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1.  DWARF TILLER1, a WUSCHEL-Related Homeobox Transcription Factor, Is Required for Tiller Growth in Rice 
PLoS Genetics  2014;10(3):e1004154.
Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that DWARF TILLER1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a WUSCHEL-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice.
Author Summary
Plant architecture is important for crop yield. In most plants, branches grow smaller than the main shoot, largely due to the ‘apical dominance’. However, in several cereal crops, including rice, wheat, and barley, the branches (tillers) have a height and size indistinguishable from the main shoot. The genetic basis of uniform tiller growth has remained elusive. We identified DWARF TILLER1, a WUSCHEL-related homeobox (WOX) transcription factor, as a positive regulator of tiller growth. Most dwt1 mutant plants show normal main shoot but dwarf tillers and reduced panicle size. Tiller growth in dwt1 appears to be inhibited by the main shoot, as removal of the main shoot releases the first tiller. The non-elongating internodes in dwt1 show reduced cell number and cell size, while DWT1 was mainly expressed in the panicles but not internodes, suggesting that DWT1 plays a long distance regulatory role in promoting internode elongation. Genome-wide expression analysis revealed that the expression of genes related to cell division and elongation, as well as to homeostasis and signaling of cytokinin and gibberellin were affected in dwt1 un-elongated internodes. This study reveals that a WOX transcription factor controls the growth uniformity of tillers and the main shoot in rice.
PMCID: PMC3952828  PMID: 24625559
2.  Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage 
BMC Plant Biology  2010;10:82.
Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.
For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.
The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.
PMCID: PMC3095354  PMID: 20444255
3.  Genome scale transcriptome analysis of shoot organogenesis in Populus 
BMC Plant Biology  2009;9:132.
Our aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species Populus, due to its genome sequence and amenability to in vitro manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.
We analyzed gene expression during poplar in vitro dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, Populus tremula × P. alba) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.
The major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.
PMCID: PMC2784466  PMID: 19919717
4.  Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation 
Annals of Botany  2007;100(4):699-709.
Background and Aims
The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm.
TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets.
Key Results
Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm.
The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.
PMCID: PMC2749624  PMID: 17670751
Bactris gasipaes; tissue culture; clonal propagation; Picloram
5.  A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro 
Plant Signaling & Behavior  2013;8(3):e23479.
Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.
PMCID: PMC3676518  PMID: 23333958
Arabidopsis; microRNA; shoot regeneration; calli; ARF10; miR160
6.  Transcript profiling of cytokinin action in Arabidopsis roots and shoots discovers largely similar but also organ-specific responses 
BMC Plant Biology  2012;12:112.
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.
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.
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.
PMCID: PMC3519560  PMID: 22824128
7.  Transcription of DWARF4 Plays a Crucial Role in Auxin-Regulated Root Elongation in Addition to Brassinosteroid Homeostasis in Arabidopsis thaliana 
PLoS ONE  2011;6(8):e23851.
The expression of DWARF4 (DWF4), which encodes a C-22 hydroxylase, is crucial for brassinosteroid (BR) biosynthesis and for the feedback control of endogenous BR levels. To advance our knowledge of BRs, we examined the effects of different plant hormones on DWF4 transcription in Arabidopsis thaliana. Semi-quantitative reverse-transcriptase PCR showed that the amount of the DWF4 mRNA precursor either decreased or increased, similarly with its mature form, in response to an exogenously applied bioactive BR, brassinolide (BL), and a BR biosynthesis inhibitor, brassinazole (Brz), respectively. The response to these chemicals in the levels of β-glucuronidase (GUS) mRNA and its enzymatic activity is similar to the response of native DWF4 mRNA in DWF4::GUS plants. Contrary to the effects of BL, exogenous auxin induced GUS activity, but this enhancement was suppressed by anti-auxins, such as α-(phenylethyl-2-one)-IAA and α-tert-butoxycarbonylaminohexyl-IAA, suggesting the involvement of SCFTIR1-mediated auxin signaling in auxin-induced DWF4 transcription. Auxin-enhanced GUS activity was observed exclusively in roots; it was the most prominent in the elongation zones of both primary and lateral roots. Furthermore, auxin-induced lateral root elongation was suppressed by both Brz application and the dwf4 mutation, and this suppression was rescued by BL, suggesting that BRs act positively on root elongation under the control of auxin. Altogether, our results indicate that DWF4 transcription plays a novel role in the BR-auxin crosstalk associated with root elongation, in addition to its role in BR homeostasis.
