Search

SUMMARY: Petal size is determined by cell division and cell expansion. Jasmonic acid (JA) has been reported to be associated with floral development, but its regulatory mechanism affecting petal size remains unclear. Here, we reveal the vital role of JA in regulating petal size and the duration of the cell division phase via the key JA signaling component RhMYC2. We show that RhMYC2 expression is induced by exogenous treatment with methyl jasmonate and decreases from stage 0 to stage 2 of flower organ development, corresponding to the cell division phase. Furthermore, silencing RhMYC2 shortened the duration of the cell division phase, ultimately accelerating flowering opening and resulting in smaller petals. In addition, we determined that RhMYC2 controls cytokinin homeostasis in rose petals by directly activating the expression of the cytokinin biosynthetic gene LONELY GUY3 (RhLOG3) and repressing that of the cytokinin catabolism gene CYTOKININ OXIDASE/DEHYDROGENASE6 (RhCKX6). Silencing RhLOG3 shortened the duration of the cell division period and produced smaller petals, similar to RhMYC2 silencing. Our results underscore the synergistic effects of JA and cytokinin in regulating floral development, especially for petal size in roses. [ABSTRACT FROM AUTHOR]

SUMMARY: Strigolactones (SLs) are key regulators of shoot growth and responses to environmental stimuli. Numerous studies have indicated that nitrogen (N) limitation induces SL biosynthesis, suggesting that SLs may play a pivotal role in coordinating systemic responses to N availability, but this idea has not been clearly demonstrated. Here, we generated triple knockout mutants in the SL synthesis gene TaDWARF17 (TaD17) in bread wheat and investigated their phenotypic and transcriptional responses under N limitation, aiming to elucidate the role of SLs in the adaptation to N limitation. Tad17 mutants display typical SL mutant phenotypes, and fail to adapt their shoot growth appropriately to N. Despite exhibiting an increased tillering phenotype, Tad17 mutants continued to respond to N limitation by reducing tiller number, suggesting that SLs are not the sole regulators of tillering in response to N availability. RNA‐seq analysis of basal nodes revealed that the loss of D17 significantly altered the transcriptional response of N‐responsive genes, including changes in the expression profiles of key N response master regulators. Crucially, our findings suggest that SLs are required for the transcriptional downregulation of cytokinin (CK) synthesis and signalling in response to N limitation. Collectively, our results suggest that SLs are essential for the appropriate morphological and transcriptional adaptation to N limitation in wheat, and that the repressive effect of SLs on shoot growth is partly mediated by their repression of CK synthesis. Significance Statement: This research investigates the key role of strigolactones (SLs) in bread wheat tillering and adaptation to nitrogen limitation. Our findings using TaDWARF17 mutants reveal that SLs, while not the sole regulators of tillering under nitrogen limitation, crucially mediate the transcriptional downregulation of cytokinin synthesis and signalling, underscoring the importance of SLs in coordinating morphological and transcriptional responses to nitrogen availability in wheat. [ABSTRACT FROM AUTHOR]

Summary Plant root system is significantly influenced by high soil levels of ammonium nitrogen, leading to reduced root elongation and enhanced lateral root branching. In Arabidopsis, these processes have been reported to be mediated by phytohormones and their downstream signaling pathways, while the controlling mechanisms remain elusive in crops. Through a transcriptome analysis of roots subjected to high/low ammonium treatments, we identified a cytokinin oxidase/dehydrogenase encoding gene, CKX3, whose expression is induced by high ammonium. Knocking out CKX3 and its homologue CKX8 results in shorter seminal roots, fewer lateral roots, and reduced sensitivity to high ammonium. Endogenous cytokinin levels are elevated by high ammonium or in ckx3 mutants. Cytokinin application results in shorter seminal roots and fewer lateral roots in wild‐type, mimicking the root responses of ckx3 mutants to high ammonium. Furthermore, CKX3 is transcriptionally activated by type‐B RR25 and RR26, and ckx3 mutants have reduced auxin content and signaling in roots under low ammonium. This study identified RR25/26‐CKX3‐cytokinin as a signal module that mediates root responses to external ammonium by modulating of auxin signaling in the root meristem and lateral root primordium. This highlights the critical role of cytokinin metabolism in regulating rice root development in response to ammonium. [ABSTRACT FROM AUTHOR]

Summary Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H2S and cytokinin (CTK) and the mechanisms by which H2S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC‐ESI‐MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2wild‐type (WT), OE CKX2 and ckx2/CKX2Cys(C)62alanine(A) transgenic lines were isolated with the ckx2 background. H2S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S‐LCD/oasa1 transgenic lines to investigate the effect of endogenous H2S on RSA, indicating that H2S reduces the gravitropic set‐point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H2S to regulate RSA in vivo. In conclusion, this study demonstrated that H2S negatively regulates CTK content and affects RSA by persulfidation of CKX2Cys62 in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Summary: Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity.Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa).PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type‐A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development.Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type‐A RR during wood formation in poplar. [ABSTRACT FROM AUTHOR]

SUMMARY: Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156‐SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans‐zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156‐targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2‐mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild‐type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156‐SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants partly optimize their water recruitment from the growth medium by directing root growth toward a moisture source, a phenomenon termed hydrotropism. The default mechanism of downward growth, termed gravitropism, often functions to counteract hydrotropism when the water‐potential gradient deviates from the gravity vector. This review addresses the identity of the root sites in which hydrotropism‐regulating factors function to attenuate gravitropism and the interplay between these various factors. In this context, the function of hormones, including auxin, abscisic acid, and cytokinins, as well as secondary messengers, calcium ions, and reactive oxygen species in the conflict between these two opposing tropisms is discussed. We have assembled the available data on the effects of various chemicals and genetic backgrounds on both gravitropism and hydrotropism, to provide an up‐to‐date perspective on the interactions that dictate the orientation of root tip growth. We specify the relevant open questions for future research. Broadening our understanding of root mechanisms of water recruitment holds great potential for providing advanced approaches and technologies that can improve crop plant performance under less‐than‐optimal conditions, in light of predicted frequent and prolonged drought periods due to global climate change. Significance Statement: Understanding the mechanisms that plant roots utilize to resist the strong tendency to grow downward in order to grow toward a moisture source, holds great potential for unveiling research approaches and technologies to improve crop performance. Herein, we assess the complex interplay between major regulators of root growth, development, and responses to myriad internal and external cues, that function to attenuate downward root growth, thereby enabling the root to reach water. [ABSTRACT FROM AUTHOR]

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity. Synopsis: In the Arabidopsis root vascular tissue, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. This work shows that vascular development requires ARGONAUTE10 to sequester miRNA165/6, thus protecting HD-ZIP III transcripts from degradation. AGO10 controls formative cell divisions in the centre of the root stele, with its loss leading to ectopic xylem strands, increased procambial cell number, and enhanced root growth. Consistent with a role in protecting HD-ZIP III transcripts from miRNA165/6-mediated degradation, AGO10 is mostly expressed in the centre of the meristematic stele. AGO10 promotes HD-ZIP III activity to suppress cytokinin responses in the stele. AGO10 mutants outperform the wild type under water-limiting conditions. However, AGO10 is required for phenotypic robustness upon reduced carbon availability. AGO10 sequesters miRNA165/6 to suppress cytokinin-dependent responses in the root apical meristem. [ABSTRACT FROM AUTHOR]

