Genome-wide identification and expression analysis of the phosphatase 2A family in rubber tree (Hevea brasiliensis)

The protein phosphatase 2As (PP2As) play a key role in manipulating protein phosphorylation. Although a number of proteins in the latex of laticifers are phosphorylated during latex regeneration in rubber tree, information about the PP2A family is limited. In the present study, 36 members of the HbPP2A family were genome-wide identified. They were clustered into five subgroups: the subgroup HbPP2AA (4), HbPP2AB’ (14), HbPP2AB'' (6), HbPP2AB55 (4), and HbPP2AC (8). The members within the same subgroup shared highly conserved gene structures and protein motifs. Most of HbPP2As possessed ethylene- and wounding-responsive cis-acting elements. The transcripts of 29 genes could be detected in latex by using published high-throughput sequencing data. Of the 29 genes, seventeen genes were significantly down-regulated while HbPP2AA1-1 and HbPP2AB55α/Bα-1were up-regulated by tapping. Of the 17 genes, 14 genes were further significantly down-regulated by ethrel application. The down-regulated expression of a large number of HbPP2As may attribute to the enhanced phosphorylation of the proteins in latex from the tapped trees and the trees treated with ethrel application.


Introduction
Protein phosphatase 2A (PP2A), a class of protein serine/threonine phosphatases, play a key role in manipulating protein phosphorylation by removing phosphate groups from the modified proteins [1][2]. The PP2A holoenzyme consists of three subunits: the structural subunit (A), the regulatory subunit (B), and the catalytic subunit (C) [3][4]. The subunit A comprises a series of conserved alpha-helical repeats, providing a scaffold for the binding of B and C; the subunit B determines substrate specificity; the subunit C dimerizes with A to form an active conformation and serves as a platform for interactions with B [5]. Based on their structural characteristics, the B subunits are further classified into B, B 0 , and B@ [6]. The large diversification of subunits confers on PP2A important roles in various biological processes [7]. Overexpression of StPP2Ac2b in Solanum tuberosum shows higher tuber induction rates compared PLOS ONE | https://doi.org/10.1371/journal.pone.0228219 February 5, 2020 1 / 14 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 to wild-type stolons [8]. In maize, over-expression ZmPP2AA1 improves low phosphate tolerance by remodeling the root system [9]. In Arabidopsis, the pp2a-b 0 γ and pp2a-b 0 θ mutants display lowered susceptibility towards the bacterial pathogen Pseudomonas syringae [10]. The rubber tree is the world's main source of natural rubber (cis-1, 4-polyisoprene) [11]. As an important industrial raw material, natural rubber is synthesized by the laticifers located in the inner bark of the rubber tree [12]. The milky laticifer cytoplasm, named latex, contains 20%-40% natural rubber. In natural rubber production, the milky latex is obtained by successive tapping, a process of artificially severing the laticifer rings every 2-3 days [13]. Latex exploitation by tapping enhances latex regeneration and the efficiency of natural rubber biosynthesis by activating jasmonate signalling in laticifers [14]. To increase the tapping efficiency, ethrel is applied to increase the rubber yield per tapping to economize on tapping workers. Prolongation of the duration of latex flow mainly increases the rubber yield per tapping by increasing the level of a 44 kDa protein in the cytosol of laticifer cells [15]. Ethrel itself, however, inhibits rubber biosynthesis [14].
Available data show that ethrel application induces the phosphorylation of a number of proteins in latex [16], which may be the mechanism behind changes in the efficiency of rubber biosynthesis. Although several protein kinase gene families such as mitogen-activated protein kinase (MPK) [17] and calcium-dependent protein kinase (CPK) [18][19] have been identified in rubber tree, information on the protein phosphatase gene families is limited. In the present study, 36 members of the HbPP2A gene family were genome-wide identified based on the latest version of the H. brasiliensis genome [20], and their structures and phylogenetics, and the ethrel-induced expression patterns were analyzed.

Plant materials and treatments
Ten-year-old virgin trees and regularly tapped trees of the H. brasiliensis clone CATAS7-33-97 were grown on the Experimental Farm of the Chinese Academy of Tropical Agricultural Sciences (CATAS) on Hainan Island. The ten-year-old trees were regularly tapped using a half spiral pattern every 3 days (S/2, d/3). The latex samples from ten tapped trees or virgin trees were combined as one sample. For ethrel treatment, the regularly tapped rubber trees had 1.5% ethrel applied on the tapping panel, and we collected the latex samples after 24 h. Three biological replications were performed.

RNA isolation and cDNA synthesis
Total RNA was extracted from samples using RNAplant Plus reagent (TIANGEN Biotech Co., Ltd., Beijing, China) and evaluated using a NanoDrop 2000 (Thermo Scientific Inc., USA). Approximately 1 μg of RNA was used for reverse transcription, and cDNA was synthesized using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific Inc., USA).

qRT-PCR analysis
The primers for the HbPP2A genes were designed using the Primer Premier 5 software (S1 Table). The experiment was performed with the CFX384 real-time PCR system (Bio-Rad, USA) using the SYBR Prime Script RT-PCR Kit (TaKaRa, Dalian). HbUBC2b was used as the standard control for gene normalization [22]. Three biological replicates were measured.

Statistical analysis
Changes in target gene relative expression levels across hormone treatments and tapping experiments were assessed by the 2 -ΔΔCt method. Expression level differences among different treatments were tested for significance by Duncan's test using SPSS Statistics 17.05. The capital letter or lowercase represented p < 0.01 or p < 0.05, respectively.

