Planta

, Volume 241, Issue 1, pp 57–67 | Cite as

Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128

  • Yanlin Pan
  • Xin Ma
  • Hanwen Liang
  • Qian Zhao
  • Dengyun Zhu
  • Jingjuan Yu
Original Article

Abstract

Main conclusion

pF128 drivesGUS specifically expressed in transgenic seeds of foxtail millet and Zea mays with higher activity than the constitutiveCaMV35S promoter and the maize seed-specific19Z promoter.

Abstract

Foxtail millet (Setaria italica), a member of the Poaceae family, is an important food and fodder crop in arid regions. Foxtail millet is an excellent C4 crop model owing to its small genome (~490 Mb), self-pollination and availability of a complete genome sequence. F128 was isolated from a cDNA library of foxtail millet immature seeds. Real-time PCR analysis revealed that F128 mRNA was specifically expressed in immature and mature seeds. The highest F128 mRNA level was observed 5 days after pollination and gradually decreased as the seed matured. Sequence analysis suggested that the protein encoded by F128 is likely a protease inhibitor/seed storage protein/lipid-transfer protein. The 1,053 bp 5′ flanking sequence of F128 (pF128) was isolated and fused to the GUS reporter gene. The corresponding vector was then transformed into Arabidopsis thaliana, foxtail millet and Zea mays. GUS analysis revealed that pF128 drove GUS expression efficiently and specifically in the seeds of transgenic Arabidopsis, foxtail millet and Zea mays. GUS activity was also detected in Arabidopsis cotyledons. Activity of pF128 was higher than that observed for the constitutive CaMV35S promoter and the maize seed-specific 19 Zein (19Z) promoter. These results indicate that pF128 is a seed-specific promoter. Its application is expected to be of considerable value in plant genetic engineering.

Keywords

Foxtail millet Lipid-transfer proteins Promoter Embryo Endosperm transfer cell 

Introduction

Many factors affect the successful engineering of genetically modified (GM) crops. Constitutive overexpression of transgenes in plant tissues may sometimes lead to undesirable pleiotropic effects in transgenic plants and correct expression of specific genes has long been a concern. Therefore, the identification of gene promoters leading to specific expression patterns is crucial for the development of new GM plant generations (Zavallo et al. 2010).

The overall contribution of millets to global food production is small (1.5 % of cereal production), although they remain essential for the sustainability of food production in many developing countries, particularly in drought-prone areas. Foxtail millet (Setaria italica) is one of the most important millet crops. It is better adapted to dry, adverse soils than most other grain crops, such as maize and sorghum (Panaud 2006). It is often cultivated under harsh environmental conditions, such as in salinity- and drought-prone regions of India, China and Japan. It has the characteristics of drought tolerance, wide adaptability, self-pollination, rich genetic diversity and a small diploid genome (1C genome size = 490 Mb) (Doust et al. 2009). In recent years, a foxtail millet genetic transformation system has been established and refined (Liu et al. 2007; Qin et al. 2008). Considerable progress has been made on whole-genome sequencing of foxtail millet (Bennetzen et al. 2012; Zhang et al. 2012; Jia et al. 2013).

The most abundantly expressed genes in seeds code for seed storage proteins, oleosins, late embryogenesis-abundant (LEA) proteins and enzymes implicated in carbohydrate and lipid biosynthesis (Vicente-Carbajosa and Carbonero 2005). Seed-specific promoters can be divided into two categories: seed storage protein (SSP) gene promoters and non-storage protein (non-SSP) gene promoters. The former mainly comprises four groups: promoters of globulin, prolamin, glutelin and napins. Examples include the promoter of globulin-1 (Glb1) from maize (Zea mays) (Belanger and Kriz 1989; Hood et al. 2003), tobacco (Nicotiana tobacum.) (Liu and Kriz 1996) and rice (Oryza sativa) (Hwang et al. 2002), the promoter of prolamine Hor31 (D-Hordein) from barley (Hordeum vulgare) (Horvath et al. 2000), the promoters of glutelin Ax, 1Dx5 from wheat (Triticum aestivum) (Alvarez et al. 2000) and the promoter of Gt-1 from rice (Rascn-Cruz et al. 2004). Promoters of napin B and cBcNA1 (Radke et al. 1988; Kridl et al. 1991) from Brassica napus have also been identified. A number of genes encoding non-SSPs are abundantly expressed in seeds, including oleosins (Huang 1996), LEA proteins (Fujiwara et al. 2002), oleate desaturases (Chung et al. 2008) and sucrose synthases (Rasmussen and Donaldson 2006). The protein 19 Zein is a maize SSP. The 19 Zein mRNA is present from 10 days after pollination (DAP) through endosperm maturation. The mRNA level increases dramatically from 10 to 16 DAP, and then decreases as the seed matures (Kriz et al. 1987). The p19Z promoter has been activated in seeds of transgenic Petunia plants, showing maximum activity at 9–10 DAP and thereafter activity decreases during later stages of seed development (Quattrocchio et al. 1990), and in protoplasts isolated from maize endosperm suspension cultures and from native endosperms 10 DAP (Giovinazzo et al. 1992).

