Light-sensitive short hypocotyl genes confer symbiotic nodule identity in the legume Medicago truncatula.

Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.

From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.

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.

A cell wall-localized cytokinin/purine riboside nucleosidase is involved in apoplastic cytokinin metabolism in Oryza sativa.

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.

Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice.

One-step and two-step pathways are proposed to synthesize cytokinin in plants. The one-step pathway is mediated by LONELY GUY (LOG) proteins. However, the enzyme for the two-step pathway remains to be identified. Here, we show that quantitative trait locus GY3 may boost grain yield by more than 20% through manipulating a two-step pathway. Locus GY3 encodes a LOG protein that acts as a 5'-ribonucleotide phosphohydrolase by excessively consuming the cytokinin precursors, which contrasts with the activity of canonical LOG members as phosphoribohydrolases in a one-step pathway. The residue S41 of GY3 is crucial for the dephosphorylation of iPRMP to produce iPR. A solo-LTR insertion within the promoter of GY3 suppressed its expression and resulted in a higher content of active cytokinins in young panicles. Introgression of GY302428 increased grain yield per plot by 7.4% to 16.3% in all investigated indica backgrounds, which demonstrates the great value of GY302428 in indica rice production.

Phytohormone Crosstalk of Cytokinin Biosynthesis and Signaling Family Genes in Moso Bamboo (Phyllostachys edulis).

Cytokinin is widely involved in the regulation of plant growth, but its pathway-related genes have not been reported in Moso bamboo. In this study, a total of 129 candidate sequences were identified by bioinformatic methods. These included 15 IPT family genes, 19 LOG family genes, 22 HK family genes, 11 HP family genes and 62 RR family genes. Phylogenetic analysis revealed that the cytokinin pathway was closely related to rice, and evolutionary pattern analysis found that most of the genes have syntenic relationship with rice-related genes. The Moso bamboo cytokinin pathway was evolutionarily conservative and mainly underwent purifying selection, and that gene family expansion was mainly due to whole-gene duplication events. Analysis of transcriptome data revealed a tissue-specific expression pattern of Moso bamboo cytokinin family genes, with auxin and gibberellin response patterns. Analysis of co-expression patterns at the developmental stages of Moso bamboo shoots revealed the existence of a phytohormone co-expression pattern centered on cytokinin signaling genes. The auxin signaling factor PheARF52 was identified by yeast one-hybrid assay as regulating the PheRR3 gene through a P-box element in the PheRR3 promoter region. Auxin and cytokinin signaling crosstalk to regulate Moso bamboo growth. Overall, we systematically identified and analyzed key gene families of the cytokinin pathway in Moso bamboo and obtained key factors for auxin and cytokinin crosstalk, laying the foundation for the study of hormone regulation in Moso bamboo.

The mRNA decapping machinery targets LBD3/ASL9 to mediate apical hook and lateral root development.

Multicellular organisms perceive and transduce multiple cues to optimize development. Key transcription factors drive developmental changes, but RNA processing also contributes to tissue development. Here, we report that multiple decapping deficient mutants share developmental defects in apical hook, primary and lateral root growth. More specifically, LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3)/ASYMMETRIC LEAVES 2-LIKE 9 (ASL9) transcripts accumulate in decapping deficient plants and can be found in complexes with decapping components. Accumulation of ASL9 inhibits apical hook and lateral root formation. Interestingly, exogenous auxin application restores lateral roots formation in both ASL9 over-expressors and mRNA decay-deficient mutants. Likewise, mutations in the cytokinin transcription factors type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs) ARR10 and ARR12 restore the developmental defects caused by over-accumulation of capped ASL9 transcript upon ASL9 overexpression. Most importantly, loss-of-function of asl9 partially restores apical hook and lateral root formation in both dcp5-1 and pat triple decapping deficient mutants. Thus, the mRNA decay machinery directly targets ASL9 transcripts for decay, possibly to interfere with cytokinin/auxin responses, during development.

Loss-function mutants of OsCKX gene family based on CRISPR-Cas systems revealed their diversified roles in rice.

