Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Utilizing Potato Virus X to Monitor RNA Movement.

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

Unique features of the grapevine VvK5.1 channel support novel functions for outward K+ channels in plants.

Grapevine (Vitis vinifera L.), one of the most important fruit crops, is a model plant for studying the physiology of fleshy fruits. Here, we report on the characterization of a new grapevine Shaker-type K+ channel, VvK5.1. Phylogenetic analysis revealed that VvK5.1 belongs to the SKOR-like subfamily. Our functional characterization of VvK5.1 in Xenopus oocytes confirms that it is an outwardly rectifying K+ channel that displays strict K+ selectivity. Gene expression level analyses by real-time quantitative PCR showed that VvK5.1 expression was detected in berries, roots, and flowers. In contrast to its Arabidopsis thaliana counterpart that is involved in K+ secretion in the root pericycle, allowing root to shoot K+ translocation, VvK5.1 expression territory is greatly enlarged. Using in situ hybridization we showed that VvK5.1 is expressed in the phloem and perivascular cells of berries and in flower pistil. In the root, in addition to being expressed in the root pericycle like AtSKOR, a strong expression of VvK5.1 is detected in small cells facing the xylem that are involved in lateral root formation. This fine and selective expression pattern of VvK5.1 at the early stage of lateral root primordia supports a role for outward channels to switch on cell division initiation.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.

The mobile RNAs, StBEL11 and StBEL29, suppress growth of tubers in potato.

KEY MESSAGE: We demonstrate that RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5 in potato. Both these RNAs appear to inhibit tuber growth by repressing the activity of target genes of StBEL5 in potato. Moreover, upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in selective organs. There are thirteen functional BEL1-like genes in potato that encode for a family of transcription factors (TF) ubiquitous in the plant kingdom. These BEL1 TFs work in tandem with KNOTTED1-types to regulate the expression of numerous target genes involved in hormone metabolism and growth processes. One of the StBELs, StBEL5, functions as a long-distance mRNA signal that is transcribed in leaves and moves into roots and stolons to stimulate growth. The two most closely related StBELs to StBEL5 are StBEL11 and -29. Together these three genes make up more than 70% of all StBEL transcripts present throughout the potato plant. They share a number of common features, suggesting they may be co-functional in tuber development. Upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short-days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in specific organs. Using a transgenic approach and heterografting experiments, we show that both these StBELs inhibit growth in correlation with the long distance transport of their mRNAs from leaves to roots and stolons, whereas suppression lines of these two RNAs exhibited enhanced tuber yields. In summary, our results indicate that the RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5. Both these RNAs appear to inhibit growth in tubers by repressing the activity of target genes of StBEL5.

Transcriptome portrait of cellulose-enriched flax fibres at advanced stage of specialization.

Functional specialization of cells is among the most fundamental processes of higher organism ontogenesis. The major obstacle to studying this phenomenon in plants is the difficulty of isolating certain types of cells at defined stages of in planta development for in-depth analysis. A rare opportunity is given by the developed model system of flax (Linum usitatissimum L.) phloem fibres that can be purified from the surrounding tissues at the stage of the tertiary cell wall deposition. The performed comparison of the whole transcriptome profile in isolated fibres and other portions of the flax stem, together with fibre metabolism characterization, helped to elucidate the general picture of the advanced stage of plant cell specialization and to reveal novel participants potentially involved in fibre metabolism regulation and cell wall formation. Down-regulation of all genes encoding proteins involved in xylan and lignin synthesis and up-regulation of genes for the specific set of transcription factors transcribed during tertiary cell wall formation were revealed. The increased abundance of transcripts for several glycosyltransferases indicated the enzymes that may be involved in synthesis of fibre-specific version of rhamnogalacturonan I.

Targets of the StBEL5 Transcription Factor Include the FT Ortholog StSP6A.

The BEL1-like family of transcription factors is ubiquitous in plants and plays important roles in regulating development. They function in tandem with KNOTTED1 types to bind to a double TTGAC motif in the upstream sequence of target genes. StBEL5 of potato (Solanum tuberosum) functions as a mobile RNA signal that is transcribed in leaves, moves down into stolons in response to short days, and induces tuber formation. Despite their importance, however, very little is known about the targets of BEL1-like transcription factors. To better understand this network, we made use of a phloem-mobile BEL5 induction model, an ethanol-inducible system coupled with RNA sequencing analysis, and a screen for tandem TTGAC cis-elements in the upstream sequence to catalog StBEL5 target genes. Induction of StBEL5 activated several genes that are also induced by StSP6A (S. tuberosum SELF-PRUNING 6A), a FLOWERING LOCUS T coregulator that functions as a signal for tuberization. Both enhancement and suppression of StBEL5 expression were also closely linked to StSP6A transcriptional activity. Site mutagenesis in tandem TTGAC motifs located in the upstream sequence of StSP6A suppressed the short day-induced activity of its promoter in both young tubers and leaves. The expression profile of StBEL5 induced in stolons from plants grown under long-day conditions revealed almost 10,000 differentially expressed genes, including important tuber marker genes and genes involved in cell growth, transcription, floral development, and hormone metabolism. In a random screen of 200 differentially expressed targets of StBEL5, 92% contained tandem TTGAC motifs in the upstream sequence within 3 kb of the transcription start site.

