Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Involvement of the ABA-and H_(2)O_(2)-Mediated Ascorbate-Glutathione Cycle in the Drought Stress Responses of Wheat Roots

Abscisic acid(ABA),hydrogen peroxide(H_(2)O_(2)) and ascorbate(AsA)–glutathione(GSH)cycle are widely known for their participation in various stresses.However,the relationship between ABA and H_(2)O_(2) levels and the AsA–GSH cycle under drought stress in wheat has not been studied.In this study,a hydroponic experiment was conducted in wheat seedlings subjected to 15%polyethylene glycol(PEG)6000–induced dehydration.Drought stress caused the rapid accumulation of endogenous ABA and H_(2)O_(2) and significantly decreased the number of root tips compared with the control.The application of ABA significantly increased the number of root tips,whereas the application of H_(2)O_(2) markedly reduced the number of root tips,compared with that under 15%PEG-6000.In addition,drought stress markedly increased the DHA,GSH and GSSG levels,but decreased the AsA levels,AsA/DHA and GSH/GSSG ratios compared with those in the control.The activities of the four enzymes in the AsA–GSH cycle were also markedly increased under drought stress,including glutathione reductase(GR),ascorbate peroxidase(APX),monodehydroascorbate reductase(MDHAR)and dehydroascorbate reductase(DHAR),compared with those in the control.However,the application of an ABA inhibitor significantly inhibited GR,DHAR and APX activities,whereas the application of an H_(2)O_(2) inhibitor significantly inhibited DHAR and MDHAR activities.Furthermore,the application of ABA inhibitor significantly promoted the increases of H_(2)O_(2) and the application of H_(2)O_(2) inhibitor significantly blocked the increases of ABA,compared with those under 15% PEG-6000.Taken together,the results indicated that ABA and H_(2)O_(2) probably interact under drought stress in wheat;and both of them can mediate drought stress by modulating the enzymes in AsA–GSH cycle,where ABA acts as the main regulator of GR,DHAR,and APX activities,and H_(2)O_(2) acts as the main regulator of DHAR and MDHAR activities.

Silicon Mitigates Aluminum Toxicity of Tartary Buckwheat by Regulating Antioxidant Systems

Aluminum (Al) toxicity is a considerable factor limiting crop yield and biomass in acidic soil. Tartary buckwheatgrowing in acidic soil may suffer from Al poisoning. Here, we investigated the influence of Al stress on the growthof tartary buckwheat seedling roots, and the alleviation of Al stress by silicon (Si), as has been demonstrated inmany crops. Under Al stress, root growth (total root length, primary root length, root tips, root surface area, androot volume) was significantly inhibited, and Al and malondialdehyde (MDA) accumulated in the root tips. At thesame time, catalase (CAT) and ascorbate peroxidase activities, polyphenols, flavonoids, and 1,1-diphenyl-2-picrylhydrazyl(DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free-radical scavenging abilitywere significantly decreased. After the application of Si, root growth, Al accumulation, and oxidative damage wereimproved. Compared to Al-treated seedlings, the contents of ·O2− and MDA decreased by 29.39% and 25.22%,respectively. This was associated with Si-induced increases in peroxidase and CAT enzyme activity, flavonoidcompounds, and free-radical scavenging (DPPH and ABTS). The application of Si therefore has positive effectson Al toxicity in tartary buckwheat roots by reducing Al accumulation in the roots and maintaining oxidationhomeostasis.

