Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development.

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions.

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts.

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-D-glucose analogue (+)-streptol, systemically supplied by mature Ca. Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria-Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

H2O2 response gene 1/2 are novel sensors or responders of H2O2 and involve in maintaining embryonic root meristem activity in Arabidopsis thaliana.

Signal molecule hydrogen peroxide (H2O2) plays critical roles in various processes of plant development. However, H2O2 signaling network, especially the responders that sense and respond to the H2O2 signal remain largely unknown. Here we report two homologous genes H2O2 Response Gene 1 and 2 (HRG1/2) in Arabidopsis that could quickly respond to exogenous or endogenous H2O2. Knockdown of HRG1/2 facilitated seed germination while overexpression of HRG1/2 greatly retarded seed germination. ROS level in HRG1 overexpression roots was significantly lower than that in HRG1/2 mutants after H2O2 treatment. The expression level of enzymatic antioxidant DHAR3 was upregulated in HRG1 overexpression plants, suggesting that DHAR3 is downstream of HRG1. That the root meristem length and cell number were significantly reduced in hrg1-1 and hrg2-1 plants upon H2O2 treatment compared to that of HRG1 overexpression plants also approves the idea that HRGs function in H2O2 removal. Further evolutionary analysis indicates that this is a dicotyledon-specific pathway responsive to H2O2. Together, this work reveals HRG1/2 as novel H2O2 responders involved in ROS scavenging to ensure embryonic root meristem activity. These findings provide valuable clues for the of H2O2 signaling and root meristem regulation.

Coumarin Interferes with Polar Auxin Transport Altering Microtubule Cortical Array Organization in Arabidopsis thaliana (L.) Heynh. Root Apical Meristem.

Coumarin is a phytotoxic natural compound able to affect plant growth and development. Previous studies have demonstrated that this molecule at low concentrations (100 µM) can reduce primary root growth and stimulate lateral root formation, suggesting an auxin-like activity. In the present study, we evaluated coumarin's effects (used at lateral root-stimulating concentrations) on the root apical meristem and polar auxin transport to identify its potential mode of action through a confocal microscopy approach. To achieve this goal, we used several Arabidopsis thaliana GFP transgenic lines (for polar auxin transport evaluation), immunolabeling techniques (for imaging cortical microtubules), and GC-MS analysis (for auxin quantification). The results highlighted that coumarin induced cyclin B accumulation, which altered the microtubule cortical array organization and, consequently, the root apical meristem architecture. Such alterations reduced the basipetal transport of auxin to the apical root apical meristem, inducing its accumulation in the maturation zone and stimulating lateral root formation.

CLAVATA3, a plant peptide controlling stem cell fate in the meristem.

CLAVATA3 (CLV3) is a peptide signal initially identified in the analysis of clv mutants in the model plant Arabidopsis thaliana, as a regulator of meristem homeostasis and floral organ numbers. CLV3 homologs are widely conserved in land plants, collectively called CLV3/ESR-related (CLE) genes. A 12-amino acid CLE peptide with hydroxyproline residues was identified in Zinnia elegans cell culture system, in which cells secrete a CLE peptide called tracheary element differentiation factor (TDIF) into the culture medium. Mature CLV3 peptide is also a post-translationally modified short peptide containing additional triarabinosylation on a hydroxyproline residue. Genetic studies have revealed the involvement of leucin-rich repeat receptor-like kinases (LRR-RLKs) in CLV3 signaling, including CLV1/BAM-CIK, CLV2-CRN and RPK2, although the mechanisms of signal transduction and integration via crosstalk is still largely unknown. Recent studies on bryophyte model species provided a clue to understand evolution and ancestral function of CLV signaling in land plants. Fundamental understanding on CLV signaling provided an opportunity to optimize the crop yield traits using a novel breeding technology with CRISPR/Cas genome editing.

Iso-anchorene is an endogenous metabolite that inhibits primary root growth in Arabidopsis.

