Sucrose synthase can play important roles in sink strength and it was suggested that CitSUS1 may have a role in supplying UDP-glucose and fructose for cell wall synthesis during the cell division stage while CitSUSA and SUS1 may supply substrates for sucrose synthesis during maturation . Two isozymes of sucrose synthase, each active during different stages of development , were also reported in pear . Our results support the notion of a balance between the action of sucrose synthases and the regulation of invertase activities. The accumulation of arabinose, galactose, xylose, and ribose, important for cell wall synthesis was correlated with proteins associated with cell wall metabolism such as cellulose synthase, pectin methylesterases, b-chitinase, PR4, b-1,3-glucanase, polygalacturonase inhibiting protein, and UDP-glucose 6-dhydrogenase. The role of phosphoenolpyruvate carboxylase in the metabolism of malate and citrate is intriguing. Pyruvate is generally the major product of glycolysis arising from PEP via the activity of pyruvate kinase. However, plant cells canconvert PEP to malate via oxaloacetate in reactions catalysed by PEP carboxylase and malate dehydrogenase . The resulting malate may be utilized as a respiratory substrate in the TCA cycle, 5 gallon plastic pots or be converted to pyruvate via the activity of malic enzyme. As shown in potato tubers with reduced NAD-dependent malic enzyme activity, the conversion of malate to pyruvate can influence glycolytic flux .

Studies in tomato and grape fruits suggested the occurrence of gluconeogenesis in fruits, particularly during the ripening stage when sugars are accumulating rapidly. A correlation between citrate, malate, and oxaloacetate loss and the activities of PEPC and PEPCK and gluconeogenesis in fruits was demonstrated . Both of these proteins were up-regulated during citrus fruit development. Our results suggest that organic acid and amino acid accumulation shifted toward sugar synthesis during the later stage of citrus fruit development. The notion of a metabolic shift during fruit maturation is supported by work on grape, strawberry, and tomato fruit maturation . Gene expression analysis of maturing grapes showed that a decrease in expression of transcripts associated with organic acid accumulation was accompanied by the increased expression of genes associated with the TCA cycle and genes encoding enzymes mediating sugar accumulation . The observed increase in SPS and SPP activity in the later stages of fruit development, concomitant with the rapid accumulation of sucrose, suggest that this sugar is also being synthesized in citrus juice sac cells during fruit development and ripening.Transcription factors play important roles in plant development and stress resistance by regulating related gene expression, and usually classified into different families on the basis of their conserved DNA-binding structure.

Basic leucine zipper family are ubiquitous TFs in plants where they participate in a great deal of physiological and biochemical processes including but not limited to light signaling and photomorphogenesis , flower development and fertility , seed maturation and germination , senescence and abiotic stress responses . bZIP family are characterized by two functionally distinct motifs located on a contiguous α-helix: a basic region for DNA binding and a leucine zipper for dimerization . Initially, AtbZIPs were systematically divided into ten subfamilies, referred to as group A to I, and S . Subsequently, the classification was further extended to 13 groups . Among these subfamilies, the S group is the largest bZIP cluster and divided into S1, S2 and S3 three subgroups . Particularly, the group S1 has five members, AtbZIP1 , AtbZIP2 , AtbZIP11, AtbZIP44 and AtbZIP53 , and they harbor an unusually long 5’ leader containing a highly conserved upstream open-reading frame that encodes a sucrose-controlled peptide, which can give rise to sucrose-induced repression of translation and involves in nutrition/energy/stress signaling cascades . This structure feature has been used to regulate plant sugar accumulation, amino acid metabolism, stress resistance, growth and development. For almost all fruits and some vegetables, sugar content and sweetness increasing is valued. Sagor et al. proposed a novel strategy that fruit-specific expression of the S1-bZIP main open reading frames , without the SIRT responsive uORFs, could produce sweeter tomato fruits. Moreover, group S1-bZIPs could efficiently form specific heterodimers with bZIP members of group C in planta.

