Proline accumulation and transcriptional regulation of proline by huanghengdong

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       reports



    Proline accumulation and transcriptional regulation of proline
    biothesynthesis and degradation in Brassica napus
    Xingning Xue, Aihua Liu & Xuejun Hua*
    Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, China



    To understand the molecular mechanism underlying proline ac-           clizes spontaneously into pyrroline-5-carboxylate (P5C), which
                                                        1
    cumulation in Brassica napus, cDNAs encoding Δ -pyrroline-5-           is further reduced to proline by P5C reductase (P5CR). Proline
    carboxylate synthetase (BnP5CS), ornithine δ-aminotransferase          degradation is the reverse process of proline biosynthesis cata-
    (BnOAT) and proline dehydrogenase (BnPDH) were isolated and            lyzed by two mitochondrial enzymes, proline dehydrogenase
    characterized. Southern blot analysis of BnP5CSs in B. napus and       (PDH) and P5C dehydrogenase (P5CDH).
    its diploid ancestors suggested a gene loss may have occurred             Although stress-induced proline accumulation is evolutio-
    during evolution. The expression of BnP5CS1 and BnP5CS2 was            narily conserved in a wide range of plants, its regulatory mech-
    induced, while the expression of BnPDH was inhibited under             anism is subject to considerable variation (6). In most plant spe-
    salt stress, ABA treatment and dehydration, prior to proline           cies studied, proline accumulation during stress is the result of
    accumulation. The upregulation of BnOAT expression was only            reciprocal action of increased biosynthesis and inhibited degra-
    detected during prolonged severe osmotic stress. Our results in-       dation (6). However, in tomato, dramatic elevation of free pro-
    dicate that stress-induced proline accumulation in B. napus re-        line content during salt stress is not correlated with the ex-
    sults from the reciprocal action of activated biosynthesis and in-     pression of either of P5CS genes (7). In Medicago sativa, pro-
    hibited proline degradation. Whether the ornithine pathway is          line accumulation under salt stress results from the decreased
    activated depends on the severity of stress. During development,       expression of two PDHs rather than the increased expression of
    proline content was high in reproductive organs and was accom-         P5CS (8). Furthermore, the disparate glutamate and ornithine
    panied by markedly high expression of BnP5CS and BnPDH,                pathways contribute differently to stress-induced proline accu-
    suggesting possible roles of proline during flower development.        mulation in different plants. In Vigna aconitifolia, the glutamate
    [BMB reports 2009; 42(1): 28-34]                                       pathway is dominant under salt stress (9). In Arabidopsis thali-
                                                                           ana, the ornithine pathway works together with the glutamate
                                                                           pathway to promote proline accumulation in young plantlets
    INTRODUCTION                                                           during salt stress, whereas only the glutamate pathway is acti-
                                                                           vated in adult plants (10). In Medicago truncatula, both path-
    Proline accumulation is one of the most widespread metabolic           ways seem to contribute to the salt-induced proline accumu-
    responses of plants to osmotic stress (1), and is thought to play      lation whatever the development stage (11).
    positive roles under stressful conditions such as a component             Brasscia napus, an amphidiploid species, accumulates pro-
    of antioxidative defense system (2), stabilizer of subcellular         line under osmotic stress (12). In addition, proline accumulation
    structures and macromolecular (3), regulator of cellular redox         in canola leaf disks is well documented (13). To understand the
    potential (4), or component of signal transduction pathways            molecular mechanism underlying proline accumulation in B.
    that regulate stress responsive genes (5).                             napus, we have isolated and characterized the cDNAs of
       In plants, proline can be derived from two different pre-           BnP5CS, BnOAT and BnPDH. The relationship between proline
    cursors: glutamate and ornithine. Glutamate is reduced to glu-         accumulation and the transcript level of BnP5CS, BnOAT and
                                                                1
    tamic-γ-semialdehyde (GSA) catalyzed by bifunctional Δ -py-            BnPDH in both seedlings and adult plants was studied.
    rroline-5-carboxylate synthetase (P5CS), while ornithine is con-
    verted to GSA by ornithine δ-aminotransferase (OAT). GSA cy-           RESULTS AND DISCUSSION

