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The senescence of retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD), a leading cause of blindness in the world. HSP90 is a predominant chaperone that regulates cellular homeostasis under divergent physio-pathological conditions including senescence. However, the role of HSP90 in senescent RPE cells still remains unclear. Here, we reported that HSP90 acts as a senomorphic target of senescent RPE cells in vitro. Using H2O2-induced senescent ARPE-19 cells and replicative senescent primary RPE cells from rhesus monkey, we found that HSP90 upregulates the expression of IKKα, and HIF1α in senescent ARPE-19 cells and subsequently controls the induction of distinct senescence-associated inflammatory factors. Senescent ARPE-19 cells are more resistant to the cytotoxic HSP90 inhibitor IPI504 (IC50 = 36.78 μM) when compared to normal ARPE-19 cells (IC50 = 6.16 μM). Administration of IPI504 at 0.5–5 μM can significantly inhibit the induction of IL-1β, IL-6, IL-8, MCP-1 and VEGFA in senescent ARPE-19 and the senescence-mediated migration of retinal capillary endothelial cells in vitro. In addition, we found that inhibition of HSP90 by IPI504 reduces SA-β-Gal’s protein expression and enzyme activity in a dose-dependent manner. HSP90 interacts with and regulates SA-β-Gal protein stabilization in senescent ARPE-19 cells. Taken together, these results suggest that HSP90 regulates the SASP and SA-β-Gal activity in senescent RPE cells through associating with distinctive mechanism including NF-κB, HIF1α and lysosomal SA-β-Gal. HSP90 inhibitors (e.g. IPI504) could be a promising senomorphic drug candidate for AMD intervention.

Inhibition of GSK-3 activity by novel small molecules enhances somatic embryogenesis efficiency by activating the brassinosteroid pathway, an innovation with high potential to improve in vitro plant regeneration for crop and forest breeding techniques., Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. However, in many crop and forest species, somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, a representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis genes FUS3, LEC2, and AGL15. Plant GSK-3 kinase BIN2 is a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using GSK-3β inhibitors that could be extended to other species.

Genome-wide analysis in Arabidopsis revealed that light controls CK homeostasis during early stages of adventitious root initiation (ARI). CK and JA cooperatively inhibit ARI by inducing the transcription of the transcription factor RAP2.6L., Adventitious rooting is a de novo organogenesis process that enables plants to propagate clonally and cope with environmental stresses. Adventitious root initiation (ARI) is controlled by interconnected transcriptional and hormonal networks, but there is little knowledge of the genetic and molecular programs orchestrating these networks. Thus, we have applied genome-wide transcriptome profiling to elucidate the transcriptional reprogramming events preceding ARI. These reprogramming events are associated with the down-regulation of cytokinin (CK) signaling and response genes, which could be triggers for ARI. Interestingly, we found that CK free base (iP, tZ, cZ, and DHZ) content declined during ARI, due to down-regulation of de novo CK biosynthesis and up-regulation of CK inactivation pathways. We also found that MYC2-dependent jasmonate (JA) signaling inhibits ARI by down-regulating the expression of the CYTOKININ OXIDASE/DEHYDROGENASE1 (CKX1) gene. We also demonstrated that JA and CK synergistically activate expression of the transcription factor RELATED to APETALA2.6 LIKE (RAP2.6L), and constitutive expression of this transcription factor strongly inhibits ARI. Collectively, our findings reveal that previously unknown genetic interactions between JA and CK play key roles in ARI.

In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat. Abbreviations: 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), IAA (Indole-3-acetic acid), AgNPs (silver nanoparticles), CuNPs (copper nanoparticles)

Plant aquaporins and hormones regulate root water uptake in mildly stressed poplar together with the participation of fungal aquaporins from the ectomycorrhizal fungus Laccaria bicolor., The relatively better performance of mycorrhizal plants subjected to drought stress has commonly been linked to improved root water uptake through the fungal regulation of plant aquaporins and hormones. In this study, we examined the role of ectomycorrhizal fungi in plant water relations and plant hormonal balance under mild drought using split-root seedlings of Populus trichocarpa × deltoides either with or without inoculation with Laccaria bicolor. The root compartments where the drought treatment was applied had higher ABA and lower cytokinin tZR contents, and greater expression of the plant aquaporins PtPIP1;1, PtPIP1;2, PtPIP2;5, and PtPIP2;7. On the other hand, the presence of L. bicolor within the roots down-regulated PtPIP1;4, PtPIP2;3, and PtPIP2;10, and reduced the abundance of PIP2 proteins. In addition, expression of the fungal aquaporins JQ585595 and JQ585596 were positively correlated with root ABA content, while tZR content was positively correlated with PtPIP1;4 and negatively correlated with PtPIP2;7. The results demonstrate a coordinated plant–fungal system that regulates the different mechanisms involved in water uptake in ectomycorrhizal poplar plants.

