In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome  and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The  Mitochondria are at the heart of energy production in eukaryotic cells, producing and regulating ATP production through the oxidative phosphorylation (OXPHOS) pathway. Mitochondria are hypothesized to originate from an endosymbiotic event occurring ∼1.5 billion years ago in which an archaea-type host engulfed an α-proteobacterium–like ancestor. This hypothesis stems from analyses of mitochondrial genes and their genomic organization and distribution , 3. The Saccharomyces cerevisiae (yeast) mtDNA is an 85-kb linear DNA that was sequenced in 1998  was the first genome to be completely sequenced in 1981 . It is a in 1998  and show in 1998 , 7, 8, u in 1998 . The remS. cerevisiae and human mtRNAP complexes, and in several aspects, yeast has remained a model system for biochemistry and genetics  is encoded by RPO41, and it requires only one initiation factor, MTF1, for promoter-specific transcription. All of the transcriptional machinery proteins are nuclear-encoded and have a 20–30-amino acid mitochondrial localization sequence for entry into the mitochondria. In both yeast and human mitochondrial systems, a successful transcription event produces a polycistronic RNA transcript, processed to produce mature RNAs. In literature, human mtRNAP is referred to by its gene name, POLRMT or h-mtRNAP, whereas the yeast mtRNAP is referred to as RPO41 mostly.The mitochondria's transcription machinery is a multicomponent system consisting of the catalytic mtRNAP subunit and several accessory transcription factors. This machinery catalyzes all the major transcription stages, including promoter recognition, promoter-specific transcription initiation, elongation, and termination. Each of these events and the steps within them is subject to regulation. The human mtRNAP (h-mtRNAP) is encoded by A). Analysis of nascent transcripts by PRO-Seq and GRO-Seq methods confirmed the transcription initiation sites on h-mtDNA and showed quantitative differences between light- and heavy-strand transcription efficiency among different human cell lines . The −7 A, −3 C, +1 to +3 AAA, and +5 A are conserved positions in all three promoters. This region interacts with TFB2M, which is required for transcription by all three promoters. The significant differences in the transcription initiation mechanism at the three promoters likely arise from the promoter sequences' differences and their requirement for TFAM. In vitro studies indicate that LSP and HSP1 require TFAM for optimal transcription , which interestingly is conserved in promoters of the distantly related yeast Kluyveromyces lactis . The ∼800 amino acids of the C-terminal domain (CTD) of y-mtRNAP and T7 RNAP have a 28% sequence identity, and there is 41% sequence identity between y-mtRNAP and h-mtRNAP. The CTD's basic structure resembles the classic “right hand” shape composed of the thumb, palm, and fingers domains  . The promoter-binding elements in T7 RNAP make extensive base-specific interactions with the T7 promoter, and T7 RNAP does not require transcription factors. The promoter-binding elements in mtRNAPs show fewer base-specific interactions and more reliance on initiation factors for promoter-specific transcription. The dependence of mtRNAPs on transcription factors adds layers of gene regulation that is necessary to synchronize mitochondrial energy production to cellular demand.The h-mtRNAP and y-mtRNAP show a significant structural and amino acid sequence similarity to each other and T7 RNAP  Fig. 2BBFig. 2B.Fig. 2C) , 38, 39.Fig. 2C) . The y-mFig. 2C) , but theIn vivo studies indicate that the deletion of N-terminal ∼185 aa does not affect transcription initiation but decreases expression of y-mtDNA genes and destabilizes the y-mtDNA , which is not present in T7 RNAP. The NTE in h-mtRNAP harbors two pentatricopeptide repeat (PPR) domains that interact with the promoter DNA and a tether-helix that interacts with TFAM in the initiation complex . Biochem y-mtDNA . Biochem y-mtDNA . Thus, NC and TFB2M and MTF1 are both essential transcription factors of h-mtRNAP and y-mtRNAP, respectively. TFB2M was discovered based on its amino acid sequence homology to MTF1 , 48 and Figs. 2TFAM is abundantly present in human mitochondria, in amounts sufficient to coat the mtDNA, and it plays essential roles in mtDNA packaging and nucleoid formation , 55, 56.in vivo, given that TFAM is bound everywhere on h-mtDNA and TFAM:h-mtRNAP complex formation is not promoter-specific   , 52, 64,ctively)  and catactively) , 27. A rctively) . FurtherC). TFB2M interacts with the promoter region from position −8 to +2, which includes the initiation bubble region. The fingers and palm domain of the CTD in h-mtRNAP contact the downstream DNA. TFB2M directly interacts with h-mtRNAP in the initiation complex; the CTD of TFB2M interacts with the TFB2M-hairpin in h-mtRNAP  (C) and is a conserved feature observed in all single-subunit RNAPs (The two components of the yeast transcription initiation complex do not recognize the promoter sequence independently as the human system. The y-mtRNAP binds both promoter and nonpromoter sequences with similar affinities (∼60 nmKd) . MTF1, omKd)  52). Wh. WhmKd)mKd) . PromotemKd) C and is it RNAPs , 36, 37.C and D) (B). The CTD insertion in y-mtRNAP interacts with the downstream DNA around +11 bp. The CTD insertion interactions stabilize the bent conformation of the DNA around the initiation site, whereas MTF1 stabilizes the DNA at the opposite end (Footprinting studies show that y-mtRNAP protects ∼30 bp of promoter DNA from approximately −15 to +15 . Unlike C and D) . The strC and D) B. The CTsite end . PerhapsC), but the crystal structure shows a disordered thumb-tip in the h-mtRNAP , and the NTD of MTF1 contacts the thumb domain of y-mtRNAP , 73. TheFig. 2h-mtRNAP . Comparih-mtRNAP .in vitro transcription studies show that mutations in the −4 to −1 base pairs in LSP decrease runoff RNA synthesis (DNA bending and untwisting converts the closed complex to an open complex in which base pairs from −4 to +2 with respect to the transcription start site at +1 are melted . All of ynthesis . AdditioC and D). The base-specific interactions of MTF1 with the promoter are consistent with the existing promoter DNA mutational studies   . InteresA and B) , 88. IntA and B) , and thiA and B) . Any claC). The fingers domain is a mobile region near the active site involved in RNA synthesis . The second metal ion, which was not observed in this structure, would be bound close to the 3′-OH of the 2-mer RNA. The Y-helix in the fingers domain wedges against the downstream junction of the initiation bubble, and this conformation is consistent with its role in aiding downstream DNA strand separation for RNA elongation, as observed in the elongation complex structures of T7 RNAP and h-mtRNAP . Fluorescence studies have provided evidence for DNA scrunching in both single-subunit and multisubunit RNAPs  remains single-stranded but gets scrunched into an NT-loop B. Fluoreit RNAPs , 92, 93.it RNAPs , 69, 84. NT-loop . The NT-2+NTP has a scrunched DNA conformation, whereas the DNA in the PmIC state is not scrunched. PmIC and IC states' coexistence in the cryo-EM experiment suggests a constant switching between the two states through dissociation and rebinding of 2-mer RNA and NTP ); hence, the C-tail is in a position to clash with the growing RNA:DNA hybrid and NT-loop to trigger abortive synthesis. Biochemical studies showed reduced abortive synthesis upon MTF1 C-tail deletion, consistent with this model 2-3 sequences, and these RNA primers are elongated to kilobase-sized RNA products . Several nucleoid proteins, including mitochondrial ribosomal protein L12 (MRPL12), leucine-rich pentatricopeptide repeat–containing protein (LRPPRC), TEFM, and MTERF1, act as secondary factors that can control the function of h-mtRNAP, with some of these findings discussed above. Interestingly, homologous proteins in yeast playing similar roles have not yet been defined.One of the most direct modes of transcriptional control in the mitochondria is through the modulation of the RNAP's access to mtDNA or its activity. H-mtRNAP is regulated by proteins that directly interact with mtDNA, termed nucleoid proteins. Nucleoid formation promotes mtDNA stability and may allow for genome regulation. Nearly 60 nucleoid proteins have been identified , 134 thapath C) and represents another form of mitochondrial transcription regulation . However, TFAM binding to the promoter region is also needed for transcription initiation; therefore, altering levels of TFAM or the ability of TFAM to bind to mtDNA is a dominant mode of transcriptional control. Structural features of mtDNA, including G-quadruplex formation and mtDNA methylation, may impact TFAM binding.In mammalian systems, the initiation factors are essential for transcription; as such, regulation of TFAM and TFB2M represent another major mechanism of transcriptional control in mitochondria. TFAM is the most abundant protein component of mitochondrial nucleoids in mammals. TFAM coats and packages mtDNA, with higher TFAM:mtDNA ratios indicative of tighter packaging and reduced accessibility of the transcription and replication machinery  . CR-sulfoxide reductase B2). These enzymes are centrally localized to protect the mitochondrial genome and preserve the replication and transcription machinery in times of stress, and additional studies are necessary to determine the importance of mtDNA proximity to their function. Like TFAM, TFB2M is also phosphorylated. Phosphorylation of TFB2M on two key threonine residues decreases the binding affinity of TFB2M for both mitochondrial promoters and its ability to carry out transcription in vitro . Nuclear respiratory factors 1 and 2 (NRF-1/2) and peroxisome proliferator–activated receptor coactivator 1α (PGC-1α) are widely established as “master regulators” of mitochondrial biogenesis and control the expression of many mitochondrial proteins, including h-mtRNAP . Many such factors have been identified in previous studies and are discussed in prior reviews; these include thyroid hormone receptors, glucocorticoid receptors, cAMP-response element binding proteins, NF-κB subunits, myocyte enhancer factor 2 (MEF2D)  , and sig (MEF2D)  and 180. (MEF2D) , 182. Wein silico analysis, indicating the possibility of direct regulation of mtDNA transcription by AR (The androgen receptor (AR) and estrogen receptors (ERα and ERβ) are ligand-dependent nuclear transcription factors that bind androgen and estrogen steroid hormones, respectively, and play diverse roles in cell function and development , 184. ARon by AR . Like ARon by AR  and 188.on by AR , 188.The NF-κB transcription factor family includes several transcription factors and their inhibitors, which