Anthocyanin-less (white) instead of black/red (coloured) fruits develop in grapevine cultivars without functional VviMYBA1 and VviMYBA2 genes, which conditions the colour of wines that can be produced. To evaluate whether this genetic variation has additional consequences on fruit ripening and composition, we performed microenvironment, transcriptomics and metabolomics comparisons of developing grapes between near-isogenic white- and black-berried somatic variants of Garnacha and Tempranillo cultivars. Berry temperature was up to 3.5ºC lower in white- compared to black-berried Tempranillo. An RNA-seq study combined with targeted and untargeted metabolomics revealed that ripening fruits of white-berried variants were characterized by the upregulation of photosynthesis and other light-responsive genes and their higher accumulation of specific terpene aroma precursors, fatty acid-derived aldehyde volatiles and phenylpropanoid precursor amino acids. MYBA1-MYBA2 function proved essential for flavonol trihydroxylation in black-berried somatic variants, which were also characterized by enhanced pathogen defence gene expression in berry skin and increased accumulation of C6-derived alcohol and ester volatiles and GABA. Collectively, our results indicate that anthocyanin depletion entails side effects on grape composition by altering the berry internal microenvironment and the partition of the phenylpropanoid pathway. These findings show how fruit colour can condition other fruit features, such as flavour potential and stress homeostasis.

Glutathione (GSH) is a ubiquitous, abundant, and indispensable thiol for plants that participates in various biological processes, such as scavenging reactive oxygen species, redox signaling, storage and transport of sulfur, detoxification of harmful substances, and metabolism of several compounds. Therefore knowledge of GSH metabolism is essential for plant science. Nevertheless, GSH degradation has been insufficiently elucidated, and this has hampered our understanding of plant life. Over the last five decades, the γ-glutamyl cycle has been dominant in GSH studies, and the exoenzyme γ-glutamyl transpeptidase has been regarded as the major GSH degradation enzyme. However, recent studies have shown that GSH is degraded in cells by cytosolic enzymes such as γ-glutamyl cyclotransferase or γ-glutamyl peptidase. Meanwhile, a portion of GSH is degraded after conjugation with other molecules, which has also been found to be carried out by vacuolar γ-glutamyl transpeptidase, γ-glutamyl peptidase, or phytochelatin synthase. These findings highlight the need to re-assess previous assumptions concerning the γ-glutamyl cycle, and a novel overview of the plant GSH degradation pathway is essential. This review aims to build a foundation for future studies by summarizing current understanding of GSH/glutathione conjugate degradation.

As sulfur is part of many essential protein cofactors such as iron-sulfur clusters, molybdenum cofactors, or lipoic acid, its mobilization from cysteine represents a fundamental process. The abstraction of the sulfur atom from cysteine is catalysed by highly conserved pyridoxal 5'-phosphate-dependent enzymes called cysteine desulfurases. The desulfuration of cysteine leads to the formation of a persulfide group on a conserved catalytic cysteine and the concomitant release of alanine. Sulfur is then transferred from cysteine desulfurases to different targets. Numerous studies have focused on cysteine desulfurases as sulfur-extracting enzymes for iron-sulfur cluster synthesis in mitochondria and chloroplasts but also for molybdenum cofactor sulfuration in the cytosol. Despite this, knowledge about the involvement of cysteine desulfurases in other pathways is quite rudimentary, particularly in photosynthetic organisms. In this review, we summarize current understanding of the different groups of cysteine desulfurases and their characteristics in terms of primary sequence, protein domain architecture, and subcellular localization. In addition, we review the roles of cysteine desulfurases in different fundamental pathways and highlight the gaps in our knowledge to encourage future work on unresolved issues especially in photosynthetic organisms.

Since the discovery of an autonomous iron-sulfur cluster (Fe-S) assembly machinery in mitochondria, significant efforts to examine the nature of this process have been made. The assembly of Fe-S clusters occurs in two distinct steps with the initial synthesis of [2Fe-2S] clusters by a first machinery followed by a subsequent assembly into [4Fe-4S] clusters by a second machinery. Despite this knowledge, we still have only a rudimentary understanding of how Fe-S clusters are transferred and distributed among their respective apoproteins. In particular, demand created by continuous protein turnover and the sacrificial destruction of clusters for synthesis of biotin and lipoic acid reveal possible bottlenecks in the supply chain of Fe-S clusters. Taking available information from other species into consideration, this review explores the mitochondrial assembly machinery of Arabidopsis and provides current knowledge about the respective transfer steps to apoproteins. Furthermore, this review highlights biotin synthase and lipoyl synthase, which both utilize Fe-S clusters as a sulfur source. After extraction of sulfur atoms from these clusters, the remains of the clusters probably fall apart, releasing sulfide as a highly toxic by-product. Immediate refixation through local cysteine biosynthesis is therefore an essential salvage pathway and emphasizes the physiological need for cysteine biosynthesis in plant mitochondria.