Cytokinins are one of the most important phytohormones and play essential roles in multiple life processes in planta. Root-derived cytokinins are transported to the shoots via long-distance transport. The mechanisms of long-distance transport of root-derived cytokinins remain to be demonstrated. In this study, we report that OsABCG18, a half-size ATP-binding cassette transporter from rice (Oryza sativa L.), is essential for the long-distance transport of root-derived cytokinins. OsABCG18 encodes a plasma membrane protein and is primarily expressed in the vascular tissues of the root, stem, and leaf midribs. Cytokinin profiling, as well as [14C]trans-zeatin tracer, and xylem sap assays, demonstrated that the shootward transport of root-derived cytokinins was significantly suppressed in the osabcg18 mutants. Transport assays in tobacco (Nicotiana benthamiana) indicated that OsABCG18 exhibited efflux transport activities for various substrates of cytokinins. While the mutation reduced root-derived cytokinins in the shoot and grain yield, overexpression of OsABCG18 significantly increased cytokinins in the shoot and improved grain yield. The findings for OsABCG18 as a transporter for long-distance transport of cytokinin provide new insights into the cytokinin transport mechanism and a novel strategy to increase cytokinins in the shoot and promote grain yield., OsABCG18 is essential for long-distance transport of cytokinins from root to shoot, and promotes grain yield in rice.

Enhanced cytokinin signaling results in delayed defoliation and a reduced response to ethylene in cotton treated with thidiazuron and ethephon, with crosstalk between cytokinin and ethylene signaling playing an important regulatory role., Abscission is a process that allows plants to shed tissues or organs via cell separation, and occurs throughout the life cycle. Removal of leaves through the use of chemical defoliants is very important for mechanical harvesting of cotton (Gossypium hirsutum). However, our knowledge of the molecular mechanisms of the defoliation response involved is limited. In this study, RNA-seq was conducted in order to profile the differentially expressed genes (DEGs) between cultivars X50 (sensitive to chemical defoliants) and X33 (relatively insensitive) at different time points after treatment with thidiazuron and ethephon (TE). A total of 2434 DEGs were identified between the two cultivars across the different time-points. Functional categories according to GO and KEGG analyses revealed that plant hormone signal transduction and zeatin biosynthesis were involved in the response to TE. Cytokinin oxidase/dehydrogenase (CKX) genes and ethylene-related genes were up-regulated following TE treatment, and were associated with increased level of ethylene, especially in cultivar X50. Down-regulation of GhCKX3 resulted in delayed defoliation and a reduced ethylene response. The results show that crosstalk between cytokinin and ethylene regulates cotton defoliation, and provide new insights into the molecular mechanisms underlying the mode of action of defoliants in cotton.

