Emerging roles of plant transcriptional gene silencing under heat.

Plants continuously endure unpredictable environmental fluctuations that upset their physiology, with stressful conditions negatively impacting yield and survival. As a contemporary threat of rapid progression, global warming has become one of the most menacing ecological challenges. Thus, understanding how plants integrate and respond to elevated temperatures is crucial for ensuring future crop productivity and furthering our knowledge of historical environmental acclimation and adaptation. While the canonical heat-shock response and thermomorphogenesis have been extensively studied, evidence increasingly highlights the critical role of regulatory epigenetic mechanisms. Among these, the involvement under heat of heterochromatic suppression mediated by transcriptional gene silencing (TGS) remains the least understood. TGS refers to a multilayered metabolic machinery largely responsible for the epigenetic silencing of invasive parasitic nucleic acids and the maintenance of parental imprints. Its molecular effectors include DNA methylation, histone variants and their post-translational modifications, and chromatin packing and remodeling. This work focuses on both established and emerging insights into the contribution of TGS to the physiology of plants under stressful high temperatures. We summarized potential roles of constitutive and facultative heterochromatin as well as the most impactful regulatory genes, highlighting events where the loss of epigenetic suppression has not yet been associated with corresponding changes in epigenetic marks.

Transcriptomic and physiological shade avoidance responses in potato (Solanum tuberosum) plants.

Plants detect competitors in shaded environments by perceiving a reduction in photosynthetically active radiation (PAR) and the reduction between the red and far-red light (R:FR) ratio and blue photons. These light signals are detected by phytochromes and cryptochromes, which trigger shade avoidance responses such as shoot and petiole elongation and lead to increased susceptibility to pathogen attack. We studied morphological, anatomical, and photosynthesis differences in potato plants (Solanum tuberosum var. Spunta) exposed to sunlight or simulated shade in a greenhouse. We found that simulated shade strongly induced stem and internode elongation with a higher production of free auxin in stems and a lower production of tubers. The mesophyll thickness of the upper leaves of plants grown in simulated shade was lower, but the epidermis was wider compared with the leaves of plants cultivated in sunlight. In addition, the photosynthesis rate was lower in the upper leaves exposed to nonsaturated irradiances and higher in the basal leaves at saturated irradiances compared with control plants. RNA-seq analysis showed that 146 and 155 genes were up- and downregulated by shade, respectively. By quantitative reverse transcription polymerase chain reaction, we confirmed that FLOWERING LOCUS T (FT), WRKY-like, and PAR1b were induced, while FLAVONOL 4-SULFOTRANSFERASE was repressed under shade. In shaded plants, leaves and tubers were more susceptible to the necrotrophic fungus Botrytis cinerea attack. Overall, our work demonstrates configurational changes between growth and defense decisions in potato plants cultivated in simulated shade.

Rearranged Endogenized Plant Pararetroviruses as Evidence of Heritable RNA-based Immunity.

Eukaryotic genomics frequently revealed historical spontaneous endogenization events of external invading nucleic acids, such as viral elements. In plants, an extensive occurrence of endogenous plant pararetroviruses (EPRVs) is usually believed to endow hosts with an additional layer of internal suppressive weaponry. However, an actual demonstration of this activity remains speculative. We analyzed the EPRV component and accompanying silencing effectors of Solanum lycopersicum, documenting that intronic/intergenic pararetroviral integrations bearing inverted-repeats fuel the plant's RNA-based immune system with suitable transcripts capable of evoking a silencing response. A surprisingly small set of rearrangements explained a substantial fraction of pararetroviral-derived endogenous small-interfering (si)RNAs, enriched in 22-nt forms typically associated with anti-viral post-transcriptional gene silencing. We provide preliminary evidence that such genetic and immunological signals may be found in other species outside the genus Solanum. Based on molecular dating, bioinformatics, and empirical explorations, we propose that homology-dependent silencing emerging from particular immuno-competent rearranged chromosomal areas that constitute an adaptive heritable trans-acting record of past infections, with potential impact against the unlocking of plant latent EPRVs and cognate-free pararetroviruses.

Functional analysis of PHYB polymorphisms in Arabidopsis thaliana collected in Patagonia.