Endosperm transfer cells (ETCs) are involved in material exchange and mediate efficient uptake of nutrients from adjacent maternal vascular tissue to the endosperm (Zheng and Wang 2010; Motto et al. 2012). Many ETC-specific genes encode low molecular weight cysteine-rich proteins with N-terminal hydrophobic signal peptides (Hueros et al. 1995, 1998; Maitz et al. 2000; Serna et al. 2001; Cai et al. 2002; Guti rrez-Marcos et al. 2004; Li et al. 2008; Kovalchuk et al. 2009; Smirnova and Kochetov 2012). One class of these proteins is the lipid-transfer proteins (LTPs), which are defined by their ability to facilitate transfer of phospholipids between membranes in vitro. Although the biological function of LTPs remains elusive, several roles have been proposed and studied, including cutin formation, embryogenesis, pathogen defense responses, symbiosis and adaption to abiotic stresses (Kader 1996). Expression patterns reported for different plant LTP genes are complex and are largely temporally and spatially controlled (Kader 1997). In recent years, some LTPs have been reported to be expressed only in seeds. For example, LTP2 is expressed in barley seeds and the LTP2 promoter confers aleurone-specific expression in transgenic rice (Kalla et al. 1994). ZmEBE-1 and ZmEBE-2 are expressed in the embryo sac before pollination, and in both ETCs and embryo surrounding region (ESR) cells of the developing endosperm following pollination (Magnard et al. 2003). When the promoter of the sunflower (Helianthus annuus) LTP HaAP10 (Zavallo et al. 2010) was isolated and analyzed in transgenic Arabidopsis plants, GUS activity was detected upon seed maturity, but not in vegetative tissues with the exception of early seedling cotyledons. The promoters of OsPR602 and OsPR9a are active during early grain development in transgenic rice and barley (Li et al. 2008). The promoters of the homologs of OsPR602 in wheat, namely TdPR61 and TaPR60, are active in the ETCs, ESR and embryo (Kovalchuk et al. 2012).

In this study, we cloned and characterized the promoter of the seed-specific gene F128 from foxtail millet ‘Jigu 11’. F128 is an ortholog of TaPR60 from hexaploid wheat and OsPR602 from rice. The protein encoded by F128 may be a protease inhibitor/SSP/LTP. The spatial and temporal activity of the F128 promoter was analyzed by stable expression in Arabidopsis, Z. mays and S. italica, and by transient expression in S. italica and Z. mays using promoter–GUS reporter constructs. The F128 promoter drove GUS expression in the embryo and basal endosperm transfer layers (BETLs) in both maize and foxtail millet. However, the promoter exhibited less specific expression in Arabidopsis, which showed GUS activity in the cotyledons of seedlings. pF128 activity was higher than that of the constitutive promoter CaMV35S and maize seed-specific 19Z promoter. The potential use of pF128 in plant genetic engineering is discussed.

Materials and methods

RT-PCR analysis

The RNA levels of F128, in different millet tissues, were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was isolated from shoots of 3-day-old germinated seeds, roots and leaves of 14-day-old seedlings, stems at flowering, inflorescences before pollination, mature anthers and developing seeds at 5, 10, 15, 20 and 25 DAP using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Primers for amplification of the F128 gene (GenBank accession no. AY327511) and the endogenous reference Actin gene (GenBank accession no. AF288226) used for qRT-PCR and semi-qRT-PCR are listed in Supplemental Table S1. The qRT-PCR analysis was performed using the Ultra SYBR Mixture (CWBIO, Beijing, China) on a Lightcycler 480 Real Time PCR System (Roche, Indianapolis, IN, USA). All reactions were performed in triplicate. The cycling parameters were as follows: 94 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. The 2−ΔΔCT method (Livak and Schmittgen 2001) was used to calculate relative gene expression levels; these were normalized to the Actin expression level. Semi-quantitative RT-PCR cycling parameters were as follows: 94 °C for 1 min, followed by 25 cycles of 94 °C for 1 min, 60 °C for 30 s and 72 °C for 30 s.

