Zeatin: The 60th anniversary of its identification.

While various labs had shown cell division-inducing activity in a variety of plant extracts for over a decade, the identification of zeatin (Z) in 1964, the first known naturally occurring cytokinin, belongs to Letham and co-workers. Using extracts from maize (Zea mays), they were the first to obtain crystals of pure Z and in sufficient quantity for structural determination by MS, NMR, chromatography, and mixed melting-point analysis. This group also crystallized Z-9-riboside (ZR) from coconut (Cocos nucifera) milk. However, their chemical contributions go well beyond the identification of Z and ZR and include two unambiguous syntheses of trans-Z (to establish stereochemistry), the synthesis of 3H-cytokinins that facilitated metabolic studies, and the synthesis of deuterated internal standards for accurate mass spectral quantification. Letham and associates also unequivocally identified Z nucleotide, the 7-and 9-glucoside conjugates of Z, and the O-glucosides of Z, ZR, dihydro Z (DHZ) and DHZR as endogenous compounds and as metabolites of exogenous Z. Their contributions to the role of cytokinins in plant physiology and development were also substantial, especially the role of cytokinins moving in the xylem. These biological advances are described and briefly related to the genetic/molecular biological contributions of others that established that plants have an absolute requirement for cytokinin.

The trans-zeatin-type side-chain modification of cytokinins controls rice growth.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

Flavin mononucleotide regulated photochemical isomerization and degradation of zeatin.

cis-Zeatin (cZ), a cytokinin often overlooked compared to trans-zeatin (tZ), can now be controlled in live cells and plants through a new biocompatible reaction. Using flavin photosensitizers, cZ can be isomerized to tZ or degraded, depending on the presence of a reducing reagent. This breakthrough offers a novel approach for regulating plant growth through chemical molecules.

Wheat cis-zeatin-O-glucosyltransferase cZOGT1 interacts with the Ca2+-dependent lipid binding protein TaZIP to regulate senescence.

Premature senescence is an important factor affecting wheat yield and quality. Wheat yield can be increased by delaying senescence and prolonging the effective photosynthetic time. Previously, we found that the cis-zeatin-O-glucosyltransferase (cZOGT1) gene plays an important role in the stay-green wheat phenotype. In this study, cZOGT1-overexpressing lines exhibited a delayed senescence phenotype, despite a significant reduction in the total cytokinin content. Further, we found that cZOGT1 interacted with the Ca2+-dependent lipid binding protein TaZIP (cZOGT1-interacting protein), and that a high level of cZOGT1 expression led to the suppression of TaZIP expression, which in turn, reduced abscisic acid (ABA) content. The synergistic reduction in cytokinins and ABA levels eventually caused the stay-green phenotype in cZOGT1-overexpressing lines. This study provides a new theoretical basis to explain the mechanism underlying the wheat stay-green phenotype and provides a genetic resource for wheat molecular-design breeding.

Physiological and Transcriptomic Analysis Provides Insights into Low Nitrogen Stress in Foxtail Millet (Setaria italica L.).

Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop that is well adapted to nutrient-poor soils. However, our understanding of how different LN-tolerant foxtail millet varieties adapt to long-term low nitrogen (LN) stress at the physiological and molecular levels remains limited. In this study, two foxtail millet varieties with contrasting LN tolerance properties were investigated through analyses of physiological parameters and transcriptomics. The physiological results indicate that JG20 (high tolerance to LN) exhibited superior biomass accumulation both in its shoots and roots, and higher nitrogen content, soluble sugar concentration, soluble protein concentration, zeatin concentration in shoot, and lower soluble sugar and soluble protein concentration in its roots compared to JG22 (sensitive to LN) under LN, this indicated that the LN-tolerant foxtail millet variety can allocate more functional substance to its shoots to sustain aboveground growth and maintain high root activity by utilizing low soluble sugar and protein under LN conditions. In the transcriptomics analysis, JG20 exhibited a greater number of differentially expressed genes (DEGs) compared to JG22 in both its shoots and roots in response to LN stress. These LN-responsive genes were enriched in glycolysis metabolism, photosynthesis, hormone metabolism, and nitrogen metabolism. Furthermore, in the shoots, the glutamine synthetase gene SiGS5, chlorophyll apoprotein of photosystem II gene SiPsbQ, ATP synthase subunit gene Sib, zeatin synthesis genes SiAHP1, and aldose 1-epimerase gene SiAEP, and, in the roots, the high-affinity nitrate transporter genes SiNRT2.3, SiNRT2.4, glutamate synthase gene SiGOGAT2, fructose-bisphosphate aldolase gene SiFBA5, were important genes involved in the LN tolerance of the foxtail millet variety. Hence, our study implies that the identified genes and metabolic pathways contribute valuable insights into the mechanisms underlying LN tolerance in foxtail millet.

Phloroglucinol Promotes Fucoxanthin Synthesis by Activating the cis-Zeatin and Brassinolide Pathways in Thalassiosira pseudonana.

Phloroglucinol improves shoot formation and somatic embryogenesis in several horticultural and grain crops, but its function in microalgae remains unclear. Here, we found that sufficiently high concentrations of phloroglucinol significantly increased fucoxanthin synthesis, growth, and photosynthetic efficiency in the microalga Thalassiosira pseudonana. These results suggested that the role of phloroglucinol is conserved across higher plants and microalgae. Further analysis showed that, after phloroglucinol treatment, the contents of cis-zeatin and brassinolide in T. pseudonana increased significantly, while the contents of trans-zeatin, N6-isopentenyladenine (iP), auxin, and gibberellin were unaffected. Indeed, functional studies showed that the effects of cis-zeatin and brassinolide in T. pseudonana were similar to those of phloroglucinol. Knockout of key enzyme genes in the cis-zeatin synthesis pathway of T. pseudonana or treatment of T. pseudonana with a brassinolide synthesis inhibitor (brassinazole) significantly reduced growth and fucoxanthin content in T. pseudonana, and phloroglucinol treatment partially alleviated these inhibitory effects. However, phloroglucinol treatment was ineffective when the cis-zeatin and brassinolide pathways were simultaneously inhibited. These results suggested that the cis-zeatin and brassinolide signaling pathways are independent regulators of fucoxanthin synthesis in T. pseudonana and that phloroglucinol affects both pathways. Thus, this study not only characterizes the mechanism by which phloroglucinol promotes fucoxanthin synthesis but also demonstrates the roles of cis-zeatin and brassinolide in T. pseudonana. IMPORTANCE Here, we demonstrate that phloroglucinol, a growth promoter in higher plants, also increases growth and fucoxanthin synthesis in the microalga Thalassiosira pseudonana and therefore may have substantial practical application for industrial fucoxanthin production. Phloroglucinol treatment also induced the synthesis of cis-zeatin and brassinolide in T. pseudonana, and the cis-zeatin and brassinolide signaling pathways were implicated in the phloroglucinol-driven increases in T. pseudonana growth and fucoxanthin synthesis. Thus, our work clarified the molecular mechanism of phloroglucinol promoting the growth and fucoxanthin synthesis of Thalassiosira pseudonana and suggested that cis-zeatin and brassinolide, in addition to phloroglucinol, have potential utility as inducers of increased microalgal fucoxanthin production.

[Winter pruning boosts growth and yield of Lonicera japonica by regulating plant hormone content].

