Identification, sequence alignment and phylogenetic analysis of tomato MIOX genes
Four Arabidopsis MIOX protein sequences were downloaded from the TAIR database (https://www.arabidopsis.org) as queries for homologous sequences in the tomato genome database (http://solgenomics.net/) as described in methods. Keywords like ‘myo-inositol oxygenase’ and ‘MIOX’ were used to do a homologous search in the tomato database. All the retrieved sequences were aligned to remove redundant sequences after a similarity comparison. Based on domain analysis as described earlier in the methods, a total of 5 protein sequences were identified as unique MIOX proteins in tomato, which contains MIOX motifs or MIOX domains (IPR007828, and PF05153). The nucleotide sequences, amino acids, theoretical isoelectric point (pi), molecular weight (Mw), sequence length (aa), and other relevant information were obtained from the SGN or ExPASy-ProtParam database (Table 1). MIOX2 contains the maximum number of 1053 bp of nucleotides and 350 amino acids with 40.87 kDa molecular weight and 5.68 pI. While MIOX3 contains a minimum number of 819 bp of nucleotides and 272 aa with 31.67 kDa Mw and 5.31 pI; MIOX1, MIOX4, and MIOX5 contain 999, 954 and 879 bp of nucleotides and 332, 317 and 292 aa, respectively. The MIOX five members were 66-80 % identical at the amino acid level. MIOX genes were localized mostly in the cytosol and nuclear along with other cell organelles such as endoplasmic reticulum, vacuolar membrane, chloroplast, mitochondria, cytoskeleton, and plasma membrane (Table 1).
MIOX protein sequences of tomato and other species were aligned, and a phylogenetic tree was constructed. Multiple sequence alignment and phylogenetic analysis revealed close linkages of S. Lycopersicum with other species MIOX proteins. Among all, MIOX1 and MIOX4 proteins shared the highest levels (70 % identical at the amino acid level) of similarity (Fig. 1S).
Gene Structure, chromosomal locations, conserved motifs and expression profiles of MIOX genes
Tomato MIOX protein sequences were aligned (Fig. S2). The phylogenetic tree was constructed using MEGA5.0 with 1000 bootstrap replicates, which revealed that 5 MIOX were clustered into two groups. MIOX1 and MIOX5 were placed in one group, and MIOX2 and MIOX4 placed in another group together with MIOX3 (Fig. 1a). The MIOX genes structure was determined using the GSDS database. The analysis revealed that all MIOX genes contain intron regions. The number of introns varies among MIOX genes, ranging from 1 to 10. MIOX1 contains a maximum of 10 intron regions, followed by MIOX4, which contains eight intronic regions (Fig. 1b), while MIOX3 contains seven intronic regions. However, MIOX2 and MIOX5 both contain only one intronic region.
The conserved motifs of MIOX genes were identified using the MEME database. Motifs 1-4 are MIOX motifs, and most of these motifs are present in all MIOX genes, indicating that these MIOX proteins contain MIOX functional domains (Fig. 1c). Although motifs 5-7 were undetected in any known functional domains according to searches using Pfam and NCBI databases. Moreover, motifs 4 was absent in only the MIOX5 gene, but all other MIOX motifs are present in MIOX5 as well. The occurrence of a similar type of conserved motifs potentially indicates functional similarity among MIOX family members.
MIOX protein sequences were used to retrieve the chromosomal locations of genes in the tomato genome. The analysis revealed that MIOX genes are distinctly spread among the tomato chromosomes. Chromosome 6, 10, and 11 each contain one MIOX gene, while chromosome 12 contains two MIOX genes (Fig. S3).