                                                                           Isolation and characterization of BnP5CS1, BnP5CS2,
    *Corresponding author. Tel: 86-10-62836251; Fax: 86-10-62836251;       BnOAT and BnPDH
    E-mail: xjhua@ibcas.ac.cn                                              The isolation of BnP5CS, BnOAT and BnPDH cDNAs was car-
                                                                           ried out as described in Materials and Methods. The 2551 bp
    Received 11 April 2008, Accepted 17 July 2008
                                                                           BnP5CS1 cDNA harbors a 2154 bp open reading frame encod-
    Keywords: Brassica napus, Osmotic stress, Ornithine δ–aminotrans-
                                                                           ing a protein of 717 amino acids with a predicted molecular
                                    1
    ferase, Proline dehydrogenase, Δ -pyrroline-5-carboxylate synthetase   weight of approximately 77.8 kDa and isoelectric point of

28 BMB reports                                                                                                             http://bmbreports.org
                                                                                                      Proline accumulation in Brassica napus
                                                                                                                         Xingning Xue, et al.




5.96. Protein sequence analysis showed that BnP5CS1 shared
sequence identities of 96% with A. thaliana P5CS1, 77% with
Actinidia (Ac) deliciosa and M. truncatula P5CS1, 76% with
Mesembryanthemum (Me) crystallinum, 75% with V. aconitifo-
lia and Vitis (Vi) vinifera, 74% with Oryza sativa and 72% with
M. sativa P5CS1 with A. thaliana P5CS1 (96%), Ac. deliciosa
(77%), M. truncatula P5CS1 (77%), Me. crystallinum (76%), V.
aconitifolia (75%), Vi. vinifera (75%), O. sativa (74%) and M.
sativa P5CS1 (72%).
    The 2529 bp BnP5CS2 cDNA harbors a 2184 bp open read-
ing frame. The encoded protein is 727 amino acids with a calcu-
lated molecular weight of 78.7 kDa and an isoelectric point of
6.85. Protein sequence analysis revealed a high degree of iden-
tity to BnP5CS2 from A. thaliana P5CS2 (93%), Vi. vinifera
(77%), Ac. deliciosa (77%), V. unguiculata (76%), Lycopersicon
esculentum tomPro2 (75%) and O. sativa (74%).
    Multiple protein sequence alignment of BnP5CS1 and
BnP5CS2 with P5CS of other plant species indicated the ex-
istence of conserved domains such as the putative ATP and
NAPDH binding site, two leucine zipper motifs presumably in-
volved in intramolecular interaction between two functional
domains: the conserved Glu-5-kinase domain and the con-
served GSA-DH domains.
    BnOAT cDNA is 1615-bp-long and consists of one open
reading frame of 1431 bp, which encoded a protein of 476
amino acids with calculated molecular weight of 52 kDa and
isoelectric point of 7.17. Protein sequence analysis revealed
high identity with OAT cloned from B. rapa (96%), A. thaliana
(91%), O. sativa (69%), M. truncatula (68%) and V. aconitifo-
lia (45%). The putative binding site for the OAT cofactor pyr-
idoxal phosphate was found in position 232-301. Although           Fig. 1. Southern blot analysis of P5CSs in B. napus, B. campestris and
                                                                   B. oleracea. Thirty micrograms of genomic DNA was digested with
there was no typical mitochondrial leader sequence, the N-ter-     the restriction enzymes indicated and probed with 1158 bp BnP5CS1
minal residues displayed common composition with the mi-           cDNA containing 3’UTR (A) and full-length BnP5CS2 cDNA (B).
tochondrial transit peptide. In addition, PSORT software
(http://psort.nibb.ac.jp/form.html) predicted with 92% certainty
a mitochondrial matrix location of BnOAT. The high amino           to test this possibility using genomic DNA extracted from B.
acid identity with OAT from A. thaliana and M. truncatula sug-     napus and its diploid ancestors: B. campestris and B. oleracea.
gests that BnOAT is also a δ-form OAT.                                A number of bands of varying intensities were observed for
    BnPDH cDNA contains a single open reading frame of             each digestion in all the species, when hybridized with
1497 bp. The deduced protein is 498 amino acids with a cal-        BnP5CS1 (Fig. 1A). Similar results were observed with the
culated molecular weight of 55 kDa and an iselectric point of      Southern blot of BnP5CS2 (Fig. 1B). These results suggested
6.77. Protein sequence comparison revealed various identities      the presence of multiple copies of a P5CS-related gene in the
with Arabidopsis thaliana PDH1 (89%), A. thaliana PDH2             three Brassica species. The number of bands observed in B.
(74%), M. sativa (55%), Nicotiana tabacum PDH1 (54%) and           napus was not significantly more than that of two diploid an-
N. tabacum PDH2 (55%). Similar to AtPDH (14), the local-           cestors, suggesting that B. napus probably harbors a similar
ization of BnPDH in the mitochondrial inner membrane was           number of P5CS genes to the diploid species B. campestris
predicted by PSORT software with 76% certainty.                    and B. oleracea, and a gene loss may have happened during
                                                                   evolution after B. napus emerged.
Genomic Southern blot analysis of proline related gene
Two P5CSs exist in most plants studied including A. thaliana       Proline accumulation and the expression profiles of related
(15), M. sativa (16), L. esculentum (7) and M. truncatula (11),.   gene in young plants
B. napus is an amphidiploid species. It is possible that it con-   To understand whether proline accumulation is organ specific,
tains more copies of homologous genes compared to other            we analyzed the proline contents in roots, stems and leaves of
diploid Brasscia species. Southern blot analysis was performed     B. napus seedlings treated with 200 mM NaCl for 24 h (Fig.

