Specialized Vacuoles of Myrosin Cells: Chemical Defense Strategy in Brassicales Plants

Specialized Vacuoles of Myrosin Cells: Chemical Defense Strategy in Brassicales Plants Abstract Plant vacuoles display many versatile functions. Vacuoles in vegetative tissues are generally involved in protein degradation, and are called lytic vacuoles. However, vegetative vacuoles in specialized cells can accumulate large concentrations of proteins, such as those in idioblast myrosin cells along veins in the order Brassicales, which store large amounts of myrosinases (thioglucoside glucohydrolase and thioglucoside glucohydrolase). Myrosinases cleave the bond between sulfur and glucose in sulfur-rich compounds (glucosinolates) to produce toxic compounds (isothiocyanates) when plants are damaged by pests. This defense strategy is called the myrosinase–glucosinolate system. Recent studies identified atypical myrosinases, PENETRATION 2 (PEN2) and PYK10, along with key components for development of myrosin cells. In this review, we discuss three topics in the myrosinase–glucosinolate system. First, we summarize the complexity and importance of the myrosinase–glucosinolate system, including classical myrosinases, atypical myrosinases and the system that counteracts the myrosinase–glucosinolate system. Secondly, we describe molecular machineries underlying myrosin cell development, including specific reporters, cell lineage, cell differentiation and cell fate determination. The master regulators for myrosin cell differentiation, FAMA and SCREAM, are key transcription factors involved in guard cell differentiation. This indicates that myrosin cells and guard cells share similar transcriptional networks. Finally, we hypothesize that the myrosinase–glucosinolate system may have originated in stomata of ancestral Brassicales plants and, after that, plants co-opted this defense strategy into idioblasts near veins at inner tissue layers. Myrosinase–Glucosinolate Chemical Defense System The myrosinase–glucosinolate system is a plant defense strategy to protect against herbivores. This defense system is characteristic of Brassicales plants as both myrosinases and glucosinolates are primarily generated by the order Brassicales (Rask et al. 2000, Halkier and Gershenzon 2006, Wittstock and Burow 2010, Shirakawa et al. 2016a). Under normal developmental conditions, myrosinases are sequestered from glucosinolates because they are localized in two different specialized cells, myrosin cells and glucosinolate-accumulating cells, respectively (Koroleva et al. 2000, Rask et al. 2000, Shroff et al. 2008, Kissen et al. 2009, Nintemann et al. 2018). Herbivore feeding breaks these cells open and the free myrosinases convert glucosinolates into toxic isothiocyanates by cleaving the bond between sulfur and glucose (Fig. 1A) (Rask et al. 2000, Halkier and Gershenzon 2006, Wittstock and Burow 2010, Shirakawa et al. 2016a). We summarize classical and atypical myrosinases in Arabidopsis thaliana, describe insect and bacterial myrosinases, and explore countervailing strategies against the myrosinase–glucosinolate system by insects and pathogens. For more detailed information about glucosinolates, see Wittstock and Halkier (2002), Grubb and Abel (2006), Hopkins et al. (2009), Sønderby et al. (2010), Jørgensen et al. (2015), Chezem and Clay (2016) and Burow and Halkier (2017). Fig. 1 View largeDownload slide Myrosinase–glucosinolate defense system. (A) Chemical reactions of the myrosinase–glucosinolate system. Myrosinase cleaves a bond between a sulfur and a glucose to produce aglycone, which is unstable. Unstable aglycones are converted into isothiocyanates by Lossen-like rearrangement. (B) A section of a cauline leaf from Arabidopsis thaliana. A myrosin cell vacuole (arrow) is filled with smooth materials that have higher electron densities than the vacuoles in an adjacent mesophyll cell. Scale bar = 5 µm (C) An enlarged image of the boxed area in (B). Dots of the anti-TGG2 immunogold label are observed in the vacuole of myrosin cells. Scale bar = 1 µm. (D) Aligned amino acid sequences of myrosinases. Typical myrosinases (TGG1/2/4/5) have Q and atypical myrosinases (PYK10 and PEN2) have E (asterisk). Atypical myrosinases (PYK10 and PEN2) have two basic amino acid residues (cross marks). (E) ER bodies and peroxisomes. A confocal image of the epidermal cells in Arabidopsis petioles expressing peroxisome-localized DsRed–PTS1 (magenta) and ER-localized GFP (green). ER bodies are football-shaped structures with bright green signals (arrows). Scale bar = 10 µm. Fig. 1 View largeDownload slide Myrosinase–glucosinolate defense system. (A) Chemical reactions of the myrosinase–glucosinolate system. Myrosinase cleaves a bond between a sulfur and a glucose to produce aglycone, which is unstable. Unstable aglycones are converted into isothiocyanates by Lossen-like rearrangement. (B) A section of a cauline leaf from Arabidopsis thaliana. A myrosin cell vacuole (arrow) is filled with smooth materials that have higher electron densities than the vacuoles in an adjacent mesophyll cell. Scale bar = 5 µm (C) An enlarged image of the boxed area in (B). Dots of the anti-TGG2 immunogold label are observed in the vacuole of myrosin cells. Scale bar = 1 µm. (D) Aligned amino acid sequences of myrosinases. Typical myrosinases (TGG1/2/4/5) have Q and atypical myrosinases (PYK10 and PEN2) have E (asterisk). Atypical myrosinases (PYK10 and PEN2) have two basic amino acid residues (cross marks). (E) ER bodies and peroxisomes. A confocal image of the epidermal cells in Arabidopsis petioles expressing peroxisome-localized DsRed–PTS1 (magenta) and ER-localized GFP (green). ER bodies are football-shaped structures with bright green signals (arrows). Scale bar = 10 µm. Classical myrosinases Myrosinase is a thioglucoside glucohydrolase (TGG) (Rask et al. 2000). The Arabidopsis genome has a total of six TGG genes (TGG1−TGG6). TGG1 and TGG2 are highly expressed in above-ground tissues to protect plants against herbivores; TGG4 and TGG5 are expressed specifically in the root tip (Fu et al. 2016); and TGG3 and TGG6 are expressed in pollen (Wang et al. 2009). The physiological roles of TGG3−TGG6 are unknown. Here, we designate TGG1−TGG6 as classical myrosinases that are characterized by an evolutionarily conserved glutamine (Q) residue for binding to the glucose ring (Fig. 1D) (Rask et al. 2000, Nakano et al. 2014). Recent studies suggest that this conserved residue is essential for binding to the glucose of aliphatic glucosinolates including sinigrin, which is widely used to test for myrosinase activity (see below). TGG1 and TGG2 are expressed specifically in two different cells: myrosin cells along leaf veins and stomatal guard cells (Fig. 2A) (Xue et al. 1995, Andreasson et al. 2001, Husebye et al. 2002, Barth and Jander 2006, Ueda et al. 2006, Shirakawa et al. 2016b). TGG1 and TGG2 are abundant proteins in aerial parts of Arabidopsis plants (Ueda et al. 2006) and are accumulated at much higher levels in myrosin cells than in guard cells (Shirakawa et al. 2014b). Myrosin cells store large amounts of TGG1 and TGG2 in the vacuoles, which show higher electron densities than the lytic vacuoles of surrounding mesophyll cells (Fig. 1B, C) (Andreasson et al. 2001, Ueda et al. 2006, Shimada et al. 2018). Hence, the vacuoles of myrosin cells can be classified as protein storage vacuoles in the vegetative tissues. Fig. 2 View largeDownload slide Patterning of myrosin cells and vascular cells in the wild type and syp22. (A) Rosette leaf of a transgenic plant [wild-type (WT) background] expressing the myrosin cell marker ProTGG2:VENUS-2sc. Myrosin cells were visualized by fluorescence (left), and leaf veins of the same leaf area were observed by dark-field microscopy (right). Boxed areas are enlarged in the respective lower panels. (B) A rosette leaf of a syp22 plant expressing ProTGG2:VENUS-2sc contains a large number of myrosin cells and shows limited development of vascular cells. Scale bars = 1 mm. Fig. 2 View largeDownload slide Patterning of myrosin cells and vascular cells in the wild type and syp22. (A) Rosette leaf of a transgenic plant [wild-type (WT) background] expressing the myrosin cell marker ProTGG2:VENUS-2sc. Myrosin cells were visualized by fluorescence (left), and leaf veins of the same leaf area were observed by dark-field microscopy (right). Boxed areas are enlarged in the respective lower panels. (B) A rosette leaf of a syp22 plant expressing ProTGG2:VENUS-2sc contains a large number of myrosin cells and shows limited development of vascular cells. Scale bars = 1 mm. Atypical myrosinases Recent work identified two atypical myrosinases: PENETRATION 2 (PEN2) and PYK10 (Bednarek et al. 2009, Nakano et al. 2017). The conserved glutamine (Q) in classical myrosinases is replaced by glutamic acid (E) in both PEN2 and PYK10 (Fig. 1D) (Nakano et al. 2014), and both can cleave the bond between sulfur and glucose in glucosinolates. How do atypical myrosinases recognize their substrate, glucosinolate, without the conserved glutamine? Three-dimensional structure modeling suggested that two basic amino acids conserved in atypical myrosinases are involved in binding to glucosinolates instead of a conserved glutamine residue in classical myrosinases (Fig. 1D) (Nakano et al. 2017). Interestingly, atypical myrosinases are not accumulated in myrosin cells. PYK10 is distributed in roots and PEN2 is accumulated in leaf epidermal cells. Atypical myrosinases differ from classical myrosinases in terms of their subcellular localization: PEN2 and PYK10 are localized in peroxisomes and endoplasmic reticulum (ER) bodies, respectively (Fig. 1E) (Matsushima et al. 2003, Lipka et al. 2005). PEN2 activates indole glucosinolates to produce antifungal isothiocyanates, which are thought to be secreted from cells via the PENETRATION 3 (PEN3) ABC transporter (Stein et al. 2006, Bednarek et al. 2009, Bednarek 2012). PYK10 prefers indole glucosinolates to aliphatic glucosinolates (Nakano et al. 2017). These results expand the definition of myrosinases and raise questions about the enzymatic specificity for indole and aliphatic glucosinolates. Structural analyses of the myrosinase–glucosinolate complex will provide answers to this question. For more detailed information about the biochemistry and evolution of atypical myrosinases, see Nakano et al. (2014), Piasecka et al. (2015) and Pastorczyk and Bednarek (2016). Pests and pathogens have evolved factors that neutralize the myrosinase–glucosinolate system After the myrosinase–glucosinolate system emerged in Brassicales, pests and pathogens evolved an anti-myrosinase–glucosinolate system. Plutella xylostella (diamondback moth) is a crucifer specialist insect that produces glucosinolate sulfatase (GSS) to detoxify glucosinolates (Ratzka et al. 2002). GSS was isolated from the gut contents of Plutella. GSS hydrolysis of glucosinolates produces desulfo-glucosinolates, which are not myrosinase substrates. It is unknown whether GSS is specifically conserved in specialist herbivores (Ratzka et al. 2002). To validate the co-evolutionary history of defense and neutralizing systems in plants and pests, the genomic and amino acid sequences of GSS should be comprehensively analyzed in both specialist and generalist herbivores. Pathovars of Pseudomonas syringae Psm E4326 and Pst DC3000 have a unique strategy to counteract the myrosinase–glucosinolate defense system (Fan et al. 2011). The Survival in Arabidopsis extracts (Sax) gene confers resistance to aliphatic isothiocyanates in pathogenic P. syringae. Pst DC3000 quintuple mutants lacking SaxA/B/F/D/G genes could not grow in young Arabidopsis leaves but could grow in young leaves of Arabidopsis myb28 myb29 mutants, which did not produce aliphatic glucosinolates. The accumulated amounts of the major glucosinolates were unchanged in plants overexpressing SaxA, suggesting that SaxA inhibits aliphatic isothiocyanate production after glucosinolate breakdown (Fan et al. 2011). These combined results suggest that the myrosinase–glucosinolate defense system forms part of an arms race between Brassicales plants and their pests and pathogens. Myrosinases in other species Myrosinases have recently been identified in insects and bacteria (Bridges et al. 2002, Beran et al. 2014, Albaser et al. 2016). The specialist herbivore Phyllotreta striolata (flea beetle) expresses myrosinases and ingests glucosinolates from Brassicales plants (Beran et al. 2014). Sequence analyses suggest that insect and bacterial myrosinases evolved independently from plant myrosinases (Nakano et al. 2017). The physiological roles of isothiocyanates from insects and bacteria are still largely unknown. Myrosin Cell Development in Arabidopsis Our understanding of myrosin cell development has grown markedly during the past 5 years. Here, we summarize reporter genes, cell lineages, cell differentiation and cell fate determination in myrosin cells. The biggest advance is identification of the master transcription factors for myrosin cell differentiation, which are the basic helix–loop–helix (bHLH) transcription factors FAMA, SCREAM (SCRM) and SCRM2 (Li and Sack 2014, Shirakawa et al. 2014b). All three transcription factors were previously identified as master regulators of guard cell differentiation (Ohashi-Ito and Bergmann 2006, Kanaoka et al. 2008). This indicates that myrosin cell development is regulated by a transcriptional network similar to that involved in guard cell development. For more detailed information about these two developmental programs, see Lau and Bergmann (2012), Han and Torii (2016) and Shirakawa et al. (2016a). Reporter genes for myrosin cells An authentic reporter gene for myrosin cells is TGG1. Multiple methods, including in situ hybridization of TGG1 mRNA (Xue et al. 1995), analysis of the TGG1 gene promoter (Husebye et al. 2002, Barth and Jander 2006) and immunohistochemistry of the TGG1 protein (Andreasson et al. 2001, Husebye et al. 2002, Ueda et al. 2006) identified elongated and irregularly shaped idioblasts along veins in Arabidopsis leaves. However, TGG1 is also expressed in guard cells in above-ground organs (Husebye et al. 2002, Barth and Jander 2006). Several new reporters of myrosin cells have been identified within the past decade (Table 1). The expression patterns of three reporters, ProTGG2:GUS, MYR001:GUS (ProVSR1:GUS) and ProbHLH090:GUS-GFP, are specific to the myrosin cell lineage (Barth and Jander 2006, Shirakawa et al. 2014a, Shirakawa et al. 2014b). These lines are useful for isolating intact myrosin cells from whole plants in future experiments [e.g. fluorescence-activated cell sorting (FACS) analysis]. The biological functions of several of these genes are still largely unknown. Studies of their loss-of-function mutants may shed light on novel functions of myrosin cells. Table 1 Specific reporter lines for myrosin cells Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  MC, myrosin cells; GC, guard cells; ME, mesophyll cells. aProTGG2:GUS is expressed in only in myrosin cells, while ProTGG2:VENUS-2sc is expressed in both myrosin cells and guard cells. bA GAL4 GFP (green fluorescent protein) enhancer trap line. Table 1 Specific reporter lines for myrosin cells Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  MC, myrosin cells; GC, guard cells; ME, mesophyll cells. aProTGG2:GUS is expressed in only in myrosin cells, while ProTGG2:VENUS-2sc is expressed in both myrosin cells and guard cells. bA GAL4 GFP (green fluorescent protein) enhancer trap line. Lineage of myrosin cells Myrosin cells are distributed along veins in Arabidopsis (Fig. 2A), Cardamine (Fig. 3A) and Nasturtium (Kissen et al. 2009, Shirakawa et al. 2014c, Shirakawa et al. 2016b). Myrosin cells lie side by side with vascular precursor cells and phloem cells (Andreasson et al. 2001, Shirakawa et al. 2014c, Shirakawa et al. 2016b); therefore, it was hypothesized that myrosin cells belong to a vascular cell lineage. However, recent studies reveal that myrosin cells did not differentiate from vascular and vascular precursor cells (Li and Sack 2014, Shirakawa et al. 2014b, Shirakawa et al. 2016b). These studies carefully compared expression patterns of myrosin cell reporters (TGG2, FAMA and E1728) and vascular cell reporters (AtHB8, Q0990 and J1721), and showed that myrosin cells differentiate from isodiametric small cells that are part of the ground meristem (ground meristem cells) (Fig. 3B, 3C). Ground meristem cells are a stem cell-like cell population located in the inner tissues of leaf primordia, and are mother cells for vascular precursor cells and mesophyll cells (Sawchuk et al. 2007, Sawchuk et al. 2008). Plants sort ground meristem cells into different cell types, including myrosin precursor cells, vascular precursor cells and mesophyll cells. Myrosin precursor cells (i.e. FAMA-expressing isodiametric ground meristem cells) mature into large, isolated and irregularly shaped idioblast myrosin cells (Fig. 3) (Li and Sack 2014, Shirakawa et al. 2014b). Fig. 3 View largeDownload slide Distribution of myrosin cells and expression pattern of FAMA in leaf inner tissue layers. (A) Coomassie brilliant blue staining of a mature Cardamine schinziana leaf. Myrosin cells distributed along leaf veins are stained blue. Myrosin cell shapes are not uniform, but they are elongated along leaf veins. Scale bar = 100 µm. (B) A 3-D image of the inner leaf tissue of leaf primordia expressing ProFAMA:GFP (white). The 3-D image was reconstructed and deconvolved. GFP signals are observed in both cell nuclei and the cytosol. FAMA-expressing cells form network structures along veins. Scale bar = 20 µm. (C) Enlarged image of the boxed area in (A). Three isodiametric small cells (ground meristem cells) started to express FAMA. Arrowheads indicate nuclei. Scale bar = 5 µm. Fig. 3 View largeDownload slide Distribution of myrosin cells and expression pattern of FAMA in leaf inner tissue layers. (A) Coomassie brilliant blue staining of a mature Cardamine schinziana leaf. Myrosin cells distributed along leaf veins are stained blue. Myrosin cell shapes are not uniform, but they are elongated along leaf veins. Scale bar = 100 µm. (B) A 3-D image of the inner leaf tissue of leaf primordia expressing ProFAMA:GFP (white). The 3-D image was reconstructed and deconvolved. GFP signals are observed in both cell nuclei and the cytosol. FAMA-expressing cells form network structures along veins. Scale bar = 20 µm. (C) Enlarged image of the boxed area in (A). Three isodiametric small cells (ground meristem cells) started to express FAMA. Arrowheads indicate nuclei. Scale bar = 5 µm. Master regulators of myrosin cell differentiation The bHLH transcription factor FAMA was identified as a master regulator of myrosin cell development (Li and Sack 2014, Shirakawa et al. 2014b, Shirakawa et al. 2016a). FAMA starts to be expressed in isodiametric small cells that are morphologically indistinguishable from surrounding cells (Fig. 3B, C). No myrosin cells and no accumulation of TGG1 and TGG2 are observed in fama mutants (Li and Sack 2014, Shirakawa et al. 2014b). Conversely, plants overexpressing FAMA make a large number of myrosin cells (Shirakawa et al. 2014b). SCRM and SCRM2 are binding partners of FAMA, and are redundantly essential for myrosin cell development (Shirakawa et al. 2014b). These three transcription factors were previously identified as master regulators of guard cell differentiation, particularly transition from guard mother cells into guard cells (Ohashi-Ito and Bergmann 2006, Kanaoka et al. 2008, Lau and Bergmann 2012, Han and Torii 2016). These results indicate that common cell differentiation pathways may be shared between myrosin cells and guard (mother) cells. For example, both cell types accumulate myrosinases (TGG1 and TGG2), although there are some differences in accumulation levels (Zhao et al. 2008, Shirakawa et al. 2014b). Genes homologous to FAMA, SPEECHLESS and MUTE (MacAlister et al. 2007, Pillitteri et al. 2007) are not required for myrosin cell development (Li and Sack 2014, Shirakawa et al. 2014b). A total of 32 genes were identified as candidate factors downstream of FAMA (Shirakawa et al. 2014b). These genes are highly up-regulated in both chemically induced FAMA overexpression lines (Hachez et al. 2011) and syp22 mutants containing numerous myrosin cells (Ueda et al. 2006; see below). As expected, one of these genes, bHLH090, is expressed specifically in the myrosin cell lineage (Table 1) (Shirakawa et al. 2014b). To identify the transcriptional network of the FAMA–SCRM/2 complex, we should examine expression patterns and perform mutant analyses of these 32 genes. Analysis of downstream targets of FAMA will shed light on the molecular mechanisms of how protein storage vacuoles of myrosin cells accumulate large amounts of the TGG1 and TGG2 proteins even in the vegetative tissues, which generally develop lytic vacuoles responsible for protein degradation. The FAMA-downstream factors could include vacuolar transporters, vacuolar channel proteins and vacuolar trafficking components. Future work will involve comprehensive analyses of cis-regulatory elements and binding sites of the FAMA–SCRM/2 transcriptional complex in target gene promoters. For additional hypothetical transcriptional mechanisms for FAMA in different cell types, see Shirakawa et al. (2016a). Myrosin cell fate determination How do plants determine the differentiation of ground meristem cells into myrosin precursor cells, vascular precursor cells and mesophyll cells before FAMA is expressed? The molecular mechanism underlying cell fate determination in myrosin cells was identified by analysis of mutants with increased numbers of myrosin cells. Loss of vacuolar trafficking machineries [SYNTAXIN OF PLANTS 22 (SYP22), VACUOLAR PROTEIN SORTING 9 A (VPS9A) and CONTINUOUS VASCULAR RING (COV1)] induced overproduction of myrosin cells (Fig. 2) (Shirakawa et al. 2014a, Shirakawa et al. 2014c). The accumulated myrosin cells in these mutants are not distributed randomly; most are still distributed near veins, although they establish a much denser network (Ueda et al. 2006, Shirakawa et al. 2014c). These network structures contain myrosin cells along veins, as do wild-type plants, and myrosin cells branch from them (Fig. 2). The myrosin cells in mutants are distributed along the abaxial side of leaves, similar to wild-type plants. Analysis of FAMA expression patterns and levels in syp22 mutants revealed that increased numbers of myrosin cells were not induced by accelerated division of myrosin cells, but rather at the fate determination step of myrosin cells from the pool of ground meristem cells. In syp22 mutants, more isodiametric