PMCID: PMC3166115  PMID: 21909364
8.  Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation 
Journal of Experimental Botany  2014;65(8):2219-2230.
The peptide RALF regulates cell expansion via an as-yet-unknown mechanism. Here, we provide evidence that AtRALF1 may be negatively regulating cell expansion by interfering with the brassinosteroid signalling pathway.
Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide’s mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF’s mechanism of action could be to interfere with the BR signalling pathway.
PMCID: PMC3991750  PMID: 24620000
Root development; brassinolide; peptide hormone.
9.  Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb. 
This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.
PMCID: PMC3550381  PMID: 23572914
In vitro propagation; Pyridone alkaloid; Cerpegin; Callus; Ceropegia juncea
10.  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.
PMCID: PMC3558495  PMID: 23382651
11.  BZR1 and BES1 participate in regulation of glucosinolate biosynthesis by brassinosteroids in Arabidopsis  
Journal of Experimental Botany  2013;64(8):2401-2412.
The effect of 24-epibrassinolide (EBR) on glucosinolate biosynthesis in Arabidopsis thaliana was investigated in the present study by using mutants and transgenic plants involved in brassinosteroid (BR) biosynthesis and signal transduction, as well as glucosinolate biosynthesis. The results showed that EBR significantly decreased the contents of major aliphatic glucosinolates including glucoiberin (S3), glucoraphanin (S4), and glucoerucin (T4), as well as the indolic glucosinolates glucobrassicin (IM) and neoglucobrassicin (1IM). In addition, a significantly higher level of glucosinolates accumulated in the BR-deficient mutant cpd and a dramatically lower glucosinolate content in the transgenic plant DWF4-ox overexpressing the BR biosynthetic gene DWF4 compared with their related wild-types, confirmed the repressing effect of BR on glucosinolate biosynthesis. BRI1, the receptor of BR signal transduction, was involved in regulation of glucosinolate biosynthesis by BR. Furthermore, the observation of reduced content of glucosinolates and lower expression levels of glucosinolate biosynthetic genes in 35S-BZR1/bzr1-1D and bes1-D plants compared with the corresponding wild-types suggested that BZR1 and BES1, two important components in BR signal transduction, are responsible for the inhibiting role of BR in glucosinolate biosynthesis. The disappearance of the repressing effect of BR on glucosinolate content in the myb28, myb34, and myb122 mutants indicated that these three MYB factors are important for the regulation of BR in glucosinolate biosynthesis.
PMCID: PMC3654425  PMID: 23580754
Arabidopsis thaliana; brassinosteroids; BZR1; BES1; glucosinolates; MYB.
12.  Development of a genotype independent and transformation amenable regeneration system from shoot apex in rice (Oryza sativa spp. indica) using TDZ 
3 Biotech  2012;2(3):233-240.
Agrobacterium-mediated transformation of indica rice has been established in only a limited number of cultivars because the regeneration of plants from transformed embryogenic calli is highly cultivar-specific. Establishment of a highly efficient plant regeneration system from shoot apex explants applicable to many cultivars of indica rice will accelerate the application of transformation technology in breeding programs and functional genomics study. We established an efficient shoot multiplication and plant regeneration system from shoot apices of indica rice using thidiazuron (TDZ) as a plant growth regulator. Shoot apices cultured on MS basal medium devoid of plant growth regulators formed solitary shoots in 90% of cultures. Addition of TDZ or benzylaminopurine to regeneration medium significantly influenced formation of multiple shoots directly from shoot apex explants without an intervening callus stage. Best shoot proliferation response (10.3 shoots per explant) was recorded when shoot apices were cultured on media supplemented with 4 mg/l TDZ. No synergistic effect on shoot proliferation was observed when indole-3-acetic acid and indole-3-butyric acid were supplemented to media containing 4 mg/l TDZ. The regeneration system was efficient in evoking multiple shoot proliferation in eight different cultivars of indica rice. Shoots were rooted in MS basal medium and plantlets were acclimatized with 100% survival rate. The shoot apex explants of all the eight cultivars of indica rice were found competent to Agrobacterium-mediated transformation while explants from IR-64 showed highest transient GUS expression. This variety-independent transformation amenable regeneration system from shoot apices may widely be applicable for genetic transformation of indica varieties.