http://bmbreports.org                                                                                                           BMB reports     29
    Proline accumulation in Brassica napus
    Xingning Xue, et al.




     Fig. 2. Proline content and the expression of BnP5CS1, BnP5CS2, BnOAT and BnPDH in 3-week-old plants during different treatments.
     The proline content was measured in different tissues after treatment with 200 mM NaCl for 24 h (A) and in whole seedlings treated
     with different concentrations of NaCl for 24 h (B). After treatment with 250 mM NaCl and 100 μM ABA and 20% PEG, proline content
     (C, D) and the expression of genes on proline metabolic pathway (E, F) were analyzed. Values marked with different letters represent sig-
     nificant differences between the treatments (one-way ANOVA, P < 0.05).


    2A). In the absence of stress, the highest proline content was          environmental stimuli in B. napus, 3-week-old seedlings were
    found in leaves, while in roots it was the lowest. After NaCl           exposed to 250 mM NaCl, 100 μM ABA and 20% PEG for dif-
    stress, proline content in different organs all showed approx-          ferent periods of time. Fig. 2C shows that significant increase
    imately 3-fold increases. It was interesting to observe this pro-       in proline content was measured after 24 h of NaCl and ABA
    line gradient along the height of seedlings from top to bottom.         treatment reaching approximately 3- and 5-fold increase, re-
    This kind of distribution was also measured in A. thaliana (17)         spectively, after 48 h. More dramatic increase in proline con-
    and M. truncatula (11). Furthermore, proline accumulation in            tent was observed in seedlings exposed to 20% polyethylene
    response to stress seems to be somewhat uniform in different            glycol (PEG), where proline started to accumulate at 6 h and
    organs. Therefore, whole seedling was used in the following             reached up to 25-fold at 48 h. (Fig. 2D).
    experiments.                                                               The gene expression in proline metabolic pathway was ex-
       To follow the correlation between the severity of stress and         amined by quantitative reverse transcription-polymerase chain
    the level of proline accumulation, 3-week-old seedlings were            reaction (RT-PCR) using gene-specific primers. The expression
    treated for 24 h with 0, 10, 50, 100, 150, 200, 250 and 300             of both BnP5CS1 and BnP5CS2 were rapidly up-regulated
    mM NaCl and the proline content was determined. As shown                within 2 h of treatment with NaCl and ABA, preceding the pro-
    in Fig. 2B, marked proline accumulation was observed after              line accumulation, but the induction was higher for BnP5CS1
    treatment with NaCl concentrations exceeding 200 mM, and                than for BnP5CS2. The induction of BnP5CS1 persisted
    increased further with the NaCl concentration increases.                throughout the course of the treatments, whereas that of
       To investigate the proline accumulation in response to other         BnP5CS2 was transient and was maintained at a relatively low


