Adventitious Root Regeneration:Molecular Basis and Influencing Factors

Plant regeneration is a self-repair of the plant body in response to adverse conditions or damaged structures,and root regeneration allows the plant body to better adapt to its environment by supplementing the roots’structure.Previous research has shown that adventitious roots can be made to occur from scratch in two ways.Studies that simulate adventitious root regeneration through natural conditions allow the regeneration process to be broadly divided into three stages:the perception of early signals,the massive accumulation of auxin,and the transformation of cell fate.The strength of regeneration,in turn,is influenced by wounding,stress,hormones,etc.This study mainly reviews the molecular mechanisms of de novo adventitious roots and discusses how the environment,hormones,and its own development in Arabidopsis thaliana.

Additional far-red light promotes adventitious rooting of doubleroot-cutting grafted watermelon seedlings

Root regeneration is an important factor influencing the healing rate of graft union and the survival of double-root-cutting grafting.To date,little information is available on how to enhance root regeneration of rootstock in grafted watermelon(Citrullus lanatus)seedlings.In this study,the effects of different light treatments on root regeneration were determined.This revealed that addition of far-red light(Fr)could significantly expedite root formation in the rootstock.Moreover,the results of transcriptome analysis revealed that plant hormone pathway and auxinrelated genes were greatly induced by Fr,especially for auxin-response proteins(including CmIAA11,CmIAA17,and CmAUX28),Small auxinup RNA genes(including CmSAUR20 and CmSAUR50)and the auxin efflux transporter(CmPIN3).In addition,the expression of Phytochrome Interacting Factor(PIFs),such as CmPIF1,CmPIF3 and CmPIF7,was remarkably increased by Fr.These genes may act together to activate auxinrelated pathways under Fr treatment.Based on the results of HPLC-MS/MS analysis,the concentrations of different auxin-types in adventitious root were significantly influenced by Fr.Furthermore,the better growth of rootstock root displayed superior vasculature transport activity of the graft union with Fr treatment,which was determined by the acid magenta dyeing experiment.Therefore,all the results suggested that Fr could induce AR formation in rootstocks,which may be associated with the auxin accumulation by regulating the transcriptional level of auxinrelated and PIF genes.The findings of this study demonstrated a practicable way to shorten the healing period of graftings and improve the quality of grafted watermelon seedlings,which will provide a theoretical basis for the speeding development of industrialized seedlings production.

Sensory regeneration in dorsal root avulsion

Brachial as well as lumbosacral plexus avulsion injuries are usually caused by high kinetic traumas,such as car-pedestrian,car and motorcycle accidents or falls from great heights.Traction forces affecting the head and shoulders or extremities pull the spinal nerve sleeves away from the spinal cord and rupture the postganglionic spinal root from the cord.In so called central avulsion injuries,the spinal root is avulsed at the peripheral nervous system （CNS interface between the central and and PNS）. This results not only in the disconnection of the root from the cord but also in a longitudi- nal spinal cord injury. The complexity of the injury leads to degen- eration of the spinal root and a marked inflammatory response of the spinal cord followed by the formation of a glial scar （Kachra- manoglou et al., 2011）.

Targeting cell surface receptors for axon regeneration in the central nervous system

Axon regeneration in the CNS is largely unsuccessful due to excess inhibitory extrinsic factors within lesion sites together with an intrinsic inability of neurons to regrow following injury. Recent work demonstrates that forced expression of certain neuronal transmembrane receptors can recapitulate neuronal growth resulting in successful growth within and through inhibitory lesion environments. More specifically, neuronal expression of integrin receptors such as alpha9beta1 integrin which binds the extracellular matrix glycoprotein tenascin-C, trk receptors such as trk B which binds the neurotrophic factor BDNF, and receptor PTPσ which binds chondroitin sulphate proteoglycans, have all been show to significantly enhance regeneration of injured axons. We discuss how reintroduction of these receptors in damaged neurons facilitates signalling from the internal environment of the cell with the external environment of the lesion milieu, effectively resulting in growth and repair following injury. In summary, we suggest an appropriate balance of intrinsic and extrinsic factors are required to obtain substantial axon regeneration.