Lonicera japonica is a ubiquitous medicinal species in China.Winter pruning has long been used to improve its quality and yield, but the mechanism is rarely studied.Therefore, in this study, the growth phenotypes of L.japonica processed with different pruning methods were observed and the yield-and quality-boosting mechanism of pruning was analyzed.Specifically, the young shoots of the three-year old L.japonica were cut to different degrees(heavy pruning, mild pruning, and no pruning, respectively) in winter in 2020 and 2021, respectively, and the growth phenotypes, hormone content, and gene expression of the lateral buds at the sprouting stage and young shoots at the anthesis stage in the next year were analyzed.The result showed that the length, flower bud number, internode length, and node number of young shoots in the next year were in the order of heavy pruning>mild pruning>no pruning.The content of auxin and zeatin in apical buds of young shoots at the anthesis stage was the highest in the heavy pruning group, followed by the mild pruning group, and coming in the third was the no pruning group.The content of auxin and zeatin in lateral buds at the sprouting stage was in the order of no pruning>mild pruning>heavy pruning.Transcriptome analysis of the lateral buds at sprouting stage yielded the differentially expressed genes related to auxin and cytokinin, such as Lj1A1163T36, Lj3A719T115, Lj7C657T7, Lj9C505T15, and Lj9A505T70.In conclusion, the growth phenotypes of young shoots of L.japonica processed with different pruning methods in winter were related to the difference in hormone content in the apical buds.Therefore, winter pruning influenced the content of auxin and cytokinin in new shoots of L.japonica and further regulated the expression of hormone-related genes, thereby promoting shoot growth and increasing the yield of L.japonica.

Metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions revealed by integration analysis.

Metabolites, phytohormones, and genes involved in dehydration responses/tolerance have been predicted in several plants. However, metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions remain unclear. Here, we analyzed the organ specificity of metabolites, phytohormones, and gene transcripts and revealed the characteristics of their regulatory networks in dehydration-treated soybeans. Our metabolite/phytohormone analysis revealed the accumulation of raffinose, trehalose, and cis-zeatin (cZ) specifically in dehydration-treated roots. In dehydration-treated soybeans, raffinose, and trehalose might have additional roles not directly involved in protecting the photosynthetic apparatus; cZ might contribute to root elongation for water uptake from the moisture region in soil. Our integration analysis of metabolites-genes indicated that galactinol, raffinose, and trehalose levels were correlated with transcript levels for key enzymes (galactinol synthase, raffinose synthase, trehalose 6-phosphate synthase, trehalose 6-phosphate phosphatase) at the level of individual plants but not at the organ level under dehydration. Genes encoding these key enzymes were expressed in mainly the aerial parts of dehydration-treated soybeans. These results suggested that raffinose and trehalose are transported from aerial plant parts to the roots in dehydration-treated soybeans. Our integration analysis of phytohormones-genes indicated that cZ and abscisic acid (ABA) levels were correlated with transcript levels for key enzymes (cytokinin nucleoside 5'-monophosphate phosphoribohydrolase, cytokinin oxidases/dehydrogenases, 9-cis-epoxycarotenoid dioxygenase) at the level of individual plants but not at the organ level under dehydration conditions. Therefore, processes such as ABA and cZ transport, among others, are important for the organ specificity of ABA and cZ production under dehydration conditions.

Responses to Systemic Nitrogen Signaling in Arabidopsis Roots Involve trans-Zeatin in Shoots.

Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N deprivation.

Genome-wide analysis of lncRNA and mRNA expression and endogenous hormone regulation during tension wood formation in Catalpa bungei.