30 BMB reports                                                                                                              http://bmbreports.org
                                                                                                       Proline accumulation in Brassica napus
                                                                                                                          Xingning Xue, et al.




level after 6 h of treatments. BnOAT expression, on the other
hand, was not significantly altered by ABA treatment, and was
even slightly inhibited under NaCl treatment. Expression of
BnPDH was markedly inhibited after 12 h of both treatments
and kept at low level during the rest period of the treatment
(Fig. 2E).
    Correlating with proline level, the highest induction of
BnP5CS transcript were observed under the PEG treatment,
where the maximum induction of BnP5CS1 and BnP5CS2 tran-
scripts were 132-fold and 33-fold, respectively, after 12 h of
treatment. Noticeably, the BnOAT transcripts were also in-
duced approximately 4-fold after 12 h of PEG treatment. On
the contrary, the expression of BnPDH was strongly inhibited
within 1 h following treatment and remained low during the
course of the treatment (Fig. 2F).
   These results indicate that in B. napus the glutamate path-
way is dominant in salt stress- and ABA-induced proline accu-
mulation, where BnP5CS1 plays more important roles than
does BnP5CS2. Under osmotic stress, however, the ornithine
pathway seems also to join the glutamate pathway in promot-
ing proline accumulation during prolonged osmotic stress (Fig.
2F). Suppression of proline degradation may also play an im-
portant role in stress-induced proline accumulation. So far, the     Fig. 3. The proline content and the expression of BnP5CS1, BnP5CS2,
involvement of OAT in stress-induced proline accumulation is         BnOAT and BnPDH in different organs of 12-week-old plants. (A) The
                                                                     proline content of different organs of B. napus treated with 200 mM
somehow contradictory. Among three plant species studied,            NaCl for one week. (B) The expression of related genes in different
while there was apparently no induction of OAT transcripts in        organs of B. napus under normal growth condition. Values marked
young seedlings of V. aconitifolia treated with 400 mM NaCl          with different letters represent significant differences between the
                                                                     treatments (one-way ANOVA, P<0.05).
(9), its upregulation in A. thaliana young seedlings (10) and M.
truncatula (11) was clearly observed. Our results demonstrate
that BnOAT is induced at a later stage of severe stress when
proline has already started to accumulate, suggesting that           was observed in flowers and the second in flower buds com-
BnOAT’s involvement in proline accumulation might depend             pared to the expression in other organs (Fig. 3B).
on the severity of the stresses.                                        The high proline content and the strong expression of P5CS
                                                                     and PDH in flower buds and flowers have also been reported
Proline accumulation and the expression profiles of related          in other plant species (11, 18, 19), and led to the proposition of
gene in adult plants                                                 the potential roles of proline turnover in flower development.
To investigate proline accumulation in adult plants, we de-          Recently, characterization of AtP5CS1- overexpressing and
termined the proline content in different organs of 12-week-         AtP5CS1 knock-out Arabidopsis mutants led to the proposal
old plants with or without NaCl treatment for 7 days. Under          that proline plays a key role in flower transition, bolting and co-
normal growth conditions, the proline content in reproductive        florescence formation (20). Moreover, AtP5CS2 was found to
organs was generally higher than that in the vegetative organs       be one of the four early targets of CONSTANS (21).
with the highest proline content being observed in flower               Strong BnOAT expression in leaves does not result in the
buds. In contrast, roots contained the lowest amount of proline      accumulation of proline. It is possible that P5C or proline syn-
among all the organs studied. After 7 days of 200 mM NaCl            thesized in leaves by the ornithine pathway is transported to
treatment, the proline level showed remarkable enhancement           the flower bud. This was supported by the fact that the high
in every organ. Although the enhancement was higher in vege-         expression of ProT1 and LeproT1 encoding proline transporter
tative organs than in reproductive organs, the highest proline       in A. thaliana (22) and tomato (23) is found in flowers.
level after stress was found in flowers while the lowest still ob-   Therefore, both proline biosynthesis and transport may con-
served in roots (Fig. 3A).                                           tribute to proline accumulation in flower buds and flowers,
   Quantitative RT-PCR revealed both BnP5CS1 and BnP5CS2             which may, in turn, up-regulate proline degradation to provide
to be similarly regulated, and showed the strongest expression       the energy for flower development.
in flower buds, correlating to the high proline level in this
organ. BnOAT expression, however, was highest in leaves.             Free amino acid content in young plants and adult plants
With regards to the BnPDH transcripts, the highest expression        To get a comprehensive overview of the variation of all amino