Carotenoid-derived regulatory metabolites and hormones are generally known to arise through the oxidative cleavage of a single double bond in the carotenoid backbone, which yields mono-carbonyl products called apocarotenoids. However, the extended conjugated double bond system of these pigments predestines them also to repeated cleavage forming dialdehyde products, diapocarotenoids, which have been less investigated due to their instability and low abundance. Recently, we reported on the short diapocarotenoid anchorene as an endogenous Arabidopsis metabolite and specific signaling molecule that promotes anchor root formation. In this work, we investigated the biological activity of a synthetic isomer of anchorene, iso-anchorene, which can be derived from repeated carotenoid cleavage. We show that iso-anchorene is a growth inhibitor that specifically inhibits primary root growth by reducing cell division rates in the root apical meristem. Using auxin efflux transporter marker lines, we also show that the effect of iso-anchorene on primary root growth involves the modulation of auxin homeostasis. Moreover, by using liquid chromatography-mass spectrometry analysis, we demonstrate that iso-anchorene is a natural Arabidopsis metabolite. Chemical inhibition of carotenoid biosynthesis led to a significant decrease in the iso-anchorene level, indicating that it originates from this metabolic pathway. Taken together, our results reveal a novel carotenoid-derived regulatory metabolite with a specific biological function that affects root growth, manifesting the biological importance of diapocarotenoids.

Signaling network regulating plant branching: Recent advances and new challenges.

Auxin alone or supplemented with cytokinins and strigolactones were long considered as the main player(s) in the control of apical dominance (AD) and correlative inhibition of the lateral bud outgrowth, the processes that shape the plant phenotype. However, past decade data indicate a more sophisticated pathways of AD regulation, with the involvement of mobile carbohydrates which perform both signal and trophic functions. Here we provide a critical comprehensive overview of the current status of the AD problem. This includes insight into intimate mechanisms regulating directed auxin transport in axillary buds with participation of phytohormones and sugars. Also roles of auxin, cytokinin and sugars in the dormancy or sustained growth of the lateral meristems were assigned. This review not only provides the latest data on implicated phytohormone crosstalk and its relationship with the signaling of sugars and abscisic acid, new AD players, but also focuses on the emerging biochemical mechanisms, at first positive feedback loops involving both sugars and hormones, that ensure the sustained bud growth. Data show that sugars act in concert with cytokinins but antagonistically to strigolactone signaling. A complex bud growth regulating network is demonstrated and unresolved issues regarding the hormone-carbohydrate regulation of AD are highlighted.

Putrescine Depletion Affects Arabidopsis Root Meristem Size by Modulating Auxin and Cytokinin Signaling and ROS Accumulation.

Polyamines (PAs) dramatically affect root architecture and development, mainly by unknown mechanisms; however, accumulating evidence points to hormone signaling and reactive oxygen species (ROS) as candidate mechanisms. To test this hypothesis, PA levels were modified by progressively reducing ADC1/2 activity and Put levels, and then changes in root meristematic zone (MZ) size, ROS, and auxin and cytokinin (CK) signaling were investigated. Decreasing putrescine resulted in an interesting inverted-U-trend in primary root growth and a similar trend in MZ size, and differential changes in putrescine (Put), spermidine (Spd), and combined spermine (Spm) plus thermospermine (Tspm) levels. At low Put concentrations, ROS accumulation increased coincidently with decreasing MZ size, and treatment with ROS scavenger KI partially rescued this phenotype. Analysis of double AtrbohD/F loss-of-function mutants indicated that NADPH oxidases were not involved in H2O2 accumulation and that elevated ROS levels were due to changes in PA back-conversion, terminal catabolism, PA ROS scavenging, or another pathway. Decreasing Put resulted in a non-linear trend in auxin signaling, whereas CK signaling decreased, re-balancing auxin and CK signaling. Different levels of Put modulated the expression of PIN1 and PIN2 auxin transporters, indicating changes to auxin distribution. These data strongly suggest that PAs modulate MZ size through both hormone signaling and ROS accumulation in Arabidopsis.

Sucrose interferes with endogenous cytokinin homeostasis and expression of organogenesis-related genes during de novo shoot organogenesis in kohlrabi.

