The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3′-end and 5′-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.


Introduction
M. pinnata (Pongamia Pinnata) belongs to the semi-mangrove plant which is an intervenient species between halophytes and glycophytes.It is the only mangrove plant in the leguminous family.Although M. pinnata does not have the salty gland that mangrove plants have, it can endure salt stress to a certain degree [1].Therefore, the mechanism of M. pinnata salt-tolerance resembles glycophytes.It will play a vital role in improving salt-tolerance of crop plants by genetic modification, esp.leguminous plants, if the mechanism of M. pinnata salt-tolerance is clarified.Therefore, the analysis on the salt-tolerant transcriptome of M. pinnata has been performed utilizing Illumina sequencing in our lab.A series of genes with changed transcriptional level were observed [2], including those enzymes participating in plant secondary metabolism.Among them, the mRNA level of enzymes involved in flavonoid biosynthesis obtained the most remarkable change.Some of them showed enhanced transcription levels both in leaves and roots, such as phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI), while the mRNA levels of flavanone 3-hydroxylase (F3H) and dihydroflavonol reductase (DFR) declined in roots after sea-water treatment (data not shown).It has been reported that the PAL enzyme is related to plants' disease resistance [3,4]; CHS and CHI are the two key enzymes in plant flavonoid biosynthesis and were confirmed to be associated with UV protection [5][6][7]; while F3H and DFR, locating in the later steps next to CHS and CHI in phenylpropanoid biosynthesis pathway (Figure 1), are mainly responsible for the biosynthesis of anthocyanins which possess of photoprotection function [8].The variations in their mRNA expression imply that CHS and CHI enzymes or their catalyzed flavonoids are closely associated with M. pinnata salt tolerance since both were improved in mRNA expression by salt stress.
Flavonoids have been reported to function in protecting plants against drought stress [9], toxic metal [6,10] and damage caused by UV [5][6][7]11]; however, their roles in plant salt-tolerance have been seldom reported.The responses of plant to salt stress are similar to those reactions to drought stress.Therefore, we would like to know the function of flavonoids in plant salt tolerance and the mechanism of the flavonoid biosynthesis in affecting or regulating plant salt tolerance.Moreover, we want to explore if there are common factors that could co-regulate the expression of enzymes in flavonoid biosynthesis pathway to increase or decrease when plants face salt stress.The CHI enzyme catalyzes the cyclization of chalcone into (2S)-naringenin which is the substrate both for flavones and anthocyanins.Once the biosynthesis of anthocyanins has been repressed, the metabolic flux will tend to the direction of flavone biosynthesis, indicating that flavones may be more vital to salt tolerance than anthocyanins in M. pinnata.Hence, the MpCHI was selected as a starting point to carry out this project.
In total, 12 putative MpCHIs have been annotated in the transcriptome sequencing assay in our lab.Only one of them was salt inducible, esp. in leaf, and chosen for the purpose of conducting this research.In this study, the QRT-PCR method was applied to further confirm that the CHI transcript level in M. pinnata leaves were dramatically improved 4 h after NaCl (500 mM) treatment.The MpCHI full length cDNA was obtained by RACE method and cloned into pYES2 yeast expression vector to generate plasmid pYES2-MpCHI.The pYES2-MpCHI transformed salt-sensitive S. cerevisiae deletion mutant strains: Δnha1 [12] and Δnhx1 [13] increased in tolerance to NaCl (1.2 M) in contrast to the pYES2-vector, without enzyme substrate fed into the culture medium, indicating one possibility that the MpCHI could regulate the response of M. pinnata to salt stress directly through changing its mRNA level or protein level.

The Transcription of MpCHI Is Up Regulated by 500 mM NaCl
To further confirm if the mRNA expression of MpCHI is salt inducible, the QRT-PCR assay was conducted to detect the relative transcription level of MpCHI in salt-treated M. pinnata roots and leaves (Figure 2).It was observed that the MpCHI transcription level was significantly enhanced (p < 0.01, n = 4) in leaves at 4 h (19 fold), 8 h (9 fold) and 12 h (6 fold) after salt-treatment, with the highest MpCHI mRNA amount at 4 h, verifying that the MpCHI cloned in this study is associated with salt-tolerance in M. pinnata.The reason why mRNA quantity decreased at 8 h and 12 h compared to 4 h might be that the M. pinnata plants had gradually adapted themselves to salt stress.This is consistent with the observation on M. pinnata leaf morphology when 20-d young plants were subjected to seawater treatment.The M. pinnata leaves wilted at 2 h and began to recover at 8 h (data not shown).In roots, slightly but significantly (p < 0.01, n = 4) incremental transcription quantity was also observed at 12 h (2 fold) after salt-treatment, indicating the expression of the MpCHI mainly occur in leaves.This is reasonable since the location of CHI in the flavonoid biosynthesis pathway is closed to the anthocyanin production which is mainly formed in plant overground tissues.