http://bmbreports.org                                                                                                            BMB reports     31
    Proline accumulation in Brassica napus
    Xingning Xue, et al.




    Table 1. Free amino acid content in 3-week-old seedlings treated with 100 μM ABA or 250 mM NaCl for 24 h, and in the different organs of
    12-week-old flowering plants. Values marked with different letters represent significant differences between the treatments (one-way ANOVA, P<
    0.05). ND, not detected.

                                                                 Concentration (μmol /g DW)

                          3-week-old seedlings                                              12-week-old flowering plants
    Amino
     acid
                Control          ABA             NaCl     Young leaves Mature leaves     Shoots        Roots      Flower buds   Flowers       Siliques

     Pro 1.82±0.09a          6.73±0.13b       6.43±1.39b  2.08±0.26b    0.87±0.09a     1.43±0.13ab       ND       18.15±0.26d 6.77±0.69c 1.95±0.04ab
                   a                  a                a           a             a              a             a             c           c          b
     Asp 8.90±0.41           3.49±1.24       11.57±3.61   4.32±1.09     3.34±1.69      2.29±0.26     0.80±0.18    14.09±0.19 16.04±1.77 9.92±1.05
                   ab                 a                b            b            b              ab            a             d           d          c
     Glu 23.11±0.48         15.94±2.48       28.58±2.14 10.60±1.36      9.24±1.97      8.33±1.33     2.34±0.37    32.93±0.71 29.63±3.87 19.20±0.37
     Gly 30.64±2.53a        29.91±5.93a      13.72±1.60a  4.46±0.47c    2.26±0.40b     0.32±0.00a        ND        1.67±0.07ab 6.53±0.80d 1.73±0.00ab
                   a                  a                a           a             a              a             a             b           c          b
     Ala 8.59±2.64           5.05±3.03        6.90±1.40   4.43±0.51     3.93±0.79      2.13±0.22     3.70±0.45    11.17±0.17 20.04±2.75 8.70±0.06
                   a                  a                a           ab            bc             ab            a             bc          d          c
     Val 4.48±0.38           4.65±0.13        4.05±0.04   1.75±0.13     2.82±0.09      1.88±0.17     0.59±0.04     4.99±0.04 16.52±2.43 5.55±0.09
                                                                   a
      Ile     ND             1.68±0.00            ND      0.76±0.08     1.11±0.27a     0.73±0.03a
                                                                                                         ND        1.52±0.00 7.05±1.18 1.83±0.08a
                                                                                                                            a           b
                                                                   a             a              a                           a           b
     Leu      ND             1.03±0.04            ND      0.27±0.04     0.62±0.14      0.35±0.03         ND        0.99±0.15 5.07±0.88       ND
                                                                   a             a                                                      b
     Tyr      ND                 ND               ND      0.30±0.03     0.61±0.11          ND            ND           ND       1.77±0.28     ND
     Phe      ND                 ND               ND      1.15±0.12a    2.15±0.51ab    1.18±0.15a        ND           ND       2.66±0.24b
                                                                                                                                             ND
                   b                  a                c                                        a                           ab          b          a
     His  3.13±0.03          2.71±0.13        3.67±0.00       ND            ND         0.50±0.03         ND        6.54±0.16 9.28±1.29 2.87±0.10
                   a                  a                a           a             a              a                           a           b          a
     Lys  3.42±0.48          2.67±0.27        3.80±0.72   0.34±0.07     0.31±0.12      0.64±0.05         ND        0.79±0.03 4.31±0.62 1.09±0.07
     Arg 78.87±12.8a        39.81±1.87a      68.45±12.03a 0.98±0.11a    0.58±0.17a     7.12±1.03b        ND        6.20±0.40b 5.83±1.23b 16.30±0.57c
     Met      ND                 ND               ND          ND            ND             ND            ND           ND           ND        ND
     Cys      ND                 ND               ND          ND            ND             ND            ND           ND           ND        ND



