Multi-omics-driven development of alternative crops for natural rubber production

Natural rubber(NR)is an irreplaceable biopolymer of economic and strategic importance owing to its unique physical and chemical properties.The Parárubber tree(Hevea brasiliensis(Willd.ex A.Juss.)Müll.Arg.)is currently the exclusive commercial source of NR,and it is primarily grown in plantations restricted to the tropical and subtropical areas of Southeast Asia.However,current Parárubber production barely meets the sharply increasing global industrial demand for rubber.Petroleum-based synthetic rubber(SR)has been used to supplement the shortage of NR but its industrial performance is not comparable to that of NR.Thus,there is an urgent need to develop new productive rubber crops with broader environmental adaptability.This review summarizes the current research progress on alternative rubberproducing plants,including horticultural plants(Taraxacum kok-saghyz Rodin and Lactuca L.species),woody plants(Parthenium argentatum A.Gray and Eucommia ulmoides Oliv.),and other plant species with potential for NR production.With an emphasis on the molecular basis of NR biosynthesis revealed by a multi-omics approach,we highlight new integrative strategies and biotechnologies for exploring the mechanism of NR biosynthesis with a broader scope,which may accelerate the breeding and improvement of new rubber crops.

Recoloring tomato fruit by CRISPR/Cas9-mediated multiplex gene editing

Fruit color is an important horticultural trait,which greatly affects consumer preferences.In tomato,fruit color is determined by the accumulation of different pigments,such as carotenoids in the pericarp and f lavonoids in the peel,along with the degradation of chlorophyll during fruit ripening.Since fruit color is a multigenic trait,it takes years to introgress all color-related genes in a single genetic background via traditional crossbreeding,and the avoidance of linkage drag during this process is difficult.Here,we proposed a rapid breeding strategy to generate tomato lines with different colored fruits from red-fruited materials by CRISPR/Cas9-mediated multiplex gene editing of three fruit color-related genes(PSY1,MYB12,and SGR1).Using this strategy,the red-fruited cultivar‘Ailsa Craig’has been engineered to a series of tomato genotypes with different fruit colors,including yellow,brown,pink,light-yellow,pink-brown,yellow-green,and light green.Compared with traditional crossbreeding,this strategy requires less time and can obtain transgene-free plants with different colored fruits in less than 1 year.Most importantly,it does not alter other important agronomic traits,like yield and fruit quality.Our strategy has great practical potential for tomato breeding and serves as a reference for improving multigene-controlled traits of horticultural crops.

Pollen-Expressed Transcription Factor 2 Encodes a Novel Plant-Specific TFIIB-Related Protein that Is Required for Pollen Germination and Embryogenesis in Arabidopsis

Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors （GTFs）, including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 （TBP2） and bind to the double- stranded DNA （dsDNA） as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases （RNAPs）, implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.

Rice Protein Tagging Project:A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

In September 1997,the International Rice Genome Sequencing Project was launched.This initiative pooled the resources of ten nations to obtain the first complete rice genome sequence,and promoted rice research and breeding into the post-genomics era(Li et al.,2018).In 2008,an internationally coordinated project named "RICE2020" was proposed to systematically and fully characterize all rice genes,transcripts,and proteins(Zhang et al.,2008).While genes and their transcripts can be readily characterized by sequencing-and PCR-based methods,the characterization of protein dynamics including protein levels,subcellular localizations,post-transla-tional modifications,and interactions with macromolecules(e.g.,proteins,DNA,RNA,carbohydrates,and lipids)and small molecules(e.g.,metabolites and ligands)is much more challenging and usually requires antibodies that specifically recognize the protein of interest.Because it is very difficult to systematically produce reliable antibodies for the specific recognition of individual plant proteins,a common practice is to transform a tag-fused open reading frame(ORF)of a gene to the corresponding loss-of-function mutant plants.However,such an ectopically expressed tagged protein may not fully reca-pitulate the properties of the endogenous protein due to the random insertion of the transgene,even when the transgene is expressed under the endogenous gene promoter.A preferred so-lution is to genetically label the coding sequence of the gene of interest,at its in vivo locus,with a sequence encoding a fluores-cent protein tag or an affinity tag such as FLAG or HA.Such "in-locus" protein tagging,as we are naming it here,has been carried out genome-wide in yeast,Caenorhabditis elegans,fly,and mammalian cultured cells,greatly facilitating the characterization of proteins in these organisms(Jiang et al.,2018).In 2017,a Genome Tagging Project was launched in mice,aiming to label every protein using a CRISPR/Cas9-based "artificial spermatids"method(Jiang et al.,2018).Significant funding and efforts have been put into this project,which is expected to provide valuable mouse resources to accelerate biomedical research.In higher plants,in-locus tagging of proteins has been extremely challenging technically.

Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.

Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis

The shoot meristem generates the entire shoot system and is precisely maintained throughout the life cycle under various environmental challenges.In this study,we identified a prion-like domain(PrD)in the key shoot meristem regulator SHOOT MERISTEMLESS(STM),which distinguishes STM from other related KNOX1 proteins.We demonstrated that PrD stimulates STM to form nuclear condensates,which are required for maintaining the shoot meristem.STM nuclear condensate formation is stabilized by selected PrD-containing STM-interacting BELL proteins in vitro and in vivo.Moreover,condensation of STM promotes its interaction with the Mediator complex subunit MED8 and thereby enhances its transcriptional activity.Thus,condensate formation emerges as a novel regulatory mechanism of shoot meristem functions.Furthermore,we found that the formation of STM condensates is enhanced upon salt stress,which allows enhanced salt tolerance and increased shoot branching.Our findings highlight that the transcription factor partitioning plays an important role in cell fate determination and might also act as a tunable environmental acclimation mechanism.

SnoRNP is essential for thermospermine-mediated development in Arabidopsis thaliana

Polyamines have been discovered for hundreds of years and once considered as a class of phytohormones.Polyamines play critical roles in a range of developmental processes.However,the molecular mechanisms of polyamine signaling pathways remain poorly understood.Here,we measured the contents of main types of polyamines,and found that endogenous level of thermospermine(T-Spm)in Arabidopsis thaliana is comparable to those of classic phytohormones and is significantly lower than those of putrescine(Put),spermidine(Spd),and spermine(Spm).We further found a nodule-like structure around the junction area connecting the shoot and root of the T-Spm biosynthetic mutant acl5 and obtained more than 50 suppressors of acl5 nodule structure(san)through suppressor screening.An in-depth study of two san suppressors revealed that NAP57 and NOP56,core components of box H/ACA and C/D snoRNPs,were essential for T-Spm-mediated nodule-like structure formation and plant height.Furthermore,analyses of rRNA modifications showed that the overall levels of pseudouridylation and 2′-O-methylation were compromised in san1 and san2 respectively.Taken together,these results establish a strong genetic relationship between rRNA modification and T-Spm-mediated growth and development,which was previously undiscovered in all organisms.

An activated form of NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to trigger cell death in rice

A key event that follows pathogen recognition by a resistance(R)protein containing an NB-ARC(nucleotide-binding adaptor shared by Apaf-1,R proteins,and Ced-4)domain is hypersensitive response(HR)-type cell death accompanied by accumulation of reactive oxygen species and nitric oxide.However,the integral mechanisms that underlie this process remain relatively opaque.Here,we show that a gain-offunction mutation in the NB-ARC protein RLS1(Rapid Leaf Senescence 1)triggers high-light-dependent HR-like cell death in rice.The RLS1-mediated defense response is largely independent of salicylic acid accumulation,NPR1(Nonexpressor of Pathogenesis-Related Gene 1)activity,and RAR1(Required for Mla12 Resistance 1)function.A screen for suppressors of RLS1 activation identified RMC(Root Meander Curling)as essential for the RLS1-activated defense response.RMC encodes a cysteine-rich receptor-like secreted protein(CRRSP)and functions as an RLS1-binding partner.Intriguingly,their co-expression resulted in a change in the pattern of subcellular localization and was sufficient to trigger cell death accompanied by a decrease in the activity of the antioxidant enzyme APX1.Collectively,our findings reveal an NBARC-CRRSP signaling module that modulates oxidative state,the cell death process,and associated immunity responses in rice.