Summary: Gibberellins (GA) have a profound influence on the formation of lateral root organs. However, the precise role this hormone plays in the cell‐specific events during lateral root formation, rhizobial infection and nodule organogenesis, including interactions with auxin and cytokinin (CK), is not clear.We performed epidermal‐ and endodermal‐specific complementation of the severely GA‐deficient na pea (Pisum sativum) mutant with Agrobacterium rhizogenes. Gibberellin mutants were used to examine the spatial expression pattern of CK (TCSn)‐ and auxin (DR5)‐responsive promoters and hormone levels.We found that GA produced in the endodermis promote lateral root and nodule organogenesis and can induce a mobile signal(s) that suppresses rhizobial infection. By contrast, epidermal‐derived GA suppress infection but have little influence on root or nodule development. GA suppress the CK‐responsive TCSn promoter in the cortex and are required for normal auxin activation during nodule primordia formation.Our findings indicate that GA regulate the checkpoints between infection thread (IT) penetration of the cortex and invasion of nodule primordial cells and promote the subsequent progression of nodule development. It appears that GA limit the progression and branching of IT in the cortex by restricting CK response and activate auxin response to promote nodule primordia development. [ABSTRACT FROM AUTHOR]

SUMMARY: Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N‐ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone‐inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen‐containing compounds such as β‐alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH‐mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions. [ABSTRACT FROM AUTHOR]

Summary: Sugar transporter proteins (STPs) play critical roles in regulating plant stress tolerance, growth, and development. However, the role of STPs in regulating crop yield is poorly understood.This study elucidates the mechanism by which knockout of the sugar transporter OsSTP15 enhances grain yield via increasing the tiller number in rice.We found that OsSTP15 is specifically expressed in the shoot base and vascular bundle sheath of seedlings and encodes a plasma membrane‐localized high‐affinity glucose efflux transporter. OsSTP15 knockout enhanced sucrose and trehalose‐6‐phosphate (Tre6P) synthesis in leaves and improved sucrose transport to the shoot base by inducing the expression of sucrose transporters. Higher glucose, sucrose, and Tre6P contents were observed at the shoot base of stp15 plants. Transcriptome and metabolome analyses of the shoot base demonstrated that OsSTP15 knockout upregulated the expression of cytokinin (CK) synthesis‐ and signaling pathway‐related genes and increased CK levels.These findings suggest that OsSTP15 knockout represses glucose export from the cytoplasm and simultaneously enhances sugar transport from source leaves to the shoot base by promoting the synthesis of sucrose and Tre6P in leaves. Subsequent accumulation of glucose, sucrose, and Tre6P in the shoot base promotes tillering by stimulating the CK signaling pathway. [ABSTRACT FROM AUTHOR]

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants' metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms by which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programmes. Here, investigating the interplay between TOR‐mediated development and TOR‐mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR‐inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK‐mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signalling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signalling, executing signalling cues in both defense and development. Thus, differential regulation of TOR or TOR‐mediated processes could regulate the required outcome of development‐defense prioritisation. Summary Statement: Target of Rapamycin (TOR) is positioned at the interface between development and defense, but little is known about the mechanisms by which TOR may regulate shifts between them. Here, we demonstrate that TOR likely acts in concert with cytokinin and gibberellin to execute signalling cues in both defense and development. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) are a class of growth‐promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA‐IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate‐IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single‐ and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA‐deficiency results in cytokinesis defects, and both iptA‐deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development. [ABSTRACT FROM AUTHOR]

Summary: Grain size is one of the important traits in wheat breeding programs aimed at improving yield, and cytokinins, mainly involved in cell division, have a positive impact on grain size. Here, we identified a novel wheat gene TaMADS‐GS encoding type I MADS‐box transcription factor, which regulates the cytokinins signalling pathway during early stages of grain development to modulate grain size and weight in wheat. TaMADS‐GS is exclusively expressed in grains at early stage of seed development and its knockout leads to delayed endosperm cellularization, smaller grain size and lower grain weight. TaMADS‐GS protein interacts with the Polycomb Repressive Complex 2 (PRC2) and leads to repression of genes encoding cytokinin oxidase/dehydrogenases (CKXs) stimulating cytokinins inactivation by mediating accumulation of the histone H3 trimethylation at lysine 27 (H3K27me3). Through the screening of a large wheat germplasm collection, an elite allele of the TaMADS‐GS exhibits higher ability to repress expression of genes inactivating cytokinins and a positive correlation with grain size and weight, thus representing a novel marker for breeding programs in wheat. Overall, these findings support the relevance of TaMADS‐GS as a key regulator of wheat grain size and weight. [ABSTRACT FROM AUTHOR]

SUMMARY: Root hairs are crucial in the uptake of essential nutrients and water in plants. This study showed that a zinc finger protein, GIS3 is involved in root hair growth in Arabidopsis. The loss‐of‐function gis3 and GIS3 RNAi transgenic line exhibited a significant reduction in root hairs compared to the wild type. The application of 1‐aminocyclopropane‐1‐carboxylic acid (ACC), an exogenous ethylene precursor, and 6‐benzyl amino purine (BA), a synthetic cytokinin, significantly restored the percentage of hair cells in the epidermis in gis3 and induced GIS3 expression in the wild type. More importantly, molecular and genetic studies revealed that GIS3 acts upstream of ROOT HAIR DEFECTIVE 2 (RHD2) and RHD4 by binding to their promoters. Furthermore, exogenous ACC and BA application significantly induced the expression of RHD2 and RHD4, while root hair phenotype of rhd2‐1, rhd4‐1, and rhd4‐3 was insensitive to ACC and BA treatment. We can therefore conclude that GIS3 modulates root hair development by directly regulating RHD2 and RHD4 expression through ethylene and cytokinin signals in Arabidopsis. Significance Statement: In this paper, we demonstrated that zinc finger protein GIS3 is the key regulator to control root hair development in Arabidopsis. Further experiments demonstrated that GIS3, RHD2, and RHD4 are required for root hair development in response to ethylene and cytokinin signaling. More importantly, the molecular and genetic experiments reveal that GIS3 directly binds to the promoter of RHD2 and RHD4 to regulate root hair elongation in Arabidopsis. These results will provide a novel function of zinc finger protein interacting with hormone signal to control root hair development and a better understanding of the network of zinc finger protein interact with root hair‐related genes to control root hair development in Arabidopsis. [ABSTRACT FROM AUTHOR]

SUMMARY: Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell‐ and subcellular‐type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle‐specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence‐Activated multi‐Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine‐derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high‐performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC‐(ESI+)‐HRMS/MS) for metabolomics and CTK profiling. Across LPS‐treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS‐induced cell stress, suggesting a role for CTKs in intra‐ and extracellular signaling of mammalian cells. [ABSTRACT FROM AUTHOR]