Cytokinin (CTK) is an important plant hormone that promotes cell division, controls cell differentiation, and regulates a variety of plant growth and development processes. Cytokinin oxidase/dehydrogenase (CKX) is an irreversible cytokinin-degrading enzyme that affects plant growth and development by regulating the dynamic balance of CTKs synthesis and degradation. There are presumed 11 members of the CKX gene family in rice (Oryza sativa L.), but limited members have been reported. In this study, based on CRISPR-Cas9 and CRISPR-Cas12a genome-editing technology, we established a complete set of OsCKX1-OsCKX11 single-gene mutants, as well as double-gene and triple-gene mutants of different OsCKXs gene combinations with high similarity. The results revealed that CRISPR-Cas12a outperformed Cas9 to generate biallelic mutations, multi-gene mutants, and more diverse genotypes. And then, we found, except the reported OsCKX2, OsCKX4, OsCKX9 and OsCKX11, OsCKX5, OsCKX6, OsCKX7, and OsCKX8 also had significant effects on agronomic traits such as plant height, panicle size, grain size, and grain number per panicle in rice. In addition, the different loss-of-function of the OsCKX genes also changed the seed appearance quality and starch composition. Interestingly, by comparing different combinations of multi-gene mutants, we found significant functional redundancy among OsCKX gene members in the same phylogenetic clade. These data collectively reveal the diversified regulating capabilities of OsCKX genes in rice, and also provide the valuable reference for further rice molecular breeding.

Arrest, Senescence and Death of Shoot Apical Stem Cells in Arabidopsis thaliana.

Shoot stem cells act as the source of the aboveground parts of flowering plants. A precise regulatory basis is required to ensure that plant stem cells show the right status during the stages of proliferation, senescence and cell death. Over the past few decades, the genetic circuits controlling stem cell fate, including the regulatory pathways of establishment, maintenance and differentiation, have been largely revealed. However, the morphological changes and molecular mechanisms of the final stages of stem cells, which are represented by senescence and cell death, have been less studied. The senescence and death of shoot stem cells are under the control of a complex series of pathways that integrate multiple internal and external signals. Given the crucial roles of shoot stem cells in influencing plant longevity and crop yields, researchers have attempted to uncover details of stem cell senescence and death. Recent studies indicate that stem cell activity arrest is controlled by the FRUITFULL-APETALA2 pathway and the plant hormones auxin and cytokinin, while the features of senescent and dead shoot apical stem cells have also been described, with dynamic changes in reactive oxygen species implicated in stem cell death. In this review, we highlight the recent breakthroughs that have enriched our understanding of senescence and cell death processes in plant stem cells.

Transcriptome Analysis Revealed Hormone Pathways and bZIP Genes Responsive to Decapitation in Sunflower.

Decapitation is an essential agricultural practice and is a typical method for analyzing shoot branching. However, it is unclear exactly how decapitation controls branching. In this study, the decapitation of sunflower plants led to the development of lateral buds, accompanied by a decrease in indole-3-acetic acid (IAA) and abscisic acid (ABA) levels and an increase in cytokinin (CK) levels. Additionally, 82 members of the HabZIP family were discovered and categorized into 9 groups, using phylogenetic and conservative domain analysis. The intron/exon structure and motif compositions of HabZIP members were also investigated. Based on tissue-specific expression and expression analysis following decapitation derived from the transcriptome, several HabZIP members may be involved in controlling decapitation-induced bud outgrowth. Therefore, it is hypothesized that the dynamic variations in hormone levels, in conjunction with particular HabZIP genes, led to the development of axillary buds in sunflowers following decapitation.

[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.

The end of flowering: interactions between cytokinin and regulatory genes.

Although the molecular regulation of global proliferative arrest (GPA) in arabidopsis (Arabidopsis thaliana) has been studied extensively, the precise role of the different contributors and their interconnections requires further research. A recent contribution by Merelo et al. now provides evidence that repression of cytokinin (CK) signaling affects the promotion of GPA.

Root electrotropism in Arabidopsis does not depend on auxin distribution but requires cytokinin biosynthesis.