Arabidopsis thaliana shows a wide range of natural genetic variation in light responses. Shade avoidance syndrome is a strategy of major adaptive significance that includes seed germination, elongation of vegetative structures, leaf hyponasty, and acceleration of flowering. Previously, we found that the southernmost Arabidopsis accession, collected in the south of Patagonia (Pat), is hyposensitive to light and displays a reduced response to shade light. This work aimed to explore the genetic basis of the shade avoidance response (SAR) for hypocotyl growth by QTL mapping in a recently developed 162 RIL population between Col-0 and Pat. We mapped four QTL for seedling hypocotyl growth: WL1 and WL2 QTL in white light, SHADE1 QTL in shade light, and SAR1 QTL for the SAR. PHYB is the strongest candidate gene for SAR1 QTL. Here we studied the function of two polymorphic indels in the promoter region, a GGGR deletion, and three non-synonymous polymorphisms on the PHYB coding region compared with the Col-0 reference genome. To decipher the contribution and relevance of each PHYB-Pat polymorphism, we constructed transgenic lines with single or double polymorphisms by using Col-0 as a reference genome. We found that single polymorphisms in the coding region of PHYB have discrete functions in seed germination, seedling development, and shade avoidance response. These results suggest distinct functions for each PHYB polymorphism to the adjustment of plant development to variable light conditions.

Dual role of specific promoter tandem repeats integrating epigenetic silencing with heat response.

Satellites are ubiquitous noncoding tandemly repeated sequences, yet knowledge about their biological relevance is still scarce. In plants, the few described cases point to roles in heterochromatin biology and gene regulation; however, a direct link to plant stress responses is yet to be uncovered. We present evidence that particular non-centromere tandem repeats may display a central regulatory role in the intersection between epigenetic silencing and gene expression in dynamic environments. Within the projected promoter of Arabidopsis thaliana's imprinted SDC locus, a transcriptional gene silencing targeted tandem-repeated area largely mediates epigenetic suppression and imprinting. Here, we show that this area, possibly acting as a cis-element/enhancer, appears necessary and sufficient for SDC's heat transcriptional activity in vegetative tissues. Our results indicate that these particular noncoding tandem repeats may be genic and exhibit dual roles, not only as silencers at normal temperatures but also facilitating expression upon stress. An unusual adaptive form of abiotic transcriptional control unrelated to canonical heat signaling is implied, emphasizing a potential importance of genomic satellites for plant environmental epigenetics.

Ultra-High-Density QTL Marker Mapping for Seedling Photomorphogenesis Mediating Arabidopsis Establishment in Southern Patagonia.

Arabidopsis thaliana shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, Arabidopsis has a high potential for the identification of genes underlying ecologically important complex traits, thus providing new insights on genome evolution. Previous research suggested that distinct light responses were crucial for Arabidopsis establishment in a peculiar ecological niche of southern Patagonia. The aim of this study was to explore the genetic basis of contrasting light-associated physiological traits that may have mediated the rapid adaptation to this new environment. From a biparental cross between the photomorphogenic contrasting accessions Patagonia (Pat) and Columbia (Col-0), we generated a novel recombinant inbred line (RIL) population, which was entirely next-generation sequenced to achieve ultra-high-density saturating molecular markers resulting in supreme mapping sensitivity. We validated the quality of the RIL population by quantitative trait loci (QTL) mapping for seedling de-etiolation, finding seven QTLs for hypocotyl length in the dark and continuous blue light (Bc), continuous red light (Rc), and continuous far-red light (FRc). The most relevant QTLs, Rc1 and Bc1, were mapped close together to chromosome V; the former for Rc and Rc/dark, and the latter for Bc, FRc, and dark treatments. The additive effects of both QTLs were confirmed by independent heterogeneous inbred families (HIFs), and we explored TZP and ABA1 as potential candidate genes for Rc1 and Bc1QTLs, respectively. We conclude that the Pat × Col-0 RIL population is a valuable novel genetic resource to explore other adaptive traits in Arabidopsis.

Unraveling the impact on agronomic traits of the genetic architecture underlying plant-density responses in canola.