Promoter isolation and plasmid construction

The 5′-UTR of F128 was amplified from foxtail millet by thermal asymmetric interlaced PCR. The procedure was in accordance with that described by Liu and Whittier (1995) with slight modifications. Primers used for amplification of pF128 and the thermal cycling conditions are given in Supplemental Tables S1 and S2. A ~1.2 kb fragment was amplified and sequenced (Invitrogen, Shanghai, China). The fragment contained 150 bp of the 5′ F128 cDNA coding sequence. Next, a 1,053 bp fragment upstream from the translational start site of F128 (pF128) was amplified using the primers YB128 s and YB128a, and the 19Z promoter was amplified using the primers p19zs and p19za (Supplemental Table S1). The fragments pF128 and p19Z were confirmed by sequencing, and then cloned into the pBI121 and pCAMBIA2300 plasmids, replacing the CaMV 35S promoter, to construct the promoter and GUS fusion vector used for transformation. Schematic diagrams of the constructs are shown in Supplemental Fig. S1. The constructs pBIpF128 and pBIp19Z were used for Arabidopsis transformation, pCAMBIA2300-F128-GUS, pCAMBIA2300-19Z-GUS and pCAMBIA2300-35S-GUS for foxtail millet transformation and pF128::GUS, p19Z::GUS and p35S::GUS for maize transformation.

Transformation and regeneration procedures

Arabidopsis thaliana transformation

The Arabidopsis thaliana ecotype Columbia-0 was transformed with Agrobacteriumtumefaciens strain GV3101 harboring pBIp128 or pBIp19Z using the floral dip method (Clough and Bent 1998). Putative transgenic Arabidopsis plants were selected on Murashige and Skoog (MS) culture medium containing 50 mg/l kanamycin. Resistant transgenic plants were confirmed by PCR using the primers gus1 and gus2 (Supplemental Table S1). The T2 generation of three independent lines for each promoter was used for subsequent analysis.

Foxtail millet transformation

The constructs pCAMBIA2300-F128::GUS, pCAMBIA2300-19Z::GUS and pCAMBIA2300-35S::GUS were introduced into foxtail millet calli induced from immature inflorescences mediated by A. tumefaciens strain LBA4404 (Qin et al. 2008; Wang et al. 2011). Following co-cultivation at 22 °C in the dark for 3 days, explants were washed with 500 mg/L cefotaxime sodium salt, rinsed with sterile distilled water and transferred to an induction medium (MS medium supplemented with 500 mg/l cefotaxime sodium salt, 2 mg/l 2,4-D, 1 g/l proline, 0.8 g/l casein, 5 mg/l AgNO3 and 30 g/l sucrose) for a 7-day recovery period. The calli were then transferred to the same medium supplemented with 80 mg/l kanamycin for 2 weeks and then to a medium containing 90 mg/l kanamycin for a further 2 weeks to select resistant calli; this procedure was performed in the dark at 28 °C. After 4 weeks of selection, resistant calli were maintained on a differentiation medium (MS medium supplemented with 250 mg/l cefotaxime sodium salt, 2 mg/l 6-BA, 0.5 mg/l NAA, 30 g/l sucrose, pH 5.8) under a 16 h light/8 h dark photoperiod. Developing shoots, approximately 3-cm long, were transferred to a root induction medium (1/2 MS medium supplemented with 0.5 mg/l NAA and 30 g/l sucrose, pH 5.8). After 2–3 weeks, plantlets with vigorous roots were transferred to pots and grown in a greenhouse. The regenerated plants were further confirmed by PCR using the primers gus1 and nosp2 (Supplemental Table S1). Independent T1 lines of the F128 promoter, 19Z promoter and 35S promoter were used for subsequent analysis.

Maize transformation

The constructs pF128::GUS, p19Z::GUS and p35S::GUS were transformed into calli induced from immature embryos of the maize hybrid Z31 × Q31 by particle bombardment. Resistant calli were screened on NB medium containing 20 mg/l hygromycin, as described by Wang et al. (2006). Transgenic plants were confirmed by PCR using the primers gus1 and gus2. Independent T1 transgenic lines of the F128 promoter, 19Z promoter and 35S promoter were used for subsequent analysis.

