Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.


Introduction
Starch is the main carbohydrate consumed for human nutrition, and is a major component of cereals, tubers, legumes, and fruits. Starch consists of a mixture of two different carbohydrates, namely amylose (20%-30%) and amylopectin (70%-80%) [1][2][3]. Amylose is a linear polymer of glucose residues joined together by a-1, 4glucosidic bonds, and its synthesis is mainly catalyzed through the activity of granule-bound starch synthase (GBSS). GBSS transfers the glucosyl residue from ADP-Glu to glucan substrates to produce relatively long-chain amylose molecules [4]. The amylose content directly affects the texture and taste of cereals grains [5]. The physical structure of amylopectin has an important impact on the crystalline properties of maize [5], and its synthesis requires soluble starch synthases (SSs), starch branching enzymes (SBEs), and starch debranching enzymes (DBEs) [4].
GBSS proteins are crucial in regulating the formation of amylose. Inhibition of GBSSI expression by RNAi interference resulted in an amylose-free transgenic sweet potato [6]. GBSS activity and amylose content also decreased significantly after RNA silencing of GBSSI gene expression in the endosperm of wheat [7]. A full-length sense cDNA encoding sweet potato GBSSI, driven by the CaMV 35S promoter, was introduced into sweet potato by Agrobacterium tumefaciens-mediated transformation. One of the resulting 26 transgenic plants had an absence of amylose in the tuberous roots [8]. Recently, through regulated GBSS gene expression in sweet potato, starch has been produced with varying amylose-amylopectin ratios. Low-amylose starch may be used in the food industry, while high-amylose starch is useful in the candy industry and for synthesizing plastics [9].
GBSS proteins are also known as waxy proteins [10]. In monocots, GBSS is divided into two families, i.e. GBSSI and GBSSII. GBSSI transcripts are predominantly found in endosperm, embryos and pollen, while GBSSII transcripts are expressed in non-storage tissues such as leaves, stems, roots, and other organs [11]. In contrast, all GBSS genes in eudicots belong to the GBSSII family and their expression was consistent with the pattern observed for GBSSII genes in monocots. This suggests that the GBSSI subfamily has been lost during the evolutionary process leading to eudicots [1]. To date, the sequence and expression of GBSSI genes has been characterized from maize [12], rice [13], barley [5], wheat [11], pea [14], potato [15], and sweet potato [8]. GBSSII genes were isolated from rice [13], wheat [11], apple, peach, and orange [1]. However, the complete sequence and expression patterns of these genes from banana fruit have not been reported yet. To facilitate further studies of banana fruit starch, it is essential to characterize the banana GBSSI and GBSSII genes in banana.
Banana (Musa spp.) is the main staple food of the tropics. Its fruits are vital for food security in many tropical and subtropical counties and banana is also among the most popular fruits in industrialized countries [16][17]. Starch is the principal component of green banana fruit and is present at levels of approximately 61.3-76.5%, which is sufficient for industrial-scale purification of starch [12]. Recently, a complete genome sequence has been released for banana (http://banana-genome.cirad.fr/). This genome sequence database provides unique opportunities for the genome-wide investigation of genes involved in banana fruit starch synthesis. In this study, we investigated the changes in amylose content, GBSS enzyme activity and GBSS protein from preharvest to postharvest of banana (Musa acuminata L. AAA group cv. Brazilian) fruit. Six MaGBSS genes were cloned, and their sequence characteristics, chromosomal location, and expression patterns were studied in different tissues and at different stages of fruit development or storage. The number, size, and shape of starch granules in banana fruit during development and storage were also observed.

Changes in total starch content, amylose content and GBSS activity
Total starch content increased gradually during the development of banana fruit but decreased after harvest ( Fig. 1A and 1B). Pulp amylose content increased from 0 d to 50 d of fruit development and declined at the 60 d time point. The amylose content also decreased gradually from 0 d to 30 d of storage ( Fig. 1C and 1D). An increase in GBSS activity occurred from 0 d to 50 d of development but declined at 60 d, while GBSS activity decreased sharply from 0 d to 30 d of storage ( Fig. 1E and 1F).