GhNAC83 inhibits corm dormancy release in Gladiolus hybridus by promoting ABA signaling and inhibiting CK biosynthesis., Corm dormancy is an important trait for breeding in many bulb flowers, including the most cultivated Gladiolus hybridus. Gladiolus corms are modified underground stems that function as storage organs and remain dormant to survive adverse environmental conditions. Unlike seed dormancy, not much is known about corm dormancy. Here, we characterize the mechanism of corm dormancy release (CDR) in Gladiolus. We identified an important ABA (abscisic acid) signaling regulator, GhPP2C1 (protein phosphatase 2C1), by transcriptome analysis of CDR. GhPP2C1 expression increased during CDR, and silencing of GhPP2C1 expression in dormant cormels delayed CDR. Furthermore, we show that GhPP2C1 expression is directly regulated by GhNAC83, which was identified by yeast one-hybrid library screening. In planta assays show that GhNAC83 is a negative regulator of GhPP2C1, and silencing of GhNAC83 promoted CDR. As expected, silencing of GhNAC83 decreased the ABA level, but also dramatically increased cytokinin (CK; zeatin) content in cormels. Binding assays demonstrate that GhNAC83 associates with the GhIPT (ISOPENTENYLTRANSFERASE) promoter and negatively regulates zeatin biosynthesis. Taken together, our results reveal that GhNAC83 promotes ABA signaling and synthesis, and inhibits CK biosynthesis pathways, thereby inhibiting CDR. These findings demonstrate that GhNAC83 regulates the ABA and CK pathways, and therefore controls corm dormancy.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host’s root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses., This work was supported by the Spanish Ministry of Economy and Competitiveness grant nos RyC 2014-16158, AGL2016-78002-R, and PID2019-108595RB-I00/AEI/10.13039/501100011033 to NSC, and through the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ (SEV-2015-0533). We also acknowledge financial support from the CERCA Programme/Generalitat de Catalunya, from the Austrian Academy of Sciences through the Gregor Mendel Institute (WB and SBS) and from funding for CTA and HMB by the United States Department of Agriculture (USDA) grant no. COL00781., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Structural components of second messenger signaling (nucleotides and associated enzyme systems) within plant and animal cells have more in common than the hormones that initiate metabolic and functional changes. Neurotransmitters and hormones of mammalian pharmacologic classes relate to purine nucleotides in respect of chemical structure and the molecular changes they initiate. This study compares the molecular structures of purine nucleotides with compounds from the abscisic acid, auxin, brassinosteroid, cytokinin, gibberellin, and jasmonate classes by means of a computational program. The results illustrate how phytohomones relate to each other through the structures of nucleotides and cyclic nucleotides. Molecular similarity within the phytohormone structures relates to synergism, antagonism and the modulation of nucleotide function that regulates germination and plant development. As with the molecular evolution of mammalian hormones, cell signaling and cross-talk within the phytohormone classes is purine nucleotide centered.

CIPK25, a calcium-regulated protein kinase, plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development., Co-ordination of auxin and cytokinin activities determines root meristem size during post-embryonic development. Calcineurin B-like proteins (CBLs) and their interacting protein kinases (CIPKs) constitute signaling modules that relay calcium signals. Here we report that CIPK25 is involved in regulating the root meristem size. Arabidopsis plants lacking CIPK25 expression displayed a short root phenotype and a slower root growth rate with fewer meristem cells. This phenotype was rescued by restoration of CIPK25 expression. CIPK25 interacted with CBL4 and -5, and displayed strong gene expression in the flower and root, except in the cell proliferation domain in the root apical meristem. Its expression in the root was positively and negatively regulated by auxin and cytokinin, respectively. The cipk25 T-DNA insertion line was compromised in auxin transport and auxin-responsive promoter activity. The cipk25 mutant line showed altered expression of auxin efflux carriers (PIN1 and PIN2) and an Aux/IAA family gene SHY2. Decreased PIN1 and PIN2 expression in the cipk25 mutant line was completely restored when combined with a SHY2 loss-of-function mutation, resulting in recovery of root growth. SHY2 and PIN1 expression was partially regulated by cytokinin even in the absence of CIPK25, suggesting a CIPK25-independent cytokinin signaling pathway(s). Our results revealed that CIPK25 plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development.

CUC1 and CUC2 regulate ovule and seed number by modulating the expression of genes involved in the balance between active and inactive forms of glycosylated cytokinin., Seeds derive from ovules upon fertilization and therefore the total number of ovules determines the final seed yield, a fundamental trait in crop plants. Among the factors that co-ordinate the process of ovule formation, the transcription factors CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 and the hormone cytokinin (CK) have a particularly prominent role. Indeed, the absence of both CUC1 and CUC2 causes a severe reduction in ovule number, a phenotype that can be rescued by CK treatment. In this study, we combined CK quantification with an integrative genome-wide target identification approach to select Arabidopsis genes regulated by CUCs that are also involved in CK metabolism. We focused our attention on the functional characterization of UDP-GLUCOSYL TRANSFERASE 85A3 (UGT85A3) and UGT73C1, which are up-regulated in the absence of CUC1 and CUC2 and encode enzymes able to catalyse CK inactivation by O-glucosylation. Our results demonstrate a role for these UGTs as a link between CUCs and CK homeostasis, and highlight the importance of CUCs and CKs in the determination of seed yield.