play roles in signaling pathways involved in inflammation, cell differentiation, and cell survival . STAT prThe TEA domain family transcription factors (TEAD1–4) are downstream transcription factors of the Hippo signaling pathway and are essential in cell proliferation. A member of this family, TEAD4, is involved in energy homeostasis in early embryonic development , 196 andMDM2 is well-known for its regulation of the nuclear transcription factor p53, an established tumor suppressor. However, MDM2 also has p53-independent functions, including binding to nuclear chromatin and altering transcription . RecentlThe four-member FoxO transcription factor family is comprised of nutrient-sensing proteins that control the transcription of nuclear genes involved in energy homeostasis, glucose metabolism, apoptosis, and cell cycle arrest. Recently, one member of this family, FoxO1, was shown to bind the mtDNA D-loop under basal metabolic conditions. Nutrient restriction in white and beige adipocytes led to FoxO1 shuttling from the mitochondria to the nucleus and binding to nuclear DNA, with a concomitant decrease in FoxO1 binding to mtDNA and transcription. This shuttling depended on specific nutrient restriction–induced reactive oxygen species patterns . The relpath F). In some cellular systems, TFAM was identified in nuclei and suggested to control nuclear gene expression (The reliance of the mitochondria upon the nucleus is quite clear. Interestingly, many mitochondrial proteins have been shown to affect nuclear gene expression. Importantly, such retrograde signaling often depends on certain metabolic states or redox balance within the cell, highlighting how global cellular conditions function in interconnecting the nucleus and the mitochondria , path F.pression , 204. Thpression . A uniqupression .+]/[ATP] and [NADH]/[ATP] ratios, and regulate expression of NAD+/H capped mitochondrial RNAs in response (Beyond mitochondrial proteins known to play dual roles in the mitochondria and nucleus, studies point to the importance of the mitochondria in modifying chromatin and nuclear DNA. Mitochondrial metabolism provides many of the requisite substrates for these epigenetic modifications, including acetyl-CoA, formate, ATP, and α-ketoglutarate. The perturbation of mitochondrial function results in altered epigenetic and transcriptional profiles within the nucleus , 210. Foresponse . FurtherThe mitochondria are hubs for many critical processes, from ATP production to cell signaling and apoptosis. Their many cellular roles, multifaceted communication with the nucleus, and the vast array of pathologies associated with their dysfunction underscore the importance of a better understanding of mitochondrial functions and regulation. Although mtRNAP is architecturally simpler than the nuclear RNAPs, mtDNA transcription must respond to the cell's energy demands, continually changing and requiring coordination between nuclear and mitochondrial DNA transcription. Our understanding of mitochondrial transcription regulation in mammalian systems is growing, with recent research illuminating aspects of regulation by transcription factors, metabolite sensing, and nucleoid dynamics. Although this research area is of great interest, we have very little understanding of its biochemical and molecular basis, which is essential for enabling new therapeutics for the myriad of mitochondrial-related diseases.Detailed studies of mitochondrial transcription and the interplay between the mitochondria and nucleus enable a deeper understanding of diseases and disorders caused by disrupted mitochondrial functions. There are increasing numbers of inherited mutations in mtDNA and nuclear genes for mitochondrial proteins associated with mitochondrial disorders involving defective ATP production, leading to diverse and often devastating effects on the heart, nervous system, and skeletal muscle . ExampleHere, we highlighted recent advances in understanding the structure, function, and regulation of mtDNA transcription. Parallel studies of yeast and human mtDNA transcription machinery have significantly been informative in revealing the basic transcription initiation mechanisms, adding clarity regarding the transcription factors' roles that critically control mtDNA transcription events. Despite this recent progress, many questions remain. We have limited information on the mechanism of various transcription steps, starting from assembly on specific promoters and culminating in the formation of a fully processed RNA transcript. We lack structures of the intermediate complexes in the initiation pathway and have limited knowledge of the dynamics of initiation, elongation, and termination reactions. A mechanistic framework is foundational for understanding how the mtDNA transcriptional machinery is regulated and controlled to sensitively respond to the cell's energy needs.


Endogenous retroviruses (ERVs) in mammals are closely related to infectious retroviruses and utilize host tRNAs as a primer for reverse transcription and replication, a hallmark of long terminal repeat (LTR) retroelements. Their dependency on tRNA makes these elements vulnerable to targeting by small RNAs derived from the 3′-end of mature tRNAs (3′-tRFs), which are highly expressed during epigenetic reprogramming and potentially protect many tissues in eukaryotes. Here, we review some key functions of ERV reprogramming during mouse and human development and discuss how small RNA-mediated silencing maintains genome stability when ERVs are temporarily released from heterochromatin repression. In particular, we take a closer look at the tRNA primer binding sites (PBS) of two highly active ERV families in mice and their sequence variation that is shaped by the conflict of successful tRNA priming for replication versus evasion of silencing by 3′-tRFs. Retroviridae genus, while LTR-retrotransposons prevalent in other phyla such as the Gypsy and Copia superfamilies are Metaviridae and Pseudoviridae, respectively [Reverse transcription and long terminal repeat (LTR) retroelements are ancient components of eukaryotic genomes . In factectively ,5,6. Allectively ,8. ERVs ectively ,10,11. Eectively ,15,16,17With few exceptions, LTR-retroelements use host tRNA to prime reverse transcription and copy their RNA into DNA for insertion into the genome  18,19,2,21. tRNAERVs are usually embedded in repressive heterochromatin, but importantly become active during epigenetic reprogramming in development and disease. Mammals undergo genome-wide epigenetic reprogramming in the embryo right after fertilization and in the germline to obtain totipotency and set aside cells for the next generation ,26,27. EThe propensity of ERVs to attract diverse silencing machineries that act upon specific transposon families at different stages of development make them ideal epigenetic switches . An estiMetaviridae gypsy-elements [Retroviridae ERV insertions mediate imprint establishment at murine loci such as retrotransposon-like 1 (Rtl1), Rasgrf1, Impact, and Slc38a4 [The epigenetic state of ERVs and transposable elements in general can not only lead to developmental stage- and cell-type-specific expression but also establish epigenetic alleles or “epialleles” that result in differential expression between isogenic offspring ,53. Epiaelements . Lineage Slc38a4 ,59,60. T Slc38a4 . Imprint Slc38a4 ,62,63. ISimilar to epigenetic reprogramming in development, ERV reactivation has been observed in other tissues with high epigenetic plasticity, particularly in the course of disease ,64,65. TRasgrf1 locus in mouse is mediated by piRNAs that target an ERV sequence [When transposable elements are released from repressive chromatin during epigenetic reprogramming in development and disease, small RNA-mediated silencing mechanisms become crucial to limit transposition and maintain genome integrity ,72,73. Asequence .Muridae, endogenous small interfering RNAs (endo-siRNAs) target transposon mRNA and protect oocytes [PIWI gene that is lacking in several rodents and produce “oocyte short piRNAs” that target HERVs [Rtl1 imprinted gene in mouse placenta [In the female germline of  oocytes ,79,80,81 oocytes . Human oet HERVs . Howeveret HERVs . Hundredet HERVs ,86,87. Aplacenta . MiRNAs placenta ,89 and rplacenta ,91. HoweSmall RNAs expressed in the germline are well-known to restrict transposable elements, but it is less clear how transposition is avoided during the first wave of reprogramming that enables totipotency in the pre-implantation embryo. DICER-dependent silencing of IAP and MERV-L transcripts was detected up to the eight-cell stage in mouse embryos . HoweverTetrahymena PIWI protein Twi12 [tRFs are a novel class of small non-coding, regulatory RNAs with distinct types, length, and diverse biological functions: 3′-tRFs and 5′-tRFs that are not the reverse complement of each other, stress-induced tRNA halves, internal fragments, and tRFs of precursor tRNAs ,96. Sevein Twi12 , suggestin Twi12 . Minihelin Twi12 , and spein Twi12 ,110. Thein Twi12 .Given the variable expression of ERVs throughout development and even between genetically identical individuals, small RNAs arguably serve well to sense and adjust ERV expression. Small RNA mediated host defense often scales with transposable element burden and acts at different levels: piRNAs in mammals are produced from single-stranded precursor RNA of clusters of transposon sequences already in the host genome, and siRNAs are produced from double-stranded RNA of transposon transcripts ,111,112.Gammaretroviridae) and ETn/MusD (Betaretroviridae) are among the most active transposons in mouse with the majority of novel insertions being non-autonomous copies carried over by a few coding family members [Xenopus, Drosophila, and human [Genome sequencing projects have given insight into the spread of repetitive elements over evolutionary time and into which transposons cause polymorphisms when comparing strains or individuals. In mice, ERVs are highly active causing an estimated 10% of germline mutations in today’s inbred laboratory strains ,113. The members ,114,115. members ,116. Two members ,118. The members  and havend human .Phe-GAA) [Lys-UUU, and are targeted by 3′-tRFLys-UUU [3-UUU and inhibitory 3′-tRFs are derived from this exact sequence. HIV and MLV only use cytoplasmic tRNAs that have passed rigorous quality control by the cell and are capable of being charged with the correct amino acid [Retroviridae such as HIV, RSV, MLV, and avian sarcoma leukosis virus (ASLV) [3-UUU tRNA primer through interaction with Lysyl-tRNA synthetase and prevent aminoacylation of the tRNA [Retroviridae like RSV follow this route, while others like MLV do not package tRNA synthetases [The most highly enriched molecules in viral particles or VLPs are the tRNA primer isotype used for reverse transcription, for example, lysine