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Broad Hormonal Responses Induced by Aluminum in Roots of Dwarf Transgenics of Solanum lycopersicum L. cv “Micro-Tom”

The spatial pattern distribution of plant hormones in response to aluminum (Al) toxicity in roots remains to be shown. This study was performed to assess the root hormonal accumulation and gene expression in response to Al toxicity in five transgenic miniature dwarf tomatoes cv. Micro-Tom (MT). MT and MT transgenics to acid indole acetic, cytokinin, gibberellin, abscisic acid and ethylene were cultivated in nutrient solutions containing different Al concentrations. Root growth elongation was measured and cellular damage was visualized by staining Evans’s blue. The GUS reporter gene staining technique was used to visualize hormonal changes in MT apex root tissues. Data indicated that the MT is sensitive to Al that induced significant growth inhibition and cellular damage. Al concentration of 27 μM was significantly toxic, inducing root apex darkening and inhibition of root development. The qualitative evaluation of GUS reporter gene expression showed intense crosstalk among all hormones studied, underscoring the complexity of signaling induced by Al in apex roots. Results point out to a major understanding of the hormonal signaling in response to Al toxicity, which may induce a change of root growth and architecture with growth inhibition and cell constraints modulated by all different hormones evaluated.

THE ROOTS AND FRUITS OF CHINESE SPRING FESTIVAL

Traditional festivals forge a nation’s cultural identity and solidarity.Through opulent customs and celebratory activities,they convey the collective psyche,emotions,and aspirations of a nation.As a key part of Chinese culture,traditional Chinese festivals reinforce the bond among Chinese people and preserve our cultural roots.Of all Chinese festivals,the Spring Festival is the most important,both culturally and historically.

Effects of plant roots on soil preferential pathways and soil matrix in forest ecosystems

To characterize effects of plant roots on preferential flow（PF）,we measured root length density（RLD）and root biomass（RB） in Jiufeng National Forest Park,Beijing,China.Comparisons were made for RLD and RB between soil preferential pathways and soil matrices.RLD and RB declined with the increasing soil depth（0–10,10–20,20–30,30–40,40–50,50–60 cm） in all experimental plots.RLD was greater in soil preferential pathways than in the surrounding soil matrix and was 69.5,75.0 and72.2 % for plant roots of diameter（d） ／1,1 ／ d ／ 3 and3 ／ d ／ 5 mm,respectively.Fine root systems had the most pivotal influence on soil preferential flow in this forest ecosystem.In all experimental plots,RB content was the sum of RB from soil preferential pathways and the soil matrix in each soil depth.With respect to 6 soil depth gradient（0–10,10–20,20–30,30–40,40–50,50–60 cm） in each plot,the number of soil depth gradient that RB content was greater in soil preferential pathways than in the soil matrix was characterized,and the proportion was68.2 % in all plots.

Transcriptome analysis reveals the effects of alkali stress on root system architecture and endogenous hormones in apple rootstocks

Soil alkalinity is a major factor that restricts the growth of apple roots.To analyze the response of apple roots to alkali stress, the root structure and endogenous hormones of two apple rootstocks, Malus prunifolia (alkali-tolerant) and Malus hupehensis (alkali-sensitive), were compared. To understand alkali tolerance of M. prunifolia at the molecular level, transcriptome analysis was performed. When plants were cultured in alkaline conditions for 15 d, the root growth of M. hupehensis with weak alkali tolerance decreased significantly. Analysis of endogenous hormone levels showed that the concentrations of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in M. hupehensis under alkali stress were lower than those in the control. However, the trend for IAA and ZR in M. prunifolia was the opposite. The concentration of abscisic acid (ABA) in the roots of the two apple rootstocks under alkali stress increased, but the concentration of ABA in the roots of M. prunifolia was higher than that in M. hupehensis. The expression of IAA-related genes ARF5, GH3.6, SAUR36, and SAUR32 and the Cytokinin (CTK)-related gene IPT5 in M. prunifolia was higher than those in the control, but the expression of these genes in M. hupehensis was lower than those in the control. The expression of ABA-related genes CIPK1 and AHK1 increased in the two apple rootstocks under alkali stress, but the expression of CIPK1 and AHK1 in M. prunifolia was higher than in M. hupehensis. These results demonstrated that under alkali stress, the increase of IAA, ZR, and ABA in roots and the increase of the expression of related genes promoted the growth of roots and improved the alkali tolerance of apple rootstocks.

