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Over-expression of the AtGA2ox8 gene decreases the biomass

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481471

Over-expression of the AtGA2ox8 gene decreases the biomass accumulation and lignification in rapeseed (Brassica napus L.)*

Xiao-ying ZHAO§1, Deng-feng ZHU§1,2, Bo ZHOU1, Wu-sheng PENG3, Jian-zhong LIN1, Xing-qun HUANG1, Re-qing HE1, Yu-hong ZHUO3, Dan PENG1, Dong-ying TANG1, Ming-fang LI2, Xuan-ming LIU??1 (1Bioenergy and Biomaterial Research Center, Institute of Life Science and Technology,

State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China) (2Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China)

(3Academy of Seed Industry of Hunan Yahua, Changsha 410001, China)

?E-mail: sw_xml@https://www.wendangku.net/doc/c07929730.html,

Received Apr. 29, 2010; Revision accepted June 11, 2010; Crosschecked June 13, 2010

Abstract: Gibberellin 2-oxidase (GA 2-oxidase) plays very important roles in plant growth and development. In this study, the AtGA2ox8 gene, derived from Arabidopsis (Arabidopsis thaliana), was transformed and over-expressed in rapeseed (Brassica napus L.) to assess the role of AtGA2ox8 in biomass accumulation and lignification in plants. The transgenic plants, identified by resistant selection, polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) analyses, and green fluorescence examination, showed growth retardation, flowering delay, and dwarf stature. The fresh weight and dry weight in transgenic lines were about 21% and 29% lower than those in wild type (WT), respectively, and the fresh to dry weight ratios were higher than that of WT. Quantitative measurements dem-onstrated that the lignin content in transgenic lines decreased by 10%–20%, and histochemical staining results also showed reduced lignification in transgenic lines. Quantitative real-time PCR analysis indicated that the transcript levels of lignin biosynthetic genes in transgenic lines were markedly decreased and were consistent with the reduced lig-nification. These results suggest that the reduced biomass accumulation and lignification in the AtGA2ox8 over- expression rapeseed might be due to altered lignin biosynthetic gene expression.

Key words: Rapeseed, AtGA2ox8, Biomass, Lignification, Gibberellins

doi:10.1631/jzus.B1000161 Document code: A CLC number: S565.4; Q812

1 Introduction

Gibberellins (GAs) are endogenous plant hor-mones playing very important roles in plant growth and regulating many aspects of plant development, including seed germination, shoot growth, flower induction, stimulation of cell elongation and cell di-vision, hypocotyl elongation, fruit maturation, and leaf expansion (Kende and Zeevaart, 1997; Harberd et al., 1998; Hedden and Proebsting, 1999). Only a few of the 126 presently known GAs, such as GA1, GA3, GA4, and GA7, have been shown to have biological activity (Hedden and Phillips, 2000).

GA metabolism takes place in three different cellular compartments, i.e., plastids, endoplasmic reticulum (ER), and cytosol (Hedden and Phillips, 2000; Olszewski et al., 2002; Sun and Gubler, 2004). Multiple enzymes including ent-copalyl diphosphate synthase, ent-kaurene synthase, P450 monooxy-genases, and dioxygenases are involved in GA me-tabolism and catabolism. Two dioxygenases, GA 20- oxidase and GA 3β

-hydroxygenase, catalyze the last

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https://www.wendangku.net/doc/c07929730.html,/jzus; https://www.wendangku.net/doc/c07929730.html,

E-mail: jzus@https://www.wendangku.net/doc/c07929730.html,

? Corresponding author

§ The two authors contributed equally to this work

* Project supported by the National High-Tech R & D Program (863) of China (No. 2007AA10Z127) and the National Natural Science Foundation of China (No. 30800080)

? Zhejiang University and Springer-Verlag Berlin Heidelberg 2010

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481 472

few steps in the synthesis of bioactive GA. Another dioxygenase, GA 2-oxidase, catalyzes GA catabolism of bioactive GA or their precursors (Thomas et al., 1999; Hedden and Phillips, 2000; Helliwell et al., 2001; Olszewski et al., 2002; Zhao et al., 2007b).