SDS-PAGE and western blotting analysis of GBSS protein
To determine whether banana fruit starch contains GBSS protein, starch granules in banana pulp were isolated; GBSS protein was purified and analyzed on SDS-PAGE gels ( Fig. 2A). The 55.0 kDa GBSS protein was migrated in the pulps by western blotting analyses (Fig.2B). The expression of GBSS protein was gradually up-regulated during the development of banana fruit, but a decrease was observed from 0 d to 30 d of storage (Fig. 2B). These SDS-PAGE and western blotting results are consistent with the changes in amylose content ( Fig. 1C and 1D) and GBSS activity ( Fig. 1E and 1F) during banana development and storage.
Amino acid sequence analysis showed that the MaGBSSI-1 had the closest relationship with MaGBSSI-2 and shared 82% amino acid identity, followed by MaGBSSI-4 and MaGBSSI-3 (Fig. 3). MaGBSSII-1 had the closest relationship with MaGBSSII-2 and shared 84% amino acid identity, followed by Vitis vinifera (XP002278470) (Fig. 3). A phylogenetic tree showed that six MaGBSS amino acid sequences from banana fruit are very

Expression of MaGBSSI and MaGBSSII genes in banana tissues
Q-RTPCR revealed significant differences in the expression of MaGBSSI and MaGBSSII in different banana tissues (Fig. 4). MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1, and MaGBS-SII-2 were upregulated in vegetative tissue such as root, stem, leaf, and bract. In contrast, MaGBSSI-3 was highly expressed in reproductive tissues such as flower, peel, and pulp, but was weakly expressed in root, stem, and leaf. These results suggest that MaGBSSI-3 might be involved in amylose metabolism in reproductive tissues.

Expression of six MaGBSS genes and scanning electron microscopy (SEM) of starch granules at different development stages of banana fruit
The expression of MaGBSSI and MaGBSSII genes at different development stages of banana fruit was determined by Q-RTPCR. Expression levels of MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 at earlier stages of banana development (from 0 d to 30 d) were higher than the later stages (from 30 d to 60 d). In contrast, MaGBSSI-3 was weakly expressed at the early stages but was highly upregulated (approximately 50-fold) at 50 d of development (Fig. 5A). These results suggest that the MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 might be involved in the early stages (0-30 d) of starch granule-filling, and MaGBSSI-3 might be involved in the later stages (30-60 d) of starch granule-filling during the development of banana fruit (Fig. 5A).
Starch granules within banana fruit at 0 d, 30 d, and 60 d of fruit development were observed using SEM. Granules could scarcely be observed at 0 d, but the number and size of ovalshaped granules significantly increased at 30 d of development. In comparison to the granules at 30 d, the number and shape of granules at 60 d was similar, but they were significantly larger (Fig. 5B). These results suggest that the first 30 d of fruit development may be critical for settling the final number and shape of starch granules in banana fruit. 30-60 d of fruit development appears to be the rapid filling phase of starch granules. These results are consistent with the expression profile of MaGBSSI-3 during the development of banana fruit. Starch granules were also observed in mature banana fruit using SEM after 0 d, 5 d, and 10 d of storage. Starch granules were detected at the first time point (0 d) and the number of granules decreased significantly thereafter. Fewer granules were observed at 5 d of storage and granules could not be detected by 10 d of storage (Fig. 6B). These results suggest that starch granules are

