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In the classical formulation of Munch (1930), plasmodesmata are considered to form simple cytoplasmic bridges between neighbouring plant cells to create the symplasm. This concept has dominated, if not monopolized, the thinking of plant biologists and in particular plant physiologists over the last few decades. Recent advances in ultrastructural, physiological and molecular studies on plasmodesmata indicate that this simple view is in need of revision. Structurally, the higher plant plasmodesma has been revealed to be a supramolecular complex consisting of membranes and proteins. Functionally, evidence is at hand that this complex structure appears to have evolved not only to control the size exclusion limit for intercellular diffusion of metabolites and small molecules, but also to potentiate and regulate intercellular trafficking of macromolecules, including proteins and nucleic acids. In this regard, plasmodesmal transport may share parallel regulatory mechanisms with nucleocytoplasmic transport. Based on these findings, we advance the hypothesis that plants function as supracellular, rather than multicellular, organisms. As such, the dynamics of the plant body, including cell differentiation, tissue formation, organogenesis and specialized physiological function(s), is subject to plasmodesmal regulation. Plasmodesmata presumably accomplish such regulatory roles by trafficking informational molecules which orchestrate both metabolic activity and gene expression. Current and future studies on the evolutionary origin(s) of plasmodesmata are likely to provide valuable information in terms of the genetic and molecular basis for the supracellular nature of plants. Contents Summary 435 I. Introduction 436 II. Plasmodesmal formation, structure and biochemistry 436 III. Evolution of plasmodesmata 445 IV. Symplasmic dynamics 452 V. Plasniodesmal trafficking of macromolecules: parallels with nucleocytoplasmic transport 457 VI. Role of plasmodesmata in plant development 464 VII. Concluding remarks 469 Acknowledgements 470 References 470.

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle-derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.

Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure., Author summary Potato spindle tuber viroid (PSTVd) is a small, circular non-coding RNA that systemically infects host plants. All functions required for replication as well as cell-to-cell and systemic spread are mediated by the RNA genome, whose 2D structure consists of multiple base paired stems and loops. However, a wealth of evidence indicates that most loops in RNA molecules are structured by non-canonical base pairs and other interactions. Here, we show that PSTVd loop 27 forms a 3D structure that is essential for replication. Analysis of an exceptional mutant revealed that loop 27 is also required for transit from infected epidermal cells to underlying palisade mesophyll cells, but is not needed for transport in the reverse direction or into other cell types. Thus, loop 27 is necessary for epidermal exit but not entry, and requirements for transit between epidermal cells and palisade mesophyll cells are unique and directional. PSTVd moves between cells through specialized structures known as plasmodesmata. Combined with previous findings, these studies suggest that plasmodesmal gates interconnecting most and perhaps all plant cell types are unique, allowing precise regulation of RNA transport and the establishment of distinct cellular boundaries.

Intercellular RNA trafficking has been shown as a widely-existing phenomenon that has significant functions in many aspects of biology. Viroids, circular noncoding RNAs that cause plant diseases, have been a model to dissect the role of RNA structural motifs in regulating intercellular RNA trafficking in plants. Recent studies on potato spindle tuber viroid (PSTVd) showed that the RNA motif loop 19 is important for PSTVd to spread from palisade to spongy mesophyll in infected leaves. Here, we performed saturated mutational analysis to uncover all possible functional variants of loop 19 and exploit this data to pinpoint to a three-dimensional structural model of this motif. Interestingly, we found that two distinct structural motifs can replace loop 19 and retain the systemic trafficking capacity. One of the alternative structures rapidly emerged from the inoculation using a loop 19 abolished mutant that is not capable of systemic trafficking. Our observation indicates the flexibility of multiple structural arrangements interchangeably exerting similar function at a particular RNA locus. Taken together, this study deepens the understanding of RNA structural motifs-regulated viroid RNA trafficking, which has broad implications for studying RNA intercellular trafficking as well.

Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

Caveolin-1 and flotillin-1 are considered as markers of lipid rafts which can be regarded as sorting platforms for targeted transport of transmembrane proteins and are involved in fundamental cellular events such as signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 90 human hepatocellular carcinoma (HCC) and adjacent noncancerous tissues (ANT) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 overexpression was found in HCC tissues compared to ANT and was confirmed by immunohistochemistry. Raft-associated Akt signaling pathway components involved in the regulation of cell survival were altered by western blotting in HCC microdomain-enriched subcellular fractions purified from paired HCC and ANT samples. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of tissues, which allows for the possibility of tissue-type-specific targeting for cell survival.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) technology are two classical reprogramming methods. Donor cell types can affect the reprogramming results in the above two methods. We here used porcine embryonic fibroblasts (PEFs) and adult porcine ear skin fibroblasts (APEFs) and adipose-derived stem cells (ADSCs) as donor cells for SCNT and source cells for iPSCs to study their in vitro developmental capability and colony-formation efficiency, respectively. For SCNT, fusion and cleavage rate has no significant difference among PEFs, ADSCs and APEFs. The rate and total cell number of blastocysts in the APEF group were significant lower than that in PEFs and ADSCs. For transcription factor-mediated reprogramming, the reprogramming efficiency of ADSCs were significantly higher than PEFs and APEFs and there is no significant difference between PEFs and APEFs. Furthermore, PEFs, APEFs and ADSCs can be used to generate iPSCs. Fianlly, somatic cloned pigs could still be successfully generated from APEFs, suggesting terminally differentiated aging adult somatic cells could be reprogrammed into a totipotent state. Considering the easy availability of animal tissue and the costs of establishing cell lines, aging porcine ear fibroblasts can support nuclear transfer-mediated and transcription factor-based reprogramming.

WD repeat-containing protein 5 (WDR5), a member of conserved WD40 protein family, is an essential component of the mixed lineage leukemia (MLL) complexes, which are crucial for numerous key biological processes including methylation of histone H3 lysine 4 (H3K4), self-renewal of embryonic stem cells, and formation of induced pluripotent stem cells. The expression pattern and functional role of WDR5 during porcine preimplantation embryonic development, however, remain unknown. Our results showed that the transcripts and protein of WDR5 exhibited stage-specific expression pattern in porcine early embryos. Moreover, blastocyst rate and total cell number per blastocyst were reduced by RNAi-mediated silencing of WDR5 or pharmacological inhibition of WDR5. Knockdown of WDR5 also disturbed the expression of several pluripotency genes. Interestingly, tri-methylation of H3K4 (H3K4me3) level was dramatically increased by WDR5 depletion. Further analysis revealed that loss of MLL3 phenocopied WDR5 knockdown, triggering increased H3K4me3 level. Simultaneously, WDR5 depletion significantly decreased the levels of histone H4 lysine 16 acetylation (H4K16ac) and its writer males absent on the first (MOF). Last but not least, WDR5 knockdown induced DNA damage and DNA repair defects during porcine preimplantation development. Taken together, results of described studies establish that WDR5 plays a significant role in porcine preimplantation embryos probably through regulating key epigenetic modifications and genome integrity.

Porcine viral diarrhea is an acute and highly contagious enteric disease of pigs that causes huge economic losses worldwide. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are the main pathogens responsible for piglet viral diarrhea. However, currently there is no specific drug available for the effective treatment of viral diarrhea. Therefore, it is necessary to seek an effective method to diminish PEDV and TGEV infection rates. RNA interference has been applied successfully to inhibit the virus replication. It provides a potential strategy for breeding resistant pigs. In this study, four promoters and four short hairpin RNA (shRNA) vectors with LoxP sites at each end of the selectable marker genes were constructed to target PEDV and TGEV. These vectors were then transfected into porcine fetal fibroblasts, G418 resistant transfectants were confirmed by PCR and transgenic SCNT porcine blastocysts were obtained. These results have paved the way for future production of marker-free transgenic resistant to PEDV and TEGV pigs by SCNT.

Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from0.5-mm follicles lacked the ability to resume meiosis. More than 90% of these oocytes remained in the germinal vesicle (GV) stage after in vitro culture (IVC) for 18 h. Oocytes isolated from0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study.