Pulsed electrical stimulation protects neurons in the dorsal root and anterior horn of the spinal cord after peripheral nerve injury

Most studies on peripheral nerve injury have focused on repair at the site of injury, but very few have examined the effects of repair strategies on the more proximal neuronal cell bodies. In this study, an approximately 10-mm-long nerve segment from the ischial tuberosity in the rat was transected and its proximal and distal ends were inverted and sutured. The spinal cord was subjected to pulsed electrical stimulation at T10 and L3, at a current of 6.5 m A and a stimulation frequency of 15 Hz, 15 minutes per session, twice a day for 56 days. After pulsed electrical stimulation, the number of neurons in the dorsal root ganglion and anterior horn was increased in rats with sciatic nerve injury. The number of myelinated nerve fibers was increased in the sciatic nerve. The ultrastructure of neurons in the dorsal root ganglion and spinal cord was noticeably improved. Conduction velocity of the sciatic nerve was also increased. These results show that pulsed electrical stimulation protects sensory neurons in the dorsal root ganglia as well as motor neurons in the anterior horn of the spinal cord after peripheral nerve injury, and that it promotes the regeneration of peripheral nerve fibers.

In vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity

Ciliary neurotrophic factor has neuroprotective effects mediated through signal transducer and Janus kinase（JAK） 2/activator of transcription 3（STAT3） and phosphatidylinositol 3-kinase（PI3 K）/Akt signaling pathways.Whether ciliary neurotrophic factor is neuroprotective for glutamate-induced excitotoxicity of dorsal root ganglion neurons is poorly understood.In the present study,the in vitro neuroprotective effects of ciliary neurotrophic factor against glutamate-induced excitotoxicity were determined in a primary culture of dorsal root ganglion neurons from Wistar rat embryos at embryonic day 15.Whether the JAK2/STAT3 and PI3 K/Akt signaling pathways were related to the protective effects of ciliary neurotrophic factor was also determined.Glutamate exposure inhibited neurite outgrowth,cell viability,and growth-associated protein 43 expression and promoted apoptotic neuronal cell death,all of which were reversed by the administration of exogenous ciliary neurotrophic factor.Additionally,preincubation with either JAK2 inhibitor AG490 or PI3 K inhibitor LY294002 blocked the neuroprotective effect of ciliary neurotrophic factor.These data indicate that the two pathways JAK2/STAT3 and PI3 K/Akt play major roles in mediating the in vitro neuroprotective effects of ciliary neurotrophic factor on dorsal root ganglion neurons with glutamate-induced neurotoxicity.

Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury

Paired immunoglobulin-like receptor B（Pir B） is a functional receptor of myelin-associated inhibitors for axonal regeneration and synaptic plasticity in the central nervous system, and thus suppresses nerve regeneration. The regulatory effect of Pir B on injured nerves has received a lot of attention. To better understand nerve regeneration inability after spinal cord injury, this study aimed to investigate the distribution of Pir B（via immunofluorescence） in the central nervous system and peripheral nervous system 10 days after injury. Immunoreactivity for Pir B increased in the dorsal root ganglia, sciatic nerves, and spinal cord segments. In the dorsal root ganglia and sciatic nerves, Pir B was mainly distributed along neuronal and axonal membranes. Pir B was found to exhibit a diffuse, intricate distribution in the dorsal and ventral regions. Immunoreactivity for Pir B was enhanced in some cortical neurons located in the bilateral precentral gyri. Overall, the findings suggest a pattern of Pir B immunoreactivity in the nervous system after unilateral spinal transection injury, and also indicate that Pir B may suppress repair after injury.

