Identification and expression of the trehalose-6-phosphate synthase gene family members in tomato exposed to different light spectra

Light is the source of energy for plants. Light wavelengths, densities and irradiation periods act as signals directing morphological and physiological characteristics during plant growth and development. To evaluate the effects of light wavelengths on tomato growth and development, Solanum lycopersicum (cv. micro-Tom) seedlings were exposed to different light-quality environments, including white light and red light supplemented with blue light (at ratios of 3:1 and 8;1, respectively). Tomatoes grown under red light supplemented with blue light displayed significantly shorter stem length, a higher number of flower buds and rate of fruit set, but an extremely late flowering compared to white-light-grown plants. To illustrate the mechanism underlying the inhibition of stem growth and floral transition mediated by red/blue light, 10 trehalose-6-phosphate synthase (TPS) genes were identified in tomato, and bioinformatics analysis was performed. qRT-PCR analysis showed that SlTPSs were expressed widely throughout plant development and SlTPS1 was expressed at extremely high levels in stems and buds. Further analysis of several flowering-associated genes and microRNAs showed that the expressions of SlTPS1, SlFT and miR172 were significantly downregulated in tomato grown under red and blue light compared with those grown under white light, whereas miR156 transcript levels were increased. A regulatory model underlying vegetative growth and floral transition regulated by light qualities is presented. Our data provide evidence that light quality strongly affects plant growth and phase transition, most likely via the TPS1-T6P signaling pathway.


IntRoduCtIon
Plants are exposed to various environmental conditions constantly.The most dramatic variation is in the daily rhythm under a light-dark cycle.Irradiative changes could be perceived by red-and blue-light photoreceptors and gene expression was regulated in plants in response to various light environments [1,2].Phytochromes are biliprotein photoreceptors that respond to different light wavelengths; they are responsive to changes in light quality and quantity, light direction and period length, enabling plants to respond optimally to changed light conditions [3].Light acts as signals regulating seed germination, seedling establishment, the proper development of photosynthetic machinery, the architecture of the vegetative plant, the timing of flowering, tuberization, bud dormancy, and potentially the allocation of resources to root, stem, leaf, and storage organs.The phytochromes also display regulatory functions mediating light responses, such as growth inhibition, leaflet or organelle movement and phototropism.The recent discovery of phytochrome-related proteins in photosynthetic cyanobacteria and nonphotosynthetic eubacteria has opened new avenues for investigating biliprotein photosensory function [3].
The transition of plants from vegetative to reproductive development is crucial for their successful reproduction.Several external and endogenous factors were found to participate in phase transition, such as day length, temperature, hormonal status, and carbohydrate availability [4].Energy status, which was indicated by light density and periods, was found to affect plant growth and development [5].Trehalose-6-phoshate (T6P) is the metabolic precursor of the non-reducing disaccharide trehalose.It is the product of the condensation reaction of uridine diphosphate (UDP)-glucose and glucose-6-phosphate (G6P), which is catalyzed by trehalos-6-phoshate synthase (TPS) [6].
T6P, found only in trace amounts in most plants, has been suggested as the signaling molecule that transduces messages of carbohydrate availability to other signaling pathways [7].TPS1 loss-of-function mutants are embryonic-lethal [8].Embryo defects in tps1 mutants can be rescued by TPS1 overexpression in seeds, but the seedlings develop slowly and senesce before entering into the reproductive phase [9].Elevating TPS1 in tps1-2 mutants results in a delayed floral transition [10].These results indicated that TPS1 is required for the initiation of flowering.Further evidence showed that the T6P pathway regulated floral transition at two sites in the plant: the leaves and the shoot apical meristem (SAM) [11].In the leaves, TPS1 induces the FLOWER-ING LOCUS T (FT) gene, which is then transported to the SAM and promotes floral formation; T6P acts as an indicator of a plant's carbon status in the SAM [11].
To study the effects of different combinations of light wavelengths on tomato growth and development, micro-Tom (Solanum lycopersicum) seedlings were exposed to different light-quality environments, including white light and red LEDs supplemented with blue light.Interestingly, tomato grown under red LEDs supplemented with 1:3 or 1:8 blue light displayed shorter stem length and an extremely late flowering compared to those of white-light-grown plants.To further explore the regulatory mechanism, 10 SlTPS that may participate in energy fluency were identified and investigated at the transcription level during the processes of plant vegetative and reproductive development and in response to changes in light quality.Our data provided the evidence that different light qualities have strongly significant effects on plant growth and phase transition.