    acids under stress, the free amino acid content of B. napus                  gests an important role of proline during flower development.
    seedlings treated with ABA and NaCl for 24 h was examined.
    Proline was the only amino acid whose content increased                      MATERIALS AND METHODS
    more than 2-fold. Proline content increased 3-4-folds after ABA
    and NaCl treatments. The level of glutamate and arginine, two                Plant growth and stress treatment
    precursors for proline biosynthesis, was not significantly en-               Seedlings were grown on 1/2 MS medium with 2% sucrose
                                                                                                                                o
    hanced, suggesting that precursor availability is probably not               and 0.8% agar in a growth chamber at 25 C under a 16 h
    limiting for proline accumulation during salt stress. Interestingly,         photoperiod. Three-week-old seedlings were treated with 1/2
    the level of the most amino acids, except for proline, de-                   MS liquid medium supplemented with 250 mM NaCl, 100 µM
    creased after ABA treatment, leading to a smaller free amino                 ABA or 20% PEG. After treatment, the seedlings were frozen
                                                                                                                      o
    acid pool. This decline of amino acid pool was not observed                  in liquid nitrogen and stored at -80 C prior to analysis.
    after NaCl treatment (Table 1).                                                 For 12-week-old plants, seeds were germinated and grown
        The content of free amino acids in different organs of adult             in pots filled with a mixture of nutrimental soil and vermiculite
                                                                                                         o     o
    plants was also measured. As expected, the proline content in                in a green house at 20 C-25 C under a 16 h photoperiod. Salt
    flower buds and flowers was significantly higher than that in                treatment was imposed by irrigating plants with 1/4 Hogland
    vegetative organs (Table 1). However, unlike in Arabidopsis                  solution containing no or 200 mM NaCl for one week. The
    (19), proline was not the most abundant amino acid in re-                    plants were then harvested and frozen in liquid nitrogen for
    productive organs. Glutamate was more abundant than proline                  subsequent analysis.
    both in reproductive organs and vegetative organs, while the
    level of arginine was only higher than proline in siliques.                  RNA isolation and cDNA synthesis
    Although the level of asparagine and alanine in flower buds                  Total RNAs were isolated as described previously (18). Total
    was lower than proline, they accumulated more in flowers and                 cDNA was synthesized from RNA (1 μg) template with
                                                                                            TM
    siliques than proline.                                                       SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad,
        In conclusion, salt stress-induced proline accumulation in B.            CA, USA) according to the manufacturer’s instructions.
    napus results from the reciprocal action of activated biosyn-
    thesis and inhibited proline degradation. In addition, the orni-             Cloning of cDNA for P5CS, OAT and PDH in Brassica napus
    thine pathway seems to contribute to the proline accumulation                BnP5CS1 and BnP5CS2 cDNAs were cloned by RT-PCR using
    under prolonged osmotic stress. During development, a high                   the gene-specific primers derived from sequences AF314811
    rate of proline turnover in flower buds and flowers, as evi-                 and AF314812 in GenBank. For cloning of BnOAT cDNA, sev-
    denced by up-regulation of both BnP5CSs and BnPDH, sug-                      en B. napus expressed sequence tag (EST) sequences were ob-


