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1.  Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane 
PLoS Biology  2013;11(1):e1001474.
Shoot branching is regulated by competition between branches to export the phytohormone auxin into the main stem. The phytohormone strigolactone balances shoot system growth by making auxin export harder to establish, thus modulating the auxin transport network.
Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.
Author Summary
Plants can adapt their form to suit the environment in which they are growing. For example, genetically identical plants can develop as a single unbranched stem or as a highly ramified bush. This broad developmental potential is possible because the shoot system is produced continuously by growing tips, known as shoot meristems. Meristems produce the stem and leaves of a shoot, and at the base of each leaf, a new meristem is formed. This meristem can remain dormant as a small bud or activate to produce a branch. Thus, the shoot system is a community of shoot meristems, the combined activity and inactivity of which shape shoot form. Here we provide evidence that growth is balanced across the Arabidopsis shoot system by competition between the shoot meristems. This competition is likely mediated by the requirement of meristems to export the plant hormone auxin in order to activate bud outgrowth. In our model, auxin in the main stem, exported from active branches, can prevent auxin export by dormant buds, thus preventing their activation. Our findings show that a second hormone, strigolactone, increases the level of competition between branches by making auxin export harder to establish. Together, these hormones balance growth across the shoot system, adjusting it according to the environmental conditions in which a plant is growing.
doi:10.1371/journal.pbio.1001474
PMCID: PMC3558495  PMID: 23382651
2.  N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression 
PLoS ONE  2013;8(11):e77863.
Background
N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.
Methodology/Principal Findings
Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.
Conclusion/Significance
NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.
doi:10.1371/journal.pone.0077863
PMCID: PMC3817199  PMID: 24223735
3.  The auxin signalling network translates dynamic input into robust patterning at the shoot apex 
We provide a comprehensive expression map of the different genes (TIR1/AFBs, ARFs and Aux/IAAs) involved in the signalling pathway regulating gene transcription in response to auxin in the shoot apical meristem (SAM).We demonstrate a relatively simple structure of this pathway using a high-throughput yeast two-hybrid approach to obtain the Aux/IAA-ARF full interactome.The topology of the signalling network was used to construct a model for auxin signalling and to predict a role for the spatial regulation of auxin signalling in patterning of the SAM.We used a new sensor to monitor the input in the auxin signalling pathway and to confirm the model prediction, thus demonstrating that auxin signalling is essential to create robust patterns at the SAM.
The plant hormone auxin is a key morphogenetic signal involved in the control of cell identity throughout development. A striking example of auxin action is at the shoot apical meristem (SAM), a population of stem cells generating the aerial parts of the plant. Organ positioning and patterning depends on local accumulations of auxin in the SAM, generated by polar transport of auxin (Vernoux et al, 2010). However, it is still unclear how auxin is distributed at cell resolution in tissues and how the hormone is sensed in space and time during development. A complex ensemble of 29 Aux/IAAs and 23 ARFs is central to the regulation of gene transcription in response to auxin (for review, see Leyser, 2006; Guilfoyle and Hagen, 2007; Chapman and Estelle, 2009). Protein–protein interactions govern the properties of this transduction pathway (Del Bianco and Kepinski, 2011). Limited interaction studies suggest that, in the absence of auxin, the Aux/IAA repressors form heterodimers with the ARF transcription factors, preventing them from regulating target genes. In the presence of auxin, the Aux/IAA proteins are targeted to the proteasome by an SCF E3 ubiquitin ligase complex (Chapman and Estelle, 2009; Leyser, 2006). In this process, auxin promotes the interaction between Aux/IAA proteins and the TIR1 F-box of the SCF complex (or its AFB homologues) that acts as an auxin co-receptor (Dharmasiri et al, 2005a, 2005b; Kepinski and Leyser, 2005; Tan et al, 2007). The auxin-induced degradation of Aux/IAAs would then release ARFs to regulate transcription of their target genes. This includes activation of most of the Aux/IAA genes themselves, thus establishing a negative feedback loop (Guilfoyle and Hagen, 2007). Although this general scenario provides a framework for understanding gene regulation by auxin, the underlying protein–protein network remains to be fully characterized.
In this paper, we combined experimental and theoretical analyses to understand how this pathway contributes to sensing auxin in space and time (Figure 1). We first analysed the expression patterns of the ARFs, Aux/IAAs and TIR1/AFBs genes in the SAM. Our results demonstrate a general tendency for most of the 25 ARFs and Aux/IAAs detected in the SAM: a differential expression with low levels at the centre of the meristem (where the stem cells are located) and high levels at the periphery of the meristem (where organ initiation takes place). We also observed a similar differential expression for TIR1/AFB co-receptors. To understand the functional significance of the distribution of ARFs and Aux/IAAs in the SAM, we next investigated the global structure of the Aux/IAA-ARF network using a high-throughput yeast two-hybrid approach and uncover a rather simple topology that relies on three basic generic features: (i) Aux/IAA proteins interact with themselves, (ii) Aux/IAA proteins interact with ARF activators and (iii) ARF repressors have no or very limited interactions with other proteins in the network.
