Genome-wide identification and evolutionary analysis of neutral/alkaline invertases in Brassica rapa

Abstract Among the several sucrose-catabolizing enzymes, neutral/alkaline invertases (NINs) play crucial roles in developmental processes as well as environmental stress responses in higher plants. Despite the fact that NINs are essential enzymes for plant life, the NIN family and their evolutionary relationships are poorly understood. Therefore, in this study, we identified 11 NINs in the Brassica rapa (Chinese cabbage; BraNINs) genome, and analyzed the evolutionary mechanisms of BraNIN genes. Evolution analysis suggested that the BraNIN genes were duplicated via a segmental duplication event originating 15.81–45.25 million years ago. Furthermore, two segmental duplicated pairs (BraNIN5/6 and BraNIN7/8) were subject to negative selection. Furthermore, expression analysis of BraNINs using RNA-sequencing data suggested various functions of BraNINs during responses to drought stress. Taken together, our comparative genomic analysis of NIN genes in B. rapa provides information that will assist future studies on sucrose metabolism in sinks and sources of higher plants.


Introduction
Sucrose plays crucial roles as a major carbohydrate transported from photosynthetic source leaves to heterotrophic sink tissues, in growth, development and responses to various stresses [1][2][3], and acts as an important signal molecule that regulates the activation and inactivation of genes and microRNA expression levels [4][5][6]. The efficient use of sucrose as a carbon and energy source depends on its cleavage catalyzed by either invertases to glucose and fructose or its conversion by sucrose synthases to UDP-glucose and fructose [3].
In higher plants, invertases are further classified into two subfamilies that display characteristic pH optima for activity [7]. Acidic invertases (optimum pH 3.5-5.5) are cell wall or vacuole invertases, whereas neutral/alkaline invertases (NINs) with optimum pH from 6.8 to 8.0 are localized to the cytosol, mitochondria, plastids and nuclei [7,8]. Cell wall and vacuole invertases have been suggested to play important roles in physiological processes, including growth, development and responses to environmental stresses [2,3,9]. Acidic invertases are transcriptionally regulated by an array of signals [10,11]; however, the activity of acidic invertases is controlled by proteinaceous inhibitors, all of which are known as small inhibitors of b-fructosidases with sizes ranging from 15 to 23 kDa [12]. This suggests that the post-transcriptional modulation of acidic invertases is required for sugar unloading to sink tissues [13]. Although accumulating evidence has supported the physiological function of acidic invertases in higher plants, very limed information is available on the function of NINs, primarily because of protein instability and low expression and enzymatic activity [8]. Since the time that genes encoding nine NINs were identified in the Arabidopsis genome [14], several NINs have been identified from the genomes of a range of species, including Oryza sativa [14], Populus trichocarpa [15], Vitis vinifera [16], Lotus japonicas [17], Malus Â domestica Borkh [18], Saccharum officinarum [19] and Capsicum annuum [20]. In S. officinarum, transcripts of NINs were more abundant than acidic invertases in response to abiotic stresses [19]. Furthermore, the expression of CaNINV5 (C. annuum alkaline/neutral invertase 5) was increased during pepper fruit development [20], suggesting that NINs may also play important roles in physiological processes.
In this study, we conducted an in-depth in silico analysis of Chinese cabbage (Brassica rapa), a model crop for genomic study of the Brassica species, using public databases coupled with bioinformatics tools. To investigate the evolutionary relationships of B. rapa NINs (BraNINs), we evaluated their expansion and evolutionary mechanisms by their duplication. In addition, we analyzed the expression levels of BraNINs using the transcriptomes of Chinese cabbage leaves and roots under drought conditions. Taken together, our results further expand our knowledge of the physiological functions of NINs, and offer further insight into the evolutionary mechanisms of BraNINs.

Phylogenetic and gene duplication analysis of B. rapa NIN genes
The protein sequences of BraNIN genes were aligned by ClustalW and used for phylogenetic analysis using the Neighbor-Joining method in MEGA 7.0 software, with 100 replicates by default.

