Strigolactone Acts Downstream of Auxin to Regulate Bud Outgrowth in Pea and Arabidopsis

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.     

Concepts and terminology of apical dominance

Apical dominance is the control exerted by the shoot apex over lateral bud outgrowth. The concepts and terminology associated with apical dominance as used by various plant scientists sometimes differ, which may lead to significant misconceptions. Apical dominance and its release may be divided into four developmental stages: (I) lateral bud formation, (II) imposition of inhibition on lateral bud growth, (III) release of apical dominance following decapitation, and (IV) branch shoot development. Particular emphasis is given to discriminating between Stage III, which is accompanied by initial bud outgrowth during the first few hours of release and may be promoted by cytokinin and inhibited by auxin, and Stage IV, which is accompanied by subsequent bud outgrowth occurring days or weeks after decapitation and which may be promoted by auxin and gibberellin. The importance of not interpreting data measured in Stage IV on the basis of conditions and processes occurring in Stage III is discussed as well as the correlation between degree of branching and endogenous auxin content, branching mutants, the quantification of apical dominance in various species (including Arabidopsis ), and apical control in trees.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制。最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制。通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf l,budl)突变体。突变体植株的表型包括顶端优势丧失、株型矮小,表明budl突变体存在生长素代谢、运输或信号传导的缺陷。一个对生长素特异反应的启动子驱动的报告基因在budl中表达模式改变。生长素敏感性和运输能力的测定表明这两个过程在budl中均正常。以上结果显示budl表型是生长素代谢缺陷的结果。遗传分析表明BUDI为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离。     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制.最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制.通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf 1, bud1 )突变体.突变体植株的表型包括顶端优势丧失、株型矮小,表明bud1 突变体存在生长素代谢、运输或信号传导的缺陷.一个对生长素特异反应的启动子驱动的报告基因在bud1 中表达模式改变.生长素敏感性和运输能力的测定表明这两个过程在 bud1中均正常.以上结果显示bud1 表型是生长素代谢缺陷的结果.遗传分析表明BUD1 为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离.     

The gravity-regulated growth of axillary buds is mediated by a mechanism different from decapitation-induced release

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.     

Execution of the auxin replacement apical dominance experiment in temperate woody species

The classic Thimann-Skoog or auxin replacement apical dominance test of exogenous auxin repression of lateral bud outgrowth was successfully executed in both seedlings and older trees of white ash, green ash, and red oak under the following conditions: (1) decapitation of a twig apex and auxin replacement were carried out during spring flush, (2) the decapitation was in the previous season's overwintered wood, and (3) the point of decapitation was below the upper large irrepressible lateral buds but above the lower repressible lateral buds. Although it has been suggested that neither auxin, the terminal bud, nor apical dominance have control over the outgrowth of the irrepressible buds during spring flush, there is evidence in the present study that indicates that such control over the repressible buds exists. In seedlings, second-order branching, which resulted from decapitation of elongating current shoots, was also inhibited by exogenous auxin in the three species. Hence, the auxin replacement experiments did work on year-old proleptic buds (of branches of older trees) that would have entered the bud bank and also on current buds of seedlings. Cytokinin treatments were ineffectual in promoting bud growth.     

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance. Received: 12 December 1996 / Accepted: 7 January 1997     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin‐mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.     

Apical Dominanace in Xanthium strumarium: A DISCUSSION IN RELATION TO CURRENT HYPOTHESES OF CORRELATIVE INHIBITION

The influence of the spectral distribution of illumination onthe gibberellin, cytokinin, auxin, and abscisic acid levelsand the correlation with the degree of branching in Xanthiumstrumarium is presented and discussed. Gibberellins do not appearto play a major role in apical dominance but may be importantfor bud extension following the initial release from dominance.The cytokinin level was much higher in inhibited buds than inreleased buds. It is suggested that the cytokinins present wereprobably not able to participate in bud growth because of anauxin-induced accumulation of abscisic acid in the buds themselves.The concentration of abscisic acid as measured by bioassay andgas-liquid chromatography was between 50 and 250 times thatoccurring in all other plants parts examined. This level felldramatically following release from apical dominance by decapitation.The results are discussed in relation to current hypothesesof apical dominance.     

Auxin dynamics after decapitation are not correlated with the initial growth of axillary buds

One of the first and most enduring roles identified for the plant hormone auxin is the mediation of apical dominance. Many reports have claimed that reduced stem indole-3-acetic acid (IAA) levels and/or reduced basipetal IAA transport directly or indirectly initiate bud growth in decapitated plants. We have tested whether auxin inhibits the initial stage of bud release, or subsequent stages, in garden pea (Pisum sativum) by providing a rigorous examination of the dynamics of auxin level, auxin transport, and axillary bud growth. We demonstrate that after decapitation, initial bud growth occurs prior to changes in IAA level or transport in surrounding stem tissue and is not prevented by an acropetal supply of exogenous auxin. We also show that auxin transport inhibitors cause a similar auxin depletion as decapitation, but do not stimulate bud growth within our experimental time-frame. These results indicate that decapitation may trigger initial bud growth via an auxin-independent mechanism. We propose that auxin operates after this initial stage, mediating apical dominance via autoregulation of buds that are already in transition toward sustained growth.     

Trehalose 6‐phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.)

Trehalose 6‐phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2‐oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration‐dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.     

The Roles of Auxin in Regulating “Shoot Branching” of Cremastra appendiculata

Cremastra appendiculata (D. Don) Makino is a mainly vegetative propagation terrestrial orchid that is a typical representative of the warm-temperate vegetation in China. In this experiment, we investigated the growth and development process of C. appendiculata leaf buds and examined their biochemical components (proteins, auxin, and cytokinin) to gain insight into the “shoot branching” of C. appendiculata pseudobulb string. The results showed that the metabolic activity of C. appendiculata pseudobulbs became lower with the increase of pseudobulb age. However, biennial and triennial pseudobulbs have higher auxin levels than annual pseudobulbs in the intact plant (P < 0.05). After decapitation, the auxin rapidly reduces in biennials. The reduction of auxin level promotes cytokinin biosynthesis, which makes the biennial dormant buds start to germinate 18 days after decapitation. These data and phenomena suggested that auxin plays important roles in regulating shoot branching of C. appendiculata, although further studies are needed to consolidate this viewpoint. Our data indirectly support the classical apical dominance theory whereby biennial pseudobulbs are strongly dependent on reduced auxin to initiate leaf bud outgrowth.

     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

An Arabidopsis kinase cascade influences auxin‐responsive cell expansion

Mitogen‐activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin‐related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1‐1 as a mutant that displays hypersensitivity in auxin‐responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin‐responsive cell expansion assays, suggesting that this MPK cascade affects auxin‐influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho‐like GTPases from Plants (ROP) small GTPase family. Similar to mpk1‐1 and mkk3‐1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin‐responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin‐responsive cell expansion are mediated through phosphorylation‐dependent modulation of ROP activity. Our data suggest a MKK3 ? MPK1 ? RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion.