

0.4

0.6

0.8

1

Root Stem Leaf

Rel

ativ

e ex

pres

sion

rat

io

Fig. 2 Expression levels of MhGLB1 in different tissues of PYTC

seedlings. Total RNA prepared from roots, stems and mature leaves

2-week-old seedlings grown hydroponically with Hoagland’s nutrient

solution was subjected to quantitative real-time PCR. The experi-

ments were repeated three times

8078 Mol Biol Rep (2012) 39:8075–8082

123

Expression of MhGLB1 under hypoxic stress

The effects of hypoxia on MhGLB1 mRNA were also

evaluated by subjecting seedlings to an anaerobic envi-

ronment. MhGLB1 mRNA accumulation was induced

within 12 h, with a subsequent decrease in MhGLB1

mRNA levels (Fig. 5).

Transgenic MhGLB1 tomato plants have higher

waterlogging tolerance than wild type

MhGLB1 transcripts in transgenic lines were detected by

quantitative real-time PCR analysis of young tissues and

signals were obvious in transgenic lines, including T2-1

and T2-5 (Fig. 6). Both transgenic and WT tomato plants

were treated under waterlogging for 2, 24, 48, and 96 h.

The photosynthetic rate, stomatal conductance and tran-

spiration rate were measured. The results indicated that

compared with the WT plants, the photosynthetic rate of

transgenic plants decreased slowly under treatment. When

treated for 24 h, the photosynthetic rate, stomatal conduc-

tance and transpiration rate of WT plants decreased 86,

86.8 and 90.7 %, respectively (Fig. 7a, b, c), while that of

Stem

0

0.2

0.4

0.6

0.8

1

Hours of NO 3¯ treatment (h)

b

Root

0

2

4

6

8

Hours of NO3¯ treatment (h)

a

Leaf

0

2

4

6

8

10

0 2 4 6 8 10 12

0 2 4 6 8 10 12

0 2 4 6 8 10 12

Hours of NO3¯ treatment (h)

NO3¯

Control

c

Rel

ativ

e ex

pres

sion

ratio

Rel

ativ

e ex

pres

sion

ratio

Rel

ativ

e ex

pres

sion

ratio

Fig. 3 Expression patterns of MhGLB1 in different tissues in

response to NO3. a Expression of MhGLB1 in roots of seedlings

in response to NO3. b Expression of MhGLB1 in leaves of seedlings in

response to NO3. c Expression of MhGLB1 in stems of seedlings in

response to NO3. The seedlings were treated with 10 mM KNO3 for

2, 6 or 12 h, and the control plants were treated with 10 mM KCl. The

experiments were repeated three times

b

0

0.02

0.04

0.06

0.08

Con

tent

of N

O(µ

mol

g-1

FW

)

a

0

0.2

0.4

0.6

0.8

1

Control c-PTIO+ABA ABA

Control c-PTIO+ABA ABA

Rel

ativ

e ex

pres

sion

rat

io

Fig. 4 The effect of ABA on the expression level of MhGLB1 and

NO content. a Expression of MhGLB1 in roots of PYTC seedlings in

response to ABA. b The content of NO in roots of PYTC seedlings in

response to ABA

0

0.5

1

1.5

2

2.5

0 12 24

Hours of treatment (h)

Rel

ativ

e ex

pres

sion

rat

io

Normoxia

Hypoxia

Fig. 5 The effect of hypoxic stress on expression level of MhGLB1.

Total RNA was isolated from roots. The experiments were repeated

three times

Mol Biol Rep (2012) 39:8075–8082 8079

123

transgenic plants decreased 40.1, 72.5 and 78.7 %,

respectively (Fig. 7a, b, c).

Discussion

Previously, sHbs had only been identified in animals.

However, Hbs have been characterized from all kingdoms

of life and known to function as oxygen carriers, oxygen

sensors, and in oxygen storage; in NO detoxification, and

in peroxidase activity. NsHbs encoding genes in plants

have been isolated, but few GLB1s in woody plants have

been detected. PYTC is an important rootstock for apple

production in China and exhibits many valuable properties.

To obtain an increased understanding of MhGLB1

expression, we identified and isolated nsHbs cDNA, called

MhGLB1, from young roots of PYTC. The phylogenetic

tree showed MhGLB1 has nearest homologous relationship

with pear GLB1. It will be as the PYTC nsHb-1s hemo-

globin encoding gene.

Our study detected MhGLB1 mRNA in roots, leaves and

stems, with the highest gene expression in root tissue

(Fig. 2). In Arabidopsis, the nsHb-1s are active in germi-

nating seedlings and can be induced by hypoxia and by

increased sucrose addition [14]. Barley shows nsHb-1s

expressed in the aleurone layers and roots subjected to low

oxygen stress [8], and expression is not regulated directly

by oxygen availability, but by ATP or some consequence

of ATP action [15]. Under normal growth conditions in

rice, nsHb-1s and -2s are expressed in leaves, however only

nsHb-1s are expressed in roots [16]. Our results and pre-

vious research together suggest that this gene is expressed

in different tissues (i.e. roots, stems and leaves), but

exhibits tissue-specific expression in other plant species.

