MOLECULAR AND GENETIC APPROACHES IN PLANT GROWTH AND DEVELOPMENT

MOLECULAR AND GENETIC APPROACHES IN PLANT GROWTH AND DEVELOPMENT

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  MOLECULAR AND GENETIC APPROACHESIN PLANT GROWTH AND DEVELOPMENT Study of internal molecular structure and gene behavior Submitted by S.ADHIYAMAAN (2017603401) I-M.Sc.,VEGETABLE SCIENCE DEPT. OF VEGETABLE CROPS HC & RI, TNAU, CBE.- 641 003
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  CLASSIFICATION Tissue – groupof cells, similar in origin, form and function
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  The plant body 1.RAM 2. SAM 3. LEAF 4. FLOWER 5. FRUIT 6. SEED
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   Meristems isodiametric are populationsof small, (having equal dimensions on all sides) cells with embryonic characteristics •Vegetative meristems are self-perpetuating •Not only do they produce the tissues that will form the body of the root or stem, but they also continuously regenerate themselves Meristems
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  Some basic celltypes 2. Collenchyma 3. Sclerids 4. Bulliform cells 1. Parenchyma
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  1. Parenchyma Young parenchymatissue cut parallel with the epidermis of Euphorbia pulcherrima (poinsettia). Note the cell contents. Note the nucleus and chloroplasts
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  2. Collenchyma Apium petiole,collenchyma Collenchyma is the typical supporting tissue of the primary plant body. It develops from parenchyma. The cell walls are unevenly thickened. It is common in organs like stems, petioles, laminae or roots. Apium is celery – and it is the petiole that you eat!
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  3. Sclerids Thickening ofthe cell wall Parenchyma Developing sclerid Sclerenchyma cells are the principal supporting cells in plant parts that have ceased elongation. Sclerenchyma fibres are the source material for many fabrics, e.g., flax, hemp and jute.
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  4. Bulliform cellsTransverse section of grass leaf Poa praetense. During drought water is lost from the thin walled bulliform cells and the two sides of the leaf blade fold up toward each other so the leaf is less exposed to sunlight and is heated less. Once adequate water is available, turgor increases, and the leaves
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  Root Apical Meristem
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  Primary Root Morphology Lateralroots form primary meristems in mature regions of roots, • form new roots (organs). • Lateral or branch roots arise from the pericycle in mature regions of the root.
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  • Most celldivisions in the root tip are transverse, or anticlinal, with the plane of cytokinesis oriented at right angles to the axis of the root (such divisions tend to increase root length). • There are relatively few periclinal divisions (mostly at tip), in which the plane of division is parallel to the root axis (such divisions tend to increase root diameter).
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  The quiescent centeris composed of a group of four cells, also known as the center cells in the Arabidopsis root meristem. The quiescent-center cells in the Arabidopsis root usually do not divide after embryogenesis.
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  Root Apical Meristems(RAM) Contain Several Types of Stem Cells  Cortical–endodermal stem cells  Columella stem cells  Root cap–epidermal stem cells  Stele stem cells
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  1.The cortical–endodermal stemcells form a ring of cells that surround the quiescent center. These stem cells generate the cortical and endodermal layers They undergo one anticlinal division (i.e., perpendicular to the longitudinal axis); then these daughters cells divide periclinally (i.e., parallel to the longitudinal axis) to establish the files that become the cortex and the endodermis
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  2. The columellastem cells are the cells immediately above (apical to) the central cells. They divide anticlinally and periclinally to generate a sector of the root cap known as the columella. 3. The root cap–epidermal stem cells are in the same tier as the columella stem cells but form a ring surrounding them.  Periclinal divisions of the same stem cells, followed by subsequent anticlinal divisions of the derivatives, produce the lateral root cap.
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  4. The stelestem cells are a tier of cells just behind the quiescent- center cells. These cells generate the pericycle and vascular tissues.