The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis., Author Summary Auxin regulates plant growth and development through auxin signaling, which begins with the interaction of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein co-receptor. Auxin binding to the co-receptor complex triggers ubiquitination and 26S proteasome degradation of Aux/IAA proteins, leading to a downstream signaling cascade that induces the expression of auxin-responsive genes. Auxin signaling is manipulated by plant pathogens to maximize their own multiplication, but the underlying mechanisms are poorly understood. Here we report that the P2 capsid protein encoded by Rice dwarf virus (RDV) sabotages auxin signaling by interacting with the rice Aux/IAA protein, OsIAA10, thereby shielding it from degradation and causing infected plants to display typical RDV symptoms including dwarfism, excessive tillering and stunted crown roots. Importantly, these symptoms are phenocopied by transgenic rice plants overexpressing OsIAA10 or its degradation-resistant mutant. Conversely, down-regulating OsIAA10 expression in rice led to milder RDV infection. Together these findings reveal a novel mechanism by which RDV reprograms auxin signaling, leading to enhanced viral infection.

MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca2+-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca2+-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species.

To establish systemic infection, plant viruses must be localized to the correct subcellular sites to accomplish replication and then traffic from initially infected cells into neighboring cells and even distant organs. Viruses have evolved various strategies to interact with pre-existing cellular factors to achieve these functions. In this review we discuss plant virus intracellular, intercellular and long-distance movement, focusing on the host cellular factors involved. We emphasize that elucidating viral movement mechanisms will not only shed light on the molecular mechanisms of infection, but will also contribute valuable insights into the regulation of endogenous macromolecular trafficking.

Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 5′-CGA-3′...5′-GAC-3′ flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.

Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, cellular antiviral response, and cell-to-cell communication. The mechanisms regulating this trafficking remain to be understood. A testable hypothesis is that many RNAs have distinct structural motifs to direct trafficking across various cellular boundaries. The identification of structural motifs in a noncoding viroid RNA required for trafficking between specific cell types in a plant provides compelling genetic evidence to support this hypothesis. Here we discuss the unique features of viroids as well as experimental data to demonstrate these RNAs as simple and highly tractable models to elucidate the general mechanisms of RNA trafficking.

There is now solid evidence that cell-to-cell trafficking of certain proteins and RNAs plays a critical role in trans-cellular regulation of gene expression to coordinate cellular differentiation and development. Such trafficking also is critical for viral infection and plant defense. The mechanisms of trafficking remain poorly understood. Although some proteins may move between cells by diffusion, many proteins and RNAs move in a highly regulated fashion. Regulation is likely achieved through interactions between distinct protein or RNA motifs and cellular factors. Some motifs and factors have been identified. One of the major focuses for future studies is to identify all motifs and their cognate factors and further elucidate their roles in trafficking between specific cells. With increasing information from such studies, we should be able to develop an understanding of the mechanisms that regulate trafficking of various proteins and RNAs across all and specific cellular boundaries. On the basis of such mechanistic knowledge, we can further investigate how the trafficking machinery has evolved to regulate developmental and physiological processes in a plant, how pathogens have co-evolved to use this machinery for systemic spread in a plant, and how plants use this machinery for counter- defense.

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid . We present our findings and discuss their biological implications.

Background Secondary, phased small interfering RNAs (phasiRNAs) derived from protein-coding or noncoding loci (PHAS) are emerging as a new type of regulators of gene expression in plants. However, the evolution and function of these novel siRNAs in plant species remain largely unexplored. Results We systematically analyzed PHAS loci in 23 plant species covering major phylogenetic groups spanning alga, moss, gymnosperm, basal angiosperm, monocot, and dicot. We identified over 3,300 PHAS loci, among which ~1,600 were protein-coding genes. Most of these PHAS loci were novel and clade- or species-specific and showed distinct expression patterns in association with particular development stages, viral infection, or abiotic stresses. Unexpectedly, numerous PHAS loci produced phasiRNAs from introns or exon–intron junction regions. Our comprehensive analysis suggests that phasiRNAs predominantly regulate protein-coding genes from which they are derived and genes from the same families of the phasiRNA-deriving genes, in contrast to the dominant trans-regulatory mode of miRNAs. The stochastic occurrence of many PHAS loci in the plant kingdom suggests their young evolutionary origins. Conclusions Our study discovered an unprecedented diversity of protein-coding genes that produce phasiRNAs in a wide variety of plants, and set a kingdom-wide foundation for investigating the novel roles of phasiRNAs in shaping phenotype diversities of plants.