Transient expression analysis

Maize kernels were harvested at 10, 12 and 18 DAP. After surface sterilization with 70 % ethanol for 1 min, the kernels were cut longitudinally into two halves and incubated on a co-culture medium (N6 medium supplemented with 0.69 g/l proline, 0.2 g/l casein, 30 g/l sucrose, 38 mg/l glycine, 0.4 mol/l mannitol and 2 mg/l 2,4-D) until ready for bombardment with DNA-coated gold particles (Bio-Rad, Hercules CA, USA). The coating and bombardment were performed in accordance with the method described by Wang et al. (2006). Following bombardment, tissue samples were incubated for 24 h at 28 °C in the dark on co-culture medium. Subsequently, the tissue samples were used for GUS staining.

Immature seeds of foxtail millet harvested at 3, 5, 10, 15 and 20 DAP. After surface sterilization with 70 % ethanol for 1 min, the seeds were cut longitudinally into two halves and incubated on co-culture medium (MS medium supplemented with 1 g/l proline, 0.8 g/l casein, 15 g/l glucose, 2 mg/l 2,4-D and 100 μmol/l AS) until ready for infection by A. tumefaciens LBA4404 harboring the promoter–GUS construct. Then, tissue samples were incubated for 3–5 days at 22 °C in the dark on co-culture medium. Subsequently, the tissue samples were used for GUS staining.

GUS activity analysis

GUS activity in transgenic Arabidopsis, foxtail millet and maize was analyzed by histochemical staining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc; Bio Vectra, Oxford, CT, USA). Root, stem, leaf, flower, the whole grain and grain sections at different development stages were immersed in GUS staining buffer (1 mM X-Gluc solution in 100 mM sodium phosphate buffer [pH 7.0], 0.5 M EDTA, 5 mM FeK3(CN)6, 5 mM K4Fe(CN)6, and 0.1 % Triton X-100). Following vacuum infiltration, samples were incubated at 37 °C until staining was observed. Tissues were incubated in 70 % ethanol for decolorization and observed under a dissecting microscope (Olympus SEX16, Tokyo, Japan). Samples were embedded in paraffin wax and 10 μm sections were prepared as described by Weigel and Glazebrook (2002). Photographs were taken using a digital camera (Canon, Tokyo, Japan). Fluorometric measurement of GUS activity was recorded as described by Jefferson et al. (1987). 4-Methylumbelliferone β-D-glucuronide hydrate (MUG) (Sigma, St Louis, MO, USA) was used as the substrate. Fluorescence was detected using a fluorescence spectrophotometer F-4500 (Hitachi, Tokyo, Japan). GUS protein concentrations were determined using the method described by Bradford (1976), and GUS enzyme activity was expressed as nanomoles of 4-MU produced per gram protein per minute. Statistical analysis of GUS activity among groups was performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Average values were calculated after data normalization of the mock-treated plant.

Results

Characterization of F128 expression in foxtail millet

The F128 gene was cloned from foxtail millet ‘Jigu 11’. The gene contained 577 nucleotides and the deduced protein comprised 109 amino acid residues. Analysis with SignalP 3.0 (Bendtsen et al. 2004) indicated the presence of an N-terminal hydrophobic signal peptide of 24 amino acid residues and implied that F128 is most likely secreted at the apoplast. After cleavage of the N-terminal hydrophobic signal peptide, F128 produced a mature protein of 85 amino acids, with a predicted molecular mass of 9.1 kDa. Previous analysis revealed that F128 mRNA was detected in 5, 10, 15, 20 and 25 DAP seeds and not detected in other tissues including the seedling, root, stem, leaf and inflorescence (Xue et al. 2004). In the present study, to confirm the F128 expression pattern, semi-qRT-PCR of grains at 5, 10, 15, 20 and 25 DAP was performed using primers based on the coding region of F128. The quantity of F128 mRNA peaked at 5 DAP and gradually decreased during subsequent seed development (Fig. 1a). The qRT-PCR analysis of the root, stem, leaf, shoot, inflorescence, anther and grain at 5, 10, 15, 20 and 25 DAP showed that F128 mRNA was only detected in the grain (Fig. 1b) with the same expression pattern as indicated by semi-qRT-PCR analysis. The F128 mRNA was detected in the endosperm at 10, 15 and 20 DAP and in the embryo at 10 DAP, with the highest quantity detected at 10 DAP in the endosperm (Fig. 1c).
Fig. 1

Expression of F128 in a immature seeds of foxtail millet by semi-quantitative RT-PCR, b different tissues of foxtail millet and c the embryo and endosperm 10, 15 20 DAP by qRT-PCR. Roots, stems, leaves, shoots, inflorescences and anthers were collected from eight to ten plants and pooled, and grains at each developmental stage were collected from the middle of several spikes from each of three to four plants and pooled. Each data point is the mean of three replicate RT-PCR reactions. DAP days after pollination, EN endosperm, EM embryo