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  Genes associated with RaM HOBBIT(HBT) genes MONOPTEROS (MP) gene is required for formation of the embryonic primary root as well as vascular development SHORT ROOT (SHR) genes SCARECROW (SCR) genes
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  Anatomy of dicotroot after meristematic growth (Differentation) Permanent tissue
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  Shoot Apical Meristem(SAM) • The vegetative shoot apical meristem generates the stem, as well as the lateral organs attached to the stem (leaves and lateral buds). • The shoot apical meristem typically contains a few hundred to a thousand cells, although the Arabidopsis shoot apical meristem has only about 60 cells.
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  • The shootapex consists of the apical meristem plus the most recently formed leaf primordia • The shoot apical meristem is the undifferentiated cell population only and does not include any of the derivative organs • The shoot apical meristem is a flat or slightly mounded region (100 to 300 μm in diameter)
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  Apical meristems Indeterminate growth e.g.,many tomato varieties Determinate growth e.g., Brinjal
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  Shoot organization -Phytomeres Phytomer = modular unit of the Shoot
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  Shoot organization Protoderm Apical Meristem Primary Meristems Protoderm Procambium Ground Meristem
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  Shoot apical meristem- Importance • Center of postembryonic growth & development • Source of all primary meristems – Protoderm, ground meristem & procambium • Source of – Leaves – Branches – Tendrils – Thorns • Self-renewing mass of cells ≈ stem cells • Balance cell division and cell differentiation
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  Apical Meristem Examples Equisetum Conifers Angiosperms
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  Shoot apical meristemorganization L1 = tunica L2 = tunica Peripheral Zone Pith or Rib Meristem Central Zone Stem Cells Organizing Center L3 = corpus
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  Anatomy of dicotstem after meristematic growth (Differentation) Permanent tissue
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  Gene Expression inthe Apical Embryo Domain WUSCHEL (WUS), CLAVATA (CLV) AND SHOOT MERISTEMLESS (STM)
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  WUS, CLV and STMexpression in the shoot apex WUS gene 1. Organizing Center of Central Cells (just a few cells) 2. Molecular: Encodes homeodomain protein 3. Molecular Genetic: Induces Expression of CLV3 4. Developmental: WUS specifies stem cells of the SAM, i.e. maintains stem cells and maintains their identity. CLV3 gene 1. Stem cells of Central Zone 2. Molecular: Encodes peptide secreted in extracellular space 3. Molecular Genetic: Inhibits WUS expression. 4. Developmental: CLV3 restricts size of Central Cells, i.e. CLV3 restricts size of the stem cell population. STM gene 1. Through SAM apical “dome” of cells: central zone and peripheral zone. 2. Molecular:Encodes homeodomain protein 3. Molecular Genetic: Blocks organ formation genes (AS1, AS2) 4. Developmental: Prevents premature differentiation of cells from Peripheral Zone… thus prevents premature organ initiation.
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  Leaf Development
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  Leaf Development Stage 1:Organogenesis Stage 2: Development of suborgan domains Stage 3: Cell and tissue differentiation
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  LEAF PRIMORDIUM
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  Leaf development
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  These cells differentiatein a genetically determined pattern that is characteristic of the species ……..but to some degree modified in response to the environment
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  The Arrangement ofLeaf Primordia is Genetically Programmed •There are five main types of phyllotaxy 137.5= Golden angle Nature by Numbers.mp4 Fibonacci series -integer sequence
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  A complete flowerhas four whorls -Calyx, corolla, androecium, and gynoecium An incomplete flower lacks one or more of these whorls Calyx = Consists of flattened sepals Corolla = Consists of fused petals Androecium = Collective term for stamens -A stamen consists of a filament and an anther Gynoecium = Collective term for carpels -A carpel consists of an ovule, ovary, style, and stigma Flower Structure
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  activation inhibition Activation of FloralMeristem Identity Genes Adult meristem Floral meristem Temperature- dependent pathway Autonomous pathway Flower- repressing genes Flower- promoting genes Vernalization Autonomous gene expression Repression of Floral Inhibitors Cold Light- dependent pathway Gibberellin- dependent Gibberellin pathway CO Light LFY AP1 ABC floral organ identity genes Floral organ development
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  Model for Flowering Thefour flowering pathways lead to an adult meristem becoming a floral meristem. They activate or repress the inhibition of floral meristem identity genes (LFY – LEAFY and AP1 - APETALA1) These two genes turn on floral organ identity genes
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  Elliot Meyerowitz ABC Model– Flower development
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  Model for Flowering TheABC model proposes that three organ identity gene classes specify the four whorls 1.Class A genes alone – Sepals 2.Class A and B genes together – Petals 3.Class B and C genes together – Stamens 4.Class C genes alone – Carpels When any one class is missing, abnormal floral organs occur in predictable positions
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  Arabidopsis showing 4floral organs Carpels-C Stamens - B&C Petals-A&B Sepals-A
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  Testing the ABCModel • Researchers tested the ABC model and found it to be supported, with the modification that two of the proteins act to inhibit the production of each other. • They also found that the DNA sequences of floral organ identity genes all contain a segment that encodes a DNA- binding domain called a MADS box. • They suggested that MADS-box genes are part of the regulatory cascade that controls the floral organ identity genes.