Plant materials and light treatments
Tomato (Solanum lycopersicum cv.Micro-Tom) plants were grown in a standard culture chamber under the following conditions: 16/8 h day/night cycle, 23ºC, 80% relative humidity.The light density was adjusted to 2000 µmol s -1 m -2 via the methods for determining light density according to Thimijan [12].Seeds were sterilized, germinated in 1/2 MS medium for 7 days, and then seedlings of similar sizes were transplanted to pots containing nutrient soil.
To evaluate the effects of light quality (spectral distribution of light) on tomato growth, the seedlings were exposed to different light conditions: white light and red light supplemented with blue light (red/bluelight photosynthetic photon flux density (PPFD) ratios were 3:1 and 8:1, respectively).Three types of LEDs were used: InGaN/YAG white LEDs (color temperature of 6500K), red gallium-aluminum-arsenide (GaAlAs) LEDs and InGaN blue LEDs.Red and blue LEDs have a peak emission at 660 nm and 450-470nm, respectively.Seedlings under white light were designated as the control.

Identification of tomato tPs genes
The TPS full-length cDNA sequences of Arabidopsis were obtained from the nucleotide database of NCBI (http://www.ncbi.nlm.nih.gov/nucleotide/) and the corresponding amino acid sequences were also obtained.To identify new homologues in tomato, the complete cDNA and protein sequences of TPS gene families in Arabidopsis were subjected to BLASTN and TBLASTN searches against SGN tomato WGS chromosomes (version SL2.40) (http://solgenomics. net/tools/blast) [13].Taken together, 10 potential TPS genes in tomato were identified from the currently available genomic databases.

Bioinformatic analyses of tomato tPs genes
After searching for SlTPSs, the bioinformatics tool FGENESH (http://linux1.softberry.com/berry) was used to analyze and predict those unknown SlTPSs.A conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/) was used for functional annotation of the proteins.Deduced amino acid sequences of SlTPSs were aligned with the homologous proteins in Arabidopsis using ClustalX 2.0 software in the default setting.A phylogenetic tree was constructed using the MEGA (version 5.0) software by the neighbor-joining (N-J) method.

RnA isolation and quantitative real-time PCR
Different tissues and fruits from different developmental stages were harvested, frozen in liquid nitrogen and stored at -80°C.Total RNA was isolated using Trizol Reagent (Invitrogen, USA) according to the manufacturer's instructions.Total RNA used for cDNA synthesis was treated with DNase I (Fermentas, Thermo, USA) to remove contaminating genomic DNA.First-strand cDNA was reverse transcribed from 2 μg of total RNA using the RevertAid™ First Strand cDNA synthesis kit (Fermentas, Thermo, USA) according to the manufacturer's instructions.Quantitative real-time PCR (qRT-PCR) was conducted on a CFX Connect Detection system (Bio-Rad, USA).PCR amplification reactions were performed in a 20-μL mixture containing 10 μL of FastSYBR Mixture (CWBIO, China), 2.5 ng of total RNA and 1 μL each of the forward and reverse primers (5 μM).PCR detection were performed by the threestep method: incubation at 95°C for 5 min, followed by 40 cycles at 95°C for 5 s, 60°C for 5 s and 72°C for 5 s.Relative expression levels were calculated based on the 2 -ΔΔCt method.Actin (Slactin-51, accession number Q96483) was used as the reference gene for expression analysis of 10 TPS and FT genes.
To investigate the expression of miRNAs, poly(A) was added to the total RNA using Escherichia coli poly(A) polymerase (NEB, UK) at 37°C for 30min and then reverse transcription was performed and adaptor was added with an oligo(dT) adapter primer (AAGCAGTGGTATCAACGCAGAG-TACTTTTTTTTTTTTTTTTTTTTTTVN) [14].U6 (accession number X51447.1) was used as reference gene for expression analysis of miRNA.Gene accession numbers and primers for qRT-PCR are listed in Table 1.For all qRT-PCR experiments, at least three biological replicates were performed and each reaction was run in triplicate.