PMCID: PMC3433876
Agrobacterium; Indica rice; Plant regeneration; Shoot apices; Thidiazuron
13.  Induced Expression of AtLEC1 and AtLEC2 Differentially Promotes Somatic Embryogenesis in Transgenic Tobacco Plants 
PLoS ONE  2013;8(8):e71714.
Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene’s induction of somatic embryogenesis.
PMCID: PMC3741171  PMID: 23951228
14.  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.
PMCID: PMC3158056  PMID: 21876682
15.  In vitro regeneration of wild chervil (Anthriscus sylvestris L.) 
Anthriscus sylvestris (L.) Hoffm. (Apiaceae) is a common wild plant that accumulates the lignan deoxypodophyllotoxin. Deoxypodophyllotoxin can be hydroxylated at the C-7 position in recombinant organisms yielding podophyllotoxin, which is used as a semi-synthetic precursor for the anticancer drugs, etoposide phosphate and teniposide. As in vitro regeneration of A. sylvestris has not yet been reported, development of a regeneration protocol for A. sylvestris would be useful as a micropropagation tool and for metabolic engineering of the plant. Calli were induced from hypocotyl explants and transferred to shoot induction medium containing zeatin riboside. Regenerated shoots were obtained within 6 mo and were transferred onto growth regulator-free root induction medium containing 1% sucrose. Regenerated plants transferred to soil and acclimatized in a greenhouse. Plants were transferred to the field with a 100% survival rate. Regenerated plants flowered and were fully fertile. This is the first report of complete regeneration of A. sylvestris via shoot organogenesis from callus.
PMCID: PMC3375414  PMID: 22719203
Organogenesis; Micropropagation; Regeneration; Tissue culture; Deoxypodophyllotoxin
16.  Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal 
Withania somnifera (L.) Dunal, is an important medicinal plant being the source of extremely important compounds like withanolides and withaferin. Influence of different plant growth regulators (PGRs) were evaluated for induction of callus, callus mediated regeneration and production of secondary metabolites in them. Explants for callusing were collected from plants grown in vitro and maximum callusing (98 %) was obtained on MS medium supplemented with a combination of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 mg l-1) and kinetin (KN) (0.2 mg l−1). Among different types of calli, best shoot regeneration was observed on green, compact calli produced on MS medium with a combination of 6-benzylamino purine (BAP) and indole butyric acid (IBA). MS medium supplemented with BAP (2 mg l−1) showed highest frequency (98 %) of shoot bud regeneration. The micro-shoots were efficiently rooted on MS media supplemented with 0.5 mg l−1 IBA. Rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium in greenhouse for acclimatization. Presence of withanolide A and withaferin A in calli was validated through high performance thin layer chromatography (HPTLC). It was interesting to observe that the PGRs showed significant influence on the secondary metabolites production in callus and 2,4-D having the least effect. Histological studies revealed the origin of shoot tip in the callus during regeneration.
PMCID: PMC3550684  PMID: 24381443
Callusing; High performance thin layer chromatography; Histology; Plant growth regulator; Shoot bud regeneration; Withaferin A; Withanolide A
17.  Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration 
At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.
PMCID: PMC4009429  PMID: 24808900
shoot regeneration; rRNA biosynthesis; WD40 repeat protein; CUC1; RID2; RID3
18.  Early Events in Agrobacterium‐mediated Genetic Transformation of Citrus Explants 
Annals of Botany  2004;94(1):67-74.
• Background and Aims Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so‐called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co‐cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co‐cultivation medium as possible enhancers of gene transfer was also studied.
• Methods A highly responsive citrus genotype and well‐established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co‐cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry.
• Key Results It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium‐inoculated and non‐inoculated explants. Furthermore, co‐ cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S‐phase, the stage in which cells are more prone to integrate foreign DNA.
• Conclusions Use of proper co‐cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.
PMCID: PMC4242373  PMID: 15145796
Agrobacterium tumefaciens; callus; cambium; cell cycle; citrange (Citrus sinensis × Poncirus trifoliata); citrus; competence; genetic transformation; sweet orange (C. sinensis); regeneration; S‐phase; transgenic
19.  Reprogramming of H3K27me3 Is Critical for Acquisition of Pluripotency from Cultured Arabidopsis Tissues 
PLoS Genetics  2012;8(8):e1002911.