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  What are MADSbox genes? • The MADS box is a highly conserved sequence motif found in a family of transcription factors. • The conserved domain was recognized after the first four members of the family, which were MCM1, AGAMOUS, DEFICIENS and SRF (serum response factor). The name MADS was constructed form the "initials" of these four "founders". • Length of the MADS-box reported by various researchers varies somewhat, but typical lengths are in the range of 168 to 180 base pairs.
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  THE ABC MODELFOR FLORAL ORGAN IDENTITY APETALA 1
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  No sepals and petals No stamen Nocarpels and stamen
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  The Quartet Modelof flower development Quaternary complexes of MADS domain proteins
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  Fruit Development The fruitis a specialized organ, which provides suitable environment for seed maturation and, often dispersal mechanisms. Fruit development can generally be considered to occur in four phases: Fruit set phase Rapid cell division A cell expansion phase ripening/maturation phase GA is a triggering signal
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  Genes Arabidopsis fruits developfrom two fused carpels and are specialized capsules called siliques FRUITFULL (FUL) is necessary for proper valve development and represses SHATTERPROOF 1/2 (SHP 1/2) (Gu et al., 1998;Ferrándiz et al., 2000a). SHATTERPROOF 1/2 (SHP1/2) are necessary for valve margin development (Liljegren et al., 2000). REPLUMLESS (RPL) is necessary for replum development and represses SHP1/2 (Roeder et al., 2003)
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  SHP1/2 activate INDEHISCENT(IND) and ALCATRAZ (ALC), which are both necessary for the differentiation of the dehiscence zone between the valves and replum (Girin et al., 2011; Groszmann et al., 2011). FUL, SHP1/2, RPL, IND, SPT, and ALC all belong to large transcription factor families. FUL and SHP1/2 belong to the MADS-box family (Gu et al., 1998; Liljegren et al., 2000)
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  MADS-box family MADS-box genesrepresent a highly conserved gene family of putative transcription factors in plants The proteins encoded by these genes are characterized by a highly conserved domain, which consists of 56 amino acids K-box involved in protein-protein interactions. The MADS-box and K-box are separated by a weakly conserved Intervening (I) region, and a few MADS-box genes have an amino-terminal extension (N). The Carboxyl-terminal (C) region may function as a transcriptional activation domain.
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  Seed development
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  The seed developsfrom the ovule and contains the embryo and endosperm, surrounded by the maternally derived seed coat. Ovules are borne by both the angiosperms (true flowering plants) and the gymnosperms (which include the conifers)
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  Maternal tissues appearto have an important influence on seed development. An arabidopsis mutant called aberrant testa shape (ats) that lacks one of the 2 integuments also lacks several cell layers in the testa (3 layers vs. 5 normally). The seed are abnormally shaped in this mutant and seed shape shows maternal effect . Therefore, the seed coat and not the embryo determines the shape of the seed, and the embryo just grows to fill in the shape determined by the testa
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  Maternal gene The maternalgene called FBP7 is specifically expressed in the ovule and seed coat and is required for normal ovule development. A mutant screen on a sterile line identified 3 genes that regulate seed development. SeedS develop on theSe mutantS in the abSence of fertilization. They are called fis for fertilization independent seeds. The genes appear important for control of seed development by fertilization.