Identification and characterization of neutral/ alkaline invertases in Chinese cabbage
To identify the NIN genes, we analyzed the B. rapa genome database (Brassica rapa FPsc v1.3) using previously identified NIN sequences in Arabidopsis and apple. Subsequently, the redundant sequences were removed, resulting in a total of 11 putative NIN genes ( Table 1). The full-length coding sequences of BraNINs ranged from 1623 bp (BraNIN8) to 1959 bp (BraNIN3) encoding 540 to 652 amino acids. These putative BraNINs have a calculated molecular weight ranging from 61.6 to 73.7 kDa and a theoretical pI ranging from 5.68 to 8.68 (Table 1). In addition, a phylogenetic tree with 11 BraNIN protein sequences was constructed using the Neighbor-Joining method, to investigate the phylogenic relationships among NIN family members in Chinese cabbage. As shown in Figure 1A, the phylogenetic tree divided the BraNINs into two major groups that differed consistently at eight amino acid residues in the conserved motifs (C273V, C277S, Y287H, Y289H, V388L, S389Q, R460P and V471T based on the amino acid numbering of BraNIN5, Figure 1C). The a group contains BraNIN1-4, encoded by six exons with conserved location, whereas the b group BraNINs (BraNIN5-11) had a different number of exons ( Figure  1B), indicating that the a and b groups arose from distinct ancestral genes [18].
Since it has been assumed that plant NINs accumulate in the cytoplasm, the subcellular localization analysis of rice and Poncirus trifoliate NINs demonstrated that plant NINs located in organelles including mitochondria, plastids and chloroplasts [23,24]. Based on subcellular location analyses, plant NINs belonging to the a group were predicted to have mitochondrial or chloroplast localizations, whereas the other NINs were predicted to be cytosolic proteins [14,23,25]. Similarly, the prediction of subcellular localization using computational analysis indicated that a group BraNINs were located in mitochondria or chloroplasts (Table 1). In Arabidopsis, sucrose hydrolysis by chloroplastic A/N-Inv (Arabidopsis NIN) is required for controlling chloroplast-cytosolic carbon partitioning [26], suggesting that BraNINs might also be involved in the controlling carbon balance between cytoplasm and chloroplasts.
Intron phase analysis of BraNIN genes revealed that their first exons flanked by intron phase 0 ( Figure 1B) defined that introns positioned between two codons [27], similar to other NIN genes in higher plants including sugar cane [19] and apple [18]. In the a group, these genes were observed to present conserved intron-exon structures, whereas BraNIN genes of the b group exhibited different intron-exon structures and different number of exons. This suggests that a and b groups of BraNIN genes derived from different ancestral genes, and the conserved intron phases in genes of a group suggest stability during evolution.

Evolutionary patterns of the BraNIN family
Gene duplication resulting from unequal crossing over, retrotransposition or chromosomal duplication has been suggested as the main reason for the generation of new genes and gene family expansion [28]. In addition, it has been suggested that tandem and segmental duplications are the major sources of diversity for the evolution of large gene families in plants [29]. Chromosomal location and phylogenetic analyses of BraNIN genes and proteins (Table 1 and Figure 1A) indicated that the expansion of BraNINs is not due to tandem duplication. In poplar (P. trichocarpa), segmental duplication has been identified to play a leading role in the expansion of the NIN family [30]. Similarly, duplication analysis regarding the identification of chromosomal homologous segments within the genome showed two pairs of segmental duplicated paralogs (BraNIN5/6 and BraNIN7/8) (Figure 2) [30], suggesting that the segmental expansion in the Populus NIN family underwent a duplication event more recently than did the BraNIN family. In the BraNIN family, the Ka/Ks ratio of the two segmental duplication pairs was lower than 1 (Table 2), suggesting that these segmental duplication pairs were subjected to negative selection [31].

Expression analysis of BraNINs in response to drought stress
Drought or water deficit is the dominant factor affecting the growth and development of crops. Agricultural drought has become a major problem in global agricultural production. The activity of NIN in higher plants is affected by abiotic stresses including wounding, drought, salinity and low temperature [32]. In addition, PtrA/NINV (Poncirus trifoliata alkaline/neutral invertase)-overexpression improves tolerance to drought stress [24], suggesting that NINs play an important role when plants are subjected to abiotic stresses. To gain insight into the potential functions of BraNIN genes during responses to drought stress, we analyzed RNA-seq data of drought treated-Chinese cabbage. As shown in Figure 3, the transcript level of most BraNIN genes was down-regulated, whereas an  These gene pairs were identified at the terminal nodes of the gene tree shown in Figure 1A.
The number of synonymous sites (S), number of non-synonymous sites (N), synonymous substitution rate (Ks), and non-synonymous substitution rate (Ka) are presented for each pair. The data of the duplication events were estimated according to T ¼ Ks/ 2k. Mya, million years ago.
increased level of BraNIN1, BraNIN4 and BraNIN5 transcripts was observed in leaves and roots, when plants were treated with drought stress. Furthermore, BraNIN3 was down-regulated in the leaves but upregulated in the roots, and BraNIN6 was up-regulated in the leaves but down-regulated in the roots under drought-stress conditions. This suggested that BraNIN1, BraNIN4 and BraNIN5 might be important BraNIN family members in terms of drought responses in Chinese cabbage, since they were up-regulated by drought stress, suggesting a divergence in the function of BraNINs in response to drought stress.

Conclusions
In this study, based on genome-wide analysis, we conducted a detailed analysis on the NIN gene family in B. rapa, and developed new insights into how these genes have evolved in B. rapa. These data will support a solid foundation for further understanding of the underlying evolutionary mechanisms in NIN genes in higher plants. An in-depth analysis of BraNIN transcription pattern under drought stress provides an important starting point for future efforts to understand the physiological function of BraNINs.
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