In 1963, Skoog isolated a cytokinin-like cell division promoter from immature maize kernels and named it zeatin. Two years later, at the proposal of Skoog and others, zeatin was renamed cytokinin (CK). With the deepening of research on cytokinins, researchers have found that cytokinins are generally produced in plant roots. They can not only promote cytoplasmic division but also facilitate the differentiation and growth of various tissues, and have a synergistic effect with plant auxins.
I. Biosynthesis and Degradation of Cytokinins
Based on structural differences, cytokinins are mainly divided into two categories: isoprenoid-type cytokinins and aromatic cytokinins. In plants, isoprenoid-type cytokinins are widely distributed and abundant, while aromatic cytokinins are relatively low in content. Therefore, the focus here is on isoprenoid-type cytokinins, mainly including isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ).
1. Biosynthesis of Cytokinins
- Biosynthesis of cis-zeatin (cZ): Dimethylallyl diphosphate (DMAPP) synthesized through the mevalonic acid (MVA) pathway in the cytoplasm reacts with tRNA under the action of tRNA-isopentenyltransferase (tRNA-IPT) to form cis-zeatin riboside monophosphate (cZRMP). cZRMP then loses the phosphate group and ribose group via cytokinin phosphoribohydrolase (LOG) to finally generate cZ.
- Biosynthesis of isopentenyladenine (iP): DMAPP synthesized through the methylerythritol phosphate (MEP) pathway in plastids reacts with ATP/ADP under the action of ATP/ADP-isopentenyltransferase (ATP/ADP-IPT) to produce two iP nucleotides, iPRTP and iPRDP. iPRTP/iPRDP are dephosphorylated to form iPRMP, which is transported to the cytoplasm through an unknown pathway. In the cytoplasm, iPRMP generates iP under the action of LOGL5/GY3 (a type of LOG enzyme).
- Biosynthesis of trans-zeatin (tZ): tZ is mainly synthesized in the vascular tissue cells of roots. Its synthesis mechanism involves the transport of iPRTP/iPRDP/iPRMP from plastids to the endoplasmic reticulum (ER) through an unknown mechanism. The side chain is oxidatively modified by cytochrome P450 monooxygenases (CYP735A1/A2) to generate tZRTP/tZRDP/tZRMP. The tZ nucleotides are transported to the cytoplasm via an unknown pathway and form tZ under the action of LOG enzymes.
- Biosynthesis of dihydrozeatin (DZ) (speculative as the mechanism is not fully clarified): tZRMP in the ER generates DZRMP under the action of reductase, which is then transferred to the cytoplasm through an unknown pathway and converted to DZ by LOG enzymes.
2. Degradation of Cytokinins
- Irreversible Degradation: Cytokinins can be catalyzed by cytokinin oxidase/dehydrogenase (CKX) to degrade into aldehydes and adenine.
- Partial Irreversible Inactivation: Cytokinins can be converted into conjugated forms (e.g., zeatin riboside) through glycosylation, acetylation, etc. In most cases, conjugated cytokinins can be reversed by degradation via specific enzymes, except for N-glycosylation occurring at the N7 or N9 position of the purine ring, which is irreversible. The rest can be reversed by β-glucosidase and other means. Conjugated cytokinins are relatively stable and participate in the storage and transport of cytokinins.
II. Transport of Cytokinins
Early studies generally believed that cytokinins are mainly synthesized in roots and then transported to shoots. However, with in-depth research, it has been found that cytokinin synthesis is not limited to roots but can occur in various cell types of both roots and shoots. These hormones reach their target cells through local and long-distance transport mechanisms.
1. Local Transport
Local transport of cytokinins involves various transporters, such as ABC family transporters (e.g., ABCI19, ABCI20, and ABCI21) expressed in the ER, which are involved in intercellular transport of cytokinins. PUP transporters (e.g., PUP7 and PUP21) and cell wall-localized CK/PN nucleotidases (e.g., OsCK/PN) jointly participate in the cell wall transport of cytokinins.
2. Long-Distance Transport
tZ synthesized in roots is transported to shoots through the xylem, while iP synthesized in shoots is transported to roots through the phloem, which constitutes the long-distance transport of cytokinins. Current studies have found that ABCG14 of the ABC transporter family mediates the outward transport of cytokinins across the plasma membrane. This gene is mainly expressed in roots and is crucial for the transport of cytokinins from roots to shoots.
A 2021 study published in Plant Physiology (IF=6.6) titled “Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins” first used semi-thin sections to confirm that AtABCG14 in Arabidopsis is expressed in the phloem companion cells and xylem tracheids of above-ground tissue veins. Through experiments expressing AtABCG14 in atabcg14 mutants using specific promoters and grafting experiments with atabcg14 mutants, the study proved that the expression of AtABCG14 in the phloem of above-ground tissues is essential for the long-distance transport of root-derived cytokinins.
Further, using grafted materials of atabcg14 and wild-type plants, methods such as isotope tracing, hormone determination in xylem sap, phloem sap, and intercellular space sap were employed to clarify the molecular mechanism of the acropetal long-distance transport of root-derived cytokinins. tZ first switches to sieve tubes for transport, is unloaded into the intercellular space by AtABCG14 localized in sieve tube companion cells, and then reaches target cells through the apoplastic transport pathway. Excess CK is transported back to roots through sieve tubes.
