A RHOse by any other name:a comparative analysis of animal and plant Rho GTPases

Rho GTPases 是作为许多的关键管理者扮演细胞的进程的分子的开关，包括房间运动，形态发生，主机防卫，房间部门和基因表示。Rho GTPases 在所有真核细胞的王国被发现。植物缺乏清楚的相当或相同的事物到在酵母和动物发现的常规 Rho GTPases；相反，他们随着时间的过去开发了一个唯一的亚科， ROP，是也知道皇家装甲兵。象 ROP 一样蛋白质的起源看起来先于陆地植物的外观。这评论试图讨论 ROP/RAC 的进化并且比较植物 ROP 和动物 Rho GTPases，集中于 GTPases 和他们的下游的受动器的规定的类似和差别。

Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA 156 Module by the Homeodomain Proteins PENNYWISE and POUND-FOOLISH in Arabidopsis

The morphology of inflorescences is regulated in part by the temporal and spatial events that regulate flower specification. In Arabidopsis, an endogenous flowering time pathway mediated by a subset of SQUAMOSA PROMOTER- BINDING PROTEIN-LIKE （SPL） transcription factors, including SPL3, SPL4, and SPL5, function to specify flowers by activating floral meristem identity genes. During shoot development, SPL3, SPL4, and SPL5 are post-transcriptionally regulated by microRNA156 （miR156）. The photoperiod regulated florigenic signal, FLOWERING LOCUS T （FT）, promotes floral induction, in part by activating SPL3, SPL4, and SPL5. In turn, these SPLs function in parallel with FT to specify flower meristems. Two related BELLl-like homeobox genes PENNYWISE （PNY） and POUND-FOOLISH （PNF） expressed in the shoot apical meristem are absolutely required for the specification of floral meristems. Genetic studies show that the floral specification function of FT depends upon PNYand PNF; however, the interplay between these homeodomain proteins and SPLs is not known. In this manuscript, we show that the photoperiodic floral induction of SPL3, SPL4, and SPL5 is dependent upon PNY and PNE Further, PNY and PNF also control SPL3, SPL4, and SPL5 expression by negatively regulating miR156. Lastly, ectopic expres- sion of SPL4 partially rescues the pny pnf non-flower-producing phenotype, while overexpression of SPL3 or SPL5 in pny pnf plants was unable to restore flower specification. These results suggest that： （1） SPL3, SPL4, and SPL5 function is dependent upon PNY and PNF, or （2） expression of multiple SPLs is required for floral specification in pny pnf plants.

ORA59 and EIN3 interaction couples jasmonate-ethylene synergistic action to antagonistic salicylic acid regulation of PDF expression

Hormonal crosstalk is central for tailoring plant responses to the nature of challenges encountered. The role of antagonism between the two major defense hormones, salicylic acid （SA） and jasmonic acid （JA）, and modulation of this interplay by ethylene （ET） in favor of JA signaling pathway in plant stress responses is well recognized, but the underlying mechanism is not fully understood. Here, we show the opposing function of two transcription factors, ethylene insensitive3 （EIN3） and EIN3-Like1 （EIL1）, in SA-mediated suppression and JA- mediated activation of PLANT DEFENSINI.2 （PDFI.2）. This functional duality is mediated via their effect on protein, not transcript levels of the PDF1.2 transcriptional activator octadecanoid-responsive Arabidopsis59 （ORA59）. Specifically, JA induces ORA59 protein levels independently of EIN3/EIL1, whereas SA reduces the protein levels dependently of EIN3/EIL1. Co-infiltration assays revealed nuclear co-localization of ORA59 and EIN3, and split- luciferase together with yeast-two-hybrid assays established their physical interaction. The functional ramification of the physical interaction is EIN3-dependent degradation of ORA59 by the 26S proteasome. These findings allude to SA-responsive reduction of ORA59 levels mediated by EIN3 binding to and targeting of ORA59 for degrada4tion, thus nominating ORA59 pool as a coordination node for the antagonistic function of ET/JA and SA.

Nanoparticle-Mediated Genetic Engineering of Plants

Genetic engineering of plants is at the core of sustainability efforts,natural product synthesis,and agricultural crop improvement.The past several decades have brought remarkable progress in biotechnology with the improvement of genome editing and sequencing tools,which stand to advance plant synthetic biology and bioengineering.In agriculture,genetic engineering can be employed to create crops that have in creased yields and nu tritio nal value,are resista nt to herbicides,in sects,diseases,and abiotic stresses,in cludi ng drought and heat .In pharmaceuticals and therapeutics,genetically engineered plants can be used to synthesize valuable small-molecule drugs and recombinant proteins.

Uridylation and adenylation of RNAs

The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone m RNAs, m RNA fragments, U6 sn RNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area.

Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants

Dear Editor,CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated)-based genome editing is a powerful and widely adopted technology for introducing specific mutations in animal and plant systems.

RNA Quality Control as a Key to Suppressing R Silencing of Endogenous Genes in Plants

RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs （dsRNAs） by RNA-DEPENDENT RNA POLYMERASEs （RDRs） and proceeds through small interfering （si） RNA-directed, ARGONAUTE （AGO）-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate posttranscriptional gene silencing of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome profiles. The tug of war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways.

A Resource for Inactivation of MicroRNAs Using Short Tandem Target Mimic Technology in Model and Crop Plants

microRNAs (miRNAs)are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression,leading to gene silencing.We previously developed short tandem target mimic (STTM)technology to deactivate endogenous miRNAs in Arabidopsis.Here,we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis,tomato,rice,and maize,providing a resource for the functional interrogation of miRNAs.We not only revealed the functions of several miRNAs in plant development,but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development.RNA-seq and small RNAseq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation,secondary metabolism,and ion-channel activity-associated electrophysiology,demonstrating that STTM technology is an effective approach for studying miRNA functions.To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks,we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTMinduced miRNA knockdown plants.

Enrichment process of lanthanum as a nonessential trace element in leaf cells of lettuce(Lactuca sativa L.)

Rare earth elements(REEs)as nonessential trace elements are enriched in living organisms and threaten their health.To early detect and reduce REE enrichment in living organisms,scientists are focused on clarifying the enrichment process of REEs in living organisms and its risks.However,the enrichment process of REEs in edible plant cells has remained unclear.Herein,by using interdisciplinary methods and techniques,the enrichment process of lanthanum(La(Ⅲ))in the leaf cells of lettuce(Lactuca sativa L.)was investigated.(1)When La(Ⅲ)exposure dose is 0.5-5μmol/L,La(Ⅲ)is enriched outside the plasma membrane(PM).In this zone,La(Ⅲ)is bound to vitronectin-like protein(VN)to form La-VN complexes;(2)When La(Ⅲ)exposure dose is 5-20μmol/L,besides the zone outside the PM,La(Ⅲ)is also enriched on the PM and bound to arabinogalactan proteins(AGPs)to form La-AGPs complexes;(3)When La(Ⅲ)exposure dose is 20-140μmol/L,besides the zone outside and on the PM,La(Ⅲ)is enriched inside the PM;(4)When La(Ⅲ)exposure dose is 60-140μmol/L,malondialdehyde content(an important indicator of invisible damage)significantly increases.Thus,as La(Ⅲ)exposure dose increases,La(Ⅲ)gradually migrates from outside the PM to the PM and inside the PM,enriching in these zones in turn.The enriched La(Ⅲ)will cause invisible damage to lettuce leaf cells and even enter human bodies along food chains.These results provide references for investigating the enrichment process of REEs in plants and its environmental risks,and finding strategies to early detect and reduce REE enrichment in plants.

RIP1 （ROP Interactive Partner 1）/ICR1 Marks Pollen Germination Sites and May Act in the ROP1 Pathway in the Control of Polarized Pollen Growth

Rho family small GTPases are universal signaling switches in the control of cell polarity in eukaryotic cells. Their polar distribution to the cell cortex is critical for the execution of their functions, yet the mechanism for this distribution is poorly understood. Using a yeast two-hybrid method, we identified RIP1 （ROP interactive partner 1）, which belongs to a family of five members of novel proteins that share a C-terminal region that interacts with ROP. When expressed in Arabidopsis pollen, green fluorescence protein GFP-tagged RIP1 was localized to the nucleus of mature pollen. When pollen grains were hydrated in germination medium, GFP-RIP1 switched from the nucleus to the cell cortex at the future pollen germination site and was maintained in the apical cortex of germinating pollen and growing pollen tubes. RIP1 was found to interact with ROP1 in pollen tubes, and the cortical RIP1 localization was influenced by the activity of ROP1. Overexpression of RIP1 induced growth depolarization in pollen tubes, a phenotype similar to that induced by ROP1 overexpression. Interestingly, RIP1 overexpression enhanced GFP-ROP1 recruitment to the plasma membrane （PM） of pollen tubes. Based on these observations, we hypothesize that RIP1 is involved in the positive feedback regulation of ROP1 localization to the PM, leading to the establishment of a polar site for pollen germination and pollen tube growth.