In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn) and embryonic flower2 (emf2) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP–chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.
Author Summary
Callus formation is a necessary step in regenerating a new plant from detached plant tissues, and the nature of the callus is similar to that of the root meristem. In this study, we intended to address the molecular basis that directs different plant tissues to form the root-meristem-like callus. We found that leaves and cotyledons, but not roots, of PRC2 mutants curly leaf-50 swinger-1 and embryonic flower2 lost the ability to form a callus. Using ChIP–chip analysis, we identified genes that were changed markedly in the histone H3 lysine 27 trimethylation (H3K27me3) levels during callus formation from leaf blades. Among these genes, a number of leaf-regulatory genes were repressed through PRC2-mediated H3K27me3. Conversely, certain auxin pathway genes and many root-regulatory genes were derepressed through H3K27 demethylation. Our data indicate that genome-wide H3K27me3 reprogramming, through the PRC2-mediated H3K27me3 and the H3K27 demethylation pathways, is critical in directing cell fate transition.
PMCID: PMC3426549  PMID: 22927830
20.  Comparative Genomic Analysis of Transgenic Poplar Dwarf Mutant Reveals Numerous Differentially Expressed Genes Involved in Energy Flow 
In our previous research, the Tamarix androssowii LEA gene (Tamarix androssowii late embryogenesis abundant protein Mrna, GenBank ID: DQ663481) was transferred into Populus simonii × Populus nigra. Among the eleven transgenic lines, one exhibited a dwarf phenotype compared to the wild type and other transgenic lines, named dwf1. To uncover the mechanisms underlying this phenotype, digital gene expression libraries were produced from dwf1, wild-type, and other normal transgenic lines, XL-5 and XL-6. Gene expression profile analysis indicated that dwf1 had a unique gene expression pattern in comparison to the other two transgenic lines. Finally, a total of 1246 dwf1-unique differentially expressed genes were identified. These genes were further subjected to gene ontology and pathway analysis. Results indicated that photosynthesis and carbohydrate metabolism related genes were significantly affected. In addition, many transcription factors genes were also differentially expressed in dwf1. These various differentially expressed genes may be critical for dwarf mutant formation; thus, the findings presented here might provide insight for our understanding of the mechanisms of tree growth and development.
PMCID: PMC4200758  PMID: 25192286
comparative genomic analysis; dwarf; photosynthesis; poplar
21.  A Fasciclin-Like Arabinogalactan-Protein (FLA) Mutant of Arabidopsis thaliana, fla1, Shows Defects in Shoot Regeneration 
PLoS ONE  2011;6(9):e25154.
The fasciclin-like arabinogalactan-proteins (FLAs) are an enigmatic class of 21 members within the larger family of arabinogalactan-proteins (AGPs) in Arabidopsis thaliana. Located at the cell surface, in the cell wall/plasma membrane, they are implicated in many developmental roles yet their function remains largely undefined. Fasciclin (FAS) domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms, but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants. Recent studies have started to elucidate the role of FLAs in Arabidopsis development. FLAs containing a single FAS domain are important for the integrity and elasticity of the plant cell wall matrix (FLA11 and FLA12) and FLA3 is involved in microspore development. FLA4/SOS5 with two FAS domains and two AGP domains has a role in maintaining proper cell expansion under salt stressed conditions. The role of other FLAs remains to be uncovered.
Method/Principal Findings
Here we describe the characterisation of a T-DNA insertion mutant in the FLA1 gene (At5g55730). Under standard growth conditions fla1-1 mutants have no obvious phenotype. Based on gene expression studies, a putative role for FLA1 in callus induction was investigated and revealed that fla1-1 has a reduced ability to regenerate shoots in an in vitro shoot-induction assay. Analysis of FLA1p:GUS reporter lines show that FLA1 is expressed in several tissues including stomata, trichomes, the vasculature of leaves, the primary root tip and in lateral roots near the junction of the primary root.
The results of the developmental expression of FLA1 and characterisation of the fla1 mutant support a role for FLA1 in the early events of lateral root development and shoot development in tissue culture, prior to cell-type specification.
PMCID: PMC3178619  PMID: 21966441
22.  Adventitious Shoot Regeneration from Leaf Explant of Dwarf Hygro (Hygrophila polysperma (Roxb.) T. Anderson) 
The Scientific World Journal  2013;2013:680425.
