Plant Hormone Biology: Auxin, Cytokinin, and Cellular Regulation

Posted on Jun 2, 2025 in Biology

Important Terminology

  • AUX1: Auxin influx carrier
  • PIN1: Facilitates vertical auxin transport from shoot to root.
  • PIN3: Involved in lateral auxin transport, particularly inward at the shoot.
  • PINOID: A kinase that regulates polarized trafficking of PIN proteins.
  • ABCB1: Mediates auxin movement in the shoot apical meristem and root tip.
  • ABCB19: Mediates auxin movement to the root tip.
  • ABCB4: Regulates auxin levels in root hairs.
  • NPA, TIBA, and 1-NOA: Specific inhibitors of auxin efflux.
  • SCFTIR1: A ubiquitin E3 ligase complex that acts as an auxin receptor, inducing AUX/IAA ubiquitination.
  • AUX/IAA: Auxin-related gene repressor.
  • Endocrine: Hormone action takes place some distance from its site of synthesis.
  • Paracrine: Hormone action takes place close to the site of synthesis.
  • Kinase: An enzyme that catalyzes phosphorylation of a substrate.
  • Phosphatase: An enzyme that catalyzes dephosphorylation of a substrate.

Plant Cell Wall Structure and Synthesis

Chemical Bonds in the Cell Wall

  • Hydrogen Bond (H-bond): Essential for cellulose assembly.
  • Ionic Bond: Involved in borate ester linkages in pectin.
  • Covalent Bond: Facilitates xyloglucan rearrangement by xyloglucan endotransglucosylase.

Cell Wall Composition

  • Primary Wall: Contains cellulose, hemicelluloses, pectin, and structural proteins.
  • Secondary Wall: Contains similar components to the primary wall, but may also include lignin.

Cell Wall Synthesis and Construction

  • Cellulose and callose are synthesized at the plasma membrane.
  • Matrix polysaccharides (hemicelluloses and pectins) are synthesized in the Golgi complex.
  • Enzymes and structural proteins are synthesized in the Rough ER.
  • Most glycoproteins are glycosylated in the Golgi complex.

Auxin Transport Mechanisms

Cellular Auxin Transport

  • Protonated auxin (IAAH) is co-transported with H+ from the cell wall (pH 5) into the cytoplasm (pH 7), or diffuses into the cell passively.
  • In the cytosol, auxin is mostly in its anionic (deprotonated) form (IAA–) and cannot diffuse across the plasma membrane.
  • Auxin can only be transported out of the cell via PIN1, which is located primarily at the basal end of the cell.
  • This is an example of apoplastic transport, as auxin travels between cells via the wall, not via plasmodesmata.

Auxin and Gravitropism

  • Statolith location depends on gravity.
  • Statoliths trigger IAA transport.
  • This redistribution inhibits growth on the lower side and stimulates growth on the upper side.

Auxin and Phototropism

  • Blue light is detected by phototropins 1 and 2 at the shoot tip.
  • Autophosphorylated phototropins interact with auxin transport and regulatory proteins, inducing lateral movement of auxin transporters.
  • Auxin accumulates in the shaded side of the shoot, causing cell elongation and growth towards the light source.

Acid Growth Hypothesis

Acid growth refers to the expansion of cells which is observed when the pH of the wall is decreased, either through auxin-induced acidification of the wall (which is hypothesized to result from activation and synthesis of plasma membrane H+-ATPases), or through immersion of the plant in an acidic buffer in experimental situations. Acidification of the wall to pH <5 results in activation of wall proteins called expansins (which loosen cellulose-hemicellulose bonds). By removing the hemicellulose “tethers,” cellulose microfibrils are able to separate from each other, allowing for expansion of the cell.

Plant Hormone Biosynthesis

Ubiquitin Mechanism in Hormone Regulation

  • Ubiquitin is a small polypeptide, which is adenylylated (and thus activated) by ubiquitin-activating enzyme E1 (using ATP).
  • Activated ubiquitin is then transferred to ubiquitin-conjugating enzyme E2.
  • A protein destined for degradation will be bound to ubiquitin ligase E3, which binds E2, allowing the transfer of ubiquitin to the target protein.
  • Multiple ubiquitin molecules may be added to the target protein. This allows targeting of the protein to the proteasome, where it is degraded.