The genes encoding the multi-functional GA 2- oxidase have been cloned from various species, such as runner bean (Phaseolus coccineus) (Thomas et al., 1999; Appleford et al., 2007), Arabidopsis (Arabi-dopsis thaliana) (Thomas et al., 1999; Schomburg et al., 2003), pea (Pisum staivum) (Lester et al., 1999; Martin et al., 1999), rice (Oryza sativa) (Sakamoto et al., 2001; 2004), poplar (Populus tremula) (Busov et al., 2003), spinach (Spinacia oleracea) (Lee and Zeevaart, 2002; 2005), and lettuce (Lactuca sativa) (Nakaminami et al., 2003). Phylogenetic analysis divides the GA 2-oxidase family into three classes according to their amino acid sequences (Lee and Zeevaart, 2005). C19-GAs (GA1 and GA4) and C19-GA precursors (GA20 and GA9) can be converted to inactive GAs by members of classes I and II, re-spectively (Sakamoto et al., 2004; Lee and Zeevaart, 2005; Lo et al., 2008), and C20-GAs (GA12 and GA53) can be 2β-hydroxylated by members of class III, in-cluding SoGA2ox3, AtGA2ox7, and AtGA2ox8 (Schomburg et al., 2003; Lee and Zeevaart, 2005; Lo et al., 2008). The 2β-hydroxylated C20-GA precursors cannot be converted to active GAs, resulting in a decrease in active GA levels.

The over-expression of the genes encoding AtGA2-oxidase causes a dwarf phenotype and de-layed flowering in Arabidopsis (Schomburg et al., 2003; Wang et al., 2004; Zhao et al., 2007a; Rieu et al., 2008). The heterologous expression of AtGA2- oxidases also resulted in growth retardation in wheat (Hedden and Phillips, 2000), rice (Sakamoto et al., 2001), tobacco (Schomburg et al., 2003), bahiagrass (Agharkar et al., 2007), and Solanum species (Dijkstra et al., 2008). Apart from the effect of ex-pression of AtGA2-oxidases on plant elongation, Biemelt et al. (2004) reported that biomass produc-tion was decreased in AtGA2ox1 plants, and that the transgenic plants have altered lignification. Moreover, they showed that GA have different effects on xylem and pitch cell formation.

Both the rapeseed (Brassica napus L.) and Arabidopsis are cruciferous plants. Zhao et al. (2007a) and Schomburg et al. (2003) reported that over- expression of AtGA2ox8 causes reduced bioactive GA levels in Arabidopsis. To investigate the correlation of AtGA2ox8 gene to biomass characteristics and lignin biosynthesis, we introduced it into rapeseed. Here, we show that heterologous expression of AtGA2ox8 gene in rapeseed causes growth retardation, flowering delay, dwarf stature, and reduced biomass accumulation and lignification.

2 Materials and methods

2.1 Plant materials and genetic transformation

The cultivar of rapeseed (Brassica napus L.) in this study was N529, provided by the Academy of Seed Industry of Hunan Yahua, Changsha, China. AtGA2ox8 transgenic plants were prepared by the first amplifying AtGA2ox8 (GenBank accession No. AL021960, accession number in The Arabidopsis Information Resource (TAIR) website is At4g21200) gene from Arabidopsis cDNA using a reverse- transcription polymerase chain reaction (RT-PCR) method with primers AtGA2ox8-F (5′-TCCCCCG GGATGGATCCACCATTCAACGAAATAT-3′) and AtGA2ox8-R (5′-CCGCTCGAGTTAGTAGACGTG TTAAGGAACCAGGAA-3′) (restriction sites are underlined), and then subcloning it into the Sma I and Xho I sites of pEGAD vector downstream of the CaMV35S promoter. The constructs were introduced into Agrobacterium tumefaciens strain GV3101 by the electroporation method, as described in our pre-vious study (Lin et al., 2009). When the rapeseed plants begin to form boll and produce flora inflo-rescences, the healthy flora inflorescences grown at the top of rapeseed plants were selected and im-mersed into a plastic bag with Agrobacterium in-oculum for 30 s, and then covered with an agricul-tural parchment bag to maintain high humidity. One week later, the agricultural parchment bags were uncovered and the flower buds at the top of dipped inflorescences were removed. When the siliques became brown and dry, the seeds from the dry siliques were collected and stored at ?20 °C for screening.

2.2 Transgenic line screening

Transgenic plants were first selected using her-bicide Basta (1:800, v/v). Total genomic DNA was

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481473

extracted from Basta-resistant transgenic lines using hexadecyltrimethylammonium bromide (CTAB) method (Murray and Thompson, 1980). The detection of Basta R gene was carried out by PCR analysis with primers Bar-F (5′-CTACATCGAGACAAGCACG GT-3′) and Bar-R (5′-CTGAAGTCCAGCTGCCAG AA-3′). The PCR program was as follows: 94 °C for 5 min, and then 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, and a final extension of 72 °C for 5 min. The PCR products were analyzed using 1.0% (w/v) agarose gel electrophoresis.

