Arabidopsis leaf-expressed AGAMOUS-LIKE 24 mRNA systemically specifies floral meristem differentiation.

Plants can record external stimuli in mobile mRNAs and systemically deliver them to distal tissues to adjust development. Despite the identification of thousands of mobile mRNAs, the functional relevance of mobile mRNAs remains limited. Many mobile mRNAs are synthesized in the source cells that perceive environmental stimuli, but specifically exert their functions upon transportation to the recipient cells. However, the translation of mobile mRNA-encoded protein in the source cells could locally activate downstream target genes. How plants avoid ectopic functions of mobile mRNAs in the source cells to achieve tissue specificity remains to be elucidated. Here, we show that Arabidopsis AGAMOUS-LIKE 24 (AGL24) is a mobile mRNA whose movement is necessary and sufficient to specify floral organ identity. Although AGL24 mRNA is expressed in vegetative tissues, AGL24 protein exclusively accumulates in the shoot apex. In leaves, AGL24 proteins are degraded to avoid ectopically activating its downstream target genes. Our results reveal how selective protein degradation in source cells provides a strategy to limit the local effects associated with proteins encoded by mobile mRNAs, which ensures that mobile mRNAs specifically trigger systemic responses only in recipient tissues.

Selective Targeting of Mobile mRNAs to Plasmodesmata for Cell-to-Cell Movement.

Many plant mRNAs move from cell to cell or long distance to execute non-cell-autonomous functions. These mobile mRNAs traffic through the phloem to regulate many developmental processes, but despite the burgeoning discovery of mobile mRNAs, little is known about the mechanism underlying the intracellular sorting of these mRNAs. Here, we exploited a fluorescence-based mRNA labeling system, using the bacteriophage coat protein MS2, fused to GFP (MS2-GFP) and an MS2 recognition site in the RNA of interest, to visualize the intracellular trafficking of mobile mRNAs in living plant cells of Nicotiana benthamiana We first improved this system by using the nuclear localization sequence from FD, which substantially reduced the fluorescent background of MS2-GFP in the cytoplasm. The modified system allowed us to observe the cytoplasmic fluorescent foci dependent on MS2-binding sites. Coexpressing the MS2-GFP system with a virus movement protein, which is a plasmodesmata (PD)-localized nonspecific RNA-binding protein, targeted cytoplasmic fluorescent foci to the PD, suggesting that the cytoplasmic fluorescent foci contain mRNA and MS2-GFP. Our ex vivo RNA imaging revealed that mobile but not nonmobile mRNAs were selectively targeted to PD. Real-time images of intracellular translocation revealed that the translocation of mRNA and organelles in the transvacuolar strands may be governed by the same mechanism. Our study suggests that PD targeting of mRNA is a selective step in determining mRNA cell-to-cell movement of mRNAs.

Mobility of Antiflorigen and PEBP mRNAs in Tomato-Tobacco Heterografts.

Photoperiodic floral induction is controlled by the leaf-derived and antagonistic mobile signals florigen and antiflorigen. In response to photoperiodic variations, florigen and antiflorigen are produced in leaves and translocated through phloem to the apex, where they counteract floral initiation. Florigen and antiflorigen are encoded by a pair of homologs belonging to FLOWERING LOCUS T (FT)- or TERMINAL FLOWER1 (TFL1)-like clades in the phosphatidylethanolamine-binding domain protein (PEBP) family. The PEBP gene family contains FT-, TFL1-, and MOTHER OF FT AND TFL1 (MFT)-like clades. Evolutionary analysis suggests that FT- and TFL1-like clades arose from an ancient MFT-like clade. The protein movement of the PEBP family is an evolutionarily conserved mechanism in many plants; however, the mRNA movement of the PEBP family remains controversial. Here, we examined the mRNA movement of PEBP genes in different plant species. We identified a tobacco (Nicotiana sylvestris) CENTRORADIALIS-like1 gene, denoted NsCET1, and showed that NsCET1 is an ortholog of the Arabidopsis (Arabidopsis thaliana) antiflorigen ATC In tobacco, NsCET1 acts as a mobile molecule that non-cell-autonomously inhibits flowering. Grafting experiments showed that endogenous and ectopically expressed NsCET1 mRNAs move long distances in tobacco and Arabidopsis. Heterografts of tobacco and tomato (Solanum lycopersicum) showed that, in addition to NsCET1, multiple members of the FT-, TFL1-, and MFT-like clades of tobacco and tomato PEBP gene families are mobile mRNAs. Our results suggest that the mRNA mobility is a common feature of the three clades of PEBP-like genes among different plant species.