BACKGROUND: Phytohormones are the key factors regulating vascular development in plants, and they are also involved in tension wood (TW) formation. Although the theory of hormone distribution in TW formation is widely supported, the effects of endogenous hormones on TW formation have not yet been assessed. In this study, TW formation was induced in Catalpa bungei by artificial bending. The phytohormone content of TW, opposite wood (OW) and normal wood (NW) was determined using liquid chromatography-mass spectrometry (LC-MS), and transcriptome sequencing was performed. The hormone content and related gene expression data were comprehensively analyzed. RESULTS: The results of analyses of the plant hormone contents indicated significantly higher levels of cis-zeatin (cZ), indoleacetic acid (IAA) and abscisic acid (ABA) in TW than in OW. Genes involved in the IAA and ABA synthesis pathways, such as ALDH (evm. MODEL: group5.1511) and UGT (evm. MODEL: scaffold36.20), were significantly upregulated in TW. and the expression levels of ARF (evm. MODEL: group5.1332), A-ARR (evm. MODEL: group0.1600), and TCH4 (evm. MODEL: group2.745), which participate in IAA, cZ and Brassinolide (BR) signal transduction, were significantly increased in TW. In particular, ARF expression may be regulated by long noncoding RNAs (lncRNAs) and the HD-ZIP transcription factor ATHB-15. CONCLUSIONS: We constructed a multiple hormone-mediated network of C. bungei TW formation based on hormone levels and transcriptional expression profiles were identified during TW formation.

Root-derived trans-zeatin cytokinin protects Arabidopsis plants against photoperiod stress.

Recently, a novel type of abiotic stress caused by a prolongation of the light period-coined photoperiod stress-has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signalling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signalling system to protect from the negative consequences of strong photoperiod stress.

Role of cis-zeatin in root responses to phosphate starvation.

Phosphate (Pi) is an essential nutrient for all organisms. Roots are underground organs, but the majority of the root biology studies have been done on root systems growing in the presence of light. Root illumination alters the Pi starvation response (PSR) at different intensities. Thus, we have analyzed morphological, transcriptional and physiological responses to Pi starvation in dark-grown roots. We have identified new genes and pathways regulated by Pi starvation that were not described previously. We also show that Pi-starved plants increase the cis-zeatin (cZ) : trans-zeatin (tZ) ratio. Transcriptomic analyses show that tZ preferentially represses cell cycle and PSR genes, whereas cZ induces genes involved in cell and root hair elongation and differentiation. In fact, cZ-treated seedlings show longer root system as well as longer root hairs compared with tZ-treated seedlings, increasing the total absorbing surface. Mutants with low cZ concentrations do not allocate free Pi in roots during Pi starvation. We propose that Pi-starved plants increase the cZ : tZ ratio to maintain basal cytokinin responses and allocate Pi in the root system to sustain its growth. Therefore, cZ acts as a PSR hormone that stimulates root and root hair elongation to enlarge the root absorbing surface and to increase Pi concentrations in roots.

Effect of exogenous abscisic acid on morphology, growth and nutrient uptake of rice (Oryza sativa) roots under simulated acid rain stress.

MAIN CONCLUSION: Application of proper ABA can improve acid tolerance of rice roots by balancing endogenous hormones and promoting nutrient uptake. Abscisic acid (ABA) has an important signaling role in enhancing plant tolerance to environmental stress. To alleviate the inhibition on plant growth and productivity caused by acid rain, it is crucial to clarify the regulating mechanism of ABA on adaptation of plants to acid rain. Here, we studied the effects of exogenously applied ABA on nutrients uptake of rice roots under simulated acid rain (SAR) stress from physiological, biochemical and molecular aspects. Compared to the single SAR treatment (pH 4.5 or 3.5), exogenous 10 µM ABA alleviated the SAR-induced inhibition of root growth by balancing endogenous hormones (abscisic acid, indole-3-acetic acid, gibberellic acid and zeatin), promoting nutrient uptake (nitrate, P, K and Mg) in rice roots, and increasing the activity of the plasma membrane H+-ATPase by up-regulating expression levels of genes (OSA2, OSA4, OSA9 and OSA10). However, exogenous 100 µM ABA exacerbated the SAR-caused inhibition of root growth by disrupting the balance of endogenous hormones, and inhibiting nutrient uptake (nitrate, P, K, Ca and Mg) through decreasing the activity of the plasma membrane H+-ATPase. These results indicate that proper concentration of exogenous ABA could enhance tolerance of rice roots to SAR stress by promoting nutrients uptake and balancing endogenous hormones.

Methylation effect on IPT5b gene expression determines cytokinin biosynthesis in apple rootstock.