To investigate whether the transgene is ex-pressed in the transgenic plants, some lateral roots were excised from the Basta-resistant transgenic lines. The green fluorescent protein (GFP) fusion proteins were examined using inverted fluorescence micro-scope (Nikon TE2000-U, Japan).

2.3 GA3 treatment

To investigate the response of hypocotyl elon-gation to exogenous GA3, about 100 seeds were sown on filter paper saturated with 100 μmol/L GA3 (Shanghai Solvent) in a plate and grown under con-tinuous white light in a temperature-controlled growth chamber at 22 °C. Hypocotyl lengths of more than twenty 6-d-old seedlings were measured manu-ally following the method of Lin et al. (1998), and the standard deviations (SDs) of the measurements were calculated from three independent experiments with 20 samples each.

2.4 Greenhouse evaluation of growth and biomass characteristics

Transgenic plants and wild-type plants were grown in a greenhouse. The stem heights of the plants were evaluated in the flowering and maturation pe-riods. Leaves were counted in the maturation period. Fresh weight and dry weight of the whole plant, in-cluding the roots, were determined after harvest ac-cording to the method of Biemelt et al. (2004).

The seed oil content and the glucosinolate, eru-cic acid, and oleic acid contents in seed oil of rape-seed were determined by near-infrared reflectance (NIR) spectroscopy after harvest (Panford and de Man, 1990).

Each value of all the experiments represents the mean±SD of 10 to 15 samples each. 2.5 Histochemical detection and quantitative de-termination of lignin

Histochemical detection was carried out on the hand-cut cross sections of stems from the second, eighth, and seventeenth internodes (counting from the bottom up to the top) of the plants grown in green-house during the flowering period. The sections were stained using phloroglucinol-HCl reagent, and then analyzed on an inverted microscope (Nikon TE2000-U, Japan) according to the methods described previously (Campbell and Ellis, 1992; Biemelt et al., 2004). Each experiment was repeated three times with at least three samples each.

The lignin contents in about 100 mg fresh weight of the apical, middle, and basal parts of the stems of both the AtGA2ox8 transgenic plants and the wild-type plants grown in the greenhouse during the flowering period were determined by acetyl bromide ultraviolet spectrophotometry (280 nm) according to the methods described previously (Fukushima and Dehority, 2000; Fukushima and Hatfield, 2001; Biemelt et al., 2004). Each value was obtained from two independent experiments with three samples each.

2.6 Semi-quantitative RT-PCR analysis

To investigate whether the AtGA2ox8 gene was over-expressed in the transgenic rapeseed plant, about 100 seeds were sown on filter paper saturated with Murashige and Skoog (MS) salt liquid medium in a plate and grown under continuous white light in a temperature-controlled growth chamber at 22 °C. Total RNA was isolated from 6-d-old seedlings using Puprep RNAeasy mini kit (Ambiogen Life Tech Ltd., China). The transcript level of AtGA2ox8 was ana-lyzed using semi-quantitative RT-PCR as described previously (Zhao et al., 2007a). The rapeseed Actin (ACT)(European Molecular Biology Laboratory (EMBL) accession No. AF111812) gene was used as an internal control. The PCR started with a denatura-tion stage at 95 °C for 5 min, which was then followed by 26 (for the ACT) or 35 (for the AtGA2ox8)cycles with each cycle composed of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The RT-PCR reactions for each experiment were repeated at least three times in three independent trials. The sequences of the primers

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481

474 were: AtGA2ox8-F (5′-CGGAATCAGAGGCATTA GC-3′), AtGA2ox8-R (5′-CCACCTTTGGGTTCGTC AT-3′), ACT -F (5′-TCCCTCAGCACTTTCCAACA G-3′), and ACT -R (5′-AAGGACCAGAGCATCATC ACAAG-3′).