As a core member of polycomb repressive complex 2, the transcription and enzyme activity of enhancer of zeste homolog 2 (Ezh2) is directly involved in the trimethylation of lysine 27 on histone H3. In this study, the fluorescence intensity of H3K27me3 in mouse in vivo morulae and blastocysts was compared by indirect immunofluorescence staining. We found that demethylation of H3K27me3 occurred during the blastocyst stage. Real-time polymerase chain reaction was performed to investigate Ezh2 expression in oocytes and in preimplantation embryos. Ezh2 expression peaked during the zygote stage and gradually decreased from the 2-cell stage, exhibiting an inverse pattern when compared with Oct4 and Sox2 mRNA in mouse preimplantation embryos. To understand the role of development-related genes on the transcription of mouse Ezh2, a promoter assay was performed in NIH/3T3 cells. Ezh2 expression was markedly suppressed by Oct4 and Sox2 alone in a dose-dependent manner, while Ezh2 promoter activity in co-transfection with Nanog, Klf-4, and c-Myc groups showed no significant change as compared with the control. Our data suggest that the demethylation of H3K27me3 is caused by the degressive expression and activity of Ezh2 in blastocysts, leading to increased expression of developmentally important transcription factors. We also observed negative effects of Oct4 and Sox2 on the transcription of Ezh2 and identified Oct4 and Sox2 as novel negative regulators of Ezh2 at the post-translation level in a mouse preimplantation embryo.

The plasmodesmata and phloem form a symplasmic network that mediates direct cell–cell communication and transport throughout a plant. Selected endogenous RNAs, viral RNAs, and viroids traffic between specific cells or organs via this network. Whether an RNA itself has structural motifs to potentiate trafficking is not well understood. We have used mutational analysis to identify a motif that the noncoding Potato spindle tuber viroid RNA evolved to potentiate its efficient trafficking from the bundle sheath into mesophyll that is vital to establishing systemic infection in tobacco (Nicotiana tabacum). Surprisingly, this motif is not necessary for trafficking in the reverse direction (i.e., from the mesophyll to bundle sheath). It is not required for trafficking between other cell types either. We also found that the requirement for this motif to mediate bundle sheath-to-mesophyll trafficking is dependent on leaf developmental stages. Our results provide genetic evidence that (1) RNA structural motifs can play a direct role in mediating trafficking across a cellular boundary in a defined direction, (2) the bundle sheath–mesophyll boundary serves as a novel regulatory point for RNA trafficking between the phloem and nonvascular tissues, and (3) the symplasmic network remodels its capacity to traffic RNAs during plant development. These findings may help further studies to elucidate the interactions between RNA motifs and cellular factors that potentiate directional trafficking across specific cellular boundaries.

The wide variety of RNAs produced in the nucleus must be localized correctly to perform their functions. However, the mechanism of this localization is poorly understood. We report here the differential subnuclear localization of RNA strands of opposite polarity derived from the replicating Potato spindle tuber viroid (PSTVd). During replication, (+)- and (−)-strand viroid RNAs are produced. We found that in infected cultured cells and plants, the (−)-strand RNA was localized in the nucleoplasm, whereas the (+)-strand RNA was localized in the nucleolus as well as in the nucleoplasm with distinct spatial patterns. Furthermore, the presence of the (+)-PSTVd in the nucleolus caused the redistribution of a small nucleolar RNA. Our results support a model in which (1) the synthesis of the (−)- and (+)-strands of PSTVd RNAs occurs in the nucleoplasm, (2) the (−)-strand RNA is anchored in the nucleoplasm, and (3) the (+)-strand RNA is transported selectively into the nucleolus. Our results imply that the eukaryotic cell has a machinery that recognizes and localizes the opposite strands of an RNA, which may have broad ramifications in the RNA regulation of gene expression and the infection cycle of pathogenic RNAs and in the development of RNA-based methods to control gene expression as well as pathogen infection.

Viroids are small, nontranslatable pathogenic RNAs that replicate autonomously and traffic systemically in their host plants. We have used in situ hybridization to analyze the trafficking pattern of Potato spindle tuber viroid (PSTVd) in tomato and Nicotiana benthamiana. When PSTVd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. PSTVd was absent from shoot apical meristems. In the flowers of infected plants, PSTVd was present in the sepals, but was absent in the petals, stamens, and ovary. The replicative form of PSTVd was detected in the phloem. Our data demonstrate that (i) PSTVd traffics long distance in the phloem and this trafficking is likely sustained by replication of the viroid in the phloem, and (ii) PSTVd trafficking is governed by plant developmental and cellular factors. The dependency of PSTVd and other viroids on cellular mechanisms for RNA trafficking makes them excellent tools to study such mechanisms.

Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.

Summary Most viroids replicate in the nuclei of infected plant cells. Nuclear import of the incoming RNA thus represents a key control point for establishment of a systemic infection. However, little is known about the mechanisms by which viroids are transported into the nucleus. We have characterized nuclear import of infectious, fluorescein-labeled potato spindle tuber viroid (F-PSTVd) in permeabilized tobacco BY2 cells. Import was observed for F-PSTVd but not for mRNA fragments of the same size or two viroids believed to replicate in the chloroplasts. Import of F-PSTVd was inhibited by addition of a 10-fold excess of non-fluorescent PSTVd but not by similar amounts of control RNAs. Import was not inhibited by pre-incubation with GTP-γ-S or GDP-β-S, however. Disruption of microtubules and actin filaments with oryzalin or cytochalasin D did not inhibit F-PSTVd import. Taken together, our results indicate that (i) PSTVd possesses a sequence and/or structural motif for nuclear import and (ii) the import is a cytoskeleton-independent process that is mediated by a specific and saturable receptor. Insensitivity to GTP-γ-S and GDP-β-S treatment suggests that PSTVd import is not coupled to Ran GTPase cycle, which mediates nuclear transport of many proteins and nucleic acids. To our knowledge, our studies are the first to examine the mechanisms of nuclear transport of RNA in plants.

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.

During plant morphogenesis, groups of cells differentiate to form specialized tissues possessing distinct structures and functions. Cell specialization is a result of specific gene expression at the individual cell level. Coordination of differential gene expression among cells requires that cells communicate with one another. Plasmodesmata provide a cytoplasmic pathway for direct intercellular communication. Recent discoveries that macromolecules such as transcription factors, viral proteins, and plant defense-related proteins can traffic through plasmodesmata suggest that intercellular protein trafficking is potentially an important means to regulate plant developmental processes, physiological functions, plant-pathogen interactions, and plant defense reactions. Thus, elucidating the specific functions and mechanisms of intercellular protein trafficking has broad implications in understanding how a plant develops and functions at the molecular level. This review is to provide an update on this rapidly developing area of plant biology, with emphasis on the discussion of possible mechanisms underlying intercellular protein trafficking.

Summary Previous micro-injection studies showed that some recombinant viral movement proteins and plant proteins produced in and purified from Escherichia coli could traffic from cell to cell. However, the relevance of these findings obtained by micro-injecting proteins produced in E. coli to the real functions of these proteins when produced in planta has been questioned. In this study, specific gene constructs were delivered by biolistic bombardment into tobacco (Nicotiana tabacum var Samsun) leaf epidermis for in planta production of the green fluorescent protein (GFP) and various fusions between the cucumber mosaic virus 3a movement protein (3a MP) and GFP. Free GFP remained in cells producing it. In contrast, 3a MP:GFP fusion protein moved from approximately half of the cells producing it into neighboring cells. The movement also occurred at 4°C. A mutant 3a MP:GFP was incapable of cell-to-cell movement in all cases. A 3a MP:GUS fusion protein produced in this manner also moved from cell to cell. Our data provide direct evidence that specific viral proteins produced in planta can be transported between cells. Furthermore, our data suggest that the CMV 3a MP contains a signal for transport. Our approach is simple and efficient and has many potential applications in studying plasmodesma-mediated macromolecular transport.

Summary The role of actin filaments in regulating plasmodesmal transport has been studied by microinjection experiments in mesophyll cells of tobacco (Nicotiana tabacum L. cv. Samsun). When fluorescent dextrans of various molecular sizes were each co-injected with specific actin filament perturbants cytochalasin D (CD) or profilin into these cells, dextrans up to 20 kilodalton (kDa) moved from the injected cell into surrounding cells within 3–5 min. In contrast, when such dextrans were injected alone or co-injected with phalloidin into the mesophyll cells, they remained in the injected cells. Phalloidin co-injection slowed down or even inhibited CD- or profilin-elicited dextran cell-to-cell movement. Dextrans of 40 kDa or larger were unable to move out of the injected cell in the presence of CD or profilin. These data suggest that actin filaments may participate in the regulation of plasmodesmal transport by controlling the permeability of plasmodesmata.