Summary: MicroRNAs (miRNAs) are important regulators of plant biological processes, including soybean nodulation. One miRNA, miR4407, was identified in soybean roots and nodules. However, the function of miR4407 in soybean is still unknown.MiR4407, unique to soybean, positively regulates lateral root emergence and root structures and represses a root‐specific ISOPENTENYLTRANSFERASE (GmIPT3). By altering the expression of miR4407 and GmIPT3, we investigated the role of miR4407 in lateral root and nodule development.Both miR4407 and GmIPT3 are expressed in the inner root cortex and nodule primordia. Upon rhizobial inoculation, miR4407 was downregulated while GmIPT3 was upregulated. Overexpressing miR4407 reduced the number of nodules in transgenic soybean hairy roots while overexpressing the wild‐type GmIPT3 or a miR4407‐resistant GmIPT3 mutant (mGmIPT3) significantly increased the nodule number. The mechanism of miR4407 and GmIPT3 functions was also linked to autoregulation of nodulation (AON), where miR4407 overexpression repressed miR172c and activated its target, GmNNC1, turning on AON. Exogenous CK mimicked the effects of GmIPT3 overexpression on miR172c, supporting the notion that GmIPT3 regulates nodulation by enhancing root‐derived CK.Overall, our data revealed a new miRNA‐mediated regulatory mechanism of nodulation in soybean. MiR4407 showed a dual role in lateral root and nodule development. [ABSTRACT FROM AUTHOR]

Summary: The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question.SlTCP24 and SlTCP29, two plant‐specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29.SlTCP24 and SlTCP29 could bind to the TCP‐binding cis‐element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double‐gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin.This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study. [ABSTRACT FROM AUTHOR]

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED‐like homeobox (KNOXII) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ‐mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (orthologue of Arabidopsis Class M KNOX) and NO APICAL MERISTEM (orthologue of Arabidopsis CUP‐SHAPED COTYLEDON), are necessary for MtKNOX4‐mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula. [ABSTRACT FROM AUTHOR]

Cytokinin (CTK) has an important regulatory effect on plant morphology, physiology, and yield, and is one of the main factors regulating nitrogen absorption, transport, and metabolism. This paper reviews the research progress in nitrogen absorption, transport and metabolism, cytokinin synthesis, transport and signal transduction, etc., focusing on the physiological mechanisms by which CTK and nitrogen collaborate to regulate the root–shoot relationship and its impact on agronomic traits of crops. The synthesis of trans‐zeatin (tZ) and trans‐zeatin riboside (tZR) is generally regulated by root nitrogen, which is transported them to the sprouts, regulates nitrogen allocation and metabolism, and affects photosynthetic characteristics and yield. In young shoots, nitrogen upregulates the synthesis of isopentenyl, adenine (iP), and iP‐nucleoside (iPR) and transports to roots through the phloem, reducing nitrogen uptake and transport, affecting root morphology. Based on this, the role of, CTK in coordinating relationships between source repositories and enhancing injection. We conducted an analysis on how various cultivation methods impact CTK metabolism and its relationship with plant growth. Additionally, we discussed the challenges associated with applying CTK functions to paddy field production and suggested future research directions. [ABSTRACT FROM AUTHOR]

SUMMARY: Ethylene‐responsive factors (ERFs) have diverse functions in the regulation of various plant developmental processes. Here, we demonstrate the dual role of an Arabidopsis ERF gene, AtERF19, in regulating reproductive meristem activity and flower organ size through the regulation of genes involved in CLAVATA–WUSCHEL (CLV–WUS) and auxin signaling, respectively. We found that AtERF19 stimulated the formation of flower primordia and controlled the number of flowers produced by activating WUS and was negatively regulated by CLV3. 35S::AtERF19 expression resulted in significantly more flowers, whereas 35S::AtERF19 + SRDX dominant‐negative mutants produced fewer flowers. In addition, AtERF19 also functioned to control flower organ size by promoting the division/expansion of the cells through activating Small Auxin Up RNA Gene 32 (SAUR32), which positively regulated MYB21/24 in the auxin signaling pathway. 35S::AtERF19 and 35S::SAUR32 resulted in similarly larger flowers, whereas 35S::AtERF19 + SRDX and 35S::SAUR32‐RNAi mutants produced smaller flowers than the wild type. The functions of AtERF19 were confirmed by the production of similarly more and larger flowers in 35S::AtERF19 transgenic tobacco (Nicotiana benthamiana) and in transgenic Arabidopsis which ectopically expressed the orchid gene (Nicotiana benthamiana) PaERF19 than in wild‐type plants. The finding that AtERF19 regulates genes involved in both CLV–WUS and auxin signaling during flower development significantly expands the current knowledge of the multifunctional evolution of ERF genes in plants. The results presented in this work indicate a dual role for the transcription factor AtERF19 in controlling the number of flowers produced and flower organ size through the regulation of genes involved in CLV–WUS and auxin signaling, respectively. Our findings expand the knowledge of the roles of ERF genes in the regulation of reproductive development. Significance Statement: The results presented in this work indicate a dual role for the transcription factor AtERF19 in controlling the number of flowers produced and flower organ size through the regulation of genes involved in CLAVATA–WUSCHEL and auxin signaling, respectively. Our findings expand the knowledge of the roles of ERF genes in the regulation of reproductive development. [ABSTRACT FROM AUTHOR]

Summary: An environmentally responsive root system is crucial for plant growth and crop yield, especially in suboptimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density while simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture.Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here, we describe a search for proteins that regulate root system architecture (RSA) by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co‐purifying proteins.Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here, we show that, in Arabidopsis, AZG1 is a cytokinin (CK) import protein that co‐localizes with and stabilizes PIN1, linking auxin and CK transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1‐dependent under salt stress.This report therefore identifies a potential point for auxin:cytokinin crosstalk, which shapes RSA in response to NaCl. [ABSTRACT FROM AUTHOR]

Although root nodules are essential for biological nitrogen fixation in legumes, the cell types and molecular regulatory mechanisms contributing to nodule development and nitrogen fixation in determinate nodule legumes, such as soybean (Glycine max), remain incompletely understood. Here, we generated a single‐nucleus resolution transcriptomic atlas of soybean roots and nodules at 14 days post inoculation (dpi) and annotated 17 major cell types, including six that are specific to nodules. We identified the specific cell types responsible for each step in the ureides synthesis pathway, which enables spatial compartmentalization of biochemical reactions during soybean nitrogen fixation. By utilizing RNA velocity analysis, we reconstructed the differentiation dynamics of soybean nodules, which differs from those of indeterminate nodules in Medicago truncatula. Moreover, we identified several putative regulators of soybean nodulation and two of these genes, GmbHLH93 and GmSCL1, were as‐yet uncharacterized in soybean. Overexpression of each gene in soybean hairy root systems validated their respective roles in nodulation. Notably, enrichment for cytokinin‐related genes in soybean nodules led to identification of the cytokinin receptor, GmCRE1, as a prominent component of the nodulation pathway. GmCRE1 knockout in soybean resulted in a striking nodule phenotype with decreased nitrogen fixation zone and depletion of leghemoglobins, accompanied by downregulation of nodule‐specific gene expression, as well as almost complete abrogation of biological nitrogen fixation. In summary, this study provides a comprehensive perspective of the cellular landscape during soybean nodulation, shedding light on the underlying metabolic and developmental mechanisms of soybean nodule formation. [ABSTRACT FROM AUTHOR]