The results of our interaction analysis suggest a model for the Aux/IAA-ARF signalling pathway in the SAM, where transcriptional activation by ARF activators would be negatively regulated by two independent systems, one involving the ARF repressors, the other the Aux/IAAs. The presence of auxin would remove the inhibitory action of Aux/IAAs, but leave the ARF repressors to compete with ARF activators for promoter-binding sites. To explore the regulatory properties of this signalling network, we developed a mathematical model to describe the transcriptional output as a function of the signalling input that is the combinatorial effect of auxin concentration and of its perception. We then used the model and a simplified view of the meristem (where the same population of Aux/IAAs and ARFs exhibit a low expression at the centre and a high expression in the peripheral zone) for investigating the role of auxin signalling in SAM function. We show that in the model, for a given ARF activator-to-repressor ratio, the gene induction capacity increases with the absolute levels of ARF proteins. We thus predict that the differential expression of the ARFs generates differences in auxin sensitivities between the centre (low sensitivity) and the periphery (high sensitivity), and that the expression of TIR1/AFB participates to this regulation (prediction 1). We also use the model to analyse the transcriptional response to rapidly changing auxin concentrations. By simulating situations equivalent either to the centre or the periphery of our simplified representation of the SAM, we predict that the signalling pathway buffers its response to the auxin input via the balance between ARF activators and repressors, in turn generated by their differential spatial distributions (prediction 2).
To test the predictions from the model experimentally, we needed to assess both the input (auxin level and/or perception) and the output (target gene induction) of the signalling cascade. For measuring the transcriptional output, the widely used DR5 reporter is perfectly adapted (Figure 5) (Ulmasov et al, 1997; Sabatini et al, 1999; Benkova et al, 2003; Heisler et al, 2005). For assaying pathway input, we designed DII-VENUS, a novel auxin signalling sensor that comprises a constitutively expressed fusion of the auxin-binding domain (termed domain II or DII) (Dreher et al, 2006; Tan et al, 2007) of an IAA to a fast-maturating variant of YFP, VENUS (Figure 5). The degradation patterns from DII-VENUS indicate a high auxin signalling input both in flower primordia and at the centre of the SAM. This is in contrast to the organ-specific expression pattern of DR5::VENUS (Figure 5). These results indicate that the signalling pathway limits gene activation in response to auxin at the meristem centre and confirm the differential sensitivity to auxin between the centre and the periphery (prediction 1). We further confirmed the buffering capacities of the signalling pathway (prediction 2) by carrying out live imaging experiments to monitor DII-VENUS and DR5::VENUS expression in real time (Figure 5). This analysis reveals the presence of important temporal variations of DII-VENUS fluorescence, while DR5::VENUS does not show such global variations. Our approach thus provides evidence that the Aux/IAA-ARF pathway has a key role in patterning in the SAM, alongside the auxin transport system. Our results illustrate how the tight spatio-temporal regulation of both the distribution of a morphogenetic signal and the activity of the downstream signalling pathway provides robustness to a dynamic developmental process.
A comprehensive expression and interaction map of auxin signalling factors in the Arabidopsis shoot apical meristem is constructed and used to derive a mathematical model of auxin signalling, from which key predictions are experimentally confirmed.
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
doi:10.1038/msb.2011.39
PMCID: PMC3167386  PMID: 21734647
auxin; biosensor; live imaging; ODE; signalling
4.  ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis 
PLoS Genetics  2014;10(1):e1003954.
Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.
Author Summary
Plant architecture is one of the key factors that affect plant survival and productivity. It is well established that the plant hormone auxin plays an essential role in organ initiation and pattern formation, thus affecting plant architecture. We found that a putative MATE (multidrug and toxic compound extrusion) transporter, ADP1, which was expressed in the meristematic regions, through regulating the level of auxin biosynthesis, controls lateral organ outgrowth so as to maintain normal architecture in Arabidopsis. The more ADP1 was expressed, the less levels of local auxin were detected in the meristematic regions of the plant, resulting in increased growth rate and a greater number of axillary branches and flowers. The reduction of auxin levels is probably due to decreased level of auxin biosynthesis in the local meristematic regions. Down-regulated expression of ADP1 and its three closely related genes caused plants to grow slower and to produce less lateral organs. Our results indicated that ADP1-mediated regulation of the local auxin levels in meristematic regions is an essential determinant for plant architecture by restraining the outgrowth of lateral organs.
doi:10.1371/journal.pgen.1003954
PMCID: PMC3879159  PMID: 24391508
5.  Apical dominance in saffron and the involvement of the branching enzymes CCD7 and CCD8 in the control of bud sprouting 
BMC Plant Biology  2014;14:171.
Background
In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs.
Results
The CsCCD7 and CsCCD8 genes are the first ones isolated and characterized from a non-grass monocotyledonous plant. CsCCD7 and CsCCD8 expression showed some overlapping, although they were not identical. CsCCD8 was highly expressed in quiescent axillary buds and decapitation dramatically reduced its expression levels, suggesting its involvement in the suppression of axillary bud outgrowth. Furthermore, in vitro experiments showed also the involvement of auxin, cytokinin and jasmonic acid on the sprouting of axillary buds from corms in which the apical bud was removed. In addition, CsCCD8 expression, but not CsCCD7, was higher in the newly developed vascular tissue of axillary buds compared to the vascular tissue of the apical bud.