MIOX gene expression pattern in different organs
RNA-seq data was examined to reveal the expression pattern of different organs during the different developmental stages of tomato. The analysis showed that MIOX genes were expressed differently in all the examined tissues of cultivar Heinz (transgenic tomato) and pimpinellifolium (wild tomato) (Fig. 2a). MIOX1 was highly expressed from flowering to the fruiting stage of cultivar Heinz, while MIOX2 expression was observed only in leaves of both the cultivar (Heinz) and wild (pimpinellifolium) tomato. MIOX3 exhibited very low expression, while MIOX5 revealed a high expression pattern at the flowering stage of cultivar Heinz. MIOX4 recorded high expression during flowering and early fruit stage with moderate expression in roots and leaves of cultivar Heinz. To further confirm the RNA-seq expression analysis, we performed an RT-qPCR to examine the expression pattern of MIOX4 in different organs of tomato cultivar S. lycopersicum cv. Alisa Craig. The analysis revealed that MIOX4 was highly expressed in the stem, flower, young, and green fruits. The expression level was, however, not high in roots, leaves, breaker, and red ripe fruits (Fig. 2b).
MIOX overexpression altered the expression of AsA-related genes and AsA concentration in both leaves and fruits
MIOX4 sequence was isolated according to S. lycopersicum full-length cDNA and transformed in tomato Alisa Craig to generate the transgenic lines as described in methods. The AsA content and expression of AsA-related genes were examined in the wild-type and MIOX4 overexpression transgenic lines (Fig. 3). The AsA level in leaves of MIOX overexpression lines showed a significant increase compared with the wild-type, and maximum AsA was observed in MIOX-M7 and MIOX17 transgenic lines (Fig. 3a). The substantial amount of ascorbate in MIOX transgenic lines signified that the biosynthesis of AsA might be subjected to feedback inhibition in tomato leaves and thus, limiting the increase of AsA up to a certain level. The total AsA and reduced AsA content in fruits of MIOX overexpression lines increased significantly compared with the wild-type (Fig. 3b); whereas, the overall increase in fruit ascorbate content was much lower than in leaves (Fig. 3).
In addition to the increased expression of the MIOX4 in the overexpression lines, the transcript level of most AsA biosynthesis and recycling genes showed a slight significant increase in fruits of two transgenic lines (Fig. 3c). In fruits, except PMI, GLDH, DHAR, AO, Capx, and AOBP, the other AsA-related structural genes such as GMP2, GME2, GPP1, GGP, MDHAR, MIOX, and tAPX showed a significant increase in MIOX4 overexpression lines. These findings indicate that MIOX4 transgenic lines contribute to the biosynthesis of AsA in tomato leaves and fruits in the D-Man/L-Gal pathway. This AsA biosynthesis could be subjected to feedback inhibition. Overexpression of the MIOX4 gene increases the AsA content in transgenic lines but down-regulates the expression of some structural genes by feedback inhibition.
Total AsA content in leaves and fruits of MIOX overexpression lines altered by Synthetic Precursor Inositol (MI)
Previously reported results indicated that; an increase of AsA content in MIOX4 transgenic lines is associated with the AsA biosynthesis pathway in leaves and fruits. Therefore, inositol (MI), a substance representing the AsA biosynthesis pathway was chosen to do the feeding experiment, H2O was used as a control. In leaves, MI feeding increased the AsA content in both MIOX transgenic lines and Alisa Craig. This increase was significant in both MIOX transgenic lines. On the contrary, the AsA content recorded no difference among Alisa Craig and transgenic lines in the mature green fruit stage. MI plays a particular role in AsA biosynthesis, suggesting that myo-inositol pathway was the main contributor to AsA accumulation in tomato leaves.
Meanwhile, MI feeding increased the AsA content in breaker and red ripe fruits of transgenic lines. However, the AsA content in the fruit of AC showed no change after feeding with MI (Fig. 4). These findings suggest that myo-inositol pathway contributes to AsA biosynthesis in tomato breaker and red ripe fruits in transgenic lines. MIOX overexpression may have improved the AsA biosynthesis pathway in which the myo-inositol pathway plays a role in leaves and fruits and consequently alters AsA content. Therefore, the increase in the AsA content of transgenic lines could be ascribed to the MIOX overexpression gene, which might have increased the overall MIOX activity.
