A rice LSD1-like-type ZFP gene OsLOL5 enhances saline-alkaline tolerance in transgenic Arabidopsis thaliana, yeast and rice

Plant materials and stress treatment

A. thaliana (ecotype: Columbia) seeds for SA treatment were provided by the Environmental Research
Center of Northeast Forestry University, Harbin City, Heilongjiang Province, China.
aast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin City,
Heilongjiang province, China. Nine-day-old rice seedlings were used for 150 mmol L
^-1
NaCl, 30 mmol L
^-1
NaHCO
₃
, and 5 mmol L
^-1
H
₂
O
₂
stress treatments. Leaf and root samples were collected after treatment and immediately
frozen in quid nitrogen. RNA was extracted using an RNeasy Plant Mini Kit (Qiagen,
Dusseldorf, Germany) and then stored at –80 °C in the Northeast Institute of Geography
and Agroecology, Chinese Academy of Sciences, Harbin City, Heilongjiang Province,
China. Each stress treatment was repeated six times.

Cloning of OsLOL5 gene

The full-length OsLOL5 cDNA sequence was obtained by RT-PCR using primer pair OsLOL5-P1, which was designed
based on the gene sequence in GenBank (AJ620677, http://www.ncbi.nlm.nih.gov/nuccore/40809630?report=genbank). The total RNA was isolated from four leaves from rice seedlings of LJ11 using Trizol
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand
cDNA was synthesized using a SuperScript^III
reverse transcriptase kit. The specific primer pair OsLOL5-P1 was designed with Primer
Premier 5.0 (Premier Biosoft, Palo Alto, USA) and used for full-length ampfication
of the gene, which was cloned into the pMD18-T(Takara Biotechnology in DAAN) vector
and confirmed through sequencing (Invitrogen, Shanghai, China).

Subcellular localization of OsLOL5 by transient expression in onion epidermal tissue

To determine the subcellular location of the OsLOL5 protein, the PCR product generated
by primer pair OsLOL5-P2 was used to construct the PBI121::OsLOL5::GFP expression vector (Fig. 1). The PBI121-OsLOL5::GFP fusion plasmids were coated onto 20 ml of 50 mg?·?mL
^?1
gold particles with 2.5 M CaCl
₂
and 0.1 mol L
^-1
spermidine and mixed rigorously using a vortex for 2 min. Plasmid-coated particles
were dehydrated using 75 and 95 % ethanol prior to bombardment. Single-layer epidermal
sheaths peeled from onion bulbs were placed on 1/2 MS plates and subjected to particle
bombardment using the standard procedure provided by the manufacturer. Plasmid-coated
gold particles were accelerated with a helium burst at 1100 psi in a PDS-1000/He instrument
(Bio-Rad, Hercules, California, USA). Plates containing transfected onion tissues
were wrapped in foil and incubated in the dark overnight (16–20 h) at room temperature
20].

Fig. 1. Schematic of the expression vectors PBI121::OsLOL5::GFP, pCXSN::OsLOL5, and pYES2::OsLOL5. 35S Promoter, Cauliflower Mosaic Virus 35S Promoter; Tnos, nos-terminator; PGAL1, Galactokinase promoter; CYC1 TT, CYC1 terminator; and GFP, green fluorescent protein

OsLOL5 gene expression analysis

Total RNA was extracted from roots and leaves after treatment of the seedlings with
150 mmol L
^-1
NaCl, 30 mmol L
^-1
NaHCO
₃
, or 5 mmol L
^-1
H
₂
O
₂
after different periods of time (0 h as control group, 12, 24, and 48 h), respectively.
First-strand cDNA was synthesized from 1 ?g of total RNA with SuperScript®III reverse
transcriptase and oligo-dT primers (Invitrogen, Shanghai, China) according to the
manufacturer’s instructions. cDNA was diluted with nuclease-free water to 1:10. Aliquots
of the same cDNA sample were used for real-time PCR with primer pair OsLOL5–P3. The OsActin1 gene was used as an internal control. PCR was performed in a 25 ?L reaction mixture
containing 400 nmol L
^-1
of each primer, 1?×?SYBR Green qPCR master mix (Agilent Technologies, Santa Clara,
CA, USA), and approximately 30 ng of cDNA. Real-time PCR was performed on the Agilent
Stratagene MxPro-Mx3000p (Agilent Technologies, Santa Clara, CA, USA) using the following
procedure: 30 s at 95 °C for denaturation, followed by 40 cycles of 5 s at 94 °C,
30 s at 60 °C, and 40s at 72 °C. Relative transcript abundance was calculated according
to the manufacturer’s instructions. The specificity of each primer pair was verified
by determining the melting curves at the end of each run, and sequencing the amplified
bands from gel electrophoresis.