expression patterns of sltPs genes in different tissues and organs
To investigate the potential functions of SlTPS genes, their expression profiles were determined by qRT-PCR in different tissues including root, stem (St), leaf (L), bud, flower (F) and four stages of fruit development.It is apparent that SlTPSs were widely expressed throughout the plant development (Fig. 2).Compared with other tissues, the 10 SlTPS genes in fruits were expressed at relatively high levels, and their expression patterns during fruit development and ripening could be divided into three types.The transcripts of four genes including Solyc07g062140.2.1, Solyc02g071590.1.1,Solyc08g076650.2.1 and Soly- TPS genes expressed at extremely high levels in stems, roots and leaves, respectively (Fig. 2).

effects of light quality on floral transition in tomato
The vegetative and reproductive growth of tomato under different irradiation sources after a 50-day cul-ture after germination was investigated.The morphological and physiological characteristics of at least 30 plants, including flowering time, length of internodes, number of flower buds and the rate of fruit set, were observed and collected.The results showed that the flowering time was significantly delayed in tomato  seedlings grown under red light supplemented with blue light compared with those grown under white light.The tomato grown under red and blue light (at a ratio of 3:1) flowered at approximately 46 days post germination (dpg), and 40 dpg in plants under white light (Table 2).Meanwhile, the flowering ratio significantly differed under different irradiation sources and various blue to red LED ratios.Statistical analysis showed that 13/15 buds flowered under white light at 50 dpg, but only 2/15 buds flowered under 75% red and 25% blue LEDs (3:1).There were no flowers observed in seedlings under red and blue LEDs (8:1 ratio) (Table 3).However, these seedlings restated their floral transition later when transferred to white light conditions at 50 dpg.In terms of vegetative growth, the lengths of two internodes below the first flowering branch were distinctly shortened in seedlings under 75% red and 25% blue LEDs compared to controls (Fig. 3).Interestingly, we observed that the plants grown under red and blue LEDs (3:1 ratio) produced more flower buds and the rate of fruit set was significantly increased compared to those in seedlings under white light treatment (Fig. 4).

expression of flowering-associated genes and microRnAs
TPS gene families play important roles in plant growth regulation, especially TPS1.According to the abovementioned results, Solyc02g071590.those grown under white light, whereas miR156 transcript levels were obviously increased (Fig. 5).