Transcriptional analysis of phloem-associated cells of potato.

BACKGROUND: Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species. RESULTS: Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome. CONCLUSIONS: As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

Phloem-specific expression of the lectin gene from Allium sativum confers resistance to the sap-sucker Nilaparvata lugens.

Rice production is severely hampered by insect pests. Garlic lectin gene (ASAL) holds great promise in conferring protection against chewing (lepidopteran) and sap-sucking (homopteran) insect pests. We have developed transgenic rice lines resistant to sap-sucking brown hopper (Nilaparvata lugens) by ectopic expression of ASAL in their phloem tissues. Molecular analyses of T0 lines confirmed stable integration of transgene. T1 lines (NP 1-2, 4-3, 11-6 & 17-7) showed active transcription and translation of ASAL transgene. ELISA revealed ASAL expression was as high as 0.95% of total soluble protein. Insect bioassays on T2 homozygous lines (NP 18 & 32) revealed significant reduction (~74-83%) in survival rate, development and fecundity of brown hoppers in comparison to wild type. Transgenics exhibited enhanced resistance (1-2 score) against brown hoppers, minimal plant damage and no growth penalty or phenotypic abnormalities.

MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena.

MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.

The impact of the long-distance transport of a BEL1-like messenger RNA on development.

BEL1- and KNOTTED1-type proteins are transcription factors from the three-amino-loop-extension superclass that interact in a tandem complex to regulate the expression of target genes. In potato (Solanum tuberosum), StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction. StBEL5 messenger RNA originates in the leaf, and its movement to stolons is induced by a short-day photoperiod. Here, we report the movement of StBEL5 RNA to roots correlated with increased growth, changes in morphology, and accumulation of GA2-oxidase1, YUCCA1a, and ISOPENTENYL TRANSFERASE transcripts. Transcription of StBEL5 in leaves is induced by light but insensitive to photoperiod, whereas in stolon tips growing in the dark, promoter activity is enhanced by short days. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor, binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. The discovery of an inverted tandem motif in the StBEL5 promoter with TTGAC motifs on opposite strands may explain the induction of StBEL5 promoter activity in stolon tips under short days. Using transgenic potato lines, deletion of one of the TTGAC motifs from the StBEL5 promoter results in the reduction of GUS activity in new tubers and roots. Gel-shift assays demonstrate BEL5/POTH1 binding specificity to the motifs present in the StBEL5 promoter and a double tandem motif present in the StGA2-oxidase1 promoter. These results suggest that, in addition to tuberization, the movement of StBEL5 messenger RNA regulates other aspects of vegetative development.

YSL16 is a phloem-localized transporter of the copper-nicotianamine complex that is responsible for copper distribution in rice.

Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with (65)Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport.

Recycling of Solanum sucrose transporters expressed in yeast, tobacco, and in mature phloem sieve elements.

The plant sucrose transporter SUT1 (from Solanum tuberosum, S. lycopersicum, or Zea mays) exhibits redox-dependent dimerization and targeting if heterologously expressed in S. cerevisiae (Krügel et al., 2008). It was also shown that SUT1 is present in motile vesicles when expressed in tobacco cells and that its targeting to the plasma membrane is reversible. StSUT1 is internalized in the presence of brefeldin A (BFA) in yeast, plant cells, and in mature sieve elements as confirmed by immunolocalization. These results were confirmed here and the dynamics of intracellular SUT1 localization were further elucidated. Inhibitor studies revealed that vesicle movement of SUT1 is actin-dependent. BFA-mediated effects might indicate that anterograde vesicle movement is possible even in mature sieve elements, and could involve components of the cytoskeleton that were previously thought to be absent in SEs. Our results are in contradiction to this old dogma of plant physiology and the potential of mature sieve elements should therefore be re-evaluated. In addition, SUT1 internalization was found to be dependent on the plasma membrane lipid composition. SUT1 belongs to the detergent-resistant membrane (DRM) fraction in planta and is targeted to membrane raft-like microdomains when expressed in yeast (Krügel et al., 2008). Here, SUT1-GFP expression in different yeast mutants, which were unable to perform endocytosis and/or raft formation, revealed a strong link between SUT1 raft localization, the sterol composition and membrane potential of the yeast plasma membrane, and the capacity of the SUT1 protein to be internalized by endocytosis. The results provide new insight into the regulation of sucrose transport and the mechanism of endocytosis in plant cells.

