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#plant cell biology

17 public questions tagged with this topic.

Plant tissue culture 2

Cybridization is mainly useful for transfer of:

Cybridization primarily useful for transfer of cytoplasmic traits encoded by mitochondrial or plastid genomes which are generally maternally inherited and cannot be introgressed through nuclear hybridization alone. Key agricultural examples include cytoplasmic male sterility CMS caused by chimeric mitochondrial open reading frames atp6 orf138 orf79 interfering with pollen development essential for hybrid seed production in maize rice sunflower Brassica onion; triazine herbicide resistance due to psbA D1 protein mutation in chloroplast photosystem II; and traits like cold tolerance linked to mitochondrial respiration efficiency. By fusing protoplast of elite high yielding cultivar nucleus donor with CMS donor cytoplasm inactivated nucleus donor protoplast, cybrid combines elite nuclear background determining yield quality with sterile cytoplasm enabling controlled cross pollination without self pollination. Mitochondrial genomes often recombine after fusion generating novel CMS patterns, while chloroplasts normally segregate maintaining single parental type. This avoids linkage drag associated with sexual backcrossing introducing undesirable nuclear genes. Thus cybridization revolutionized hybrid breeding providing male sterile female lines at commercial scale for many vegetable and oilseed crops, facilitating hybrid vigor exploitation.

Ref: Bravo & Evans 2011 cybrid CMS; Kumar et al., Front Plant Sci cytoplasmic traits.

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Plant tissue culture 2

Fusion of cytoplasm of two cells with nucleus from one parent is called:

Fusion product containing cytoplasm of two cells but nucleus from only one parent is termed cybrid cytoplasmic hybrid, distinction from true somatic hybrid which contains both nuclei yielding allotetraploid. Cybrids arise when protoplasts of two sources fuse forming heterokaryon with two nuclei and mixed organelles followed by selective loss or inactivation of one nucleus. Inactivation can be engineered by pre treating one partner with gamma irradiation X ray or metabolic inhibitor iodoacetate that damages nucleus while preserving cytoplasm viability. During subsequent mitosis heterokaryon resolves uninucleate cybrid retaining nuclear genome of one parent determining morphological traits while cytoplasmic organelles mitochondria chloroplasts may be biparental, recombined, or sorted singly. This outcome is deliberately exploited to transfer extra nuclear encoded traits. Unlike somatic hybrid with combined chromosome sets, cybrid is diploid nuclear background with novel cytoplasm enabling creation of novel cytoplasmic genomic interactions. Molecular analysis using mitochondrial RFLP chloroplast SSR confirms organelle identity. Cybrid formation is major tool for cytoplasmic genetic engineering overcoming strict maternal inheritance barriers in sexual crossing impossible for organelle traits.

Ref: Sidorov et al., 1981 cybrid concept; Gleba 1979 Cytoplasmic inheritance.

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Plant tissue culture 2

Electrofusion induces fusion by:

Electrofusion induces protoplast fusion by applying controlled electrical pulses that create reversible membrane breakdown at points of contact allowing lipid bilayers to merge and cytoplasms to coalesce, offering higher fusion frequency viability and selectivity than chemical PEG method. Protocol consists of two electrical phases: first low strength high frequency alternating current 50 to 200 kilohertz field causes dielectrophoresis polarizing protoplasts forming induced dipoles aligning them in pearl chains bringing membranes into intimate contact at poles. Second short high intensity direct current pulses 0.5 to 2 kilovolts per centimeter lasting microseconds generate transient pores dielectric breakdown in contacting membranes that upon resealing fuse into single membrane encircling two protoplasts. Post fusion alternating current field maintained briefly stabilizes heterokaryons. Advantages include ability to monitor individual pair fusion under microscope enabling one to one fusion selection, reduced chemical toxicity preserving division capacity, and high heterokaryon yield up to 60 percent. Parameters voltage duration number of pulses optimized per species to avoid Joule heating irreversible electroporation and cell death. Electrofusion combined with fluorescence activated sorting and microfluidic devices now facilitates precise cybrid and somatic hybrid production for citrus potato Brassica rootstock improvement and organelle transfer programs.

Ref: Zimmermann & Vienken 1982 electrofusion; Tempelaar et al., Plant Sci electroporation.

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Plant tissue culture 2

Common fusogen used for induced protoplast fusion is:

Common chemical fusogen employed to induce protoplast fusion is polyethylene glycol PEG polymer with molecular weight typically 1500 to 6000 daltons applied at concentration 15 to 40 percent weight per volume combined with calcium ions 50 millimolar and alkaline pH 9 to 10. PEG induces fusion through multiple mechanisms: it is highly hydrophilic binding water molecules dehydrating intermembrane space reducing hydration repulsion between negatively charged phospholipid bilayers, promotes close apposition of membranes via calcium bridging between phosphate head groups and creates molecular disorder increasing membrane fluidity and probability of lipid mixing and coalescence upon gradual dilution washing out PEG. Efficiency of heterokaryon formation ranges 10 to 30 percent depending on species and concentration exposure time 15 to 30 minutes. Although PEG causes some cytotoxicity protein denaturation requiring thorough washing after treatment, it remains cheapest most accessible method for somatic hybridization compared to electrofusion equipment requiring electroporators. Other chemical fusogens include high calcium alkaline pH alone and inactivated Sendai virus with fusogenic hemagglutinin proteins. PEG fusion nonspecifically generates multinucleate products but selection via fluorescence markers or complementation enables recovery of desired hybrids for crop improvement.