Arabidopsis cyclophilins direct intracellular transport of mobile mRNA via organelle hitchhiking.

Plants convert external cues into mobile mRNAs to synchronize meristematic differentiation with environmental dynamics. These mRNAs are selectively transported to intercellular pores, plasmodesmata (PD), for cell-to-cell movement. However, how plants recognize and deliver mobile mRNAs to PD remains unknown. Here we show that mobile mRNAs hitchhike on organelle trafficking to transport towards PD. Perturbed cytoskeleton organization or organelle trafficking severely disrupts the subcellular distribution of mobile mRNAs. Arabidopsis rotamase cyclophilins (ROCs), which are organelle-localized RNA-binding proteins, specifically bind mobile mRNAs on the surface of organelles to direct intracellular transport. Arabidopsis roc mutants exhibit phenotype alterations and disruptions in the transport of mobile mRNAs. These findings suggest that ROCs play a crucial role in facilitating the systemic delivery of mobile mRNAs. Our results highlight that an RNA-binding protein-mediated hitchhiking system is specifically recruited to orient plant mobile mRNAs for intercellular transport.

Arabidopsis thaliana CENTRORADIALIS homologue (ATC) acts systemically to inhibit floral initiation in Arabidopsis.

Floral initiation is orchestrated by systemic floral activators and inhibitors. This remote-control system may integrate environmental cues to modulate floral initiation. Recently, FLOWERING LOCUS T (FT) was found to be a florigen. However, the identity of systemic floral inhibitor or anti-florigen remains to be elucidated. Here we show that Arabidopsis thaliana CENTRORADIALIS homologue (ATC), an Arabidopsis FT homologue, may act in a non-cell autonomous manner to inhibit floral initiation. Analysis of the ATC null mutant revealed that ATC is a short-day-induced floral inhibitor. Cell type-specific expression showed that companion cells and apex that express ATC are sufficient to inhibit floral initiation. Histochemical analysis showed that the promoter activity of ATC was mainly found in vasculature but under the detection limit in apex, a finding that suggests that ATC may move from the vasculature to the apex to influence flowering. Consistent with this notion, Arabidopsis seedling grafting experiments demonstrated that ATC moved over a long distance and that floral inhibition by ATC is graft transmissible. ATC probably antagonizes FT activity, because both ATC and FT interact with FD and affect the same downstream meristem identity genes APETALA1, in an opposite manner. Thus, photoperiodic variations may trigger functionally opposite FT homologues to systemically influence floral initiation.

Long-distance movement of Arabidopsis FLOWERING LOCUS T RNA participates in systemic floral regulation.

The finding of mRNA acting as a systemic information molecule is one of the most exciting discoveries in recent plant biology. However, evidence demonstrating the functional significance of non-cell autonomous RNA remains limited. Recent analyses of Arabidopsis and rice revealed FLOWERING LOCUS T (FT) protein as a systemic florigenic signal. However, whether the FT RNA also participates in systemic floral regulation remains controversial. By using Arabidopsis cleft-grafting experiments, we showed that the RNA of Arabidopsis FT undergoes long-distance movement from the stock to the scion apex in both FT transformants and non-transformants. In addition, the sequences of FT RNA are sufficient to target a cell-autonomous RNA for long-distance movement. Therefore, FT RNA is a bona fide non-cell autonomous RNA. To examine the systemic action of FT RNA, we uncoupled the movement of FT RNA from protein by fusing FT with RED FLUORESCENT PROTEIN (RFP). When RFP-FT protein was retained in companion cells, the detection of RFP-FT RNA correlates with floral promotion in the scion. Further depletion of the translocated RFP-FT RNA by RNAi or artificial miRNA against FT delayed the floral promotion, indicating that the translocated FT RNA acts as a part of the systemic floral signaling. Our results indicate that both FT RNA and protein move long distance and act redundantly to integrate the photoperiodic signals.

Development of a split fluorescent protein-based RNA live-cell imaging system to visualize mRNA distribution in plants.