The molecular mechanism of dwarfing in the apple rootstock is poorly understood, and has been attributed to low root cytokinin biosynthesis levels. Here we identified differences in root trans-zeatin content and expression of cytokinin metabolic pathway genes between dwarfing and vigorous rootstocks. Specifically, a stable low expression of IPT5b was identified in the dwarfing rootstocks. Bisulfite sequencing showed that two CpG islands (CpG2 and CpG4) in the IPT5b promoter region showed higher methylation levels in the M9 rootstock (dwarfing) compared to the Mr (vigorous) rootstock. Furthermore, IPT5b expression increased when M9 rootstocks were treated with 5-azaC, a methylation blocker, indicating that methylation levels influence IPT5b expression. In conclusion, we found low IPT5b expression with high level methylations in promoter region, leading to poor root trans-zeatin biosynthesis in the M9 rootstock, which may induce dwarfing.

A significant positive correlation between endogenous trans-zeatin content and total arsenic in arsenic hyperaccumulator Pteris cretica var. nervosa.

A pot experiment was conducted to compare the content of endogenous trans-zeatin (Z), plant arsenic (As) uptake and physiological indices in the fronds of As-hyperaccumulator (Pteris cretica var. nervosa) and non-hyperaccumulator (Pteris ensiformis). Furthermore, a stepwise regression method was used to study the relationship among determined indices, and the time-course effect of main indices was also investigated under 100mg/kg As stress with time extension. In the 100-200mg/kg As treatments, plant height showed no significant difference and endogenous Z content significantly increased in P. cretica var. nervosa compared to the control, but a significant decrease of height and endogenous Z was observed in P. ensiformis. The concentrations of As (III) and As (V) increased significantly in the fronds of two plants, but this increase was much higher in P. cretica var. nervosa. Compared to the control, the contents of chlorophyll and soluble protein were significantly increased in P. cretica var. nervosa but decreased in P. ensiformis in the 200mg/kg As treatment, respectively. A significant positive correlation was found between the contents of endogenous Z and total As in P. cretica var. nervosa, but such a correlation was not found in P. ensiformis. Additionally, in the time-course effect experiment, a peak value of each index was appeared in the 43rd day in two plants, except for chlorophyll in P. ensiformis, but this value was significantly higher in P. cretica var. nervosa than that in P. ensiformis. In conclusion, a higher endogenous Z content contributed to As accumulation of P. cretica var. nervosa under As stress.

Crystallographic and CD probing of ligand-induced conformational changes in a plant PR-10 protein.

Plant pathogenesis-related class 10 (PR-10) proteins are a family of abundant proteins initially identified as elements of the plant defense system. The key structural feature suggesting PR-10 functionality is a huge hydrophobic cavity created in the protein interior by a scaffold composed of an extended ß-sheet wrapped around a long and flexible C-terminal α-helix. Several crystallographic and NMR studies have shown that the cavity can accommodate a variety of small molecule ligands, including phytohormones. The article describes ∼1.3 Å resolution crystal structures of a Lupinus luteus PR-10 isoform LlPR-10.1A, in its free form and in complex with trans-zeatin, a naturally occurring plant hormone belonging to the cytokinin group. Moreover we present the structure of the same protein where the saturation with zeatin is not complete. This set of three crystal structures allows us to track the structural adaptation of the protein upon trans-zeatin docking, as well as the sequence of the ligand-binding events, step-by-step. In addition, titration of LlPR-10.1A with trans-zeatin monitored in solution by CD spectra, confirmed the pattern of structural adaptations deduced from the crystallographic studies. The ligand-biding mode shows no similarity to other zeatin complexes of PR-10 proteins. The present work, which describes the first atomic models of the same PR-10 protein with and without a physiological ligand, reveals that the conformation of LlPR-10.1A undergoes a significant structural rearrangement upon trans-zeatin binding.

Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots.

