Significance analysis was performed by using student’s t-test (P ≤ 0.05), where unlike letters represent significant differences between treatments within a group. For root anatomy, samples were fixed in FAA solution (1 L) containing; 100 mL of 35% formaldehyde, 50 mL of glacial acetic acid, 520 mL of 96% ethanol, and 330 mL of dH2O. The average temperature and relative humidity at this site during the study (13–24 September 2008) were 17.6 ± 4.3 °C and 86.1 ± 16.0 %, as measured hourly in the subcanopy (mean of 2 HOBO Pro … LS and WL performed gibberellin quantification. Il conçoit tout ou partie d'un produit microtechnique ainsi que les outillages de validation de processus. Rev. Harnessing potato and sweetpotatos power for food security, nutrition and climate resilience. GA3 treatment was found to promote secondary xylem formation, as evident in 2W and 5W cross-sections of ARs (Figure 5B). doi: 2210.1105/tpc.2104.024190, Scofield, S., Murray, J. To look for genes involved in the regulation of root xylem development and vascular lignification following GA3 application, we identified the following sweetpotato orthologues of the respective Arabidopsis genes: IbNAC075 (upstream regulator of VND7) (Endo et al., 2015), IbVND7 (involved in xylem vessel differentiation) (Yamaguchi et al., 2008), IbSND2 (regulator of genes involved in secondary wall development) (Hussey et al., 2011), IbXND1 (negative regulator of xylem formation) (Tang et al., 2018), IbVNI2 (transcriptional repressor of VND7) (Yamaguchi et al., 2010), and IbVNI2-like (Table 1) and their transcript profiles were investigated (Figure 9). 8, e25504. 11, 3194–3205. Plant Vascular Tissues-connecting tissue comes in all shapes. doi: 510.1146/annurev.arplant.1154.031902.134938, Bolduc, N., Hake, S. (2009). 4, 1028–1034. Sections were stained with safranin and fast green, and represent six to eight roots sampled from individual plants. For example, 1.9- and 2.9- as well as 1.7- and 2.5-fold reduction in AR number and length was observed at 2W, with GA3 and PB treatments, respectively (Figures 2A, B). Paclobutrazol application, opposed to GA3, caused significant down-regulation of IbNAC075 transcript levels. In addition, GA3 application caused down-regulation in expression of IbGBSS and IbSP (2.1- and 2.6-fold, respectively, in 5W ARs). [PhD thesis]. 55, 652–664. Paclobutrazol treatment did not cause a significant change in expression in either of the tested carbohydrate metabolism genes at 2W of development, while expression of IbSuSy, IbAGPa1 and IbAGPb1A was reduced when tested in 5W ARs (Figure 12). Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones. A. At this early phase of root development most xylem vessels were primary xylem (protoxylem and metaxylem) and a significant increase in protoxylem and metaxylem number at 1W, by both GA3 and PB treatments, was observed (Figure 5B). doi: 510.1016/S1360-1385(1000)01790-01798. A., Preiss, J. Protoplasma. Another link was demonstrated between the BP gene and GA, showing that BP may negatively regulate GA (Bolduc and Hake, 2009). The list of all sweetpotato contigs together with the respective Arabidopsis genes is presented in Table 1. It should be noted that Liebsch et al. Pro-texx (RI = 1.495) Technicon Resin (RI = 1.62) Uv-inert (RI = 1.517) XAM (RI = 1.52). Lire la transcription. Elevated gibberellin enhances lignin accumulation in celery (Apium graveolens L.) leaves. – 84 semaines en établissement scolaire / 2 semaines d’examens Total xylem vessel number was calculated by counting the number of protoxylem, metaxylem and secondary xylem elements in root sections. : Personnalisation de la pendulette aux couleurs du Crédit Agricole, Notre participation au concours « je filme le métier qui me plaît », Les activités de l’Association Sportive du lycée Diderot. In tobacco, overexpression of a KNOTTED-type protein caused decreased expression of a GA biosynthesis gene (Tanaka-Ueguchi et al., 1998). Genes Dev. doi: 10.1016/j.tplants.2014.02.002. Expression of IbSuSy and AGPb1A was down-regulated 2.2- and 4.0-fold, respectively, as early as 2W, and 8.7- and 24.7-fold, respectively, in 5W ARs. 10:1320. doi: 10.3389/fpls.2019.01320. Colour-coded images were analysed with Image-Pro Plus version 2.0.0.260 (Media Cybernetics, Inc., USA; Fig. This gene expression pattern parallels the elevated lignification observed in tissue sections (Figure 6). Exogenous gibberellin enhances secondary