The MpCHI Shares High Homology with Leguminous CHIs
In order to verify if the MpCHI cDNA cloned here is correct, the putative MpCHI nucleotide sequence was analyzed using NCBI BLAST.The result indicated that both the MpCHI aminoacid sequence and nucleotide sequence (the open reading frame, ORF) (Figure S1) shares 85% homology with soybean CHI (GenBank: AF276302.1)peptide sequence and ORF nucleotide sequence.Sequence alignment was also carried out to compare the putative MpCHI ORF nucleotide sequence and other 27 plant CHI ORFs by phylogenic tree view (Figure 3), which demonstrated that the MpCHI is a close relative of leguminous plant CHIs.The peptide sequence alignment (Figure 4) showed that the aminoacid residues of substrate-binding site and reactive site in MpCHI enzyme are the same as other leguminous CHIs reported previously [14].

The pYES2-MpCHI Transformed Yeast Mutants Showed Improved Salt-Tolerance
Yeast has been successfully used as a model in exploring the function of plant flavonoid metabolic enzymes [15].When soybean CHIs were expressed in yeast, proteins with CHI enzyme activity were obtained [15].Hence, we utilized yeast to preliminarily probe the relationship between the MpCHI and salt response.Here, two S. cerevisiae salt-sensitive mutants (BY4742, Δnha1 and Δnhx1) were transformed using plasmid pYES2-MpCHI to see the effect of MpCHI on their salt tolerance.Both of the pYES2-MpCHI transformed Δnha1 and Δnhx1 S. cerevisiae strains grew faster (3.5-7.5 fold) than their corresponding pYES2-vector transformed control whether grown on agar plates (Figure 5a) or liquid medium (Figure 5b) containing 1.2 M NaCl.We tried to transform wild-type (WT) yeast (BY4742) using pYES2-MpCHI, while the difference in salt resistance between the pYES2-vector transformed WT yeast control and the pYES2-MpCHI transformed WT yeast is not significant, because the WT yeast itself can resist NaCl-stress to a high concentration, even to 2.0-M NaCl.However, the yeast droplet could not be absorbed onto synthetic complete drop-out (SC) agar plates when the concentration of NaCl in SC medium increased to 2.5 M (data not shown).Hence, we tried to transform salt-sensitive S. cerevisiae deletion mutants, Δnha1 (BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YLR138w::kanMX4), Δnhx1 (BY4742; Mat α; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR456w:kanMX4) and Δhog1 (BY4742; Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YLR113w::kanMX4).Both NHA1 and NHX1 (NHA2) are involved in sodium and potassium efflux through the plasma membrane.They play significant roles in osmotolerance to heavy hypertonic stress [12,13].We observed that the Δnha1 yeast decreased in tolerance to dehydration and osmotic stress [12,16] and Δnhx1 decreased in the tolerance to 1-M NaCl [13].HOG1 is a mitogen-activated protein kinase and participates in osmoregulation by controlling the reallocation of RNA polymerase II in response to osmotic stress [17,18].It was reported that the growth of Δhog1 was arrested in the presence of NaCl (0.4 M) [19].The pYES2-MpCHI transformed Δhog1 did not show higher tolerance to either 400 mM or 800 mM NaCl than the pYES2-vector transformed Δhog1 control and could not even grow on 800 mM NaCl SC agar plates.Hence, the MpCHI could not reverse the effect aroused by HOG1 deletion and partially complement the deficiency in salt tolerance caused by NHA1 and NHX1 deletion (Figure S2), suggesting that MpCHI may has functional homology with NHA1 and NHX1 rather than HOG1.We also isolated a salt-inducible M. pinnata CHS (MpCHS) cDNA and introduced it into the same yeast mutants that MpCHI has transformed (data not shown).However, the MpCHS transformed yeast mutants only showed slightly faster growth than the empty vector transformants, suggesting that the MpCHI perhaps plays a more important role in salt tolerance in M. pinnata than the MpCHS does.
It has been verified that chalcone isomerase is unique in plants [13].Therefore, there are no substrates for CHI in yeast.This assay insinuates that the MpCHI enzyme may participate in regulating the response of yeast to salt stress directly by three possibile manners.Firstly, it may play an osmotic regulation function in yeast transformants; secondly, it might help yeast transformants to reject the entering of salt ions; thirdly, the MpCHI may help to pump out the extra salt ions that have accessed to transgenic yeast.In Arabidopsis, three AtCHIs bearing fatty-acids binding function have been isolated [20].Hence, the MpCHI could perhaps bind the membrane fatty acid of yeast cells and then influence the fluidity of cell membranes, finally either rejecting or pumping out the salt ions.To expound the exact mechanism about how MpCHI affects the salt tolerance in transgenic yeast or M. pinnata, further research is being performed in our lab.