2.7 Quantitative real-time PCR analysis

To detect the mRNA level of lignin biosynthetic gene, total RNA was isolated from apical parts of stems of rapeseed during the flowering period using Puprep RNAeasy mini kit (Ambiogen Life Tech Ltd.). The amount of mRNA was analyzed using quantita-tive real-time PCR as described previously (Wang et al ., 2009). The PCR was performed in Mx3000P (Stratagene), and started with a denaturation stage at 95 °C for 10 min, which was then followed by 40 cycles with each cycle composed of 95 °C for 30 s, 60 °C for 20 s, and 72 °C for 20 s. The PCR reactions were repeated three times from three independent experiments. The ACT gene of rapeseed was used as an internal control. The sequences of primers used in this study are listed in Table 1. 3 Results 3.1 Transformation and molecular characterization The AtGA2ox8 gene was introduced into rape-seed by Agrobacterium -mediated floral dip trans-formation as stated in Section 2.1. The expression cassette contains the GFP report gene, the target gene (AtGA2ox8), and the herbicide Basta-resistant (Basta R ) select maker gene. AtGA2ox8 and GFP were driven by the CaMV35S promoter and would express fusion protein in the transgenic plants (Fig. 1a in p.478). Ten Basta-resistant independent lines were obtained after the first screening with herbicide Basta (Fig. 1b). The Basta R gene was detected by PCR analysis in all Basta-resistant plants but not in the wild-type plants (partly in Fig. 1c), indicating that the transgene was indeed integrated into the genomic DNA. To determine whether the transgene is normally expressed in the transgenic plants, the GFP fusion proteins were ex-amined using inverted fluorescence microscope, and

strong green fluorescence was detected in the root of transgenic lines (partly in Fig. 1d). No green fluo-rescence was observed in root of the wild-type plant (Fig. 1e). These data confirmed that transgenic plants were obtained. Two independent transgenic lines, 35S::GFP-AtGA2ox8-2 and 35S::GFP-AtGA2ox8-3, were then randomly selected for the further detection of the target gene expression using semi-quantitative RT-PCR analysis. The results showed that the AtGA2ox8 signals of the two independent transgenic lines were quite strong, whereas the corresponding signal of the wild type was hardly detectable (Fig. 2). These results indicated that the AtGA2ox8 gene was normally over-expressed in the two independent transgenic lines.

Table 1 Primer sequences used for quantitative real-time PCR analysis Gene name EMBL accession No. Forward and reverse (5′-3′) PAL1 DQ341309 Forward: CAAAGCGATTCACGGAGGTAA; reverse: CGCTCCTTTGAAACCGTAGTC PAL2 AY795080 Forward: TTGGATTACGGATTCAAAGGA; reverse: CGAGATGAGTCCCAAAGAGTT C4H DQ485129 Forward: TGACTTTAAGTATGTGCCGTTTG; reverse: GGACCTTGGCTTCATTACGAT 4CL CX190902 Forward: TAATCCGAATCTTTACTTCCACAG; reverse: GCAACCGTCACTTTACACCTCT HCT CD832138 Forward: CTCAAGGCTAAAGCCAAGGAG; reverse: TGACGTTGCCAAAGTAACCAG CCR CD844319 Forward: GGTGGAAGTTTAGGTCATTAGAAGA; reverse: CCAATAGTAGACTTGAGGAGGTGAA CAD CD814354 Forward: TTATGTCCTGGTTGGTTTCCC; reverse: CACCCTTTCAATAGCTTCGTTT ACT AF111812 Forward: TCCCTCAGCACTTTCCAACAG; reverse: ACACTCACCACCACGAACCAG

Fig. 2 Semi-quantitative RT-PCR analysis of AtGA2ox8 gene in the transgenic rapeseed

Total RNA was isolated from 6-d-old seedlings of the wild type (WT) and the two independent transgenic lines grown

on filter paper saturated with MS salt liquid medium in a

plate under continuous white light in a temperature- controlled growth chamber at 22 °C. The transcript levels are shown as gel images, and the ACT is used as an internal

control. Each experiment was repeated at least three times in

three independent trials and similar results were obtained. Lane 1: WT; Lane 2: 35S::GFP-AtGA2ox8-2; Lane 3: 35S::GFP-AtGA2ox8-3 AtGA2ox8

ACT

1 2 3

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481475

3.2 Effect of over-expression of AtGA2ox8 in rapeseed on plant growth

Transgenic seeds were sown in plates to inves-tigate the hypocotyl phenotype. Compared with wild type, the over-expression of AtGA2ox8 in rapeseed inhibited the hypocotyl elongation and led to shorter hypocotyls (Figs. 3a and 3b). Furthermore, the phe-notype of 35S::GFP-AtGA2ox8 could be rescued by exogenous GA3 (Figs. 3c and 3d). These results are consistent with our previous reports that the over-expression of AtGA2ox8 in Arabidopsis resulted in short hypocotyl (Zhao et al., 2007a), and the ga1 mutant impaired in the ent-copalyl diphosphate syn-thase gene caused short hypocotyl (Sun et al., 1992; Alabadi et al., 2004). Therefore, these observations strongly suggested that AtGA2ox8could induce a GA-deficient phenotype in different species.