Cross-talk between phytohormones and sugars is intensely involved in plant metabolism, growth and regeneration. We documented alterations in cytokinin (CK) homeostasis in four developmental stages during de novo shoot organogenesis (DNSO) of kohlrabi (Brassica oleracea var. gongylodes cv. Vienna Purple) seedlings induced by exogenous CKs, trans-zeatin (transZ) and thidiazuron (TDZ), added together with elevated sucrose concentration (6% and 9%). Significant impact of CK and sucrose treatment and their interaction was recorded in all investigated stages, including plantlet development before calli formation (T1 and T2), calli formation (T3) and shoot regeneration (T4). Results showed remarkable increase in total CK levels for transZ treatment, particularly with 9% sucrose. This trend was observed for all physiological and structural groups of CKs. Application of TDZ contributed to little or no increase in CK levels regardless of sucrose concentration. Analysis of expression profiles of organogenesis-related genes involved in auxin transport, CK response, shoot apical meristem formation and cell division revealed that higher sugar concentration significantly downregulated the analysed genes, particularly in T3. This continued on TDZ, but transZ induced an opposite effect with 9% sucrose in T4, increasing gene activity. Our results demonstrated that phytohormone metabolism might be triggered by sucrose signalling in kohlrabi DNSO.

Latrunculin B facilitates gravitropic curvature of Arabidopsis root by inhibiting cell elongation, especially the cells in the lower flanks of the transition and elongation zones.

Gravitropism plays a critical role in the growth and development of plants. Previous reports proposed that the disruption of the actin cytoskeleton resulted in enhanced gravitropism; however, the mechanism underlying these phenomena is still unclear. In the present study, real-time observation on the effect of Latrunculin B (Lat B), a depolymerizing agent of microfilament cytoskeleton, on gravitropism of the primary root of Arabidopsis was undertaken using a vertical stage microscope. The results indicated that Lat B treatment prevented the growth of root, and the growth rates of upper and lower flanks of the horizontally placed root were asymmetrically inhibited. The growth of the lower flank was influenced by Lat B more seriously, resulting in an increased differential growth rate between the upper and lower flanks of the root. Further analysis indicated that Lat B affected cell growth mainly in the transition and elongation zones. Briefly, the current data revealed that Lat B treatment inhibited cell elongation, especially the cells in the lower flanks of the transition and elongation zones, which finally manifested as the facilitation of gravitropic curvature of the primary root.

Toxicogenetic of tebuconazole based fungicide through Lactuca sativa bioassays.

The rampant use of pesticides can cause serious environmental problems. They can be contaminating surface water and groundwater, affecting the surrounding micro and macro biota. In this sense, this work aimed to evaluate the effects of a tebuconazole-based fungicide through endpoints accessed in Lactuca sativa bioassays. Germinated-seeds with roots upon 2 mm were treated with a fungicide containing Tebuconazole (TBZ) as active compound. The final concentration of TBZ in the tested solutions were 0.025 (C1); 0.05 (C2); 0.1 (C3); 0.2 (C4) and 0.4 g/L (C5). L. sativa roots were exposed for 24 h to these solutions and Petri dishes containing the treated seeds were kept in incubation chamber at 24 °C. Two positive controls (PC,) the herbicide trifluralin (0.84 mg/L) and Methanesulfonate (4 ×10-4 mol/L), were applied. Distilled water was negative control (NC). The following endpoints were analyzed: root growth (RG), cytogenotoxic potential by cell cycle analysis, induction of DNA damage through TUNEL and comet assays. The obtained data were submitted to one-way variance analysis (ANOVA) and then to Tukey or Kruskal Wallis (P < 0.05) tests. The concentrations (C1, C2, C4 and C5) affected negatively the RG of L. sativa, in comparison with the NC. The mitotic index was reduced by 25% from NC to C1 and in the rest of treatments it did not present significant modifications. However, from C3 to C5 great amount of chromosome alterations were observed, in comparison with the NC. TBZ-based fungicide also induced DNA fragmentation as measured by TUNEL and comet assays. Thus, TBZ-based fungicide in some concentrations can have phytotoxic, cytotoxic and genotoxic effects in roots and meristematic cells of L. sativa.

