Vascular supply
IV. Article : Dynamics of strigolactone function in pea : soumis dans Molecular Plant
Dun EA, de Saint Germain A, Rameau C, Beveridge CA.
Résumé:
Les strigolactones (SL), ou des dérivés de strigolactones ont été récemment identifiés comme inhibiteurs endogènes de la ramification des plantes. Cependant, certaines caractéristiques physiologiques essentielles de la dynamique de l'action des SL restaient à caractériser. Nous montrons dans cet article que l’application successive de SL directement sur les bourgeons axillaires de chaque nœud le long de la tige, peut inhiber totalement la ramification. Nous avons trouvé que les SLs, jusqu'à un certain point peuvent modérer la croissance continue des branches en plus de la levée de dormance initiale. L'efficacité des SL à inhiber la croissance des bourgeons et des branches est en corrélation avec la capacité des SL à réguler l'expression de PsBRC1, un facteur de transcription impliqué dans la régulation de la croissance des bourgeons axillaires, en aval des SL. Conformément à un rôle dynamique de l'hormone, l'inhibition de la croissance des bourgeons par les SL n’empêche pas le bourgeon de répondre à une décapitation plus tardive, alors même que le traitement aux SL inhibe la levée de dormance des bourgeons si elles sont appliquées immédiatement après la décapitation. Comme prévu à partir de l’hypothèse de l’existence d’un réseau de contrôle de la ramification chez les plantes, l’application exogène de SL provoque un rétrocontrôle négatif sur l’expression des gènes de biosynthèse de SL dans les 2 heures. Finalement, ces résultats révèlent de nouvelles connaissances sur la dynamique du fonctionnement des SL et permettent de soutenir l'hypothèse que les SLs ou un dérivé de SL fonctionnent de façon dynamique sur l’inhibition de la ramification.
129
INTRODUCTION
Shoot branching is an important developmental process contributing to plant yield. It is regulated by a myriad of factors internal and external to axillary buds including hormonal, genetic, environmental, positional and developmental factors (Dun et al., 2009a; Leyser, 2009). For many years the phytohormones auxin and cytokinin (CK) have been implicated in the control of bud outgrowth, auxin as a repressor (e.g. Thimann and Skoog, 1933; Thimann and Skoog, 1934) and CK as a promoter (e.g., Sachs and Thimann, 1967). Strigolactones (SLs) were recently discovered to also function in the repression of bud outgrowth (Gomez‐Roldan et al., 2008; Umehara et al., 2008), however their role in the shoot remains to be completely physiologically characterized. Various other roles for SLs in plant development are now being unraveled (e.g. root hair growth, root growth, secondary growth, adventitious root formation; Kapulnik et al., 2011; Ruyter‐Spira et al., 2011; Agusti et al., 2011; Rasmussen et al., 2012, reviewed in this issue by Brewer et al., 2012).
Prior to the discovery that SLs function in shoot branching inhibition, SLs were not known to have an endogenous function within the plant. SLs were instead known as chemicals exuded into the rhizosphere that stimulate beneficial symbioses with arbuscular mycorrhizae (Akiyama et al., 2005) and promote the germination of parasitic weed seeds including Striga spp. and Orobanche spp. (Cook et al., 1972). This function of SLs in the rhizosphere helped in the discovery of SL mediated branching inhibition, as increased branching mutants in rice (Oryza sativa) and pea (Pisum sativum) that were thought to lack an unknown branch‐inhibiting hormone showed reduced mycorrhizal associations and their root exudates exhibited decreased capacity for stimulation of parasitic weed seed germination (Gomez‐Roldan et al., 2008; Umehara et al., 2008; reviewed in Dun et al., 2009a).