Salt-Treatment on M. pinnata Young Plants
M. pinnata seeds (Shenzhen Garden show park, China) were soaked in tap water at 28 °C in a growth cabinet until radicle appeared.These germinated seeds were then planted in soil for further 20-d growth and then submitted to salt (500-mM NaCl) and tap-water treatment.M. pinnata roots and leaves were sampled at different times (0, 2, 4, 6, 8 h) after treatment.Four plants were treated at each time point.

Real-Time Quantitative Reverse Transcription PCR (QRT-PCR)
Total RNAs were extracted from Salt-treated M. pinnata roots and leaves mentioned above using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the instructions of manufacturer.RNA (2 μg) was used for the first-strand synthesis (20 μL reaction system) after treated by DNase following the instructions of the SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA).The RT product (1 μL) was applied as a PCR template to perform QRT-PCR in the ABI 7300 Real Time PCR System using the SYBR ® Premix Ex Taq™ (TaKaRa, Otsu, Japan) following the instructions of manufacturer with M. pinnata Actin (MpActin) as an internal control.Quadruple reactions were conducted.The same experiment was repeated 3 times and coincident results were obtained.The relative mRNA expression level was calculated and statistically analyzed using delta-delta-Ct method and U-test respectively, with non-treated samples as outer control.Primers CHI-IF (5'-CGGTGGCATCACTCGCCACC-3') and CHI-IR (5'-TGCGGCGGCTTCAGCATCGCC-3') were applied to amplify MpCHI; Primers ActinF (5'-AGAGCAGTTCTTCAGTTGAG-3') and ActinR (5'-TCCTCCAATCCAGACACTAT-3') were used for MpActin amplification.

MpCHI Full Length cDNA Clone
The 3'-end and 5'-end RACE were performed according to the instruction of FirstChoice ® RLM-RACE Kit (Roche).A nested PCR method was carried out to obtain the 3'-end and 5'-end of MpCHI cDNA.The gene-specific primer CHI-OF (5'-GATTACCTCTTCAGCCTCCGGC-3') and the 3'-RACE outer primer provided by the manufacturer were used to do the first-round 3'-RACE PCR, whose product was then diluted 10 times and 1 μL of the diluted PCR product was applied as the template to do the second-round PCR by using the gene specific primer CHI-IF and the 3'-RACE outer primer.Both PCRs were finished under a temperature program of 94 °C, 30 s; 65 °C, 30 s and 72 °C, 90 s; running for 10 cycles; and then another temperature program of 94 °C, 30 s; 58 °C, 30 s and 72 °C, 90 s; running for 20 cycles.The PCR product from the second-round reaction was purified from argrose gel and then cloned into a T-vector (TaKaRa, Otsu, Japan) for DNA sequencing to obtain the 3'-end of MpCHI cDNA.
The Gene-specific primer CHI-OR (5'-CATGGTCTCCAACACTGCCTC-3') and the 5'-RACE outer primer provided by manufacturer were used to do the first-round 5'-RACE PCR, whose product was then diluted 10 times and 1 μL was used as the template to do the second-round PCR by using the gene specific primer CHI-IR and the 5'-RACE outer primer.Both PCRs were finished under a temperature program of 94 °C, 30 s and 72 °C, 120 s; running for 5 cycles; and then 94 °C, 30 s; 65 °C, 30 s and 72 °C, 90 s; running for 10 cycles; after that, a final temperature program of 94 °C, 30 s; 58 °C, 30 s and 72 °C, 90 s; running for 15 cycles.The PCR product from the 2nd-round reaction was diluted 10 times and 1 μL of the diluted PCR product was applied as the template to do the third-round 5'-RACE PCR using gene specific primer CHI-IR2 (5'-GAAGTCGATGGCCTCTA CCAACTGTTC-3') and 5'-RACE outer primer, under a temperature program of 94 °C, 30 s; 65 °C, GAL1 promoter [21,22].Subsequently, the plasmid pYES2-MpCHI was mobilized into two salt-sensitive yeast deletion mutants (Δnha1 and Δnhx1) which were purchased from EUROpean Saccharomyces Cerevisiae ARchive for Functional Analysis (EUROSCARF) using LiAc/SS carrier DNA/PEG yeast transformation method [23].The plasmid pYES2 was also introduced into Δnha1 and Δnhx1 to generate two vector controls.The presence and expression of MpCHI were confirmed by PCR and RT-PCR (Figure S3) respectively using primer WH01 and WH02, with yeast actin (YFL039C) as control.Yeast RNAs were extracted using Yeast RNAiso Kit (TaKaRa, Otsu, Japan) Primers used for amplifying yeast actin are yACT1F (5'-CTACAACGAATTGAGAGTTGCC-3') and yACT1R (5'-AACCAGCGTAAATTGGAACGAC-3').

