The C2H2 Zinc-Finger protein ELR1 negatively regulates cytokinin metabolism and signaling pathway in rice.

The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis

The two-component signaling systems (TCSs), which mediate the histidine-aspartate signaling, control diverse biological processes of many organisms, including cell division, cell growth and proliferation, and responses to environmental stimuli and growth regulators. We have provided in planta evidence that the cytokinin (CK) responsive TCS mediates abscisic acid (ABA) and osmotic stress responses. By using loss-of-function approach we have demonstrated that the three cytokinin (CK) receptor histidine kinases AHK2, AHK3 and AHK4/CRE1 act as negative regulators in ABA, drought and high salinity stress signalings. Genome-wide expression profiling of the stress-tolerant double mutant suggested that CK receptor kinases mediate osmotic stress response in both an ABA-dependent and ABA-independent manner. Additionally, we showed evidence as for the role of CK in mediating stress responses, judging from the fact that AHK4 requires the CK to function as a negative regulator in osmotic stress response. Our results suggested that cross-talks exist among CK, ABA and osmotic stress signaling pathways, and that CK signaling and CK metabolism may play crucial roles not only in plant growth and development but also in osmotic stress signaling.

Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole-3-acetic acid (IAA)-amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA-amino acid conjugates, and MsGH3.5-overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type-B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.

Inflorescence development in cereals, including such important crops as rice, maize, and wheat, directly affects grain number and size and is a key determinant of yield. Cytokinin regulates meristem size and activity and, as a result, has profound effects on inflorescence development and architecture. To clarify the role of cytokinin action in inflorescence development, we used the NanoString nCounter system to analyze gene expression in the early stages of rice panicle development, focusing on 67 genes involved in cytokinin biosynthesis, degradation, and signaling. Results point toward key members of these gene families involved in panicle development and indicate that the expression of many genes involved in cytokinin action differs between the panicle and vegetative tissues. Dynamic patterns of gene expression suggest that subnetworks mediate cytokinin action during different stages of panicle development. The variation of expression during panicle development is greater among genes encoding proteins involved in cytokinin metabolism and negative regulators of the pathway than for the genes in the primary response pathway. These results provide insight into the expression patterns of genes involved in cytokinin action during inflorescence development in a crop of agricultural importance, with relevance to similar processes in other monocots. The identification of subnetworks of genes expressed at different stages of early panicle development suggests that manipulation of their expression could have substantial effects on inflorescence architecture.

The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s (della1 della2) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation.

Plant hormones modulate plant growth, development, and defense. However, many aspects of the origin and evolution of plant hormone signaling pathways remain obscure. Here, we use a comparative genomic and phylogenetic approach to investigate the origin and evolution of nine major plant hormone (abscisic acid, auxin, brassinosteroid, cytokinin, ethylene, gibberellin, jasmonate, salicylic acid, and strigolactone) signaling pathways. Our multispecies genome-wide analysis reveals that: (1) auxin, cytokinin, and strigolactone signaling pathways originated in charophyte lineages; (2) abscisic acid, jasmonate, and salicylic acid signaling pathways arose in the last common ancestor of land plants; (3) gibberellin signaling evolved after the divergence of bryophytes from land plants; (4) the canonical brassinosteroid signaling originated before the emergence of angiosperms but likely after the split of gymnosperms and angiosperms; and (5) the origin of the canonical ethylene signaling pathway postdates shortly the emergence of angiosperms. Our findings might have important implications in understanding the molecular mechanisms underlying the emergence of land plants.