Nitric oxide reduces the aluminum-binding capacity in rice root tips by regulating the cell wall composition and enhancing antioxidant enzymes.

Plant cell wall, the first interface or barrier for toxic ions entering into protoplast, suffers from risk. Nitric oxide (NO) plays an important role in plant growth and responses to abiotic stresses, however, it is not clear whether NO is connected with the response of cell wall to aluminum (Al) tolerance in rice (Oryza sativa L.). In this study, we found that the application of 50 µM Al induces nitrate reductase (NR) activity and endogenous NO production, but not nitric oxide synthase (NOS) activity in two rice genotypes. Pretreatment with 100 µM NO donor (sodium nitroprusside, SNP) reduced Al-induced inhibition of root elongation by 32.3% and 91.7%, and Al accumulation in root-tip by 38.4% and 44.3% in Nipponbare and Zhefu802, respectively. The addition of SNP significantly decreased Al-induced accumulation of pectin, hemicellulose 1 and hemicellulose 2 by 43.1%, 13.1% and 19.2% in Zhefu802 and by 16.9%, 13.4% and 14.0% in Nipponbare, compared with roots treated with Al alone, as well as pectin methylesterase (PME) activity. Therefore, the content of Al absorbed in cell walls was decreased, indicating that the Al-induced structure damage to cell walls was alleviated. Furthermore, the activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) treated by Al were all increased by SNP pretreatment, and the lipid peroxidation and damage to plasma membrane of root tips detected with Schiff's reagent and Evans blue reduced. In contrast, the effect was abolished when NO scavenger (cPTIO), and NR inhibitor (NaN3), were added. These results indicated that by regulating the Al-binding capacity to cell walls and lipid peroxidation, the structure of cell walls can be stabilized and that Al toxicity in rice can be alleviated with increased NO.

Chemotaxonomy of the ethnic antidote Aristolochia indica for aristolochic acid content: Implications of anti-phospholipase activity and genotoxicity study.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochia indica L. (Aristolochiaceae) is a common medicinal plant described in many traditional medicine as well as in Ayurveda used against snakebites. Besides, the plant has also been reported traditionally against fever, rheumatic arthritis, madness, liver ailments, dyspepsia, oedema, leishmaniasis, leprosy, dysmenorrhoea, sexual diseases etc. The plant is known to contain its major bioactive constituent aristolochic acid (AA) known for its anti-snake venom, abortifacient, antimicrobial and antioxidant properties. MATERIALS AND METHODS: This present work describes a validated, fast and reproducible high performance thin layer chromatography (HPTLC) method to estimate AA from the roots of 20 chemotypes of A. indica procured from 20 diverse geographical locations from the state of West Bengal, India. Further, an evidence-based approach was adopted to investigate the reported anti-venom activity of the aqueous extracts of the A. indica roots by assessing its phospholipase A2 (PLA2) inhibitory properties since PLA2 is a major component of many snake-venoms. Finally, the cytotoxicity and genotoxicity of the aqueous root extract of the Purulia (AI 1) chemotype were assessed at various concentrations using Allium cepa root meristematic cells. RESULTS: The highest amount of AA (7643.67 µg/g) was determined in the roots of A. indica chemotype collected from Purulia district followed by the chemotypes collected from Murshidabad, Jalpaiguri and Birbhum districts (7398.34, 7345.09 and 6809.97 µg/g respectively). This study not only determines AA in the plants to select pharmacologically elite chemotypes of A. indica, but it also identifies high AA producing A. indica for further domestication and propagation of the plants for pharmacological and industrial applications. The method was validated via analyzing inter-day and intra-day precision, repeatability, reproducibility, instrumental precision, limit of detection (LOD) and limit of quantification (LOQ) and specificity. Chemotypes with high AA content exhibited superior anti-PLA2 activity by selectively inhibiting human-group PLA2. Moreover, A. indica root extract significantly inhibited mitosis in Allium cepa root tips as a potent clastogen. CONCLUSIONS: The present quick, reproducible and validated HPTLC method provides an easy tool to determine AA in natural A. indica plant populations as well as in food and dietary supplements, a potential antivenin at one hand and a possible cause of aristolochic acid nephropathy (AAN) at another. Besides, the cytotoxic and mitotoxic properties of the root extracts should be used with caution especially for oral administration.