The majority of the SL biosynthesis and response pathway in plants remains to be determined. To date, mutant based approaches have identified several steps in the SL biosynthesis pathway. These have utilized the ramosus (rms) mutants in pea, more axillary
growth (max) mutants in Arabidopsis thaliana, decreased apical dominance (dad) mutants in Petunia hybrida (petunia) and dwarf (d) and high tillering dwarf (htd) mutants in rice
(reviewed in Dun et al., 2009a; Beveridge and Kyozuka, 2010; Domagalska and Leyser, 2011). These mutants show increased branching and are generally shorter in stature than their
131 wild‐type counterparts (e.g. Beveridge et al., 1996; Napoli, 1996; Stirnberg et al., 2002; Ishikawa et al., 2005; Zou et al., 2006), and can be classified as either SL deficient or SL response mutants based on SL measurements and responses.
The proposed biosynthetic pathway starts with a carotenoid precursor, likely β‐ carotene. Based on recent in vitro biochemical studies, DWARF27 (D27), a plastid‐localised iron‐containing protein (Lin et al., 2009), is the first enzyme in the SL pathway and functions as an isomerase to convert trans‐β‐carotene to 9‐cis‐β‐carotene (Alder et al., 2012). CAROTENOID CLEAVAGE DIOXYGENASE (CCD) 7 and CCD8 in the plastid, encoded by
RMS5/MAX3/DAD3/D17/HTD1 and RMS1/MAX4/DAD1/D10 respectively (Sorefan et al.,
2001; Booker et al., 2004; Snowden et al., 2005; Johnson et al., 2006; Zou et al., 2006; Arite et al., 2007; Drummond et al., 2009) then act sequentially to produce carlactone, a mobile intermediate in the SL pathway (Alder et al., 2012). A cytochrome P450, MAX1, likely acts after CCD7 and CCD8 in the synthesis of SLs (Booker et al., 2005). An α/β‐fold hydrolase encoded by D14/D88/HTD2 and an F‐box protein encoded by RMS4/MAX2/D3 may act downstream of SLs and are required for the SL branching inhibition response (Stirnberg et al., 2002; Ishikawa et al., 2005; Johnson et al., 2006; Gomez‐Roldan et al., 2008; Umehara et al., 2008; Arite et al., 2009; Gao et al., 2009; Liu et al., 2009).
BRANCHED1 (BRC1), a TCP (TB1, CYCLOIDEA, PCF domain) transcription factor
strongly expressed in non‐growing axillary buds, likely functions downstream of SL response to regulate bud outgrowth (Aguilar‐Martínez et al., 2007; Finlayson, 2007; Brewer et al., 2009; Braun et al., 2012). Evidence for this includes that the increased branching observed in the Arabidopsis and pea brc1 mutants cannot be reduced by SL treatment (Brewer et al., 2009; Braun et al., 2012), and that BRC1 expression in the bud is down‐regulated in SL mutants and up‐regulated by SL treatment (Aguilar‐Martínez et al., 2007; Finlayson, 2007; Braun et al., 2012; Dun et al., 2012). However, BRC1 likely integrates multiple bud outgrowth regulatory pathways, not just the SL pathway, at the bud, as its expression is regulated by multiple bud growth regulatory factors especially CK (Aguilar‐Martínez et al., 2007; Finlayson et al., 2010; Braun et al., 2012; Dun et al., 2012).
The SL biosynthesis and response pathway is not a simple linear pathway but rather involves feedback regulatory mechanisms (Dun et al., 2009b). If SL response is depleted, such as via mutation of SL biosynthesis or response genes, the expression of CCD7 and CCD8 is enhanced (e.g. Bainbridge et al., 2005; Foo et al., 2005; Snowden et al., 2005; Johnson et
133 al., 2006; Arite et al., 2007; Drummond et al., 2009; Dun et al., 2009b; Hayward et al., 2009) and the export of xylem‐sap CK (X‐CK) is reduced (Beveridge et al., 1997a; Beveridge et al., 1997b; Morris et al., 2001; Foo et al., 2007). In pea, it is suggested that this feedback regulation involves a novel long‐distance signal that moves in the direction of shoot‐to‐root and requires RMS2 (sequence unknown; Beveridge, 2000; Dun et al., 2009b). There is some evidence that a similar novel signalling system may occur in Arabidopsis, but it is minor compared with the role of auxin (Hayward et al., 2009).
