Taken together, our findings provide insight into the mechanisms underlying sweetpotato storage-root formation and provide a valuable database of genes for further research. Int. Phloroglucinol-HCl staining was done according to an established protocol by Mitra and Loque (2014), where deparaffinized samples were stained with a freshly prepared Ph-HCl stain and observed immediately under the microscope (Ph-HCl causes sample deterioration). For transportation or brief storage, place the leaves between sheets of wet toweling paper and keep in a closed container such as a tin can or a Mason jar. Paclobutrazol application, as opposed to GA3, did not induce fiber formation (Figure 5C). In Aspen, it was suggested that GA has a role in regulating early stages of xylem differentiation during wood formation (Israelsson et al., 2005). Minimum 16 cuttings were planted for each treatment group and each sampling time. Botanical microtechnique. Quantitative measurement of starch in very small amounts of leaf tissue. By clicking OK, you are confirming that this image is only to be used for the rights in the existing license. Transcription switches for protoxylem and metaxylem vessel formation. PloS One 9, e105726. (1950). Plant Cell. – 22 semaines de formation en entreprise. Lignin biosynthesis (being the linking of monolignol units) depends on the monolignol biosynthesis pathway, starting with deamination of phenylalanine by phenylalanine ammonia-lyase (PAL; the main enzyme of the phenylpropanoid pathway) (Boerjan et al., 2003). BTS CIM Conception et Industrialisation en Microtechniques (2017). Several KNOX gene sequences were previously identified by us to be upregulated in initiating sweetpotato SRs (Firon et al., 2013), including two Arabidopsis BREVIPEDICELLUS (BP) orthologues. Botanical microtechnique. Exogenous application of PB, caused the opposite effect, reducing the levels of all tested bioactive GAs by more than 2-fold. 55, 652–664. Exogenous gibberellin altered morphology, anatomic and transcriptional regulatory networks of hormones in carrot root and shoot. Plant Physiol. The expression of all tested positive regulators (Figure 9A) was induced by GA3 treatment as evident in ARs sampled at 5W after planting, while expression of IbNAC075 was upregulated earlier, already at 2W (Figure 9A). Cross sections of SR collected at 5W of development, representing six independent biological replicates (plants), are given in (B) as viewed under a confocal microscope focusing on starch granules. Lignin biosynthesis starts with deamination of phenylalanine by PAL, followed by a series of reactions involving C4H, 4CL, HCT, CCoAOMT, and CAD (Kao et al., 2002; Raes et al., 2003). The experiment was conducted during April 2017. Significance analysis was performed by using student’s t-test (P ≤ 0.05), where unlike letters represent significant differences between treatments within a sampling group. Compounds were eluted with 0.2 M formic acid in acetonitrile and the eluate was dried in vacuo. By clicking OK, you are confirming that this image is only to be used for the rights in the existing license. Les inscriptions se font via la plateforme eCandidat, Xavier WALLERANDResponsable pédagogiquexavier.wallerand@univ-fcomte.fr, Nadia MESSAOUDISecrétariatnadia.messaoudi@univ-fcomte.fr03 81 66 68 04, Contact : Scolarité de l'IUT de Besançon-Vesoul. In accordance with increased root xylem development and lignification levels, GA3-treated plants exhibited induced transcript levels of the sweetpotato orthologues of Arabidopsis NAC075 and VND7 (IbNAC075 and IbVND7) genes, a corresponding increase in expression levels of IbSND2, as well as downregulation of IbVNI2 and IbXND1. Janick, J. GAs were separated isocratically at 80% of 10 mM formic acid/water (A, v/v) and 20% of methanol (B) at a flow rate of 0.35 mL/min for 1 min, followed by consecutive linear gradient to 70 and 90% of B over 9 min. Lateral stem length was recorded at three, four, and five weeks (3W, 4W, and 5W, respectively) after planting. doi: 210.1093/pcp/pcu1134, Eriksson, M. E., Israelsson, M., Olsson, O., Moritz, T. (2000). This cascade leads to upregulation of lignin biosynthesis genes, causing high lignification of the AR and development of a non-SR. Il conçoit tout ou partie d'un produit microtechnique ainsi que les outillages de validation de processus. Contribution of gibberellins to the formation of Arabidopsis seed coat through starch degradation. Pro-texx (RI = 1.495) Technicon Resin (RI = 1.62) Uv-inert (RI = 1.517) XAM (RI = 1.52). 