Salt-Tolerance Assay on the pYES2-MpCHI Transformed Δnha1 and Δnhx1 Yeast Mutants
The pYES2-MpCHI and pYES2 transformed Δnha1 and Δnhx1 S. cerevisiae were firstly cultured in tubes containing 5 mL of SC liquid selection medium without uracil for 30 h at 30 °C, shaking at a speed of 250 rpm/min.One milliliter of each of the above yeast cultures was then transferred into 150-mL flasks with 50 mL of SC liquid medium for further 6 h incubation.The absorption value of OD 600 nm was then measured.Each yeast culture was diluted to OD 600 nm = 0.01 and 5 μL of the diluted culture was dripped onto the SC agar plates (Figure 5a) with or without NaCl (1.2 M) and liquid medium (Figure 5b) containing NaCl (1.2 M) to compare the growth rate, with galactose (2%) as a carbon source and inducing factor in gene expression.Agar plates were photographed 2 days after the salt-treatment assay and yeast cultured in liquid medium were measured the absorption value of OD 600 nm at 28, 32, 35 and 38 h after treatment.

Conclusions
In the present study, we isolated a new 666-bp MpCHI cDNA clone which has not been previously identified.Both its nucleotide and peptide sequence share 85% homology with soybean.The MpCHI protein possessed the substrate binding and reactive site as other leguminous CHIs have, suggesting the accuracy of MpCHI cDNA obtained in this study.Salt-sensitive yeast transformed by the plasmid pYEST2-MpCHI increased in salt tolerance, indicating the possibility that the MpCHI may be involved in M. pinnata salt tolerance in a direct manner.Future works should be carried out to introduce the MpCHI into plants to observe its effect on plant salt tolerance.The co-regulation manner among salt-inducible or salt-repressed enzymes in M. pinnata flavonoid biosynthesis is also an interesting project that we will perform in the future.

Figure 2 .
Figure 2. Quantitative real-time PCR analysis of MpCHI expression under salt stress.Leaf and root samples were collected at 0, 4, 8, and 12 h after 500 mM NaCl treatment.** p < 0.01 by U test.Bars represent SD (n = 4).

Figure 3 .
Figure 3. Phylogeny of plant chalcone isomerase (CHI) genes, based on 27 different plant CHI and M. pinnata CHI (MpCHI, underlined) nucleotide sequences cloned in this study, indicating MpCHI is a close relative of leguminous CHIs, with two soy bean actin cDNAs as control (Bottom G. max NM_001252731.2and G. max NM_001253024.2).The accession numbers for the genes are displayed on the tree.Bottom scale shows nucleotide substitutions.

Figure 4 .
Figure 4. Peptide sequence alignment to compare MpCHI protein with four other leguminous CHIs.Residues of the (2S)-naringenin binding (green) and residues of the active site hydrogen bond network (purple) are highlighted according to previous report.Other conserved residues are marked in blue.