Impact of disturbance from roadworks on Pteleopsis suberosa regeneration in roadside environments in Burkina Faso, West Africa

The seedling population structure of Pteleopsis suberosa and their regeneration mechanisms were investigated in four roadside environments （graded, adjacent, intermediate and ungraded areas） along paved and unpaved roads in West Africa. A total of 203 quadrats of 2 m × 5 m in size were surveyed and placed along transects parallel to the roads. Within each quadrat, the total number of seedlings and the number of living shoots per seedling base were recorded. Regeneration mechanisms were determined by assessing basal and aerial sprouts and excavating the root systems below ground level. The results show that the total seedling density and the densities of single- and multi-stemmed individuals varied significantly （p 〈 0.05） among the four roadside environments. However, all seedlings were produced asexually; root suckers were predominant （98%） compared to water sprout （1%） and coppices （less than 1%）. This study demonstrates that an intermediate level of soil disturbance from grading along paved and unpaved roads may stimulate P. suberosa regeneration by root suck- ering. Road type （paved and unpaved） did not affect seedling density, but was a highly significant variable in relation to the coppicing ability of P. suberosa populations in roadside sites. In conclusion, P. suberosa is a disturbance-tolerant species which can proliferate mainly by root suckering after roadwork disturbance.

End-to-side neurorrhaphy repairs peripheral nerve injury：sensory nerve induces motor nerve regeneration

End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve.It involves suturing the distal stump of the disconnected nerve（recipient nerve） to the side of the intimate adjacent nerve（donor nerve）.However,the motor-sensory specificity after end-to-side neurorrhaphy remains unclear.This study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves after end-to-side neurorrhaphy.Thirty rats were randomized into three groups：（1） end-to-side neurorrhaphy using the ulnar nerve（mixed sensory and motor） as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve;（2） the sham group：ulnar nerve and cutaneous antebrachii medialis nerve were just exposed;and（3） the transected nerve group：cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied.At 5 months,acetylcholinesterase staining results showed that 34% ± 16% of the myelinated axons were stained in the end-to-side group,and none of the myelinated axons were stained in either the sham or transected nerve groups.Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%.In contrast,no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment.These results confirmed that motor neuron regeneration occurred after cutaneous nerve end-to-side neurorrhaphy.

Evaluation on Chinese Bread Wheat Landraces for Low pH and Aluminum Tolerance Using Hydroponic Screening

Aluminum （Al） toxicity often takes place in acidic soils with a pH of 5.5 or lower. Breeding and cultivation of Al tolerance wheat can partially protect wheat escaping from Al toxicity. The scarcity of the tolerant sources impedes the wheat breeding. In order to find new Al tolerance sources, we screened 173 bread wheat landraces from Tibet of China using hydroponic screening. It was indicated that： （1） There were diversities on the root regenerate length （RRL）. The RRL of a large of landraces were longer than 7.00 cm in pH 7 （58.38%） and pH 4.5 （66.47%）, but shorter than 5.00 cm in pH 4.5 ＋50μM Al^3＋ （80.93%）. The low pH showed either promotion or restraining effects depend on landraces, but Al toxicity under low pH only showed restraining effects on the root elongation. （2） There were also diversities on root tolerance index of low pH （RTI 1） or root aluminum tolerance index （RTI2） among cultivars. The RTI1 varied from a narrow range but with relatively high value （0.8722-1.2953） in comparison with that of RTI2 （0.3829-1.0058）, and the RTI1 of approximately 60% landraces was higher than 1.0000, the RTI2 of only 19.07% landraces was higher than 0.7000, suggesting that Al toxicity acted as an important factor for the reduction of the root elongation under acidic soils. （3） The RTI 1 of many wheats was higher than 1.0000, and As2256 and As2295 were the most tolerant for low pH, with RTI1 1.2953 and 1.2925, respectively. （4） Based on RTI2, seven wheats showed similar or higher tolerance to Al toxicity than Chinese Spring （CS）, a known tolerance wheat. Much better tolerance existed in landraces of As1543 and As1242, which can be used as the new parents for Al tolerant breeding.