Auxin is also implicated in the SL pathway as it has been demonstrated to positively regulate the expression of CCD7 and/or CCD8 SL biosynthesis genes in pea, Arabidopsis, rice and chrysanthemum (Dendranthema grandiflorum); exogenous treatment with auxin enhances the expression of CCD7 and/or CCD8, and removal of the apical source of auxin via decapitation leads to a substantial depletion while replacement with exogenous auxin can restore CCD7 and/or CCD8 expression to levels observed in intact wild‐type plants (Sorefan et al., 2001; Foo et al., 2005; Johnson et al., 2006; Zou et al., 2006; Arite et al., 2007; Hayward et al., 2009; Liang et al., 2010). The depletion in SL biosynthesis gene expression, and presumably SL content, after decapitation likely in part accounts for the decapitation‐ induced bud outgrowth response. Indeed, decapitation‐induced outgrowth at the node below the site of decapitation can be repressed by directly treating the bud with GR24, a synthetic SL (Brewer et al., 2009). A cautionary note is worthwhile with respect to this decapitation response. Morris et al. (2005) and Renton et al. (2012) have demonstrated that auxin changes after decapitation are too slow to be responsible for the initiation of bud outgrowth that occurs a considerable distance from the site of decapitation. Furthermore, we have not explored carefully whether SL can prevent the earliest stage of bud outgrowth observed very soon after decapitation.
The bud outgrowth promoter CK is another major contributor to the decapitation response. Auxin depletion causes enhanced expression of ADENOSINE PHOSPHATE‐
ISOPENTYLTRANSFERASE (IPT) CK biosynthesis genes, local stem CK content and X‐CK
supplied from the roots to the shoot and depleted expression of CK metabolism genes; these changes are reversed by re‐supply of apical auxin to the plant (Bangerth, 1994; Tanaka et al., 2006; Werner et al., 2006; Shimizu‐Sato et al., 2009). It is likely that auxin, CK and SL co‐ ordinately regulate bud outgrowth after decapitation. Indeed, recent studies have indicated
135 that CK and SL coordinate bud outgrowth in pea by converging at the bud to regulate expression of PsBRC1 (Braun et al., 2012; Dun et al., 2012).
Phenotypic mutant studies indicate that SLs can be synthesised in root and/or shoot tissue, can move upwards in the direction of root to shoot only, and that bud outgrowth inhibition requires SL response local to the bud (e.g. Napoli, 1996; Beveridge et al., 1997a; Turnbull et al., 2000; Stirnberg et al., 2007). The inability of SLs to move downwards from shoot to root was recently confirmed by SL measurement from root tissue of grafted pea plants (Foo and Davies, 2011) and SLs were detected in Arabidopsis xylem sap samples and may therefore move upwards in the plant to inhibit branching via this mechanism (Kohlen et al., 2011). Consistent with this, the recently identified SL transporter from petunia, PDR1, is a pleiotropic drug resistance (PDR)‐type transporter and is expressed in vasculature and nodal tissues adjacent to leaf axils but not in axillary buds (Kretzschmar et al., 2012).
There are two hypotheses about the function of SLs for bud outgrowth inhibition, though they are not necessarily exclusive. One hypothesis suggests that SLs act directly in the bud to inhibit bud outgrowth (Brewer et al., 2009; Dun et al., 2012), while the other hypothesis proposes that SLs impede buds abilities to export auxin into the main stem, and hence inhibit their outgrowth (Bennett et al., 2006; Prusinkiewicz et al., 2009; Crawford et al., 2010; reviewed in Domagalska and Leyser, 2011). A recent stem auxin transport modelling study suggests that changes in auxin transport in SL mutants are more likely due to changes in the uptake and transfer of auxin to the long‐distance polar stream rather than an affect of SLs on auxin movement in the polar stream itself (Renton et al., 2012). Accordingly, both models rely on SL action in buds. Direct bud application studies using the synthetic SL, GR24, in addition to bud specific gene expression studies demonstrate that SLs can inhibit bud outgrowth directly at the bud (e.g. Gomez‐Roldan et al., 2008; Brewer et al., 2009; Braun et al., 2012; Dun et al., 2012). SLs supplied to the roots via hydroponics or growth media, or to the stem vasculature, can also lead to bud outgrowth inhibition (e.g. Gomez‐Roldan et al., 2008; Umehara et al., 2008; Brewer et al., 2009; Crawford et al., 2010), but it has yet to be determined if this is due to delivery of SLs or SL metabolites to the axillary bud or via action from a distance, such as in the stem. The following experiments address several aspects of strigolactone function that have been postulated but until now have waited testing with SL applications. These include whether SLs are perceived in leaves adjacent to buds, whether they require an apical auxin
136
137 supply for inhibition of bud outgrowth, and whether they affect only bud release or both bud release and branch growth. In so doing we also address the mechanism of how an axillary branch can undergo a transition from a readily repressible strigolactone responsive bud to a branch which essentially phenocopies the dominant main stem. We also test whether strigolactone has a dynamic role in the process of bud inhibition, release and re‐ inhibition.