Conclusions
We showed that production and transport of auxin in saffron corms could act synergistically with SLs to arrest the outgrowth of the axillary buds, similar to the control of above-ground shoot branching. In addition, jasmonic acid seems to play a prominent role in bud dormancy in saffron. While cytokinins from roots promote bud outgrowth. In addition the expression results of CsCCD8 suggest that SLs could positively regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm.
doi:10.1186/1471-2229-14-171
PMCID: PMC4077219  PMID: 24947472
Auxin; Buds; Carotenoid cleavage oxygenases; Corm; Saffron; Strigolactones
6.  Involvement of ribosomal protein RPL27a in meristem activity and organ development 
Plant Signaling & Behavior  2011;6(5):712-714.
The plant shoot apical meristem is established early during embryogenesis and subsequently gives rise to a shoot through reiterative generation of lateral organs and axillary meristems. In our recent manuscript we reported identification and characterization of a semi-dominant mutation in ribosomal protein RPL27a, which disrupts plant growth and shoot development.1 rpl27ac-1d effects on the shoot are evident from an early stage of embryo development. During embryogenesis rpl27-1d mutants are slow growing and are defective in apical patterning with a delay in establishment of the shoot meristem and outgrowth of cotyledons. Concomitant with this disturbed patterning, the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2) are misexpressed in outer cell layers of the rpl27ac-1d embryo and there is a delay in expression of the organ-patterning gene FILAMENTOUS FLOWER (FIL). Genetic interactions between rpl27ac-1d and other ribosomal protein mutants indicates rpl27ac-1d has reduced ribosome function. Our results highlight a role for ribosomal proteins in growth and development and we propose that the ribosome regulates specific patterning events during development.
doi:10.4161/psb.6.5.15070
PMCID: PMC3172845  PMID: 21448008
development; meristem; organ polarity; ribosomal protein
7.  Florigen is involved in axillary bud development at multiple stages in Arabidopsis 
Plant Signaling & Behavior  2013;8(11):e27167.
The wide variety of plant architectures is largely based on diverse and flexible modes of axillary shoot development. In Arabidopsis, floral transition (flowering) stimulates axillary bud development. The mechanism that links flowering and axillary bud development is, however, largely unknown. We recently showed that FLOWERING LOCUS T (FT) protein, which acts as florigen, promotes the phase transition of axillary meristems, whereas BRANCHED1 (BRC1) antagonizes the florigen action in axillary buds. Here, we present evidences for another possible role of florigen in axillary bud development. Ectopic overexpression of FT or another florigen gene TWIN SISTER OF FT (TSF) with LEAFY (LFY) induces ectopic buds at cotyledonary axils, confirming the previous proposal that these genes are involved in formation of axillary buds. Taken together with our previous report that florigen promotes axillary shoot elongation, we propose that florigen regulates axillary bud development at multiple stages to coordinate it with flowering in Arabidopsis.
doi:10.4161/psb.27167
PMCID: PMC4091375  PMID: 24305631
Arabidopsis; flowering; florigen; axillary bud; cotyledon; FT; TSF; LFY; BRC1
8.  Opposite Root Growth Phenotypes of hy5 versus hy5 hyh Mutants Correlate with Increased Constitutive Auxin Signaling 
PLoS Genetics  2006;2(11):e202.
The Arabidopsis transcription factor HY5 controls light-induced gene expression downstream of photoreceptors and plays an important role in the switch of seedling shoots from dark-adapted to light-adapted development. In addition, HY5 has been implicated in plant hormone signaling, accounting for the accelerated root system growth phenotype of hy5 mutants. Mutants in the close HY5 homolog HYH resemble wild-type, despite the largely similar expression patterns and levels of HY5 and HYH, and the functional equivalence of the respective proteins. Moreover, the relative contribution of HYH to the overall activity of the gene pair is increased by an alternative HYH transcript, which encodes a stabilized protein. Consistent with the enhanced root system growth observed in hy5 loss-of-function mutants, constitutively overexpressed alternative HYH inhibits root system growth. Paradoxically, however, in double mutants carrying hy5 and hyh null alleles, the hy5 root growth phenotype is suppressed rather than enhanced. Even more surprisingly, compared to wild-type, root system growth is diminished in hy5 hyh double mutants. In addition, the double mutants display novel shoot phenotypes that are absent from either single mutant. These include cotyledon fusions and defective vasculature, which are typical for mutants in genes involved in the transcriptional response to the plant hormone auxin. Indeed, many auxin-responsive and auxin signaling genes are misexpressed in hy5 mutants, and at a higher number and magnitude in hy5 hyh mutants. Therefore, auxin-induced transcription is constitutively activated at different levels in the two mutant backgrounds. Our data support the hypothesis that the opposite root system phenotypes of hy5 single and hy5 hyh double mutants represent the morphological response to a quantitative gradient in the same molecular process, that is gradually increased constitutive auxin signaling. The data also suggest that HY5 and HYH are important negative regulators of auxin signaling amplitude in embryogenesis and seedling development.
Synopsis
Genetic redundancy is the total or partial compensation of inactivation of one gene by another, usually related gene. In Arabidopsis, HY5 and HYH are highly similar, principally exchangeable genes. However, only inactivation of HY5 results in morphological defects, indicating that HY5 plays a more important role in development than HYH. Nevertheless, if inactive versions of such partially redundant genes are combined in a double mutant, the defects observed in the single mutant often worsen. Paradoxically, however, combined inactivation of HY5 and HYH leads to a defect that is opposite to inactivation of HY5 alone: compared to controls, root system growth is decreased in the double mutant, rather than enhanced as in plants only lacking HY5 activity. Through careful analysis of the double mutant defects and scans of genome-wide gene expression levels, the authors determined that the opposite root system growth of hy5 single and hy5 hyh double mutants is a morphological response to a gradually increased quantitative disturbance in the same molecular process, the physiological response to the plant hormone auxin. This example suggests that inactivation of genes that quantitatively affect the balance of a physiological process in the same manner might manifest in very different morphological changes.
doi:10.1371/journal.pgen.0020202
PMCID: PMC1657057  PMID: 17121469
9.  Aintegumenta and Aintegumenta-Like6 regulate auxin-mediated flower development in Arabidopsis 
BMC Research Notes  2011;4:176.