BACKGROUND: RNA live-cell imaging systems have been used to visualize subcellular mRNA distribution in living cells. The RNA-binding protein (RBP)-based RNA imaging system exploits specific RBP and the corresponding RNA recognition sequences to indirectly label mRNAs. Co-expression of fluorescent protein-fused RBP and target mRNA conjugated with corresponding RNA recognition sequences allows for visualizing mRNAs by confocal microscopy. To minimize the background fluorescence in the cytosol, the nuclear localization sequence has been used to sequester the RBP not bound to mRNA in the nucleus. However, strong fluorescence in the nucleus may limit the visualization of nucleus-localized RNA and sometimes may interfere in detecting fluorescence signals in the cytosol, especially in cells with low signal-to-noise ratio. RESULTS: We eliminated the background fluorescence in the nucleus by using the split fluorescent protein-based approach. We fused two different RBPs with the N- or C-terminus of split fluorescent proteins (FPs). Co-expression of RBPs with the target mRNA conjugated with the corresponding RNA recognition sequences can bring split FPs together to reconstitute functional FPs for visualizing target mRNAs. We optimized the system with minimal background fluorescence and used the imaging system to visualize mRNAs in living plant cells. CONCLUSIONS: We established a background-free RNA live-cell imaging system that provides a platform to visualize subcellular mRNA distribution in living plant cells.

Non-sterile Grafting Methods for Arabidopsis.

Mobile signals play pivotal roles in coordinating interorgan communication. Grafting provides an effective strategy to identify and explore the movement of the mobile signals. The mutant collection of Arabidopsis offers background-free living materials for examining the transport of mobile signals in vivo. In the past few years, many grafting methods have been developed to overcome the limitations of rosette-type growth and small size in Arabidopsis. Here we describe a non-sterile grafting method involving an insect pin to secure the scion to the rootstock. The scions can be grafted onto epicotyls or hypocotyls of soil-grown Arabidopsis rootstocks at a wide range of developmental stages. This grafting method provides a useful tool to analyze leaf-derived mobile signals in Arabidopsis.

The sequences of Arabidopsis GA-INSENSITIVE RNA constitute the motifs that are necessary and sufficient for RNA long-distance trafficking.

In higher plants a number of physiological processes are regulated by systemic RNA signaling molecules. This phloem-mediated remote-control system provides specific and efficient regulation to fine-tune many plant developmental programs. However, the molecular mechanism underlying long-distance movement of RNA remains to be elucidated. To this end, we examined the long-distance movement of GA-insensitive (GAI) RNA by Arabidopsis inflorescence grafting and RT-PCR analysis. Our results demonstrated that long-distance movement of RNA only occurred in specific transcripts. In addition, the sequences of GAI RNA are necessary and sufficient to target GREEN FLUORESCENT PROTEIN (GFP) RNA for long-distance movement, which indicates that the trafficking of GAI RNA is mediated by specific RNA motifs. Further analyses revealed that the motifs at coding sequences and 3' untranslated regions of GAI RNA play important roles during RNA movement. In addition, the structure of the RNA rather than its specific sequence may also be important in GAI RNA trafficking. However, the secondary structure of GAI RNA is not the only factor to target RNA for long-distance movement, because recovery of the secondary structure of movement-defective GAI RNA only partially rescued RNA movement. Taken together, our results show that long-distance movement of non-cell autonomous RNA operates by specific RNA mobile elements.

In Vivo Imaging of Mobile mRNAs in Plants Using MS2.

Multicellular organisms rely on systemic signals to orchestrate diverse developmental and physiological programs. To transmit environmental stimuli that perceived in the leaves, plants recruit many mobile molecules including mobile mRNAs as systemic signals for interorgan communication. The mobile mRNAs provide an efficient and specific remote control system for plants to cope with environmental dynamics. Upon being transcribed in local tissues, mobile mRNAs are selectively targeted to plasmodesmata for cell-to-cell and long-distance translocation. The mRNA labeling system based on the RNA-binding protein MS2 provides a useful tool to investigate intracellular trafficking of mobile mRNAs in plants. Here we describe the detailed protocol to visualize intracellular trafficking of plant mobile mRNAs by using the MS2 live-cell imaging system.

A pin-fasten grafting method provides a non-sterile and highly efficient method for grafting Arabidopsis at diverse developmental stages.