Discussion
Changes in amylose content, GBSS enzyme activity, and GBSS protein Amylose content is an important factor contributing to the yield and quality of banana fruit. Slack and Wainwright [18] reported that amylose content increased gradually during the development of barley grain, and the GBSS enzyme plays a key role in amylose synthesis [19]. In this study, changes in amylose content and GBSS enzyme activity were detected during banana fruit development and storage. Amylose content and GBSS enzyme activity increased gradually during the development of banana fruit but decreased significantly during storage ( Fig. 1C and 1D; Fig. 1E and 1F). Regulating amylose content and GBSS enzyme activity is thus a promising method to enhance banana fruit yield and quality.
In our study, a GBSS protein purified from banana fruit was approximately 55.0 kDa ( Fig. 2A and 2B). This molecular weight is similar to those GBSS proteins purified from rice (56 kDa) [20], maize (58 kDa) [21], and cowpea (58 kDa) [22]. SDS-PAGE and western blotting analyses indicated that banana GBSS proteins accumulated during the development of banana fruit and their levels decreased during fruit storage ( Fig.2A and 2B). These results are consistent with the changes in amylose content and GBSS enzyme activity from banana fruit.

Expression of GBSS genes in banana tissues
The wheat GBSSI gene is exclusively expressed in reproductive tissues such as endosperm, embryos, and pollen, while the GBSSII gene is expressed in non-storage tissues including leaf, stem, root, and pericarp [11]. Within the eudicots, GBSS genes identified in apple, peach, and orange are expressed in both vegetative and reproductive tissues such as leaves, flowers, and fruits [1]. GBSSI in Amaranthus cruentus is expressed in storage-unrelated organs, such as leaf, stem, petiole, and root [10]. The pea GBSSI gene is expressed in leaves, pod, roots, and embryos, but not in flowers or stipules [25]. In our study, MaGBSSI-1, MaGBSSI-2, and MaGBSSI-4 were upregulated expression in vegetative organs, such as root, stem, leaf, and bract (Fig.4). The expression pattern is similar to GBSSI genes in other eudicots [10,25]. The banana MaGBSSI-3 gene was abundantly expressed in reproductive tissues such as flower, peel, and pulp, while the MaGBSSII-1 and MaGBSSII-2 genes were highly expressed in vegetative organs such as root, stem, leaf and bract (Fig.4). The expression pattern of GBSS is more similar to GBSSI and GBSSII gene in monocots [11]. These results suggest that amylose accumulation in banana vegetative organs may be correlated with abundant expression of MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1 and MaGBSSII-2 in root, stem, leaf and bract (Fig.4). However, amylose accumulation in banana reproductive organs may require the activity of MaGBSSI-3 in flower, peel, and pulp (Fig. 4).

Expression of GBSS genes at different stages of banana fruit development
In Amaranthus cruentus, GBSSI were strongly expressed in the middle and mid-late stages of seed development and decreased thereafter [10]. GBSSI-1 expression in wheat endosperm may control starch synthesis at the post transcriptional level [5]. In apple fruit, GBSSII genes are highly expressed during all developmental stages. The GBSSII-2 gene in peach is expressed only during the early development of the fruit and the GBSSII-2 gene in orange is weakly expressed throughout fruit development [1]. The six GBSS genes in our study can be divided into two groups according to their temporal expression patterns. The earlyexpressing genes (MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBS-SII-1, and MaGBSSII-2) are expressed in the early stage (0-30 d) of starch granule formation, and are similar to the GBSSII-2 gene in peach fruit [1]. The expression level of MaGBSSI-1 and MaGBSSI-2 in the early stages of banana fruit development was approximately 5-fold higher than that of MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2. This result suggests that MaGBSSI-1 and MaGBSSI-2 might play an important role in early starch accumulation in banana fruit. The second group consists of a single, late-expressing  (MaGBSSI-3), which is expressed in the later stage (30-60 d) of starch granule formation during the development of banana fruit (Fig.5A). These findings suggest that the six MaGBSS genes cloned from banana fruit contribute to starch accumulation at different stages of development and at varying levels.