Potato genes for putative cytokinin receptors were cloned and expressed. The proteins displayed properties of cytokinin receptors, and their expression in planta was organ specific and sensitive to growth conditions and sucrose content., Potato is the most economically important non-cereal food crop. Tuber formation in potato is regulated by phytohormones, cytokinins (CKs) in particular. The present work studied CK signal perception in potato. The sequenced potato genome of doubled monoploid Phureja was used for bioinformatic analysis and as a tool for identification of putative CK receptors from autotetraploid potato cv. Désirée. All basic elements of multistep phosphorelay required for CK signal transduction were identified in the Phureja genome, including three genes orthologous to three CK receptor genes (AHK 2–4) of Arabidopsis. As distinct from Phureja, autotetraploid potato contains at least two allelic isoforms of each receptor type. Putative receptor genes from Désirée plants were cloned, sequenced and expressed, and the main characteristics of encoded proteins were determined, in particular their consensus motifs, modelled structure, ligand-binding properties, and ability to transmit CK signals. In all studied aspects the predicted sensor histidine kinases met the requirements for genuine CK receptors. Expression of potato CK receptors was found to be organ-specific and sensitive to growth conditions, particularly to sucrose content. Our results provide a solid basis for further in-depth study of CK signaling system and biotechnological improvement of potato.

In a two-branched pea system, growth and IAA export from branches are controlled by a xylem cytokinin feedback loop regulated by apical auxin., A model system of 10–12 day-old, two-branched (2-B) pea (Pisum sativum L. cv. Adagumsky) seedlings was used to study the roles of endogenous auxin indole-3-acetic acid (IAA) and cytokinins (CKs) in the interaction between the shoots. The IAA export activity (IEA) from shoots was 2-fold higher in one-branched (1-B) plants with one shoot removed than in the 2-B plants, while tZ-type cytokinin contents in xylem sap were 4-fold greater in the 1-B plants than in 2-B plants. Exogenous 6-benzylaminopurine introduced into the vascular stream of one shoot enhanced its IEA. Therefore, xylem cytokinin appears to control both growth and IEA in branches. In the hypocotyls of 1-B and 2-B plants, IAA contents were equal in both cases, while the levels of tZ-type cytokinins were different. These data do not agree with the well-supported role of auxin in down-regulating CK content. The observed paradox may be explained by assuming that a steady-state IAA level in the hypocotyls is feedback regulated via xylem cytokinin, which controls the delivery of IAA from the shoots. As a result, the level of IAA in the hypocotyl is most likely maintained at a threshold below which a decrease in auxin content can switch on CK synthesis that will increase xylem cytokinin levels, thereby stabilizing the level of IAA in the hypocotyl. Therefore, our results suggest that correlative inhibition in the 2-B pea system is a function of an IAA/CK feedback loop, in which cytokinin essentially acts as a second messenger for IAA.

Auxin-dependent cell expansion is crucial for initiation of fiber cells in cotton (Gossypium hirsutum), which ultimately determines fiber yield and quality. However, the regulation of this process is far from being well understood. In this study, we demonstrate an antagonistic effect between cytokinin (CK) and auxin on cotton fiber initiation. In vitro and in planta experiments indicate that enhanced CK levels can reduce auxin accumulation in the ovule integument, which may account for the defects in the fiberless mutant xu142fl. In turn, supplementation with auxin can recover fiber growth of CK-treated ovules and mutant ovules. We further found that GhPIN3a is a key auxin transporter for fiber-cell initiation and is polarly localized to the plasma membranes of non-fiber cells, but not to those of fiber cells. This polar localization allows auxin to be transported within the ovule integument while specifically accumulating in fiber cells. We show that CKs antagonize the promotive effect of auxin on fiber cell initiation by undermining asymmetric accumulation of auxin in the ovule epidermis through down-regulation of GhPIN3a and disturbance of the polar localization of the protein., Cytokinin antagonizes the promotive effect of auxin on cotton fiber initiation by disrupting the localization of GhPIN3a, which mediates asymmetric accumulation of auxin in the ovule epidermis.

The formation of the haustorium in holoparasitic plants as demonstrated in Phelipanche ramosa is initiated through perception of a host-exudated cytokinin and a cytokinin signaling response., The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.