Ref: Kao & Michayluk 1974 PEG fusion; NCBI protoplast fusion review.

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Plant tissue culture 2

Dead protoplasts are stained red by:

Dead protoplasts are stained red by propidium iodide PI nucleic acid intercalating fluorescent dye that distinguishes membrane compromised cells. PI is cationic molecule unable to cross intact lipid bilayer of viable protoplasts due to charge and size exclusion, so viable population excludes dye remaining unstained. In dead cells membrane integrity lost through enzymatic damage or physical stress, PI diffuses entering cytoplasm nucleus where it intercalates between double stranded DNA and RNA base pairs with strong affinity, enhancing red fluorescence emission at 617 nm when excited green light 535 nm. Under fluorescence microscope dead protoplasts show bright red nucleus clearly differentiated from viable green FDA positive cells in dual viability test. This assay allows rapid quantitative assessment of protoplast isolation success and cytotoxicity of fusogen treatments. Compared to colorimetric dyes like Evans blue which stains dead cells blue but requires bright field observation, PI provides fluorescent contrast compatible with flow cytometric analysis sorting live cells for downstream culture fusion transformation. Maintaining membrane integrity crucial because only viable protoplasts can regenerate wall divide form microcallus and participate in heterokaryon formation during somatic hybridization procedures.

Ref: Jones 1987 PI dead staining; Molecular Probes Handbook viability dyes.

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Plant tissue culture 2

Viable protoplasts fluoresce green when stained with:

Viable protoplasts fluoresce bright green when stained with fluorescein diacetate FDA, vital fluorescent dye used routinely for viability assessment before fusion or transformation to ensure batch quality above 80 percent. FDA itself is non fluorescent lipophilic molecule that diffuses across intact plasma membrane permeable only to living cells with intact barrier. Inside cytoplasm active non specific esterases present exclusively in living cells hydrolyze ester bonds releasing fluorescein highly fluorescent polar molecule that accumulates inside membrane intact vesicle because its negative charge prevents efflux, emitting green fluorescence 520 nm upon excitation blue light 490 nm under fluorescence microscope. Dead protoplasts with compromised membranes lack esterase activity and cannot retain fluorescein thus remain non fluorescent and also allow entry of counter stain propidium iodide marking nucleus red. Assay provides rapid quantitative estimation of viability percentage and metabolic vigor as fluorescence intensity correlates with esterase activity and capacity to regenerate wall and divide in culture medium with hormones. Dual staining FDA propidium iodide enables simultaneous live dead discrimination via flow cytometry sorting heterokaryons for somatic hybridization applications.

Ref: Widholm 1972 FDA viability; Larkin 1976 fluorescein diacetate Plant Physiol.

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Cell Organelles

Which of the following plastids is involved in pigment synthesis?

Plastids constitute a family of interrelated organelles arising from undifferentiated proplastids, with interconversion regulated by light, developmental cues and nuclear-encoded transcription factors like GLK. Leucoplasts are non-pigmented plastids predominantly found in roots, seeds, tubers and non-green tissues, serving as biosynthetic hubs rather than photosynthetic factories. Unlike chloroplasts and chromoplasts, they lack extensive thylakoid grana and photosynthetic apparatus but retain double envelope and stromal enzymes for fatty acid, amino acid, starch and secondary metabolite synthesis. Subclasses include amyloplasts for starch, elaioplasts for lipids and proteinoplasts for proteins. Importantly, colorless leucoplasts synthesize monoterpenes, diterpenes and isoprenoid precursors through plastidial methylerythritol phosphate, MEP, pathway providing geranylgeranyl diphosphate substrates for carotenoid and chlorophyll pigment production that later accumulate after differentiation into chromoplasts and chloroplasts. Chromoplasts then actively synthesize and store carotenoids such as lycopene, beta-carotene and lutein for fruit and flower coloration via phytoene synthase. This division highlights metabolic channeling among plastid types with leucoplasts supplying upstream steps.

Ref: Taiz et al., Plant Physiology and Development, 6th ed., Chapter 1: Plastid Types and Terpenoid Synthesis.

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Protein Sorting and Trafficking

What signal is required for targeting proteins to the thylakoid lumen?