ground meristem cells start to express FAMA at the very young leaf stage. Further experiments showed that auxin distribution selected FAMA-expressing isodiametric ground meristem cells from the pool of ground meristem cells. Auxin distribution was regulated by vesicle trafficking-mediated localization of PIN-FORMED 1 (PIN1), which is an efflux carrier of auxin (Shirakawa et al. 2014c). Changes in auxin distribution in syp22 mutants induce simpler vascular pattern phenotypes in addition to the phenotype of increased numbers of myrosin cells (Fig. 2) (Shirakawa et al. 2009, Shirakawa et al. 2010). These results suggest that the auxin flow/level may be a switch for cell fate determination of myrosin precursor cells and vascular precursor cells from the population of ground meristem cells. Future studies should establish real-time imaging of cell differentiation. Evolutionary Origin of Myrosin Cells Hypothesis: the myrosinase–glucosinolate system originated in stomata Recent studies indicate that FAMA–SCRM/2 heterodimers are common master regulators of the development of both myrosin cells and guard cells. This hypothesis could be used to determine which specialized cells first acquired the FAMA–TGG transcriptional cascade. Land plants acquired guard cells before myrosin cells. Guard cells are present in moss and hornwort, suggesting that stomata are one of the oldest innovations of plants when adapting to land (Bowman, 2011). FAMA and SCRM/2 functions are conserved from moss because PpSMF1 and PpSCREAM1, which are FAMA and SCRM/2 orthologs in Physcomitrella patens, function in guard cell differentiation in moss (Chater et al. 2016, Chater et al. 2017). In contrast, TGGs are an innovation of Brassicales plants (Rask et al. 2000). We hypothesize that TGG1 is first connected with the FAMA-mediated transcriptional cascade in guard cells of the ancestral Brassicales plants (Fig. 4). Guard cell vacuoles in a basal Brassicales plant, Carica papaya, accumulate ‘myrosin grains’ that are thought to be aggregates of large amounts of myrosinases (Jorgensen 1995). This is similar to Arabidopsis myrosin cell vacuoles, whereas Arabidopsis guard cell vacuoles accumulate lower levels of myrosinases. Fig. 4 View largeDownload slide Origin of the myrosinase–glucosinolate system. FAMA–SCRMs activate myrosinase genes, whereas MYBs–MYCs transcribe genes encoding glucosinolate biosynthetic enzymes. Myrosinases and glucosinolates are stored in the same guard cell. However, they are separated in different organelles or in different compartments within the same organelle. Myrosinases hydrolyze glucosinolates in a stimuli-dependent manner to produce isothiocyanate, which has a role in defense against bacteria and/or the regulation of stomatal opening and closing. Fig. 4 View largeDownload slide Origin of the myrosinase–glucosinolate system. FAMA–SCRMs activate myrosinase genes, whereas MYBs–MYCs transcribe genes encoding glucosinolate biosynthetic enzymes. Myrosinases and glucosinolates are stored in the same guard cell. However, they are separated in different organelles or in different compartments within the same organelle. Myrosinases hydrolyze glucosinolates in a stimuli-dependent manner to produce isothiocyanate, which has a role in defense against bacteria and/or the regulation of stomatal opening and closing. The original myrosinase–glucosinolate defense system accumulated myrosinases and glucosinolates in the same cells because plants that acquired either myrosin cells or glucosinolate-accumulating cells, but not both, do not become more adaptive, and it is statistically challenging to invent these two different cell types simultaneously. Therefore, it is suggested that ancestral Brassicales plants accumulated glucosinolates in stomatal guard cells and produced isothiocyanates, which were secreted to the extracellular region by proteins such as PEN3 because stomata are the major entry site of bacteria into leaves (Fig. 4) (Melotto et al. 2006). Brassicales plants may have acquired myrosin cells during evolution. It has not been determined how and when FAMA expression began in ground meristem cells of inner leaf tissues to generate myrosin cells along leaf veins. The myrosin cell is a synapomorphy in Brassicales, which can be used to distinguish among clades. Therefore, myrosin cell differentiation is a useful model for studying species evolution. Concluding Remarks and Future Perspectives In this review, we summarized recent advances in our knowledge of myrosin cell physiology and developmental biology. Recent studies revealed that the myrosinase–glucosinolate defense system in plants, bacteria and pests has complex and divergent evolution. Detailed analysis of reporter genes and auxin signaling pathways identified the lineage and cell fate determination of myrosin cells. The shared master transcription factors in myrosin cells and guard cells unexpectedly indicated that they shared common molecular mechanisms and transcriptional networks. Plants evolved various kinds of idioblasts during evolution (Foster, 1956, Hagel et al. 2008, Hara et al. 2015). These cells accumulate useful secondary metabolites. For example, Catharanthus roseus produces terpenoid indole alkaloids that are used as antitumor drugs, such as vinblastine and vincristine (Yamamoto et al. 2016). Therefore, research on plant idioblast development and secondary metabolites provides useful benefits for human health. A recent study performed chemical genetics analysis to identify compounds that can modify plant development (e.g. the stomatal number) (Nemhauser and Torii 2016, Sakai et al. 2017, Ziadi et al. 2017). Similar approaches may discover agents that modulate the number of idioblasts and the production of secondary metabolites. Idioblasts have shapes and contents that differ from those of the surrounding homogenous cells. Guard cells and trichomes can be categorized as idioblasts (Foster, 1956), which represent a mixed population of specialized plant cells. It remains unclear whether diverse idioblasts have common cell characteristics. Future work will address the challenging question of whether guard cells and myrosin cells share common molecular pathways that regulate mutual cell characteristics, and whether myrosin cells employee specific molecules in their development. Given the complexity of cell development and the application of idioblasts including myrosin cells, this field is likely to flourish for years to come. Funding This work was supported by the Japan Society for the Promotion of Science [grant Nos. 22000014 and 15H05776 to I.H-.N. and No. 24005453 to M.S]. Acknowledgments We thank Dr. Haruko Ueda of Konan University for providing photographs of ER bodies. Disclosures The authors have no conflicts of interest to declare. References Albaser A., Kazana E., Bennett M.H., Cebeci F., Luang-In V., Spanu P.D., et al.   ( 2016) Discovery of a bacterial glycoside hydrolase family 3 (GH3) β-glucosidase with myrosinase activity from a Citrobacter strain isolated from soil. J. Agric. Food Chem . 64: 20– 27. 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Specialized Vacuoles of Myrosin Cells: Chemical Defense Strategy in Brassicales Plants

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy082
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Abstract