To evaluate the growth characteristics, the rape-seed seedlings were planted in a greenhouse. The phenotypes were investigated during different growth periods. The AtGA2ox8 transgenic lines exhibited growth retardation, flowering delay, and dwarf phe-notype. The stem heights of the AtGA2ox8 over- expression lines were only about 70% of those of the wild-type plants (Fig. 4). Moreover, the AtGA2ox8over-expression lines have shorter leaves (data not shown). Compared with the wild-type plants, the flowering time in the AtGA2ox8 over-expression lines was delayed for approximately one week (Fig. 4a).

3.3 Evaluation of biomass characteristics of transgenic lines

The biomass characteristics of the transgenic plants during maturation period were also analyzed (Table 2). As far as the number of leaves is concerned, there was almost no difference between the transgenic plants and the wild type. In order to assess the impact of the transgenic plants on biomass production, the fresh weight and dry weight of the whole plants were determined. As shown in Table 2, significant differ-ences in biomass production between the wild type and the transgenic lines were observed. Compared with the wild type, the fresh weight and dry weight in the transgenic lines decreased by about 21% and 29%, respectively. The fresh to dry weight ratios for wild type plants, 35S::GFP-AtGA2ox8-2, and 35S::GFP- AtGA2ox8-3 were 10.1, 11.2, and 11.3, respectively. Moreover, the 1000-seed weight in the transgenic plants was significantly increased, though the seed yield per plant was only slightly increased.

1 2 3 1 2 3 L

(

)

1 2 3 L

(

)

1 2 3

*** ***

(a) (b)

(c)

(d)

Fig. 3 Response of AtGA2ox8

transgenic plants to GA3

(a) & (b) Six-day-old seedlings of

both the wild type (WT) and the

two independent transgenic lines

and their hypocotyl lengths are

shown, respectively; (c) & (d) GA3-

rescuable phenotype of 6-d-old

seedlings of the two independent

transgenic lines and their hypocotyl

lengths are shown, respectively.

The data represent the mean values

obtained from three independent

experiments with 20 samples each.

The error bars represent the SDs.

***P<0.001, significant difference

between the WT and transgenic

plants. Plant: 1. WT; 2. 35S::

GFP-AtGA2ox8-2; 3. 35S::GFP-

AtGA2ox8-3

?GA3 +GA3 +GA3

Plant

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481

476 The determination of the seed oil content and the glucosinolate, erucic acid, and oleic acid contents in seed oil revealed that the two transgenic lines produced 3% or 6% more seed oil and 15%–25% less glucosi-nolate in seed oil than the wild type. The erucic acid content in seed oil was higher in the transgenic lines as compared to the wild type. However, there was almost no difference in the oleic acid content between the transgenic lines and the wild type. These results dem-onstrated that AtGA2ox8 reduced the biomass accu-mulation and also affected the yield and quality of seed oil in rapeseed. 3.4 Effect of over-expression of AtGA2ox8 in

rapeseed on lignification

It has been reported that the change in fresh to dry weight ratio might result from the altered composition

or deposition of the structure-forming substances like cellulose and/or lignin, and that higher fresh to dry weight ratio of plants usually led to a relatively low lignin content in plants (Biemelt et al ., 2004). Lignifi-cation is also a characteristic feature of secondary growth and increases with maturity (Israelsson et al ., 2005). To determine the different levels of lignification in the transgenic lines and the wild type, different parts of stems, i.e., apical, middle, and basal parts, were col-lected and their lignin contents were measured. For

both the transgenic lines and the wild type, the lignin content increased gradually from the apical parts to the

basal parts (Fig. 5). Lignin content comparison revealed

that the two independent transgenic lines, 35S::GFP-

AtGA2ox8-2 and 35S::GFP-AtGA2ox8-3, produced

about 10% and 20% lower lignin contents in stems than

the wild-type plants, respectively.

1 2 3 1209060300

S t e m h e i g h t (c m )

** ** 1 2 3 120

9060300S t e m h e i g h t (c m ) **

1 2 3

(a) (b) (c) (d) Plant Plant

1 2 3

Plant

Plant Fig. 4 Comparison of the stem heights of both the AtGA2ox8

transgenic plants and the wild

type (WT) grown in a greenhouse

during different growth periods

(a) & (c) Plants of the WT and the two independent transgenic lines during the flowering (a) and matu-ration (c) periods, respectively; (b)

& (d) Stem heights of plants during the flowering (b) and maturation

(d) periods, respectively. The error

bars represent the SDs of 10 to 15 samples each. ** P <0.01, significant difference between the WT and transgenic plants. Plant: 1. WT; 2. 35S::GFP-AtGA2ox8-2; 3. 35S::