Salt stress negatively affects plant growth by impairing biochemical and physiological processes. Appropriate modulation of cytokinin (CK) metabolism and signaling can improve salt tolerance in plants. Protection of the photosynthetic apparatus, promotion of antioxidant systems, improvement of plant growth and differentiation, and crosstalk with stress-related phytohormones are important mechanisms that may contribute to cytokinin-mediated enhancement of salt tolerance. CKs mainly trigger plant environmental stress responses through the regulation of gene expression. A two-component system is employed to transduce the cytokinin signal to the target genes. CKs are perceived by membrane-localized histidine kinase receptors. The signal is transduced through a His-Asp phosphorelay (Histidine-aspartate phosphorelays) to activate a family of transcription factors in the nucleus. CKs cause organ specific responses in plants. This hormone is a negative regulator of root growth. Root-specific overexpression of CKX (cytokinin oxidase/dehydrogenase) gene can enhance root growth, nutrient uptake and salt tolerance. In contrast, increasing cytokinin level (by overexpression of IPT genes) promotes shoot growth of salt stressed plants, by inducing the expression of genes that are involved in photosynthesis, chlorophyll levels, photochemical quenching, photochemical efficiency, electron transport rates and CO2 assimilation. This chapter focuses on the cytokinin metabolism, transport and signaling, and discusses the role of this phytohormone in regulating changes in gene expression and physiological processes to mediate salt tolerance in plants. KeywordsCytokinin signalingGene expressionPhysiological processesSalinity

3 - Cytokinins

Crosstalk between the cytokinin and MAX2 signaling pathways in growth and callus formation of Arabidopsis thaliana

Cell–cell communication is critical for tissue and organ development. In plants, secretory CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides function as intercellular signaling molecules in various aspects of tissue development including vascular development. However, little is known about intracellular signaling pathways functioning in vascular development downstream of the CLE ligands. We show that CLE peptides including CLE10, which is preferentially expressed in the root vascular system, inhibit protoxylem vessel formation in Arabidopsis roots. GeneChip analysis displayed that CLE10 peptides repressed specifically the expression of two type-A Arabidopsis Response Regulators (ARRs), ARR5 and ARR6, whose products act as negative regulators of cytokinin signaling. The arr5 arr6 roots exhibited defective protoxylem vessel formation. These results indicate that CLE10 inhibits protoxylem vessel formation by suppressing the expression of type-A ARR genes including ARR5 and ARR6. This was supported by the finding that CLE10 did not suppress protoxylem vessel formation in a background of arr10 arr12, a double mutant of type-B ARR genes. Thus, our results revealed cross-talk between CLE signaling and cytokinin signaling in protoxylem vessel formation in roots. Taken together with the indication that cytokinin signaling functions downstream of the CLV3/WUS signaling pathway in the shoot apical meristem, the cross-talk between CLE and cytokinin signaling pathways may be a common feature in plant development.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

Using mutant plants of Arabidopsis thaliana, participation of the genes involved in abscisic acid (ABA) and cytokinins (CKs) metabolism and signaling in plant defense responses to heat shock (HS) was investigated. The magnitude of the stress action was assessed with biochemical indicators, such as accumulation of proline and malonic dialdehyde (MDA) and changes in the content of gene transcripts of heat shock proteins (HSPs) (HSP90.1 and HSP90.5) and transcription factor of HS (HSFA2) as well as stress-inducible genes, markers of oxidative stress (AOX1a, RD29, PRODH1, and P5CS1). Mutants with inactivated genes of ABA synthesis and, especially, signal perception exhibited lower thermo-resistance and accumulated elevated amounts of CK metabolism and signal transduction genes. In contrast, plants with inactivated components of CK synthesis and signal perception displayed increased tolerance to high temperatures and reduced levels of mRNA of oxidative stress genes as compared to wild type (WT) plants. However, enhancement of HS contributed to decrease of thermo-resistance of CKs receptor mutants up to the level of WT plants. Under hyperthermia, the stimulation of the plant defense mechanisms was accompanied by downregulation of the expression of CK metabolism and signal transduction genes (IPT3, CKX1, ARR5, AHK2, and AHK4) and ABA catabolism gene (CYP707A1) and upregulation of the expression of ABA synthesis and signal perception genes (ABA3 and ABI2). In the mutants insensitive to ABA, CYP707A1 gene was upregulated under the HS, while the expression of CK receptor gene expression did not reliably change. The results indicate that the response of the plants to elevated temperatures was determined not only by strength and the duration of the stress but the state of their ABA and CK metabolic and signaling systems as well.

A decoy receptor derived from alternative splicing fine-tunes cytokinin signaling in Arabidopsis

The Antagonistic Action of Abscisic Acid and Cytokinin Signaling Mediates Drought Stress Response in Arabidopsis

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.