Plant Hormone Synthesis Sites

  • Auxin: Shoot apical meristem, young leaves.
  • Gibberellins (GA): Immature seeds, developing fruits, shoot apical meristem, young leaves.
  • Cytokinins (CK): Root apical meristem, young leaves, young developing seeds and fruits.
  • Ethylene: Senescing leaves and flowers, ripening fruits.
  • Abscisic Acid (ABA): Mature and dormant seeds, dormant buds, root apex and tip, wilting leaves.

Indole-3-Acetic Acid (IAA) Pathways

  • IPA pathway
  • TAM pathway
  • IAN pathway
  • Tryptophan pathway

IAA Metabolism and Regulation

  • Reversible (Storage): Conjugation
  • Irreversible (Degradation): Oxidation
  • Conversion to IBA
  • Compartmentalization in chloroplasts, ER, nucleus

Hormone Biosynthesis Pathway Types

  • Amino Acid Pathways
    • Tryptophan → IAA
    • Methionine → Ethylene
  • Isoprenoid Pathways
    • IPP → Cytokinin, Brassinosteroid, Gibberellin, Abscisic Acid, Strigolactone
  • Lipid Pathways
    • α-Linolenic Acid → Jasmonic Acid

Auxin Response Genes

  • Primary Response (Early) Genes: Involved in three main functions: transcription of secondary response genes, signaling, and auxin conjugation/catabolism.
  • Secondary Response (Late) Genes: Typically involved in stress responses.

Plant Physiology: Experimental Evidence

Experimental Evidence for Auxin’s Role

Apical Dominance: Lateral bud growth is suppressed by auxin synthesized in the shoot apical meristem (terminal bud). Experimental evidence shows that removing the terminal bud of a bean plant leads to lateral bud growth, but applying auxin to the removal site suppresses this growth, demonstrating auxin’s sufficiency in maintaining apical dominance.

Lateral and Adventitious Root Formation: Auxin induces the formation of lateral and adventitious roots. For instance, in the alf1 mutant, a higher concentration of endogenous IAA is associated with the formation of significantly more lateral and adventitious roots compared to a wild-type plant.

Fruit Development: Auxin promotes fruit development. Experimental evidence, such as studies on strawberries, shows that achenes promote strawberry growth. If achenes are removed, the strawberry is much smaller; however, this stunting can be reversed by applying auxin, indicating that achenes mediate fruit development through auxin.

Auxin and Cytokinin Interactions

Auxin Signaling Pathway: AUX/IAA and ARF Roles

Both AUX/IAA and ARF proteins are transcription factors important for auxin signal transduction. AUX/IAA is a repressor protein that prevents the activity of ARF transcription factors and is part of the auxin receptor complex. Auxin binds partially to AUX/IAA and partially to TIR1 in the receptor complex. Upon auxin binding, AUX/IAA is ubiquitinated and degraded, freeing ARF to transcribe auxin-regulated genes. Therefore, AUX/IAA is targeted for ubiquitination and degradation.

Auxin Synthesis and Leaf Vascular Development

Auxins are synthesized in meristems and young, dividing tissues (e.g., young leaves). They induce vascular differentiation, such as xylem formation. Polar auxin transport in the leaf is arranged to promote leaf vasculature formation, with PIN1 transporting auxin to predict sites of leaf vein formation. Auxin’s role in vascular differentiation is also relevant in the plant’s response to wounding.

Determining Endogenous Auxin Levels: Promoter-Reporter Assay

By fusing a reporter gene (e.g., GUS or GFP) to an auxin-activated promoter, the endogenous level of auxin in a plant can be inferred by correlating auxin levels with the expression level of the promoter-reporter construct. A commonly used promoter is pDR5, modified from an auxin-responsive promoter in soybean. High auxin levels activate the pDR5 promoter at a high rate, leading to high expression of the fused GUS or GFP reporter gene.

Experimental Evidence for Auxin’s Physiological Roles

Experimental evidence for auxin’s role in these phenomena is substantial:

  • Apical Dominance: Auxin produced in the shoot apical meristem suppresses lateral bud growth. Removing the terminal bud releases lateral buds, but applying exogenous auxin restores suppression.
  • Lateral and Adventitious Roots: Auxin promotes their formation. Mutants with higher endogenous IAA levels, like alf1, exhibit increased lateral and adventitious root development.
  • Fruit Development: Auxin mediates fruit growth. In strawberries, achenes (which produce auxin) promote fruit development; their removal stunts growth, which can be reversed by auxin application.