Cadmium (Cd) uptake from polluted soils inhibits plant growth and disturbs physiological processes, at least partly due to disturbances in the cellular redox environment. Although the sulfur-containing antioxidant glutathione is important in maintaining redox homeostasis, its role as an antioxidant can be overruled by its involvement in Cd chelation as a phytochelatin precursor. Following Cd exposure, plants rapidly invest in phytochelatin production, thereby disturbing the redox environment by transiently depleting glutathione concentrations. Consequently, a network of signalling responses is initiated, in which the phytohormone ethylene is an important player involved in the recovery of glutathione levels. Furthermore, these responses are intricately connected to organellar stress signalling and autophagy, and contribute to cell fate determination. In general, this may pave the way for acclimation (e.g. restoration of glutathione levels and organellar homeostasis) and plant tolerance in the case of mild stress conditions. This review addresses connections between these players and discusses the possible involvement of the gasotransmitter hydrogen sulfide in plant acclimation to Cd exposure.

Soil contamination with trace metals and metalloids can cause toxicity to plants and threaten food safety and human health. Plants have evolved sophisticated mechanisms to cope with excess trace metals and metalloids in soils, including chelation and vacuolar sequestration. Sulfur-containing compounds, such as glutathione and phytochelatins, play a crucial role in their detoxification, and sulfur uptake and assimilation are regulated in response to the stress of toxic trace metals and metalloids. This review focuses on the multi-level connections between sulfur homeostasis in plants and responses to such stresses, especially those imposed by arsenic and cadmium. We consider recent progress in understanding the regulation of biosynthesis of glutathione and phytochelatins and of the sensing mechanism of sulfur homeostasis for tolerance of trace metals and metalloids in plants. We also discuss the roles of glutathione and phytochelatins in controlling the accumulation and distribution of arsenic and cadmium in plants, and possible strategies for manipulating sulfur metabolism to limit their accumulation in food crops.

Improving and stabilizing the quality of seed proteins are of growing interest in the current food and agroecological transitions. Sulfur is a key determinant of this quality since it is essential for the synthesis of sulfur-rich proteins in seeds. A lack of sulfur provokes drastic changes in seed protein composition, negatively impacting the nutritional and functional properties of proteins, and leading in some cases to diseases or health problems in humans. Sulfur also plays a crucial role in stress tolerance through the synthesis of antioxidant or protective molecules. In the context of climate change, questions arise regarding the trade-off between seed yield and seed quality with respect to sulfur availability and use by crops that represent important sources of proteins for human nutrition. Here, we review recent work obtained in legumes, cereals, as well as in Arabidopsis, that present major advances on: (i) the interaction between sulfur nutrition and environmental or nutritional stresses with regard to seed yield and protein composition; (ii) metabolic pathways that merit to be targeted to mitigate negative impacts of environmental stresses on seed protein quality; and (iii) the importance of sulfur homeostasis for the regulation of seed protein composition and its interplay with seed redox homeostasis.

Under conditions of sulfur deprivation, O-acetylserine (OAS) accumulates, which leads to the induction of a common set of six genes, called OAS cluster genes. These genes are induced not only under sulfur deprivation, but also under other conditions where OAS accumulates, such as shift to darkness and stress conditions leading to reactive oxygen species (ROS) or methyl-jasmonate accumulation. Using the OAS cluster genes as a query in ATTED-II, a co-expression network is derived stably spanning several hundred conditions. This allowed us not only to describe the downstream function of the OAS cluster genes but also to score for functions of the members of the co-regulated co-expression network and hence the effects of the OAS signal on the sulfate assimilation pathway and co-regulated pathways. Further, we summarized existing knowledge on the regulation of the OAS cluster and the co-expressed genes. We revealed that the known sulfate deprivation-related transcription factor EIL3/SLIM1 exhibits a prominent role, as most genes are subject to regulation by this transcription factor. The role of other transcription factors in response to OAS awaits further investigation.

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.

Fruits of Fragaria species usually have an appealing bright red color due to the accumulation of anthocyanins, water-soluble flavonoid pigments. Octoploid cultivated strawberry (Fragaria x ananassa) is a major horticultural crop for which fruit color and associated nutritional value are main breeding targets. Great diversity in fruit color intensity and pattern is observed not only in cultivated strawberry but also in wild relatives such as its octoploid progenitor F. chiloensis or the diploid woodland strawberry F. vesca, a model for fruit species in the Rosaceae. This mini-review examines our understanding of fruit color formation in strawberry and how ongoing developments will advance it. Natural variations of fruit color as well as color changes during fruit development or in response to several cues have been used to explore the anthocyanin biosynthetic pathway and its regulation. So far, the successfull identification of causal genetic variants has been largely driven by the availability of high-throughput genotyping tools and high-quality reference genomes of F. vesca and F. x ananassa. The current completion of haplotype-resolved genomes of F. x ananassa combined with QTL mapping will accelerate the exploitation of the untapped genetic diversity of fruit color and help translate the findings into strawberry improvement.