Efficient foraging by plant roots relies on the ability to sense multiple physical and chemical cues in soil and to reorient growth accordingly (tropism). Root tropisms range from sensing gravity (gravitropism), light (phototropism), water (hydrotropism), touch (thigmotropism), and more. Electrotropism, also known as galvanotropism, is the phenomenon of aligning growth with external electric fields and currents. Although root electrotropism has been observed in a few species since the end of the 19th century, its molecular and physical mechanisms remain elusive, limiting its comparison with the more well-defined sensing pathways in plants. Here, we provide a quantitative and molecular characterization of root electrotropism in the model system Arabidopsis (Arabidopsis thaliana), showing that it does not depend on an asymmetric distribution of the plant hormone auxin, but instead requires the biosynthesis of a second hormone, cytokinin. We also show that the dose-response kinetics of the early steps of root electrotropism follows a power law analogous to the one observed in some physiological reactions in animals. Future studies involving more extensive molecular and quantitative characterization of root electrotropism would represent a step toward a better understanding of signal integration in plants and would also serve as an independent outgroup for comparative analysis of electroreception in animals and fungi.

ARResting cytokinin signaling for salt-stress tolerance.

Cytokinins (CKs) are primarily known as a prominent type of plant hormones with pleiotropic functions such as the control of the cell division and morphogenesis. CKs are also well known to orchestrate plant responses to many types of environmental stresses. More specifically, CKs were previously shown to negatively regulate the response to salinity stress. However, the molecular mechanisms underlying this physiological process have not been investigated in detail. In a new report, Yan and colleagues show that salt stress interrupts the CK transduction pathway by promoting the degradation of some CK signaling modules. This represents an unprecedented advancement in our comprehension of how plants are able to inhibit their own development under stress conditions by interfering with the cell signaling circuitry of a growth hormone.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Stage Specificity, the Dynamic Regulators and the Unique Orchid Arundina graminifolia.

Orchids take years to reach flowering, but the unique bamboo orchid (Arundina graminifolia) achieves reproductive maturity in six months and then keeps on year round flowering. Therefore, studying different aspects of its growth, development and flowering is key to boost breeding programs for orchids. This study uses transcriptome tools to discuss genetic regulation in five stages of flower development and four tissue types. Stage specificity was focused to distinguish genes specifically expressed in different stages of flower development and tissue types. The top 10 highly expressed genes suggested unique regulatory patterns for each stage or tissue. The A. graminifolia sequences were blasted in Arabidopsis genome to validate stage specific genes and to predict important hormonal and cell regulators. Moreover, weighted gene co-expression network analysis (WGCNA) modules were ascertained to suggest highly influential hubs for early and late stages of flower development, leaf and root. Hormonal regulators were abundant in all data sets, such as auxin (LAX2, GH3.1 and SAUR41), cytokinin (LOG1), gibberellin (GASA3 and YAB4), abscisic acid (DPBF3) and sucrose (SWEET4 and SWEET13). Findings of this study, thus, give a fine sketch of genetic variability in Orchidaceae and broaden our understanding of orchid flower development and the involvement of multiple pathways.

WHITE AND LESION-MIMIC LEAF1, encoding a lumazine synthase, affects reactive oxygen species balance and chloroplast development in rice.

The riboflavin derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential cofactors for enzymes in multiple cellular processes. Characterizing mutants with impaired riboflavin metabolism can help clarify the role of riboflavin in plant development. Here, we characterized a rice (Oryza sativa) white and lesion-mimic (wll1) mutant, which displays a lesion-mimic phenotype with white leaves, chlorophyll loss, chloroplast defects, excess reactive oxygen species (ROS) accumulation, decreased photosystem protein levels, changes in expression of chloroplast development and photosynthesis genes, and cell death. Map-based cloning and complementation test revealed that WLL1 encodes lumazine synthase, which participates in riboflavin biosynthesis. Indeed, the wll1 mutant showed riboflavin deficiency, and application of FAD rescued the wll1 phenotype. In addition, transcriptome analysis showed that cytokinin metabolism was significantly affected in wll1 mutant, which had increased cytokinin and δ-aminolevulinic acid contents. Furthermore, WLL1 and riboflavin synthase (RS) formed a complex, and the rs mutant had a similar phenotype to the wll1 mutant. Taken together, our findings revealed that WLL1 and RS play pivotal roles in riboflavin biosynthesis, which is necessary for ROS balance and chloroplast development in rice.