Isolation and analysis of the F128 promoter

The 1,053 bp fragment upstream from the translational start site of the foxtail millet F128 gene was cloned. Analysis using PLACE software (Lescot et al. 2002; http://www.dna.Affrc.go.jp/PLACE/signalscan.html) revealed several putative seed-specific cis-elements in the F128 promoter (Table 1), including an RY/G-box (a complex containing two RY repeats and a G-box), E-box, three DPBFCOREDCDC3 motifs, ACGTOSGLUB1 and AACACOREOSGLUB1. The RY motif is important for high-level gene expression in seeds and inhibits gene expression in non-seed tissue components. The RY/G-box of the napA gene in Brassica napus is required for seed-specific expression (Ezcurra et al. 1999; 2000). The E-box is a plant transcription element important in the regulation of temporal and spatial expression of seed-specific genes (Stalberg et al. 1996). The DPBFCOREDCDC3 elements are likely required for embryo-specific expression. This element can be bound by bZIP transcription factors which regulate a subset of late embryogenesis-abundant genes (Soderman et al. 2000). ACGTOSGLUB1 is essential for the expression of the β-phaseolin gene during embryogenesis in tobacco and Arabidopsis (Washida et al. 1999; Wu et al. 2000). The AACACOREOSGLUB1 motif is involved in the control of endosperm-specific expression of glutelin genes (Wu et al. 2000).
Table 1

Putative seed-specific cis-elements in the F128 promoter sequence

cis-Element

Consensus

Detected in the pF128 sequence

Motif position

Reference

RY repeat

CATGCA

CATGCA

−221 to −226

Ezcurra et al. (1999)

−251 to −256

Ezcurra et al. (2000)

−687 to −692

 

G-box

CANNTG

CACGTG

−322 to −327

Ezcurra et al. (1999)

CATTTG

−232 to −237

Stalberg et al. (1996)

−292 to −297

−762 to −767

DPBFCOREDCDC3

ACACGTG

ACACGTG

−202 to −208

Kim et al. (1997)

−262 to −268

Soderman et al. (2000)

−307 to −313

 

ACGTOSGLUB1

GTACGTG

GTACGTG

−854 to −860

Wu et al. (2000)

AACACOREOSGLUB1

AACAAAC

AACAAAC

−698 to −704

Wu et al. (2000)

Characterization of F128 promoter in Arabidopsis thaliana

To characterize the F128 promoter, pF128 (CN 101063139A) was fused with GUS (pBIpF128) and transformed into Arabidopsis. Additionally, the 19Z promoter fused with GUS (pBIp19Z) was transformed into Arabidopsis and used as a control. Fifty-four ArabidopsispBIpF128 transgenic lines and 14 pBIp19Z transgenic lines were confirmed by PCR. Three independent T2 lines carrying the single-copy insertion were selected from each pBIpF128 and pBIp19ZArabidopsis transgenic line for GUS histochemical staining. In pBIpF128 lines, no GUS expression was detected in the root, stem, leaf or flower (Fig. 2a–d). GUS expression was detected in the 10, 15 and 20 DAP immature seeds and mature seeds, and the GUS staining signal gradually increased in intensity as the seed matured (Fig. 2e–h). Seedlings of the transgenic Arabidopsis lines did not display GUS activity in other organs except the cotyledons (Fig. 2i–o). Quantitative analysis of GUS enzyme activity also revealed the trend for increased GUS activity as the seed matured (Fig. 2t). The 19Z promoter exhibited a similar GUS staining pattern to that of the F128 promoter in Arabidopsis cotyledons at different growth stages (Fig. 2p–s). However, the GUS staining signal was weaker in the seed at 15 DAP for pBI19Z (Fig. 2j) than that of pBIpF128 (Fig. 2i). The GUS staining signal in pBIpF128Arabidopsis plants gradually increased as the seed matured, whereas the quantity of F128 mRNA in foxtail millet decreased as the seed matured (Fig. 1). This inconsistency may be caused by differences in mRNA stability and/or in GUS protein stability in these species.
Fig. 2

Spatial and temporal GUS expression in transgenic Arabidopsis thaliana. ad Root, stem, leaf and flower of the pBIpF128 transformant; eh immature and mature seeds of the pBIpF128 transformant at 10, 15 and 20 DAP; ipBIpF128 transgenic silique and immature seeds at 15 DAP; jpBIp19Z transgenic silique and immature seeds at 15 DAP; k seeds from an untransformed plant at 15 DAP as a negative control; lo various growth stages of ArabidopsispBIpF128 seedlings; ps various growth stages of Arabidopsis pBIp19Z seedlings. The histochemical GUS assay was performed during seedling development in three representative homozygous transgenic Arabidopsis lines carrying each promoter construct. The results are shown for one representative line. t Quantitative analysis of GUS activity in different tissues of ArabidopsispBIpF128. 10 DAP, 15 DAP and 20 DAP, immature seeds; MS mature seeds, DAP days after pollination