66, 1113–1121. Plants were irrigated with 100 mL of either water (half field capacity; “control” treatment), an aqueous solution of 50 ppm GA3, or 5 ppm PB (“GA” and “PB” treatments, respectively) on every third day, until two weeks (2W) after planting. Stat. Gibberellin is known to regulate diverse plant developmental processes throughout the life cycle, like stem elongation and seed germination (Gupta and Chakrabarty, 2013). Altered phenylpropanoid metabolism in the maize LcExpressed sweet potato (Ipomoea batatas) affects storage root development. Solidworks, c'est un logiciel de FAO. Data were processed using MassLynxTM software with TargetLynxTM program (version 4.1, Waters, USA). (2002). Pre-pay for multiple images and download on demand. Ann. Control (treated with water); GA3, application of 50 ppm gibberellic acid 3 for two weeks; PB, application of 5 ppm paclobutrazol for two weeks. Nat. 156, 391–400. doi: 210.1104/pp.1103.036988, Boerjan, W., Ralph, J., Baucher, M. (2003). Poursuite d'études possible, notamment en licence pro (pour 1 an). At 5W, those ARs that did not develop into SRs exhibited reduced number of protoxylem and metaxylem vessels and elevated number of secondary xylem vessels, following GA3 treatment as compared to control (Figure 5B). Behav. Exogenous application of GA3 caused a significant reduction in most tested RSA parameters, measured during the first two weeks after planting (SR initiation phase). Protoplasma. Au départ, on part du dessin sur Solidworks. Botany -- Anatomy; Botany -- Morphology; Microscopy -- Technique; Microscopes -- Technique; Botany; Plants -- anatomy & histology; Microscopy. A study in the tuberous-root formation of sweet potato. Furthermore, GA3 treatment caused significant downregulation of carbohydrate metabolism and starch biosynthesis genes including genes encoding the small and large subunits (IbAGPa1 and IbAGPb1A) of the regulatory step in starch biosynthesis, conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate by AGPase (Ballicora et al., 2004). (2017). doi: 810.1007/s00709-00016-00995-00706, Wang, H., Yang, J., Zhang, M., Fan, W., Firon, N., Pattanaik, S., et al. In order to follow the effect of gibberellin on lignification, we have used auto-fluorescence imaging (being a nonspecific tool indicative of lignin and various phenolics (Donaldson and Knox, 2012) as well as phloroglucinol staining (indicative of the 4-O-linked hydroxycinnamyl aldehyde structures in lignins) (Pomar et al., 2002) and results are presented in Figure 6. Contact : scoliut25@univ-fcomte.fr ou 03.81.66.68.08 / 68.21 / 68.22. The knotted1-like homeobox gene BREVIPEDICELLUS regulates cell differentiation by modulating metabolic pathways. 9, 3884. doi: 3810.1038/s41467-41018-06430-41468, Taylor-Teeples, M., Lin, L., de Lucas, M., Turco, G., Toal, T. W., Gaudinier, A., et al. The experimental setup and conditions used are detailed in Supplementary Figure S1. , is a colorless, neutral medium in which most standard stains are well preserved. Plant Biol. PloS One 9, e107384. doi: 10.1007/s00709-00018-01341-00703. CTRL, Control (treated with water); GA3, application of 50 ppm gibberellic acid 3 for two weeks; PB, application of 5 ppm paclobutrazol for two weeks. The effect of different levels of KNOX activity should be further studied in a variety of systems in order to better understand their function. Since starch accumulation is an important indication for formation of a SR, we investigated starch content at 2W (in ARs) and 5W (in ARs and SRs) after planting in nontreated and treated (GA3- and PB-treated) plant roots (Figure 7A). The effluent was introduced into electrospray ion source of Xevo® TQ-S triple quadrupole mass spectrometer (Waters MS Technologies, Manchester, UK), having the capillary voltage of 3 kV. Curr. Representative cross-sections of adventitious roots sampled at two and five weeks (2W and 5W, respectively) after planting. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. (2005). The GA signaling pathway starts by binding of GA to its receptor, GA Insensitive Dwarf1 (GID1), which enhances an interaction between GID1 and DELLA that causes DELLA degradation and de-repression of the pathway (Sun, 2011). : JoVE. Interestingly, gibberellin downregulated root expression levels of orthologues of the Arabidopsis BREVIPEDICELLUS transcription factor (IbKN2 and IbKN3), regulator of meristem maintenance. The remained pellet was re-extracted with 1 mL of the extraction solvent without adding internal standards, sonicated, rotated for 1 h at 4°C, and centrifuged for 10 min at 17,000 rpm (at 4°C). The NAC domain repressor, VND-INTERACTING 2 (VNI2) negatively regulates xylem vessel formation/differentiation and represses VND7-induced expression of vessel-specific genes (Yamaguchi et al., 2010). We have previously suggested that major events involved in SR initiation are the reduction of carbon flow towards phenylpropanoid synthesis and its delivery into carbohydrate metabolism and starch biosynthesis (Firon et al., 2013). Adventitious root primordia formation and development in stem nodes of ‘Georgia Jet’ sweetpotato, Ipomoea batatas. Là, je recrée une pièce. 135, 254–265. Rev. – Technicien en optique de précision. In the system of carrot SR formation, it was recently found that exogenous GA enhanced lignin accumulation and induced the transcript levels of lignin biosynthesis genes, including PAL, 4CL, CCoAOMT, and CAD (Wang et al., 2017). En fin de semestre 4, il passent 12 semaines en entreprise. Conditions d'admission, le contenu de la formation, les matières enseignées et les débouchés après la formation bac pro Microtechniques A., Preiss, J. Significance analysis was performed by using student’s t-test (P ≤ 0.05), where unlike letters represent significant differences between treatments within a group. Il choisit et optimise les procédés et processus mis en Suvre, analyse, synthétise et corrige. While, 2.3- and 4.9- as well as 3.0- and 5.5-fold reduction in LR number and LR cumulative length, was detected following GA3 and PB treatments, respectively (Figures 2C, D). BMC Plant Biol. It was further suggested that in ARs with a prevalence of arrested/non-emerged LR primordia, the adjacent stellar tissue becomes lignified, rendering it incapable of undergoing swelling due to the lack of vascular and anomalous cambia development (Villordon and Clark, 2014). Enhancing plant growth and fiber production by silencing GA 2-oxidase. This is followed by a series of reactions, involving the following enzymes: cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p-hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT), and cinnamyl alcohol dehydrogenase (CAD) (Raes et al., 2003). In accordance with this hypothesis, GA3 application caused a significant reduction in transcript levels of both IbKN2 and IbKN3 genes. VS and NF conceived and designed the experiments, analyzed the data, and wrote the manuscript. (A) Representative cross-sections of adventitious roots (AR) sampled at one, two, and five weeks (1W, 2W, and 5W, respectively) after planting. Collège et Lycée LES PERSEVERANTS - 97100 BASSE TERRE - 2nde à Terminales STMG - Bac Pro Commerce - Secrétariat Lycée Professionnel BOC CALMET - 97110 POINTE A PITRE - Bac Pro et BTS Tertiaires Lycée Technique et Professionnel BEL AIR - 97122 BAIE-MAHAULT - Tertiaire - Bac Pro - Bac STMG - BTS - Licence Pro. For following the effect of GA3 and PB treatments on plant development, lateral stem length (originated from node 10) was measured at 3 weeks (3W), 4W, and 5W after planting, using 16 plants (biological replicates) (Supplementary Figure S1). The mechanisms underlying this developmental switch are still unclear. Before loading samples, the column was activated with 100% MeOH, washed with mQ water, and equilibrated with 5% of NH4OH. Column was thermostated at 50°C. (2014) stressed the importance of root architecture in root (sweetpotato, cassava) and tuber (potato) crop productivity. Autofluorescence was detected under UV excitation at 365 nm with a PMT detector in range of 415–490 nm. The negative regulator of GA, IbGAI, showed variable responses to GA3 and PB, being opposite in 5W ARs (reduced expression by GA3 and upregulation by PB). Microscopic