The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36,091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system.

Metabolic changes and associated cytokinin signals in response to nitrate assimilation in roots and shoots of Lolium perenne.

The efficiency of inorganic nitrogen (N) assimilation is a critical component of fertilizer use by plants and of forage production in Lolium perenne, an important pasture species worldwide. We present a spatiotemporal description of nitrate use efficiency in terms of metabolic responses and carbohydrate remobilization, together with components of cytokinin signal transduction following nitrate addition to N-impoverished plants. Perennial ryegrass (L. perenne cv. Grasslands Nui) plants were grown for 10 weeks in unfertilized soil and then treated with nitrate (5 mM) hydroponically. Metabolomic analysis by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry revealed a dynamic interaction between N and carbon metabolism over a week-long time course represented by the relative abundance of amino acids, tricarboxylic acid intermediates and stored water-soluble carbohydrates (WSCs). The initial response to N addition was characterized by a rapid remobilization of carbon stores from the low-molecular weight WSC, along with an increase in N content and assimilation into free amino acids. Subsequently, the shoot became the main source of carbon through remobilization of a large pool of high-molecular weight WSC. Associated quantification of cytokinin levels and expression profiling of putative cytokinin response regulator genes by quantitative reverse transcription polymerase chain reaction support a role for cytokinin in the mediation of the response to N addition in perennial ryegrass. The presence of high levels of cis-zeatin-type cytokinins is discussed in the context of hormonal homeostasis under the stress of steady-state N deficiency.

Cytokinins can act as suppressors of nitric oxide in Arabidopsis.

Maintaining nitric oxide (NO) homeostasis is essential for normal plant physiological processes. However, very little is known about the mechanisms of NO modulation in plants. Here, we report a unique mechanism for the catabolism of NO based on the reaction with the plant hormone cytokinin. We screened for NO-insensitive mutants in Arabidopsis and isolated two allelic lines, cnu1-1 and 1-2 (continuous NO-unstressed 1), that were identified as the previously reported altered meristem program 1 (amp1) and as having elevated levels of cytokinins. A double mutant of cnu1-2 and nitric oxide overexpression 1 (nox1) reduced the severity of the phenotypes ascribed to excess NO levels as did treating the nox1 line with trans-zeatin, the predominant form of cytokinin in Arabidopsis. We further showed that peroxinitrite, an active NO derivative, can react with zeatin in vitro, which together with the results in vivo suggests that cytokinins suppress the action of NO most likely through direct interaction between them, leading to the reduction of endogenous NO levels. These results provide insights into NO signaling and regulation of its bioactivity in plants.

Putative zeatin O-glucosyltransferase OscZOG1 regulates root and shoot development and formation of agronomic traits in rice.

As a ubiquitous reaction, glucosylation controls the bioactivity of cytokinins in plant growth and development. Here we show that genetic manipulation of zeatin-O-glucosylation regulates the formation of important agronomic traits in rice by manipulating the expression of OscZOG1 gene, encoding a putative zeatin O-glucosyltransferase. We found that OscZOG1 was preferentially expressed in shoot and root meristematic tissues and nascent organs. The growth of lateral roots was stimulated in the overexpression lines, but inhibited in RNA interference lines. In shoots, knockdown of OscZOG1 expression by RNA interference significantly improved tillering, panicle branching, grain number per panicle and seed size, which are important agronomic traits for grain yield. In contrast, constitutive expression of OscZOG1 leads to negative effects on the formation of the grain-yielding traits with a marked increase in the accumulation levels of cis-zeatin O-glucoside (cZOG) in the transgenic rice plants. In this study, our findings demonstrate the feasibility of improving the critical yield-determinant agronomic traits, including tiller number, panicle branches, total grain number per panicle and grain weight by downregulating the expression level of OscZOG1. Our results suggest that modulating the levels of cytokinin glucosylation can function as a fine-tuning switch in regulating the formation of agronomic traits in rice.