Spatial and temporal activity of the F128 promoter in transgenic foxtail millet

To analyze pF128 activity in foxtail millet, pCAMBIA2300-F128::GUS was transformed into callus induced from the immature inflorescence of foxtail millet and 13 transgenic lines were obtained. Additionally, pCAMBIA2300-19Z::GUS and pCAMBIA2300-35S::GUS were transformed into foxtail millet as controls, for which 11 and 20 transgenic lines were obtained, respectively. Three transgenic lines that showed positive GUS staining for each promoter were analyzed further.

GUS signals were not detected in the root, stem, leaf, rachilla or inflorescence of pCAMBIA2300-F128::GUS plants (data not shown). GUS staining was detected in all tissues of the seed at 10 DAP, including the embryo and endosperm, and represented the peak signal intensity observed (Fig. 3a). The strongest GUS staining signal was detected in the aleurone layer, embryo and BETLs (Fig. 3j), and the signal intensity decreased in 15 DAP seeds (Fig. 3b). Signals were not detected in the embryo and endosperm, with the exception of the BETL, in 20 DAP seeds (Fig. 3c). In pCAMBIA2300-19Z::GUS plants, GUS staining was detected mainly in the endosperm of 10 DAP seeds (Fig. 3d, k), and in the BETL of 15 DAP seeds (Fig. 3e). No signal was detectable in 20 DAP seeds (Fig. 3f). In pCAMBIA2300-35S::GUS plants, GUS staining was detected in the whole seed at 10 DAP (Fig. 3g), but at a much lower intensity than that observed for the F128 promoter, and no signal was detected in 15 and 20 DAP seeds (Fig. 3h, i). Quantitative fluorometric assays were performed on three independent lines of each promoter–GUS reporter construct (Fig. 3l). GUS activities controlled by the F128 promoter were higher than those driven by the 19Z and 35S promoters.
Fig. 3

Analysis of GUS expression in T1 transgenic foxtail millet. ac GUS expression driven by pF128 immature seeds at 10, 15 and 20 DAP; df driven by p19Z immature seeds at 10, 15 and 20 DAP; gi driven by p35S immature seeds at 10, 15 and 20 DAP. j, k Longitudinal section (10 μm thick) of immature seed at 10 DAP driven by pF128 (j) and p19Z (k). Bars 1 mm. l Quantative fluorometric assays of GUS activity for each promoter–reporter construction were performed on three independent lines of foxtail millet immature seeds at 10 DAP (P < 0.05). EN endosperm, EM embryo, AL aleurone layer, BETL base of endosperm cell layer, DAP days after pollination

With the advantage of no effect of the insertion sites, the transient expression was used to analyze promoter activity. Foxtail millet immature seeds at 3, 5, 10, 15, 20 and 25 DAP were infected with A. tumefaciens LBA4404 harboring pCAMBIA2300-F128::GUS and co-cultured for 3 days. The GUS staining patterns were similar to those of the stable expression system (Fig. 4). GUS expression was detected in the entire embryo sac and lodicule of 3 and 5 DAP seeds (Fig. 4a, b), and also in the whole seed at 10 DAP, including the embryo and endosperm (Fig. 4c). As the seed matured, the GUS staining signal weakened and was mainly detected in the embryo and BETL (Fig. 4d, e). GUS activity was no longer detectable in mature seeds at 25 DAP (Fig. 4f).
Fig. 4

Transient expression of pF128::GUS in immature seeds of foxtail millet. a 3 DAP, b 5 DAP, c 10 DAP, d 15 DAP, e 20 DAP and f 25 DAP. ES embryo sac, EN endosperm, EM embryo, BETL base of endosperm cell layer, DAP days after pollination

Spatial and temporal activity of the F128 promoter in transgenic maize

Maize seed proteins mainly lack essential amino acids, such as lysine and tryptophan, which limits the use of these proteins. Seed-specific expression of high-lysine proteins is an effective strategy for improvement of maize protein quality. Therefore, evaluation of pF128 activity in maize potentially provides an applied promoter for maize quality improvement.