MaGBSSI-3 might be involved in regulating the quantity and size of starch granules in banana fruit
In cereal mutants, debranching enzymes (principally isoamylases) have an effect on granule number and form [26]. Buleon et al. [27] reported that, in the GBSS -defective (sta2) mutant Chlamydomonas reinhardtii cells, starch granules are smaller (0.7-1.5 pm) and have more regular shapes. In potato tubers, SSIII activity also alters granule shape [14]. Slack and Wainwright [18] reported that small granules arise at an early stage of development within immature tissue, while large granules are found in mature tissue during the development of barley grain. However, the influence of GBSS activity on starch granules in banana fruit has not been reported yet. In this study, scanning electron microscopy analysis showed that this change in the quantity and size of starch granules was consistent with pattern of MaGBSSI-3 expression during the development of banana fruit (Fig.6), suggesting that MaGBSSI-3 might regulate the quantity and size of starch granules in banana fruit.
In conclusion, amylose content, GBSS enzyme activity, and GBSS protein levels gradually increased during banana fruit development, but they decreased during banana fruit storage. Fulllength cDNAs encoding MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 were 1851 bp, 1851 bp, 675 bp, 1845 bp, 2265 bp, and 906 bp, respectively. Among the six MaGBSS genes, only MaGBSSI-3 was highly expressed in reproductive tissues and at the late developmental stages of banana fruit. Scanning electron microscopy analysis showed that the level of MaGBSSI-3 expression is correlated with the quantity and size of starch granules in banana fruit during development and storage. These results suggest that up-regulated expression of MaGBSSI-3 might be a key factor regulating amylose metabolism by affecting the variation of GBSS levels and the quantity and size of starch granules in banana fruit. Further work is required to elucidate how the different expression levels of the MaGBSSI-3 gene at different times could result in changes of the quality and size of starch granules of banana fruit.

Plant materials
Banana (M. acuminata L. AAA group cv. Brazilian) fruits were obtained from the banana plantation at the Institute of Tropical Bioscience and Biotechnology (Chengmai, Hainan province, China). Root, stem, leaf, bract, flower, peel, and pulp tissues were collected separately using tweezers and frozen immediately in liquid nitrogen, then stored at 280uC until further analysis. Pulps of 0 d, 10 d, 20 d, 30 d, 40 d, 50 d, and 60 d after emergence from the pseudostem were collected, immediately frozen in liquid nitrogen and stored at 280uC for expression analysis.
Banana hands were separated into individual fingers representing the same developmental stage. The group of banana fingers was kept at 22uC and allowed to ripen naturally. In accordance with the banana ripening stages [28], fruits were incubated for 0 d, 5 d, 10 d, 15 d, 20 d, 25 d, and 30 d after harvest, then were frozen in liquid nitrogen and stored at 280uC until further analysis. All experiments were repeated three times.
Determination of total starch content, amylose content, and GBSS enzyme activity Banana pulp was immersed in 0.5% sodium bisulfite solution for 10 min to prevent browning, and then dried at 40uC for 24 h. Pulp was ground and centrifuged. The residue was suspended in 5 mL 80% Ca(NO 3 ) 2 in a boiling water bath for 10 min, and then centrifuged for 4 min at 4,000 rpm. The supernatant was transferred to a 20 mL volumetric flask, and the residue was extracted two times with 80% Ca(NO 3 ) 2 , yielding a combined extract volume of 20 mL. All experiments were repeated three times. The total starch content was detected following the method of Yang et al. [29].
13 mg total starch was placed in a 10 mL graduated, stoppered test tube, to which was added 1.0 mL of 1M NaOH solution. The amylose content of banana pulp was determined following the method described by Yang et al. [29]. GBSS activity was detected according to the procedure of Nakamura et al. [24].