The SmMYB36-SmERF6/SmERF115 module regulates the biosynthesis of tanshinones and phenolic acids in salvia miltiorrhiza hairy roots

Tanshinone and phenolic acids are the most important active substances of Salvia miltiorrhiza,and the insight into their transcriptional regulatory mechanisms is an essential process to increase their content in vivo.SmMYB36 has been found to have important regulatory functions in the synthesis of tanshinone and phenolic acid;paradoxically,its mechanism of action in S.miltiorrhiza is not clear.Here,we demonstrated that SmMYB36 functions as a promoter of tanshinones accumulation and a suppressor of phenolic acids through the generation of SmMYB36 overexpressed and chimeric SmMYB36-SRDX(EAR repressive domain)repressor hairy roots in combination with transcriptomic-metabolomic analysis.SmMYB36 directly down-regulate the key enzyme gene of primary metabolism,SmGAPC,up-regulate the tanshinones biosynthesis branch genes SmDXS2,SmGGPPS1,SmCPS1 and down-regulate the phenolic acids biosynthesis branch enzyme gene,SmRAS.Meanwhile,SmERF6,a positive regulator of tanshinone synthesis activating SmCPS1,was up-regulated and SmERF115,a positive regulator of phenolic acid biosynthesis activating SmRAS,was down-regulated.Furthermore,the seven acidic amino acids at the C-terminus of SmMYB36 are required for both self-activating domain and activation of target gene expression.As a consequence,this study contributes to reveal the potential relevance of transcription factors synergistically regulating the biosynthesis of tanshinone and phenolic acid.

The SmNPR4-SmTGA5 module regulates SA-mediated phenolic acid biosynthesis in Salvia miltiorrhiza hairy roots

Phenolic acids are the main bioactive compounds in Salvia miltiorrhiza,which can be increased by salicylic acid(SA)elicitation.However,the specific molecular mechanism remains unclear.The nonexpresser of PR genes 1(NPR1)and its family members are essential components of the SA signaling pathway.Here,we report an NPR protein,SmNPR4,that showed strong expression in hairy root after SA treatment,acting as a negative moderator of SA-induced phenolic acid biosynthesis in S.miltiorrhiza(S.miltiorrhiza).Moreover,a basic leucine zipper family transcription factor SmTGA5 was identified and was found to interact with SmNPR4.SmTGA5 activates the expression of phenolic acid biosynthesis gene SmTAT1 through binding to the as-1 element.Finally,a series of biochemical assays and dual gene overexpression analysis demonstrated that the SmNPR4 significantly inhibited the function of SmTGA5,and SA can alleviate the inhibitory effect of SmNPR4 on SmTGA5.Overall,our results reveal the molecular mechanism of salicylic acid regulating phenolic acid biosynthesis in S.miltiorrhiza and provide new insights for SA signaling to regulate secondary metabolic biosynthesis.

Fusarium pseudograminearum and F.culmorum affect the root system architecture of bread wheat

Yield losses of bread wheat due to crown rot can be more severe when drought conditions occur during the grain-filling period.Root architecture characteristics are important for soil exploration and belowground resource acquisition and are essential for adaptation to water-limited environments.Traits such as root angle,length and density have been strongly associated with acquisition efficiency and contribute to yield stability of the crop.The impact of crown rot pathogens on wheat root architecture is poorly understood.We examined differences in root angle,length and number,as well as dry root weight of the crown rot-susceptible bread wheat cultivar,Livingston inoculated with one of two crown rot pathogens Fusarium culmorum or Fusarium pseudograminearum in a transparent-sided root observation chamber.Significant adverse impacts on plant health and growth were revealed by visual discolouration of the leaf sheaths;fresh and dry shoot weight;leaf area of the oldest and the youngest fully expanded leaf and leaf number.Values of most recorded root system measurements were reduced when inoculated with either F.culmorum or F.pseudograminearum.In contrast,root angle was increased in the presence of F.culmorum but was not significantly changed by F.pseudograminearum.The development of whiteheads and grain losses in bread wheat caused by crown rot have previously been associated with blockages of the vascular systems.The method employed here was able to identify differences in the pathogen impacts on roots,which were not detected using previous systems.This research indicates that in the presence of F.culmorum and F.pseudograminearum infection,not only reductions in the size and biomass of the shoot system but also changes in the length,biomass and architecture of the root system could play an important role in yield loss.