Predicting Lateral Root Phenotypes in Mutant Plants

Predict the lateral root phenotype of the following mutant plants, explaining the cellular or molecular basis for the phenotype:

  • a) 35S::DFL1-expressing plant: DFL1 overexpression leads to increased conjugation of IAA to amino acids, reducing the pool of free auxin. Since auxin is essential for lateral root formation, these plants will exhibit fewer lateral roots.
  • b) LAX3 T-DNA insertion plant: The LAX3 gene encodes an auxin influx carrier expressed in cortical and epidermal cells, facilitating auxin transport for wall loosening and lateral root emergence. In a lax3 knockout mutant, these cells cannot expand effectively, resulting in fewer fully-developed lateral roots, though the number of lateral root primordia would not be reduced.
  • c) 35S::AUX1-expressing plant: The AUX1 gene encodes an auxin influx carrier expressed in the pericycle, where it promotes cell division via auxin influx. Overexpression would lead to more pericycle cells dividing to form lateral root primordia, thus resulting in more lateral roots.

Auxin’s Control Over Lateral Bud Growth Hormones

Auxin, synthesized in the shoot apical meristem (SAM), is the primary determinant of lateral bud growth (apical dominance). Its presence signals whether lateral bud growth is needed. Auxin inhibits lateral bud growth by up-regulating strigolactones (which inhibit lateral bud growth) and down-regulating cytokinins (which promote lateral bud growth). When SAM is removed, auxin synthesis decreases, perceived by AXR1 in the xylem and interfascicular sclerenchyma. AXR1 regulates SCF ubiquitin E3 ligase complexes, modulating auxin responses and cross-talk with other hormones. Reduced auxin leads to reduced strigolactone synthesis, promoting lateral bud growth. AXR1 also promotes cytokinin response by facilitating ARR5 proteolysis.

Comparing Auxin and Cytokinin Storage and Catabolism

Both auxin and cytokinin can be conjugated to various molecules for inactive storage or irreversibly degraded by oxidation.

  • Similarities: Both hormones undergo conjugation (reversible storage) and oxidation (irreversible degradation) via transferases/hydrolases.
  • Differences: Auxin can be conjugated to myo-inositol, glucose, methyl groups, amino acids, peptides, glycoproteins, or glucans. Cytokinins, derived from adenine, can be conjugated into riboside or ribotide forms for translocation or cell-to-cell transport, respectively, and also to sugars (glycosides) or alanine. The riboside/ribotide forms are unique to cytokinins due to their adenine origin, which is common in RNA.

Plant Pathogens and Cytokinin Synthesis

Plant pathogens, such as Agrobacterium tumefaciens, synthesize cytokinins (and auxins) to manipulate host plant hormone levels, affecting cell division and differentiation to facilitate invasion. Agrobacterium carries auxin and cytokinin biosynthesis genes on its T-DNA, which is inserted into the plant chromosome. The plant then uses its own resources to transcribe these genes. The resulting hormone imbalance promotes proliferation of de-differentiated cells (tumors) that synthesize opines, which are carbon and nitrogen sources beneficial only to the bacteria, not the plant host.

Auxin and Cytokinin Relationship in Apical Dominance

Auxin, produced in the shoot, is transported to cells adjacent to axillary (lateral) buds. There, it maintains apical dominance by repressing local cytokinin synthesis through down-regulation of IPT genes (cytokinin biosynthetic enzymes) and up-regulation of CKX genes (cytokinin oxidases), increasing cytokinin catabolism. When the shoot apical meristem is removed, the auxin signal is absent, promoting cytokinin production in cells near the axillary bud. Cytokinin then moves into the bud, promoting cell division and releasing it from dormancy.

Auxin:Cytokinin Ratio and Callus Differentiation

The auxin:cytokinin ratio significantly influences cellular differentiation, as seen in callus tissue and Agrobacterium tumefaciens infections. Predict and explain the phenotype of callus tissues expressing the following constructs:

  • a) 35S::CKX1 (CYTOKININ OXIDASE 1) expression: This construct increases cytokinin catabolism, reducing cellular cytokinin concentration. This elevates the auxin:cytokinin ratio, leading to callus cells differentiating into root cells.
  • b) 35S::YUC1 (YUCCA 1) expression: YUC1 is an auxin biosynthetic gene. Its expression increases auxin biosynthesis, thereby increasing the auxin:cytokinin ratio. Similar to the 35S::CKX1 mutant, callus cells will differentiate into root cells.