Overexpression of the Auxin Receptor AFB3 in Arabidopsis Results in Salt Stress Resistance and the Modulation of NAC4 and SZF1.

Soil salinity is a key problem for crop production worldwide. High salt concentration in soil negatively modulates plant growth and development. In roots, salinity affects the growth and development of both primary and lateral roots. The phytohormone auxin regulates various developmental processes during the plant's life cycle, including several aspects of root architecture. Auxin signaling involves the perception by specialized receptors which module several regulatory pathways. Despite their redundancy, previous studies have shown that their functions can also be context-specific depending on tissue, developmental or environmental cues. Here we show that the over-expression of Auxin Signaling F-Box 3 receptor results in an increased resistance to salinity in terms of root architecture and germination. We also studied possible downstream signaling components to further characterize the role of auxin in response to salt stress. We identify the transcription factor SZF1 as a key component in auxin-dependent salt stress response through the regulation of NAC4. These results give lights of an auxin-dependent mechanism that leads to the modulation of root system architecture in response to salt identifying a hormonal cascade important for stress response.

Toxicity assessment of zinc sulfate: A commonly used compound.

Because zinc sulfate (ZnSO4) is widely used in many fields such as biomedicine, electronics, and chemistry, it is important to evaluate its toxic effects. In this study, the cyto-genotoxic effects of ZnSO4 on meristematic cells in the root tip of Allium cepa L. were investigated. After calculating the effective concentration (EC50 = 70 ppm) of ZnSO4, A. cepa root tip cells were suspended for 24, 48, 72, and 96 h in solutions of 35 ppm (EC50/2), 70 ppm (EC50), and 140 ppm (EC50 × 2) concentrations. Using the counts of dividing cells, the mitotic index (MI) was calculated. Chromosome aberration index (CAI) was determined from percentages of abnormal cells. When the obtained data were statistically evaluated, it was determined that all application concentrations caused a significant decrease in MI and an increase in CAI compared to the control group (distilled water). It was concluded that increased ZnSO4 dose concentrations and exposure times caused cytotoxicity and genotoxicity in the root cells of A. cepa L.

Hydrogen sulfide negatively regulates cd-induced cell death in cucumber (Cucumis sativus L) root tip cells.

BACKGROUND: Hydrogen sulfide (H2S) is a gas signal molecule involved in regulating plants tolerance to heavy metals stress. In this study, we investigated the role of H2S in cadmium-(Cd-) induced cell death of root tips of cucumber seedlings. RESULTS: The results showed that the application of 200 µM Cd caused cell death, increased the content of reactive oxygen species (ROS), chromatin condensation, the release of Cytochrome c (Cyt c) from mitochondria and activated caspase-3-like protease. Pretreatment of seedlings with 100 µM sodium hydrogen sulfide (NaHS, a H2S donor) effectively alleviated the growth inhibition and reduced cell death of root tips caused by Cd stress. Additionally, NaHS + Cd treatment could decrease the ROS level and enhanced antioxidant enzyme activity. Pretreatment with NaHS also inhibited the release of Cyt c from the mitochondria, the opening of the mitochondrial permeability transition pore (MPTP), and the activity of caspase-3-like protease in the root tips of cucumber seedling under Cd stress. CONCLUSION: H2S inhibited Cd-induced cell death in cucumber root tips by reducing ROS accumulation, activating the antioxidant system, inhibiting mitochondrial Cyt c release and reducing the opening of the MPTP. The results suggest that H2S is a negative regulator of Cd-induced cell death in the root tips of cucumber seedling.

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

The HK5 and HK6 cytokinin receptors mediate diverse developmental pathways in rice.

The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice (Oryza sativa) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.