Identification of the HAK gene family reveals their critical response to potassium regulation during adventitious root formation in apple rootstock

Adventitious root formation is a bottleneck during vegetative proliferation.Potassium(K^(+))is an essential macronutrient for plants.K^(+)accumulation from the soil and its distribution to the different plant organs is mediated by K^(+)transporters named K^(+)transporter(KT),K^(+)uptake(KUP),or high-affinity K^(+)(HAK).This study aimed to identify members of the HAK gene family in apples and to characterize the effects of K^(+)supply on adventitious root formation and on the expression of HAK genes and the genes that putatively control auxin transport,signaling,and cell fate during adventitious root formation.In this study,34 HAK genes(MdHAKs)were identified in the apple(Malus×domestica‘Golden Delicious’)genome.A phylogenetic analysis divided MdHAKs into four clusters(Ⅰ,Ⅱ,Ⅲ,andⅣ),comprising 16,1,4,and 13 genes,respectively.The syntenic relationships revealed that 62.5%of the total MdHAK genes arise from genomic duplication events.Chromosome location,domain structure,motif analysis,and physico-chemical characteristics were subsequently investigated.Furthermore,the application of K^(+)indicated the emergence of adventitious roots at 8 d and produced more adventitious roots at 16 d than the K^(+)-free control(CK)treatment.In addition,various MdHAKs showed root-specific expression in B9 apple rootstock stem cuttings and enhanced expression during the initiation and emergence stages of adventitious root formation in response to K^(+)treatment.Additionally,K^(+)treatment enhanced the expression levels of MdPIN1,MdPIN2,and MdAUX1.Further data indicated that a higher expression of MdWOX11,MdLBD16,and MdLBD29 and of cell cycle-related genes contributed to the auxin-stimulated adventitious root formation in response to K^(+).

The Building of Grass-roots Agricultural Technology Extension System

Based on the survey of 154 farmers in Guiyang City, we analyze the basic situation of building of grass-roots agricultural technology extension system in Guiyang City. Then we point out some problems in the building of grass-roots agricultural technology extension system in Guiyang City: the function of grass-roots agricultural technology extension center weakens; the content of agricultural technology extension is difficult to adapt to farmers' needs for technology; the extension mode of agricultural technology departments does not adapt to the needs of modern agriculture. In order to perfect the building of grass-roots agricultural technology extension system, the countermeasures and recommendations are put forth as follows: strengthening the input of funds, and ensuring that the basic work of public welfare agricultural technology extension is smoothly carried out; innovating upon the system, and improving the function of grass-roots agricultural technology extension center; implementing management on agricultural technology extension personnel's performance, and promoting the extension efficiency; strengthening the building of extension team in rural areas, and cultivating high-quality agricultural technology extension personnel; exploring the advanced service mode to meet farmers' needs.