Out of step： The function of TALE homeodomain transcription factors that regulate shoot meristem maintenance and meristem identity

The indeterminate growth pattern displayed by shoots is mediated by the proper maintenance of the shoot meristem. Meristem maintenance is dependent upon the balance of stem cell perpetuation in the central zone （CZ） and organogenesis in the peripheral zone （PZ）. Although the mechanisms that coordinate CZ and PZ function is not understood, meristem cell fate is likely achieved by the spatial interplay between gene regulatory networks and hormone signaling pathways. During shoot maturation, the identity of the shoot meristem as well as the lateral organs are transformed during the vegetative and reproductive transitions. Studies in model plant systems indicate that three amino acid extension （TALE） homeodomain proteins integrate signaling events that transform the identity of the shoot meristem and establish reproductive patterns of growth. This review will highlight the function of TALE homeodomain transcription factors that regulate shoot meristem cell fate and also function with phase specific regulators to maintain shoot meristem identity.

Protein arginine methyltransferase 3 fine-tunes the assembly/disassembly of pre-ribosomes to repress nucleolar stress by interacting with RPS2B in arabidopsis

Ribosome biogenesis,which takes place mainly in the nucleolus,involves coordinated expression of preribosomal RNAs(pre-rRNAs)and ribosomal proteins,pre-rRNA processing,and subunit assembly with the aid of numerous assembly factors.Our previous study showed that the Arabidopsis thaliana protein arginine methyltransferase AtPRMT3 regulates pre-rRNA processing;however,the underlying molecular mechanism remains unknown.Here,we report that AtPRMT3 interacts with Ribosomal Protein S2(RPS2),facilitating processing of the 90S/Small Subunit(SSU)processome and repressing nucleolar stress.We isolated an intragenic suppressor of atprmt3-2,which rescues the developmental defects of atprmt3-2 while produces a putative truncated AtPRMT3 protein bearing the entire N-terminus but lacking an intact enzymatic activity domain We further identified RPS2 as an interacting partner of AtPRMT3,and found that loss-of-function rps2a2b mutants were phenotypically reminiscent of atprmt3,showing pleiotropic developmental defects and aberrant pre-rRNA processing.RPS2B binds directly to pre-rRNAs in the nucleus,and such binding is enhanced in atprmt3-2.Consistently,multiple components of the 90S/SSU processome were more enriched by RPS2B in atprmt3-2,which accounts for early pre-rRNA processing defects and results in nucleolar stress.Collectively,our study uncovered a novel mechanism by which AtPRMT3 cooperates with RPS2B to facilitate the dynamic assembly/disassembly of the 90S/SSU processome during ribosome biogenesis and repress nucleolar stress.

Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2

Stress-induced retrograde signal transmission from the plastids to the nucleus has long puzzled plant biologists.To address this,we performed a suppressor screen of the ceh1 mutant,which contains elevated 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate(MEcPP)levels,and identified the gain-of-function mutant impα-9,which shows reversed dwarfism and suppressed expression of stress-response genes in the ceh1 background despite heightened MEcPP.Subsequent genetic and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in Arabidopsis SKP1-like 1(ASK1)abundance,a pivotal component in the proteasome degradation pathway.This increase in ASK1 prompts the degradation of IMPα-9.Moreover,we uncovered a protein-protein interaction between IMPα-9 and TPR2,a transcriptional co-suppressor and found that a reduction in IMPα-9 levels coincides with a decrease in TPR2 abundance.Significantly,the interaction between IMPα-9 and TPR2 was disrupted in impα-9 mutants,highlighting the critical role of a single amino acid alteration in maintaining their association.Disruption of their interaction results in the reversal of MEcPP-associated phenotypes.Chromatin immunoprecipitation coupled with sequencing analyses revealed that TPR2 binds globally to stress-response genes and suggested that IMPα-9 associates with the chromatin.They function together to suppress the expression of stress-response genes under normal conditions,but this suppression is alleviated in response to stress through the degradation of the suppressing machinery.The biological relevance of our discoveries was validated under high light stress,marked by MEcPP accumulation,elevated ASK1 levels,IMPα-9 degredation,reduced TPR2 abundance,and subsequent activation of a network of stress-response genes.In summary,our study collectively unveils fresh insights into plant adaptive mechanisms,highlighting intricate interactions among retrograde signaling,the proteasome,and nuclear transport machinery.

Hemipteran and dipteran pests: Effectors and plant host immune regulators

Hemipteran and dipteran insects have behavioral,cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant＇s defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.

Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

Arabidopsis DXO1 possesses deNADding and exonuclease activities and its mutation affects defense-related and photosynthetic gene expression^FA

RNA capping and decapping tightly coordinate with transcription,translation,and RNA decay to regulate gene expression.Proteins in the DXO/Rai1 family have been implicated in mRNA decapping and decay,and mammalian DXO was recently found to also function as a decapping enzyme for NAD+-capped RNAs(NAD-RNA).The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein,AtDXO1.Here we show that AtDXO1 possesses both NAD-RNA decapping activity and 5?-3?exonuclease activity but does not hydrolyze the m7G cap.The atdxo1 mutation increased the stability of NAD-RNAs and led to pleiotropic phenotypes,including severe growth retardation,pale color,and multiple devel-opmental defects.Transcriptome profiling analysis showed that the atdxo1 mutation resulted in upregulation@of defense-related genes but downregulation of photo-synthesis-related genes.The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation.However,the various phenotypes associated with the atdxo1 mutant could be complemented by an enzymatically inactive AtDXO1.The atdxo1 mutation ap-parently enhances post-transcriptional gene silencing by elevating levels of siRNAs.Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic mo-lecular functions in mediating RNA processing and decay.