xylem development and lignification in carrot taproot. The mechanisms underlying this developmental switch are still unclear. Similar findings were reported recently in carrot taproot, demonstrating increased lignification following GA3 treatment (Wang et al., 2017). The inhibitory effect of GA3 on expression levels of all tested genes (except IbPGM) was evident also in SRs. Bot. All microscopic observations and image acquisitions were performed by a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany), equipped with a solid-state laser with 405 nm light, HC PL APO CS 10x/0.40 objective (Leica, Wetzlar, Germany), and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). Such an interaction between GA and KNOX may affect the balance between lignification, on one hand, and meristem development, on the other hand, and may be highly relevant to the issue of SR formation. J. Bot. |, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi, https://www.frontiersin.org/articles/10.3389/fpls.2019.01320/full#supplementary-material, http://cipotato.org/annualreport2017/wp-content/uploads/sites/7/2018/09/ANNUAL-REPORT-2017.pdf, http://dx.doi.org/110.1016/j.plaphy.2016.1006.1005, Creative Commons Attribution License (CC BY). Thus, GA3 and PB showed antagonistic effects causing elongated or stunted stem growth, respectively, throughout sweetpotato plant development. A. H. (2006). (2010). The following six sweetpotato genes, known to be involved in carbohydrate metabolism and starch biosynthesis were studied for their expression behavior following GA3 and PB treatments: Sucrose synthase (IbSuSy), phosphoglucomutase (IbPGM), ADP-glucose pyrophosphorylase small and large subunits (IbAGPa1 and IbAGPb1A, respectively), granule-bound starch synthase (IbGBSS), and starch phosphorylase (IbSP) (Figure 12). Roots were thus harvested at the phase of SR initiation (1W and 2W) and at the time when SRs were already formed (5W) and analyzed for AR anatomical structure and xylem development. BMC Plant Biol. (2014) stressed the importance of root architecture in root (sweetpotato, cassava) and tuber (potato) crop productivity. Compounds were quantified by multiple ion monitoring mode (MRM) as described (Kolachevskaya et al., 2017). Ed. Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. doi: 10.4161/psb.4.11.9875, Zhong, R., Demura, T., Ye, Z. H. (2006). Root system architecture parameters, including root system FW, DW, volume, and surface area, were further recorded at 5W after planting, when SR formation was already detected. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Root architecture and root and tuber crop productivity. 46, 1317–1325. The work was based upon previous findings from our laboratory (in collaboration with the laboratories of A. Villordon and D. LaBonte) demonstrating that (i) the initial ARs that develop from stem-cutting possess the necessary requirements for forming SRs (Villordon et al., 2009); (ii) SR initiation phase spans the first two weeks after planting (Villordon et al., 2009). . The expression of the negative regulators, IbXND1 and IbVNI2, was, however, downregulated by GA3 (5.2- and 1.7-fold, respectively, in 5W ARs) (Figure 9B). For the study of normal structure, select healthy, representative plants. Copyright complaints  ~   Lignification is considered part of the plant’s developmental program, being coordinated with the production of cellulose, hemicellulose, and other polysaccharides during secondary wall formation (Bonawitz and Chapple, 2010 and references therein). In another model, the camera is held on a bracket attached to the ocular tube, and the camera is swung into position over the ocular (Fig. 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). However, it is obvious that the microscope cannot be used conveniently for visual work when fastened to the base of the camera shown in Fig. Nat. We thank the Chief Scientist of The Ministry of Agriculture and Rural Development, Israel, Grant Agreement No. The results show that GA3 application influenced sweetpotato plant development by increasing stem elongation, caused a significant increase in levels of root bioactive GAs, decreased the number of ARs as well as the number and length of LRs, and reduced significantly the number of SRs. Gibberellin signaling: biosynthesis, catabolism, and response pathways. HortSci. doi: 1710.1104/pp.1108.121533, Kao, Y., Harding, S. A., Tsai, C. J. Following sectioning, root sections were deparaffinized in a histoclear solution, rehydrated with ethanol dilution series, and used further for histochemical staining and autofluorescence imaging. doi: 310.1046/j.1365-1313X.1998.00217.x, Tanaka, M. (2016). (2017). 