In angiosperms, NO3 is not only a nutrient but also

serves as a signal for regulating gene expression, such as

glutamine synthetase (GLN) and asparagine synthetase

(ASN) [17]. Rapid induction of MhGLB1 transcripts in

roots within 2 h following addition of NO3 and down-

regulation after 6 h (Fig. 3a), suggests NO3 sensitivity to

the regulatory mechanisms of MhGLB1 expression. How-

ever, decreased MhGLB1 induction and subsequent accu-

mulation was observed in stems and leaves relative to root

tissues (Fig. 3b, c). We hypothesize a signal transition

process from roots to stems to leaves operates throughout

induction. Two nsHb-1s genes, ORYsaGLB1a and ORY-

saGLB1b, were strongly induced by NO3. NO3 began to

induce accumulation of both ORYsaGLB1a and ORY-

saGLB1b transcripts after 1–2 h of treatment, with amounts

peaking at 4–8 h, followed by a decrease in accumulation.

In this study, our results together with previous studies

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

WT T2-1 T2-5

Rel

ativ

e ex

pres

sion

rat

io

MhGLB1

LeGLB1

Fig. 6 Quantitative real-time PCR analysis expression of MhGLB1 in

transgenic tomato plants. LeGLB1, the GLB1 gene of tomato.

Quantitative real-time PCR analysis was performed using total

RNA from the young leaves of the plants. The experiments were

repeated three times

c

0

1

2

3

4

Hours of treatment (h)

Tra

nspi

ratio

n ra

te

(mm

ol m

-2 s

-1) T2-1

T2-5

WT

0

02468

101214161820

Hours of treatment (h)

Pho

tosy

nthe

tic R

ate

µ mol

CO

2 m

-2 s

-1

a

b

0

20

40

60

80

100

0 20 40 60 80 100

20 40 60 80 100

0 20 40 60 80 100

Hours of treatment (h)

Sto

mat

al c

ondu

ctan

ce

(mm

ol H

2O m

-2 s

-1)

-2

Fig. 7 Changes in leaf photosynthesis rates, stomatal conductance

and transpiration rate in transgenic tomato plants and WT under

waterlogging treatment. a Change of leaf photosynthesis rate in

transgenic tomato and WT plants. b Change of stomatal conductance

in transgenic tomato and WT plants. c Change of transpiration rate in

transgenic tomato and WT plants

8080 Mol Biol Rep (2012) 39:8075–8082

123

suggest that GLB1 gene could respond to NO3 signals at the

transcriptional level in some species.

In this study, MhGLB1 mRNA accumulated under ABA

treatment and was higher than the c-PTIO pretreatment and

control. Significant differences were not detected between

c-PTIO pretreatment and the control. These results indi-

cated that the effects of ABA on MhGLB1 maybe activated

by NO (Fig. 4).

The accumulation of MhGLB1 mRNA was detected

within 12 h, and subsequently declined under hypoxic

stress. GLB1 mRNA gene expression is induced in isolated

barley aleurone layers exposed to anaerobic conditions, and

in the roots of flood-stressed barley plants [8]. These results

indicated that MhGLB1 gene could respond to hypoxic

stress, and it maybe play an important role in protecting

plants from hypoxic stress.

Overexpression of a candidate gene in a model plant

species, such as Arabidopsis and tomato, may provide

important information for understanding gene function. In

this research, we introduced MhGLB1 under the control of

the 35S CaMV promoter into tomato plants. Two inde-

pendent kanamycin-resistant transformants were identified

by quantitative real-time PCR.

In the present study, compared with wild-type tomato,

MhGLB1 overexpression enhanced tomato tolerance under

waterlogging. The photosynthetic rate of transgenic plants

decreased slowly, whereas the wild-type photosynthetic

rate declined significantly (Fig. 7a). Stomatal conductance

and transpiration rate for wild-type plants declined by a

large margin, and a slower decrease was observed in

transgenic plants (Fig. 7b, c). Overexpression of a GLB1

protects Arabidopsis thaliana from the effects of severe

hypoxia [18]. Increased tolerance of transgenic plants

under waterlogging was supported by hemoglobin but

regulated by NO levels. Previous studies have demon-

strated that NO is formed during hypoxia in alfalfa root

cultures and the levels of NO detected were inversely

related to the levels of nsHb-1s expression in tissues [4].

During the last 2 years, an increasing number of reports

have implicated nsHbs as the key enzymatic system for NO

scavenging in plants, indicating that the function of

hemoglobin may well be to protect against nitrosative

stress and to modulate NO signaling functions [19]. In

transgenic alfalfa root cultures, extracts from lines over-

expressing hemoglobin had approximately twice the NO

conversion rate of either control or antisense lines under

normoxic conditions. Only the control line showed a sig-

nificant increase in the rate of NO degradation when placed

under anaerobic conditions [20]. Nonsymbiotic hemoglo-

bin AHB1 was shown to scavenge NO through production

of S-nitrosohemoglobin and reduce NO emission under

hypoxic stress in Arabidopsis thaliana [21]. Our data in

conjunction with other studies has led us to conclude that

the MhGLB1 gene has an important role in hypoxia. Future

studies should obtain the overexpression or antisense

MhGLB1 gene in PYTC plants to confirm these results.

This approach will precisely clarify the roles of the

MhGLB1 gene and serve to elucidate the potential appli-

cation of MhGLB1 in enhancing plant stress tolerance.

Acknowledgments This work was supported by the Earmarked

Fund for Modern Agro-industry Technology Research System.

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