for HIV-1, representing 70% of all packaged tRNAs . IAP elePhe-GAA) . ETn/MusFLys-UUU ,24. tRNAFLys-UUU ,121, so ino acid . Primer s (ASLV) . In the the tRNA . Some Rethetases ,124. In thetases . Althougthetases ,126. Thithetases ,24.There are a number of techniques to quantify active retrotransposition and probe its regulation. RNA levels are often used as a proxy for transposon activity, but transcription is only one step in the life cycle of transposons and does not necessarily reflect mobility and mutagenic burden, for example, if transposition is inhibited post-transcriptionally by small RNAs. LTR-retrotransposon activity can be experimentally assessed almost every step of the way. Capped, spliced RNA levels reflect mRNA before translation ,48,127, in trans, and perhaps explains the much higher copy numbers for ETn over MusD in murine genomes. ERVs with cis-preference, such as IAP elements, bind and retrotranspose their own RNA in cis, at much higher frequencies than RNA from other elements [Retrotransposition assays with an autonomous and a non-autonomous element allow to dissect the effect of PBS mutations and endogenous 3′-tRFs on ERV activity. A coding-competent MusD element with a scrambled PBS can no longer prime its own reverse transcription but can still replicate its non-autonomous partner, ETn . Withoutelements . For sucelements . TherefoRetroviridae. The Retroviridae HIV and Moloney MLV copy the primer tRNA sequence during second strand synthesis and therefore produce dsDNA and insertions that carry their parent PBS on one strand and the tRNA copy on the other strand (Pseudoviridae copia transposon yeast 1 (Ty1) and Metaviridae gypsy Ty3 elements copy the PBS during reverse transcription to both strands and therefore strictly inherit their PBS sequence [1,2-CUU tRNA as a primer (1,2 signature and its PBS sequence was added to the ETnIIbeta alignment for comparison (Pro1,2 can adapt to use tRNAGln-GUC [One would expect a trade-off between a perfect PBS for tRNA priming but susceptibility to tRF silencing. PBS sequences that allow tRNA priming but reduce tRF silencing would be most “successful” and accumulate in the pool of full-length ERVs. We collected the PBS sequences of four ERV families, whose activity has been tested in retrotransposition assays, and compared them for mismatches with the perfect PBS . Over tir strand  [133,134sequence . The PBSa primer . Indeed,mparison b. SimilaAGln-GUC . Of note, the majority of PBS mutations are found at certain positions . G to A 3-UUU tRF3b downregulates MusD6 RNA and protein levels although it has two mismatches with its PBS [Lys3-UUU did not affect expression levels of ETn or MusD when transfected, but instead abrogated RT priming and were highly sensitive to mismatches with the PBS irrespective of the seed sequence [Which mutations in the PBS would benefit ERVs? Fragments of the tRF3b type, both transfected and endogenous, show all the hallmarks of miRNA silencing ,101,102. its PBS . For miR its PBS ,144. How its PBS . Similar its PBS ,101. The its PBS . Less issequence . This su3-UUU primer tRNA to the PBS [Retroviridae [What else explains the pattern of mutations that we see at the PBS of these ERVs? If most mutations reduce silencing by tRFs, the exact positions of these mutations could be driven by whether they still allow tRNA binding and reverse transcription of the ERV. There are several lines of evidence regarding which PBS sequences allow successful tRNA priming. A number of ERVs have been cloned and found active in retrotransposition assays . Studies the PBS . However the PBS . A recen the PBS . Of noteoviridae . When ta3-tRFs have been found in response to HIV-1 infection in T-cells [Due to the high mutation rate of retroviruses but conservation of the PBS, several studies suggested that destroying the PBS is the ultimate “cure” to disable LTR-retroelements ,148. Thi T-cells . Do 3′-tUsing CENSOR and REPBASE, the following sequences were compiled , and theAfter reformatting to BED format, internal ERV sequences were merged to their corresponding LTR using the BEDtools merge function within 500 bases, on the same strand . Sequenc


Occurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3’ends, thus greatly expanding the diversity of mRNAs and of proteins derived from a single gene. As a key molecular mechanism, APA is involved in various gene regulation steps including mRNA maturation, mRNA stability, cellular RNA decay, and protein diversification. APA is frequently dysregulated in cancers leading to changes in oncogenes and tumor suppressor gene expressions. Recent studies have revealed various APA regulatory mechanisms that promote the development and progression of a number of human diseases, including cancer. Here, we provide an overview of four types of APA and their impacts on gene regulation. We focus particularly on the interaction of APA with microRNAs, RNA binding proteins and other related factors, the core pre-mRNA 3’end processing complex, and 3’UTR length change. We also describe next-generation sequencing methods and computational tools for use in poly(A) signal detection and APA repositories and databases. Finally, we summarize the current understanding of APA in cancer and provide our vision for future APA related research. The maturation of nascent RNAs is a key step in transcription. For mRNA, the maturation of messenger RNA precursors (pre-mRNAs), involving the processing of 3’termini, is critical for mRNA function and stability . In the INK4b, p16INK4a, and ARF [Recent studies have shown that the global regulation of APA and the resulting distinct transcripts are involved in various aspects of tumorigenesis and cancer progression . Differe and ARF . Estroge and ARF . Such a  and ARF , while t and ARF .This review provides a general summary of four types of APA and their effects on gene regulation. We focus on APA regulatory mechanisms, including the interaction of APA with microRNAs, RNA binding proteins and other related factors, the core pre-mRNA 3’end processing complex, and 3’UTR length change. We also introduce high-throughput sequencing methods and computational tools for poly(A) signal detection and related corresponding additions to APA databases. Finally, we summarize recent research on APA in cancer and provide our vision for future APA related research.APA is a phenomenon that generates various transcript isoforms with different 3’termini from the same gene. It is observed in all eukaryotes species as an important mechanism of gene regulation. APA was first discovered in 1980 in the genes encoding immunoglobulin M (IgM) and dihydrofolate reductase (DHFR) , 17. OveTandem 3’UTR-APA occurs in the 3’UTR and can change the structure of 3’UTRs or generate various isoforms of RNAs with different 3’UTR lengths Fig. a. The loAmong these mechanisms, some are relevant to the progression and invasion of tumors. For example, GALNT5 uaRNA (a UTR-associated RNA) is a lncRNA derived from the 3’UTR of GALNT5. It promotes the proliferation of gastric cancer by interacting with the molecular chaperone HSP90 . miRNA-2UR-APA occurs upstream of the last exon, a location far removed from the 3’UTR. It can be further divided into three subclasses, namely alternative terminal exon APA, intronic APA, and internal exon APA. Alternative terminal exon APA occurs as a consequence of alternative splicing Fig. b 23, 2424. Both miRNAs are a type of trans-acting element that can bind to the 3’UTR of mRNA and regulate gene expression at a post-transcriptional level –40. TheyThe interaction between RNA and protein is essential for regulating gene expression at the post-transcriptional level Fig. c and d. RBPs contain specific RNA-binding domains (RBDs). These provide preferential selection of binding sites and targets and interact with RNA through these recognition regions. These RBDs include the RNA recognition motif (RRM), the K homology domain (KH), DEAD motif, double-stranded RNA-binding motif (DSRM), CCCH tandem zinc-finger domain, and Pumilio p-homology and Fem-3 mRNA binding factor (PUF) domains , 54–56. As can be seen from the above studies, the interaction between RBPs and the 3’UTR is deeply involved in PTGR and mRNA stability. It is often difficult to disassociate disease from transcription and translation. The regulation of RBP-RNA binding is a very important pathogenic mechanism of disease. For example, cold-inducible RNA binding protein  is a stress-induced protein involved in cancer. CIRP can bind to the transcripts of pro-survival genes, which contain RNA signature motifs in their 3’UTRs, and stabilize them. In ectopic mouse xenograft models of human breast cancer and melanomas, CIRP promotes tumor growth by increasing the expression level of HIF-1α. Immunohistochemical analysis shows that CIRP is over expressed in the stroma and hypoxic areas of human tumors . FurtherDrosophila melanogaster, embryonic-lethal abnormal visual protein (ELAV) can be recruited to RNA polymerase II (Pol II) at promoter regions with GAGA sequences and then suspend Pol II. ELAV increases the expression of long 3’UTR isoforms during neurogenesis by inhibiting proximal PAS usage [RBPs and RNAs assemble into a dynamic RNP complex. This plays an important role in RNA maturation, regulation, and transportation. Mutations in the heterogeneous nuclear RNPs (hnRNPs) cause amyotrophic lateral sclerosis (ALS) , 67. SurAS usage , 71. AllUR-APA plays an important role in generating truncated transcripts. For example, Singh et al. showed that intronic APA isoforms, as widely expressed in immune cells and as participants in the development of B cells, lead to the production of truncated proteins lacking functional C-terminal domains. Furthermore, the number of intronic APA isoforms is decreased in multiple myeloma cells. This may contribute to the progression of multiple myelomas and is a factor associated with shorter progression-free survival . A termiOn the other hand, the diversification of protein can also be a key part of gene versatility. For example, there are two isoforms of immunoglobulin M (IgM) heavy chain mRNA. The longer one, with the distal PAS usage in the 3’end of the third exon, is appropriate for membrane-binding, while the shorter one, with the proximal PAS in a composite terminal exon usage, is involved in secretion. Different mRNAs also predominate at different stages of immunocyte development, the longer ones at the lymphocyte stages and the shorter one at the secretion stages . AnotherThe core pre-mRNA 3’end processing complex contains four subcomplexes, namely cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CSTF), and cleavage factors I and II (CFI and CFII). These play a critical roles in APA formation and regulation , CPSF2 , CPSF3 , CPSF4 , FIP1 , and WDR33. It has been found that CPSF1 plays a key role in pre-mRNA 3’end formation. Recent