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  All three genesshow parent-of-origin effects (Genome imprinting) FIE = fertilization independent endosperm, encodes a WD type POLYCOMB protein MEDEA encodes a SET domain type POLYCOMB protein FIS2 = fertilization independent seed2, encodes a zinc finger protein The maternally inherited gene is expressed and required but the paternally inherited gene is not expressed or required for seed development.
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  POLYCOMB proteins areinvolved in chromatin structure and regulate (repress) the expression of genes in big portions of the genome. ABA is necessary to induce the expression of genes involved in maturation and desiccation tolerance Case study FLOWERING LOCUS T genes control onion b
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  FLOWERING LOCUS Tgenes control onion bulb formation and flowering Robyn Lee1, Samantha Baldwin2, Fernand Kenel2, John McCallum2 & Richard Macknight1 Received 20 Jun 2013 | Accepted 6 Nov 2013 | Published 3 Dec 2013 1 Department of Biochemistry, University of Otago, 9016 Dunedin, PO Box 56 Dunedin, New Zealand. 2 Breeding and Genomics, New Zealand Institute for Plant and Food Research, Private Bag 4704, 8140 Christchurch, New Zealand.
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  Introduction • Onions areone of the oldest vegetables known to mankind and have been cultivated for more than 4,700 years. • Vernalization has only been studied at the molecular level in a few species and, in all cases, results in the inhibition of a repressor, thereby allowing FLOWERING LOCUS T (FT) to be expressed. • Flower signal molecule – Florigen
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  Introduction • FT isproduced in the phloem companion cells of the leaf and is transported to the apical meristem cells. • where it forms a complex with 14-3-3 proteins and the bZIP transcription factor FD. • This complex, known as the florigen activation complex, is thought to translocate to the nucleus where it activates the floral meristem identity genes, thereby inducing flowering. • A recent exciting discovery is that the potato FT orthologue StSP6A is involved in the SD induction of tuberization
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  Methods- Plant materials •Doubled haploid onion lines CUDH2150 and CUDH2107 were obtained from the Cornell University. • Seed lots were sown in spring either directly as seed or as transplants in Jiffy 7 plug (Jiffy Corp) and grown in Black Magic seed-raising mix (Yates Orica, New Zealand). • Onion and Arabidopsis plants were grown under either LD (16 h light: 8 h dark) or SD (8 h light: 16 h dark) photoperiod in plant growth rooms maintained at 20 ºC with 30–40% humidity and a light intensity of - 115 mmolm-2 s-1 .
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  Methods-Identification and cloningof onion FT-like genes. • Onion FT-like sequences were identified from our transcriptome data deposited at NCBI BioProject using TBLASTN. • Full-length cDNA sequences were obtained from six partial FT-like sequences (AcFT1-6) using the Invitrogen GeneRacer RNA ligase mediated rapid amplification of 50 and 30 cDNA ends (RLM-RACE) kit and these have been deposited in GenBank Nucleotide core database under accession codes KC485348-53. • Full-length PCR products of each FT were cloned from ‘CUDH2150’ using pCR8/GW/TOPO TA(primer) Cloning Kit (Invitrogen). • Correct orientation was confirmed by sequencing. The TOPO-based clones were then linearized with either XbaI or XhoI, and recombined into pB2GW7 with LR Clonase (Invitrogen), thereby producing a binary vector containing CaMV 35S:AcFT.