32 BMB reports                                                                                                                       http://bmbreports.org
                                                                                                     Proline accumulation in Brassica napus
                                                                                                                        Xingning Xue, et al.




tained by blast in Brassica DB (http://brassica.bbsrc.ac.uk/)       curve was run after PCR cycles. Detection of PCR products
with AtOAT cDNA. Assembling these EST sequences gave two            was done using the SYBR Green Real-time PCR Master Mix
contigs highly homologous to the 5’ end and the 3’ end of           (Toyobo, Tokyo, Japan) following the manufacturer’s recom-
AtOAT cDNA respectively. Two primers, 5’-ATGGCAGCAGC                mendations. Reactions were repeated four times for each
CACGAGAC-3’ and 5’-AATGTCGAAACAACTAACTCTG-3’ were                   sample. Relative gene expression was calculated using the
                                                                     -ΔΔC
then designed according to the sequences of two contigs to          2 T method (25).
obtain the 1615 bp long BnOAT cDNA (genebank accession
no. EU375566).                                                      Amino acid analysis
   For BnPDH cDNA, we obtained 21 B. napus EST sequences            Plant materials were ground into power in liquid nitrogen and
showing high homology to AtPDH1 by blast in Brasscia DB. A          lyophilized. The dried powder was extracted with the extract-
1497 bp fragment was amplified by PCR with two primers              ing buffer consisting of eight volumes of ethanol and two vol-
5’-ATGGCAACCCGTCTCCTCC-3’ and 5’-TCACGCAATTCCA                      umes of water, and processed as described previously (19).
GCGATTAG-3’, designed according to the single contig as-            The supernatant was then dried down in a Speed Vac and re-
sembled by the EST sequences (GenBank accession no.                 suspended in 200 mM lithium citrate buffer (pH 2.2). Amino
EU375567).                                                          acid analysis was carried out on a SYKAM 433-D Amino Acid
                                                                    Analyzer.
Southern blot analysis
Genomic DNA was isolated from leaves of 3-week-old B. na-           Acknowledgements
pus, B. oleracea and B. campestris seedlings as described pre-      We thank Dr. Maurice Moloney, Dr. Ed Yeung and Dr.
viously (18). Thirty micrograms of genomic DNA was digested         Mingjuan Tang for kindly providing seeds of B. napus (cv.
overnight with EcoRI, EcoRV and XbaI for hybridization with         Westar). This work is supported by the Chinese National Key
BnP5CS1, and with DraI, EcoRV and NdeI for hybridization            Basic Research Project (#2006CB100100) from the Ministry of
with BnP5CS2. The digestions were separated on 1% agarose           Science and Technology of China and the Hundred Talent
                                     +
gel and transferred onto Hybond-N nylon membrane (Amer-             Program of Chinese Academy of Science.
sham Pharmacia Biotech, Piscataway, NJ, USA). The mem-
                                   o
brane filter was hybridized at 42 C with 1158 bp BnP5CS1            REFERENCES
cDNA fragment or 2184 bp BnP5CS2 cDNA and labeled with
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                                                o
0.5 × SSC, 0.1% sodium dodecyl sulfate at 65 C for 30 min.              thesis and osmoregulation in plants. Plant J. 4, 215-223.
The hybridization, washing and immunostaining were per-              2. Molinaria, H., Marura, C., Darosb, E., Camposa, M.,
                                                                        Carvalhoa, J., Filhob, J., Pereirac, L. and Vieiraa, L. (2007)
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Prime DNA Labeling and Detection Starter Kit I, Roche                   transgenic sugarcane (Saccharum spp.): osmotic adjust-