Summary: Parallel veins are characteristic of monocots, including grasses (Poaceae). Therefore, how parallel veins develop as the leaf grows in the medial–lateral (ML) dimension is a key question in grass leaf development.Using fluorescent protein reporters, we mapped auxin, cytokinin (CK), and gibberellic acid (GA) response patterns in maize (Zea mays) leaf primordia. We further defined the roles of these hormones in ML growth and vein formation through combinatorial genetic analyses and measurement of hormone concentrations.We discovered a novel pattern of auxin response in the adaxial protoderm that we hypothesize has important implications for the orderly formation of 3° veins early in leaf development. In addition, we found an auxin transport and response pattern in the margins that correlate with the transition from ML to proximal–distal growth. We present evidence that auxin efflux precedes CK response in procambial strand development. We also determined that GA plays an early role in the shoot apical meristem as well as a later role in the primordium to restrict ML growth.We propose an integrative model whereby auxin regulates ML growth and vein formation in the maize leaf through control of GA and CK. [ABSTRACT FROM AUTHOR]

BACKGROUND: Microalgae can biosynthesize phytohormones that promote plant growth and development. These compounds can potentially replace synthetic agricultural inputs that are harmful to the environment. The main plant growth‐promoting phytohormones present in microalgae are auxins and cytokinins. The objective of this study was to evaluate the influence of culture conditions (autotrophic and heterotrophic) on the concentrations of the endogenous phytohormones indole‐3‐acetic acid (IAA) and trans‐zeatin (tZ) in the microalgae Chlorella fusca LEB 111, Chlorella vulgaris LEB 112, Scenedesmus obliquus LEB 117, Synechococcus nidulans LEB 115 and Spirulina sp. LEB 18. RESULTS: All microalgae produced IAA and tZ under the three evaluated conditions (autotrophic light/dark (12/12 h), autotrophic continuous light (24 h) and heterotrophic). The IAA concentrations ranged from 1.94 to 56.45 nmol g−1, whereas those of tZ ranged from 0.06 to 35.52 pmol g−1. The cultivation conditions increased the carbohydrate and protein concentrations. Continuous light favored the production of carbohydrates in the studied cyanobacteria (Synechococcus nidulans LEB 115 and Spirulina sp. LEB 18). However, the light/dark condition (12/12 h) afforded the highest protein concentrations for green algae and cyanobacteria. There were no statistically significant differences in the lipid content of the strains studied. CONCLUSIONS: The results of this study are valuable and indicate that the cultivation conditions and species used in this study may be applied in sustainable agriculture, providing alternatives that may contribute to minimizing the environmental impact of synthetic chemical inputs in agriculture. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Low temperatures limit the geographic distribution and yield of plants. Hormones play an important role in coordinating the growth and development of plants and their tolerance to low temperatures. However, the mechanisms by which hormones affect plant resistance to extreme cold stress in the natural environment are still unclear.In this study, two winter wheat varieties with different cold resistances, Dn1 and J22, were used to conduct targeted plant hormone metabolome analysis on the tillering nodes of winter wheat at 5 °C, −10 °C and −25 °C using an LC–ESI–MS/MS system. We screened 39 hormones from 88 plant hormone metabolites and constructed a partial regulatory network of auxin, jasmonic acid and cytokinin.GO analysis and enrichment of KEGG pathways in different metabolites showed that the 'plant hormone signal transduction' pathway was the most common. Our study showed that extreme low temperature increased the most levels of auxin, cytokinin and salicylic acid, and decreased levels of jasmonic acid and abscisic acid, and that levels of auxin, jasmonic acid and cytokinin in Dn1 were higher than those in J22. These changes in hormone levels were associated with changes in gene expression in synthesis, catabolism, transport and signal transduction pathways. These results differ from the previous hormone regulation mechanisms, which were mostly obtained at 4 °C.Our results provide a basis for further understanding the molecular mechanisms by which plant endogenous hormones regulate plant freezing stress tolerance. [ABSTRACT FROM AUTHOR]

SUMMARY: Root growth dynamics is an outcome of complex hormonal crosstalk. The primary root meristem size, for example, is determined by antagonizing actions of cytokinin and auxin. Here we show that RAV1, a member of the AP2/ERF family of transcription factors, mediates cytokinin signaling in roots to regulate meristem size. The rav1 mutants have prominently longer primary roots, with a meristem that is significantly enlarged and contains higher cell numbers, compared with wild‐type. The mutant phenotype could be restored on exogenous cytokinin application or by inhibiting auxin transport. At the transcript level, primary cytokinin‐responsive genes like ARR1, ARR12 were significantly downregulated in the mutant root, indicating impaired cytokinin signaling. In concurrence, cytokinin induced regulation of SHY2, an Aux/IAA gene, and auxin efflux carrier PIN1 was hindered in rav1, leading to altered auxin transport and distribution. This effectively altered root meristem size in the mutant. Notably, CRF1, another member of the AP2/ERF family implicated in cytokinin signaling, is transcriptionally repressed by RAV1 to promote cytokinin response in roots. Further associating RAV1 with cytokinin signaling, our results demonstrate that cytokinin upregulates RAV1 expression through ARR1, during post‐embryonic root development. Regulation of RAV1 expression is a part of secondary cytokinin response that eventually represses CRF1 to augment cytokinin signaling. To conclude, RAV1 functions in a branch pathway downstream to ARR1 that regulates CRF1 expression to enhance cytokinin action during primary root development in Arabidopsis. Significance Statement: Interplay of auxin and cytokinin is known to regulate primary root meristem size in Arabidopsis, and here we show that RAV1 augments cytokinin signaling to control primary root meristem size. Cytokinin regulates RAV1 expression through ARR1, and RAV1 downregulates CRF1 expression to promote cytokinin actions and antagonize auxin response to determine proper root meristem size during post‐embryonic root development. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effect of a soil ammonia‐oxidizing bacterial (AOB) strain on Italian ryegrass (Lolium multiflorum Lam.) regrowth. Potted Italian ryegrass was used as test material. We isolated and screened an AOB strain from the soil for use in inoculation of other soils to increase nitrification. The sequence of the two isolated and screened AOB strains (S2_8_1 and S2_7_25) were 100% similar to that of Ensifer sesbaniae and Acinetobacter pittii. The nitrification inhibitor 3,4‐dimethylpyrazole phosphate (DMPP) was also added to the soil to inhibit nitrification. The experimental design involving the inoculation of the soils with the AOB strain included seven treatments: (1) regrowth without any additives, (2) regrowth with blank enrichment medium added to the soil, (3) regrowth with S2_8_1 strain inoculation, (4) regrowth with S2_7_25 strain inoculation, (5) regrowth with DMPP added to the soil, (6) regrowth with DMPP added to the soil and with S2_8_1 strain inoculation, and (7) regrowth with DMPP added to the soil and with S2_7_25 strain inoculation. Compared with regrowth treatment without any additives, the regrowth treatment with S2_8_1 and S2_7_25 inoculations significantly increased leaf biomass by 23.5–48.6%, the leaf net photosynthetic rate by 27.9–48.5%, the rhizosphere soil nitrification rate by 43.0–144.2%, leaf cytokinin concentrations by 23.7–24.9%, and xylem sap cytokinin concentrations by 32.5–43.2%. The increase of nitrate nitrogen content in rhizosphere soil induced by S2_8_1 and S2_7_25 promoted the transfers of cytokinin from roots to leaves, resulting in the increase of cytokinin concentration in leaves. This increase in leaf cytokinin concentrations improved Italian ryegrass regrowth. However, the S2_8_1 and S2_7_25‐induced increases in the nonrhizosphere soil nitrification rate were not closely related to Italian ryegrass regrowth. The tested AOB strains have considerable potential to increase Italian ryegrass regrowth. [ABSTRACT FROM AUTHOR]