RESULTS
Inhibition of branch growth in SL deficient pea plants
Previous experiments in pea have examined the ability of direct treatments of the synthetic SL, GR24, in inhibiting the outgrowth of one axillary bud at a particular node of SL deficient mutant plants (e.g. Gomez‐Roldan et al., 2008; Dun et al., 2009b). As SL can endogenously inhibit the outgrowth of all axillary buds of wild‐type pea plants, we determined if this is also the case when the synthetic SL GR24 is exogenously applied to all axillary buds. This needs to be shown, as growing axillary buds or branches can inhibit the growth of other buds or branches and hence a small inhibitory effect at one bud in mutant plants may be reinforced by other growing buds and is not representative of the case in wild‐ type plants where all buds are inhibited simultaneously. Axillary buds of SL deficient rms1 plants were treated on two consecutive days as each leaf opened (two treatments per bud in total). Wild‐type non‐treated plants were grown alongside as a comparison. Overall, two single treatments of 1 µM GR24 to rms1 buds all along the stem were effective at inhibiting bud growth relative to controls, except for the bud at nodes 2 and 11 which exhibited a small amount of growth relative to wild‐type control plants (Fig. 1). Since previous experiments have shown that direct GR24 treatment is effective at inhibiting outgrowth of
rms1 buds at node 2 when other buds were left untreated (e.g. Nelson et al., 2011), it is
likely that the buds at node 2 grew out in this case due to release from inhibition by GR24. In contrast with our treatment method, wild‐type plants have a continuous supply of SL.
GR24 is perceived in the axillary bud, not adjacent stipules
The RMS4 F‐box protein is required for SL inhibition of branching, as the increased branching phenotype of rms4 mutant plants is not affected by treatment with exogenous GR24 (Gomez‐Roldan et al., 2008). Previous expression analyses found that RMS4 is more strongly expressed in the stipules than in the roots, node or apex of pea plants (Johnson et
138
139 al., 2006). Experiments conducted previously to test SL inhibition of bud outgrowth have either supplied SL to the stem vasculature, via hydroponics, or within a 5 or 10 µL volume applied to the axillary buds. The larger volume bud treatments completely cover small axillary buds and also make contact with the stipules. To rule out the possibility that SL can be perceived in the stipules to inhibit bud outgrowth, we treated the stipules or the bud specifically with a small 4 µL volume of rms1 plants with or without GR24 being careful to ensure that the stipule treatments did not contact the axillary bud and vice versa. In contrast to bud treatments, treatment of GR24 to the stipules did not significantly reduce rms1 bud length (Fig. 2). In addition, GR24 treatment to the stipules had no effect on final stipule size (data not shown). These data indicate that SL is not perceived in the stipules for bud outgrowth inhibition. The relatively high expression of RMS4 in the stipules might instead relate to a different developmental role for SLs/RMS4, such as modulation of leaf senescence, light signalling or feedback regulation of SL content (e.g. Woo et al., 2001; Mayzlish‐Gati et al., 2010).
