The miR166-SlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato.

Fruit set is inhibited by adverse temperatures, with consequences on yield. We isolated a tomato mutant producing fruits under non-permissive hot temperatures and identified the causal gene as SlHB15A, belonging to class III homeodomain leucine-zipper transcription factors. SlHB15A loss-of-function mutants display aberrant ovule development that mimics transcriptional changes occurring in fertilized ovules and leads to parthenocarpic fruit set under optimal and non-permissive temperatures, in field and greenhouse conditions. Under cold growing conditions, SlHB15A is subjected to conditional haploinsufficiency and recessive dosage sensitivity controlled by microRNA 166 (miR166). Knockdown of SlHB15A alleles by miR166 leads to a continuum of aberrant ovules correlating with parthenocarpic fruit set. Consistent with this, plants harboring an Slhb15a-miRNA166-resistant allele developed normal ovules and were unable to set parthenocarpic fruit under cold conditions. DNA affinity purification sequencing and RNA-sequencing analyses revealed that SlHB15A is a bifunctional transcription factor expressed in the ovule integument. SlHB15A binds to the promoters of auxin-related genes to repress auxin signaling and to the promoters of ethylene-related genes to activate their expression. A survey of tomato genetic biodiversity identified pat and pat-1, two historical parthenocarpic mutants, as alleles of SlHB15A. Taken together, our findings demonstrate the role of SlHB15A as a sentinel to prevent fruit set in the absence of fertilization and provide a mean to enhance fruiting under extreme temperatures.

Genetic basis of phenotypic plasticity and genotype × environment interactions in a multi-parental tomato population.

Deciphering the genetic basis of phenotypic plasticity and genotype × environment interactions (G×E) is of primary importance for plant breeding in the context of global climate change. Tomato (Solanum lycopersicum) is a widely cultivated crop that can grow in different geographical habitats and that displays a great capacity for expressing phenotypic plasticity. We used a multi-parental advanced generation intercross (MAGIC) tomato population to explore G×E and plasticity for multiple traits measured in a multi-environment trial (MET) comprising optimal cultural conditions together with water deficit, salinity, and heat stress over 12 environments. Substantial G×E was observed for all the traits measured. Different plasticity parameters were estimated by employing Finlay-Wilkinson and factorial regression models and these were used together with genotypic means for quantitative trait loci (QTL) mapping analyses. In addition, mixed linear models were also used to investigate the presence of QTL × environment interactions. The results highlighted a complex genetic architecture of tomato plasticity and G×E. Candidate genes that might be involved in the occurrence of G×E are proposed, paving the way for functional characterization of stress response genes in tomato and for breeding climate-adapted cultivars.

Glutathione application affects the transcript profile of genes in Arabidopsis seedling.

Glutathione (GSH), a tripeptide thiol compound has multiple functions in plants. Recent works suggested that GSH plays a regulatory role in signaling in plants as part of their adaptation to stress. To better understand the role of GSH as a regulatory molecule, 14 days old Arabidopsis thaliana seedlings were treated with 5mM of GSH for 4h. Changes in gene expression patterns were studied by cDNA microarray analysis. The expression of 453 genes was significantly changed compared to the untreated control, of which 261 genes were up-regulated and 192 genes were down-regulated. Genes from several groups were affected, including those of sulfur metabolism, degradation and synthesis of macromolecules and transcription factors. Up-regulation of genes involved in responses to biotic stresses, or in jasmonate or salicylic acid synthesis and their signaling, suggests that GSH triggers genes that help protect the plants during stresses. In addition, GSH down regulated genes involved in plant growth and development, like those involved in cell wall synthesis and its extension, and genes associated with auxin and cytokinins response, which are related to growth and development of the plants. The results suggest that GSH might have a role in response to biotic stress by initiating defense responses and modifying plants' growth and development in an effort to tune their sessile lifestyle of plants to environmental constraints.

ROS and redox signalling in the response of plants to abiotic stress.

The redox state of the chloroplast and mitochondria, the two main powerhouses of photosynthesizing eukaryotes, is maintained by a delicate balance between energy production and consumption, and affected by the need to avoid increased production of reactive oxygen species (ROS). These demands are especially critical during exposure to extreme environmental conditions, such as high light (HL) intensity, heat, drought or a combination of different environmental stresses. Under these conditions, ROS and redox cues, generated in the chloroplast and mitochondria, are essential for maintaining normal energy and metabolic fluxes, optimizing different cell functions, activating acclimation responses through retrograde signalling, and controlling whole-plant systemic signalling pathways. Regulation of the multiple redox and ROS signals in plants requires a high degree of coordination and balance between signalling and metabolic pathways in different cellular compartments. In this review, we provide an update on ROS and redox signalling in the context of abiotic stress responses, while addressing their role in retrograde regulation, systemic acquired acclimation and cellular coordination in plants.

Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis.

Thiamin and thiamin pyrophosphate (TPP) are well known for their important roles in human nutrition and enzyme catalysis. In this work, we present new evidence for an additional role of these compounds in the protection of cells against oxidative damage. Arabidopsis (Arabidopsis thaliana) plants subjected to abiotic stress conditions, such as high light, cold, osmotic, salinity, and oxidative treatments, accumulated thiamin and TPP. Moreover, the accumulation of these compounds in plants subjected to oxidative stress was accompanied by enhanced expression of transcripts encoding thiamin biosynthetic enzymes. When supplemented with exogenous thiamin, wild-type plants displayed enhanced tolerance to oxidative stress induced by paraquat. Thiamin application was also found to protect the reactive oxygen species-sensitive ascorbate peroxidase1 mutant from oxidative stress. Thiamin-induced tolerance to oxidative stress was accompanied by decreased production of reactive oxygen species in plants, as evidenced from decreased protein carbonylation and hydrogen peroxide accumulation. Because thiamin could protect the salicylic acid induction-deficient1 mutant against oxidative stress, thiamin-induced oxidative protection is likely independent of salicylic acid signaling or accumulation. Taken together, our studies suggest that thiamin and TPP function as important stress-response molecules that alleviate oxidative stress during different abiotic stress conditions.

Identification of new protein substrates for the chloroplast ATP-dependent Clp protease supports its constitutive role in Arabidopsis.

The ATP-dependent Clp protease in plant chloroplasts consists of a heterogeneous proteolytic core containing multiple ClpP and ClpR paralogues. In this study, we have examined in detail the only viable knockout mutant to date of one of these subunits in Arabidopsis thaliana, ClpR1. Loss of ClpR1 caused a slow-growth phenotype, with chlorotic leaves during early development that later partially recovered upon maturity. Analysis of the Clp proteolytic core in the clpR1 mutant (clpR1-1) revealed approx. 10% of the wild-type levels remaining, probably due to a relative increase in the closely related ClpR3 protein and its partial substitution of ClpR1 in the core complex. A proteomic approach using an in organello proteolytic assay revealed 19 new potential substrates for the chloroplast Clp protease. Many of these substrates were constitutive enzymes involved in different metabolic pathways, including photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis, whereas others function in housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling processes. In contrast, degradation of the stress-related chloroplast proteins Hsp21 (heat-shock protein 21) and lipoxygenase 2 was unaffected in the clpR1-1 line and thus not facilitated by the Clp protease. Overall, we show that the chloroplast Clp protease is principally a constitutive enzyme that degrades numerous stromal proteins, a feature that almost certainly underlies its vital importance for chloroplast function and plant viability.

Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination.

Within their natural habitat plants are subjected to a combination of different abiotic stresses, each with the potential to exacerbate the damage caused by the others. One of the most devastating stress combinations for crop productivity, which frequently occurs in the field, is drought and heat stress. In this study we conducted proteomic and metabolic analysis of Arabidopsis thaliana plants subjected to a combination of drought and heat stress. We identified 45 different proteins that specifically accumulated in Arabidopsis in response to the stress combination. These included enzymes involved in reactive oxygen detoxification, malate metabolism, and the Calvin cycle. The accumulation of malic enzyme during the combined stress corresponded with enhanced malic enzyme activity, a decrease in malic acid, and lower amounts of oxaloacetate, suggesting that malate metabolism plays an important role in the response of Arabidopsis to the stress combination. Cytosolic ascorbate peroxidase 1 (APX1) protein and mRNA accumulated during the stress combination. When exposed to heat stress combined with drought, an APX1-deficient mutant (apx1) accumulated more hydrogen peroxide and was significantly more sensitive to the stress combination than wild type. In contrast, mutants deficient in thylakoid or stromal/mitochondrial APXs were not more sensitive to the stress combination than apx1 or wild type. Our findings suggest that cytosolic APX1 plays a key role in the acclimation of plants to a combination of drought and heat stress.