Abstract Plant vacuoles display many versatile functions. Vacuoles in vegetative tissues are generally involved in protein degradation, and are called lytic vacuoles. However, vegetative vacuoles in specialized cells can accumulate large concentrations of proteins, such as those in idioblast myrosin cells along veins in the order Brassicales, which store large amounts of myrosinases (thioglucoside glucohydrolase and thioglucoside glucohydrolase). Myrosinases cleave the bond between sulfur and glucose in sulfur-rich compounds (glucosinolates) to produce toxic compounds (isothiocyanates) when plants are damaged by pests. This defense strategy is called the myrosinase–glucosinolate system. Recent studies identified atypical myrosinases, PENETRATION 2 (PEN2) and PYK10, along with key components for development of myrosin cells. In this review, we discuss three topics in the myrosinase–glucosinolate system. First, we summarize the complexity and importance of the myrosinase–glucosinolate system, including classical myrosinases, atypical myrosinases and the system that counteracts the myrosinase–glucosinolate system. Secondly, we describe molecular machineries underlying myrosin cell development, including specific reporters, cell lineage, cell differentiation and cell fate determination. The master regulators for myrosin cell differentiation, FAMA and SCREAM, are key transcription factors involved in guard cell differentiation. This indicates that myrosin cells and guard cells share similar transcriptional networks. Finally, we hypothesize that the myrosinase–glucosinolate system may have originated in stomata of ancestral Brassicales plants and, after that, plants co-opted this defense strategy into idioblasts near veins at inner tissue layers. Myrosinase–Glucosinolate Chemical Defense System The myrosinase–glucosinolate system is a plant defense strategy to protect against herbivores. This defense system is characteristic of Brassicales plants as both myrosinases and glucosinolates are primarily generated by the order Brassicales (Rask et al. 2000, Halkier and Gershenzon 2006, Wittstock and Burow 2010, Shirakawa et al. 2016a). Under normal developmental conditions, myrosinases are sequestered from glucosinolates because they are localized in two different specialized cells, myrosin cells and glucosinolate-accumulating cells, respectively (Koroleva et al. 2000, Rask et al. 2000, Shroff et al. 2008, Kissen et al. 2009, Nintemann et al. 2018). Herbivore feeding breaks these cells open and the free myrosinases convert glucosinolates into toxic isothiocyanates by cleaving the bond between sulfur and glucose (Fig. 1A) (Rask et al. 2000, Halkier and Gershenzon 2006, Wittstock and Burow 2010, Shirakawa et al. 2016a). We summarize classical and atypical myrosinases in Arabidopsis thaliana, describe insect and bacterial myrosinases, and explore countervailing strategies against the myrosinase–glucosinolate system by insects and pathogens. For more detailed information about glucosinolates, see Wittstock and Halkier (2002), Grubb and Abel (2006), Hopkins et al. (2009), Sønderby et al. (2010), Jørgensen et al. (2015), Chezem and Clay (2016) and Burow and Halkier (2017). Fig. 1 View largeDownload slide Myrosinase–glucosinolate defense system. (A) Chemical reactions of the myrosinase–glucosinolate system. Myrosinase cleaves a bond between a sulfur and a glucose to produce aglycone, which is unstable. Unstable aglycones are converted into isothiocyanates by Lossen-like rearrangement. (B) A section of a cauline leaf from Arabidopsis thaliana. A myrosin cell vacuole (arrow) is filled with smooth materials that have higher electron densities than the vacuoles in an adjacent mesophyll cell. Scale bar = 5 µm (C) An enlarged image of the boxed area in (B). Dots of the anti-TGG2 immunogold label are observed in the vacuole of myrosin cells. Scale bar = 1 µm. (D) Aligned amino acid sequences of myrosinases. Typical myrosinases (TGG1/2/4/5) have Q and atypical myrosinases (PYK10 and PEN2) have E (asterisk). Atypical myrosinases (PYK10 and PEN2) have two basic amino acid residues (cross marks). (E) ER bodies and peroxisomes. A confocal image of the epidermal cells in Arabidopsis petioles expressing peroxisome-localized DsRed–PTS1 (magenta) and ER-localized GFP (green). ER bodies are football-shaped structures with bright green signals (arrows). Scale bar = 10 µm. Fig. 1 View largeDownload slide Myrosinase–glucosinolate defense system. (A) Chemical reactions of the myrosinase–glucosinolate system. Myrosinase cleaves a bond between a sulfur and a glucose to produce aglycone, which is unstable. Unstable aglycones are converted into isothiocyanates by Lossen-like rearrangement. (B) A section of a cauline leaf from Arabidopsis thaliana. A myrosin cell vacuole (arrow) is filled with smooth materials that have higher electron densities than the vacuoles in an adjacent mesophyll cell. Scale bar = 5 µm (C) An enlarged image of the boxed area in (B). Dots of the anti-TGG2 immunogold label are observed in the vacuole of myrosin cells. Scale bar = 1 µm. (D) Aligned amino acid sequences of myrosinases. Typical myrosinases (TGG1/2/4/5) have Q and atypical myrosinases (PYK10 and PEN2) have E (asterisk). Atypical myrosinases (PYK10 and PEN2) have two basic amino acid residues (cross marks). (E) ER bodies and peroxisomes. A confocal image of the epidermal cells in Arabidopsis petioles expressing peroxisome-localized DsRed–PTS1 (magenta) and ER-localized GFP (green). ER bodies are football-shaped structures with bright green signals (arrows). Scale bar = 10 µm. Classical myrosinases Myrosinase is a thioglucoside glucohydrolase (TGG) (Rask et al. 2000). The Arabidopsis genome has a total of six TGG genes (TGG1−TGG6). TGG1 and TGG2 are highly expressed in above-ground tissues to protect plants against herbivores; TGG4 and TGG5 are expressed specifically in the root tip (Fu et al. 2016); and TGG3 and TGG6 are expressed in pollen (Wang et al. 2009). The physiological roles of TGG3−TGG6 are unknown. Here, we designate TGG1−TGG6 as classical myrosinases that are characterized by an evolutionarily conserved glutamine (Q) residue for binding to the glucose ring (Fig. 1D) (Rask et al. 2000, Nakano et al. 2014). Recent studies suggest that this conserved residue is essential for binding to the glucose of aliphatic glucosinolates including sinigrin, which is widely used to test for myrosinase activity (see below). TGG1 and TGG2 are expressed specifically in two different cells: myrosin cells along leaf veins and stomatal guard cells (Fig. 2A) (Xue et al. 1995, Andreasson et al. 2001, Husebye et al. 2002, Barth and Jander 2006, Ueda et al. 2006, Shirakawa et al. 2016b). TGG1 and TGG2 are abundant proteins in aerial parts of Arabidopsis plants (Ueda et al. 2006) and are accumulated at much higher levels in myrosin cells than in guard cells (Shirakawa et al. 2014b). Myrosin cells store large amounts of TGG1 and TGG2 in the vacuoles, which show higher electron densities than the lytic vacuoles of surrounding mesophyll cells (Fig. 1B, C) (Andreasson et al. 2001, Ueda et al. 2006, Shimada et al. 2018). Hence, the vacuoles of myrosin cells can be classified as protein storage vacuoles in the vegetative tissues. Fig. 2 View largeDownload slide Patterning of myrosin cells and vascular cells in the wild type and syp22. (A) Rosette leaf of a transgenic plant [wild-type (WT) background] expressing the myrosin cell marker ProTGG2:VENUS-2sc. Myrosin cells were visualized by fluorescence (left), and leaf veins of the same leaf area were observed by dark-field microscopy (right). Boxed areas are enlarged in the respective lower panels. (B) A rosette leaf of a syp22 plant expressing ProTGG2:VENUS-2sc contains a large number of myrosin cells and shows limited development of vascular cells. Scale bars = 1 