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  • To constructthe SUC2 promoter-driven constructs, first the 35S promoter in pB2GW7 was replaced with the SUC2 promoter from pAF12. • pB2GW7 was digested with SacI/EcoRI to release the 35S promoter. • The attRI site was lost in this reaction, so it was replaced with a PCR fragment that contained SacI–XbaI sites at the 50-end and an EcoRI site at the 30-end. Ligated together, SacI/ EcoRI, this reforms pB2GW7 without the 35S promoter. • The SUC2 promoter was PCR amplified from the pAF12 vector, with SacI and XbaI sites on the 50- and 30- ends respectively, and cloned into the promoterless pB2GW7 vector at these sites. Methods-Identification and cloning of onion FT-like genes.
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  Plant transformation • SUC2:AcFTand 35S:AcFT binary vectors were transformed into Agrobacterium tumefaciens GV3101 by electroporation. Arabidopsis were transformed using the method of Martinez-Trujillo et al. as follows: • Agrobacterium harbouring the binary vector were streaked onto LB media plates with selective antibiotics and incubated at 28 ºC for 2 days. The bacteria were then scraped off the plate and resuspended in 10 ml of infiltration media (0.5 MS (Duchefa), 0.05% Silwett L-77 (Lehle Seeds), 5% sucrose) to an OD600 an droplets of this solution were pipetted onto unopened flower buds of Arabidopsis thaliana ft-1 mutants. • This was repeated three times in 2–3-day intervals. The resulting seed was sown on seed-raising mix and transformants selected by spraying the young seedlings with Basta herbicide (glufosinate ammonium; Bayer) at 120 mg/l
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  • Onions weretransformed with a binary vector containing the AcFT1 or AcFT4 cDNA under the control of the 35S promoter, along with the eGFP-ER reporter gene and the Bar selectable marker-, using the method of Eady et al. as follows: • Immature embryos from onion seeds (‘Pukekohe LongKeeper’) incubated overnight at 4 ºC were isolated and cut into -1mm lengths before transfer into 50 ml liquid P5 medium. • Agrobacterium, resuspended in 0.4 ml liquid P5 to an OD550 of 0.4–0.6, was added and embryos were vortexed for 30 s, and then placed under vacuum (B20 in Hg) for 30 min before blot drying on filter paper and transferred to P5 medium solidified with 0.4% Phytagel (Gellan Gum). • After 6 days cocultivation, embryo pieces were transferred to P5 plus 2.5 mg /1 phosphinothricin and 200 mg /1 timentin. These embryo pieces were cultured in the dark under the same conditions as described for the production of secondary embryos. Cultures were transferred to fresh medium every 2 weeks. Onion transformation
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  • After threeto four transfers, growing material was transferred to P5 plus 5mg /1 phosphinothricin and grown for a further 8 weeks. • During this time, pieces of putative transgenic tissue that reached -2mm2 were transferred to regeneration medium. • Shoot cultures were maintained for 12 weeks and developing shoots were transferred to ½ MS medium plus 20 mg /1 geneticin to induce rooting. • Rooted plants were either transferred to ½ MS plus 120 g /1 sucrose to induce bulb formation or to soil in the glasshouse (12 h 12–23 C day, 12 h 4–16 C night). • Expression of the transgenes were confirmed by reverse transcriptase–PCR using primers.
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  Analysis of geneexpression • Plant tissue was harvested into liquid nitrogen. Total RNA was extracted using the Invitrogen Plant RNA Purification Reagent, according to the manufacturer’s instructions, but with an addition 30 min incubation in the reagent for onion tissue. • RNA concentrations were determined using a NanoDrop 8000 (ThermoScientific). • Reverse transcription was carried out with 1 mg of total RNA in 20 ml volumes using Invitrogen SuperScriptIII according to the manufacturer’s instructions. • First-strand cDNA was diluted 30-fold, and 3 ml of this was used in each 10 ml reaction. • Real-time PCR was performed using the Roche LC480 machine and Light-Cycler 480 SYBR Green I Master mix (Roche). Relative gene expression levels were calculated using the 2 (2delta delta C(T)) method using Roche LC480 software. • b-tubulin was used as the reference gene.