Identification of the HbPP2A genes in rubber tree
Three AtPP2AA, nine AtPP2AB', six AtPP2AB'', two AtPP2AB55, and five AtPP2AC genes were used as queries to perform genome-wide identification of the HbPP2A family in rubber tree based on tBLASTn searches (E<1-150). The HEAT domain (PF13646), the B56 domain (PF01603), the EF-hand domain (PF13499), and the WD40 domain (PF00400) in the putative members of the HbPP2A family were respectively further validated by using the NCBI Conserved Domain Search Service. In this way, thirty-six HbPP2A genes were identified and designated based on their presumptive Arabidopsis orthologue names. They included four HbPP2AAs, fourteen HbPP2AB's, six HbPP2AB''s, four HbPP2AB55s, and eight HbPP2ACs. The length of the HbPP2A proteins ranged from 280 aa (HbPP2AC6) to 588 aa (HbPP2AA1-1 and HbPP2AA2), the molecular weights ranged from 31.56 kDa (HbPP2AC6) to 65.57 kDa (HbPP2AA2), and their predicted isoelectric points ranged from 4.79 (HbPP2AC2-2) to 8.81 (HbPP2AB'η-5) ( Table 1).

The expression patterns of HbPP2A genes in latex upon tapping and ethrel application
The expression patterns of the 29 HbPP2A genes that were expressed in latex were further analyzed by qRT-PCR upon tapping and ethrel application (Fig 6, S3 Table). In comparison with virgin trees, two genes (HbPP2AA1-1 and HbPP2AB55α/Bα-1) were up-regulated, seventeen genes were down-regulated and ten genes had not been affected in the tapped trees without ethrel application. In comparison with the tapped trees without ethrel application, HbPP2AC2-2 was significantly up-regulated, twenty-one genes were down-regulated and eight genes had not been affected in the tapped trees with ethrel application. Of the down-regulated seventeen genes by tapping, fourteen genes were further significantly down-regulated by ethrel application.
The protein phosphorylated modification is manipulated by protein kinases and phosphatases [27][28]. Ethrel application increases the level of phosphorylation of a number of proteins in latex [16]. The enhanced protein phosphorylation in latex may be associated with MPK and CPK considering that four HbMPK and eight HbCPK genes in latex were upregulated by ethrel application [17][18][19]. It may also be associated with HbPP2As as 21 of the 29 laticifer-expressed HbPP2A genes are significantly down-regulated upon ethrel application (Fig 6). The presence of ERE, a cis-acting element with a core sequence (TTTGAATT) that responses to ethylene [29][30] in the promoter regions of most HbPP2As confers their responses to ethrel application. In addition, 24 of 29 latex-expressed HbPP2As possess the wound responsive elements (WRE3, W-box, WUN-motif), suggesting their potential responsive to mechanical wounding. The cis-acting element WRE3 is absent from the 8 of 10 genes that have no response to tapping. Alternatively, the tapping-induced down-regulation of HbPP2As may mainly attribute to the wounding-induced endogenous ethylene since 14 of the 17 genes thatare down-regulated by tapping are further down-regulated by ethrel application. The wounding-induced ethylene formation is controlled by the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis (ACS) [31]. The C-terminal domain of ACS can be phosphorylated by MPK and CPK and dephosphorylated by PP2A [32][33][34]. The ACS activity is significantly activated in the Arabidopsis pp2aa1 mutant [35], suggesting that the ACS activity is positively related to the increased phosphorylation level of ACS by inhibiting AtPP2AA1. Here, the significant down-regulation of its ortholog, HbPP2AA1-2, may result in the production of endogenous ethylene by activating ACS activity.
The enhanced in vitro rubber biosynthesis of latex from tapped trees is closely associated with the up-regulation of the genes encoding the key enzymes for rubber biosynthesis by activating jasmonate signalling [14]. It may also be related to the changes in the key enzyme activity by phosphorylation modification. Available data shows that PP2As act as negative regulators for plant secondary metabolism. AtPP2AB'γ negatively regulates the methoxylation of hydroxylated indole glucosinolates and the formation of 4-hydroxyindol-3-yl-methyl glucosinolate [36]. AtP-P2AB''β acts as a posttranslational negative regulator of hydroxy-3-methyl glutaryl coenzyme A reductase activity [37]. With this respect, the tapping-caused down-regulation of HbPP2As may attribute to the enhanced rubber biosynthesis in tapped trees. Given the effects of ethrel application on enhanced phosphorylation of proteins in latex are mainly ascribed to the down-regulated expression of HbPP2As, the tapping-caused down-regulated expression of a large number of HbPP2As may result in the enhanced phosphorylation of proteins in latex from tapped trees. The effect of enhanced protein phosphorylation on rubber biosynthesis remains to be elucidated.

Conclusions
Ethrel application induces the phosphorylation of a large number of proteins in the latex from rubber trees. The down-regulated expression of the most of the genome-wide identified HbPP2A genes may attribute to the enhanced phosphorylation of proteins in the latex from ethrel treated trees. The tapping-caused down-regulated expression of seventeen HbPP2As may be related to the endogenous ethylene generation. The genome-wide identification of HbPP2A proteins will facilitate the regulation of the phosphorylized modification of rubber biosynthesis-related proteins in rubber tree.
Supporting information S1 Fig. Conserved amino acid sequences of 20