Dwarf hygro (Hygrophila polysperma) is an ornamental aquatic plant that changes its leaf colours to pinkish in high light. It is listed as a medicinal plant in medicinal plant lists of Indian states of West Bengal and Karnataka. It is also used as a screening tool for toxicities and a bioindicator to detect and control algae. The study reported in vitro adventitious shoot regeneration from leaf explants cultured on MS medium containing 0.10–1.60 mg/L Kin/TDZ with or without 0.10 mg/L IBA and 500 mg/L Amoklavin to eradicate endogenic bacterial contamination. Direct adventitious shoot regeneration started within one week from both culture mediums followed by late callus induction which was more prominent on TDZ containing media compared to Kin containing media. Addition of 0.10 mg/L IBA with both Kin and TDZ increased shoot regeneration frequency, mean number of shoots per explant, and mean shoot length. Maximum number of 16.33 and 20.55 shoots per explant was obtained on MS medium containing 0.80 + 0.10 mg/L Kin-IBA and 0.10 + 0.10 mg/L TDZ-IBA, respectively. Regenerated shoots were rooted on MS medium containing 0.20–1.00 mg/L IBA followed by successfull acclimatization in aquariums. Regenerated plantlets were also tested in jars containing distilled water that showed the pH 6–9 for the best plant growth and development.
PMCID: PMC3703325  PMID: 23853539
23.  Brassinosteroids control root epidermal cell fate via direct regulation of a MYB-bHLH-WD40 complex by GSK3-like kinases 
eLife  2014;3:e02525.
In Arabidopsis, root hair and non-hair cell fates are determined by a MYB-bHLH-WD40 transcriptional complex and are regulated by many internal and environmental cues. Brassinosteroids play important roles in regulating root hair specification by unknown mechanisms. Here, we systematically examined root hair phenotypes in brassinosteroid-related mutants, and found that brassinosteroid signaling inhibits root hair formation through GSK3-like kinases or upstream components. We found that with enhanced brassinosteroid signaling, GL2, a cell fate marker for non-hair cells, is ectopically expressed in hair cells, while its expression in non-hair cells is suppressed when brassinosteroid signaling is reduced. Genetic analysis demonstrated that brassinosteroid-regulated root epidermal cell patterning is dependent on the WER-GL3/EGL3-TTG1 transcriptional complex. One of the GSK3-like kinases, BIN2, interacted with and phosphorylated EGL3, and EGL3s mutated at phosphorylation sites were retained in hair cell nuclei. BIN2 phosphorylated TTG1 to inhibit the activity of the WER-GL3/EGL3-TTG1 complex. Thus, our study provides insights into the mechanism of brassinosteroid regulation of root hair patterning.
eLife digest
Roots anchor a plant into the ground, and allow the plant to absorb water and mineral nutrients from the soil. As roots grow and branch, they increase the surface area of root exposed to the soil—and many plant cells in the root's outer layer have a hair-like projection to further increase this surface area. Thus, root hairs are where most water and mineral nutrients are absorbed. Many factors affect whether, or not, a plant cell will develop into a root hair. These factors include both external cues (such as the mineral content of the soil) and signals from the plant itself (such as hormones).
Brassinosteroids are plant hormones that regulate the development of shoots and roots, as well as the timing of when flowers begin to develop. These hormones are detected on the outside of plant cells, and activate a signaling pathway within the cell that causes changes in gene expression. Brassinosteroids also control if a root cell will become a hair cell or not, although the mechanism behind this activity is unclear.
Here, Cheng et al. have looked at the root hairs of mutant Arabidopsis thaliana plants that have had individual genes involved in brassinosteroid signaling knocked-out. Plant biologists commonly study this plant species because it is small and grows quickly—and Arabidopsis has regular stripes of root hair cells and ‘non-hair cells’ in the outer layer of its roots. Cheng et al. reveal that brassinosteroids prevent the formation of root hairs via signaling pathways that involve proteins called GSK3-like kinases. These hormones ‘switch off’ these kinases’ activity, so knocking-out the genes that code for these kinases has the same effect as adding extra brassinosteroids to the plant roots: fewer root hair cells.