The pF128, p19Z and p35S promoters were fused with GUS and transformed into maize calli induced from immature embryos by particle bombardment. Eight pF128::GUS transgenic lines, nine p19Z::GUS transgenic lines and ten p35S::GUS transgenic lines were obtained. F128 promoter activity was detected in immature seeds at 20 and 25 DAP, with GUS activity observed in both the embryo and endosperm (Fig. 5a, b). The strongest GUS staining signal was detected in the BETL (Fig. 5a, j, k), which plays an important role in nutrient transportation to the endosperm and embryo. The F128 promoter activity gradually decreased in the endosperm, but was maintained in the embryo (Fig. 5b) at later DAP stages. GUS activity in seeds of p19Z::GUS lines (Fig. 5c, d) was as strong as that observed in the pF128::GUS lines (Fig. 5a, b). The GUS staining signal of the 35S promoter (Fig. 5e, f) was much lower than that observed for the F128 and 19Z promoters.
Fig. 5

Analysis of GUS expression in transgenic maize seeds. af GUS staining of immature seeds from T0 transgenic maize. a, bpF128::GUS immature seeds at 20 and 25 DAP; c, dp19Z::GUS immature seeds at 20 and 25 DAP; e, fp35S::GUS immature seeds at 20 and 25 DAP; gi GUS transient expression analysis. Immature maize seeds were transformed with pF128::GUS by particle bombardment and GUS staining was conducted; g immature seed at 10 DAP, h immature seed at 12 DAP, i immature seed at 18 DAP. j, k Longitudinal section (10 μm thick) of pF128::GUS immature seed at 20 DAP. Bars 1 mm. l Quantitative analysis of GUS activity in T1 transgenic maize. Each promoter–reporter construct was performed on immature seeds of two independent lines at 20 DAP. Each data point is the mean of three replicate measurements of GUS enzyme activity (P < 0.05). EN endosperm, EM embryo, BETL base of endosperm cell layer, AL aleurone layer, DAP days after pollination

GUS transient expression of the F128 promoter by biolistic bombardment was performed concurrently using immature seeds. In seeds at 10 DAP, the F128 promoter was active in the whole seed, with GUS activity detected mainly in the aleurone layer and in sub-aleurone endosperm cells (Fig. 5g); this pattern was maintained in seeds at 12 DAP (Fig. 5h). As the seed matured, the embryo volume gradually increased and GUS activity was mainly detected in the embryo at 18 DAP and was not observed in the endosperm (Fig. 5i). Quantitative analysis of GUS activity was performed on two independent lines for each promoter–GUS reporter construct (Fig. 5l). GUS activities for the F128 promoter were higher than those of lines carrying the 19Z and 35S promoters.

Discussion

F128 cDNA was isolated from a cDNA library prepared from immature seeds of foxtail millet. A blastx search of the NCBI database revealed that the putative conserved domain of the F128 protein belonged to the nsLTP-like protein family. Protein sequence by hydrophobic cluster analysis indicated that the positions of eight cysteine residues were highly conserved (Fig. S2a; marked by arrowheads), the cysteine residues in F128 could form disulfide bridges and the associated parts of the sequences could form α-helical secondary structural elements. The SWISS-MODEL tool (Arnold et al. 2006) was used to search templates for the F128 protein. The most appropriate structural template for the F128 protein was the lipid-binding protein 2rkn:A from Arabidopsis thaliana. The positional sequence identity and similarity between 2rkn:A and F128 were 26 % and 65 %, respectively. These scores reflected a high complexity of modeling, because MODELLER typically requires at least 30 % homology to obtain alignments of sufficient quality to produce an accurate structural model for a query protein sequence (Sanchez and Sali 1998).

OsPR602 and TaPR60 are homologous genes that show seed-specific expression. The proteins encoded by OsPR602 and TaPR60 are LTPs, and are mainly expressed in the early stages of rice and wheat grain development following pollination (Li et al. 2008; Kovalchuk et al. 2009). Multiple alignment of F128 protein sequences with OsPR602 and TaPR60 revealed 65.1 and 48.6 % identity, respectively, and the eight cysteine residues were highly conserved in all three proteins (Fig. S2b). The F128 protein has the typical structure of an LTP, including an N-terminal hydrophobic signal peptide of 24 amino acid residues, eight highly conserved cysteine residues, four α-helices in the secondary structure, a molecular weight of 9.1 kDa and pH 8.5. These results support the conclusion that the F128 protein belongs to the LTP family and suggest that it may play a role in seed development.