Final intra-chloroplast sorting thylakoid lumen after stroma entry requires crossing thylakoid membrane barrier maintaining proton gradient ATP synthesis. Lumenal precursors plastocyanin copper OE16 OE23 photosystem II oxygen evolving complex Rieske iron-sulfur PetC transport stroma lumen. Precursors carry bipartite targeting sequences N-terminal chloroplast transit peptide TOC-TIC entry second thylakoid transfer domain 30-70 residues positively charged N-region hydrophobic core C-region cleavage thylakoid processing peptidase. Two distinct pathways recognize transfer domain: Sec pathway transports unfolded chains SecYE channel driven SecA ATPase proteins plastocyanin apoprotein folding after transport Tat twin-arginine translocation transports fully folded proteins already assembling cofactors disulfide bonds stroma cofactor insertion before transport essential unfolded impossible. Diagnostic signature Tat substrates twin-arginine motif SRRXFLK invariant consecutive arginines RR N-region signal plus hydrophobic Phe-Leu-Lys essential recognition cpTatC Hcf106 receptor complex oligomeric pore varied size driven exclusively proton motive force Delta pH across thylakoid not ATP. SKL peroxisomal PTS1 matrix NPXY endocytic internalization PTB adaptors DXE ER export COPII unrelated thylakoid lumen sorting. Mutation arginine pair lysine abolishes import demonstrating motif necessity and energy coupling mechanism distinct.

Ref: Cline & Theg, Mol Membr Biol 2007: Twin-arginine RR motif for Tat pathway to thylakoid lumen.

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Protein Sorting and Trafficking

Which pathway is involved in the targeting of thylakoid proteins?

Thylakoid membrane harbors photosynthetic machinery requiring insertion light-harvesting polytopic proteins after chloroplast envelope import stroma. Four conserved pathways inherited cyanobacterial ancestor: Sec pathway unfolded proteins SecYEG translocase SecA ATPase, SRP pathway specialized highly hydrophobic light-harvesting chlorophyll a/b binding proteins LHCPs pigments. Stromal chloroplast SRP cpSRP54 GTPase homologous bacterial SRP54 NG M domains binding signal sequence GTP, unique cpSRP43 chaperone absent bacteria three chromodomains four ankyrin repeats specifically binding L18 motif 18 amino acids between transmembrane helices two three LHCP preventing aggregation maintaining soluble transit competent essential pigments otherwise aggregate uncontrollably. Cargo-cpSRP complex targets thylakoid membrane via cpFtsY GTPase homologous FtsY SRalpha then hands substrate Alb3 insertase Oxa1 YidC family integrating bilayer lateral gate GTP hydrolysis dependent. OXA pathway mitochondria inserts inner proteins matrix side TIM23 handles presequence translocation mitochondrial inner membrane MIA40 mediates oxidative disulfide mitochondrial IMS none relevant thylakoid. Loss cpSRP43 Arabidopsis chaos pale green phenotype severe loss LHCPs demonstrating indispensable role thylakoid assembly functional chloroplast development and photosynthesis efficiency and light harvesting.

Ref: Schuenemann, Annu Rev Plant Biol 2007: cpSRP pathway targets proteins to thylakoid membrane.

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Protein Sorting and Trafficking

What is the main function of the TIC complex in chloroplasts?

Chloroplast envelope encloses two translocon systems tandem importing majority organelle proteome 3000 proteins despite plastid genome about 100. Precursors synthesized cytosol N-terminal transit peptide enriched hydroxylated serine threonine positively charged amphipathic not hydrophobic distinct mitochondrial presequence. First receptor TOC outer envelope: Toc159 large 159 kDa GTPase selectivity filter photosynthetic versus housekeeping via acidic A-domain, Toc34 small 34 kDa regulatory, Toc75 Omp85 beta-barrel superfamily 14-strand channel 3 nm pore translocating polypeptide. After outer membrane TOC hands substrate TIC inner envelope: Tic110 main cation-selective channel large intermembrane space domain recruiting peptide stromal scaffold binding chaperones, Tic40 co-chaperone Sti1-like TPR recruiting Hsp93 ClpC AAA ATPase ATP-driven motor pulling substrate, Tic20 alternative smaller channel, Tic22 soluble intermembrane bridging, Tic236 giant forming supercomplex spanning both membranes creating direct continuous contact sites. Upon arrival stroma transit peptide cleaved stromal processing peptidase SPP metalloenzyme producing mature protein. TIC does not import across outer envelope TOC function does not direct Golgi plastids lack connections nor recycle; exclusive role inner envelope passage stromal compartment preceding further thylakoid sorting Sec SRP Tat pathways essential development and photosynthesis.

Ref: Soll & Schleiff, Nat Rev Mol Cell Biol 2004: TIC imports proteins across chloroplast inner envelope.

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Mitochondria Plastids Ribosomes and Other Organelles

The thylakoid system of a chloroplast is extensively damaged, but the stroma and chloroplast envelope remain intact. Whi

Chlorophyll occurs in the thylakoids, and stacks of thylakoids form grana, which are the sites of light reactions. Circular DNA, ribosomes and enzymes for carbohydrate and protein synthesis occur in the stroma and may remain present.

Ref: NCERT Class 11 Biology Chapter 8: Cell: The Unit of Life Mitochondria Plastids Ribosomes and Other Organelles

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