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  •Result
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  Onion has atleast six FT-like genes • Phylogram of FT-like protein sequences from different monocot plants. Four subgroups are indicated. The sequences are from • onion (Allium cepa, Ac), • rice (Oryza sativa, Os), • maize (Zea mays, Zm), • barley (Hordeum vulgare, Hv) and • spring orchid (Cymbidium goeringii, Cg).  FTs with asterisks have been shown to promote flowering.
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  Onion has atleast six FT-like genes • Complementation of the Arabidopsis ft-1 mutant with onion FT-like genes expressed under the constitutive 35S promoter (blue) or phloem-specific SUC2 promoter (yellow). • Flowering time as determined by number of rosette leaves at flowering of representative lines grown in LD. • (c) Photo of 35S:AcFT lines grown in LD, with AcFT1 flowering and floral buds visible on WT and 35S:AcFT2 plants (plants are in the same order as the panel b graph).
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  AcFT1 and AcFT4expression suggests they control bulbing. • Expression of AcFT2 in leaf tissue from seedlings with 3–4 leaves (young), plants with 10 leaves before bulb formation (mature), and bulbing plants (bulbing) and central bud tissue from bulbs (bulb tissue) non-vernalized (-V) or vernalized in the dark for 3 months (+V), leaves from flowering plants (flowering bulb). • Samples were taken from plants grown at 20 ºC under SD (8 h light: 16 h dark) and LD (16 h light: 8 h dark) photoperiods (except bulbing plants, as bulbs do not form in SD). • Data represent an average ±s.e.m. of three biological replicates, with transcripts normalized to b-tubulin. AcFT2 expression correlates with flowering.
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  AcFT1 and AcFT4control bulb formation in transgenic onions. (a) Expression of AcFT1 and AcFT4 in leaf tissue from young plants with 3–4 leaves (young), plants with -10 leaves before bulb formation (mature) and bulbs from previous season grown to have 4–5 fully expanded leaves (bulbs). AcFT1 is induced and AcFT4 is repressed by conditions that induce bulb formation Long day condition only promote bulb formation. AcFT 4 Inhibit bulb formation
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  Expression of AcFT1and AcFT4 after transfer from SD to LD • Mature onion seedlings were grown under SD for 14 weeks and then transferred to LD. • Leaf samples were taken. All plants were grown at 20 ºC under SD (8 h light: 16 h dark) or LD (16 h light: 8 h dark) photoperiods. • The data represent an average ±s.e.m. of three individual plants, with transcripts normalized to b- tubulin.
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  Overexpression of AcFT4prevents bulbing and inhibits expression of AcFT1 • a) 35S:AcFT1 plants form bulb-like structures in tissue culture (independent lines are shown in separate photographs) and in comparison • (b) 35S:AcFT4 plants that are the same age, show normal vegetative growth in tissue culture. (NO BULB) • (c) Relative expression of AcFT1, AcFT2 and the bulb marker 1-SST in representative 35S:AcFT41 and 35S:AcFT4 lines grown in tissue culture and bulb tissue from a mature wild- type plant. • (d) 35S:AcFT4 plants (NO BULB) • (e) control plants (derived from calli generated in same experiment as 35S:AcFT4 plants but not expressing GFP or AcFT4) grown in glasshouse conditions and photographed in late summer (28 Feb 2013). • (f) Expression ofAcFT4 and AcFT1 in representative 35S:AcFT4 and control plants.
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  Discussion • Our evidenceindicates that in juvenile plants and those grown under non-inductive photoperiod, AcFT4 functions to prevent the upregulation of AcFT1. • Once the plants are mature and daylength reaches a critical length, AcFT4 is downregulated and AcFT1 is upregulated leading to the initiation of bulb formation
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  • Interestingly, AcFT4also has a number of amino-acid changes that are predicted to prevent its interaction with the 14-3-3 protein that mediates the interaction between FT and FD
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  • FT genescontrol both floral induction and bulb formation in onion., also play a wider role in controlling developmental decisions.