mm. Fig. 2 View largeDownload slide Patterning of myrosin cells and vascular cells in the wild type and syp22. (A) Rosette leaf of a transgenic plant [wild-type (WT) background] expressing the myrosin cell marker ProTGG2:VENUS-2sc. Myrosin cells were visualized by fluorescence (left), and leaf veins of the same leaf area were observed by dark-field microscopy (right). Boxed areas are enlarged in the respective lower panels. (B) A rosette leaf of a syp22 plant expressing ProTGG2:VENUS-2sc contains a large number of myrosin cells and shows limited development of vascular cells. Scale bars = 1 mm. Atypical myrosinases Recent work identified two atypical myrosinases: PENETRATION 2 (PEN2) and PYK10 (Bednarek et al. 2009, Nakano et al. 2017). The conserved glutamine (Q) in classical myrosinases is replaced by glutamic acid (E) in both PEN2 and PYK10 (Fig. 1D) (Nakano et al. 2014), and both can cleave the bond between sulfur and glucose in glucosinolates. How do atypical myrosinases recognize their substrate, glucosinolate, without the conserved glutamine? Three-dimensional structure modeling suggested that two basic amino acids conserved in atypical myrosinases are involved in binding to glucosinolates instead of a conserved glutamine residue in classical myrosinases (Fig. 1D) (Nakano et al. 2017). Interestingly, atypical myrosinases are not accumulated in myrosin cells. PYK10 is distributed in roots and PEN2 is accumulated in leaf epidermal cells. Atypical myrosinases differ from classical myrosinases in terms of their subcellular localization: PEN2 and PYK10 are localized in peroxisomes and endoplasmic reticulum (ER) bodies, respectively (Fig. 1E) (Matsushima et al. 2003, Lipka et al. 2005). PEN2 activates indole glucosinolates to produce antifungal isothiocyanates, which are thought to be secreted from cells via the PENETRATION 3 (PEN3) ABC transporter (Stein et al. 2006, Bednarek et al. 2009, Bednarek 2012). PYK10 prefers indole glucosinolates to aliphatic glucosinolates (Nakano et al. 2017). These results expand the definition of myrosinases and raise questions about the enzymatic specificity for indole and aliphatic glucosinolates. Structural analyses of the myrosinase–glucosinolate complex will provide answers to this question. For more detailed information about the biochemistry and evolution of atypical myrosinases, see Nakano et al. (2014), Piasecka et al. (2015) and Pastorczyk and Bednarek (2016). Pests and pathogens have evolved factors that neutralize the myrosinase–glucosinolate system After the myrosinase–glucosinolate system emerged in Brassicales, pests and pathogens evolved an anti-myrosinase–glucosinolate system. Plutella xylostella (diamondback moth) is a crucifer specialist insect that produces glucosinolate sulfatase (GSS) to detoxify glucosinolates (Ratzka et al. 2002). GSS was isolated from the gut contents of Plutella. GSS hydrolysis of glucosinolates produces desulfo-glucosinolates, which are not myrosinase substrates. It is unknown whether GSS is specifically conserved in specialist herbivores (Ratzka et al. 2002). To validate the co-evolutionary history of defense and neutralizing systems in plants and pests, the genomic and amino acid sequences of GSS should be comprehensively analyzed in both specialist and generalist herbivores. Pathovars of Pseudomonas syringae Psm E4326 and Pst DC3000 have a unique strategy to counteract the myrosinase–glucosinolate defense system (Fan et al. 2011). The Survival in Arabidopsis extracts (Sax) gene confers resistance to aliphatic isothiocyanates in pathogenic P. syringae. Pst DC3000 quintuple mutants lacking SaxA/B/F/D/G genes could not grow in young Arabidopsis leaves but could grow in young leaves of Arabidopsis myb28 myb29 mutants, which did not produce aliphatic glucosinolates. The accumulated amounts of the major glucosinolates were unchanged in plants overexpressing SaxA, suggesting that SaxA inhibits aliphatic isothiocyanate production after glucosinolate breakdown (Fan et al. 2011). These combined results suggest that the myrosinase–glucosinolate defense system forms part of an arms race between Brassicales plants and their pests and pathogens. Myrosinases in other species Myrosinases have recently been identified in insects and bacteria (Bridges et al. 2002, Beran et al. 2014, Albaser et al. 2016). The specialist herbivore Phyllotreta striolata (flea beetle) expresses myrosinases and ingests glucosinolates from Brassicales plants (Beran et al. 2014). Sequence analyses suggest that insect and bacterial myrosinases evolved independently from plant myrosinases (Nakano et al. 2017). The physiological roles of isothiocyanates from insects and bacteria are still largely unknown. Myrosin Cell Development in Arabidopsis Our understanding of myrosin cell development has grown markedly during the past 5 years. Here, we summarize reporter genes, cell lineages, cell differentiation and cell fate determination in myrosin cells. The biggest advance is identification of the master transcription factors for myrosin cell differentiation, which are the basic helix–loop–helix (bHLH) transcription factors FAMA, SCREAM (SCRM) and SCRM2 (Li and Sack 2014, Shirakawa et al. 2014b). All three transcription factors were previously identified as master regulators of guard cell differentiation (Ohashi-Ito and Bergmann 2006, Kanaoka et al. 2008). This indicates that myrosin cell development is regulated by a transcriptional network similar to that involved in guard cell development. For more detailed information about these two developmental programs, see Lau and Bergmann (2012), Han and Torii (2016) and Shirakawa et al. (2016a). Reporter genes for myrosin cells An authentic reporter gene for myrosin cells is TGG1. Multiple methods, including in situ hybridization of TGG1 mRNA (Xue et al. 1995), analysis of the TGG1 gene promoter (Husebye et al. 2002, Barth and Jander 2006) and immunohistochemistry of the TGG1 protein (Andreasson et al. 2001, Husebye et al. 2002, Ueda et al. 2006) identified elongated and irregularly shaped idioblasts along veins in Arabidopsis leaves. However, TGG1 is also expressed in guard cells in above-ground organs (Husebye et al. 2002, Barth and Jander 2006). Several new reporters of myrosin cells have been identified within the past decade (Table 1). The expression patterns of three reporters, ProTGG2:GUS, MYR001:GUS (ProVSR1:GUS) and ProbHLH090:GUS-GFP, are specific to the myrosin cell lineage (Barth and Jander 2006, Shirakawa et al. 2014a, Shirakawa et al. 2014b). These lines are useful for isolating intact myrosin cells from whole plants in future experiments [e.g. fluorescence-activated cell sorting (FACS) analysis]. The biological functions of several of these genes are still largely unknown. Studies of their loss-of-function mutants may shed light on novel functions of myrosin cells. Table 1 Specific reporter lines for myrosin cells Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  MC, myrosin cells; GC, guard cells; ME, mesophyll cells. aProTGG2:GUS is expressed in only in myrosin cells, while ProTGG2:VENUS-2sc is expressed in both myrosin cells and guard cells. bA GAL4 GFP (green fluorescent protein) enhancer trap line. Table 1 Specific reporter lines for myrosin cells Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  Name  AGI code  Expressionpattern  Identity  Rerefernces  TGG1  At5g26000  MC, GC  Myrosinase  Barth and Jander (2006),Husebye et al. (2002)  TGG2  At5g25980  MC, GCa  Myrosinase  Barth and Jander (2006),Shirakawa et al. (2016b)  PAP17  At3g17790  MC, GC, ME  Purple acid phosphatase  