The S1/C bZIP complex leads to the strong activation of target genes involving stress stimuli and metabolic signals in SnRK1-dependent and -independent way, which facilitates a signal integration in plant transcriptional networks . It has been demonstrated that sustaining energy homeostasis is important to control plant growth process. Transient energy deprivation will always happen when plant adjust their metabolism to adapt to daily light-dark cycles as well as unpredictable environmental changes . AtbZIP1 and its heterodimerization partners AtbZIP53, AtbZIP10, and AtbZIP25 can facilitate primary carbohydrate and amino acid metabolism reprogramming to induce salt stress tolerance by ABA-independent signaling module in Arabidopsis root . In the experiment of sensory perception to strawberry flavor, sweetness intensity which dependents on sugar concentration is the strongest driver of overall liking. Especially, the reduction of sucrose would undermine the hedonic effect . Therefore, creating a sweeter strawberry germplasm is desirable. Given that enormous influence of S1-bZIPs on sugar metabolism, plant growth and development, we identified the S1-bZIP members in strawberry to give a deeper insight into their function.The sequence information of Arabidopsis S1-bZIP five members was obtained from TAIR database and were used as query probes to BLAST search against NCBI database and Fragaria vesca v1.0 genome database . S1-bZIPs were reported to harbor an unusually long 5’ leader containing a highly conserved upstream open-reading frame that encodes a sucrose-controlled peptide. Hence, uORF of FvS1-bZIPs was isolated and then aligned with uORF of AtS1-bZIPs to further ensure that identified sequences belong to S1-bZIPs in strawberry. Moreover, the evolutionary relationship of S1-bZIP homologues among strawberry, peach, rice, Arabidopsis was analyzed in MEGA v.6.0 according to neighbor-joining method with Poisson model and 1000 bootstrap replications, after sequence alignment was performed in ClustalX v.2.0 software. The physical and chemical parameters of FvS1-bZIP proteins were computed using ProtParam tool, round pot and putative subcellular location was predicted in CELLO v.2.5 program. Conserved Motifs of the FvS1-bZIPs were analyzed using the Multiple EM for Motif Elicitation web server .A total of four FvbZIP-S1 genes were identified in Fragaria vesca v1.0 genome database. As shown in Table 1, two members of gene21832 and gene15193 were mapped to chromosome 2, while gene14942 and gene02284 were respectively distributed to chromosome 1 and 5. The deduced amino acid lengths varied from 148 to 171 aa, and the predicted molecular weights ranged from 17.19 to 19.14 KDa. The gene21832 and gene15193 had the similar theoretical isoelectric point around 6.5, and the pI of gene14942 and gene02284 were 4.93 and 5.49, respectively. Instability indexes above 40 indicated that all FvbZIP-S1 proteins were unstable. Aliphatic indexes defined as the relative volume occupied by aliphatic side chains were predicted between 58.65 and 79.49. The identified proteins with negative GRAVY values within -0.58 and -0.94 were therefore hydrophilic. Subcellular localization results showed all of them were located in nuclear. To further investigate the structural diversity and predict function of FvbZIP-S1 proteins, a total of eight conserved motifs were identified using online MEME tool . All members shared motif 1–3. Motif 1 was annotated as bZIP-basic domain. .Each S1-bZIP harbored a sucrose controlled upstream open reading frame . Here, SC-uORFs of four S1-bZIP sequences in strawberry were isolated and aligned with those in Arabidopsis . The results showed that SC-uORFs were relatively conserved. The length of three SC-uORFs in gene15193, gene21832 and gene14942 were longer than that in gene02284. To analyze phylogenetic relationship of S1-bZIP proteins in strawberry, rice, peach, and Arabidopsis, the full-length amino acid sequences were aligned to construct an unrooted phylogenetic tree based on the neighbor-joining method . Phylogenetic tree was categorized into three distinct branches.