Thidiazuron (TDZ) is an active substituted phenyl urea compound that has found a significant role as a plant growth regulator. The most exciting aspect of its function is that it can mimic auxins and cytokinin but is chemically different from these two. Many theories have been put forward, and experiments performed to understand the mode of action of TDZ in callogenesis. One suggested mechanism presents that it works by inhibiting the cytokinin degrading enzymes that compete with cytokinin for an active site on the enzyme. An example is the TDZ‐induced suppressed expression of gibberellic acid (GA) biosynthesis genes encoding GA3 and GA20 oxidases. This is entailed with a slightly increased expression of GA catabolism genes encoding GA20 oxidase. Similarly, one of the recommendations is that TDZ induces the expression of specific genes and transcription regulatory sequences that are either responsible directly for callus formation or in turn induce other auxins or cytokinin for callogenesis. There is no concise review available that discusses the details of TDZ‐induced callus, specifically and other in vitro cultures in general. This review is an attempt to explore all these pathways and mechanisms involved in callogenesis in plants stimulated by TDZ. [ABSTRACT FROM AUTHOR]

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)‐fixing bacteria. Unlike earlier processes of the legume–rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence‐associated genes, we performed a dual plant‐bacteria RNA sequencing approach on Medicago truncatula–Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2‐dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence‐associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well‐known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules. [ABSTRACT FROM AUTHOR]

Delayed stem senescence (DSS) in soybean plants is defined as retarded leaf and stem maturity, even if pods mature normally. This phenomenon is a serious problem for the combine harvesting of soybean in Japan. Our previous research showed that DSS occurred more frequently in June sowing than in July sowing, and the plants sown in June were exposed to higher temperature and higher solar radiation intensity during reproductive growth stage as compared to those sown in July. We additionally observed higher levels of cytokinins, plant hormones with senescence inhibition activity, in xylem exudate after pod-setting stage (R4) under the DSS-induced condition. Thus, this study investigated the effects of the respective environmental factors, temperature and light intensity, after R4 on cytokinin levels in the xylem exudate and the occurrence of DSS. Pot experiments were conducted in the experimental field of Nihon University (Fujisawa-city, Kanagawa, Japan). In temperature treatments, soybean plants grown in the field environment were moved to an air-conditioned room at R4 stage, whereas control of light intensity was achieved by shading using cheese clothes under the field condition. The temperature control experiments revealed that high temperature after R4 promoted the DSS symptom, while low temperature after R4 inhibited the occurrence of DSS. On the other hand, decreased light intensity after R4 did not affect the severity of DSS. The cytokinin levels in the xylem exudate at R5 correlated with the severity of DSS in the temperature treatments, but the change in light intensity did not influence the cytokinin levels. These results indicated that high temperature after pod-setting stage have a profound effect on the occurrence of DSS, and this effect is mediated by the increased cytokinin levels in the xylem exudate. [ABSTRACT FROM AUTHOR]

State‐of‐the‐art technology based on organic electronics can be used as a flow‐free delivery method for organic substances with high spatial resolution. Such highly targeted drug micro applications can be used in plant research for the regulation of physiological processes on tissue and cellular levels. Here, for the first time, an organic electronic ion pump (OEIP) is reported that can transport an isoprenoid‐type cytokinin, N6‐isopentenyladenine (iP), to intact plants. Cytokinins (CKs) are plant hormones involved in many essential physiological processes, including primary root (PR) and lateral root (LR) development. Using the Arabidopsis thaliana root as a model system, efficient iP delivery is demonstrated with a biological output – cytokinin‐related PR and LR growth inhibition. The spatial resolution of iP delivery, defined for the first time for an organic compound, is shown to be less than 1 mm, exclusively affecting the OEIP‐targeted LR. Results from the application of the high‐resolution OIEP treatment method confirm previously published findings showing that the influence of CKs may vary at different stages of LR development. Thus, OEIP‐based technologies offer a novel, electronically controlled method for phytohormone delivery that could contribute to unraveling cytokinin functions during different developmental processes with high specificity. [ABSTRACT FROM AUTHOR]

SUMMARY: Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to affect drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss‐of‐function approach we showed that AHP4 possesses an important role in the response of Arabidopsis to drought. This is evidenced by the higher survival rates of ahp4 than wild‐type (WT) plants under drought conditions, which is accompanied by the downregulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4‐mediated expression of several dehydration‐ and/or abscisic acid‐responsive genes involved in modulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous abscisic acid at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. In addition, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species, reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to upregulation of genes that encode enzymes involved in reactive oxygen species scavenging and non‐enzymatic antioxidant metabolism. Overall, our findings suggest that AHP4 plays a crucial role in plant drought adaptation. Significance Statement: Loss‐of‐function analysis of the cytokinin signaling member Arabidopsis histidine phosphotransfer protein 4 revealed its negative role in Arabidopsis adaptation to drought, affecting various physiological and biochemical processes by modulating the expression of a large set of genes, including abscisic acid‐responsive genes. [ABSTRACT FROM AUTHOR]

Summary: De novo shoot organogenesis is a prerequisite for numerous applications in plant research and breeding but is often a limiting factor, for example, in genome editing approaches. Class III homeodomain‐leucine zipper (HD‐ZIP III) transcription factors have been characterized as crucial regulators of shoot specification, however up‐stream components controlling their activity during shoot regeneration are only partially identified.In a chemical genetic screen, we isolated ZIC2, a novel activator of HD‐ZIP III activity. Using molecular, physiological and hormone transport analyses in Arabidopsis and sunflower (Helianthus annuus), we examined the molecular mechanism by which the drug promotes HD‐ZIP III expression.ZIC2‐dependent upregulation of HD‐ZIP III transcription promotes shoot regeneration in Arabidopsis and is accompanied by the induction of shoot specifying factors WUS and RAP2.6L and a subset of cytokinin biosynthesis enzymes. ZIC2's effect on HD‐ZIP III expression and regeneration is based on its ability to limit polar auxin transport. We further provide evidence that chemical modulation of auxin efflux can enhance de novo shoot formation in the regeneration recalcitrant species sunflower.Activation of HD‐ZIP III transcription during shoot regeneration depends on the local distribution of auxin and chemical modulation of auxin transport can be used to overcome poor shoot organogenesis in tissue culture. [ABSTRACT FROM AUTHOR]