studies have shown that the depletion of CPSF1 can induce cell cycle arrest at the G0/G1 phase and promote cell apoptosis in ovarian cancer cells . AnotherCSTF contains three subunits, CSTF1 , CSTF2 , and CSTF3 . The CSTF complex can enhance CPSF’s recognition of upstream PASs. Specifically, CSTF1 plays a key role in the regulation of 3’end processing signal recognition. Studies have also shown that CSTF1 is involved in chromatin remodeling during DNA damage responses , 91. CSTCFI and CFII  are two core components of cleavage machinery and regulators of APA in mammals. CFI contains two small subunits of CFIm25 and two alternative large subunits of CFIm68 and/or CFIm59 . CFI is Other related factors can regulate APA and participate in the processing of its precursors, including poly(A) polymerase (PAP) complex (composed of PAPα and PAPγ), retinoblastoma-binding protein 6 (RBBP6), and others. For example, PAP is responsible for the efficient cleavage of PAS sites via the recruitment of FIP1 and CPSF1. PAP can also bind to an RBP-RNA complex called U1 small nuclear ribonucleoprotein (U1 snRNP) and inhibit polyadenylation , 102. As3’UTR shortening is a significant consequence of APA regulation Fig. a. On accWith the advent of NGS technologies, genome-wide profiling of APA sites has been performed in a variety of species, tissues, and disease states –107. TheA wide-spread shortening of 3’UTRs in mRNAs by APA has recently been discovered in cancer cells. However, the post-transcription regulation of 3’UTR lengthening has not been fully illustrated Fig. b. In 201Global 3’UTR regulation has been observed in various biological systems and processes including those of embryonic development, differentiation of myoblasts, and embryonic stem cells , 115. FoGlobal programs of APA-dependent isoform expression have been discovered in human cancers. Specific APA events have been implicated in various pathological conditions such as malignancies and autoimmune disorders. It has been hypothesized that the global regulation of polyadenylation activity might underlie the global APA profile changes. The usage of PASs is often altered in human hematological, immunological, and neurological diseases, as well as in cancers , 120. ThSince pre-mRNA isoforms with differing lengths of 3’UTRs are widely present in cells, many studies on the post-transcription regulation of pre-mRNA highlight the regulation of 3’UTR’s APA and poly(A) tail length changes. These studies not only reveal the mechanisms and factors that regulate cytoplasmic and nuclear changes in the poly(A) domain, but also clarify the relationship between these mechanisms. The relationship between 3’UTR-APA and miRNA targeting has been particularly illuminated. Similarly, relationships between deadenylation and the change of PAS usage in inflammation, or between cytoplasmic polyadenylation and the 3’UTR shortening in neurons, or relating to the alternative lengths of poly(A) tails in germ cells and tumors, have all been elucidated. As a novel mechanism for regulating various gene functions, APA has been involved in various biological processes including mammalian development, immune system function, disease pathogenesis, etc. , 124–128C. elegans. The study revealed short poly(A) tails as a conserved feature of highly expressed genes [In 2014, two different high-throughput sequencing approaches were developed to sequence the 3′-terminome. Using the first of these methods, TAIL-Seq, researchers measured the length of the poly(A) tail and found the median poly(A) length to be 50-100 nt in HeLa and NIH 313 cells . The seced genes . Subsequed genes , 133. Ined genes . More reed genes  and Polyed genes , have beed genes , these nIn parallel with the advancement of experimental methods, computational tools to detect APA have been actively developed. These are summarized in Table DaPars is a powerful tool to identify numerous APA events from standard RNA-Seq data. It employs a piecewise linear regression to model read count data of RNA-seq to identify the location of the de novo proximal poly(A) sites. Using DaPars, Xia et al. identified 1346 genes with tumor-specific APAs from 358 pairs of tumor/normal samples across seven cancer types. Compared with normal tissue samples, more than 90% of these APA genes had shorter-length isoforms in the tumor samples. This tool has been widely used to detect APA events from RNA-seq data and has also been adopted by many databases [APAtrap is capable of APA identification and quantification. Based on the mean squared error model, APAtrap can identify differential PAS usage and predict all potential poly(A) sites. When APAtrap was applied to the simulation data and real RNA-Seq data from human and Arabidopsis tissues, it showed higher accuracy than other tools in identifying APA events .DeepPASTA is a deep neural network method to detect APA events. It was the first tool to predict poly(A) sites from both sequence and RNA secondary structure data. In addition, this tool can predict the most dominant poly(A) site of a gene in a specific tissue and predict the relative abundance of two polyA sites of the same gene .Finally, scDAPA is a software package that can be used to detect APA profiles from single-cell RNA-seq data. It includes three main modules, namely 3’end annotation, APA event identification, and APA event visualization. scDAPA has a high degree of confidence for APA detection. This tool facilitates the portrait of dynamic APA profiles in different cell types from scRNA-seq data .A large quantity of APA data has been produced using NGS techniques. Using these data, several databases have been established to facilitate the research community to obtain APA information from various samples. These are summarized in Table The PolyA_DB is a database for analyzing pre-mRNA cleavage and polyA sites. It contains a large amount of data on polyA sites in humans, mice, rats, and chickens. In 2018, this database had been updated to version 3.0 (renamed as PolyA_DB 3). Based on deep sequencing data, using the 3’READS method, this version contains large volumes of data from multiple samples to supplement PAS information. The database can also be visualized by the UCSC genome browser .TC3A focuses on human cancers with large-scale RNA-Seq datasets from TCGA which contains 10,537 tumor samples across 32 cancer types and provides APA usage analysis and visualization. This atlas is based on a bioinformatics algorithm called DaPars and its updated version, DaPars2. Users can compare the PAS usage of genes between tumor and normal samples .PolyASite is a resource of PAS information generated using 3’end sequencing in humans and mice. In 2019, it was updated to version 2.0 containing new PAS datasets from worm genomes. PolyASite 2.0 integrates sequencing data generated by multiple sequencing methods  .The APAatlas contains 1,125,143 APA events from 9475 samples across a total of 53 human tissue types. It focuses on the APA events located in 3’UTR regions and provides a view of the APA landscape across tissues. APA events in the APAatlas were inferred using DaPars and SAAP-RS. Since the APAatlas includes a large amount of normal human tissue samples, compared with other databases, it contains more APA events from normal samples and provides a good opportunity for investigation of the correlation between PAS usage and gene expression .Global APA within 3’UTR has been characterized in various cancer tissues and cells. Many of these are identified to be involved in the proliferation and metastasis of cancer cells. The following describes the role of several of these important APA factors in cancer  encoded by the Nudt21 gene, belongs to the Nudix family of hydrolases . This faPoly(A) binding protein nuclear 1 (PABPN1) plays a major role in the post-transcriptional processing of RNA and in controlling the poly(A) tail length of RNA transcripts. PABPN1 binds at proximal poly(A) sites to block their cleavage. Yu et al. characterized the APA profiles of 6398 patient samples across 17 cancer types from The Cancer Genome Atlas (TCGA) and of 739 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE). They identified 1971 clinically relevant APA events and their analysis further illustrated PABPN1 as a master modulator of 3’UTR shortening. PABPN1 possess the capacity of proximal PAS binding and then alters the APA site selection . In tripHeterogeneous nuclear ribonucleoproteins C (hnRNPC) is an RNA-binding protein encoded by the HNRNPC gene in humans. hnRNPC regulates genome-wide PAS usage selection. By generating a pre-mRNA 3’end sequencing library from hnRNPC-knockdown cell lines, Gruber et al. observed that nearly 54% of PASs in exons had altered their usage from that of the control group. Mechanistically, hnRNPC binds the poly(U) motifs that are frequently located near distal poly(A) sites. HNRNPC’s binding in close proximity of distal poly(A) sites prevents them from cleavage and polyadenylation, thereby increasing genome-wide proximal PAS usage . AberranAs a part of CFII, PCF11 contains an N-terminal RNAPII C-terminal domain (CTD)-interacting domain (CID) and plays a role in transcription termination and mRNA nuclear export control , 175. LiMounting evidence is now demonstrating APA as a new layer of regulation for gene expression. The four types of APA work synergistically with miRNAs, RBPs, and other factors, to regulate gene expression and functional versatility. Due to the differential usage of PASs, various transcript isoforms can be generated in cells. These transcript isoforms are involved in multiple cellular processes including control of the cell cycle, mRNA translation efficiency, and cell proliferation and differentiation. APA is frequently dysregulated in cancer and this promotes tumorigenesis and progression by increasing the expression of oncogenes and reducing the expression of tumor suppressor genes , 176–178Many computational tools and databases have also been developed to detect APA events Tables  and 2. MExtensive APA occurs during the pathophysiology of many diseases including cancers. In these, APA events are emerging as clinical biomarkers of high potential. Most of the differentially regulated APA events result in transcript isoforms with different lengths of 3’UTRs. These are often related to a variety of clinical characteristics. These APA events are independent of commonly used molecular data  , and havAlthough recent studies have greatly enriched our knowledge of APA, we still know little about certain functions such as the differential affinity of PASs, the recruitment of the 3’end processing complex and other details on the regulation of APA factors. Continuing in-depth research on the modulation of APA regulation, the impact of APA on biological processes, and the possibility of manipulating APA in disease treatment, remains of high priority.