Rice WOX11 plays a regulatory role high in the hierarchy of control of multiple processes such as hormone signaling, stress response, redox, and metabolism during crown root development., The rice root system is mainly composed of post-embryonic shoot-borne roots called crown roots. WOX11, encoding a WUSCHEL-related homeobox domain transcription factor, is a key regulator of crown root growth and development in rice (Oryza sativa. L). In addition to specifically activating crown root development, WOX11 is also involved in lateral root initiation, root hair formation, and abiotic stresses. However, the gene regulatory network downstream of WOX11 remains largely unknown. Here, we studied the transcriptome of wox11 root tips by RNA-Seq and determined direct WOX11-binding targets by bioinformatic and biochemical analysis. The transcriptomic analysis revealed 664 differentially expressed genes, which covered a wide range of functions related to root development, cytokinin homeostasis/signaling, stress response, and redox metabolic processes. Bioinformatic analysis also revealed that the WOX11-binding motif was distributed over 41% (273/664) of the differentially expressed genes, and was mostly enriched in the promoter and intron regions. We used qRT-PCR and/or in situ hybridization to confirm co-expression of some of the WOX11-regulated genes in crown root development. We also used electrophoretic mobility shift assay and chromatin immunoprecipitation with anti-WOX11 antibody to validate direct regulation of these genes by WOX11. The analysis identified several genes that acted downstream of WOX11 in controlling crown root formation, cytokinin signaling, stress response, and redox metabolism. This work built a hierarchical regulatory model of WOX11 in rice crown root development.

Only AHP2, AHP3, and AHP5 are downstream targets of CKI1 in regulating Arabidopsis female gametophyte development., Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs). CYTOKININ INDEPENDENT 1 (CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are five HP genes in Arabidopsis, AHP1–AHP5, but it remains unknown which AHP genes act downstream of CKI1 in Arabidopsis female gametophyte development. Promoter activity analysis of AHP1–AHP5 in embryo sacs revealed AHP1, AHP2, AHP3, and AHP5 expression in the central cell. Phenotypic studies of various combinations of ahp mutants showed that triple mutations in AHP2, AHP3, and AHP5 resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, the ahp2-2 ahp3 ahp5-2/+ triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling the cki1 mutant phenotypes. These data suggest that AHP2, AHP3, and AHP5 are the major factors acting downstream of CKI1 in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.

Albinotic inflorescence stems of Arabidopsis plants overexpressing the cytokinin-responsive gene Cytokinin-induced F-box encoding (CFB) phenocopy the sterol biosynthesis mutant cas1-1 and accumulate 2,3-oxidosqualene, suggesting a link between cytokinin and sterol biosynthesis., Protein degradation by the ubiquitin–26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway.

Highlight Manipulation of cytokinin levels in tomato plants affected transpiration rate. While the hormone had no effect on stomatal movement, it increased stomatal density by promoting epidermal cell division., Previous studies on cytokinin (CK) and drought have suggested that the hormone has positive and negative effects on plant adaptation to restrictive conditions. This study examined the effect of CK on transpiration, stomatal activity, and response to drought in tomato (Solanum lycopersicum) plants. Transgenic tomato plants overexpressing the Arabidopsis thaliana CK-degrading enzyme CK oxidase/dehydrogenase 3 (CKX3) maintained higher leaf water status under drought conditions due to reduced whole-plant transpiration. The reduced transpiration could be attributed to smaller leaf area and reduced stomatal density. CKX3-overexpressing plants contained fewer and larger pavement cells and fewer stomata per leaf area than wild-type plants. In addition, wild-type leaves treated with CK exhibited enhanced transpiration and had more pavement cells and increased numbers of stomata per leaf area than untreated leaves. Manipulation of CK levels did not affect stomatal movement or abscisic acid-induced stomatal closure. Moreover, we found no correlation between stomatal aperture and the activity of the CK-induced promoter Two-Component Signaling Sensor (TCS) in guard cells. Previous studies have shown that drought reduces CK levels, and we propose this to be a mechanism of adaptation to water deficiency: the reduced CK levels suppress growth and reduce stomatal density, both of which reduce transpiration, thereby increasing tolerance to prolonged drought conditions.