BACKGROUND: Higher plants have evolved sophisticated communication systems to integrate environmental stimuli into their developmental programs. Grafting provides a powerful technique to examine transportation and systemic effects of mobile molecules. In Arabidopsis, many grafting approaches have been developed to investigate systemic molecules. However, these methods are usually limited to specific developmental stages or require sterilized conditions. To broaden the application of grafting for examining systemic signals at diverse developmental stages, we developed an Arabidopsis pin-fasten grafting method with insect pins used to assemble stocks and scions. RESULTS: We report the step-by-step protocol of Arabidopsis pin-fasten grafting. Arabidopsis wild-type or gl1-1 plants were grown under long- or short-day conditions. Insect pins were inserted into gl1-1 scions at different developmental stages for grafting onto epicotyls or hypocotyls of stocks. Successfully grafted scions with newly developed glabrous leaves were observed at 14 days after grafting. Further longitudinal sections of the graft union showed well-connected vascular tissues between grafted plants. Use of fluorescent phloem-limited dye carboxyfluorescein diacetate in grafted plants demonstrated a symplastic connection established at 6 days after grafting and almost fully developed at 8 days. CONCLUSIONS: Our method provides a simple and robust approach to grafting Arabidopsis at different developmental stages. Sterilized conditions are not required, which greatly improves the success of grafting and plant growth.

Phloem long-distance trafficking of GIBBERELLIC ACID-INSENSITIVE RNA regulates leaf development.

The phloem translocation stream contains a population of RNA molecules, suggesting plants use RNA to integrate developmental processes, at the whole-plant level. In the present study, we analyzed the role of long-distance trafficking in the delivery of transcripts from two members of the GRAS family, namely CmGAIP and GAI. These two homologs were chosen because of their involvement as transcriptional regulators in GA signaling. A combination of pumpkin, tomato and Arabidopsis was employed to examine the processes involved in long-distance delivery, to sink tissues, of RNA for engineered dominant gain-of-function pumpkin (Cmgaip) and Arabidopsis (DeltaDELLA-gai) genes. Our studies demonstrate that gai RNA entry into functional sieve elements occurs via a selective process. Both engineered mutant gai transcripts were able to exit the scion phloem and traffic cell to cell into the shoot apex. Delivery of Cmgaip and DeltaDELLA-gai RNA mediated highly reproducible changes in leaf phenotype in transgenic tomato lines grown under greenhouse conditions. Phenotypic analysis indicated that tomato leaflet morphology was influenced quite late in development. In addition, tissue sink strength did not appear to dictate gai RNA delivery, suggesting complexity in the process underlying macromolecular trafficking. These results establish that the molecular properties of the Cmgaip and DeltaDELLA-gai transcripts are compatible with the tomato cell-to-cell and long-distance macromolecular trafficking systems. An important conclusion, based on our work, is that control over GAI RNA delivery, via the phloem, may be regulated by sequence motifs conserved between plant families. We propose that RNA delivery via the phloem allows for flexibility in fine tuning of developmental programs to ensure newly developing leaves are optimized for performance under the prevailing environmental conditions.

alpha-Amylase is not required for breakdown of transitory starch in Arabidopsis leaves.

The Arabidopsis thaliana genome encodes three alpha-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large alpha-amylase (93.5 kDa) with the C-terminal half showing similarity to other known alpha-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show alpha-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial alpha-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knock-out mutants in the predicted non-plastidial alpha-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that alpha-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice.

Expression of alpha-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. In a transient expression assay, a 105-bp sugar response sequence (SRS) in the promoter of a sugar starvation highly inducible rice alpha-amylase gene, alphaAmy3, was shown previously to confer sugar response and to enhance the activity of the rice Act1 promoter in rice protoplasts. A 230-bp SRS-like sequence was also found in the promoter of another sugar starvation highly inducible rice alpha-amylase gene, alphaAmy8. The alphaAmy8 SRS contains a GA response sequence and was designated as alphaAmy8 SRS/GARS. In the present study, a transgenic approach was employed to characterize the function of the alpha-amylase gene SRSs in rice. We found that the alphaAmy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the alphaAmy8 SRS/GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the alphaAmy8 SRS/GARS is specifically active in rice embryo and endosperm and is subject to sugar repression and GA induction in rice embryos. This sugar repression was found to override GA induction of alphaAmy8 SRS/GARS activity. Our study demonstrates that the alpha-amylase transcriptional enhancers contain cis-acting elements capable of enhancing endogenous expression patterns or activating sugar-sensitive, hormone-responsive, tissue-specific, and developmental stage-dependent expression of promoters in transgenic rice. These enhancers may facilitate the design of highly active and tightly regulated composite promoters for monocot transformation and gene expression. Our study also reveals the existence of cross-talk between the sugar and GA signaling pathways in cereals and provides a system for analyzing the underlying molecular mechanisms involved.