Temporal regulation of two cytosolic phosphoglucomutases during stigma development in ornamental kale (Brassica oleracea var. acephala)

ABSTRACT

Phosphoglucomutases (PGM) (5.4.2.2.) belong to the Phosphohexomutases superfamily and are highly specific in catalyzing the interconversion of Glc-1-P to Glc-6-P. In this study, we characterize the expression and activity of two cytosolic PGMs (cPGM2 and cPGM3) stigmas of ornamental kale during flower development. In stigmas, cPGM expression and activity showed a gradual increase during stigma development with the highest activity around the time of anthesis. Blocking of cPGM activity in the stigmas using a known inhibitor, resulted in breakdown of self-incompatibility in immature S3 and S4 stigmas, but had no effect on the fully mature S5 stigmas. It is likely that cPGMs are required for accumulation of factors necessary for SI response in mature stigmas.

Lan et al. 2017, investigated changes in the proteome during different stages of stigma development and identified 79 unique proteins which are temporally regulated during stigma development.¹ Of these, 11 unique proteins that displayed an increase during stigma development were cataloged to be involved in carbohydrate and energy metabolism.¹ When these were subjected to gene ontology analysis using the Princeton GO Term Finder (http://go.princeton.edu/cgi-bin/GOTermFinder), we found a cluster of three unique proteins involved in carbohydrate metabolism (27.3%, p = 4.68E⁻⁰⁶) (cytoplasmic phosphoglucomutase 2, AT1G70730, cytoplasmic phosphoglucomutase 3, AT1G23190; UDP-glucose pyrophosphorylase 2, AT5G17310). A heatmap generated for these proteins based on their relative abundance at different developmental stages of stigma showed a gradual upregulation of these proteins during stigma development (Figure 1a). Phosphoglucomutases (PGM) (5.4.2.2.), which belong to the Phosphohexomutases superfamily, are highly specific in catalyzing the interconversion of Glc-1-P to Glc-6-P.² The UDP-glucose pyrophosphorylase (UGPase), which plays important roles in the carbohydate metabolism, catalyzes the reversible conversion of Glc-1-P to UDP-Glc (Fig. 1b).³ Glc-6-P participates primarily in the respiratory pathways, while Glc-1-P and UDP-Glc are required for the synthesis of sucrose and cell wall constituents, including cellulose and callose.^2–4 Previous knock-down of cPGM in Arabidopsis resulted in aberrant plant growth and development, as well as gametophyte (ovule and pollen) development,^5,6 but its role during stigma development or SI has not been investigated thus far. Here, we show the molecular characterization of two cytosolic phosphoglucomutases (cPGM2 and cPGM3) that were upregulated during stigma development.

Figure 1.

Expression of cPGM and UGPase, involved in carbohydrate metabolism are regulated during sigma development. A). Heatmap showing differential expression of proteins involved in carbohydrate metabolism during stigma development. The proteins are represented by homologs from Arabidopsis thaliana. Respective spot numbers on the 2D-DIGE are also indicated on the right side. cPGM2: AT1G70730, cPGM3: AT1G23190, UGPase: AT5G17310. B). Both cPGMs and UGPase participate in carbohydrate metabolic pathway.

To characterize cPGMs, cDNAs of BocPGM2 and BocPGM3 were generated from RNA extracted from stigmas of self-incompatible (S_13-bS_13-b) ornamental kale (Brassica oleracea var. acephala). The nucleotide sequences of the putative full-length cDNA of BocPGM2 and BocPGM3 were 1,922 bp and 2,066 bp respectively. The BocPGM2 contains a short 5ʹ (41 bp)- and a long 3ʹ (129 bp)-untranslated region and an open reading frame of 1,752 bp, which encoded a peptide of 583 amino acids with the predicted molecular mass of 63.4 kDa (NCBI accession: MG470768). The BocPGM3 contained a short 5ʹ (68 bp)- and a long 3ʹ (249 bp)-untranslated region and an open reading frame of 1,749 bp, which encoded a peptide of 582 amino acids with the predicted molecular mass of 63.3 kDa (NCBI accession: MG470769). The amino acid sequences of BocPGM2 and BocPGM3 were matched with unique peptides from Mass spectrometry data.¹ BocPGM2 had 94% amino acid sequences identity with BocPGM3 (Fig S1), and both BocPGM2 and BocPGM3 belonged to the cPGM cluster based on phylogeny (Fig. S2).

To test whether BocPGM2 and BocPGM3 have phosphoglucomutase activity, recombinant BocPGM2 and BocPGM3 proteins were generated in E. coli as 6XHis-tagged recombinant proteins, purified and resolved around 66 kDa on SDS-PAGE, consistent with the predicted size of the recombinant proteins (Fig. S3). When enzyme activity for these proteins were assessed by resolving them on a native-PAGE, followed by staining for enzyme activity, both recombinant BocPGM2 and BocPGM3 displayed activity that could convert Glc-1-P to Glc-6-P (Fig. 2a). The activity of the enzyme also increased in a concentration dependent manner (Fig. 2a). These results demonstrated that both BocPGM2 and BocPGM3 possess phosphoglucomutase activity in in vitro assays.

Figure 2.