A mutant (oen1) restored non‐vitreousness and produced a large cavity in the seed due to severely depleted endosperm filling. When oen1 was introgressed into inbred W64A with a normal O2 gene, the seeds appeared vitreous but had a shrunken crown. oen1 was determined to encode Shrunken1 (Sh1), a sucrose synthase . Maize contains three SUS‐encoding genes  with Sh1 contributing predominantly to the endosperm. We determined SUS activity and found a major and minor reduction in oen1 and o2, respectively. In o2;oen1-1, SUS activity was further decreased. We found all Sus gene promoters contain at least one O2 binding element that can be specifically recognized and be transactivated by O2. Sus1 and Sus2 promoters had a much stronger O2 transactivation than Sh1, consistent with their transcript reduction in o2 endosperm. Although sus1 and sus2 alone or in combination had no perceptible phenotype, either of them could dramatically enhance seed opacity and cavity in sh1, indicating that transactivation of Sus1 and Sus2 by O2 supplements SUS‐mediated endosperm filling in maize. Our findings demonstrate that O2 transcriptionally regulates the metabolic source entry for protein and starch synthesis during endosperm filling.The endosperm‐specific transcription factor Opaque2 (O2) acts as a central regulator for endosperm filling, but its functions have not been fully defined. Regular  Because many o2 mutants create an opaque mature kernel phenotype, a mild reduction in the synthesis of starch or storage proteins resulting from mutations in o2 enhancers may not cause a visible change in the phenotype of o2 seeds. A previous platform used γ‐irradiation to mutagenize a vitreous o2 variety, quality protein maize (QPM) and created many opaque QPM variants to identify o2 modifier genes  Opaque2 (O2) functions as a central player that not only transcriptionally regulates expression of most zein storage protein‐encoding genes but also directly and indirectly regulates starch synthesis  that modify the endosperm texture, creating a vitreous, hard phenotype. This mutagenesis method could identify o2 modifier genes themselves, or unrelated genes that simply exacerbate the o2 phenotype (o2 enhancers). If an o2 enhancer is mutated, endosperm modification in QPM could be disrupted and produce the opaque phenotype. Thus, we treated pollen of K0326Y, a QPM inbred line, with EMS and applied it to the associated ears. The resulting seeds were planted for self‐pollination, yielding 1500 M1 ears. One ear from this screen was found to segregate progeny kernels with normal (vitreous and plump) and mutant (opaque and shrunken) phenotypes in a 3:1 ratio . The 100‐kernel weight (KW) of oen1 in the K0326Y background (K0326Y‐oen1) was only 11.00 g, which was 36% lower than that of normal K0326Y  for two generations and then self‐pollinated for two generations. In the second backcrossing, each plant was numbered and self‐pollinated, while the pollen was used for backcrossing. Approximately one quarter of the self‐pollinated ears segregated progeny seeds with four different phenotypes, that is vitreous and plump , vitreous and shrunken , opaque and plump , and opaque and shrunken (indicative of homozygotes for o2 and oen1). Subsequently, seeds from the corresponding backcrossed ears were planted and should have produced equal amounts of four different genotypes . The WT, two single‐gene mutants and the double mutant were isolated by self‐pollinating the four different plants. The homozygous ears for each genotype resulting from one additional propagation are shown in Figure Because QPM is homozygous for o2, the oen1 and o2;oen1 double mutants had normal seed sets , thereby allowing for a little light translucency (indicated by arrows). The oen1 seeds exhibited a shrunken phenotype in the crown area, but vitreous endosperm could be formed, although it was less than WT. The double mutant seeds were dramatically shrunken and contained almost no vitreous endosperm. On a light box, the o2;oen1 seeds were even more opaque than the o2 seeds  of t Figure . A signid Figure . The conoen1 into W64A, self‐pollinated plants with the O2/O2;oen1/Oen1 genotype segregated plump and shrunken seeds at a ratio of 3 : 1 . Comparison of the single‐nucleotide polymorphism (SNP) index between the mutant and WT pools revealed a 28‐Mb region, containing nine candidate genes, near the short arm of chromosome 9  of Zm00001d045042, which could result in defective RNA splicing of this intron  to analyse the expression of Sus genes in 12‐DAP endosperms of the WT and o2. As shown in Figure Sus1 and Sus2 were dramatically reduced to a barely detectable level in o2 compared with WT. Although expression of Sh1 was downregulated in o2, the fold changes were not comparable to those for Sus1 and Sus2, consistent with our previous published RNA‐seq data  reporter gene driven by the cauliflower mosaic virus 35S promoter was used as an internal control, and the Sus gene promoters were fused with the firefly luciferase coding sequence, yielding the reporter vectors PSh1‐LUC, PSus1‐LUC and PSus2‐LUC. The effector plasmid was generated by fusing the coding region of O2 to the 35S promoter (35S‐O2). Coexpression of PSh1‐LUC, PSus1‐LUC or PSus2‐LUC with 35S‐O2 all resulted in a significant increase in LUC activity. The Sus2 promoter exhibited the strongest activation by O2 (100‐fold), followed by the Sus1 promoter (20‐fold), whereas the Sh1 promoter exhibited only a 10‐fold increase . Sh1 and Sus1 genes were found to bear one O2 box within 800‐bp promoter regions, and Sus2 had two such motifs. Oligonucleotides (50‐60 bp) labelled with FAM fluorescent molecules were used as probes to examine the binding affinity. Binding of the His‐O2 fusion protein and the His‐O2‐bZIP(aa229‐293) fusion protein to the probes could be visualized as retarded bands in the gel. The results showed that O2 and the O2‐bZIP domain protein could specifically bind to the probes from all Sus gene promoters ; each of these mutants harbour a stop codon in the coding region. Consistent with previous reports  and sus1 was created by screening their recombinants. As shown in Figure sh1 and sus1 or sus2 resulted in the seed cavity being dramatically enlarged compared with that of the sh1 single mutant. This enlargement was further enhanced in the triple mutant, which exhibited the largest hollow seed compartment among all the mutants. Similar to o2;oen1, the sh1;sus1 and sh1;sus2 double mutants and the sh1;sus1;su2 triple mutant had full seed sets on the ears. Due to severely insufficient endosperm filling, seeds in the central cob of these plants were all non‐vitreous and extremely shrunken, forming flattened kernels during seed desiccation  CWI2 and SUS function complementarily in a parallel way. Each of these genes alone is insufficient for efficient sucrose cleavage and effective endosperm filling; (ii) CWI2 and SUS work sequentially to influence endosperm development and filling. Both outcomes support the idea that the products of the two enzymes contribute to endosperm filling, but differ in location and reaction mechanisms and outcomes. The CWI2 pathway for sucrose cleavage produces twice as much hexose (fructose and glucose) as the SUS pathway (fructose and UDP‐glucose). Both mn1 and the sus triple mutant manifest dramatic reductions in endosperm size and weight, but mn1 exhibits additional developmental defects at the BETL at a relatively early stage. It is generally accepted that glucose creates signals that trigger BETL cell division and differentiation  was isolated by recurrently backcrossing to W64A (a WT inbred line) for two generations. The homozygous oen1‐1 ears were obtained by self‐pollination of BC2 seeds for two generations.The QPM line K0326Y is an 1 hybrids of oen1 and sh1‐ref. The sh1‐ref seeds were generously provided by Professor Yubin Li from the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences. To further verify the action of sh1 as an o2 enhancer, another mutant that enhances the o2 phenotype was created by EMS mutagenesis. EMS‐treated pollen of W64A was applied to the silks of W64Ao2 ears. The resulting seeds were planted for self‐pollination to screen for ears that segregated progeny with an enhanced o2 phenotype. Due to the allelism of oen1 and the new mutant, these alleles were named oen1‐1 and oen1‐2, respectively.An allelism test was carried out by gene complementation in the FA dozen mature kernels of each genotype were ground to fine powder using a tissue grinder for the analysis of the soluble sugar, starch and protein content. All measurements for each genotype were performed in at least triplicate.et al., For the measurement of soluble sugar content, 100 mg of mature kernel flour was weighed and added into 80% (vol/vol) ethanol to extract soluble sugars. The anthrone method for determining soluble sugar content was previously described  based on the activities of thermostable α‐amylase and amyloglucosidase. Each sample was measured four times.The total protein content in the mature seed flour was determined with using a Dumas rapid nitrogen determination analyser . Approximately 50 mg of seed flour was weighed into tin boats without pretreatment and pressed to pellets using a manual pressing tool. The nitrogen content was then detected by a thermal conductivity detector. Analyses were run using a standard method implemented in the instrument software, with a total analysis time of approximately 5 min. A protein factor of 6.25 was applied to calculate the average protein content. Each sample was measured three times.1F2 ears of K0326Y‐oen1 and W64A with the segregating seeds of WT and oen1 were used to clone the oen1 gene by the mapping‐by‐sequencing approach. 