GFP-AtGA2ox8-3

Table 2 Effects of over-expression of AtGA2ox8 in rapeseed on biomass accumulation

Plant Number of leaves FW (g) DW

(g)

FW/DW Seed yield per plant (g) 1000-seed weight (g) Seed oil content (%) Erucic acid content (%) Glucosinolate content (μmol/g) Oleic acid content (%)

1 30.0±1.3 620.4±20.5 61.7±5.6 10.117.7±1.8 2.36±0.09 32.5±0.60.86±0.35 40.6±2.7 62.2±0.17

2 29.5±1.2 495.5±14.5***44.2±7.6** 11.218.8±1.8 2.85±0.10***33.5±0.5* 1.30±0.50 33.5±2.7*

62.5±0.20

3 30.0±1.0 488.7±13.2

***43.5±7.3** 11.317.8±1.4 2.92±0.09***34.4±0.4** 1.35±0.49 30.8±2.8* 63.3±0.28Results are given for two independent lines and represent the mean±SD of 10 to 15 samples each. FW: fresh weight; DW: dry weight. *

P <0.05,** P <0.01, ***

P <0.001, significant differences between the WT and the two mutant lines. Plant: 1. WT; 2. 35S::GFP-AtGA2ox8-2;3. 35S::GFP-AtGA2ox8-3

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481 477

The cell walls of lignification display red or purple-red color in acidic phloroglucinol. For visually investigating the differences in lignification between the transgenic lines and the wild type, different parts of stems (the seventeenth internode, the eighth in-ternode, and the second internode) were analyzed by histochemical detection. The biomass characteristics and lignin contents of the two transgenic lines were essentially the same. Hence, only one of the two transgenic lines, 35S::GFP-AtGA2ox8-2, was se-lected for the histochemical detection, and its staining pattern is shown in Fig. 6. In general, the number of lignified vessels and the amount of lignin deposition were gradually increased from the apex to the base of the stem. The AtGA2ox8 over-expression line had fewer lignified vessels than the wild-type controls, which was in good accordance with the lignin con-tents shown in Fig. 5. These results demonstrate that the heterologous expression of AtGA2ox8 in rapeseed can result in lignin deposition reduction.

3.5 Effect of over-expression of AtGA2ox8 in

rapeseed on lignin biosynthetic gene expression

Lignin is a complex phenylpropanoid polymer, and lignin monolignols are synthesized through the phenylpropanoid pathway (Boerjan et al ., 2003). To determine whether the changes of lignin content in transgenic lines could be a result of altered genes expression, a search for the cDNA coding or the ex-pressed sequence tag (EST) sites for the enzymes in the lignin pathway was performed on the Brassica website (https://www.wendangku.net/doc/c07929730.html,/). The cDNA cod-ing sequences of seven key enzymes in lignin syn-thesis, i.e., phenylalanine ammonialyase 1 (PAL1),

phenylalanine ammonialyase 2 (PAL2), 4-coumarate: CoA ligase (4CL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD), were found. The relative expression of these genes in apical parts of the stems of the AtGA2ox8 over-expression lines and the wild-type plants was analyzed by quan-titative real-time PCR. The transcript levels of all the seven genes were found to have a significant differ-ence between the wild type and the transgenic plants,

and were markedly reduced in the AtGA2ox8 over- expression lines (Fig. 7). These transcript reductions coincided with the reduced lignification in the apical

parts of stems of the transgenic plants (Figs. 5 and 6). All these results indicate that the changes in lignifica-tion in transgenic plants might be associated with the altered expression of lignin biosynthetic genes.

4 Discussion This study investigated the over-expression of AtGA2ox8 in rapeseed by genetic engineering tech-nology. It was found that the over-expression of AtGA2ox8 could inhibit hypocotyl elongation in rapeseed. This inhibition effect can be removed by the application of GA 3 to the AtGA2ox8 transgenic lines. AtGA2ox8 acts as GA 2-oxidases that hydroxylate carbon-2 of C 20-GA precursors (e.g., GA 12), but not active GAs (Schomburg et al ., 2003; Lee and Zeevaart, 2005; Lo et al ., 2008). Therefore, active C 19-GAs such as GA 3 can be employed to rescue