Epigenetic reprogramming by glucose-TOR signaling controls plant development

Glucose,the typical photosynthetic product,functions not only as a universal nutrient but also as a regulatory signaling cue during plant development.The evolutionally conserved Target of Rapamycin(TOR)kinase is a known master regulator that can be activated by glucose in coordinating multiple developmental programs with metabolic changes(Xiong et al.,2013).However,how glucose-TOR signaling affects specific gene sets involved in numerous regulatory networks during plant developmental transition and differentiation is not well understood.

MONOCULM 3,an Ortholog of WUSCHEL in Rice,Is Required for Tiller Bud Formation

WUSCHEL （WUS） plays an essential role for the maintenance of meristem activity in dicots, but its function is still elusive in monocots. We isolated a new monoculm mutant, monoculm 3 （moc3）, in which a point mutation causes the premature termination of rice O. sativa WUS （OsWUS）. Morphological observation revealed that the formation of tiller buds was disrupted in moc3. MOC3 was localized in the nuclear and could interact with TOPLESS-RELATED PROTEINS （TPRs）. The expression of MOC3 was induced by cytokinins and defection of MOC3 affected the expression of several two-component cytokinin response regulators, OsRRs and ORRs. Our results suggest that MOC3 is required for the formation of axillary buds and has a complex relationship with cytokinins.

Tiller Bud Formation Regulators MOC1 and MOC3 Cooperatively Promote Tiller Bud Outgrowth by Activating F0N1 Expression in Rice

Tillering in rice is one of the most important agronomic traits.Rice tiller development can be divided into two main processes: the formation of the axillary bud and its subsequent outgrowth.Several genes critical for bud formation in rice have been identified by genetic studies;however,their molecular functions and relationships are still largely unknown.Here,we report that MONOCULM 1 (MOC1) and MONOCULM 3/ TILLERS ABSENT 1/STERILE AND REDUCED TILLERING 1 (MOC3/TAB1/SRT1),two vital regulators for tiller formation in rice,physically interact to regulate tiller bud outgrowth through upregulating the expression of FLORAL ORGAN NUMBER 1 (FON1),the homolog of CLAVATA1 in rice.We found that M0C3 is able to directly bind the promoter ofFONI and subsequently activate FON1 expression.MOC1 functions as a coactivator of MOC3,whereas it could not directly bind the FON1 promoter,and further activated FON1 expression in the presence of MOC3.Accordingly,FON1 is highly expressed at axillary meristems and shows remarkably decreased expression levels in mod and moc3 mutants.Loss-of-function mutants of FON1 exhibit normal bud formation but defective bud outgrowth and reduced tiller number.Collectively,these results shed light on a joint transcriptional regulatory mechanim by MOC1 and MOC3,and establish a new framework for the control of tiller bud formation and outgrowth.

Expression Patterns of ABA and GA Metabolism Genes and Hormone Levels during Rice Seed Development and Imbibition: A Comparison of Dormant and Non-Dormant Rice Cultivars

Seed dormancy is an important agronomic trait in cereals. Using deep dormant （N22）, medium dormant （ZH11）, and non-dormant （G46B） rice cultivars, we correlated seed dormancy phenotypes with abscisic acid （ABA） and gibberellin （GA） metabolism gene expression profiles and phytohormone levels during seed development and imbibition. A time course analysis of ABA and GA content during seed development showed that N22 had a high ABA level at early and middle seed developmental stages, while at late developmental stage it declined to the level of ZHll; however, its ABA/GA ratio maintained at a high level throughout seed development. By contrast, G46B had the lowest ABA content during seed development though at early developmental stage its ABA level was close to that of ZH11, and its ABA/GA ratio peaked at late developmental stage that was at the same level of ZHll. Compared with N22 and G46B, ZH11 had an even and medium ABA level during seed development and its ABA/GA ratio peaked at the middle developmental stage. Moreover, the seed development time-point having high ABA/GA ratio also had relatively high transcript levels for key genes in ABA and GA metabolism pathways across three cultivars. These indicated that the embryo-imposed dormancy has been induced before the late developmental stage and is determined by ABA/GA ratio. A similar analysis during seed imbibition showed that ABA was synthesized in different degrees for the three cultivars. In addition, water uptake assay for intact mature seeds suggested that water could permeate through husk barrier into seed embryo for all three cultivars; however, all three cultivars showed distinct colors by vanillin-staining indicative of the existence of flavans in their husks, which are dormancy inhibition compounds responsible for the husk-imposed dormancy.

A Strigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice

Plant architecture is a complex agronomic trait and a major factor of crop yield,which is affected by several important hormones.Strigolactones(SLs)are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes.Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture.However,whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive.Here,we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene,HIGH TILLERING AND DWARF 1/DWARF17(HTD1/D17),which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7(CCD7),increases tiller number and improves grain yield in rice.We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1(SD1),both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s.Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield.

Identification and Analyses of miRNA Genes in Allotetraploid Gossypium hirsutum Fiber Cells Based on the Sequenced Diploid G.raimondii Genome

The plant genome possesses a large number of microRNAs （miRNAs） mainly 21-24 nucleotides in length. They play a vital role in regulation of target gene expression at various stages throughout the whole plant life cycle. Here we sequenced and analyzed ~ 10 million non-coding RNAs （ncRNAs） derived from fiber tissue of the allotetraploid cotton （Gossypium hirsutum） 7 days post-anthesis using ncRNA-seq technology. In terms of distinct reads, 24 nt ncRNA is by far the dominant species, followed by 21 nt and 23 nt ncRNAs. Using ab initio prediction, we identified and characterized a total of 562 candidate miRNA gene loci on the recently assembled D5 genome of the diploid cotton G. raimondii. Of all the 562 predicted miRNAs, 22 were previously discovered in cotton species and 187 had sequence conservation and homology to homologous miRNAs of other plant species. Nucleotide bias analysis showed that the 9th and 1st positions were significantly conserved among different types of miRNA genes. Among the 463 putative miRNA target genes, most significant up/down-regulation occurred in 10-20 days post-anthesis, indicating that miRNAs played an important role during the elongation and secondary cell wall synthesis stages of cotton fiber development. The discovery of new miRNA genes will help understand the mechanisms of miRNA generation and regulation in cotton.

Single-Nucleotide Resolution Mapping of the Gossypium raimondii Transcriptome Reveals a New Mechanism for Alternative Splicing of Introns

Alternative splicing （AS） is a vital genetic mechanism that enhances the diversity of eukaryotic transcriptomes. Here, we generated 8.3 Gb high-quality RNA-sequencing data from cotton （Gossypium raimondii） and performed a systematic, comparative analysis of AS events. We mapped 85% of the RNA-sequencing data onto the reference genome and identified 154 368 splice junctions with 16 437 as events in 10197 genes. I ntron retention constituted the majority （40%） of all AS events in G. raimondii. Comparison across 11 eukaryote species showed that intron retention is the most common AS type in higher plants. Although transposable elements （TEs） were found in only 2.9% of all G. raimondii introns, they are present in 43% of the retained introns, suggesting that TE-insertion may be an important mechanism for intron retention during AS. The majority of the TE insertions are concentrated 0-40 nt upstream of the 3＇-splice site, substantially altering the distribution of branch points from preferred positions and reducing the efficiency of intron splicing by decreasing RNA secondary structure flexibility. Our data suggest that TE-insertion-induced changes in branch point-site distribution are important for intron retention-type AS. Our findings may help explain the vast differences in intron-retention frequencies between vertebrates and higher plants.