150 seeds of each phenotype in the BC1F2 ears, and 30 seeds of each parent, K0326Y‐oen1 and W64A, were selected and germinated. The resulting seedlings were used individually to extract genomic DNA. The individual genomic DNA for each phenotype/parent was pooled equally. Four pooled libraries with insert sizes of approximately 350–500 bp were prepared and sequenced on a HiSeq X™ Ten (Illumina) lane using 150‐base paired‐end sequencing. To identify the mutation site, the paired ‐end short reads obtained from the four samples were aligned to the reference genome of Zea mays (AGPv4.35) using BWA‐MEM software  in a 2‐mL tube at room temperature for more than 2 h. The mixture was centrifuged at 17370 g for 10 min, and 100 μL of the supernatant was transferred to a new tube and mixed with 10 μL of 10% SDS. A Concentrator Plus (Eppendorf) was used to dry the solution, and the material was then redissolved with 100 μL of ddH2O. The precipitate was extracted prior to zein buffer three additional times for non‐zein extraction. The precipitate was dried in the Concentrator Plus and vortexed with 0.5 mL of non‐zein extraction buffer  at room temperature for more than 2 h. After centrifugation at 17370 g for 10 min, the supernatant containing non‐zein proteins was transferred to a new tube. Then, 3 μL of the zein and non‐zein proteins was analysed by SDS‐PAGE (15%).Three kernels were taken from each genotype, and the embryos were removed. The endosperms were ground to a powder in a steel pot in a tissue grinder, and 50 mg of each sample was used for zein extraction with 0.5 mL of zein extraction buffer (3.75 m49EFDALFDSDKEKYAP63). Total protein in the four 12‐DAP endosperm samples was extracted as described above and separated by SDS‐PAGE (4%–20%). The proteins were electrophoretically transferred to a Bio‐Rad PVDF membrane that was activated by methanol. The membrane was processed according to the manufacturer's instructions, and the Sh1 protein was detected by the Tanon‐5200 system. The dilutions of the antibodies against Sh1 and ACTIN  were 1:1000 and 1:8000, respectively.A peptide antibody for Sh1 was prepared by Abclonal, China, in rabbits. The specific antibodies were produced using sequences unique to maize Sh1 with the following sequence: , 1% SDS and 5 mm EDTA (pH 8.0). The samples were rapidly homogenized using a tissue grinder and subsequently extracted twice using an equal volume of phenol–chloroform (pH 4.2) and an equal volume of chloroform once. The aqueous phase was transferred to TRIzol (Invitrogen) for further extraction of total RNA, followed by RNA purification using the RNeasy Mini Kit (Qiagen). A total of 5 μg of total RNA from each sample were used for reverse transcription with the Superscript III First Strand Kit (Invitrogen). RT‐qPCR was performed with SYBR Green (Takara) on a Bio‐Rad CFX‐96 thermocycler. The relative expression of these genes was calculated using the ΔΔCt method. The internal control was the maize Ubiquitin gene. All primers used in this experiment are listed in Table All materials for RNA extraction were kept at −80 °C until use. For total RNA extraction of developing seeds and endosperms, samples were placed in a 2.0‐mL microfuge tube with 200 μL of RNA extraction buffer containing 150 mEndosperms separated from fresh developing seeds were rapidly frozen in liquid nitrogen and kept at −80 °C until use. More than 20 endosperms were immersed in a steel tank cooled with liquid nitrogen and rapidly ground to a powder in a tissue grinder. 50 mg of endosperm powder was immediately placed into a tube cooled with liquid nitrogen to measure SUS activity. The Solarbio Sucrose Synthetase (SS) Assay Kit  was used for activity determination. Sucrose synthetase catalyses the conversion of fructose and UDPG to UDP and sucrose. The reaction between sucrose and resorcin results in a colour change, with a characteristic absorption peak at 480 nm, and the enzyme activity is proportional to the colour intensity. Each sample was measured six times. We used ultraviolet visible photometer (Beckman Coulter DU730) to detect the characteristic absorption peak of 480 nm. Each sample was measured six parallel experiments.2O with shaking incubation for 30 min on ice. The supernatant was then moved to a new centrifuge tube after centrifugation at 13 000 rpm for 10 min at 4 °C and filtered with 0.22 μm filter. The filtered liquid was diluted with ddH2O by 10 fold for measurement by the ion chromatography  using a CarboPac PA‐20 column  and solvent B (200 mm NaOH).Fifty mg of freshly endosperm prepared powder was extracted with 2 mL ddHet al., O2 driven by the 35S promoter. The reporter vector pGreenII 0800‐LUC was used to detect the transactivation of the three Sus gene promoters. The LUC/REN activity ratio was measured using the DLR assay system (Promega). Primers are listed in Table Isolation of Arabidopsis mesophyll protoplasts, PEG–calcium transfection of plasmid DNA, and protoplast cultivation were performed as described previously (Zhang 229‐293) and the EMSA procedure have been described previously  was used to calculate The authors have no conflict of interest to declare.YW conceived the idea and supervised the project overall. YD and YW wrote and revised the manuscript together. YD designed and performed the experiments. JW and ZZ participated in the experimental design, phenotype measurements and manuscript revision.Figure S1 The K0326Y‐oen1 mutant generated by EMS‐induced mutagenesis of K0326Y.Figure S2 The plant phenotype of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background.Figure S3 SDS‐PAGE analysis of zein (upper) and non‐zein (lower) proteins of mature kernels of WT, o2, oen1‐1, o2;oen1‐1 in the W64A backcross population.Figure S4 The phenotype of oen1‐1 in the W64A background.Figure S5 Genetic complementation test of oen1‐1 with sh1‐ref and oen1‐2 alleles.Figure S6 Kernel phenotypes of WT, o2, oen1‐2, o2;oen1‐2 in the W64A background.Figure S7 SDS‐PAGE analysis of zein (upper) and non‐zein (lower) proteins of mature seeds of WT, o2, oen1‐2, o2;oen1‐2 in the W64A background.Figure S8 SUS activity in developing endosperms of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background at 10 and 14 DAP.Figure S9 Levels of sucrose (a), glucose (b) and fructose (c) in developing endosperms of WT, o2, oen1‐1, o2;oen1‐1 in the W64A background from 8 to 24 DAP.Figure S10 Alignment analysis of amino acid sequences of the three SUS proteins.Figure S11 Kernel phenotypes of WT and single, double and triple mutants of the three Sus genes.Figure S12 Levels of sucrose (a), glucose (b) and fructose (c) in 12DAP endosperms of WT and single, double and triple mutants of the three sus genes.Table S1 Primers used in this study.Appendix S1 The promoter information of Sh1, Sus1 and Sus2.Click here for additional data file. Click here for additional data file.Supplemental Dataset File Original data and details for all statistical analyses in this study.Click here for additional data file. Click here for additional data file.


The ability to restore or replace injured tissues can be undoubtedly named among the most spectacular achievements of plant organisms. One of such regeneration pathways is organogenesis, the formation of individual organs from nonmeristematic tissue sections. The process can be triggered in vitro by incubation on medium supplemented with phytohormones. Cytokinins are a class of phytohormones demonstrating pleiotropic effects and a powerful network of molecular interactions. The present study reviews existing knowledge on the possible sequence of molecular and genetic events behind de novo shoot organogenesis initiated by cytokinins. Overall, the review aims to collect reactions encompassed by cytokinin primary responses, starting from phytohormone perception by the dedicated receptors, to transcriptional reprogramming of cell fate by the last module of multistep-phosphorelays. It also includes a brief reminder of other control mechanisms, such as epigenetic reprogramming. Being sessile organisms, plants have developed and mastered the art of adaptation. The ability to restore or replace injured tissues and structures undoubtedly can be named among their most spectacular achievements. This remarkable plasticity of the developmental and regenerative route is the function of regaining cellular totipotency . FollowiThis regenerative potential was exploited in many biotechnological  practices, including the production of transgenic plants. The number of reported in vitro regeneration protocols from a wide range of source explants is vast and covers all known modes. However, behind all these events there are extremely complex molecular regulation and genetic mechanisms responsible for cell/tissue fate. Initial cues elicit transcriptional cascades and amend concentration and distribution of endogenous plant hormones. In laboratory conditions, such processes are triggered by wounding, incubation on the appropriate medium or supplementation with phytohormones, usually auxins and cytokinins. Consequently, the inductive signals are perceived by plant cells and modify ongoing metabolic and genetic settings, and the mode of reprogramming is started. However, precise knowledge on how the stimuli modulate the developmental pattern and how they point out the definite, as well as the optimal pathway of regeneration, is still to be acquired.Cytokinins (CKs) represent one of the most important groups of plant hormones that can be used to exogenously provoke in vitro responses . Indeed,De novo shoot organogenesis is the ability to perform a new complete process of postembryonic shoot formation. Typically, only a group of explant cells displays responsiveness to inductive signals during organogenesis as well as somatic embryogenesis from mature plant organs. A routine laboratory scheme of in vitro shoot regeneration may include using an auxin-enriched medium for the explant preculture to induce callus generation and subsequent cytokinin-dependent shoot induction. Thus, although this analysis was to focus on cytokinin-triggered events, one just cannot ignore an auxin-based medium intermediate. The golden hormonal regeneration pattern by Skoog and Miller  remains the guiding determinant of the in vitro fate of the explant . A high cell dedifferentiation, as a consequence of the acquisition of morphogenic competence;response to exogenous plant hormones, understood as a determination of competent cells to form a shoot (induction);phytohormone-independent organ morphogenesis .Nevertheless, direct protocols to form shoots from explants are used just as often as the above ones, both resulting in two universal, to a certain extent, morphological steps: acquisition of pluripotency  and nonembryonic shoot organogenesis. Since a great number of excellent reviews on shoot regeneration has been published, the number of stages is higher depending on the details covered. According to some scientists, there are three general steps (given the exogenous plant hormones required and their role) including:Reviewing the process of DNSO from calli Shin et al.  resultedcis-elements in the nucleotide sequence and the screening of the trans factors involved. Brenner et al. [cis-acting cytokinin response sequences; (iv) dedicated genome analysis of the Arabidopsis transcriptome and finally (v) identification of the linkage between specific transcriptional reactions and the biological functions of these phytohormones [Arabidopsis thaliana investigating indirect organogenesis, was a source of the great majority of these findings, although nowadays some comparative analyses of engaged sequences of other species are available  [De novo shoot regeneration definitely requires cell proliferation involving the activation of the mitotic division. As has already been mentioned, cytokinins are a class of phytohormones with a wide spectrum of functions throughout the life cycle of plants, including