III. Signal Transduction of Cytokinins
The cytokinin signal transduction system is a multi-step two-component signal regulatory system composed of two types of proteins: histidine kinases and response regulators (RRs).
For example, in Arabidopsis, cytokinin receptors AHK2, AHK3, and CRE1/AHK4 (mainly present during root development) located on the plasma membrane and ER bind to auxins, triggering histidine kinase activity, which leads to the autophosphorylation of conserved histidine residues in the domain of transmitters AHPs.
Transmitters AHPs transport the activated phosphate groups from the cytoplasm to the nucleus. In the nucleus, these phosphate groups are transferred to the receiver domains of response regulators (Type B-ARRs and Type A-ARRs) and specifically bind to their conserved aspartic acid (Asp) residues, thereby activating or inhibiting the expression of auxin-regulated genes, which in turn affects plant growth and development.
A 2022 study published in Science Advances (IF=11.7) titled “Arabidopsis TIE1 and TIE2 transcriptional repressors dampen cytokinin response during root development” found through genetic transformation that both TIE1 and TIE2 are expressed in specific root regions.
To investigate the molecular mechanism by which TIE transcriptional repressors regulate root morphogenesis, researchers screened a transcription factor library and found that TIE1 can interact with key transcription factors Type B-ARRs in the cytokinin signaling pathway. Further co-immunoprecipitation and luciferase complementation experiments confirmed the in vivo interaction between transcriptional repressors (TIE1 and TIE2) and response regulators (Type B-ARR1 and Type B-ARR2). Biochemical experiments demonstrated that TIE can inhibit the transcriptional activation activity of Type B-ARRs.
Marker analysis of the cytokinin signaling pathway showed that the cell division signal is significantly enhanced in tie1 tie2 double mutants. Through a series of genetic, RNA-seq, and molecular biology methods, the researchers further proved that the root morphological changes in tie1 tie2 double mutants are due to the weakened inhibition of the activity of key transcription factor Type B-ARRs in the cell division signaling pathway, which enhances the output of cytokinin signals, thereby inhibiting primary root elongation and lateral root number.
When plants perceive cytokinin signals through cytokinin receptors AHKs, autophosphorylation first occurs on their histidine residues, followed by the transfer of phosphate groups to their aspartic acid residues. The phosphate groups on aspartic acid residues are transferred to Type B-ARRs via AHPs to activate their functions. In addition to regulating the expression of numerous genes responsive to cytokinins, Type B-ARRs also bind to the promoter regions of TIE1 and TIE2 to promote their expression. TIE1 and TIE2 interact with Type B-ARRs to inhibit their activity, promptly shutting down the signaling pathway in the absence of cytokinin signal input and avoiding excessive plant response to cytokinin signals.
IV. Functions of Cytokinins
Cytokinins are important plant hormones that play crucial roles in plant growth and development. They are involved in many physiological processes, such as plant growth and development, morphogenesis, vascular tissue differentiation, leaf senescence, regulation of apical dominance, and biotic and abiotic stress responses. The following two studies illustrate the functions of cytokinins.
1. Salt Stress
A 2023 study published in Molecular Plant (IF=17.1) titled “Cytokinin signaling promotes salt tolerance by modulating shoot chloride exclusion in maize” identified a Type A response regulator ZmRR1, which negatively regulates maize salt tolerance by negatively regulating the cytokinin signaling pathway.
On this basis, the study clarified the molecular mechanism by which cytokinins promote maize salt tolerance through regulating Cl⁻ transporters: under salt stress, the protein level of ZmRR1 decreases, relieving its inhibition on the positive regulator of cytokinin signaling ZmHP2. Subsequently, ZmHP2-mediated cytokinin signaling upregulates the expression of ZmMATE29 (encoding a vacuole-localized Cl⁻-transporting protein). ZmMATE29 sequesters Cl⁻ into the vacuoles of root cortical cells to reduce the transport of Cl⁻ from roots to above-ground parts, thereby enhancing maize salt tolerance.
2. Growth and Development
A 2021 study published in Science (IF=44.8) titled “Molecular mechanism of cytokinin-activated cell division in Arabidopsis” found through genetic screening that MYB3R1 and MYB3R4 are essential factors for maintaining stem cell activity.
ChIP-seq experiments combined with RNA fluorescence in situ hybridization proved that MYB3R1 and MYB3R4 can directly bind to the promoters of mitotic genes to promote their transcription. Subcellular localization showed that MYB3R1 is located in the nucleus, while MYB3R4 is mainly distributed in the cytoplasm.
Tracking the dynamic changes of MYB3R4 during cell division revealed that MYB3R4 is synthesized before cell division and accumulates in the cytoplasm under the regulation of nuclear export. When mitosis begins, a large amount of MYB3R4 protein is transported to the nucleus mediated by importins IMPA3 and IMPA6.
After entering the nucleus, MYB3R4 forms a dimer with MYB3R1 to activate the expression of downstream genes. Further studies found that MYB3R4 can also directly activate the transcription of IMPA3 and IMPA6 gene mRNAs.
Thus, a “positive feedback loop” is formed between MYB3R4 and IMPA3/6 to achieve the rapid nuclear localization of MYB3R4 protein within a short time (approximately 10 minutes), a mechanism that aligns with the cutting-edge plant biotechnology research and applications advanced by Dora Agri-Tech.