dIsCussIon
Light provides an energy source for the plants via photosynthesis, and reception of the light signal is crucial for optimizing plant growth and reproduction.About 150 years ago, scientists noticed that different light wavelengths could result in different energy conversion efficiency [15].In many species, red light has the highest quantum yield for CO 2 fixation among various light wavelengths in the photosynthetically active region (PAR) of the spectrum (400-700 nm, which includes green and blue light) [16,17].In view of the high photosynthetic quantum yield derived from red light, high-efficiency red LEDs have been used to pro-duce optimal light environments for photosynthesis and the growth of plants.Combinations of different light wavelengths were also proved to enhance quantum yields [18].Interestingly, consistent treatment with red light alone resulted in poor photosynthetic performance, which is converse to the opinion that red light is the most efficient energy source [19][20][21][22].Actually, utilization during plant development does not merely depend on carbon assimilation.For example, carbon starvation treatment for even short periods leads to growth inhibition, which cannot be recovered immediately after carbon supplementation [23], indicating that there might be a time difference between energy status energy sensing.The plants may evolve a regulation mechanism to perceive sugar signals and cope with the various environments.
Previous studies showed that different light quality has a distinct effect of on morphologic characteristics during plant growth and reproduction.In strawberry, stem elongation was promoted and inhibited under red and blue light, respectively.Plantlets cultured under 70% red+30% blue showed higher leaf and root number, higher plant height and root length, and greater fresh and dry weight compared to those cultured under 90% red+10% blue and 80% red+20% blue.Goins et al [20] reported that wheat grown under red LEDs alone displayed greater main culm length, fewer subtillers and a lower seed yield on harvest day compared to white-light-grown plants, while wheat grown under red LEDs supplemented with 10% blue light produced a seed yield close to that of white light.However, we observed that tomato grown under red LEDs supplemented with 1/3 blue light demonstrated shorter internodes (Fig. 3), but higher flower numbers and rates of fruit set (Fig. 4) compared to those of white-light-grown plants.These results suggest that there is an optimal threshold level for blue light for optimal growth and reproduction under a red-based light source, which might depend on plant species.It has been demonstrated that flowering time was delayed significantly under red LEDs and blue light treatment (Table 2), especially in the tomato under higher ratios of red to blue light (Table 3).
To illustrate the mechanism underlying the regulation of vegetative growth and floral transition via light wavelength, the energy signaling pathway-associated gene SlTPS1-1 was investigated at the transcription level.The results showed that SlTPS1-1 was downregulated significantly (Fig. 5), indicating that TPSrelated sugar sensing and signaling pathways were significantly suppressed in tomato plants grown under red LEDs.The FT gene integrates several external and endogenous cues controlling flowering, with FT protein moving directly from the leaves to the shoot apex, behaving as a long-distance signal [31].Previous studies suggested that the SPL gene, as downstream targets of FT, function in the control of flowering time and phase change.Here, FT was downregulated in tomato grown under red and blue light (Fig. 5), which can delay flowering via affecting SPL expression [29].miR156 was considered as an age marker in plants, downregulated with increasing plant age [29].miR172 was proved to be as a miR156 antagonist on regulating phase transition in plants [30].Compared to white light grown plants, the expressions of miR156 and miR172 were upregulated and downregulated, respectively (Fig. 5), in tomato plants grown under red LEDs supplemented with blue light.This might be the reasons that the vegetative phases were maintained and flowering was inhibited in tomato plants grown under higher ratios of red/blue light.The upregulation of miR156 in tomato grown under red/blue light would suppress the expression of SPL, the target of miR156 [32], and subsequently inhibit flowering in an FT-independent manner [29].Recent research demonstrated the correlation of sugar signaling and miR156 expression and their roles in regulating the floral pathway.It showed that T6P was able to modu-Fig.6.A hypothesized model for the regulation of vegetative growth and floral transition by light quality.When tomato plants were grown under white light, TPS1 were expressed at higher levels in order to produce abundant sugars as carbon source.Higher levels of TPS1 suppressed the expression of miR156, which could promote plant aging and lead to fast growth and floral transition.On the contrary, when tomato plants were exposed to a high ratio of red to blue light, TPS1 expression were downregulated to accommodate CO 2 assimilation because of increased net leaf photosynthesis rate, and therefore miR156 expression was upregulated.Subsequently, higher levels of miR156 suppressed the transcripts of the SPL gene and resulted in delayed flowering.miR172 as miR156, antagonizes phase transition.late the expression of the targets of miR156, partially via the miR156-dependent age-related pathway [11].
Finally, a regulatory mechanism underlying TPS1 functions on plant vegetative and reproductive growth via energy signaling pathway mediated by red/blue light, and eventually influencing the growing of the plant, was hypothesized (Fig. 6).When tomato plants were grown under white light, TPS1 were expressed at higher levels in order to produce abundant sugars as a carbon source.Higher levels of TPS1 suppressed the expression of miR156, which could promote plant aging and lead to fast growth and floral transition.On the contrary, when tomato plants were exposed to high ratios of red to blue light, TPS1 expression were downregulated to accommodate CO2 assimilation because of an increased net leaf photosynthesis rate, and therefore miR156 expression was upregulated.Subsequently, higher levels of miR156 suppressed the transcripts of SPL gene and resulted in delayed flowering.

ConClusIon
In this work, defects in phase transition including juvenile-to-adult transition and vegetative-to-reproductive phase transition were observed in tomatoes grown in environments under a high ratio of red to blue light.The key regulator in energy status sensing, SlTPS1, was downregulated in plants grown in the presence of red and blue light as compared to those grown under white light.This indicates that an imbalance in light source would affect the energy sensing pathway, thereby modulating miR156, miR172 and SlFT, and eventually inhibiting vegetative growth and floral transition.
1.1 showed homology to AtTPS1, and was named SlTPS1-1.SlTPS1-1 was strongly expressed in flower buds, suggesting a potential important role in the initiation of flowering.To reveal the molecular mechanism underlying whether floral transition was regulated by light quality, the transcripts of SlTPS1-1 and floral transition-related genes or microRNA, such as FT, miR172 and miR156, were determined by qRT-PCR in tomato plants under different light treatments.The expressions of SlTPS1-1, SlFT and miR172 were significantly downregulated in tomato grown under red and blue LEDs (at 3:1 and 8:1 ratios) compared with

Fig. 4 .
Fig. 4. Number of flower buds and rates of fruit set under different light-quality environments.Control − white-light-grown plants, Treatment − seedlings grown under red LEDs and 1:3 blue light environment.Significance was analyzed using the same methods as mentioned above.

table 2 .
Floral timing in tomatoes grown under environments with different light-quality.At least 30 plants were investigated for each treatment.