Characterizing RNA Pseudouridylation by Convolutional Neural Networks

Pseudouridine(Ψ)is the most prevalent post-transcriptional RNA modification and is widespread in small cellular RNAs and m RNAs.However,the functions,mechanisms,and precise distribution ofΨs(especially in m RNAs)still remain largely unclear.The landscape ofΨs across the transcriptome has not yet been fully delineated.Here,we present a highly effective model based on a convolutional neural network(CNN),called Pseudo Uridy Lation Site Estimator(PULSE),to analyze large-scale profiling data ofΨsites and characterize the contextual sequence features of pseudouridylation.PULSE,consisting of two alternatively-stacked convolution and pooling layers followed by a fully-connected neural network,can automatically learn the hidden patterns of pseudouridylation from the local sequence information.Extensive validation tests demonstrated that PULSE can outperform other state-of-the-art prediction methods and achieve high prediction accuracy,thus enabling us to further characterize the transcriptome-wide landscape ofΨsites.We further showed that the prediction results derived from PULSE can provide novel insights into understanding the functional roles of pseudouridylation,such as the regulations of RNA secondary structure,codon usage,translation,and RNA stability,and the connection to single nucleotide variants.The source code and final model for PULSE are available at https://github.com/mlcb-thu/PULSE.

NAD^(+)-capped RNAs are widespread in rice(Oryza sativa)and spatiotemporally modulated during development

Dear Editor,Nicotinamide adenine dinucleotide(NAD+),a nucleotidecontaining metabolite,can be incorporated as an RNA 5′cap to form NAD^(+)-capped RNAs(NAD-RNAs),which have been identified in bacteria,yeast,mammalian cells and Arabidopsis thaliana(Hu et al.,2021).Most NAD-RNAs are m RNAs encoded by the nuclear and mitochondrial genomes。

A reproducible and sensitive method for generating high-quality transcriptomes from single whitefly salivary glands and other low-input tissues

Transcriptomic studies are an important tool for understanding the molecular pathways underlying host plant use by agricultural pests,including vectors of damaging plant pathogens.Thus far,bulk RNA-Seq has been the main approach for non-model insects.This method relies on pooling large numbers of whole organisms or hundreds of individually dissected organs.The latter approach is logistically challenging,may introduce artifacts of handling and storage,and is not compatible with biological replication.Here,we tested an approach to generate transcriptomes of individual salivary glands and other low-input body tissues from whiteflies(Bemisia tabaci MEAM1),which are major vectors of plant viruses.By comparing our outputs to published bulk RNA-Seq datasets for whole whitefly bodies and pools of salivary glands,we demonstrate that this approach recovers similar numbers of transcripts relative to bulk RNA-Seq in a tissue-specific manner,and for some metrics,exceeds performance of bulk tissue RNA-Seq.Libraries generated from individual salivary glands also yielded additional novel transcripts not identified in pooled salivary gland datasets,and had hundreds of enriched transcripts when compared with whole head tissues.Overall,our study demonstrates that it is feasible to produce high quality,replicated transcriptomes of whitefly salivary glands and other low-input tissues.We anticipate that our approach will expand hypothesis-driven research on salivary glands of whiteflies and other Hemiptera,thus enabling novel control strategies to disrupt feeding and virus transmission.

The plant Mediator and its role in noncoding RNA production

Mediator,a conserved multiprotein complex in animals,plants,and fungi,is a cofactor of RNA polymerase II(Pol II).It is known to promote basal Pol II-mediated transcription as well as bridge sequence-specific transcriptional regulators and Pol II to integrate regulatory information.Pol II transcribes not only protein-coding genes but also intergenic regions to generate noncoding RNAs such as small RNAs(microRNAs and small interfering RNAs)and long noncoding RNAs.Intriguingly,two plant-specific polymerases,Pol IV and Pol V,have evolved from Pol II and play a role in the production of small interfering RNAs and long noncoding RNAs at heterochromatic regions to maintain genome stability through transcriptional gene silencing(TGS).Recent studies have defined the composition of the plant Mediator and evaluated its role in noncoding RNA production in relationship to Pol II,Pol IV and Pol V.Here,we review the functions of Mediator and that of noncoding RNAs generated by Pol II,Pol IV and Pol V in plants,and discuss a role of Mediator in epigenetic regulation via noncoding RNA production.