Background
Two related genes encoding AP2/ERF-type transcription factors, AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6), are important regulators of floral growth and patterning in Arabidopsis. Evidence suggests that these genes promote several aspects of flower development in response to auxin. To investigate the interplay of ANT, AIL6 and auxin during floral development, I have examined the phenotypic consequences of disrupting polar auxin transport in ant, ail6 and ant ail6 mutants by either genetic or chemical means.
Results
Plants containing mutations in ANT or AIL6 alone or in both genes together exhibit increased sensitivity to disruptions in polar auxin transport. Both genes promote shoot growth, floral meristem initiation and floral meristem patterning in combination with auxin transport. However, differences in the responses of ant and ail6 single mutants to perturbations in auxin transport suggest that these two genes also have non-overlapping activities in each of these developmental processes.
Conclusions
The enhanced sensitivity of ant and ail6 mutants to alterations in polar auxin transport suggests that these mutants have defects in some aspect of auxin physiology. The inability of ant ail6 double mutants to initiate flowers in backgrounds disrupted for auxin transport confirm the proposed roles for these two genes in floral meristem initiation.
doi:10.1186/1756-0500-4-176
PMCID: PMC3127960  PMID: 21645408
10.  Gibberellin mediates daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria × ananassa Duch) 
BMC Plant Biology  2009;9:18.
Background
Differentiation of long and short shoots is an important developmental trait in several species of the Rosaceae family. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of gibberellin (GA) in regulation of shoot differentiation in strawberry (Fragaria × ananassa Duch.) cv. Korona. In strawberry, differentiation of axillary buds to runners (long shoot) or to crown branches (short shoot) is promoted by long-day and short-day conditions, respectively. Formation of crown branches is a prerequisite for satisfactory flowering because inflorescences are formed from the apical meristems of the crown.
Results
We found that both prohexadione-calcium and short photoperiod inhibited runner initiation and consequently led to induction of crown branching. In both cases, this correlated with a similar decline in GA1 level. Exogenous GA3 completely reversed the effect of prohexadione-calcium in a long photoperiod, but was only marginally effective in short-day grown plants. However, transfer of GA3-treated plants from short days to long days restored the normal runner formation. This did not occur in plants that were not treated with GA3. We also studied GA signalling homeostasis and found that the expression levels of several GA biosynthetic, signalling and target genes were similarly affected by prohexadione-calcium and short photoperiod in runner tips and axillary buds, respectively.
Conclusion
GA is needed for runner initiation in strawberry, and the inhibition of GA biosynthesis leads to the formation of crown branches. Our findings of similar changes in GA levels and in GA signalling homeostasis after prohexadione-calcium and short-day treatments, and photoperiod-dependent responsiveness of the axillary buds to GA indicate that GA plays a role also in the photoperiod-regulated differentiation of axillary buds. We propose that tightly regulated GA activity may control induction of cell division in subapical tissues of axillary buds, being one of the signals determining bud fate.
doi:10.1186/1471-2229-9-18
PMCID: PMC2653492  PMID: 19210764
11.  Microdissection of Shoot Meristem Functional Domains 
PLoS Genetics  2009;5(5):e1000476.
The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.
Author Summary
All the organs of plant shoots are derived from the shoot apical meristem (SAM), a pool of plant stem cells that are both organogenic and self-sustaining. These dual SAM functions take place in distinct yet adjacent meristematic domains; leaves are derived from the peripheral zone (PZ) of the SAM whereas cells lost during organogenesis are replenished from the central zone (CZ). Deciphering the global patterns of differential gene expression within these discrete SAM functional domains is integral toward understanding the molecular-signaling networks that regulate plant development. We utilized laser-microdissection technology to isolate tissues from the SAM crown and center (SAM-proper) and from initiating leaves (P0/P1) at the SAM periphery for use in microarray comparisons of gene expression within these SAM functional domains. Nine hundred and sixty-two maize genes were differentially expressed, confirming that the distinct functions of these meristematic domains involve widespread differences in gene expression. Genes involved in cell division, cell wall biosynthesis, chromatin structure, and RNA binding are especially upregulated in initiating leaves, whereas genes regulating protein stability and DNA repair are upregulated in the SAM proper. Mutations in a D-cyclin gene that was upregulated in the P0/P1 render narrow-leafed mutant plants with defective stomatal patterning, providing functional genetic data for a previously uncharacterized maize gene.
doi:10.1371/journal.pgen.1000476
PMCID: PMC2673047  PMID: 19424435
12.  Transcript profiling of cytokinin action in Arabidopsis roots and shoots discovers largely similar but also organ-specific responses 
BMC Plant Biology  2012;12:112.