Detection of GBSS protein levels by SDS-PAGE and western blotting
Starch granules were purified from 0.5 g banana pulp each sample, according to the method of Nakamura et al. [24] with some modifications. A 10 mg sample of purified starch was suspended in sample buffer (0.5 M Tris-HCL pH 6.8, 2.5% SDS, 10% glycerol, 2% 2-mercaptoethanol) and boiled for 3 min, then centrifuged at 15,000 rpm for 5 min. The supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel and a 5% stacking gel. Gels were stained with Coomassie Blue and destained according to standard protocols.

Isolation of total RNA and synthesis of double-stranded cDNA
Total RNA was extracted from various tissues using the plant RNAout Kit (TIANDZ, Beijing, China). The integrity and concentration of isolated RNAs were examined by agarose gel electrophoresis and spectrophotometry (GelDoc-XR, Bio-RAD, Hercules, CA, USA). First-strand cDNA was synthesized in a 20.0 mL reaction mixture using 1.0 mg of total RNA, an Oligo(dT) 18

Cloning of six GBSS genes in banana fruit
GBSS homologues in banana were identified using a BLASTbased method. Primer pairs were designed to amplify each gene and their sequences are presented in Table S1. The PCR program consisted of 35 cycles of 40 s at 94uC, 40 s at 58uC, 90 s at 72uC, and a final extension for 10 min at 72uC. PCR products were purified using an agarose gel DNA Purification Kit (TaKaRa, Dalian, China) and inserted into the pMD19-T vector (TaKaRa, Dalian, China). Ligated products were transformed into E. coli DH5a competent cells. Positive recombinant plasmids were selected by the white/blue selection method and verified by colony PCR. Target DNA was then confirmed by restriction enzyme digestion and sequence determination. Full-length cDNA sequences encoding MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBS-SII-2 were submitted to GenBank.

Sequence analysis
Banana GBSS coding sequences were BLASTed against the banana genome sequence database (http://banana-genome.cirad. fr/) to recover their corresponding genomic DNA sequences. Exon lengths were calculated by alignment of genomic DNA sequences with cDNA sequences, and introns were determined according to the ''GC-AG'' rule [1].
Similarity of the full-length banana GBSSI or GBSSII cDNA sequences with other homologues in the GenBank database was performed using the BLAST program (E,0.001). The deduced amino acid sequences were aligned using the computer program Clustal W, and a homology tree was constructed with neighborjoining method using MEGA software (Arizona State University, Tempe, AZ, USA). The number for each interior branch is the percent bootstrap values calculated from 1,000 replicates.

Expression profiles of six GBSS genes in banana using quantitative reverse transcription PCR (Q-RTPCR)
Specific primer pairs were designed using Primer 5.0 software and their sequences are listed in Table S1. Expression levels of GBSS genes were quantified by Q-RTPCR using an iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and a SYBR ExScript The amplification program consisted of one cycle of 95uC for 1 min, followed by 40 cycles of 95uC for 10 s, 57uC for 15 s, and 72uC for 30 s. Melting curve analysis was performed at the end of 40 cycles to ensure proper amplification of target fragments. Fluorescence readings were collected from 60uC to 90uC at a heating rate of 0.5uC s 21 for melting curve analysis. Reaction mixtures lacking cDNA templates were run as negative controls to rule out contaminating DNA. All analyses were repeated three times using biological replicates. Relative expression levels of each gene were calculated using the 2 2DDCT method [30]. Data were analyzed using iQ5 software provided with the iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA).

Scanning electron microscopy (SEM)
Based on the expression of GBSS genes during different stages of development and storage of banana fruit, pulp was isolated after 0 d, 30 d, and 60 d of development and from the fruit 0 d, 5 d, and 10 d following storage.
The samples were fixed in stubs using double-faced tape and coated with a 10 nm-thick platinum layer in a Bal-tec MEDo020 Coating system (Kettleshulme, UK). The samples were analyzed in an FEI Quanta 600 FEG Scanning Electron Microscope (FEI Company, Oregon, USA). SEM observations were performed in the secondary electron mode operating at 15 kV.