Plant density defines vegetative architecture and the competition for light between individuals. Brassica napus (canola, rapeseed) presents a radically different plant architecture compared to traditional crops commonly cultivated at high density, and can act as a model system of indeterminate growth. Using a panel of 152 spring-type accessions and a double-haploid population of 99 lines from a cross between the cultivars Lynx and Monty, we performed genome-wide association studies (GWAS) and quantitative trait locus (QTL) mapping for 12 growth and yield traits at two contrasting plant densities of 15 and 60 plants m-2. The most significant associations were found for time to flowering, biomass at harvest, plant height, silique and seed numbers, and seed yield. These were generally independent of plant density, but some density-dependent associations were found in low-density populations. RNA-seq transcriptomic analysis revealed distinctive latent gene-regulatory responses to simulated shade between Lynx and Monty. Having identified candidate genes within the canola QTLs, we further examined their influence on density responses in Arabidopsis lines mutated in certain homologous genes. The results suggested that TCP1 might promote growth independently of plant density, while HY5 could increase biomass and seed yield specifically at high plant density. For flowering time, the results suggested that PIN genes might accelerate flowering in plant a density-dependent manner whilst FT, HY5, and TCP1 might accelerate it in a density-independent. This work highlights the advantages of using agronomic field experiments together with genetic and transcriptomic approaches to decipher quantitative complex traits that potentially mediate improved crop productivity.

Becoming a Selfish Clan: Recombination Associated to Reverse-Transcription in LTR Retrotransposons.

Transposable elements (TEs) are parasitic DNA bits capable of mobilization and mutagenesis, typically suppressed by host's epigenetic silencing. Since the selfish DNA concept, it is appreciated that genomes are also molded by arms-races against natural TE inhabitants. However, our understanding of evolutionary processes shaping TEs adaptive populations is scarce. Here, we review the events of recombination associated to reverse-transcription in LTR retrotransposons, a process shuffling their genetic variants during replicative mobilization. Current evidence may suggest that recombinogenic retrotransposons could beneficially exploit host suppression, where clan behavior facilitates their speciation and diversification. Novel refinements to retrotransposons life-cycle and evolution models thus emerge.

Evidence of developmental escape from transcriptional gene silencing in MESSI retrotransposons.

Transposable elements (TEs) are ubiquitous genomic features. 'Copy-and-paste' long-terminal-repeat (LTR) retrotransposons have been particularly successful during evolution of the plant kingdom, representing a substantial proportion of genomes. For survival in copious numbers, these TEs may have evolved replicative mobilization strategies that circumvented hosts' epigenetic silencing. Stressful circumstances are known to trigger the majority of known mobilizing plant retrotransposons, leading to the idea that most are activated by environmental signals. However, previous research revealed that plant developmental programs include steps of silencing relaxation, suggesting that developmental signals may also be of importance for thriving parasitic elements. Here, we uncover an unusual family of giant LTR retrotransposons from the Solanum clade, named MESSI, with transcriptional competence in shoot apical meristems of tomato. Despite being recognized and targeted by the host epigenetic surveillance, this family is activated in specific meristematic areas fundamental for plant shoot development, which are involved in meristem formation and maintenance. Our work provides initial evidence that some retrotransposons may evolve developmentally associated escape strategies to overcome transcriptional gene silencing in vegetative tissues contributing to the host's next generation. This implies that not only environmental but also developmental signals could be exploited by selfish elements for survival within the plant kingdom.

High-frequency recombination between members of an LTR retrotransposon family during transposition bursts.

Retrotransposons containing long terminal repeats (LTRs) form a substantial fraction of eukaryotic genomes. The timing of past transposition can be estimated by quantifying the accumulation of mutations in initially identical LTRs. This way, retrotransposons are divided into young, potentially mobile elements, and old that moved thousands or even millions of years ago. Both types are found within a single retrotransposon family and it is assumed that the old members will remain immobile and degenerate further. Here, we provide evidence in Arabidopsis that old members enter into replication/transposition cycles through high rates of intra-family recombination. The recombination occurs pairwise, resembling the formation of recombinant retroviruses. Thus, each transposition burst generates a novel progeny population of chromosomally integrated LTR retrotransposons consisting of pairwise recombination products produced in a process comparable the sexual exchange of genetic information. Our observations provide an explanation for the reported high rates of sequence diversification in retrotransposons.

Developmental Restriction of Retrotransposition Activated in Arabidopsis by Environmental Stress.

Retrotransposons (RTs) can rapidly increase in copy number due to periodic bursts of transposition. Such bursts are mutagenic and thus potentially deleterious. However, certain transposition-induced gain-of-function or regulatory mutations may be of selective advantage. How an optimal balance between these opposing effects arises is not well characterized. Here, we studied transposition bursts of a heat-activated retrotransposon family in Arabidopsis We recorded a high inter and intraplant variation in the number and chromosomal position of new insertions, which usually did not affect plant fertility and were equally well transmitted through male and female gametes, even though 90% of them were within active genes. We found that a highly heterogeneous distribution of these new retroelement copies result from a combination of two mechanisms, of which the first prevents multiple transposition bursts in a given somatic cell lineage that later contributes to differentiation of gametes, and the second restricts the regulatory influence of new insertions toward neighboring chromosomal DNA. As a whole, such regulatory characteristics of this family of RTs ensure its rapid but stepwise accumulation in plant populations experiencing transposition bursts accompanied by high diversity of chromosomal sites harboring new RT insertions.