Transcriptional landscapes of de novo root regeneration from detached Arabidopsis leaves revealed by time-lapse and single-cell RNA sequencing analyses

Detached Arabidopsis thaliana leaves can regenerate adventitious roots,providing a platformfor studying de novo root regeneration(DNRR).However,the comprehensive transcriptional framework of DNRR remains elusive.Here,we provide a high-resolution landscape of transcriptome reprogramming from wound response to root organogenesis in DNRR and show key factors involved in DNRR.Time-lapse RNA sequencing(RNA-seq)of the entire leaf within 12 h of leaf detachment revealed rapid activation of jasmonate,ethylene,and reactive oxygen species(ROS)pathways in response towounding.Genetic analyses confirmed that ethylene andROSmay serve as wound signals to promoteDNRR.Next,time-lapse RNA-seq within 5 d of leaf detachment revealed the activation of genes involved in organogenesis,wound-induced regeneration,and resource allocation in the wounded region of detached leaves during adventitious rooting.Genetic studies showed that BLADE-ON-PETIOLE1/2,which control aboveground organs,PLETHORA3/5/7,which control root organogenesis,and ETHYLENE RESPONSE FACTOR115,which controlswound-induced regeneration,are involved in DNRR.Furthermore,single-cell RNA-seq data revealed gene expression patterns in thewounded region of detached leaves during adventitious rooting.Overall,our study not only provides transcriptome tools but also reveals key factors involved in DNRR from detached Arabidopsis leaves.

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis,thereby exerting a neuroprotective effect.In the present study,we established a rat model of spinal cord contusion injury using the modified Allen＇s method.Three hours after injury,senegenin（30 mg/g） was injected into the tail vein for 3 consecutive days.Senegenin reduced the size of syringomyelic cavities,and it substantially reduced the number of apoptotic cells in the spinal cord.At the site of injury,Bax and Caspase-3 m RNA and protein levels were decreased by senegenin,while Bcl-2 m RNA and protein levels were increased.Nerve fiber density was increased in the spinal cord proximal to the brain,and hindlimb motor function and electrophysiological properties of rat hindlimb were improved.Taken together,our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.

TAA family contributes to auxin production during de novo regeneration of adventitious roots from Arabidopsis leaf explants

Many differentiated plant organs have the ability to regenerate into a new plant after detachment via de novo organogenesis. During de novo root organogenesis from Arabidopsis thaliana leaf explants, wounding first induces endogenous auxin production in mesophyll cells. Auxin is then polar transported to, and accumulates in, regenerationcompetent cells near the wound to trigger the cell-fate transition. The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS(TAA) family proteins and the YUCCA(YUC) family proteins catalyze two successive biochemical steps in auxin biogenesis, and YUCs have been shown to be involved in auxin production in mesophyll cells during de novo root organogenesis. In thisstudy, we show that the TAA family is also required for adventitious rooting. Inhibition of TAA blocked adventitious root formation from leaf explants. Intriguingly,whereas YUC1 and YUC4 have been shown to be highly induced by wounding, TAA genes retained consistent expression levels before and after leaf detachment.Therefore, we suggest that TAAs and YUCs are both required for auxin biogenesis in leaf explants, but they play different roles in regeneration. While YUC1 and YUC4 function in response to wounding to catalyze the ratelimiting step in auxin biosynthesis, TAAs probably serve as abiding and basal enzymes during de novo root organogenesis from leaf explants.

Central projections and connections of lumbar primary afferent fibers in adult rats:effectively revealed using Texas red-dextran amine tracing

Signals from lumbar primary afferent fibers are important for modulating locomotion of the hind-limbs.However,silver impregnation techniques,autoradiography,wheat germ agglutinin-horseradish peroxidase and cholera toxin B subunit-horseradish peroxidase cannot image the central projections and connections of the dorsal root in detail.Thus,we injected 3-k Da Texas red-dextran amine into the proximal trunks of L4 dorsal roots in adult rats.Confocal microscopy results revealed that numerous labeled arborizations and varicosities extended to the dorsal horn from T12–S4,to Clarke＇s column from T10–L2,and to the ventral horn from L1–5.The labeled varicosities at the L4 cord level were very dense,particularly in laminae I–Ⅲ,and the density decreased gradually in more rostral and caudal segments.In addition,they were predominately distributed in laminae I–IV,moderately in laminae V–VⅡ and sparsely in laminae VⅢ–X.Furthermore,direct contacts of lumbar afferent fibers with propriospinal neurons were widespread in gray matter.In conclusion,the projection and connection patterns of L4 afferents were illustrated in detail by Texas red-dextran amine-dorsal root tracing.