Background
The plant hormone cytokinin regulates growth and development of roots and shoots in opposite ways. In shoots it is a positive growth regulator whereas it inhibits growth in roots. It may be assumed that organ-specific regulation of gene expression is involved in these differential activities, but little is known about it. To get more insight into the transcriptional events triggered by cytokinin in roots and shoots, we studied genome-wide gene expression in cytokinin-treated and cytokinin-deficient roots and shoots.
Results
It was found by principal component analysis of the transcriptomic data that the immediate-early response to a cytokinin stimulus differs from the later response, and that the transcriptome of cytokinin-deficient plants is different from both the early and the late cytokinin induction response. A higher cytokinin status in the roots activated the expression of numerous genes normally expressed predominantly in the shoot, while a lower cytokinin status in the shoot reduced the expression of genes normally more active in the shoot to a more root-like level. This shift predominantly affected nuclear genes encoding plastid proteins. An organ-specific regulation was assigned to a number of genes previously known to react to a cytokinin signal, including root-specificity for the cytokinin hydroxylase gene CYP735A2 and shoot specificity for the cell cycle regulator gene CDKA;1. Numerous cytokinin-regulated genes were newly discovered or confirmed, including the meristem regulator genes SHEPHERD and CLAVATA1, auxin-related genes (IAA7, IAA13, AXR1, PIN2, PID), several genes involved in brassinosteroid (CYP710A1, CYP710A2, DIM/DWF) and flavonol (MYB12, CHS, FLS1) synthesis, various transporter genes (e.g. HKT1), numerous members of the AP2/ERF transcription factor gene family, genes involved in light signalling (PhyA, COP1, SPA1), and more than 80 ribosomal genes. However, contrasting with the fundamental difference of the growth response of roots and shoots to the hormone, the vast majority of the cytokinin-regulated transcriptome showed similar response patterns in roots and shoots.
Conclusions
The shift of the root and shoot transcriptomes towards the respective other organ depending on the cytokinin status indicated that the hormone determines part of the organ-specific transcriptome pattern independent of morphological organ identity. Numerous novel cytokinin-regulated genes were discovered which had escaped earlier discovery, most probably due to unspecific sampling. These offer novel insights into the diverse activities of cytokinin, including crosstalk with other hormones and different environmental cues, identify the AP2/ERF class of transcriptions factors as particularly cytokinin sensitive, and also suggest translational control of cytokinin-induced changes.
doi:10.1186/1471-2229-12-112
PMCID: PMC3519560  PMID: 22824128
13.  FINE CULM1 (FC1) Works Downstream of Strigolactones to Inhibit the Outgrowth of Axillary Buds in Rice 
Plant and Cell Physiology  2010;51(7):1127-1135.
Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 μM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 μM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice.
doi:10.1093/pcp/pcq083
PMCID: PMC2900823  PMID: 20547591
FINE CULM1 (FC1); Rice; Shoot branching; Strigolactone; TCP transcription factor; Tiller
14.  Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development 
PLoS ONE  2013;8(7):e70069.
Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.
doi:10.1371/journal.pone.0070069
PMCID: PMC3726503  PMID: 23922907
15.  Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network 
BMC Plant Biology  2012;12:169.
Background
The Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known.
Results
In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15 and the proximal promoter sequence of miR156b.
Conclusions
Enhanced miR156b expression in sk156 leads to the mutant phenotype including carotenoid levels in the seed through suppression of SPL15 and other SPL target genes. Moreover, SPL15 has a regulatory role not only for downstream components, but also for its own upstream regulator miR156b.
doi:10.1186/1471-2229-12-169
PMCID: PMC3520712  PMID: 22989211
16.  Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice 
BMC Genomics  2011;12:387.
Background
In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development.
Results
To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively.
Conclusion
Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, GTE4 and MAP, require CRL1 to be induced by auxin suggesting that they are likely directly regulated by CRL1. These genes have a function related to polarized cell growth, cell cycle regulation or chromatin remodelling. This suggests that these genes are controlled by CRL1 and involved in CR initiation in rice.
doi:10.1186/1471-2164-12-387
PMCID: PMC3163228  PMID: 21806801
17.  Roles of DgBRC1 in Regulation of Lateral Branching in Chrysanthemum (Dendranthema ×grandiflora cv. Jinba) 
PLoS ONE  2013;8(4):e61717.
The diverse plasticity of plant architecture is largely determined by shoot branching. Shoot branching is an event regulated by multiple environmental, developmental and hormonal stimuli through triggering lateral bud response. After perceiving these signals, the lateral buds will respond and make a decision on whether to grow out. TCP transcriptional factors, BRC1/TB1/FC1, were previously proven to be involved in local inhibition of shoot branching in Arabidopsis, pea, tomato, maize and rice. To investigate the function of BRC1, we isolated the BRC1 homolog from chrysanthemum. There were two transcripts of DgBRC1 coming from two alleles in one locus, both of which complemented the multiple branches phenotype of Arabidopsis brc1-1, indicating that both are functionally conserved. DgBRC1 was mainly expressed in dormant axillary buds, and down-regulated at the bud activation stage, and up-regulated by higher planting densities. DgBRC1 transcripts could respond to apical auxin supply and polar auxin transport. Moreover, we found that the acropetal cytokinin stream promoted branch outgrowth whether or not apical auxin was present. Basipetal cytokinin promoted outgrowth of branches in the absence of apical auxin, while strengthening the inhibitory effects on lower buds in the presence of apical auxin. The influence of auxin and strigolactons (SLs) on the production of cytokinin was investigated, we found that auxin locally down-regulated biosynthesis of cytokinin in nodes, SLs also down-regulated the biosynthesis of cytokinin, the interactions among these phytohormones need further investigation.
doi:10.1371/journal.pone.0061717
PMCID: PMC3629106  PMID: 23613914
18.  Simulation of Organ Patterning on the Floral Meristem Using a Polar Auxin Transport Model 
PLoS ONE  2012;7(1):e28762.