AtGA2ox8-derived short hypocotyl phenotype. The over-expression of AtGA2ox8 in rapeseed

also resulted in dwarf phenotype with delayed flow-ering, shorter stems, and shorter leaves. Nevertheless,

viable seeds were obtained from the transgenic lines. Moreover, the 1000-seed weight in the transgenic plants was significantly increased, though the seed yield per plant was only slightly increased. Similarly, heterologous over-expression of C 20-GA2ox, includ-ing Arabidopsis GA2ox8 (Schomburg et al ., 2003),

spinach GA2ox3 (Lee and Zeevaart, 2005), rice

35S::GFP-AtGA2ox8-2

35S::GFP-AtGA2ox8-3

50.0

37.5

25.0

12.5

L i g n i n c o n t e n t (m g /g F W )

A M D

*** ***

* **

Fig. 5 Lignin content in different parts of stems of both

the AtGA2ox8 transgenic plants and the wild-type (WT) plants grown in a greenhouse during the flowering period

A, M, and B symbolize apical, middle, and basal parts, respectively. The error bars represent the SDs of two in-dependent experiments with three samples each. *

P <0.05, **

P <0.01, *** P <0.001, significant difference between the

WT and transgenic plants. FW: fresh weight

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481

478 GA2ox5, and GA2ox6 (Lo et al ., 2008) in transgenic tobacco, resulted in reduced plant height, while al-lowing the production of viable seeds. The same results were also obtained from homologous over-expression of C 20-GA2ox, including rice GA2ox5 and GA2ox6, and Arabidopsis GA2ox8 in transgenic plants. In con-trast, the rice over-expressing C 19-GA2ox, including GA2ox1, completely suppressed the formation of inflorescences (Sakamoto et al ., 2001). These seem-ingly contradictory results may be explained as fol-lows based on the findings in previous studies: there are so many more C 20-GA precursors than the C 19-GA precursors and active C 19-GAs in plants that some of the C 20-GA precursors could escape from inactivation

35S::GFP-AtGA2ox8

250 μm (a) (d) Fig. 6 Histochemical detection of lignin in the stem cross sections of both the AtGA2ox8 transgenic plants and the wild type (WT) Different internodes were collected from plants during the flowering period for histochemical detection. (a), (b) & (c) Phloroglucinol-HCl stained second, eighth, and seventeenth internodes of the WT, respectively; (d), (e) & (f) Phloroglu-cinol-HCl stained second, eighth, and seventeenth internodes of

the AtGA2ox8 transgenic plants, respectively. pf, pi, and x symbolize phloem fiber, pith, and xylem, respectively. Each experiment was repeated three times using at least three samples each with similar results

Fig. 1 Transformation and transgenic rapeseed plant screening

(a) Transgene expression cassette contains the CaMV35S promoter (35S), AtGA2ox8 gene, green fluorescent protein (GFP

) reporter gene, and herbicide Basta-resistant (Basta R ) select marker gene, and the positions of enzyme sites and the T-DNA left border (LB) and right border (RB) are shown; (b) Transgenic rapeseed plants were screened by herbicide Basta. Basta S or Basta R

symbolizes Basta-susceptible or Basta-resistance, respectively; (c) PCR analysis of Basta R gene in the transgenic rapeseed. DNA was extracted from the wild type (WT) and the Basta-resistant lines, respectively. P, 2, 3, WT, and M symbolize pEGAD plasmid, 35S::GFP-AtGA2ox8-2, 35S::GFP-AtGA2ox8-3, wild type, and marker, respectively; (d) & (e) GFP fluorescence imaging of transgenic plants with inverted fluorescence microscope. The images of the lateral root cap of the transgenic plant and WT are shown in (d) and (e), respectively 1017 bp

Sma I Xho I RB 35S GFP AtGA2ox8 35S Basta R

(a) P 2 3 WT M

500 bp

200 bp (d)(e)

(b) Basta S

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481 479

by C 20-GA2oxs (Sakamoto et al ., 2001; Schomburg et al ., 2003; Lee and Zeevaart, 2005). Hence, they can be converted to active C 19-GAs by GA 20-oxidase and GA 3-oxidase (Lo et al ., 2008). To date, there has been no report about whether altered GA metabolism might change the seed oil content of rapeseed, glucosinolate and erucic acid contents in seed oil. Experimental results obtained in this work revealed that, compared to the wild-type plants, the AtGA2ox8 over-expression lines could produce significantly more seed oil content. The eru-cic acid content also increased in the seed oil of the transgenic plants, but agreed with the criteria for double-low rapeseed species (Olsen and Soerensen, 1980; Sivaraman et al ., 2004). However, the glu-cosinolate content in the seed oil of the AtGA2ox8 over-expression lines was obviously lower than that of the wild type. These preliminary results suggested that AtGA2ox8 might be useful in rapeseed breeding without affecting the yield and quality of seed oil. More detailed studies are needed, however, to fully elucidate the effect of GA on the formation and qual-ity of seed oil.