SUMMARY: Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans ‐zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6‐benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8–9 days when compared with mock‐treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type‐A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type‐B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1. Significance Statement: Cytokinins mediate various aspects of plant development including flowering time. Here we show that exogenous cytokinin application prolongs the duration of the vegetative phase by suppressing florigen expression in rice (Oryza sativa) and maize (Zea mays). Further experiments elucidated the genetic and molecular mechanisms influencing the transition from vegetative growth to the reproductive phase by the hormone treatment. [ABSTRACT FROM AUTHOR]

Summary: Organ size is an important agronomic trait. Small peptides function in various stages of plant growth, but their regulatory mechanisms in organ growth remain poorly understood.Here, we characterize a novel small peptide, AtZSP1, which positively regulates organ size in Arabidopsis. Loss‐of‐function mutant atzsp1‐1 exhibited small organs, whereas AtZSP1 overexpression plants (p35S:AtZSP1#1) produced larger organs. Differentially expressed genes in the shoots of atzsp1‐1 and p35S:AtZSP1#1 were enriched in the cytokinin pathway. Further analysis on shoots of atzsp1‐1 showed that endogenous cytokinin levels were significantly reduced, consistent with reduced expression of the cytokinin response genes ARR5/6/7 and a decrease in pARR5:GUS activity. By contrast, cytokinin levels were elevated in p35S:AtZSP1#1. These results indicate that AtZSP1 affects shoot size via changes in cytokinin levels.AtZSP1 is ubiquitously expressed and encodes a 57‐amino acid endomembrane‐associated protein that is highly conserved among plant species. AtZSP1 interacts with ROCK1 at the endomembrane. Genetic analysis confirmed that the small organs and low cytokinin levels in atzsp1‐1 shoots are partially suppressed by the rock1‐4 mutation, suggesting that AtZSP1 may function in a common pathway with ROCK1 to antagonistically regulate organ growth.Our study identified an unknown small peptide, AtZSP1, and defined its function in regulating organ size in Arabidopsis. [ABSTRACT FROM AUTHOR]

SUMMARY: Cytokinin has strong connections to development and a growing role in the abiotic stress response. Here we show that CYTOKININ RESPONSE FACTOR 2 (CRF2) is additionally involved in the salt (NaCl) stress response. CRF2 promoter‐GUS expression indicates CRF2 involvement in the response to salt stress as well as the previously known cytokinin response. Interestingly, CRF2 mutant seedlings are quite similar to the wild type (WT) under non‐stressed conditions yet have many distinct changes in response to salt stress. Cytokinin levels measured by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) that increased in the WT after salt stress are decreased in crf2, potentially from CRF2 regulation of cytokinin biosynthesis genes. Ion content measured by inductively coupled plasma optical emission spectrometry (ICP‐OES) was increased in the WT for Na, K, Mn, Ca and Mg after salt stress, whereas the corresponding Ca and Mg increases are lacking in crf2. Many genes examined by RNA‐seq analysis were altered transcriptionally by salt stress in both the WT and crf2, yet interestingly approximately one‐third of salt‐modified crf2 transcripts (2655) showed unique regulation. Different transcript profiles for salt stress in crf2 compared with the WT background was further supported through an examination of co‐expressed genes by weighted gene correlation network analysis (WGCMA) and principal component analysis (PCA). Additionally, Gene Ontology (GO) enrichment terms found from salt‐treated transcripts revealed most photosynthesis‐related terms as only being affected in crf2, leading to an examination of chlorophyll levels and the efficiency of photosystem II (via the ratio of variable fluorescence to maximum fluorescence, Fv/Fm) as well as physiology after salt treatment. Salt stress‐treated crf2 plants had both reduced chlorophyll levels and lower Fv/Fm values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis. Significance Statement: A role for CYTOKININ RESPONSE FACTOR 2 (CRF2) is found in the modulation of the salt (NaCl) stress response. This is shown in mutant studies linking CRF2 to salt stress regulation of cytokinin profiles, ionome and transcriptome expression levels, as well as the physiological response. [ABSTRACT FROM AUTHOR]

Summary: LONELY GUY (LOG) was first identified in a screen of rice mutants with defects in meristem maintenance. In plants, LOG codes for cytokinin riboside 5′‐monophosphate phosphoribohydrolase, which converts inactive cytokinin nucleotides directly to the active free bases. Many enzymes with the PGGxGTxxE motif have been misannotated as lysine decarboxylases; conversely not all enzymes containing this motif are cytokinin‐specific LOGs. As LOG mutants clearly impact yield in rice, we investigated the LOG gene family in bread wheat. By interrogating the wheat (Triticum aestivum) genome database, we show that wheat has multiple LOGs. The close alignment of TaLOG1, TaLOG2 and TaLOG6 with the X‐ray structures of two functional Arabidopsis thaliana LOGs allows us to infer that the wheat LOGs 1‐11 are functional LOGs. Using RNA‐seq data sets, we assessed TaLOG expression across 70 tissue types, their responses to various stressors, the pattern of cis‐regulatory elements (CREs) and intron/exon patterns. TaLOG gene family members are expressed variously across tissue types. When the TaLOG CREs are compared with those of the cytokinin dehydrogenases (CKX) and glucosyltransferases (CGT), there is close alignment of CREs between TaLOGs and TaCKXs reflecting the key role of CKX in maintaining cytokinin homeostasis. However, we suggest that the main homeostatic mechanism controlling cytokinin levels in response to biotic and abiotic challenge resides in the CGTs, rather than LOG or CKX. However, LOG transgenics and identified mutants in rice variously impact yield, providing interesting avenues for investigation in wheat. [ABSTRACT FROM AUTHOR]

Drought is a critical environmental factor which constrains plant survival and growth. Genetic engineering provides a credible strategy to improve drought tolerance of plants. Here, we generated transgenic poplar lines expressing the isopentenyl transferase gene (IPT) under the driver of PtRD26 promoter (PtRD26pro‐IPT). PtRD26 is a senescence and drought‐inducible NAC transcription factor. PtRD26pro‐IPT plants displayed multiple phenotypes, including improved growth and drought tolerance. Transcriptome analysis revealed that auxin biosynthesis pathway was activated in the PtRD26pro‐IPT plants, leading to an increase in auxin contents. Biochemical analysis revealed that ARABIDOPSIS RESPONSE REGULATOR10 (PtARR10), one of the type‐B ARR transcription factors in the cytokinin pathway, was induced in PtRD26pro‐IPT plants and directly regulated the transcripts of YUCCA4 (PtYUC4) and YUCCA5 (PtYUC5), two enzymes in the auxin biosynthesis pathway. Overexpression of PtYUC4 enhanced drought tolerance, while simultaneous silencing of PtYUC4/5 evidently attenuated the drought tolerance of PtRD26pro‐IPT plants. Intriguingly, PtYUC4/5 displayed a conserved thioredoxin reductase activity that is required for drought tolerance by deterring reactive oxygen species accumulation. Our work reveals the molecular basis of cytokinin and auxin interactions in response to environmental stresses, and shed light on the improvement of drought tolerance without a growth penalty in trees by molecular breeding. [ABSTRACT FROM AUTHOR]