Partial defoliation up-regulated photosynthesis and down-regulated expression of sucrose export-related genes in tomato leaves, and increased trans-zeatin riboside in the roots, implicating this cytokinin in root–shoot signaling., Photosynthetic activity is affected by exogenous and endogenous inputs, including source–sink balance. Reducing the source to sink ratio by partial defoliation or heavy shading resulted in significant elevation of the photosynthetic rate in the remaining leaf of tomato plants within 3 d. The remaining leaf turned deep green, and its area increased by almost 3-fold within 7 d. Analyses of photosynthetic activity established up-regulation due to increased carbon fixation activity in the remaining leaf, rather than due to altered water balance. Moreover, senescence of the remaining leaf was significantly inhibited. As expected, carbohydrate concentration was lower in the remaining leaf than in the control leaves; however, expression of genes involved in sucrose export was significantly lower. These results suggest that the accumulated fixed carbohydrates were primarily devoted to increasing the size of the remaining leaf. Detailed analyses of the cytokinin content indicated that partial defoliation alters cytokinin biosynthesis in the roots, resulting in a higher concentration of trans-zeatin riboside, the major xylem-translocated molecule, and a higher concentration of total cytokinin in the remaining leaf. Together, our findings suggest that trans-zeatin riboside acts as a signal molecule that traffics from the root to the remaining leaf to alter gene expression and elevate photosynthetic activity.

Cytokinin promotes photomorphogenesis independent of ethylene, acting through the two-component signaling system and requiring a functional light-signaling system., The transition of skotomorphogenesis to photomorphogenesis is induced by the perception of light, and is characterized by the inhibition of hypocotyl elongation and opening of cotyledons. Although it is known that the plant hormone cytokinin inhibits hypocotyl elongation in dark-grown Arabidopsis plants when applied in high concentrations, it is unclear to what extent this response is the result of cytokinin alone or cytokinin-induced ethylene production. Here, we show that cytokinin-induced inhibition of hypocotyl elongation is largely independent of ethylene and suggest a close connection between the cytokinin two-component system and the light-signaling networks. We show that this cytokinin signal is mainly mediated through the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE3 and the ARABIDOPSIS RESPONSE REGULATOR1 in combination with ARR12. Interestingly, mutation of CONSTITUTIVELY PHOTOMORPOGENIC1 (COP1), DE-ETIOLATED1, and CYTOKININ INSENSITIVE4/COP10 renders plants insensitive to cytokinin, and these factors are indispensable for the transcriptional response during cytokinin-induced de-etiolation, indicating that a functional light-signaling pathway is essential for this cytokinin response. In addition, the effect of cytokinin on hypocotyl elongation is strongly dependent on the light conditions, with higher light intensities causing a switch in the response to cytokinin from an inhibitor to a promoter of hypocotyl elongation.

Highlight Recent research shows that sugar availability triggers bud outgrowth. This paper further demonstrates that the effect of sucrose involves changes in the hormonal network related to bud outgrowth, and identifies potential hormones involved in sugar control., Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.

Highlight Plants with altered intracellular Pi compartmentation surprisingly show accelerated dark-induced senescence. We propose that low cytosolic phosphate concentrations are of critical importance for proper plant development., The phosphate transporter PHT4;6 locates to the trans-Golgi compartment, and its impaired activity causes altered intracellular phosphate compartmentation, leading to low cytosolic Pi levels, a blockage of Golgi-related processes such as protein glycosylation and hemicellulose biosynthesis, and a dwarf phenotype. However, it was unclear whether altered Pi homeostasis in pht4;6 mutants causes further cellular problems, typically associated with limited phosphate availability. Here we report that pht4;6 mutants exhibit a markedly increased disposition to induce dark-induced senescence. In control experiments, in which pht4;6 mutants and wild-type plants developed similarly, we confirmed that accelerated dark-induced senescence in mutants is not a ‘pleiotropic’ process associated with the dwarf phenotype. In fact, accelerated dark-induced senescence in pht4;6 mutants correlates strongly with increased levels of toxic NH4 + and higher sensitivity to ammonium, which probably contribute to the inability of pht4;6 mutants to recover from dark treatment. Experiments with modified levels of either salicylic acid (SA) or trans-zeatin (tZ) demonstrate that altered concentrations of these compounds in pht4;6 plants act as major cellular mediators for dark-induced senescence. This conclusion gained further support from the notion that the expression of the pht4;6 gene is, in contrast to genes coding for major phosphate importers, substantially induced by tZ. Taken together, our findings point to a critical function of PHT4;6 to control cellular phosphate levels, in particular the cytosolic Pi availability, required to energize plant primary metabolism for proper plant development. Phosphate and its allocation mediated by PHT4;6 is critical to prevent onset of dark-induced senescence.

Highlight Decisive role for cytokinins in stimulation of transpiration during the early heat stress response was demonstrated. Longer term positive effects of cytokinins were associated with elevation of stress- and photosynthesis-related proteins and transcripts., Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS – targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.