Analysis of sensitivity to abiotic stress in OsLOL5 transgenic yeast

To detect OsLOL5 response to NaCl, NaHCO
₃
, and H
₂
O
₂
, OsLOL5 was amplified using primer pair OsLOL5-P4, and the PCR product was cloned into pMD18-T.
OsLOL5 was cut from pMD18-T with restriction endonucleases KpnI and SphI, and ligated
into the yeast expression vector pYES2. Using the LiAc method, the plasimds pYES2 and pYES2::OsLOL5 (Fig. 1) were transformed into the Saccharomyces cerevisiae strain INVSc1.Transformed yeast strains were grown in synthetic defined medium minus
the appropriate amino acids (SD-Ura) for selective growth for the expression plasmids.
To analyze abiotic stress tolerances, the pYES2- and pYES2::OsLOL5-transformed cell cultures were adjusted to an OD
₆₀₀
of 0.6 using yeast extract-peptone-dextrose (YPD) medium. Ten-fold serial dilutions
of yeast strains were prepared, and 5-?l aliquots of each dilution were spotted on
solid YPD medium containing NaCl (0 mol L
^-1
as control group, 0.8, and 1 mol L
^-1
), NaHCO
₃
(30, 32, and 40 mmol L
^-1
), or H
₂
O
₂
(3, 3.2, and 3.4 mmol L
^-1
). All of the plates were incubated at 30 °C for 3–6 days.

Functional analysis of OsLOL5 in Arabidopsis

Using primer pair OsLOL5-P4, the OsLOL5 PCR product was ligated into the expression vector pCXSN after XcmI digestion 21]. For Arabidopsis transformation, the pCXSN::OsLOL5 (Fig. 1) vectors were first introduced into Agrobacterium tumefaciens GV3101 by electroporation. Arabidopsis cv Col-0 plants were transformed via floral dip method as previously described 22]. Arabidopsis transgenic seeds were plated on half-strength Murashige and Skoog (MS) medium containing
25 mg?·?L
^?1
hygromycin for selection. Resistant plants were used for molecular identification.
To study the function of OsLOL5 in the abiotic stress response, the transgenic T3
generation encoding OsLOL5 driven by the cauliflower mosaic virus (CaMV35S) promoter were tested with primer
pair OsLOL5-P5 and then used for the following studies. The T3 (#1–#3) and WT seeds
were sterilized and sowed in 1/2 MS medium for germination for 10 d. The seedlings
were then transferred to 1/2 MS medium containing 0 (0 mmol L
^-1
as control group), 100, 125, or 150 mmol L
^-1
NaCl or 0, 2, 4, and 6 mmol L
^-1
NaHCO
₃
. After 30 d, the growth phenotype, plant height, fresh weight, and MDA content of
the seedlings were measured.

Alkaline stress tolerance analysis of OsLOL5 in rice

For rice transformation, the pCXSN::OsLOL5 vectors were transferred into A. tumefaciens EHA105 through electroporation. OsLOL5 was transformed into O. sativa L. ssp. japonica cv. “Longjing 11” by using the Agrobacterium-mediated co-cultivation method. The transgenic T2 generation encoding OsLOL5 driven
by the cauliflower mosaic virus (CaMV35S) promoter were tested with primer pair OsLOL5-P5.
OsOLO5 expression in transgenic plants was confirmed by using Northern blot. Three
independent T2 homologous transgenic lines and the control Longjing 11 were used for
alkaline stress tolerance experiments. For alkaline treatment, concurrent buds were
transferred to the stress liquid culture medium containing 0 (0 mmol L
^-1
as control group), 5, 7.5, and 10 mmol L
^-1
NaHCO
₃
. After 21 d, the growth phenotype, root length, fresh weight, and MDA content of
seedlings were measured. Simultaneously, the expression level of oxidative stress
response genes OsAPX2 (AB053297), OsCAT (AB020502), OsCu/Zn-SOD (AK059841), and OsRGRC2 (AY136765) were analyzed. Procedures for RNA extraction and real-time PCR were similar
to those described and listed in Table 1.

Table 1. The primers used in gene clone and qRT-PCR

MDA content measurements

MDA content was determined using the previously described thiobarbituric acid reaction
23]. Absorbance levels at 532 and 600 nm were determined using a spectrophotometer. After
subtracting the non-specific absorbance at 600 nm, MDA concentration was determined
using its extinction coefficient 155 mM
^?1
?·?cm
^?1
.

Data analysis

Analysis of variance (AVONA) and multiple comparison by software data processing system
(DPS) (version 7.05).