Plasmodesmata are intercellular organelles in plants that establish cytoplasmic continuity between neighboring cells. Microinjection studies showed that plasmodesmata facilitate the cell-to-cell transport of a plant-encoded transcription factor, KNOTTED1 (KN1). KN1 can also mediate the selective plasmodesmal trafficking of kn1 sense RNA. The emerging picture of plant development suggests that cell fate is determined at least in part by supracellular controls responding to cellular position as well as lineage. One of the mechanisms that enables the necessary intercellular communication appears to involve transfer of informational molecules (proteins and RNA) through plasmodesmata.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.

The substructure of plasmodesmata in freeze-substituted tissues of developing leaves of the tobacco plant (Nicotiana tabacum L. var. Maryland Mammoth) was studied by high resolution electron microscopy and computer image enhancement techniques. Both the desmotubule wall and the inner leaflet of the plasmodesmatal plasma membrane are composed of regularly spaced electron-dense particles approximately 3 nm in diameter, presumably proteinaceous and embedded in lipid. The central rod of the desmotubule is also particulate. In plasmodesmata with central cavities, spoke-like extensions are present between the desmotubule and the plasma membrane in the central cavity region. The space between the desmotubule and the plasma membrane appears to be the major pathway for intercellular transport through plasmodesmata. This pathway may be tortuous and its dimensions could be regulated by interactions between desmotubule and plasma membrane particles.

The organization and distribution of microfilaments in freze substituted leaf tissues and root tips of tobacco plants (Nicotiana tabacum L. var. Maryland Mammoth) were investigated in detail. Three categories of microfilaments were recognized in interphase cells of all tissues including those in the root cap: (1) microfilament bundles; (2) single microfilaments; (3) cortical microtubuleassociated microfilaments. While the microfilament bundles appeared to be distributed throughout the cytoplasm, the single microfilaments were mainly confined to the cell periphery. All three categories of microfilaments were associated with various organelles. Our study indicates that the three categories of microfilaments are normal cytoskeletal components in higher plant cells. The implications of these findings are discussed.

RNA replication and systemic trafficking play significant roles in developmental regulation and host–pathogen interactions. Viroids are the simplest noncoding eukaryotic RNA pathogens and genetic units that are capable of autonomous replication and systemic trafficking and offer excellent models to investigate the role of RNA structures in these processes. Like other RNAs, the predicted secondary structure of a viroid RNA contains many loops and bulges flanked by double-stranded helices, the biological functions of which are mostly unknown. Using Potato spindle tuber viroid infection of Nicotiana benthamiana as the experimental system, we tested the hypothesis that these loops/bulges are functional motifs that regulate replication in single cells or trafficking in a plant. Through a genome-wide mutational analysis, we identified multiple loops/bulges essential or important for each of these biological processes. Our results led to a genomic map of viroid RNA motifs that mediate single-cell replication and systemic trafficking, respectively. This map provides a framework to enable high-throughput studies on the tertiary structures and functional mechanisms of RNA motifs that regulate viroid replication and trafficking. Our model and approach should also be valuable for comprehensive investigations of the replication and trafficking motifs in other RNAs.

Viroids are small, noncoding and nonencapsidated RNAs that infect plants. To establish systemic infection, viroid genomes or their derivatives must interact directly with cellular factors. There is increasing evidence that subcellular localization and systemic trafficking of viroid RNAs are regulated, likely via interactions between viroid RNA cis elements and specific cellular proteins. Here we summarize recent progress on the characterization of viroid structures and host proteins that may play important roles in trafficking. We also discuss critical issues that need to be addressed in future investigations.

RNA trafficking has broad implications in the systemic spread of infectious agents, plant defense, and the systemic regulation of gene expression. The mechanisms that regulate trafficking remain poorly understood. The non-coding, infectious viroid RNAs are emerging as highly tractable model systems for the investigation of the basic mechanisms of RNA trafficking. Recent studies on viroids have led to new insights into the direct role of RNAs in intracellular and systemic trafficking, and to the identification of cellular proteins that might play a role in RNA trafficking. Here, we discuss these areas of progress, emphasizing on the unifying principles that control the trafficking of viroid, viral and endogenous RNAs.