WOX11:the founder of plant organ regeneration

De novo organ regeneration is the process in which adventitious roots or shoots regenerate from detached or wounded organs.De novo organ regeneration can occur either in natural conditions,e.g.adventitious root regenera-tion from the wounded sites of detached leaves or stems,or in in-vitro tissue culture,e.g.organ regeneration from cal-lus.In this review,we summarize recent advances in research on the molecular mechanism of de novo organ regen-eration,focusing on the role of the WUSCHEL-RELATED HOMEOBOX11(WOX11)gene in the model plant Arabidopsis thaliana.WOX11 is a direct target of the auxin signaling pathway,and it is expressed in,and regulates the establish-ment of,the founder cell during de novo root regeneration and callus formation.WOX11 activates the expression of its target genes to initiate root and callus primordia.Therefore,WOX11 links upstream auxin signaling to downstream cell fate transition during regeneration.We also discuss the role of WOX11 in diverse species and its evolution in plants.

Tanshinone ⅡA improves functional recovery in spinal cord injury-induced lower urinary tract dysfunction

Tanshinone ⅡA, extracted from Salvia miltiorrhiza Bunge, exerts neuroprotective effects through its anti-inflammatory, anti-oxidative and anti-apoptotic properties. This study intravenously injected tanshinone ⅡA 20 mg/kg into rat models of spinal cord injury for 7 consecutive days. Results showed that tanshinone ⅡA could reduce the inflammation, edema as well as compensatory thickening of the bladder tissue, improve urodynamic parameters, attenuate secondary injury, and promote spinal cord regeneration. The number of hypertrophic and apoptotic dorsal root ganglion（L6–S1） cells was less after treatment with tanshinone ⅡA. The effects of tanshinone ⅡA were similar to intravenous injection of 30 mg/kg methylprednisolone. These findings suggested that tanshinone ⅡA improved functional recovery after spinal cord injury-induced lower urinary tract dysfunction by remodeling the spinal pathway involved in lower urinary tract control.

CRE/LOX-based analysis of cell lineage during root formation and regeneration in Arabidopsis

The root system of Arabidopsis thaliana comprises primary,lateral,and adventitious roots.Different types of roots are formed by diverse inductive cues and developmental programs.Here,we adopted the CRE/LOX system to trace cell lineage during the three types of root formation under the control of the promoter of WUSCHEL-RELATED HOMEOBOX5.The results show that the cells forming adventitious roots during de novo root regeneration from detached leaves and lateral roots from the primary root are descendants of the WOX5-expressing root primordium.During the post-embryonic growth of the primary root,some vascular and root cap cells are descendants of the WOX5-expressing stem cell niche in the root apical meristem.Overall,our data suggest that the CRE/LOX system is a useful tool to trace cell lineage in different types of root organogenesis.

Treatment with analgesics after mouse sciatic nerve injury does not alter expression of wound healingassociated genes

Animal models of sciatic nerve injury are commonly used to study neuropathic pain as well as axon regeneration. Administration of post-surgical analgesics is an important consideration for animal welfare, but the actions of the analgesic must not interfere with the scientific goals of the experiment. In this study, we show that treatment with either buprenorphine or acetaminophen following a bilateral sciatic nerve crush surgery does not alter the expression in dorsal root ganglion（DRG） sensory neurons of a panel of genes associated with wound healing. These findings indicate that the post-operative use of buprenorphine or acetaminophen at doses commonly suggested by Institutional Animal Care and Use Committees does not change the intrinsic gene expression response of DRG neurons to a sciatic nerve crush injury, for many wound healing-associated genes. Therefore, administration of post-operative analgesics may not confound the results of transcriptomic studies employing this injury model.