Wenzel et al. (2008),  VSR1  At3g52850  MC  Vacuolar sorting receptor  Shirakawa et al. (2014a)  FAMA  At3g24140  MC, GC  bHLH transcription factor  Shirakawa et al. (2014b), Li and Sack (2014,) Ohashi-Ito and Bergmann (2006)  SCRM  At3g26744  MC, GC, ME  bHLH transcription factor  Shirakawa et al. (2014b), Kanaoka et al. (2008)  bHLH090  At1g10610  MC  bHLH transcription factor  Shirakawa et al. (2014b)  E1728  b  MC, GC  –  Shirakawa et al. (2014b), Li and Sack (2014), Gardner et al. (2009)  MC, myrosin cells; GC, guard cells; ME, mesophyll cells. aProTGG2:GUS is expressed in only in myrosin cells, while ProTGG2:VENUS-2sc is expressed in both myrosin cells and guard cells. bA GAL4 GFP (green fluorescent protein) enhancer trap line. Lineage of myrosin cells Myrosin cells are distributed along veins in Arabidopsis (Fig. 2A), Cardamine (Fig. 3A) and Nasturtium (Kissen et al. 2009, Shirakawa et al. 2014c, Shirakawa et al. 2016b). Myrosin cells lie side by side with vascular precursor cells and phloem cells (Andreasson et al. 2001, Shirakawa et al. 2014c, Shirakawa et al. 2016b); therefore, it was hypothesized that myrosin cells belong to a vascular cell lineage. However, recent studies reveal that myrosin cells did not differentiate from vascular and vascular precursor cells (Li and Sack 2014, Shirakawa et al. 2014b, Shirakawa et al. 2016b). These studies carefully compared expression patterns of myrosin cell reporters (TGG2, FAMA and E1728) and vascular cell reporters (AtHB8, Q0990 and J1721), and showed that myrosin cells differentiate from isodiametric small cells that are part of the ground meristem (ground meristem cells) (Fig. 3B, 3C). Ground meristem cells are a stem cell-like cell population located in the inner tissues of leaf primordia, and are mother cells for vascular precursor cells and mesophyll cells (Sawchuk et al. 2007, Sawchuk et al. 2008). Plants sort ground meristem cells into different cell types, including myrosin precursor cells, vascular precursor cells and mesophyll cells. Myrosin precursor cells (i.e. FAMA-expressing isodiametric ground meristem cells) mature into large, isolated and irregularly shaped idioblast myrosin cells (Fig. 3) (Li and Sack 2014, Shirakawa et al. 2014b). Fig. 3 View largeDownload slide Distribution of myrosin cells and expression pattern of FAMA in leaf inner tissue layers. (A) Coomassie brilliant blue staining of a mature Cardamine schinziana leaf. Myrosin cells distributed along leaf veins are stained blue. Myrosin cell shapes are not uniform, but they are elongated along leaf veins. Scale bar = 100 µm. (B) A 3-D image of the inner leaf tissue of leaf primordia expressing ProFAMA:GFP (white). The 3-D image was reconstructed and deconvolved. GFP signals are observed in both cell nuclei and the cytosol. FAMA-expressing cells form network structures along veins. Scale bar = 20 µm. (C) Enlarged image of the boxed area in (A). Three isodiametric small cells (ground meristem cells) started to express FAMA. Arrowheads indicate nuclei. Scale bar = 5 µm. Fig. 3 View largeDownload slide Distribution of myrosin cells and expression pattern of FAMA in leaf inner tissue layers. (A) Coomassie brilliant blue staining of a mature Cardamine schinziana leaf. Myrosin cells distributed along leaf veins are stained blue. Myrosin cell shapes are not uniform, but they are elongated along leaf veins. Scale bar = 100 µm. (B) A 3-D image of the inner leaf tissue of leaf primordia expressing ProFAMA:GFP (white). The 3-D image was reconstructed and deconvolved. GFP signals are observed in both cell nuclei and the cytosol. FAMA-expressing cells form network structures along veins. Scale bar = 20 µm. (C) Enlarged image of the boxed area in (A). Three isodiametric small cells (ground meristem cells) started to express FAMA. Arrowheads indicate nuclei. Scale bar = 5 µm. Master regulators of myrosin cell differentiation The bHLH transcription factor FAMA was identified as a master regulator of myrosin cell development (Li and Sack 2014, Shirakawa et al. 2014b, Shirakawa et al. 2016a). FAMA starts to be expressed in isodiametric small cells that are morphologically indistinguishable from surrounding cells (Fig. 3B, C). No myrosin cells and no accumulation of TGG1 and TGG2 are observed in fama mutants (Li and Sack 2014, Shirakawa et al. 2014b). Conversely, plants overexpressing FAMA make a large number of myrosin cells (Shirakawa et al. 2014b). SCRM and SCRM2 are binding partners of FAMA, and are redundantly essential for myrosin cell development (Shirakawa et al. 2014b). These three transcription factors were previously identified as master regulators of guard cell differentiation, particularly transition from guard mother cells into guard cells (Ohashi-Ito and Bergmann 2006, Kanaoka et al. 2008, Lau and Bergmann 2012, Han and Torii 2016). These results indicate that common cell differentiation pathways may be shared between myrosin cells and guard (mother) cells. For example, both cell types accumulate myrosinases (TGG1 and TGG2), although there are some differences in accumulation levels (Zhao et al. 2008, Shirakawa et al. 2014b). Genes homologous to FAMA, SPEECHLESS and MUTE (MacAlister et al. 2007, Pillitteri et al. 2007) are not required for myrosin cell development (Li and Sack 2014, Shirakawa et al. 2014b). A total of 32 genes were identified as candidate factors downstream of FAMA (Shirakawa et al. 2014b). These genes are highly up-regulated in both chemically induced FAMA overexpression lines (Hachez et al. 2011) and syp22 mutants containing numerous myrosin cells (Ueda et al. 2006; see below). As expected, one of these genes, bHLH090, is expressed specifically in the myrosin cell lineage (Table 1) (Shirakawa et al. 2014b). To identify the transcriptional network of the FAMA–SCRM/2 complex, we should examine expression patterns and perform mutant analyses of these 32 genes. Analysis of downstream targets of FAMA will shed light on the molecular mechanisms of how protein storage vacuoles of myrosin cells accumulate large amounts of the TGG1 and TGG2 proteins even in the vegetative tissues, which generally develop lytic vacuoles responsible for protein degradation. The FAMA-downstream factors could include vacuolar transporters, vacuolar channel proteins and vacuolar trafficking components. Future work will involve comprehensive analyses of cis-regulatory elements and binding sites of the FAMA–SCRM/2 transcriptional complex in target gene promoters. For additional hypothetical transcriptional mechanisms for FAMA in different cell types, see Shirakawa et al. (2016a). Myrosin cell fate determination How do plants determine the differentiation of ground meristem cells into myrosin precursor cells, vascular precursor cells and mesophyll cells before FAMA is expressed? The molecular mechanism underlying cell fate determination in myrosin cells was identified by analysis of mutants with increased numbers of myrosin cells. Loss of vacuolar trafficking machineries [SYNTAXIN OF PLANTS 22 (SYP22), VACUOLAR PROTEIN SORTING 9 A (VPS9A) and CONTINUOUS VASCULAR RING (COV1)] induced overproduction of myrosin cells (Fig. 2) (Shirakawa et al. 2014a, Shirakawa et al. 2014c). The accumulated myrosin cells in these mutants are not distributed randomly; most are still distributed near veins, although they establish a much denser network (Ueda et al. 2006, Shirakawa et al. 2014c). These network structures contain myrosin cells along veins, as do wild-type plants, and myrosin cells branch from them (Fig. 2). The myrosin cells in mutants are distributed along the abaxial side of