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild‐type plants. Here, we crossed BR‐ and CK‐related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR‐response transcription factor BZR1 directly interacted with the CK‐response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR‐deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK‐deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK‐mediated and ‐independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops. [ABSTRACT FROM AUTHOR]

Societal Impact Statement The rising global population and the increasingly conspicuous effects of climate change are putting growing pressure on the agricultural industry. Food production must increase significantly with little or no access to extra land; therefore, new approaches are urgently required to increase crop productivity. The strategic application of plant growth regulators is one method of achieving this, made possible by RootTarget. RootTarget maps regional land use and resource availability, identifying locations where crops could benefit from the application of plant growth regulators. Such targeted application can contribute to creating a more resilient and sustainable farming system. Summary: Developing novel strategies for optimising crop productivity is essential to meet the requirement of doubling food production by 2050. Geographic information system (GIS) software provides the opportunity to develop models for targeted application of plant growth regulators in rice‐growing regions based on water availability and agricultural practices, in order to optimise yields.GIS was used to identify rice‐growing areas with adequate irrigation, a high evapotranspiration index or suitable sowing technique for site‐specific application of plant growth regulators. Rule‐based decisions were incorporated into a GIS model in order to identify key areas where plant growth regulators should be applied. This approach allows information sharing between multiple stakeholders including researchers, companies and farmers with the aim of maximising crop productivity.A root growth promoter (RGP) was identified that promotes root growth under optimum conditions but not when applied concurrently with a drought stress. The effect of the RGP diminishes as the severity of the stress increases. These data were used to execute queries to identify regions with a suitable drought index, growing practice and adequate irrigation. Two hundred and twenty‐nine million hectares of land, spanning four continents, were identified as having the potential to benefit from application of the RGP.Significant areas of land used for rice production were identified that could benefit from application of the RGP. This study highlights the potential to incorporate academic research into models for research dissemination to the agricultural industry and increase crop productivity. [ABSTRACT FROM AUTHOR]

Summary: The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo‐nodules even in the absence of rhizobia. Hence, a genome‐wide analysis of the cytokinin signalling, two‐component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule‐deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay‐based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea. [ABSTRACT FROM AUTHOR]

Summary: The hemiparasitic plant Phtheirospermum japonicum (Phtheirospermum) is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy‐inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium‐derived CKs and the induction of hypertrophy are still unknown.We searched for haustorium‐expressed isopentenyltransferases (IPTs) that catalyze the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter‐reporter, and further analyzed the subcellular localization, the enzymatic function and contribution to inducing hypertrophy by studying CRISPR‐Cas9‐induced Phtheirospermum mutants.PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed IPT activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite‐induced CK responses in the host. A homolog of PjIPT1a also was identified in the related weed Striga hermonthica.With PjIPT1a, we identified the IPT enzyme that induces CK responses in Phtheirospermum japonicum‐infected Arabidopsis roots. We propose that PjIPT1a exemplifies how parasitism‐related functions evolve through gene duplications and neofunctionalization. [ABSTRACT FROM AUTHOR]

Cytokinins are phytohormones that regulate plant development, growth, and responses to stress. In particular, cytokinin has been reported to negatively regulate plant adaptation to high salinity; however, the molecular mechanisms that counteract cytokinin signaling and enable salt tolerance are not fully understood. Here, we provide evidence that salt stress induces the degradation of the cytokinin signaling components Arabidopsis (Arabidopisis thaliana) response regulator 1 (ARR1), ARR10 and ARR12. Furthermore, the stress‐activated mitogen‐activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ARR1/10/12 to promote their degradation in response to salt stress. As expected, salt tolerance is decreased in the mpk3/6 double mutant, but enhanced upon ectopic MPK3/MPK6 activation in an MKK5DD line. Importantly, salt hypersensitivity phenotypes of the mpk3/6 line were significantly alleviated by mutation of ARR1/12. The above results indicate that MPK3/6 enhance salt tolerance in part via their negative regulation of ARR1/10/12 protein stability. Thus, our work reveals a new molecular mechanism underlying salt‐induced stress adaptation and the inhibition of plant growth, via enhanced degradation of cytokinin signaling components. Synopsis: MKK5‐MPK3/6 signaling positively regulates salt tolerance in Arabidopsis, via phosphorylation and degradation of cytokinin signaling components ARR1/10/12. Salinity stress results in MPK3/6‐mediated phosphorylation and degradation of ARR1/10/12 proteins.MPK3/6 act upstream of ARR1/10/12 to promote the plant's adaption to high‐salinity.MPK3/6 are not involved in the regulation of cytokinin signaling under normal growth conditions. [ABSTRACT FROM AUTHOR]

Compensatory growth of forage grass can determine its optimal biomass, which is beneficial for increasing its production. The effect of rhizosphere soil nitrification on Italian ryegrass (Lolium multiflorum Lam) regrowth was investigated on the basis of root‐produced cytokinin concentration in leaves to reveal the forage grass compensatory growth mechanism. The nitrification inhibitor 3,4‐dimethylpyrazole phosphate was added to inhibit soil nitrification. Without soil nitrification being inhibited, compared with no clipping one defoliation cycle increased the leaf photosynthetic rate by 43.82%–53.79%, increased the leaf cytokinin content by 33.48%–34.73%, and increased the cytokinin transport from roots to leaves by 28.88%–39.47%. Nitrification inhibitor decreased soil nitrification rates by 23.33%–84.17% in the rhizosphere and by 42.71%–68.29% in the bulk soil during regrowth. In the rhizosphere micro‐environment, nitrification increased soil nitrate concentration that played an important role in the transport of cytokinin from roots to leaves during regrowth, increasing the leaf cytokinin concentration. However, bulk soil nitrification rate and nitrate content had little influence on leaf cytokinin concentration during regrowth. An increase in leaf cytokinin improved the photosynthesis and the regrowth. Without soil nitrification being inhibited, total biomass at the end of regrowth period was 1.09 times higher in plants that were defoliated once than in non‐clipped plants. However, soil nitrification inhibitor and two defoliation cycles decreased Italian ryegrass regrowth. Super compensatory growth occurred in the one defoliation cycle Italian ryegrasses without adding soil nitrification inhibitor. Compensatory growth occurred in once‐defoliated Italian ryegrass with adding soil nitrification inhibitor and in twice‐defoliated Italian ryegrass without adding soil nitrification inhibitor. In conclusion, rhizosphere soil nitrification is the key factor that regulates the compensatory growth of Italian ryegrass. [ABSTRACT FROM AUTHOR]