Heat-induced release of epigenetic silencing reveals the concealed role of an imprinted plant gene.

Epigenetic mechanisms suppress the transcription of transposons and DNA repeats; however, this suppression can be transiently released under prolonged heat stress. Here we show that the Arabidopsis thaliana imprinted gene SDC, which is silent during vegetative growth due to DNA methylation, is activated by heat and contributes to recovery from stress. SDC activation seems to involve epigenetic mechanisms but not canonical heat-shock perception and signaling. The heat-mediated transcriptional induction of SDC occurs particularly in young developing leaves and is proportional to the level of stress. However, this occurs only above a certain window of absolute temperatures and, thus, resembles a thermal-sensing mechanism. In addition, the re-silencing kinetics during recovery can be entrained by repeated heat stress cycles, suggesting that epigenetic regulation in plants may conserve memory of stress experience. We further demonstrate that SDC contributes to the recovery of plant biomass after stress. We propose that transcriptional gene silencing, known to be involved in gene imprinting, is also co-opted in the specific tuning of SDC expression upon heat stress and subsequent recovery. It is therefore possible that dynamic properties of the epigenetic landscape associated with silenced or imprinted genes may contribute to regulation of their expression in response to environmental challenges.

Transcriptomic and metabolic changes associated with photorespiratory ammonium accumulation in the model legume Lotus japonicus.

The transcriptomic and metabolic consequences of the lack of plastidic glutamine (Gln) synthetase in the model legume Lotus japonicus were investigated. Wild-type and mutant plants lacking the plastidic isoform of Gln synthetase were grown in conditions that suppress photorespiration and then transferred for different lengths of time to photorespiratory conditions. Transcript and metabolite levels were determined at the different time points considered. Under photorespiratory active conditions, the mutant accumulated high levels of ammonium, followed by its subsequent decline. A coordinate repression of the photorespiratory genes was observed in the mutant background. This was part of a greater modulation of the transcriptome, especially in the mutant, that was paralleled by changes in the levels of several key metabolites. The data obtained for the mutant represent the first direct experimental evidence for a coordinate regulation of photorespiratory genes over time. Metabolomic analysis demonstrated that mutant plants under active photorespiratory conditions accumulated high levels of several amino acids and organic acids, including intermediates of the Krebs cycle. An increase in Gln levels was also detected in the mutant, which was paralleled by an increase in cytosolic Gln synthetase1 gene transcription and enzyme activity levels. The global panoramic of the transcripts and metabolites that changed in L. japonicus plants during the transfer from photorespiration-suppressed to photorespiration-active conditions highlighted the link between photorespiration and several other cellular processes, including central carbon metabolism, amino acid metabolism, and secondary metabolism.

Comparative metabolomics of drought acclimation in model and forage legumes.

Water limitation has become a major concern for agriculture. Such constraints reinforce the urgent need to understand mechanisms by which plants cope with water deprivation. We used a non-targeted metabolomic approach to explore plastic systems responses to non-lethal drought in model and forage legume species of the Lotus genus. In the model legume Lotus. japonicus, increased water stress caused gradual increases of most of the soluble small molecules profiled, reflecting a global and progressive reprogramming of metabolic pathways. The comparative metabolomic approach between Lotus species revealed conserved and unique metabolic responses to drought stress. Importantly, only few drought-responsive metabolites were conserved among all species. Thus we highlight a potential impediment to translational approaches that aim to engineer traits linked to the accumulation of compatible solutes. Finally, a broad comparison of the metabolic changes elicited by drought and salt acclimation revealed partial conservation of these metabolic stress responses within each of the Lotus species, but only few salt- and drought-responsive metabolites were shared between all. The implications of these results are discussed with regard to the current insights into legume water stress physiology.

New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress.