Biomass measurements demonstrated that there were some significant differences between the wild

type and the transgenic lines. The fresh to dry weight ratio was higher in the transgenic plants than in the wild-type plant, which might be due to the altered deposition of lignin. The AtGA2ox8 over-expression

lines produced lower lignin content than the wild-type plants. The histochemical staining results also showed that the number of lignified vessels was less in the AtGA2ox8 over-expression lines, compared to the wild-type controls. The explanations of these results are as follows: GAs stimulate xylem fiber elongation (Eriksson et al ., 2000; Israelsson et al ., 2005) and lignin accumulation (Biemelt et al ., 2004). The AtGA2ox8 could 2β-hydroxylate the C 20-GA precursors and render them unable to be converted to

active GAs. The decrease in the levels of active GAs resulted in the decrease in lignin accumulation

(Schomburg et al ., 2003; Biemelt et al ., 2004). Lignin is a complex phenylpropanoid polymer, and lignin monolignols are synthesized through the phenylpro-panoid pathway (Boerjan et al ., 2003). To elucidate the cause for the effect of GA on lignin accumulation, we analyzed the transcript levels of seven key en-zymes in the lignin pathway and found that their transcripts were lower in the apical parts of the stems of the transgenic plants than in those of the wild type. 15 12 9 6 3 0 15 12 9 6 3 0 4C L r e l a t i v e e x p r e s s i o n

15 12 9 6 3 0

1 2 3

Plant

1 2 3 Plant

1 2 3

Fig. 7 Expression analysis of lignin biosynthetic genes in both the AtGA2ox8 transgenic plants and the wild type (WT) Plants were grown in a greenhouse, and apical parts of stems were collected during the flowering period. Total RNA was isolated for quantitative real-time

PCR analysis. (a) PAL1; (b) PAL2; (c) C4H ; (d) 4CL ; (e) HCT ; (f) CCR ;

(g) CAD . Data represent mean values with SDs obtained from three inde-pendent assays. * P <0.05, ** P <0.01, ***

P <0.001, significant difference be-tween the WT and transgenic plants. Plant: 1. WT; 2. 35S::GFP-AtGA2ox8-2; 3. 35S::GFP-AtGA2ox8-3 (a) (b) (c) (d) (e) (f) (g) *** *** *** *** *** *** ** ** ** ** * * ** **

15 12 9

6 3 0

15 12 9 6 3 0 151296301512

6301 2 3 1 2 3

1 2 3 1 2 3 P A L 1 r e l a t i v e e x p r e s s i o n

Plant C A D r e l a t i v e e x p r e s s i o n

Plant Plant

Plant Plant P A L 2 r e l a t i v e e x p r e s s i o n

C 4H r e l a t i v e e x p r e s s i o n

H C T r e l a t i v e e x p r e s s i o n

C C R r e l a t i v e e x p r e s s i o n

Zhao et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(7):471-481 480

The results demonstrate that changes in lignification in AtGA2ox8 over-expression lines might be due to the altered expression of biosynthetic genes. Similar results were obtained in AtGA2ox1 over-expression tobacco (Biemelt et al., 2004). Thus, our data suggest that GAs might affect plant lignification by regulating transcript levels of lignin biosynthetic genes.

The stems of the transgenic plants with low lig-nin content are conducive to rational use of resources and environment protection in the paper industry, and also more susceptible to fiber digestion by livestock (Fukushima and Dehority, 2000; Fukushima and Hatfield, 2004). Therefore, one of the major targets for intervention by genetic manipulation in our future work may be to inhibit GA biosynthesis and obtain plants with decreased lignin content. A focus will be placed on the molecular mechanism of gibberellins- regulated lignin biosynthesis.

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Introducing editorial board member:

Prof. Xuan-ming LIU, the corresponding author of this ar-

ticle, is the academic leader of a research group on plant

molecular biology in Hunan University. He gained his PhD

in 1995. Shortly after, he obtained a position of Professor

in 1999. His study is focused on functional genomics of

plant and on the molecular mechanism of phytohormone

metabolism. In addition, he is interested in biochemical

analysis of plant. In recent years, He dedicates to studying

the function of transcription fac-

tors and receptor-like kinases

(RLKs) in plant development,

which was supported by different

kinds of foundation, such as Na-

tional High-Tech R & D Program

(863), National Natural Science

Foundation, National Science and

Technology Major Project, and

International Coordination. Prof. Xuan-ming LIU