1). The distribution of the GUS signal was analyzed using a stereomicroscope (SMZ745T, Nikon Instruments Europe B.V., Warszawa, Poland) and a digital camera (DLT-Cam PRO, Delta Optical, Nowe Osiny, Poland). Contribution of gibberellins to the formation of Arabidopsis seed coat through starch degradation. After incubation, ethanol was discarded and tubes containing ethanol-insoluble sugar (starch) were kept in an oven at 60°C for drying overnight. Secondary xylem development in Arabidopsis: a model for wood formation. Lateral root density increased more than 3-fold at 2W as compared to 1W, under all tested conditions, and no significant differences were apparent following GA3 or PB applications (Figure 2E). Plant Cell. doi: 3410.1105/tpc.3110.075036, Olszewski, N., Sun, T., Gubler, F. (2002). Plant Physiol. Table 1 Sweetpotato (Ipomoea batata) Georgia Jet orthologues/genes investigated in our study. doi: 22910.23390/ijms160922960, Gupta, R., Chakrabarty, S. K. (2013). 206 Botanical Microtechnique The inexpensive Histoslide camera lacks the observation tube. AD-F did bioinformatics analysis. BLAST and tBLASTx were used to compare sequences of candidate sweetpotato contigs/genes of GA biosynthesis and signaling, lignin biosynthesis, and upstream regulators, class I knotted 1-like and starch metabolism, derived from our published database (Firon et al., 2013) with Arabidopsis2 and NCBI3 databases. (2008). Following incubation, ethanol was discarded, fresh 80% ethanol was added and samples were incubated, as described above, this step being repeated twice. Ravi, V., Chakrabarti, S. K., Makeshkumar, T., Saravanan, R. (2014). These results point to GA, and potentially KNOX genes, as important regulators of root xylem development and lignification, on one hand, and formation of starch-accumulating cells, on the other hand, regulating SR formation and yield. Hay et al. 79, 1–24. Duan, A. Q., Feng, K., Wang, G. L., Liu, J. X., Xu, Z. S., Xiong, A. S. (2019). It should be noted that the same 12 plants, per each treatment and sampling date, were used for looking into root anatomy, quantifying root endogenous GA levels and starch levels, and for gene expression analysis (RNA extraction) as detailed below and described in Supplementary Figure S1. Bars represent mean of 16 independent biological replicates (plants) ± SE. Solidworks, c'est un logiciel de FAO. 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 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). Exogenous application of GA3 was shown in Betula platyphylla to induce xylem development and expression of secondary wall biosynthesis genes (Guo et al., 2015). Rev. If the material is to be kill. BMC Genomics 14, 460. doi: 410.1186/1471-2164-1114-1460, Geigenberger, P. (2011). Dried herbarium specimens can be softened and sectioned to make slides in which it is possible to determine the gross features of vascular arrangement or carpellary organization (Hyland, 1941). The object is focused through the microscope ocular and the camera is carefully placed upon the ocular. Botanical microtechnique. Horlogerie, appareils photo, instruments de mesure... L'univers des microtechniques inclut tous les appareils miniaturisés utilisant plusieurs technologies : micromécanique, microélectronique, optique, électrotechnique... Selon son niveau de qualification, le microtechnicien occupe un … KNOX gene function in plant stem cell niches. GA also affects sweetpotato root system architecture, reducing LR number and LR length by an unknown mechanism. Characterization of lateral root development at the onset of storage root initiation in ‘Beauregard’ sweetpotato adventitious roots. 47, 961–968. 5, 523–530. Samples stored in FAA solution (as mentioned above) were dehydrated using ethanol dilution series, followed by embedding in paraffin wax (Ruzin, 1999). Shoot meristem size is dependent on inbred background and presence of the maize homeobox gene, knotted1. The parameters included adventitious root (AR) number per plant (A), AR cumulative length per plant (B), lateral root (LR) number per plant (C), LR cumulative length per plant (D), LR density per AR (E), and root surface area per plant (F), determined at one and two weeks (1W and 2W, respectively) after planting. : JoVE. Rev. Moreover, significant reduction in AR number, AR cumulative length, LR number, and LR cumulative length per plant was observed at 2W after planting following both, GA3 and PB treatments (Figures 2A, B, C, and D, respectively). 