Highlight The relationship between an adenine isopentenyltransferase transgene, reactive oxygen species (ROS) content and the ROS-scavenging system, mechanistically contributing to improved root growth under drought stress in creeping bentgrass, is discussed., Drought stress limits root growth and inhibits cytokinin (CK) production. Increases in CK production through overexpression of isopentenyltransferase (ipt) alleviate drought damages to promote root growth. The objective of this study was to investigate whether CK-regulated root growth was involved in the alteration of reactive oxygen species (ROS) production and ROS scavenging capacity under drought stress. Wild-type (WT) creeping bentgrass (Agrostis stolonifera L. ‘Penncross’) and a transgenic line (S41) overexpressing ipt ligated to a senescence-activated promoter (SAG12) were exposed to drought stress for 21 d in growth chambers. SAG12-ipt transgenic S41 developed a more extensive root system under drought stress compared to the WT. Root physiological analysis (electrolyte leakage and lipid peroxidation) showed that S41 roots exhibited less cellular damage compared to the WT under drought stress. Roots of SAG12-ipt transgenic S41 had significantly higher endogenous CK content than the WT roots under drought stress. ROS (hydrogen peroxide and superoxide) content was significantly lower and content of total and free ascorbate was significantly higher in S41 roots compared to the WT roots under drought stress. Enzymatic assays and transcript abundance analysis showed that superoxide dismutase, catalase, peroxidase, and dehydroascorbate reductase were significantly higher in S41 roots compared to the WT roots under drought stress. S41 roots also maintained significantly higher alternative respiration rates compared to the WT under drought stress. The improved root growth of transgenic creeping bentgrass may be facilitated by CK-enhanced ROS scavenging through antioxidant accumulation and activation of antioxidant enzymes, as well as higher alternative respiration rates when soil water is limited.

Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.

Fruit-localized phytochromes and their downstream signaling cascades not only modulate chloroplast biogenesis in immature tomato fruits but also regulate sugar and carotenoid accumulation, two essential features of tomato fruit quality., Light signaling has long been reported to influence fruit biology, although the regulatory impact of fruit-localized photoreceptors on fruit development and metabolism remains unclear. Studies performed in phytochrome (PHY)-deficient tomato (Solanum lycopersicum) mutants suggest that SlPHYA, SlPHYB2, and to a lesser extent SlPHYB1 influence fruit development and ripening. By employing fruit-specific RNAi-mediated silencing of SlPHY genes, we demonstrated that fruit-localized SlPHYA and SlPHYB2 play contrasting roles in regulating plastid biogenesis and maturation in tomato. Our data revealed that fruit-localized SlPHYA, rather than SlPHYB1 or SlPHYB2, positively influences tomato plastid differentiation and division machinery via changes in both light and cytokinin signaling-related gene expression. Fruit-localized SlPHYA and SlPHYB2 were also shown to modulate sugar metabolism in early developing fruits via overlapping, yet distinct, mechanisms involving the co-ordinated transcriptional regulation of genes related to sink strength and starch biosynthesis. Fruit-specific SlPHY silencing also drastically altered the transcriptional profile of genes encoding light-repressor proteins and carotenoid-biosynthesis regulators, leading to reduced carotenoid biosynthesis during fruit ripening. Together, our data reveal the existence of an intricate PHY–hormonal interplay during fruit development and ripening, and provide conclusive evidence on the regulation of tomato quality by fruit-localized phytochromes.

Highlight To antagonize the developmental process initiated by Rhodococcus fascians and in response to the bacterial cytokinins, Arabidopsis activates its strigolactone response, partially suppressing shoot branching in the rosette., Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures—including the strigolactone response—in reaction to bacterial cytokinins to constrain the activity of R. fascians.

Highlight Comparative studies using Arabidopsis and radish (Raphanus sativus) found that cytokinin-mediated regulatory programmes in the cambium are important for the radial growth of radish roots and its variations., The root serves as an essential organ in plant growth by taking up nutrients and water from the soil and supporting the rest of the plant body. Some plant species utilize roots as storage organs. Sweet potatoes (Ipomoea batatas), cassava (Manihot esculenta), and radish (Raphanus sativus), for example, are important root crops. However, how their root growth is regulated remains unknown. In this study, we characterized the relationship between cambium and radial root growth in radish. Through a comparative analysis with Arabidopsis root expression data, we identified putative cambium-enriched transcription factors in radish and analysed their expression in representative inbred lines featuring distinctive radial growth. We found that cell proliferation activities in the cambium positively correlated with radial growth and final yields of radish roots. Expression analysis of candidate transcription factor genes revealed that some genes are differentially expressed between inbred lines and that the difference is due to the distinct cytokinin response. Taken together, we have demonstrated for the first time, to the best of our knowledge, that cytokinin-dependent radial growth plays a key role in the yields of root crops.