An intriguing phenomenon in plant development is the timing and positioning of lateral organ initiation, which is a fundamental aspect of plant architecture. Although important progress has been made in elucidating the role of auxin transport in the vegetative shoot to explain the phyllotaxis of leaf formation in a spiral fashion, a model study of the role of auxin transport in whorled organ patterning in the expanding floral meristem is not available yet. We present an initial simulation approach to study the mechanisms that are expected to play an important role. Starting point is a confocal imaging study of Arabidopsis floral meristems at consecutive time points during flower development. These images reveal auxin accumulation patterns at the positions of the organs, which strongly suggests that the role of auxin in the floral meristem is similar to the role it plays in the shoot apical meristem. This is the basis for a simulation study of auxin transport through a growing floral meristem, which may answer the question whether auxin transport can in itself be responsible for the typical whorled floral pattern. We combined a cellular growth model for the meristem with a polar auxin transport model. The model predicts that sepals are initiated by auxin maxima arising early during meristem outgrowth. These form a pre-pattern relative to which a series of smaller auxin maxima are positioned, which partially overlap with the anlagen of petals, stamens, and carpels. We adjusted the model parameters corresponding to properties of floral mutants and found that the model predictions agree with the observed mutant patterns. The predicted timing of the primordia outgrowth and the timing and positioning of the sepal primordia show remarkable similarities with a developing flower in nature.
doi:10.1371/journal.pone.0028762
PMCID: PMC3264561  PMID: 22291882
19.  Auxin at the Shoot Apical Meristem 
Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Here, we discuss the so-called shoot apical meristem (SAM), which generates all the aerial parts of the plant. It has been known for many years that auxin plays a central role in the functioning of this meristem. Auxin is not homogeneously distributed at the SAM and it is thought that this distribution is interpreted in terms of differential gene expression and patterned growth. In this context, auxin transporters of the PIN and AUX families, creating auxin maxima and minima, are crucial regulators. However, auxin transport is not the only factor involved. Auxin biosynthesis genes also show specific, patterned activities, and local auxin synthesis appears to be essential for meristem function as well. In addition, auxin perception and signal transduction defining the competence of cells to react to auxin, add further complexity to the issue. To unravel this intricate signaling network at the SAM, systems biology approaches, involving not only molecular genetics but also live imaging and computational modeling, have become increasingly important.
Auxin dynamically regulates patterning at the shoot apical meristem. Transporters and local biosynthesis are involved in the control of its distribution at the shoot apex, where it is required for formation of new buds.
doi:10.1101/cshperspect.a001487
PMCID: PMC2845202  PMID: 20452945
20.  Convergence of the 26S proteasome and the REVOLUTA pathways in regulating inflorescence and floral meristem functions in Arabidopsis 
Journal of Experimental Botany  2010;62(1):359-369.
The 26S proteasome is a large multisubunit proteolytic complex, regulating growth and development in eukaryotes by selective removal of short-lived regulatory proteins. Here, it is shown that the 26S proteasome and the transcription factor gene REVOLUTA (REV) act together in maintaining inflorescence and floral meristem (IM and FM) functions. The characterization of a newly identified Arabidopsis mutant, designated ae4 (asymmetric leaves1/2 enhancer4), which carries a mutation in the gene encoding the 26S proteasome subunit, RPN2a, is reported. ae4 and rev have minor defects in phyllotaxy structure and meristem initiation, respectively, whereas ae4 rev demonstrated strong developmental defects. Compared with the rev single mutant, an increased percentage of ae4 rev plants exhibited abnormal vegetative shoot apical and axillary meristems. After flowering, ae4 rev first gave rise to a few normal-looking flowers, and then flowers with reduced numbers of all types of floral organs. In late reproductive development, instead of flowers, the ae4 rev IM produced numerous filamentous structures, which contained cells seen only in the floral organs, and then carpelloid organs. In situ hybridization revealed that expression of the WUSCHEL and CLAVATA3 genes was severely down-regulated or absent in the late appearing ae4 rev primordia, but the genes were strongly expressed in top-layer cells of inflorescence tips. Double mutant plants combining rev with other 26S proteasome subunit mutants, rpn1a and rpn9a, resembled ae4 rev, suggesting that the 26S proteasome might act as a whole in regulating IM and FM functions.
doi:10.1093/jxb/erq277
PMCID: PMC2993919  PMID: 20797995
Arabidopsis; floral meristem; inflorescence meristem; 26S proteasome; REVOLUTA
21.  Tomato Yield Heterosis Is Triggered by a Dosage Sensitivity of the Florigen Pathway That Fine-Tunes Shoot Architecture 
PLoS Genetics  2013;9(12):e1004043.