GR24 affects outgrowth and continued growth of branches in a PsBRC1-dependent manner
Direct treatment of GR24 to axillary buds can inhibit branching in SL deficient mutant and decapitated pea plants (Fig. 1 and e.g. Gomez‐Roldan et al., 2008; Brewer et al., 2009). To investigate if axillary buds or branches become insensitive to growth inhibition by GR24, we determined the responses of growing rms1 buds of increasing size and developmental age to direct GR24 treatment. SL deficient rms1 seeds were sown at daily intervals to achieve growing buds of different sizes and stages of development. Buds at a particular node were then treated at daily intervals for two days while the buds at the nodes below the treatment node were excised to encourage the growth of the test bud. Firstly we tested
rms1 SL deficient buds at node 3 with a mean size ranging from 0.19 ± 0.01 mm up to 1.09 ±
0.08 mm at the time of treatment (Fig. 3A). As expected, control treated buds continued to grow after treatment, and their growth followed an exponential pattern as seen previously in the early stages of bud growth (e.g. Brewer et al., 2009). Treatment with GR24 was effective at significantly reducing the growth of all of these small buds in the seven days since the initial treatment; bud growth was almost completely halted by GR24 treatment to these buds (Fig. 3B).
140
141 To further test the responsiveness of buds of increasing size and developmental stage to GR24, another experiment was conducted with a similar design, except buds at node 8 with a larger mean size at the time of treatment, ranging from 0.88 ± 0.05 mm up to 14.23 ± 1.94 mm, were tested (Fig. 3C). In this case, GR24 was effective at inhibiting buds with a pre‐ treatment size up to approximately 2 mm, but only reduced rather than inhibited the growth of buds with a pre‐treatment size above 5 mm (Figs. 3C and 3D). This suggests that bud outgrowth is not a simple on/off switch but perhaps a continuum and that SL functions in controlling bud growth over this broad period.
Finally, to determine if branches ever become resistant to GR24 inhibition and to determine if the diminishing effect of SL occurs in other genetic backgrounds of pea, SL deficient rms1 seeds, on the dwarf gibberellin (GA) deficient Térèse background, were sown every second day and grown until initial bud sizes ranged from 1.62 ± 0.40 mm up to 18.78 ± 1.79 mm (Fig. 4A). These correspond to particularly advanced branches as the internode lengths are short in dwarf lines such as Térèse. Measuring bud growth 7 d after treatment revealed that small branches on older plants rms1 plants transitioned from exhibiting partial to no growth inhibition in response to GR24 treatment (Fig. 4B). This suggests that as axillary branches become dominantly growing shoots they undergo a transition into a state of SL insensitivity. We next investigated if the degree of SL responsiveness of these buds and branches could be explained by the ability of GR24 to up‐regulate expression of PsBRC1. PsBRC1 is a SL responsive transcription factor expressed strongly in non‐growing axillary buds and weakly in the main shoot tip that is thought to be necessary downstream of SLs and CK to regulate bud outgrowth (Braun et al., 2012; Dun et al., 2012). Buds and branches from equivalent plants to those described above (Fig. 4) were treated alongside and were harvested 6 h after treatment for gene expression analyses. Careful dissection of apical buds from branches was performed to avoid dilution effect. PsBRC1 expression (Fig. 4C) was most greatly elevated by GR24 in buds whose growth was greatly suppressed (Fig. 4B). In contrast, expression of
PsBRC1 was also induced, but not to the same extent, in small growing branches whose
growth was reduced by treatment with GR24. Growing branches that exhibited no growth repression by GR24 treatment showed no GR24‐induced induction of PsBRC1 expression. Therefore, the degree of SL responsiveness of PsBRC1 expression in axillary buds and branches might determine the degree of SL growth inhibition response. These results also
142
143 suggest that SL and PsBRC1 function not only in bud release but also in regulating a bud’s transition into a dominantly growing shoot as PsBRC1 expression decreased over time alongside its decreased response to GR24.
GR24 inhibition of bud outgrowth is not permanent
Previously we demonstrated that exogenous treatment of GR24 to the uppermost axillary bud of decapitated wild‐type pea plants inhibits their outgrowth (Brewer et al., 2009). Under