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). doi: 4310.1242/dev.111369, Ma, J., Aloni, R., Villordon, A., Labonte, D., Kfir, Y., Zemach, H., et al. Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in Aspen. doi: 310.1146/annurev-genet-102209-163508, Chaffey, N., Cholewa, E., Regan, S., Sundberg, B. Indeed, Villordon and Clark (2014) and Villordon et al. . Exogenous application of PB, caused the opposite effect, reducing the levels of all tested bioactive GAs by more than 2-fold. 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). 15, 1591–1604. J. Vis. These inhibitory effects of GA on sweetpotato LR development and RSA were linked with a significant reduction in SR formation (SR number) and SR characteristics (diameter and weight). (2002). At 5W, the lignified area in GA3-treated roots was represented mainly by fibers, while in PB-treated roots it was represented by proto- and meta-xylem cells. For safranin-fast green staining, deparaffinized samples were stained for 2 h in safranin-O (1%) with counter staining for 10 s in fast green (0.5%). The effect of exogenous application of GA3 and of the GA biosynthesis inhibitor PB was tested on stem growth and on root characteristics. Gibberellin metabolism and its regulation. In accordance with these developmental effects, gibberellin application upregulated expression levels of sweetpotato orthologues of Arabidopsis vascular development regulators (IbNA075, IbVND7, and IbSND2) and of lignin biosynthesis genes (IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD), while downregulating starch biosynthesis genes (IbAGPase and IbGBSS) in the roots. *Correspondence: Nurit Firon, vcfiron@volcani.agri.gov.il, Front. Differential expression and detection of transcripts in sweetpotato (Ipomoea batatas (L.) Lam.) Figure 13 Schematic presentation showing a central role for gibberellin (GA) in formation of either lignified non-storage roots or storage roots. 17.2. using cDNA microarrays. An Arabidopsis gene regulatory network for secondary cell wall synthesis. Exogenous application of GA3 to sweetpotato plants during the first two weeks of development caused elevated levels of bioactive and non-bioactive GAs in the roots together with an increase in the plant’s stem length. Exogenous application of plant growth regulators increased the total flavonoid content in Taraxacum officinale Wigg. Furthermore, it is hypothesized that environmental stress conditions will reduce SR yield by activating similar mechanisms. Malamy, J. E., Benfey, P. N. (1997). Photosyn. 19, 1855–1860. This image could have imperfections as it’s either historical or reportage. Paclobutrazol, in spite of its inhibitory effect on accumulation of bioactive GAs in the sweetpotato root system, exhibited a similar effect to GA3 with respect to enhancing significantly the number of root xylem vessels. Plant Cell. 96, 4698–4703. Plant Physiol. 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. BAC PRO MICROTECHNIQUES 1 Simon, Terminale bac pro : Ce que j'aime bien dans la microtechnique, c'est le contrôle, la maîtrise des machines. 8, 5727–5732. Dayan, J., Schwarzkopf, M., Avni, A., Aloni, R. (2010). Front. Vollbrecht, E., Reiser, L., Hake, S. (2000). It has previously been bought for a multiple re-use license which is still valid. 16, 22960–22975. Gibberellin metabolism: new insights revealed by the genes. (2018). It was shown to affect xylem formation and plant lignification in various systems, causing upregulation in expression of lignin biosynthesis genes (Biemelt et al., 2004). The results substantiate our hypothesis and mark gibberellin as an important player in regulation of sweetpotato root development, suggesting that increased fiber formation and lignification inhibit storage-root formation and yield. Expression of all tested genes, except IbPGM, was reduced by GA3 application. Plant Sig. Sweetpotato plants are propagated through stem-cuttings that produce adventitious roots (ARs) from primordia developed on stem nodes (Ma et al., 2015). Rev. Lateral stem length was recorded at three, four, and five weeks (3W, 4W, and 5W, respectively) after planting. Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation. qRT-PCR data are mean (± SE) of three independent biological replicates (each representing roots pooled from four plants). Recently, it was demonstrated that the plant hormone gibberellin (GA) is involved in root growth, secondary xylem development and lignin accumulation in carrot (Wang et al., 2015a; Wang et al., 2017). doi: 1510.1016/S0960-9822(1502)01125-01129, Hedden, P., Phillips, A. L. (2000). J. Exp. (2011). Morphological characteristics, anatomical structure and gene expression: novel insights into gibberellin biosynthesis and perception during carot growth and development. ©2019. Il établit le cahier de maintenance prévisionnelle et curative et l'informatise. Biol. – Microtechnicien, This image is a public domain image, which means either that copyright has expired in the image or the copyright holder has waived their copyright. In another model, the camera is held on a bracket attached to the ocular tube, and the camera is swung into position over the ocular (Fig. Greenhouse temperature was maintained at 25/20°C ± 3 day/night temperatures under natural conditions, with no supplemental light. 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. doi: 110.1111/j.1744-7909.2010.01018.x, Zhang, S., Zhang, D., Fan, S., Du, L., Shen, Y., Xing, L., et al. Gibberellin biosynthesis and response during Arabidopsis seed germination. Subsequently, samples were cooled down to room temperature and 4 ml of working solution, containing 0.5 ml acetate buffer (2M, pH 4.8), 2.5 ml dH2O, and 1 ml of amyloglucosidase (10 mg/ml), was added to each sample. Sampled roots for endogenous GA, starch, and gene expression analyses were plunged immediately into liquid nitrogen and stored at -80°C until use. 44, 651–655. 56, 242–254. The effect of PB on most tested root system parameters and on SR production was similar to that of GA3 in spite of its antagonistic effect on endogenous root GA levels. Cross-sections of ARs at 1W and 2W after planting show that root anatomy was influenced by both GA3 and PB treatments, exhibiting higher number of xylem vessels (Figure 5A). doi: 1510.1105/tpc.011650, Ohashi-Ito, K., Oda, Y., Fukuda, H. (2010). In accordance with this hypothesis, GA3 application caused a significant reduction in transcript levels of both IbKN2 and IbKN3 genes. Cette demande doit être formulée, en principe, dans les quinze jours qui suivent le début des cours. Contact :  scoliut25@univ-fcomte.fr ou  03.81.66.68.08 / 68.21 / 68.22. Hussey, S. G., Mizrachi, E., Spokevicius, A. V., Bossinger, G., Berger, D. K., Myburg, A. 19, 270–280. – 3eme générale, Overexpression of BP was found to cause decreased lignification and bp mutants exhibited elevated lignin levels (Mele et al., 2003). . 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. Botany -- Anatomy; Botany -- Morphology; Microscopy -- Technique; Microscopes -- Technique; Botany; Plants -- anatomy & histology; Microscopy. 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). Interestingly, gibberellin downregulated root expression levels of orthologues of the Arabidopsis BREVIPEDICELLUS transcription factor (IbKN2 and IbKN3), regulator of meristem maintenance. In the present work, GA3 may cause the opposite effect, namely reduction of carbon flow toward carbohydrate metabolism and delivery into phenylpropanoid and lignin synthesis. Paclobutrazol application, as opposed to GA3, did not induce fiber formation (Figure 5C). The significance of the current findings is illustrated by highlighting potential regulators/regulatory mechanisms that control SR formation in sweetpotato to be used for enhancing yield under variable growth conditions. doi: 10.1093/oxfordjournals.aob.a084729, Yamaguchi, M., Kubo, M., Fukuda, H., Demura, T. (2008). 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. Moreover, area covered by xylem elements (vessels and fibers) and total root area were calculated using the imagej software (Imagej 1.51a, NIH, USA, (Schneider et al., 2012), and percent root area occupied by xylem vessels (protoxylem, metaxylem, and secondary xylem) and xylem fibers was determined. Plant microtechnique and microscopy. It is prepared by dissolving the common plastic, polystyrene, in a suitable hydrocarbon solvent (usually xylene). Il conçoit tout ou partie d'un produit microtechnique ainsi que les outillages de validation de processus.

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