Overexpression of micro RNA_(40)8 enhances photosynthesis, growth, and seed yield in diverse plants

The ability of a plant to produce grain, fruit, or forage depends ultimately on photosynthesis. There have been few attempts, however, to study microRNAs, which are a class of endogenous small RNAs post-transcription- ally programming gene expression, in relation to photosynthetic traits. We focused on miR408, one of the most conserved plant miRNAs, and overexpressed it in parallel in Arabidopsis, tobacco, and rice. The transgenic plants all exhibited increased copper content in the chloroplast, elevated abundance of plastocyanin, and an induction of photosynthetic genes. By means of gas exchange and optical spectroscopy analyses, we showed that higher expression of miR408 leads to enhanced photosynthesis through improving efficiency of irradiation utilization and the capacity for carbon dioxide fixation. Consequently, miR408 hyper-accumulat- ing plants exhibited higher rate of vegetative growth. An enlargement of seed size was also observed in all three species overproducing miR408. Moreover, we conducted a 2-year-two-location field trial and observed miR408 overexpression in rice significantly increased yield, which was primarily attributed to an elevation in grain weight. Taken together, these results demonstrate that miR408 is a positive regulator of photosynthesis and that its genetic engineering is a promising route for enhancing photosynthetic performance and yield in diverse plants.

SRWD1, a novel target gene of DELLA and WRKY proteins, participates in the development and immune response of rice (Oryza sativa L.)

SRWD1, a member of the WD40 protein subfamily, is induced by salt stress in rice and its homolog in barley can bind to GAMYB, implying that SRWD1 might be involved in plant defense against environmental stress and gibberellic acid(GA) signalings. In this study, we focused on the biological functions and regulation mechanisms of SRWD1 in rice. The results showed that SRWD1 expression was repressed by GA and induced by abscisic acid(ABA). Two WRKY-family transcription factors, Os WRKY45 and Os WRKY72, were found to regulate SRWD1 expression by directly binding to the W-box region in its promoter. Transient co-expression and yeast two-hybrid analyses showed that a DELLA protein strengthened the activation of Os WRKY45 and partly relieved the suppression of Os WRKY72 by binding to them.Interestingly, both SRWD1-overexpressing transgenic plants and SRWD1-knockout mutants showed dwarf phenotypes and resistance to Xanthomonas oryzae.

Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar-related Genes

Ethylene response factor （ERF） proteins are important plant-specific transcription factors. Increasing evidence shows that ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interaction with different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activates a cluster gene expression through interacting with GCC box and dehydration responsive element （DRE）, resulting in enhanced sensitivity to abscisic acid （ABA） and tolerance to drought, and dark green leaves of mature plants, indicating that TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpression of TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides information indicating that there are many cis-acting elements, including sugar responsive elements （SURE） and W box, suggesting that TERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reaction amplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Further investigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination, root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment. Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE and activates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response. Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.

Oryza sativa mediator subunit OsMED25 interacts with OsBZR1 to regulate brassinosteroid signaling and plant architecture in rice

Brassinosteroids(BRs)are plant-specific steroid hormones which regulate plant growth,development,and adaptation.Transcriptional regulation plays key roles in plant hormone signaling.A mediator can serve as a bridge between gene-specific transcription factors and the RNA polymerase machinery,functioning as an essential component in regulating the transcriptional process.However,whether a mediator is involved in BR signaling is unknown.Here,we discovered that Oryza sativa mediator subunit 25(Os MED25)is an important regulator of rice BR signaling.Phenotypic analyses showed that the Os MED25-RNAi and osmed25 mutant presented erect leaves,as observed in BR-deficient mutants.In addition,the Os MED25-RNAi and osmed25 mutant exhibited decreased BR sensitivity.Genetic analysis indicated that Os MED25-RNAi could suppress the enhanced BR signaling phenotype of Osbzr1-D.Further biochemical analysis showed that Os MED25 interacts with Os BZR1 in vivo,and Os MED25 is enriched on the promoter of Os BZR1 target genes.RNA sequencing analysis indicated that Os MED25 affects the expression of approximately 45%of Os BZR1-regulated genes and mainly functions as a corepressor of Os BZR1.Together,these findings revealed that Os MED25 regulates rice BR signaling by interacting with Os BZR1 and modulating the expression of Os BZR1 target genes,thus expanding our understanding of the roles of mediators in plant hormone signaling.

FED:a web tool for foreign element detection of genome-edited organism

Dear Editor,Genome editing,especially the newly developed CRISPR technology,is now widely implemented for diverse medical and agricultural applications(Puchta,2018).However,for genome editing,the DNA cassettes encoding the editing components are usually assembled and delivered into the cells of organisms(Cong et al.,2013).