The superiority of hybrids has long been exploited in agriculture, and although many models explaining “heterosis” have been put forth, direct empirical support is limited. Particularly elusive have been cases of heterozygosity for single gene mutations causing heterosis under a genetic model known as overdominance. In tomato (Solanum lycopersicum), plants carrying mutations in SINGLE FLOWER TRUSS (SFT) encoding the flowering hormone florigen are severely delayed in flowering, become extremely large, and produce few flowers and fruits, but when heterozygous, yields are dramatically increased. Curiously, this overdominance is evident only in the background of “determinate” plants, in which the continuous production of side shoots and inflorescences gradually halts due to a defect in the flowering repressor SELF PRUNING (SP). How sp facilitates sft overdominance is unclear, but is thought to relate to the opposing functions these genes have on flowering time and shoot architecture. We show that sft mutant heterozygosity (sft/+) causes weak semi-dominant delays in flowering of both primary and side shoots. Using transcriptome sequencing of shoot meristems, we demonstrate that this delay begins before seedling meristems become reproductive, followed by delays in subsequent side shoot meristems that, in turn, postpone the arrest of shoot and inflorescence production. Reducing SFT levels in sp plants by artificial microRNAs recapitulates the dose-dependent modification of shoot and inflorescence production of sft/+ heterozygotes, confirming that fine-tuning levels of functional SFT transcripts provides a foundation for higher yields. Finally, we show that although flowering delays by florigen mutant heterozygosity are conserved in Arabidopsis, increased yield is not, likely because cyclical flowering is absent. We suggest sft heterozygosity triggers a yield improvement by optimizing plant architecture via its dosage response in the florigen pathway. Exploiting dosage sensitivity of florigen and its family members therefore provides a path to enhance productivity in other crops, but species-specific tuning will be required.
Author Summary
For over a century, it has been known that inbreeding harms plant and animal fitness, whereas interbreeding between genetically distinct individuals can lead to more robust offspring in a phenomenon known as hybrid vigor, or heterosis. While heterosis has been harnessed to boost agricultural productivity, its causes are not understood. Especially controversial is a model called “overdominance,” which states in its simplest form that a single gene can drive heterosis, although multiple overdominant genes can also contribute. In tomato, a mutation in just one of two copies of a gene encoding the flowering hormone called florigen causes remarkable increases in yield, but it is not known why. We show that yield increases are triggered by a fine-tuning of florigen levels that cause subtle delays in the time it takes all shoots to produce flowers. The resulting plant architecture maximizes yield in varieties that dominate the processing tomato industry. We show that while similar changes in flowering occur when one copy of florigen is mutated in the model crucifer plant Arabidopsis, yield is not increased, suggesting that, while manipulating florigen holds potential to improve crop productivity, the tuning of florigen and related genes will have to be tailored according to species.
doi:10.1371/journal.pgen.1004043
PMCID: PMC3873276  PMID: 24385931
22.  The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots 
Journal of Experimental Botany  2012;63(15):5689-5703.
Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively.
doi:10.1093/jxb/ers221
PMCID: PMC3444280  PMID: 22915742
Cell fate; competence; determination; hormonal mutants; Micro-Tom; plant development; regeneration
23.  The Making of a Compound Inflorescence in Tomato and Related Nightshades 
PLoS Biology  2008;6(11):e288.
Variation in the branching of plant inflorescences determines flower number and, consequently, reproductive success and crop yield. Nightshade (Solanaceae) species are models for a widespread, yet poorly understood, program of eudicot growth, where short side branches are initiated upon floral termination. This “sympodial” program produces the few-flowered tomato inflorescence, but the classical mutants compound inflorescence (s) and anantha (an) are highly branched, and s bears hundreds of flowers. Here we show that S and AN, which encode a homeobox transcription factor and an F-box protein, respectively, control inflorescence architecture by promoting successive stages in the progression of an inflorescence meristem to floral specification. S and AN are sequentially expressed during this gradual phase transition, and the loss of either gene delays flower formation, resulting in additional branching. Independently arisen alleles of s account for inflorescence variation among domesticated tomatoes, and an stimulates branching in pepper plants that normally have solitary flowers. Our results suggest that variation of Solanaceae inflorescences is modulated through temporal changes in the acquisition of floral fate, providing a flexible evolutionary mechanism to elaborate sympodial inflorescence shoots.
Author Summary
Among the most distinguishing features of plants are the flower-bearing shoots, called inflorescences. Despite a solid understanding of flower development, the molecular mechanisms that control inflorescence architecture remain obscure. We have explored this question in tomato, where mutations in two genes, ANANTHA (AN) and COMPOUND INFLORESCENCE (S), transform the well-known tomato “vine” into a highly branched structure with hundreds of flowers. We find that AN encodes an F-box protein ortholog of a gene called UNUSUAL FLORAL ORGANS that controls the identity of floral organs (petals, sepals, and so on), whereas S encodes a transcription factor related to a gene called WUSCHEL HOMEOBOX 9 that is involved in patterning the embryo within the plant seed. (F-box proteins are known for marking other proteins for degradation, but they can also function in hormone regulation and transcriptional activation) Interestingly, these genes have little or no effect on branching in inflorescences that grow continuously (so-called “indeterminate” shoots), as in Arabidopsis. However, we find that transient sequential expression of S followed by AN promotes branch termination and flower formation in plants where meristem growth ends with inflorescence and flower production (“determinate” shoots). We show that mutant alleles of s dramatically increase branch and flower number and have probably been selected for by breeders during modern cultivation. Moreover, the single-flower inflorescence of pepper (a species related to tomato, within the same Solanaceae family) can be converted to a compound inflorescence upon mutating its AN ortholog. Our results suggest a new developmental mechanism whereby inflorescence elaboration can be controlled through temporal regulation of floral fate.