cell division. Cytokinins influence the competent cells of the DNSO system leading to the production of a cell mass and cell fate conversion. This process involves cytokinin perception and its further signaling. It has been shown that a great part of this signaling occurs through the fine-tuned regulation of gene expression. It seems rather unlikely to succeed in isolating genetic or molecular players that are responsible only for shoot organogenesis. However, since the discovery of cytokinins, much work has been done to determine the biosynthetic enzymes, phytohormone receptors and their signal transduction pathways at various levels. The research on the regulation of gene expression included both the identification of the r et al.  pointed hormones . The resea mays) ,20,21,22Arabidopsis plants, cytokinin signaling starts with their detection by a set of three sensor receptors of a catalytic type. Discovered in 2001, the receptors, known as Arabidopsis Histidine Kinase 4 , Arabidopsis Histidine Kinase 2 and 3, are transmembrane proteins localized in the endoplasmic reticulum (predominantly) and the plasma membrane [In membrane ,24. Thesmembrane . They dimembrane .trans- and cis- configurations, followed by N9-riboside phosphate and the N9-ribosylated derivatives, while isopentenyladenine and dihydrozeatin are also presented at a reasonable, but lower, level. The aromatic types of phytohormones  are present in plant tissues as well but at smaller concentrations [trans-zeatin riboside, once transported via the xylem to other parts of a plant, requires conversion to the corresponding free base form to trigger shoot organogenesis, or whether it happens as a consequence of its direct binding to the receptor. A similar inconsistency was also observed regarding the interaction of hormone receptors with cytokinin ribosides. However, the observation of the crystal structure of the AHK4-tZ complex should shed light on the biological activity of ribosides. The analysis of the AHK4-tZ complex revealed that the positions of the N3, N9, and N7 of the adenine ring are buried in the binding pocket of AHK4. This observation supported the absence of the hormonal activity of tZ ribosylated at the N9 position because the riboside moiety did not fit into the binding pocket [Escherichia coli favor riboside ability to bind the receptor. However, investigating the competition of isopentenyl adenosine, trans-zeatin riboside and their corresponding bases, Lomin et al. [E. coli spheroplasts, ribosides were as effective as the free bases. However, when plant membranes from the leaves of N. benthamiana were used, they hardly competed for binding to the cytokinin receptors [Cytokinins, the target molecules of these receptors, occur in plant organisms as a variety of isoforms. The most prevalent among those is zeatin, which can be found in trations . The tZ trations . To a ceg pocket . Interesn et al.  showed teceptors . Hence, Zea mays, reveals the highest affinity for trans-zeatin and decreasing affinity for dihydrozeatin >isopentenyladenine >N6-benzyladenine >cis-zeatin  [In general, however, the question of affinity should be carefully explored. For example, although the es(AHKs) . TherefoThe cascade of protein phosphorylation is a chief mechanism regulating routes of signal transduction in prokaryotic and eukaryotic cells. The cytokinin signaling is structurally and functionally related to the bacterial Two-Component System (TCS), which is believed to be one of the most common systems in prokaryotic organisms ,32. It hArabidopsis histidine kinases known that could be classified as hybrids because of the presence of histidine and a receiver domain in one molecule. According to various authors, the Arabidopsis MSP system includes 11 HKs [Medicago sativa, Glycine max, Oryza sativa, Zea mays or Solanum lycopersicum) show that phosphorelay elements are quite conserved across the plant world [The model thale cress represents the most investigated plant. There are, for example, 16 s 11 HKs . They tys 11 HKs ,35. Thiss 11 HKs ,36. In as 11 HKs . In spitArabidopsis plants, cytokinin signaling engages three modules: cytokinin-binding histidine kinase receptors, a set of phosphorelay shuttle proteins responsible for transduction along the pathway cytoplasm—nucleus structures and type-B Response Regulators (RRB) that are transcription factors. The last elements of this cascade activate gene expression related to cytokinin primary responses. Additionally, a negative feedback on multistep-phosphorelay signaling is exerted by type-A response regulators [ATVHSEIRTP) of the kinase domain is performed. Four conserved boxes, known as N-, G1-, F- and G2-motifs, are believed to participate in the binding of ATP molecules. Finally, the C-terminus receiver domain, including an acceptor sequence (DD-D-K) with the key aspartate residue, mediates the transport of the phosphate moiety [Described in gulators . Apart fgulators ,38. In ggulators ,30. In be moiety , and thuA. thaliana species, all known regulatory elements were divided into three aforementioned groups basing on their structure and function [The next step is assured by a set of low molecular weight mobile Histidine Phosphotransfer Proteins (HPT). These phosphotransmitters also possess the conserved histidine residue and shuttle between cytoplasm and the nucleus transferring the phosphate signal. In thale cress, two classes of these MSP have been distinguished. One of them relates to HPTs and carries the aforementioned H residue being able to pass the signal from receptor domains towards nuclear type-B response regulators (positive regulators). The second group possesses asparagine instead of histidine and, therefore, cannot run the phosphotransfer (negative regulator) . The cytfunction . A consefunction ,40. Whenfunction . The B-Rfunction . It shoufunction .The scheme of the cytokinin signal transduction is presented below .WUSCHEL (WUS) is considered to be a critical molecular event triggering CK-induced shoot organogenesis. In the process of SAM establishment, WUS is thought to define the organizing center. Therefore, WUS expression is substantial for the de novo organization of the shoot stem cell niches that generate signals that control the balance between self-renewal processes and the production of daughter cells that can differentiate into new tissues [WUS overexpression. Since WUS mutants displayed no shoot regeneration in vitro, it is assumed that stimulation of the identity acquisition of the shoot meristematic progenitor cells requires a check point level of WUS gene expression [WUS-positive mark the shoot progenitor region during de novo shoot organogenesis [WUS, a homeodomain transcription factor, is synthesized in the organizing center (its RNA is present in cells of the OC niche) and then moved to a central zone to activate CLAVATA3 (CLV3), a specific regulator of shoot meristem development. The WUSCHEL–CLAVATA3 expressing system makes a core regulatory loop coordinating proliferation and stem cell identity in the shoot apical meristem (keeping a constant number of stem cells) [WUS migrates to the CZ, it binds to a promoter sequence of CLV3 (using three WUS-binding elements with conserved TAAT motif). The latter gene encodes a small peptide (a negative feedback regulator) that binds to CLAVATA1 (CLV1) in the extracellular space. CLV1 is a leucine-rich repeat receptor kinase typically synthesized in the rib meristem (RM) area. Its activation  mediates the downregulation of WUSCHEL transcription in OC cells [WUS TF directly suppresses the transcription of Arabidopsis RR7 and RR15 genes in the cells of the organizing center. Hence, it indirectly promotes cellular cytokinin response by blocking its cellular A-type inhibitors [WUSCHEL or CLAVAT3, the stage of shoot induction is connected with the transcriptional activation of other shoot meristem-associated genes, for example, the SHOOT MERISTEMLESS (STM) gene, which is expressed in the promeristem [WOX2 (expressed at the zygote level) transcripts in wild-type explants on shoot inducing medium [De novo shoot formation requires initiation and maintenance of the functional shoot apical meristem (SAM) with its central zone (CZ) carrying the pool of pluripotent stem cells (SC). The central zone includes three separate cell layers that form the epidermis (L1), ground tissue (L2) and vasculature L3). The induction and maintenance of the stem cell fate are conditioned by the activity of the organizing center (OC), which is situated directly underneath the SC area ,43. The pression . Clearlyogenesis . WUS, a OC cells . Additiohibitors ,47. Aparmeristem . It is cmeristem . It keepmeristem  or Ikeuc. The indmeristem  indicateg medium .YUC1, YUC4, PLETHORA  or CUP-SHAPED COTYLEDON (CUC1 and CUC2) may be earlier attached [ISOPENTENYLTRANSFERASE 3 or the LONELY GUY gene family: LOG1, LOG4, LOG5 in the case of wounding event), although, on the other hand, the CK biosynthetic gene LOG2 is upregulated in response to cytokinin. On the one hand, it potentially serves to help replenish the cytokinin pool, and probably to achieve the cytokinin amount needed to trigger the repression of their excessive biosynthesis on the other [On a side note, most subject reviews focus on indirect organogenesis, where the development of the apical shoot meristem follows auxin addition, which results in callus formation and engages auxin-induced genes . The claattached . Moreovehe other ,71. Of ccis-motif candidates, flanked by degenerate sequences [NSHB) genes. The transient bombardment test performed on tobacco leaf disks, using a rice OsNSHB2 promoter, revealed the cytokinin-dependent promoter activation. This activation was mediated by ARR1 via its binding to cis-element AGATT in the promoter sequence [The addition of cytokinin causes rapid down and upregulation of many genes . In fact, it seems that the CK-mediated de novo shoot development is the consequence of: (i) the expression of genes involved in cytokinin balance, (ii) signal transduction, (iii) activity of factors responsible for the regulation of transcription process, (iv) splicing performing and (v) chromatin remodelers’ activity . The shiequences . The corequences ,75. Howeequences . CKs endsequence ,52. The WUS is a strategic gene indispensable to shoot progenitor formation, shoot apical meristem maintenance  and shoot morphogenesis. Referring to a cytokinin-dependent de novo shoot organogenesis, it was proved that B-type RRs, the fundamental players in cytokinin signaling, were primary positive regulators of the WUSCHEL expression [WUSCHEL induction. Thus, these B-ARRs, apart from the activation of WUS transcription, take part in SAM maintenance, formation of axillary shoot meristems and in vitro regeneration of shoots. In parallel, they maintain a mutual phytohormone balance by