Decapitation Experiments Combined with the Transcriptome Analysis Reveal the Mechanism of High Temperature on Chrysanthemum Axillary Bud Formation.

Temperature is an important factor that largely affects the patterns of shoot branching in plants. However, the effect and mechanism of temperature on axillary bud development in chrysanthemum remains poorly defined. The purpose of the present study is to investigate the effect of high temperature on the axillary bud growth and the mechanism of axillary bud formation in chrysanthemum. Decapitation experiments combined with the transcriptome analysis were designed. Results showed that the axillary bud length was significantly inhibited by high temperature. Decapitation of primary shoot (primary decapitation) resulted in slower growth of axillary buds (secondary buds) under 35 °C. However, secondary decapitation resulted in complete arrest of tertiary buds at high temperature. These results demonstrated that high temperature not only inhibited axillary bud formation but also retarded bud outgrowth in chrysanthemum. Comparative transcriptome suggested differentially expressed gene sets and identified important modules associated with bud formation. This research helped to elucidate the regulatory mechanism of high temperature on axillary bud growth, especially bud formation in chrysanthemum. Meanwhile, in-depth studies of this imperative temperature signaling can offer the likelihood of vital future applications in chrysanthemum breeding and branching control.

The Effect of Exogenous Nitrate on LCO Signalling, Cytokinin Accumulation, and Nodule Initiation in Medicago truncatula.

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2/sickle, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.

Analysis of Phytohormone Signal Transduction in Sophora alopecuroides under Salt Stress.

Salt stress seriously restricts crop yield and quality, leading to an urgent need to understand its effects on plants and the mechanism of plant responses. Although phytohormones are crucial for plant responses to salt stress, the role of phytohormone signal transduction in the salt stress responses of stress-resistant species such as Sophora alopecuroides has not been reported. Herein, we combined transcriptome and metabolome analyses to evaluate expression changes of key genes and metabolites associated with plant hormone signal transduction in S. alopecuroides roots under salt stress for 0 h to 72 h. Auxin, cytokinin, brassinosteroid, and gibberellin signals were predominantly involved in regulating S. alopecuroides growth and recovery under salt stress. Ethylene and jasmonic acid signals may negatively regulate the response of S. alopecuroides to salt stress. Abscisic acid and salicylic acid are significantly upregulated under salt stress, and their signals may positively regulate the plant response to salt stress. Additionally, salicylic acid (SA) might regulate the balance between plant growth and resistance by preventing reduction in growth-promoting hormones and maintaining high levels of abscisic acid (ABA). This study provides insight into the mechanism of salt stress response in S. alopecuroides and the corresponding role of plant hormones, which is beneficial for crop resistance breeding.

Sound Waves Promote Arabidopsis thaliana Root Growth by Regulating Root Phytohormone Content.

Sound waves affect plants at the biochemical, physical, and genetic levels. However, the mechanisms by which plants respond to sound waves are largely unknown. Therefore, the aim of this study was to examine the effect of sound waves on Arabidopsis thaliana growth. The results of the study showed that Arabidopsis seeds exposed to sound waves (100 and 100 + 9k Hz) for 15 h per day for 3 day had significantly longer root growth than that in the control group. The root length and cell number in the root apical meristem were significantly affected by sound waves. Furthermore, genes involved in cell division were upregulated in seedlings exposed to sound waves. Root development was affected by the concentration and activity of some phytohormones, including cytokinin and auxin. Analysis of the expression levels of genes regulating cytokinin and auxin biosynthesis and signaling showed that cytokinin and ethylene signaling genes were downregulated, while auxin signaling and biosynthesis genes were upregulated in Arabidopsis exposed to sound waves. Additionally, the cytokinin and auxin concentrations of the roots of Arabidopsis plants increased and decreased, respectively, after exposure to sound waves. Our findings suggest that sound waves are potential agricultural tools for improving crop growth performance.