Optimizing Culture System of Ri T-DNA Transformed Roots for Citrus grandis cv. Changshou Shatian You

Genetic transformation experiments of the different explants from Citrus grandis cv. Changshou Shatian You infected with Agrobacterium rhizogenes were carried out in darkness or in light. The optimizing culture system of Ri T-DNA transformed roots for C. grandis cv. Changshou Shatian You was constructed as follows： After the ventral wounded striations on the single activation cotyledon were inoculated by A. rhizogenes A4 （logarithmic period）, they were cocultured at （25 ±2）℃ in darkness for 25-30 days; some transformed roots were generated from wounded striations of most cotyledons. The genetically transformed ratio is （83 ± 11）%. Axenic Ri T-DNA transformed roots （hairy roots） were harvested after five subcultures. Explants were activated on MT medium. The MS medium was used for subculture of transformed roots. Mass Ri T-DNA transformed roots in which the hormone was produced independently were harvested from this optimizing culture system. White, fresh Ri T-DNA transformed roots were （1.14 ±0.07） cm long, （0.73 ±0.04） mm wide, and the growth direction of transformed roots was negative geotropism.

Regulation of the growth of sprouting roots of black locust seedlings using root barrier panels

How can we regulate an invasive alien species of high commercial value?Black locust(Robinia pseudoacacia L.)has a unique capacity for seed dispersal and high germination.Field surveys indicate that black locust increases its growing area with sprouting roots and the elongation of horizontal roots at a soil depth of 10 cm.Therefore,a method to regulate the development of horizontal roots could be eff ective in slowing the invasiveness of black locust.In this study,root barrier panels were tested to inhibit the growth of horizontal roots.Since it is labor intensive to observe the growth of roots in the fi eld,it was investigated in a nursery setting.The decrease in secondary fl ush,an increase in yellowed leafl ets,and the height in the seedlings were measured.Installing root barrier panels to a depth of 30 cm eff ectively inhibit the growth of horizontal roots of young black locust.

Effects of solar radiation and fine roots on suction of Amorpha fruticose-vegetated soil

The thickness of shallow landslides is generally less than 2 m,which is of the same order of magnitude as the growth range of vegetation roots.Vegetation roots can improve the stability of shallow soil through mechanical and hydraulic effects.Therefore,the landslide process is closely related to the plant roots growing on the slope surface.Plant roots play a dominant role in the regulation of soil suction through solar radiation induced transpiration.However,little is known about the correlation between cumulative solar radiation and soil suction.Moreover,the specific effects of fine roots on the suction distribution are not clear in most previous studies.In this study,a vegetated soil of a drought-tolerant and water-tolerant shrub,namely Amorpha fruticose,was adopted.The suction and volumetric water content of bare and vegetated soils were monitored under natural conditions for 4 months.The results demonstrate that there is a nearly linear relationship between cumulative solar radiation and suction ranging from zero to 100 kPa.Regarding the modeling of the soil-plant-atmosphere interactions,this relationship could serve a significant role in calculating the root water uptake under given solar radiation conditions.In addition,higher suctions were observed at the lower layer of the vegetated soil than those at the middle layer,which is different from the results of vegetated soil from previous investigations.This is due to the fact that the root area index(RAI)of fine roots at the lower layer is twice that of the middle layer.Importantly,the higher concentration of fine roots at the lower layer of vegetated soil sample resembles the root distribution of shrub near the soil-bedrock interface on shallow bedrock landslides.The fine roots would increase soil suction through transpiration,and hence reduce the permeability and increase shear strength of landslides.Eventually,these new findings serve as a preliminary step on the evaluation of the stability of this common type of landslides.

CG hypermethylation of the bHLH39 promoter regulates its expression and Fe deficiency responses in tomato roots