repressing auxin accumulation by blocking the expression of YUCCAs encoding critical enzymes for auxin biosynthesis [Arabidopsis ARR1 and ARR12 significantly hinder root elongation; all three acting to maintain the size of SAM [Arabidopsis thaliana ARR1, ARR2, ARR10 and ARR12 directly induce the WUS factor gene and physically interact with partner microRNA165/6 targeted HD-ZIP III transcription factors: PHABULOSA, PHAVOLUTA and REVOLUTA , to spatially restrict WUS expression to shoot progenitor cells. The inactivation of all these complex compounds gives a triple mutant  with impaired ability to create shoot meristems. This loss-of-function event cannot be reversed even by a high dose of cytokinin [WUS SAM-inducing activity, is confined to the cells marked by AHK4 receptor presence [As it has already been discussed, pression . Out of pression , also duynthesis ,75. A the of SAM . In Arabytokinin . Additioytokinin . Strikinpresence ,75. ThusWUS TF. Scientific research indicates that B-type ARR12 is one of the most powerful positive regulators of shoot regeneration. It seems unquestionable that it does not contribute to dedifferentiation or callus proliferation processes but is engaged in the formation of shoot apical meristem. The loss-of-function mutant hardly regenerates shoots, while ARR12 overexpression clearly amplifies the number of the differentiated shoots  [WUS promoter, triggering the synthesis of the WUSCHEL protein [As it has already been mentioned, the B-type ARR1, ARR10 and ARR12 directly activate the homeobox d twice) ,47,77. A protein .arr1 mutant produced more shoots than the wild type (WT) plants. ARR1-related repression of callus induction and shoot regeneration is connected with the presence of the ARR12 factor. It has been hypothesized that it moderates the expression of WUS and CLV3 genes in ARR12-dependent mode and that it directly initiates INDOLE-3-ACETIC ACID INDUCIBLE17 . The fact that CLV3 expression is repressed by ARR1 suggests that it functions upstream of at least CLAVATA3. The two B-type regulators may affect WUS expression by competitively binding to the same motifs in the WUSCHEL promoter. Apparently, ARR1 counteracts the positive impact of B-ARR12 provoking inhibition of callus creation and shoot development. Thereby, ARR1 may block the regeneration of shoots. The data collected by Liu and co-workers suggest that overexpression of ARR1 slightly enhances shoot induction in the arr1, arr12 double mutant . On the other hand, the WT plants with ARR1 overexpression displayed shoot regeneration inhibition. The function of ARR12 might be altered then. Thus, a B-type network comprises response regulators of various and putatively shifting strength  [As with all B-type elements, ARR1 was usually described as a positive regulator and drew much attention . The afoive one) .WUSCHEL sequence [WUS expression [The activity of ARR10 seems to raise fewer doubts. Similar to the two above-mentioned elements, it functions locally during shoot organogenesis and directly binds to the sequence . Accordisequence  demonstrpression . The anapression ,79. Refepression , the couWUSCHEL in Arabidopsis OC of the plant shoot apical meristem was shown to be controlled by epigenetic modifications  [WUS activation is not observed, which implies the involvement of other factors in WUS repression [WUS expression in the callus tissue [Apart from all the above factors, genetic reprogramming conditioning de novo shoot organogenesis is also the function of chromatin availability. Thus, epigenetic mechanisms represent another level of controlling plant cell regenerative capacity. Since loci of regeneration-related target genes can be epigenetically repressed, there must also be a state of permissive chromatin, leading to the transcriptional activation of shoot meristem formation. Jerzmanowski  distinguistones) . At the pression ,53. Indes tissue ,61,81.Arabidopsis plants, the loss-of-function met1 mutant exhibited accelerated shoot regeneration on a shoot inducing medium with prematurely observed WUSCHEL expression [WUS locus is considered to counteract cytokinin-promoted WUSCHEL activation. A more detailed description of MET1 regulation during shoot regeneration has been provided in a publication by Liu et al. [DNA methylation in plants occurs at CG, CHG and CHH motifs . Compiling evidence shows that decreased levels of DNA methylation encourage de novo shoot formation in planta. The level of methylation is controlled, amongst other things by DNA METHYLOTRASFERASE 1 (MET1), an enzyme that may be modulated by many factors or states/events  . In modepression . MET1-meu et al. .FAS2 in SAM organization is confirmed by the studies on their mutants. Mutations in these genes lead to aberrant shoot apical meristems with an atypical expression of WUS [WUSCHEL locus in a cell cycle-determined way. Such histone marks  sustain the repressive status of different shoot regeneration-related genes , also ensuring positive control of the shoot character and shape maintenance [STM gene is expressed exclusively in the shoot apical meristem and repressed in lateral organ primordia. The expression pattern of STM is thought to be essential for SAM maintenance and its preservation is possible due to the repressive marker H3K27m3 in the STM promoter in primordia structure [Different epigenetic mechanisms also display certain crosstalk having common players. Chromatin remodelers responsible for nucleosome assembly/disassembly events, such as FASCIATA 1 (FAS1), may be directly regulated by the E2F transcription factor . The latn of WUS ,83. Thusn of WUS . On the n of WUS . High amntenance . For exatructure .WUS expression via direct binding to its promoter. Moreover, it limits WUS expression to the organizing center and thereby influences SAM organization and maintenance. Mutations in SYD abolish WUS expression and impair SAM formation [Among other epigenetic controllers, the SWI/SNF complex with the SPLAYED (SYD) and BRAHMA (BRM) factors (being ATP-dependent chromatin remodeling agents) may be found. These factors were demonstrated to be expressed in SAM and draw in shoot development. SPLAYED modulates ormation ,56.Arabidopsis accelerates activation and repression, the expression of target genes binding to their cis-regulatory sequences (both promoters and terminators) [WUS or STM.The SWI/SNF complex is a subfamily of remodeling ATPases, originally purified from yeast, and it seems conserved in different organisms . Members of this family are considered to be epigenetically engaged in plant stem cell initiation and maintenance. Just recently, it has been demonstrated that the SWI/SNF complex in inators) . Thus, cDe novo shoot regeneration is one of the conditions allowing plant adaptation and survival in an unfavorable environment. It is also an essential prerequisite for plant propagation and genetic engineering. The plant life cycle is marked by the presence of stem cells that are capable of self-renewing and conversion into founder cells of a specific tissue. This ability underlies the process of shoot organogenesis and is unquestionably linked with hormonal guidelines. Cytokinins, phytohormones having pleiotropic effects, play a pivotal role in DNSO, which is clearly visible, and their signal transduction becomes more and more elucidated. Using the multistep-phosphorelay, CKs transduce information through membrane-localized histidine kinase receptors to finally activate nuclear B-type response regulators. These transcription factors trigger the cascade of genetic events, regulating both gene players that directly take part in metabolic response and those controlling the second line of transcription factors. This results in an impressive network of connections that crosstalk with multiple different life processes and are regulated at multiple levels (including transcription or epigenesis).The aim of this review was to collect these aspects of cytokinin primary response that are responsible for de novo shoot organogenesis, but it turned out impossible in a way. The isolated analysis of genetic or molecular players responsible only for shoot organogenesis seems rather unlikely to succeed. Nevertheless, a certain fragmentary scheme of the cytokinin-dependent shoot induction has emerged. Most of the available analyses concern indirect organogenesis, explaining the role of callus intermediate and reciprocal auxin—cytokinin pathway connections. It results from the greater availability of indirect protocols. Perhaps more attention has been paid to this pathway resulting in more extensive knowledge, or it is the consequence of greater competence of most plant species to indirect regeneration, namely, it is easier to achieve in vitro. This may lead to the conclusion that, despite the complexity of indirect organogenesis, at a biochemical level it is easier for a plant to perform dedifferentiation to an unorganized state  and then to redifferentiate to a target tissue, than simplifying to shift/reprogram directly one tissue type to another. Perhaps a direct process does not mean a less complex one.Arabidopsis thaliana. Further research led to the identification of several putative orthologues of multistep-phosphorelays in other species . However, the products of the respective genes may not always have an equivalent function, and variations in elements of MSP have been described. Hence, as was mentioned, the extrapolation of molecules involved in cytokinin-dependent shoot regeneration should be careful. In addition, it seems obvious that the universality of the triggering cytokinin concerns mostly the primary response molecules. It should not matter what type of cytokinin is supplemented to the medium. However, there is no detailed research on the system variations that may occur due to the CK used, namely there is no, e.g., zeatin or benzylaminopurine-specific pathway described. The existence of differences can be inferred on the basis of plant morphology, growth rate or the presence of other markers. Many scientists reported variations in the regeneration of plant individuals  after treatment with different cytokinins. It is very likely that they resulted from the activity of molecular players involved in the cytokinin secondary response. However, it requires further analysis to determine to what extent de novo shoot organogenesis and its underlying cytokinin is a universal response, even at the primary response stage.Nevertheless, more thorough knowledge on players of cytokinin signaling mechanism in shoot regeneration may generate more questions about the process, and often requires a revision of the background theory. For example, the current studies clearly indicate that microRNAs are important factors of DNSO gene regulation, but satisfactory data on this subject are still missing from the literature . The res