Differential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant.

BACKGROUND: Lipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence. RESULTS: Based on amino acid sequence similarity to plant LOXs, a LOX was identified in tea plant and named CsLOX1, which encodes a polypeptide comprising 861 amino acids and has a molecular mass of 97.8 kDa. Heterologous expression in yeast analysis showed that CsLOX1 protein conferred a dual positional specificity since it released both C-9 and C-13 oxidized products in equal proportion and hence was named 9/13-CsLOX1. The purified recombinant CsLOX1 protein exhibited optimum catalytic activity at pH 3.6 and 25°C. Real-time quantitative PCR analysis showed that CsLOX1 transcripts were detected predominantly in flowers, up-regulated during petal senescence, and down-regulated during flower bud opening. In leaves, the gene was up-regulated following injury or when treated with methyl jasmonate (MeJA), but salicylic acid (SA) did not induce such response. The gene was also rapidly and highly induced following feeding by the tea green leafhopper Empoasca vitis, whereas feeding by the tea aphid Toxoptera aurantii resulted in a pattern of alternating induction and suppression. CONCLUSIONS: Analysis of the isolation and expression of the LOX gene in tea plant indicates that the acidic CsLOX1 together with its primary and end products plays an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders.

A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected.

The aim of this study was to investigate the role of the amino acid permease gene AAP6 in regulating phloem amino acid composition and then to determine the effects of this altered diet on aphid performance. A genotype of Arabidopsis thaliana (L.) was produced in which the function of the amino acid permease gene AAP6 (At5g49630) was abolished. Plants homozygous for the insertionally inactivated AAP6 gene had a significantly larger mean rosette width than the wild type and a greater number of cauline leaves. Seeds from the aap6 mutant were also significantly larger than those from the wild-type plants. Sieve element (SE) sap was collected by aphid stylectomy and the amino acids derivatized, separated, and quantified using Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF). In spite of the large variation across samples, the total amino acid concentration of SE sap of the aap6 mutant plants was significantly lower than that of the wild-type plants. The concentrations of lysine, phenylalanine, leucine, and aspartic acid were all significantly lower in concentration in the aap6 mutant plants compared with wild-type plants. This is the first direct demonstration of a physiological role for an amino acid transporter in regulating SE composition in vivo. The amino acid availability in sieve element sap is thought to be the major limiting factor for aphid growth and reproduction. Despite the changes in their diet, the aphid Myzus persicae (Sulzer) displayed only small changes in feeding behaviour on mutant plants when measured using the Electronic Penetration Graph (EPG) technique. Salivation by the aphid into the SE (E1 phase) was increased on mutant plants but there was no significant effect on other feeding EPG behaviours, or in the rate of honeydew production. Consistent with the small effect on aphid feeding behaviour, there was only a small effect of reduced sieve element amino acid concentration on aphid reproduction. The data are discussed in relation to the regulation of phloem composition and the role of phloem amino acids in regulating aphid performance.

Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Accumulation of BEL1-like transcripts in solanaceous species.

Although numerous RNAs have been detected in the phloem, only a few have been confirmed to move long distances. In potato, full-length mRNA of the BEL1-like transcription factor, StBEL5, moves from leaf veins through the phloem to stolon tips to activate tuber formation. BEL1-like transcription factors are ubiquitous in plants and interact with KNOTTED1-types to regulate numerous developmental processes. To explore the range of KNOTTED1- and BEL1-like mRNAs present in phloem, an analysis of the transcript profile of phloem sap was undertaken. Using a modified technique for the collection of phloem-enriched exudate from excised stems, numerous RNAs encoding these transcription factors were detected in the phloem sap from several solanceous species. All seven known BEL1-like RNAs of potato were detected in the phloem-enriched exudates of stem, whereas several stolon-abundant RNAs were not. After refining the technique to minimize the contamination from RNA arising from wounded cells, KNOTTED1-like RNAs were detected in phloem-enriched sap of potato and BEL5 RNA was detected in the sap collected from two closely related nontuber-bearing potato species and tomato. BEL5 RNA was also detected in RNA extracted from leaf veins of tobacco. The detection of these full-length mRNAs from the KNOTTED1- and BEL1-like families in phloem sap indicates that their potential role as long-distance signals seems to be much more extensive than previously known.

Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.