Iron(Fe)is an essential micronutrient for all organisms,including plants,whose limited bioavailability restricts plant growth,yield,and nutritional quality.While the transcriptional regulation of plant responses to Fe deficiency have been extensively studied,the contribution of epigenetic modulations,such as DNA methylation,remains poorly understood.Here,we report that treatment with a DNA methylase inhibitor repressed Fe deficiency-induced responses in tomato(Solanum lycopersicum)roots,suggesting the importance of DNA methylation in regulating Fe deficiency responses.Dynamic changes in the DNA methylome in tomato roots responding to short-term(12 hours)and long-term(72 hours)Fe deficiency identified many differentially methylated regions(DMRs)and DMR-associated genes.Most DMRs occurred at CHH sites under short-term Fe deficiency,whereas they were predominant at CG sites following long-term Fe deficiency.Furthermore,no correlation was detected between the changes in DNA methylation levels and the changes in transcript levels of the affected genes under either short-term or long-term treatments.Notably,one exception was CG hypermethylation at the bHLH39 promoter,which was positively correlated with its transcriptional induction.In agreement,we detected lower CG methylation at the bHLH39 promoter and lower bHLH39 expression in MET1-RNA interference lines compared with wild-type seedlings.Virus-induced gene silencing of bHLH39 and luciferase reporter assays revealed that bHLH39 is positively involved in the modulation of Fe homeostasis.Altogether,we propose that dynamic epigenetic DNA methylation in the CG context at the bHLH39 promoter is involved in its transcriptional regulation,thus contributing to the Fe deficiency response of tomato.

Hormones and carbohydrates synergistically regulate the formation of swollen roots in a Chinese cabbage translocation line

The genus Brassica contains a rich diversity of species and morphological types,including leaf,root,and oil crops,all of which show substantial phenotypic variation.Both Chinese cabbage and cabbage are typical leaf-type crops with normal roots.We created translocation lines based on interspecific crosses between Chinese cabbage and cabbage and identified qdh225,which exhibited a swollen-root phenotype.The swollen root of qdh225 contained a large number of granular substances,and the formation of its irregular morphological tissue was caused by a thickening of the phloem.Transcriptomic and metabolomic data suggested that differential expression of genes encoding nine types of enzymes involved in starch and sucrose metabolism caused changes in starch synthesis and degradation in the swollen root.These genes jointly regulated sucrose and starch levels,leading to significant enrichment of starch and soluble proteins in the swollen root and a reduction in the content of soluble sugars such as d-glucose and trehalose 6-phosphate.A significant increase in auxin(IAA)and abscisic acid(ABA)contents and a decrease in gibberellin(GA)content in the swollen root likely promoted the differential expression of genes associated with hormone signal transduction,thereby regulating the development of the swollen root.Taken together,our data suggest that accumulation of IAA and ABA and reduction in GA promote swollen root formation by regulating hormone-mediated signaling,leading to a thickening of phloem,root enlargement,and substantial accumulation of starch and soluble proteins.The latter provide materials,energy,and nutrient sources for the development of swollen roots.

Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases.Its roots,used for traditional Chinese medicine materials,are usually brick-red due to accumulation of red pigments,such as tanshinone IIA and tanshinone I.Here we report a S.miltiorrhiza line(shh)with orange roots.Compared with the red roots of normal S.miltiorrhiza plants,the contents of tanshinones with a single bond at C-15,16 were increased,whereas those with a double bond at C-15,16 were significantly decreased in shh.We assembled a high-quality chromosome-level genome of shh.Phylogenomic analysis showed that the relationship between two S.miltiorrhiza lines with red roots was closer than the relationship with shh.It indicates that shh could not be the mutant of an extant S.miltiorrhiza line with red roots.Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shh Sm2OGD3m.Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation.Consistently,in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone,15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA,tanshinone I and 1,2-dihydrotanshinone I,respectively.Thus,Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis.The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis)

Roots are essential for plant growth and development.Bamboo is a large Poaceae perennial with 1642 species worldwide.However,little is known about the transcriptional atlas that underpins root cell-type differentiation.Here,we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14279 filtered single cells derived from the basal root tips of moso bamboo.We identified four cell types and defined new cell-type-specific marker genes for the basal root.We reconstructed the developmental trajectories of the root cap,epidermis,and ground tissues and elucidated critical factors regulating cell fate determination.According to in situ hybridization and pseudotime trajectory analysis,the root cap and epidermis originated from a common initial cell lineage,revealing the particularity of bamboo basal root development.We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice.Additionally,PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap,which was different from WOX13 orthologs in Arabidopsis.Taken together,our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.