leaves, similar to wild-type plants. Analysis of FAMA expression patterns and levels in syp22 mutants revealed that increased numbers of myrosin cells were not induced by accelerated division of myrosin cells, but rather at the fate determination step of myrosin cells from the pool of ground meristem cells. In syp22 mutants, more isodiametric ground meristem cells start to express FAMA at the very young leaf stage. Further experiments showed that auxin distribution selected FAMA-expressing isodiametric ground meristem cells from the pool of ground meristem cells. Auxin distribution was regulated by vesicle trafficking-mediated localization of PIN-FORMED 1 (PIN1), which is an efflux carrier of auxin (Shirakawa et al. 2014c). Changes in auxin distribution in syp22 mutants induce simpler vascular pattern phenotypes in addition to the phenotype of increased numbers of myrosin cells (Fig. 2) (Shirakawa et al. 2009, Shirakawa et al. 2010). These results suggest that the auxin flow/level may be a switch for cell fate determination of myrosin precursor cells and vascular precursor cells from the population of ground meristem cells. Future studies should establish real-time imaging of cell differentiation. Evolutionary Origin of Myrosin Cells Hypothesis: the myrosinase–glucosinolate system originated in stomata Recent studies indicate that FAMA–SCRM/2 heterodimers are common master regulators of the development of both myrosin cells and guard cells. This hypothesis could be used to determine which specialized cells first acquired the FAMA–TGG transcriptional cascade. Land plants acquired guard cells before myrosin cells. Guard cells are present in moss and hornwort, suggesting that stomata are one of the oldest innovations of plants when adapting to land (Bowman, 2011). FAMA and SCRM/2 functions are conserved from moss because PpSMF1 and PpSCREAM1, which are FAMA and SCRM/2 orthologs in Physcomitrella patens, function in guard cell differentiation in moss (Chater et al. 2016, Chater et al. 2017). In contrast, TGGs are an innovation of Brassicales plants (Rask et al. 2000). We hypothesize that TGG1 is first connected with the FAMA-mediated transcriptional cascade in guard cells of the ancestral Brassicales plants (Fig. 4). Guard cell vacuoles in a basal Brassicales plant, Carica papaya, accumulate ‘myrosin grains’ that are thought to be aggregates of large amounts of myrosinases (Jorgensen 1995). This is similar to Arabidopsis myrosin cell vacuoles, whereas Arabidopsis guard cell vacuoles accumulate lower levels of myrosinases. Fig. 4 View largeDownload slide Origin of the myrosinase–glucosinolate system. FAMA–SCRMs activate myrosinase genes, whereas MYBs–MYCs transcribe genes encoding glucosinolate biosynthetic enzymes. Myrosinases and glucosinolates are stored in the same guard cell. However, they are separated in different organelles or in different compartments within the same organelle. Myrosinases hydrolyze glucosinolates in a stimuli-dependent manner to produce isothiocyanate, which has a role in defense against bacteria and/or the regulation of stomatal opening and closing. Fig. 4 View largeDownload slide Origin of the myrosinase–glucosinolate system. FAMA–SCRMs activate myrosinase genes, whereas MYBs–MYCs transcribe genes encoding glucosinolate biosynthetic enzymes. Myrosinases and glucosinolates are stored in the same guard cell. However, they are separated in different organelles or in different compartments within the same organelle. Myrosinases hydrolyze glucosinolates in a stimuli-dependent manner to produce isothiocyanate, which has a role in defense against bacteria and/or the regulation of stomatal opening and closing. The original myrosinase–glucosinolate defense system accumulated myrosinases and glucosinolates in the same cells because plants that acquired either myrosin cells or glucosinolate-accumulating cells, but not both, do not become more adaptive, and it is statistically challenging to invent these two different cell types simultaneously. Therefore, it is suggested that ancestral Brassicales plants accumulated glucosinolates in stomatal guard cells and produced isothiocyanates, which were secreted to the extracellular region by proteins such as PEN3 because stomata are the major entry site of bacteria into leaves (Fig. 4) (Melotto et al. 2006). Brassicales plants may have acquired myrosin cells during evolution. It has not been determined how and when FAMA expression began in ground meristem cells of inner leaf tissues to generate myrosin cells along leaf veins. The myrosin cell is a synapomorphy in Brassicales, which can be used to distinguish among clades. Therefore, myrosin cell differentiation is a useful model for studying species evolution. Concluding Remarks and Future Perspectives In this review, we summarized recent advances in our knowledge of myrosin cell physiology and developmental biology. Recent studies revealed that the myrosinase–glucosinolate defense system in plants, bacteria and pests has complex and divergent evolution. Detailed analysis of reporter genes and auxin signaling pathways identified the lineage and cell fate determination of myrosin cells. The shared master transcription factors in myrosin cells and guard cells unexpectedly indicated that they shared common molecular mechanisms and transcriptional networks. Plants evolved various kinds of idioblasts during evolution (Foster, 1956, Hagel et al. 2008, Hara et al. 2015). These cells accumulate useful secondary metabolites. For example, Catharanthus roseus produces terpenoid indole alkaloids that are used as antitumor drugs, such as vinblastine and vincristine (Yamamoto et al. 2016). Therefore, research on plant idioblast development and secondary metabolites provides useful benefits for human health. A recent study performed chemical genetics analysis to identify compounds that can modify plant development (e.g. the stomatal number) (Nemhauser and Torii 2016, Sakai et al. 2017, Ziadi et al. 2017). Similar approaches may discover agents that modulate the number of idioblasts and the production of secondary metabolites. Idioblasts have shapes and contents that differ from those of the surrounding homogenous cells. Guard cells and trichomes can be categorized as idioblasts (Foster, 1956), which represent a mixed population of specialized plant cells. It remains unclear whether diverse idioblasts have common cell characteristics. Future work will address the challenging question of whether guard cells and myrosin cells share common molecular pathways that regulate mutual cell characteristics, and whether myrosin cells employee specific molecules in their development. Given the complexity of cell development and the application of idioblasts including myrosin cells, this field is likely to flourish for years to come. Funding This work was supported by the Japan Society for the Promotion of Science [grant Nos. 22000014 and 15H05776 to I.H-.N. and No. 24005453 to M.S]. Acknowledgments We thank Dr. Haruko Ueda of Konan University for providing photographs of ER bodies. Disclosures The authors have no conflicts of interest to declare. References Albaser A., Kazana E., Bennett M.H., Cebeci F., Luang-In V., Spanu P.D., et al.   ( 2016) Discovery of a bacterial glycoside hydrolase family 3 (GH3) β-glucosidase with myrosinase activity from a Citrobacter strain isolated from soil. J. Agric. Food Chem . 64: 20– 27. 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Plant and Cell PhysiologyOxford University Press

Published: Apr 20, 2018

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