The light-dependent fluctuation of AsA content in wild type and MIOX lines
To further investigate the effect of MIOX transgenic lines on light-dependent ascorbate metabolism, we determined AsA content in the leaves under different light conditions for 24 hrs. Dynamic changes as a model of “up-down-up”, were observed in total AsA content of both wild-type and transgenic lines under the different light conditions for 24 hrs. However, AsA content in transgenic lines was significantly higher compared with the wild type (Fig. 5). These results indicate photoperiod-specific changes in AsA content, affirming that light significantly contributes to AsA accumulation in tomato. We observed that AsA content increased in leaves under light condition up to a certain level but declined in extended illumination. Generally, MIOX overexpression improved the light-induced AsA accumulation in tomato leaves.
MIOX overexpression increases AsA transport capacity in transgenic lines
The MIOX transgenic lines capability to affect AsA transport was measured as it has been reported earlier that AsA synthesis both in leaves and fruits are mostly transported from leaves to fruits [16]. Leaf petioles and fruit exudates of wild-type and MIOX transgenic lines were used to measure the AsA content. The analysis revealed that AsA content has no significant difference in leaf petioles and mature green fruit exudates of wild-type and MIOX transgenic lines. Interestingly, breaker and red ripe fruit exudates showed a significant increase of AsA content in transgenic lines compared with wild-type (Fig. 6), consistent with the AsA content in fruits. The findings indicate that the AsA content significantly increased in fruit secretion and leaf petioles of MIOX transgenic lines compared with wild-type.
MIOX overexpression improved tolerance to oxidative stress in tomato
Ascorbic acid (AsA) is an antioxidant involved in the removal of reactive oxygen species (ROS) and protects plants from oxidative damage. Mostly, increased AsA content helps plants to resist oxidative stress. To examine whether AsA biosynthesis-related MIOX transgenic lines in tomato can increase oxidative stress tolerance, 4-weeks-old transgenic lines and wild-type were exposed to oxidative stress by spraying different concentrations (0.15 and 0.30 mM) of methyl viologen (MV), which can imitate the oxidation environment, for 48 hours. The electrical conductivity (EC) showed the damage caused by MV. After MV treatments, EC in the wild-type plants showed a significant increase, whereas no significant changes were found in MIOX transgenic lines. After MV treatments, EC was significantly reduced in transgenic lines (66 % in 0.15 mM MV and 69 % in 0.30 mM MV) compared with wild-type plants (Fig. 7a).
Furthermore, DAB staining and H2DCFDA fluorescence were observed to determine the accumulation of H2O2 in leaves of wild-type and MIOX transgenic lines. DAB staining analysis showed no significant difference between Alisa Craig and MIOX transgenic lines after treatment with H2O. Significant brown spots were observed on the leaves of Alisa Craig compared with the leaves of MIOX transgenic lines after MV treatments (Fig. 7b). The H2DCFDA fluorescence results showed similar results with no significant difference between Alisa Craig and MIOX transgenic lines treated with H2O. At the same time, Alisa Craig generated brighter fluorescence than MIOX transgenic lines after MV treatments (Fig. 7c). In general, it seems that AsA related MIOX transgenic lines increased oxidative stress tolerance in tomato.
MIOX overexpression altered the water holding capacity of transgenic lines
It has been well reported that AsA is one of the most abundant water-soluble antioxidant compounds for enhancing abiotic stress tolerance in plants [17]. To evaluate MIOX transgenic lines response to stress conditions, 4-weeks-old transgenic lines, and leaves of wild-type plants were exposed to dehydration stress. The analysis revealed that after the dehydration stress, wild-type plants showed higher water loss (26 %) compared with transgenic lines (Fig. 8); thus, MIOX tomato transgenic lines exhibit increased desiccation stress tolerance.
Morphological attributes affected by MIOX overexpression
Remarkably, other than improved AsA accumulation, MIOX transgenic lines showed a noticeable difference in fruit weight, size, shape, and Brix level compared with Alisa Craig (Fig. 9). The fruit weight, the horizontal and vertical diameter of MIOX transgenic lines was significantly reduced compared to Alisa Craig (Fig. 9a-c). The Brix level of MIOX transgenic line (M7) was significantly higher than Alisa Craig (Fig. 9d). These findings demonstrate that MIOX transgenic lines could significantly affect the overall appearance, quality, and Brix level of tomato fruits.