Francesco Di Serio

A survey of nitrate and oxalate content in fresh vegetables

A survey of nitrateONO ˇU and oxalate ((COO ˇ )2) content in fresh vegetables was conducted in Bari (Italy) over 15 months (from March 1994 to May 1995). A total of 327 samples (edible portions and related sub-samples) were taken from 26 different vegetable types on the wholesale fruit and vegetable market. The data revealed that leaf vegetables (namely rocket, celery, parsley and spinach) contained higher levels of nitrate than bulb, root, shoot, inflorescence and tuber vegetables. Higher oxalate levels were found in spinach and Swiss chard. Based on consumption data for the whole population provided by the National Institute of Nutrition, daily nitrate intake from vegetables was calculated to be 71 mg. Over 30% of nitrate intake was derived from the consumption of lettuce and Swiss chard. # 1999 Society of Chemical Industry The presence of nitrate in vegetables, as in water and generally in other food products, is a serious threat to mans health, although recent research has shown that nitrate also has beneficial effects on health related to its role in the bodys mechanism against pathogenic micro-organisms. 3 The harmful effects of nitrate are related not so much to its toxicity, which is low, but to the dangerous compounds that are synthesised in the organism. Indeed, the most serious danger comes from nitrite which is produced by nitrate reduction and which can lead to methaemoglobinemia or form nitrosamines and nitrosamides by reacting with amines and amides, whose carcinogenic action is well known. 4,5

Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction

Background Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21–24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms. Methodology/Principal Findings To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (−) viroid RNAs, with a prevalence of those from the (−) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots. Conclusions/Significance These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.

Small RNAs containing the pathogenic determinant of a chloroplast‐replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus.

ABSTRACT The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

Nitrogen nutrition, yield and quality of spinach

When grown in solution culture spinach plants confirmed the preference toward NO 3 - nutrition and showed heavy toxicity to NH 4 + . In open field condition the highest yield was achieved with the ammonium sulphate in Bari (autumn-winter cycle-110 days) and with calcium nitrate in Policoro (winter-spring cycle-64 days). By increasing N level, yield, nitrates and oxalates leaf content increased. Oxalate content was not affected by nitrogen form. Remarkable differences were observed between leaf petiole and blade in nitrate (4062 vs 925 mg kg -1 of fresh mass) and oxalate (1051 vs 6999 mg kg -1 of fresh mass).

A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development

Peach latent mosaic viroid (PLMVd) is a chloroplast-replicating RNA that propagates in its natural host, peach (Prunus persica), as a complex mixture of variants, some of which are endowed with specific structural and pathogenic properties. This is the case of variant PC-C40, with an insertion of 12 to 13 nucleotides that folds into a hairpin capped by a U-rich loop, which is responsible for an albino-variegated phenotype known as peach calico (PC). We have applied a combination of ultrastructural, biochemical, and molecular approaches to dissect the pathogenic effects of PC-C40. Albino sectors of leaves infected with variant PC-C40 presented palisade cells that did not completely differentiate into a columnar layer and altered plastids with irregular shape and size and with rudimentary thylakoids, resembling proplastids. Furthermore, impaired processing and accumulation of plastid rRNAs and, consequently, of the plastid translation machinery was observed in the albino sectors of leaves infected with variant PC-C40 but not in the adjacent green areas or in leaves infected by mosaic-inducing or latent variants (including PC-C40Δ, in which the 12- to 13-nucleotide insertion was deleted). Protein gel blot and RT-PCR analyses showed that the altered plastids support the import of nucleus-encoded proteins, including a chloroplast RNA polymerase, the transcripts of which were detected. RNA gel blot and in situ hybridizations revealed that PLMVd replicates in the albino leaf sectors and that it can invade the shoot apical meristem and induce alterations in proplastids, bypassing the RNA surveillance system that restricts the entry of a nucleus-replicating viroid and most RNA viruses. Therefore, a non-protein-coding RNA with a specific structural motif can interfere with an early step of the chloroplast developmental program, leading ultimately to an albino-variegated phenotype resembling that of certain variegated mutants in which plastid rRNA maturation is also impaired. Our results highlight the potential of viroids for further dissection of RNA trafficking and pathogenesis in plants.

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.

Viroids: How to infect a host and cause disease without encoding proteins

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the satellite RNA, does not exclude alternative mechanisms.

Peach latent mosaic viroid variants inducing peach calico (extreme chlorosis) contain a characteristic insertion that is responsible for this symptomatology

The involvement of Peach latent mosaic viroid (PLMVd) in an extensive chlorosis of peach known as calico (PC) has been advanced but ultimate proof is lacking. Sequencing of 16 full-length PLMVd cDNA clones of a PC isolate revealed two groups of variants. Nine had a size (336-338 nt) similar to that of typical PLMVd variants of nonsymptomatic and mosaic-inducing isolates, whereas the other 7 were longer (348-351 nt) due to an insertion of 12-13 nt. This insertion was always found in the hairpin loop capping the hammerhead arm, had a limited sequence variability, and folded itself into a hairpin. When three PLMVd dimeric transcripts, two with and the other without the insertion, were slash-inoculated on GF-305 peach seedlings, PC symptoms were produced exclusively by the RNAs containing the insertion, which was conserved in the progeny. These data demonstrate that the agent of PC is PLMVd. Direct support that the 12- to 13-nt insertion contains the PC pathogenicity determinant was obtained by its removal through site-directed mutagenesis from one of the PC-inducing variants. Inoculations with dimeric transcripts of the resulting variant showed that it could replicate but without eliciting symptoms. Our results also suggest that the insertion emerges sporadically de novo.

Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs

As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson–Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures – either global or local – determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.