Polyphenol oxidase can cross thylakoids by both the Tat and the Sec-dependent pathways: a putative role for two stromal processing sites.

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1), a thylakoid-lumen protein encoded by a nuclear gene, plays a role in the defense of plants against both herbivores and pathogens. Although previously reported to be a Tat (twin-arginine-dependent translocation) protein, the import of PPO by isolated chloroplasts was inhibited by azide, a diagnostic inhibitor of the Sec-dependent pathway. Import of PPO inhibited thylakoid translocation of a Tat protein and did not affect translocation of Sec-dependent proteins. In contrast, a pre-accumulated iPPO competed with Sec-dependent but not with Tat proteins. A previously reported second processing step in the stroma removes a twin-Arg that is part of a 'Sec-avoidance' motif in the thylakoid targeting domain of PPO. When the second processing site was mutated, the import of the resulting precursor showed Sec-dependent characteristics. The PPO transit peptide could drive thylakoid translocation of a Tat protein in the dark. Azide inhibited the secretion of a PPO intermediate that lacks a twin-Arg to the periplasm of Escherichia coli, but had no effect on the export of the intermediate containing the twin-Arg. PPO is synthesized in plants in response to wound and pathogen-related signals and it is possible that when the Tat pathway is unable to translocate adequate amounts of newly synthesized PPO, translocation is diverted to the Sec-dependent pathway by processing the intermediate at the second site and removing the twin-Arg.

Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses.

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.

Signals from chloroplasts converge to regulate nuclear gene expression.

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast function and is essential for the photoautotrophic life-style of plants. Three retrograde signals have been described, but little is known of their signaling pathways. We show here that GUN1, a chloroplast-localized pentatricopeptide-repeat protein, and ABI4, an Apetala 2 (AP2)­type transcription factor, are common to all three pathways. ABI4 binds the promoter of a retrograde-regulated gene through a conserved motif found in close proximity to a light-regulatory element. We propose a model in which multiple indicators of aberrant plastid function in Arabidopsis are integrated upstream of GUN1 within plastids, which leads to ABI4-mediated repression of nuclear-encoded genes.

An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development.

The ATP-dependent Clp protease has been well-characterized in Escherichia coli, but knowledge of its function in higher plants is limited. In bacteria, this two-component protease consists of a Ser-type endopeptidase ClpP, which relies on the ATP-dependent unfolding activity from an Hsp100 molecular chaperone to initiate protein degradation. In the chloroplasts of higher plants, multiple isoforms of the proteolytic subunit exist, with Arabidopsis having five ClpPs and four ClpP-like proteins termed ClpR predicted in its genome. In this work we characterized an Arabidopsis mutant impaired in one subunit of the chloroplast-localized Clp protease core, ClpR1. clpR1-1, a virescent mutant, carries a pre-mature stop codon in the clpR1 gene, resulting in no detectable ClpR1 protein. The accumulation of several chloroplast proteins, as well as most of the chloroplast-localized Clp protease subunits, is inhibited in clpR1-1. Unexpectedly, some plastid-encoded proteins do not accumulate, although their transcripts accumulate to wild-type levels. Maturation of 23S and 4.5S chloroplast ribosomal RNA (cp-rRNA) is delayed in clpR1-1, and both RNAs accumulate as higher molecular weight precursors. Also, chloroplasts in clpR1-1 are smaller than in wild type and have fewer thylakoid membranes with smaller grana stacks. We propose that a ClpR1-containing activity is required for chloroplast development and differentiation and in its absence both are delayed.

Plastid-to-nucleus retrograde signaling.

Plant cells store genetic information in the genomes of three organelles: the nucleus, plastid, and mitochondrion. The nucleus controls most aspects of organelle gene expression, development, and function. In return, organelles send signals to the nucleus to control nuclear gene expression, a process called retrograde signaling. This review summarizes our current understanding of plastid-to-nucleus retrograde signaling, which involves multiple, partially redundant signaling pathways. The best studied is a pathway that is triggered by buildup of Mg-ProtoporphyrinIX, the first intermediate in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In addition, there is evidence for a plastid gene expression-dependent pathway, as well as a third pathway that is dependent on the redox state of photosynthetic electron transport components. Although genetic studies have identified several players involved in signal generation, very little is known of the signaling components or transcription factors that regulate the expression of hundreds of nuclear genes.

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate.

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1) takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8-16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.