AtbZIP1 was singly formed to be a branch. AtbZIP53 together with OsbZIP38, OsbZIP87, ppa013046 and gene02284 clustered into a group, while others classified into another branch. The strawberry gene21832 were referred to as FvbZIP11 as a consequence of it clustering with Arabidopsis homologous bZIP11-related TFs .To explore the tissue-specific expression levels of these four S1- FvbZIPs in strawberry, strawberry root, stem, leaf, flower and different stage fruits were collected for qRT-PCR . The results showed that four members could be detected in all tissues, but their expression patterns differed. Obviously, four S1-FvbZIPs had relatively lower transcript abundance in stem and leaf. The gene21832 was the most highly expressed gene among four S1-FvbZIP members in various tissues. In particular, its transcript was mainly presented during fruit development and ripening. The gene02284 had the lowest expression level in these tissues, and it most highly expressed in flower in comparison with other tissues. The gene14942 had a very significant transcript accumulation in root, while the gene15193 mainly expressed during the fruit development process and had the similar change tendency to gene21832 .The basic leucine zipper transcription factor family is one of the largest and most diverse TF families. It has been widely identified or predicted in planta, as the massive genome sequences are released, including rice , wheat , maize, barley , Arabidopsis , tomato , cucumber , grapevine , tea , watermelon and melon , three rosaceae species of strawberry, peach and apple , etc. Extensive studies have provided strong evidences that bZIP TFs are involved in many different biological processes that range from the response to various types of stress to the control of the nitrogen/carbon balance, the regulation of genes involved in the seed protein storage and others . bZIPs family can be further classified into several subfamilies. Among these members, the S1 branch belonging to bZIP S subfamily is very special. They harbor an unusually long 5’-leader region corresponding to a highly conserved sucrose controlled upstream open reading frame , which affects translation of bZIP protein . This feature has been attracting much attention in exploring S1-bZIP function. It has been documented that S1-bZIPs played an important role in plant growth and development, especially affecting accumulation of metabolites which is very vital for fruits . However, the bZIP S1 genes were just deeply studied in some model plants, like Arabidopsis, tobacco and tomato, and very little is known about them in strawberry and other fruit crops. This study characterized gene14942, gene21832, gene15193, gene02284 four S1-bZIP members from strawberry at the genome-wide level, which were respectively distributed on chromosomes 1, 2, and 5. The results showed that all FvbZIP-S1 were unstable proteins, consistent with the previous report . Moreover, they were predicted to be nuclear-localized. It was related to the basic region of 16 amino acid residues with an invariant N-x7-R/ K motif in bZIP family . Obviously, the FvbZIP-S1 had highly conserved SC-uORFs in their long 5’ UTRs, indicating they were involved in sucrose sensing such that the mORF translation was inhibited if the amount of cellular sucrose increases and vice versa. The conserved uORFs take up only a fairly small portion of all uORFs in eukaryotic transcripts, but it is a key element for gene expression at post-transcriptional level . It has been reported that they can regulate the translation of the downstream mORFs through a specific metabolite, i.e. sucrose , polyamines , and phosphocholine , in a feedback manner. However, there is little information about the mechanism that uORFs are sensitive to small molecules. The circumstantial proofs somewhat hint that uORFs possibly act as the receptor for the metabolites . The phylogenetic analysis showed strawberry S1 FvbZIPs had a closer correlation with peach and Arabidopsis S1-bZIPs than those of monocotyledonous rice, indicating the genes were established before the corresponding taxonomic lineages diverged. Moreover, those homologs clustered in the same group were possible to be involved in similar functions. Four S1-FvbZIP genes were expressed in all of the samples analyzed, but they had the different transcript abundances, suggesting they may possess functional divergence and redundancy for strawberry growth and development. Notably, the expression level of gene21832 kept greatly high during fruit development and other tissues, so we speculated the gene21832 regulated metabolism involved in fruit ripening or quality, and affected plant growth. Since Arabidopsis bZIP11-related TFs was the closest homolog of gene21832, it was denoted as FvbZIP11 to reflect this homology. The previous studies have illustrated that S1-bZIPs could be induced by different external elicitors and had the organspecific feature . AtbZIP11 transcripts are abundant in flowers and roots, but fairly low in leaf tissues; i.e., photosynthetic organs, indicating the gene plays a role in carbohydrate-consuming tissues.