Plant flower production is largely determined by the number of inflorescences, the branches produced on flower stems. Two genes identified in tomato reveal a new phase transition that may explain the mechanism of evolution of compound inflorescences in the Solanaceae family.
doi:10.1371/journal.pbio.0060288
PMCID: PMC2586368  PMID: 19018664
24.  DNA Methylation and Histone Modifications Regulate De Novo Shoot Regeneration in Arabidopsis by Modulating WUSCHEL Expression and Auxin Signaling 
PLoS Genetics  2011;7(8):e1002243.
Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants in vitro was made possible. Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes—including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase—resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.
Author Summary
Plants have a strong ability to generate organs from differentiated somatic tissues. Due to this feature, shoot regeneration in vitro has been used as an important way for producing whole plants in agriculture and biotechnology. Phytohormones and the transcription factor WUSCHEL (WUS) are essential for reprogramming during de novo shoot regeneration. Epigenetic modifications are also critical for mammalian cell differentiation and organogenesis. Here, we show that epigenetic modifications mediate the de novo shoot regeneration in Arabidopsis. Mutations of key epigenetic genes resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Bisulfite sequencing and chromatin immunoprecipitation revealed that the regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications. By transcriptome analysis, we identified that some differentially expressed genes between wild type and met1 are involved in signal transduction of the phytohormone auxin. Our results suggest that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role during de novo organogenesis.
doi:10.1371/journal.pgen.1002243
PMCID: PMC3158056  PMID: 21876682
25.  Modelling and experimental analysis of hormonal crosstalk in Arabidopsis 
An important question in plant biology is how genes influence the crosstalk between hormones to regulate growth. We have developed the first hormonal crosstalk network in Arabidopsis by iteratively combining modelling with experimental analysis.We have revealed that the POLARIS gene interacts with the ethylene receptor and regulates both auxin transport and biosynthesis.Our modelling analysis has reproduced all known mutants. With new experimental data, it has provided new insights into how the POLARIS gene regulates auxin concentration for root development in Arabidopsis, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling.Modelling and experimental analysis have revealed that a bell-shaped dose–response relationship between endogenous auxin and root length is established via POLARIS.
Hormone signalling systems coordinate plant growth and development through a range of complex interactions. The activities of plant hormones, such as auxin, ethylene and cytokinin, depend on cellular context and exhibit interactions that can be either synergistic or antagonistic. An important question regarding the understanding of those interactions is how genes act on the crosstalk between hormones to regulate plant growth.
Previously, we identified the POLARIS (PLS) gene of Arabidopsis, which transcribes a short mRNA encoding a 36-amino acid peptide that is required for correct root growth and vascular development (Casson et al, 2002). Experimental evidence shows that there is a link between PLS, ethylene signalling, auxin homeostasis and microtubule cytoskeleton dynamics (Chilley et al, 2006). Specifically, mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered sensitivity to microtubule inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. The expression of PLS is itself repressed by ethylene and induced by auxin. It was also shown that pls mutant roots are hyper-responsive to exogenous cytokinins and show increased expression of the cytokinin inducible gene ARR5/IBC6 compared with the wild type (Casson et al, 2002). Therefore, PLS may also be required for correct auxin–cytokinin homeostasis to modulate root growth.
In this study, we model PLS gene function and crosstalk between auxin, ethylene and cytokinin in Arabidopsis.
Experimental evidence suggests that PLS acts on or close to the ethylene receptor ETR1, and a mathematical model describing possible PLS–ethylene pathway interactions is developed, and used to make quantitative predictions about PLS–hormone interactions. Modelling correctly predicts experimental results for the effect of the pls gene mutation on endogenous cytokinin concentration. Modelling also reveals a role for PLS in auxin biosynthesis in addition to a role in auxin transport (Figures 1 and 4).
The model reproduces available mutants, and with new experimental data provides new insights into how PLS regulates auxin concentration, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling. Modelling further reveals that a bell-shaped dose–response relationship between endogenous auxin and root length is established via PLS.
In summary, we developed the first hormonal crosstalk model in Arabidopsis and revealed a hormonal crosstalk circuit through PLS and the downstream of ethylene signalling. Our study provides a platform to further integrate hormonal crosstalk in space and time in Arabidopsis.
An important question in plant biology is how genes influence the crosstalk between hormones to regulate growth. In this study, we model POLARIS (PLS) gene function and crosstalk between auxin, ethylene and cytokinin in Arabidopsis. Experimental evidence suggests that PLS acts on or close to the ethylene receptor ETR1, and a mathematical model describing possible PLS–ethylene pathway interactions is developed, and used to make quantitative predictions about PLS–hormone interactions. Modelling correctly predicts experimental results for the effect of the pls gene mutation on endogenous cytokinin concentration. Modelling also reveals a role for PLS in auxin biosynthesis in addition to a role in auxin transport. The model reproduces available mutants, and with new experimental data provides new insights into how PLS regulates auxin concentration, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling. Modelling further reveals that a bell-shaped dose–response relationship between endogenous auxin and root length is established via PLS. This combined modelling and experimental analysis provides new insights into the integration of hormonal signals in plants.
doi:10.1038/msb.2010.26
PMCID: PMC2913391  PMID: 20531403
hormonal crosstalk; mathematical model; POLARIS gene; root development

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