Highlight The phenotype of the E151 pea mutant, unique among known legume mutants, provides the first evidence for a promoting role for cytokinins in the development of the mycorrhizal symbiosis., In legumes, the formation of rhizobial and mycorrhizal root symbioses is a highly regulated process which requires close communication between plant and microorganism. Plant mutants that have difficulties establishing symbioses are valuable tools for unravelling the mechanisms by which these symbioses are formed and regulated. Here E151, a mutant of Pisum sativum cv. Sparkle, was examined to characterize its root growth and symbiotic defects. The symbioses in terms of colonization intensity, functionality of micro-symbionts, and organ dominance were compared between the mutant and wild type. The endogenous cytokinin (CK) and abscisic acid (ABA) levels and the effect of the exogenous application of these two hormones were determined. E151 was found to be a low and delayed nodulator, exhibiting defects in both the epidermal and cortical programmes though a few mature and functional nodules develop. Mycorrhizal colonization of E151 was intensified, although the fungal functionality was impaired. Furthermore, E151 displayed an altered lateral root (LR) phenotype compared with that of the wild type whereby LR emergence is initially delayed but eventually overcome. No differences in ABA levels were found between the mutant and the wild type, but non-inoculated E151 exhibited significantly high CK levels. It is hypothesized that CK plays an essential role in differentially mediating the entry of the two micro-symbionts into the cortex; whereas it would inhibit the entry of the rhizobia in that tissue, it would promote that of the fungus. E151 is a developmental mutant which may prove to be a useful tool in further understanding the role of hormones in the regulation of beneficial root symbioses.

Highlight The homeostasis of biologically active cytokinins was the predominant leaf-inherent factor for winter oilseed rape cultivar-differences in nitrogen starvation-induced leaf senescence and thus nitrogen efficiency., Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.

Highlight Cytokinin synthesis gene expression patterns and mutant phenotypes show the relevance of cytokinins in generating a paracrine signal to regulate lateral root spacing, which is important in shaping root system architecture., The root systems of plants have developed adaptive architectures to exploit soil resources. The formation of lateral roots (LRs) contributes to root system architecture. Roots of plants with a lower cytokinin status form LR primordia (LRP) in unusually close proximity, indicating a role for the hormone in regulating the positioning of LRs along the main root axis. Data obtained from cytokinin-synthesis mutants of Arabidopsis thaliana combined with gene expression analysis indicate that cytokinin synthesis by IPT5 and LOG4 occurring early during LRP initiation generates a local cytokinin signal abbreviating LRP formation in neighbouring pericycle cells. In addition, IPT3, IPT5, and IPT7 contribute to cytokinin synthesis in the vicinity of existing LRP, thus suppressing initiation of new LRs. Interestingly, mutation of CYP735A genes required for trans-zeatin biosynthesis caused strong defects in LR positioning, indicating an important role for this cytokinin metabolite in regulating LR spacing. Further it is shown that cytokinin and a known regulator of LR spacing, the receptor-like kinase ARABIDOPSIS CRINKLY4 (ACR4), operate in a non-hierarchical manner but might exert reciprocal control at the transcript level. Taken together, the results suggest that cytokinin acts as a paracrine hormonal signal in regulating root system architecture.

Highlight Cytokinin receptors studied in a novel plant assay system recognize cytokinin ribosides poorly, unlike cytokinin bases. Molecular modelling explained this receptor feature. Some receptors were suggested to function as pH sensors., Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N 9-ribosylation alters the bonding pattern in cytokinin–receptor interaction and prevents β6–β7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0–5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.

© The Author 2014. Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.

Key message Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. Abstract The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11–39 nmol g−1 DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121–424 pmol g−1 DW) and zeatin ryboside (cZR, 146–432 pmol g−1 DW). Surprisingly, a relatively high level (10–64 pmol g−1 DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of ‘responsive’ DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with ‘recalcitrant’ genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential. Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1686-4) contains supplementary material, which is available to authorized users.