Cheng et al. show that one of the GSK3-like kinases binds and adds phosphate groups to protein complexes that control gene expression—and this causes these protein complexes to be less active. When GSK3-like kinase activity is switched off by brassinosteroids, these complexes instead become more active and trigger the expression of genes that direct a plant cell to become a non-hair cell.
The findings of Cheng et al. reveal the pathways that allow brassinosteroids to stop plant cells in roots from becoming hair cells, and that instead encourage these cells to become non-hair cells. However, further work is needed to uncover how the striped pattern of hair cells and non-hair cells on Arabidopsis roots is established, and how brassinosteroids work with other plant hormones to control this pattern.
PMCID: PMC4005458  PMID: 24771765
brassinosteroids; GSK3-like kinases; root epidermal cell fate; EGL3; phosphorylation; TTG1; Arabidopsis
24.  Genome-Wide Analysis of a TaLEA-Introduced Transgenic Populus simonii × Populus nigra Dwarf Mutant 
A dwarf mutant (dwf1) was obtained among 15 transgenic lines, when TaLEA (Tamarix androssowii late embryogenesis abundant gene) was introduced into Populus simonii × Populus nigra by Agrobacterium tumefaciens-mediated transformation. Under the same growth conditions, dwf1 height was significantly reduced compared with the wild type and the other transgenic lines. Because only one transgenic line (dwf1) displayed the dwarf phenotype, we considered that T-DNA insertion sites may play a role in the mutant formation. The mechanisms underlying this effect were investigated using TAIL-PCR (thermal asymmetric interlaced PCR) and microarrays methods. According to the TAIL-PCR results, two flanking sequences located on chromosome IV and VIII respectively, were cloned. The results indicated the integration of two independent T-DNA copies. We searched for the potential genes near to the T-DNA insertions. The nearest gene was a putative poplar AP2 transcription factor (GI: 224073210). Expression analysis showed that AP2 was up-regulated in dwf1 compared with the wild type and the other transgenic lines. According to the microarrays results, a total of 537 genes involved in hydrolase, kinase and transcription factor activities, as well as protein and nucleotide binding, showed significant alterations in gene expression. These genes were expressed in more than 60 metabolic pathways, including starch, sucrose, galactose and glycerolipid metabolism and phenylpropanoids and flavonoid biosyntheses. Our transcriptome and T-DNA insertion sites analyses might provide some useful insights into the dwarf mutant formation.
PMCID: PMC3317382  PMID: 22489122
dwarf; mutant; poplar; microarrays; AP2; RAV
25.  Consequences of induced brassinosteroid deficiency in Arabidopsis leaves 
BMC Plant Biology  2014;14(1):309.
The identification of brassinosteroid (BR) deficient and BR insensitive mutants provided conclusive evidence that BR is a potent growth-promoting phytohormone. Arabidopsis mutants are characterized by a compact rosette structure, decreased plant height and reduced root system, delayed development, and reduced fertility. Cell expansion, cell division, and multiple developmental processes depend on BR. The molecular and physiological basis of BR action is diverse. The BR signalling pathway controls the activity of transcription factors, and numerous BR responsive genes have been identified. The analysis of dwarf mutants, however, may to some extent reveal phenotypic changes that are an effect of the altered morphology and physiology. This restriction holds particularly true for the analysis of established organs such as rosette leaves.
In this study, the mode of BR action was analysed in established leaves by means of two approaches. First, an inhibitor of BR biosynthesis (brassinazole) was applied to 21-day-old wild-type plants. Secondly, BR complementation of BR deficient plants, namely CPD (constitutive photomorphogenic dwarf)-antisense and cbb1 (cabbage1) mutant plants was stopped after 21 days. BR action in established leaves is associated with stimulated cell expansion, an increase in leaf index, starch accumulation, enhanced CO2 release by the tricarboxylic acid cycle, and increased biomass production. Cell number and protein content were barely affected.
Previous analysis of BR promoted growth focused on genomic effects. However, the link between growth and changes in gene expression patterns barely provided clues to the physiological and metabolic basis of growth. Our study analysed comprehensive metabolic data sets of leaves with altered BR levels. The data suggest that BR promoted growth may depend on the increased provision and use of carbohydrates and energy. BR may stimulate both anabolic and catabolic pathways.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0309-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4240805  PMID: 25403461
Brassinosteroids; Arabidopsis; Tricarboxylic acid cycle; Biomass; Cell expansion; Growth

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