Plasmodesmata and the phloem form a cytoplasmic network that permits direct cell-cell communication in plants. This network can mediate the trafficking of selective proteins and RNAs that may have important developmental functions. Recent work has provided evidence that protein and RNA traffic across specific interfaces of this network is regulated in a distinct manner. Progress has been made in identifying potential cellular factors that confer such regulation. These advances should promote further investigations into the mechanisms and functions of protein and RNA traffic using biochemical, cellular, genetic and molecular tools.

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.

Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamianayielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs.

Intercellular communication is essential for differentiation and development. In plants, plasmodesmata (PD) form cytoplasmic channels for direct communication. During plant development, programmed reduction in PD number and transport capacity creates the so-called symplasmic domains. Small fluorescent dyes and ions can diffuse among cells within a domain but not across domain boundaries. Such symplasmic isolation is thought to allow groups of cells to differentiate and develop into tissues with distinct structures and functions. Whether or how "symplasmically isolated" cells communicate with one another is poorly understood. One well-documented symplasmic domain is the sieve element-companion cell (SE-CC) complex in the phloem tissue. We report here that, when produced in the CC of transgenic tobacco, the 3a movement protein (3a MP) of Cucumber mosaic virus fused to green fluorescent protein (GFP) can traffic out of the SE-CC complex via PD. The extent of 3a MP:GFP traffic across the boundary between vascular and nonvascular tissues depends on organ type and developmental stage. Our findings provide experimental evidence that endogenous machinery exists for protein traffic between the symplasmically isolated SE-CC complex and neighboring cells. We suggest that PD-mediated traffic of selected macromolecules can be a mechanism for symplasmically isolated cells to communicate with one another.

The organization and distribution of microfilaments (MFs) in the preprophase bands (PPBs) of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) root tip cells were studied with high pressure freezing and freeze-substitution methods. MFs were present predominantly as single filaments interspersed among microtubules (MTs) throughout the PPB. Some MFs appeared to be associated with MTs, whereas others were not. This is the first time that MFs have been demonstrated in the PPB at the electron microscope level.

Plasmodesmata provide a cytoplasmic pathway for direct cell-to-cell communication in plants. They are highly dynamic in biogenesis and structural modifications, organize cells into symplasmic domains that serve as basic developmental units in a plant, and mediate intercellular trafficking of proteins and nucleic acids as well as small molecules. Intercellular macromolecular trafficking appears to be a significant mechanism of regulating gene expression and physiological functions among neighboring cells and between distant organs. Current data suggest that intercellular macromolecular trafficking is also likely a general function in animals. Evolutionarily, intercellular macromolecular trafficking may be a primitive rather than an advanced biological function.

Viroids are non-translatable, autonomously replicating circular RNAs that infect only plants. An important component of the viroid infection process is cell-to-cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. In this study, potato spindle tuber viroid (PSTVd) has been used as a model system to investigate the mechanism of viroid cell-to-cell transport. Infectious RNA transcripts were produced from PSTVd cDNA clones in vitro, labeled with the nucleotide-specific fluorescent dye TOTO-1 iodide, and used for micro-injection. When injected into symplasmically isolated guard cells of mature tomato and tobacco leaves, PSTVd remained in the injected cells; in contrast, PSTVd injected into symplasmically connected mesophyll cells moved rapidly from cell to cell. A 1400 nt RNA containing only vector sequences was unable to move out of the injected mesophyll cells, but when PSTVd was fused to this transcript, the fusion RNA moved from cell to cell. At the DNA level, PSTVd cDNA also appears able to mediate cell-to-cell movement of plasmid DNA. These data indicate that (i) PSTVd moves from cell to cell via plasmodesmata, and (ii) this movement may be mediated by a specific sequence or structural motif.

Cell-to-cell communication is vital to the growth and development of multicellularly structured organisms. Recent studies have demonstrated that plants have an endogenous machinery to transport macromolecules such as proteins and nucleic acids between cells through plasmodesmata. Such transport may be a novel means of cell-to-cell signalling. Here, Biao Ding discusses the mechanisms underlying this newly discovered biological function.

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.

Mature leaves of a transgenic tobacco plant (Nicotiana tabacum L var. Samsun, line A41-10) that constitutively express a yeast-derived acid invertase gene develop symptoms which are characterized by the presence of greenish-yellow and green sectors in the same leaf, and onset of early, leaf senescence. Previous studies indicated that invertase activity was two- to threefold higher in the greenish-yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish-yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase-expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.