The same promoter can display different spatial and temporal activities in different species, and the same tissue-specific promoter can be significantly spatially and temporally different in diverse cereal crops. For example, the temporal expression patterns of the OsPR602 promoter are not consistent during seed development in rice and barley, with GUS expression first detectable 3 days earlier and ceasing 6 days earlier in rice compared with barley. The spatial patterns and strength of the TdPR61 promoter driving GUS expression in the embryos of wheat, barley and rice also differ (Kovalchuk et al. 2009, 2010; Li et al. 2008). This phenomenon was also observed for the F128 promoter in Arabidopsis, foxtail millet and maize. GUS activity driven by the F128 promoter in seeds was mainly concentrated in the embryo and BETL in maize and foxtail millet, whereas in Arabidopsis staining was detected in the whole seed and increased as the seed matured. That the expression patterns of the F128 promoter in Arabidopsis thaliana are not consistent with those in maize and foxtail millet is probably due to the different structure of seeds in dicot and monocot plants. During seed development in Arabidopsis, the embryo gradually occupies the entire seed, with the endosperm gradually disappearing, whereas in monocot seeds, the endosperm constitutes most of the total seed volume. Furthermore, GUS expression was detected in the seedling cotyledons of pF128 and p19Z transgenic plants, which might be a consequence of residual β-glucuronidase protein in the seeds. The promoters of sunflower LTP HaAP10 and oleate desaturase HaFAD2-1 (Zavallo et al. 2010) showed similar results to those observed for the F128 promoter in transgenic Arabidopsis. However, the GUS activity driven by the F128 promoter was stronger and longer lasting than that driven by the HaAP10 and HaFAD2-1 promoters.

Stable expression of the F128 promoter in foxtail millet and maize, and its associated GUS expression were not consistent during seed development. This may be because of the different periods over which foxtail millet and maize seeds develop. Foxtail millet seeds mature after about 25 days, whereas maize seeds take approximately 35 days to mature. Grain filling of seeds is essentially completed by 15 DAP in foxtail millet (Li 1997), whereas 28 days are required for completion of grain grouting in maize (Ingle et al. 1965). A high level of GUS expression in foxtail millet and maize seeds occurs earlier under transient expression than under stable expression and most likely is a result of β-glucuronidase mRNA stability and GUS protein stability in cells, rather than a result of sustained expression. Similar GUS expression patterns driven by the F128 promoter were observed in foxtail millet and maize, with the highest GUS enzyme activity detected in the early stages of seed development. The GUS activity was mainly located in areas of active energy metabolism in the embryo, ETC and BETL. The BETL plays a role in the transfer of nutrients and acts as a barrier between pathogenic bacteria and the seed (Thompson et al. 2001; Offler et al. 2003). Seed size and shape are mainly determined at an early stage of cell proliferation and differentiation. Therefore, to alter grain size and shape, expression of transgenes is required before or during cellularization and in tissues involved in the transfer of nutrients to the developing endosperm, such as the ETC layers (Zee and O’Brien 1971; Zheng and Wang 2010). We also observed that the patterns of pF128 and p19Z activity are very similar in foxtail millet and maize. This is consistent with the similar patterns of F128 mRNA and 19 Zein mRNA accumulation observed during seed development.

The F128 promoter is the first seed-specific promoter reported in foxtail millet. Activities of F128 in the dicot Arabidopsis, and in the monocots foxtail millet and maize, are significantly higher than those observed for the 35S promoter and slightly higher than those for the maize 19Z promoter. Various seed-specific promoters have been employed to restrict recombinant protein expression to different parts of the seed (Stoger et al. 2005), leading to beneficial alterations in grain size, shape and composition. The 19Z promoter isolated from maize has already been applied for crop genetic breeding (Yu et al. 2004; Wang et al. 2013). The F128 promoter, which shows higher activity in Arabidopsis, foxtail millet and maize, shows great potential for use in plant molecular breeding programs and to facilitate improvements in GM crop quality.

Notes

Acknowledgments

This work was supported by the National Transgenic Major Program of China (grant no. 2008ZX08003-002, 2009ZX08009093-002, 2011ZX08003-002 and 2013ZX08003-002).

Supplementary material

425_2014_2164_MOESM1_ESM.docx (960 kb)
Supplementary material 1 (DOCX 960 kb)
425_2014_2164_MOESM2_ESM.pdf (106 kb)
Supplementary material 2 (PDF 106 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yanlin Pan
    • 1
  • Xin Ma
    • 1
  • Hanwen Liang
    • 1
  • Qian Zhao
    • 1
  • Dengyun Zhu
    • 1
  • Jingjuan Yu
    • 1
  1. 1.State Key Laboratory of Agrobiotechnology, College of Biological SciencesChina Agricultural UniversityBeijingChina

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