Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128
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.
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.
KeywordsFoxtail millet Lipid-transfer proteins Promoter Embryo Endosperm transfer cell
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 Hor3–1 (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
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.
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.
Characterization of F128 expression in foxtail millet
Isolation and analysis of the F128 promoter
Putative seed-specific cis-elements in the F128 promoter sequence
Detected in the pF128 sequence
−221 to −226
Ezcurra et al. (1999)
−251 to −256
Ezcurra et al. (2000)
−687 to −692
−322 to −327
Ezcurra et al. (1999)
−232 to −237
Stalberg et al. (1996)
−292 to −297
−762 to −767
−202 to −208
Kim et al. (1997)
−262 to −268
Soderman et al. (2000)
−307 to −313
−854 to −860
Wu et al. (2000)
−698 to −704
Wu et al. (2000)
Characterization of F128 promoter in Arabidopsis thaliana
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.
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.
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.
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.
This work was supported by the National Transgenic Major Program of China (grant no. 2008ZX08003-002, 2009ZX08009093-002, 2011ZX08003-002 and 2013ZX08003-002).
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