Polyamines (putrescine, spermidine and spermine) are traditionally implicated in the response of plants to environmental cues. Free spermine accumulation has been suggested as a particular feature of long-term salt stress, and in the model plant Arabidopsis thaliana the spermine synthase gene (AtSPMS) has been reported as inducible by abscisic acid (ABA) and acute salt stress treatments. With the aim to unravel the physiological role of free spermine during salinity, we analyzed polyamine metabolism in A. thaliana salt-hypersensitive sos mutants (salt overlay sensitive; sos1-1, sos2-1 and sos3-1), and studied the salt stress tolerance of the mutants in spermine and thermospermine synthesis (acl5-1, spms-1 and acl5-1/spms-1). Results presented here indicate that induction in polyamine metabolism is a SOS-independent response to salinity and is globally over-induced in a sensitive background. In addition, under long-term salinity, the mutants in the synthesis of spermine and thermospermine (acl5-1, spms-1 and double acl5-1/spms-1) accumulated more Na(+) and performed worst than WT in survival experiments. Therefore, support is given to a role for these higher polyamines in salt tolerance mechanisms.

Modification of OsSUT1 gene expression modulates the salt response of rice Oryza sativa cv. Taipei 309.

A metabolic depletion syndrome was discovered at early vegetative stages in roots of salt sensitive rice cultivars after prolonged exposure to 100mM NaCl. Metabolite profiling analyses demonstrate that this syndrome is part of the terminal stages of the rice salt response. The phenotype encompasses depletion of at least 30 primary metabolites including sucrose, glucose, fructose, glucose-6-P, fructose-6P, organic- and amino-acids. Based on these observations we reason that sucrose allocation to the root may modify the rice response to high salt. This hypothesis was tested using antisense lines of the salt responsive OsSUT1 gene in the salt sensitive Taipei 309 cultivar. Contrary to our expectations of a plant system impaired in one component of sucrose transport, we find improved gas exchange and photosynthetic performance as well as maintenance of sucrose levels in the root under high salinity. Two independent OsSUT1 lines with an antisense inhibition similar to the naturally occurring salt induced reduction of OsSUT1 gene expression showed these phenomena but not a more extreme antisense inhibition line. We investigated the metabolic depletion syndrome by metabolomic and physiological approaches and discuss our results with regard to the potential role of sucrose transporters and sucrose transport for rice salt acclimation.

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus.

• Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. • Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. • The exposure of plants to 10 µM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 µM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. • We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 µM Al.

Homeostatic control of polyamine levels under long-term salt stress in Arabidopsis: changes in putrescine content do not alleviate ionic toxicity.

Salt stress has been frequently studied in its first osmotic phase. Very often, data regarding the second ionic phase is missing. It has also been suggested that Putrescine or/and Spermine could be responsible for salt resistance. In order to test this hypothesis under long-term salt stress, we obtained Arabidopsis thaliana transgenic plants harboring pRD29A::oatADC or pRD29A::GUS construction. Although Putrescine was the only polyamine significantly increased after salt acclimation in pRD29A::oatADC transgenic lines, this rendered in no advantage to this kind of stress. The higher Spermine levels found in WT and transgenic lines when compared to control conditions along with no increment on Putrescine levels in WT plants under salt acclimation, leads us to analyze Spermine effect on pADC1 and pADC2 expression. Increasing levels of this polyamine inhibits these promoters expression while enhances pRD29A expression, making Spermine the polyamine responsible for salt acclimation, and the transgenic lines developed in this work suitable for studying Putrescine roles in conditions where its biosynthesis would be inhibited in the WT genotype.

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress.

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the over-expression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlated with the induction of known stress-responsive genes, and suggested that putrescine may be directly or indirectly involved in ABA metabolism and gene expression.

Comparative functional genomics of salt stress in related model and cultivated plants identifies and overcomes limitations to translational genomics.

One of the objectives of plant translational genomics is to use knowledge and genes discovered in model species to improve crops. However, the value of translational genomics to plant breeding, especially for complex traits like abiotic stress tolerance, remains uncertain. Using comparative genomics (ionomics, transcriptomics and metabolomics) we analyzed the responses to salinity of three model and three cultivated species of the legume genus Lotus. At physiological and ionomic levels, models responded to salinity in a similar way to crop species, and changes in the concentration of shoot Cl(-) correlated well with tolerance. Metabolic changes were partially conserved, but divergence was observed amongst the genotypes. Transcriptome analysis showed that about 60% of expressed genes were responsive to salt treatment in one or more species, but less than 1% was responsive in all. Therefore, genotype-specific transcriptional and metabolic changes overshadowed conserved responses to salinity and represent an impediment to simple translational genomics. However, 'triangulation' from multiple genotypes enabled the identification of conserved and tolerant-specific responses that may provide durable tolerance across species.