Enzyme activity assays and expression pattern of BocPGM2 and BocPGM3. A). Native-PAGE followed by enzyme activity staining showed that BocPGM2 and BocPGM3 are active enzymes (top panel). Input proteins were separated by Native-PAGE and stained with Coomassie brilliant blue (CBB) R250 (bottom panel). B). Purified proteins from various stigma developmental stages were separated by SDS-PAGE transferred and subjected to Western blotting using anti-BocPGM2 and anti-BocPGM3, respectively. C). Native-PAGE followed by enzyme activity staining showed that cPGM activity increased from S1 to S5 stigma developmental stages. Equal amounts of the total proteins were separated by Native-PAGE and stained with CBB R250 (bottom panel).

To explore the expression pattern of cPGM2 and cPGM3 more thoroughly during stigma development, polyclonal antibodies were raised against BocPGM2 and BocPGM3. Western blots revealed that BocPGM2 and BocPGM3 were abundant in anther, stigma, and petal, and were present at lower levels in the style and ovary (Fig. S4). To assess their expression pattern in developing stigmas, total proteins extracted from S1-S5 stage stigmas were subjected to Western blots with anti-BocPGM2 and anti-BocPGM3 antibodies respectively. In stigmas, both proteins were expressed at basal levels during early developmental stages (S1 and S2 stage), but rapidly increased from S3 and reached its highest level in the S4 and S5 stages (Fig. 2b). When PGM activity during stigma development was assessed using a native-PAGE, followed by staining for enzyme activity, the increase in cPGM protein levels during stigma maturation correlated with an increase in enzyme activity from S1 to S5 stages (Fig. 2c). These results indicate that both BocPGM2 and BocPGM3 are developmentally regulated in the stigmas and display enhanced activity over the course of stigma development, with the maximum activity in S4 and S5 stages coinciding with stigma maturity.

To investigate the role of cPGMs during pollen-pistil interactions, we used sodium orthovanadate, an inhibitor of the glycolytic pathway, which has been shown to efficiently inhibit PGM activity^7,8 to examine the role of PGMs during compatible and SI responses. Although vanadate is a known potent inhibitor of tyrosine phosphatases and efficiently blocks phosphoryl transfers, it has been specifically used as an inhibitor of PGM and thus indirectly inhibits carbohydrate metabolism.^9,10 To examine a role for PGM during pollination, flowers of self-incompatible S_13-bS_13-b stigmas were treated with vanadate (100 µM) and were either pollinated with compatible Brassica napus cv Westar or self-incompatible S_13-bS_13-b pollen followed by aniline blue assays. We did not observe any difference in either pollen attachment or pollen tube formation following compatible pollination of vanadate treated S4 and S5 stigmas (Fig. S5). Contrastingly, when similar stages were pollinated with self–incompatible pollen, vanadate-treated S3 and S4 stigmas were able to support pollen attachment and penetration, essentially resulting in a breakdown of SI response in these young stigmas. However, this response was not observed in S5 stigmas, indicating that vanadate treatment or inhibiting PGM is ineffective in breaking down SI in fully mature stigmas (Fig. 3).

Figure 3.

Vanadate treatment leads to breakdown of SI in younger stigmas (S4 stage) while mature stigmas (S5) are unaffected. Aniline blue assay to assess pollen attachment and pollen tube germination after vanadate (100 μM) treatment compared with untreated self-incompatible stigma at S3 and S4 stages following incompatible pollination (A). A significant increase in the mean number of pollen attachment (B) and pollen tubes were observed (C) in S3 and S4 stigmas (t-test, *p < 0.05, n=6). Values are presented as ± SEM.

Given the wide use of vanadate as a potent inhibitor of tyrosine phosphatases, any interpretations arising from these observations must be assumed to be a result of all the interactions that vanadate could potentially have in the stigmas and not exclusively from PGM inhibition. Till date, only a single kinase associated protein phosphatase (KAPP) has been shown to interact with SRK, the female determinant of SI response and has been hypothesized to negatively regulate SRK activity during pollination.¹¹ However, KAPP and the other proteins of the PP2C family are insensitive to vanadate, making it unlikely that KAPP could be a target of vanadate.¹² It is important to note that pollination during stage 3 and 4 during flower development is often used as a technique for forced successful self-pollination in self-incompatible cultivars of Brassica. This is due to low abundance of SRK and other SI factors at the stigmatic interface responsible for rejection as these are known to be temporally regulated in stigmas with maximal expression prior to anthesis.^13–15 Moreover, glycosylation has also been shown to be important for SRK function and self-pollen rejection.¹⁶ Since cPGM is involved in carbohydrate metabolism and cell wall formation, we speculate that the inhibition of cPGM could lead to a retarded growth of stigmatic cells, thus prolonging the stigmatic cell maturation process or slowing down accumulation of SI factors allowing stronger breakdown of SI in younger stigmas.

Taken together, through molecular characterization of the two cPGMs from ornamental kale, we have shown that cPGM expression and activity increase as the stigma matures and their activity could be essential for acquisition of a strong SI phenotype in the stigmas at maturity. A more direct evidence for their role in promoting SI could be assessed through suppressing cPGMs through either RNAi or CRISPR-Cas9-mediated genome editing to eliminate their function.

Funding Statement

This work was supported by Natural Sciences and Engineering Research Council of Canada [RT735240] and funding from University of Calgary to M.A.S. A.K. was supported by an Eyes High International Doctoral Scholarship and Faculty of Graduate Studies Doctoral Scholarship from the University of Calgary. This work was also supported by the Fundamental Research Funds for the Central Universities [No. 2572018BD01].

Conflicts of interest

No potential conflicts of interest were disclosed.

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