examination of sections was done by a light microscope (Leica, Germany) and images were digitally captured by a Nikon DS-Fi1 digital camera. Figure 1 Effect of GA3 application on sweetpotato “Georgia Jet” stem growth. Tubes were allowed to cool down at room temperature and measurements were done at 550 nm in a spectrophotometer (Pharmacia Biotech, USA). Le titulaire de cette licence professionnelle évolue dans un contexte d'ingéniérie collaboratrice. A model is suggested (Figure 13), serving as a basis for further studies. Pro. J. Biotechnol. No use, distribution or reproduction is permitted which does not comply with these terms. doi: 10.1371/journal.pone.0051502, Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., et al. Agric. The expression pattern of the following GA-related genes was followed during root development at 1W, 2W, and 5W: GA biosynthesis genes, ent-kaurene oxidase (IbKO), GA20-oxidase (IbGA20ox), and GA3-oxidase4 (IbGA3ox4) (Figure 8A); GA catabolizing genes, GA2-oxidase (IbGA2ox1, IbGA2ox3, and IbGA2ox8) (Figure 8B), and genes involved in GA signaling and regulation, GIBBERELLIN INSENSITIVE DWARF1 (IbGID1) and GIBBERELLIC ACID INSENSITIVE (IbGAI) (Figure 8C). Histochemical staining was done by either, safranin-fast green or Phloroglucinol-HCl (Ph-HCl or Weisner) staining to observe the root vascular system and lignin deposition (phloroglucinol is a good indicator of general cinnamaldehydes), respectively. The camera must be slid vertically and the post swung back to permit use of the ocular. NIH Image to ImageJ: 25 years of image analysis. doi: 22910.23390/ijms160922960, Gupta, R., Chakrabarty, S. K. (2013). qRT-PCR data are mean (± SE) of three independent biological replicates (each representing roots pooled from four plants). cultivars. doi: 710.1104/pp.006262, Kim, Y. C., Nakajima, M., Nakayama, A., Yamaguchi, I. Cette demande doit être formulée, en principe, dans les quinze jours qui suivent le début des cours. Sweetpotato yield depends on a change in the developmental fate of adventitious roots into storage-roots. Jap. Hellmann, E., Ko, D., Ruonala, R., Helariutta, Y. Res. In SRs, cambial cells proliferation occurs, forming starch-accumulating parenchyma cells in the root vascular cylinder (Villordon et al., 2009). KNOX gene function in plant stem cell niches. Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. Bars (B, C) represent mean of six to eight independent biological replicates (plants) ± SE. Thereafter, samples were reconstituted in 60 µL of initial mobile phase 10 mM formic acid: MeOH (80: 20, v/v), filtered through micro spin filters 0.2 µm (Thermo Fisher Scientific, USA) and injected onto reversed-phase UPLC CSH C18 column (100 x 2.1 mm, 1.7 µm) of Acquity UPLCTM I-Class Core System (Waters, USA). La licence professionnelle s'adresse à des titulaires d'un bac+2 qui souhaitent acquérir un niveau de qualification supérieur ou une spécialisation dans leur domaine. You cannot download or purchase for any new licenses. The inhibitory effect of GA3 on expression levels of all tested genes (except IbPGM) was evident also in SRs. In cassava roots, similar to sweetpotato, the root system consists mainly of two root types, fibrous/lignified roots that transport water and nutrients, and SRs that accumulate starch. In addition, it is concluded that the response of sweetpotato roots to PB is complex, being opposite to GA3 in some parameters (fiber formation, gene expression of IbGA20ox1, IbGA3ox4, IbGA2ox1, IbGAI, IbNAC075, IbXND1, and IbHCT) and similar to that of GA3, but attenuated, in other factors (lignification, SR number). Expression/transcript levels were measured at 1W, 2W, and 5W after planting for AR and at 5W for SR, using qRT-PCR analyses, and values were normalized relative to the expression levels of phospholipase D1a. 60, 929–946. (2014). Genes Dev. In the present study, in order to better characterize the complex events that take place during the change of the sweetpotato AR into a SR, we looked into the effect of exogenous application of GA3 on root development and root lignin accumulation, together with vascular system- and xylem-development, starch accumulation, transcript levels of genes that regulate all these processes as well as the number of SRs formed.