Summary: DWARF53 (D53) in rice (Oryza sativa) and its homologs in Arabidopsis (Arabidopsis thaliana), SUPPRESSOR OF MAX2‐LIKE 6 (SMXL6), SMXL7 and SMXL8, are well established negative regulators of strigolactone (SL) signalling in shoot branching regulation. Little is known of pea (Pisum sativum) homologs and whether D53 and related SMXLs are specific to SL signalling pathways. Here, we identify two allelic pea mutants, dormant3 (dor3), and demonstrate through gene mapping and sequencing that DOR3 corresponds to a homolog of D53 and SMXL6/SMXL7, designated PsSMXL7. Phenotype analysis, gene expression, protein and hormone quantification assays were performed to determine the role of PsSMXL7 in regulation of bud outgrowth and the role of PsSMXL7 and D53 in integrating SL and cytokinin (CK) responses. Like D53 and related SMXLs, we show that PsSMXL7 can be degraded by SL and induces feedback upregulation of PsSMXL7 transcript. Here we reveal a system conserved in pea and rice, whereby CK also upregulates PsSMXL7/D53 transcripts, providing a clear mechanism for SL and CK cross‐talk in the regulation of branching. To further deepen our understanding of the branching network in pea, we provide evidence that SL acts via PsSMXL7 to modulate auxin content via PsAFB5, which itself regulates expression of SL biosynthesis genes. We therefore show that PsSMXL7 is key to a triple hormone network involving an auxin–SL feedback mechanism and SL–CK cross‐talk. Significance Statement: Shoot branching, an important component of plant architecture, is regulated by hormonal signals such as cytokinin, strigolactones and auxin. Here the strigolactone signalling target, SMXL/D53, is characterized in Pisum sativum and we demonstrate that cytokinin regulation of PsSMXL7/D53 transcript levels is conserved in divergent plants, namely pea and rice. We further highlight the central role of PsSMXL7 in pea with evidence that it affects feedback regulation of strigolactone biosynthesis through the auxin receptor PsAFB4/5 (RMS2). [ABSTRACT FROM AUTHOR]

Summary: Cytokinins regulate diverse aspects of plant growth and development, primarily through modulation of gene expression. The cytokinin‐responsive transcriptome has been thoroughly described in dicots, especially Arabidopsis, but much less so in monocots. Here, we present a meta‐analysis of five different transcriptomic analyses of rice (Oryza sativa) roots treated with cytokinin, including three previously unpublished experiments. We developed a treatment method in which hormone is added to the media of rice seedlings grown in sterile hydroponic culture under a continuous airflow, which resulted in minimal perturbation of the seedlings, thus greatly reducing changes in gene expression in the absence of exogenous hormone. We defined a core set of 205 upregulated and 86 downregulated genes that were differentially expressed in at least three of the transcriptomic datasets. This core set includes genes encoding the type‐A response regulators (RRs) and cytokinin oxidases/dehydrogenases, which have been shown to be primary cytokinin response genes. GO analysis revealed that the upregulated genes were enriched for terms related to cytokinin/hormone signaling and metabolism, while the downregulated genes were significantly enriched for genes encoding transporters. Variations of type‐B RR binding motifs were significantly enriched in the promoters of the upregulated genes, as were binding sites for other potential partner transcription factors. The promoters of the downregulated genes were generally enriched for distinct cis‐acting motifs and did not include the type‐B RR binding motif. This analysis provides insight into the molecular mechanisms underlying cytokinin action in a monocot and provides a useful foundation for future studies of this hormone in rice and other cereals. Significance Statement: Transcriptional changes in response to exogenous cytokinin treatment have been thoroughly studied in the dicot Arabidopsis and to a lesser extent in monocots. This manuscript describes a meta‐analysis of five rice (Oryza sativa) root transcriptome datasets that address cytokinin‐induced changes in gene expression. We present a set of core cytokinin response genes in rice that consists of 291 differentially regulated genes shared by at least three datasets, which includes both previously identified and novel cytokinin‐responsive genes. [ABSTRACT FROM AUTHOR]

Summary: Cytokinins play a central role in delaying senescence, reducing oxidative damage and maintaining plant growth during drought. This study showed that the ectopic expression of ProRE‐deleted MdIPT5b, a key enzyme involved in cytokinin metabolism, increased the drought tolerance of transgenic Malus domestica (apple) callus and Solanum lycopersicum (tomato) seedlings by maintaining cytokinin homeostasis, and thus maintaining redox balance. Under restricted watering regimes, the yields of transgenic tomato plants were enhanced. Heterodimers of C/S1 bZIP are involved in the cytokinin‐mediated drought response. The heterodimers bind the ProRE of MdIPT5b promoter, thus directly suppressing gene transcription. Single C/S1 bZIP members could not independently function as suppressors. However, specific paired members (heterodimers of MdbZIP80 with MdbZIP2 or with MdbZIP39) effectively suppressed transcription. The α‐helical structure is essential for the heterodimerization of C/S1 bZIP members and for synergistic transcriptional suppression. As negative regulators of drought tolerance, suppressing either MdbZIP2 or MdbZIP39 alone does not improve the expression of MdIPT5b and did not increase the drought tolerance of transgenic apple callus. However, this could be achieved when they were co‐suppressed. The suppression of MdbZIP80 alone could improve MdIPT5b expression and increase the drought tolerance of transgenic apple callus. However, these effects were reversed in response to the cosuppression of MdbZIP80 and MdIPT5b. Similar results were also observed during delayed dark‐induced senescence in apple leaves. In conclusion, the apple C/S1 bZIP network (involving MdbZIP2, MdbZIP39 and MdbZIP80) directly suppressed the expression of MdIPT5b, thus negatively modulating drought tolerance and dark‐induced senescence in a functionally redundant manner. Significance Statement: This study proposes the condition of cytokinin homeostasis (appropriate and steady concentration), which counteracts leaf senescence and essentially improves drought tolerance. A regulatory relationship was found between the C/S1 bZIP network and cytokinins, which provides insights into cytokinin regulation under drought stress. [ABSTRACT FROM AUTHOR]

Summary: Isopentenyltransferase (IPT) in plants regulates a rate‐limiting step of cytokinin (CTK) biosynthesis. IPTs are recognized as key regulators of CTK homeostasis and phytohormone crosstalk in both biotic and abiotic stress responses. Recent research has revealed the regulatory function of IPTs in gene expression and metabolite profiles including source‐sink modifications, energy metabolism, nutrient allocation and storage, stress defence and signalling pathways, protein synthesis and transport, and membrane transport. This suggests that IPTs play a crucial role in plant growth and adaptation. In planta studies of IPT‐driven modifications indicate that, at a physiological level, IPTs improve stay‐green characteristics, delay senescence, reduce stress‐induced oxidative damage and protect photosynthetic machinery. Subsequently, these improvements often manifest as enhanced or stabilized crop yields and this is especially apparent under environmental stress. These mechanisms merit consideration of the IPTs as 'master regulators' of core cellular metabolic pathways, thus adjusting plant homeostasis/adaptive responses to altered environmental stresses, to maximize yield potential. If their expression can be adequately controlled, both spatially and temporally, IPTs can be a key driver for seed yield. In this review, we give a comprehensive overview of recent findings on how IPTs influence plant stress physiology and yield, and we highlight areas for future research. [ABSTRACT FROM AUTHOR]