Diagnostics, Indianapolis, IN, USA).                                    ment, chlorophyll fluorescence and oxidative stress.
                                                                        Physiol. Plant 130, 218-229.
Measurement of proline content                                       3. Rajendrakumar, C. S., Reddy, B. V. and Reddy, A. R.
Samples frozen in -80oC were ground in liquid nitrogen and              (1994) Proline-protein interactions: Protection of structural
free proline content was measured as described previously               and functional integrity of M4 lactate dehydrogenase.
(24).                                                                   Biochem. Biophys. Res. Commun. 201, 957-963.
                                                                     4. Hare, P. D. and Cress, W. A. (1997) Metabolic implications
                                                                        of stress-induced proline accumulation in plants. Plant
Quantitative real-time RT-PCR                                           Growth Regul. 21, 79-102.
Quantitative real-time RT-PCR was carried out with actin gene        5. Khedr, A. H. A., Abbas, M. A., Wahid, A. A. A., Quick,
as the internal standard. The gene-specific primers were as fol-        W. P. and Abogadallah, G. M. (2003) Proline induces the
lows: 5’-CGATTTGGACTTGGTGCTGA-3’ and 5’-GCCCATCC                        expression of salt-stress-responsive proteins and may im-
TCTCCTAGTCTC-3’ for BnP5CS1; 5’-CCATTATCTTCCTCCTC                       prove the adaptation of Pancratium maritimum L. to
TCAC-3’ and 5’-AACAACTGCTGTCCCAA CC-3’ for BnP5CS2;                     salt-stress. J. Exp. Bot. 54, 2553-2562.
5’-GCACGGCGGAGTTATGGA-3’ and 5’-CAGCGGAGTAAG                         6. Kavi Kishor, P. B., Sangam, S., Amrutha, R. N., Laxmi, P.
CAGCAAG-3’ for BnOAT; 5’-TGGTATGGTCCTTGGCCTTG-3’                        S., Naidu, K. R., Rao, K. R. S. S., Rao, S., Reddy, K. J.,
and 5’-ACTGAGCTAAAGTGAG ATGTA-3’ for BnPDH; and                         Theriappan, P. and Sreenivasulu, N. (2005) Regulation of
                                                                        proline biosynthesis, degradation,uptake and transport in
5’-CTGACCGTATGAGCAAAG AG-3’ and 5’-CCACGAACCA
                                                                        higher plants: Its implications in plant growth and abiotic
GAAGGCAGA-3’ for BnActin. Real-time RT-PCR was performed                stress tolerance. Curr. Sci. 88, 424-438.
using a Stratagene Mx3000P real-time PCR machine. Amplifi-           7. Fujita, T., Maggio, A., Garcia-Rios, M., Bressan, R. A. and
                        o
cation was started at 95 C for 10 min as the first step, followed       Csonka, L. N. (1998) Comparative analysis of the regu-
by 40 cycles of PCRs: denaturation at 95oC for 30 s, annealing          lation of expression and structures of two evolutionarily
                                                                                                  1
at 55oC for 30 s, and elongation at 72oC for 30 s. A melting            divergent genes for Δ -pyrroline-5-carboxylate synthetase


http://bmbreports.org                                                                                                          BMB reports     33
    Proline accumulation in Brassica napus
    Xingning Xue, et al.




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        1323-1335.                                                                LeProT1, a transporter for proline, glycine betaine, and
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                                                                                            -ΔΔC
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                                                           1
        and characterization of two different cDNAs of Δ -pyrroline-




34 BMB reports                                                                                                               http://bmbreports.org

								
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