Plant growth and development depends on sufficient nutrient availability in soils. Agricultural soils are generally nitrogen (N) deficient and thus, soils need to be supplemented with fertilizers. Ammonium (NH4  +) is a major inorganic N source. However, at high concentrations ammonium nutrition becomes a stress situation that inhibits plant growth. The cause of ammonium stress or toxicity is multi-factorial but the interaction with other nutrients is among the main determinants of plants sensitivity towards high NH4  + supply. In addition, NH4  + uptake and assimilation provokes the acidification of the cell external medium (apoplast/rhizosphere), which has a clear impact on nutrients availability. This review summarizes current knowledge, at both physiological and molecular level, of ammonium nutrition interaction with essential mineral elements absorbed as cations: macronutrients (K +, Ca 2+, Mg 2+) and micronutrients (Fe 2+/3+, Mn 2+, Cu +/2+, Zn 2+, Ni 2+). We hypothesize that considering these nutritional interactions, and soil pH, when formulating fertilizers may be key in order to boost the use of NH4  +-based fertilizers, which have less environmental impact compared to the nitrate-based ones. In addition, we are convinced that better understanding these interactions will help to identify novel targets with potential to improve crop productivity.

It becomes increasingly evident that interspecific hybridization at homoploid level or coupled with whole genome duplication (i.e., allopolyploidization) has played a major role in biological evolution. Yet, the direct impact of hybridization and allopolyploidization on genome structure and function, phenotype, and fitness remains to be fully understood. Synthetic hybrids and allopolyploids are trackable experimental systems to address this issue. Here, we resynthesized a pair of reciprocal F1 hybrids and corresponding reciprocal allotetraploids using the two diploid progenitor species, Triticum urartu (AA) and Aegilops tauschii (DD), of bread wheat (Triticum aestivum L., BBAADD). By comparing phenotypes related to growth, development and fitness, andanalyzing genome expression in both hybrids and allotetraploids in relation to parents, we find the types and trends of karyotype variation in the immediately formed allotetraploids, are correlated with both meiosis instability and chromosome- and subgenome-biased expression. We document clear advantages of allotetraploids over diploid F1 hybrids in several morphological traits including fitness, which mirror the tissue- and developmental stage-dependent subgenome-partitioning of the allotetraploids. The allotetraploids are meiotically unstable primarily due to homoeologous pairing that varies dramatically among the chromosomes. Nonetheless, the manifestation of organismal karyotype variation and occurrence of meiotic irregularity are not concordant, suggesting a role of functional constraints probably imposed by subgenome- and chromosome-biased gene expression. Our results provide new insights into the direct impacts and consequences of hybridization and allopolyploidization, which are relevant to evolution and likely informative to crop improvement by synthetic polyploid approaches.

For survival in the natural environment, plants have evolved a 'bet-hedging' strategy where individual variation is high and a range of phenotypes is produced. When faced with unpredictable environmental conditions, fluctuation in seed behaviour is a beneficial trait that allows plant species to survive, particularly if seedlings from early-germinated seeds die. However, this is not a desired trait from an agricultural perspective, where a set of uniformly growing seedlings is required. Whilst variability in seed behaviour is unavoidable, over the centuries humans have attempted to select seeds with minimum variability for agricultural use. In the model plant Arabidopsis, even non-stratified seeds in the same silique germinate variably, and it remains elusive how this variability is manifested from genes to a physiological outcome and what molecular mechanisms of bet-hedging facilitate this diversity. Will the re-introduction of valuable wild alleles into domesticated crops contribute to this variability between individual seeds by promoting bet-hedging? Recent advances have shed light on possible molecular pathways of germination that are affected at the level of single seeds and single cells. Here, we review the hormonal, molecular, and cellular mechanisms that might affect the germination outcome of individual genetically identical seeds.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support cells while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of model and crop plants have facilitated cataloguing and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall-synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

Cyanobacteria are structurally the simplest oxygenic phototrophs, but it is difficult to understand the regulation of their photosynthesis because the photosynthetic and respiratory processes share the same thylakoid membranes and cytosolic space. This review aims to summarize the molecular mechanisms and in vivo activities of electron transport in cyanobacterial thylakoid membranes based on the latest progress in photosynthesis research in cyanobacteria. Photosynthetic linear electron transport for CO2 assimilation is the dominant electron flux in the thylakoid membranes. The capacity for O2 photoreduction mediated by flavodiiron proteins is comparable to that for photosynthetic CO2 assimilation in cyanobacteria. Additionally, cyanobacterial thylakoid membranes harbour the significant electron flux of respiratory electron transport through a homologue of respiratory complex I, which is also recognized as forming part of the cyclic electron transport chain if it is coupled with photosystem I in the light. Further, O2-independent alternative electron transport through hydrogenase and nitrate reductase function with reduced ferredoxin as the electron donor. Whereas all these electron transport chains are understood individually, the regulatory complexity of the whole system remains to be uncovered in the near future.

Comment on J Exp Bot. 2023 Jun 27;74(12):3651-3666.

Due to their unique properties, nanomaterials behave peculiarly in biosystems. Regarding plants, the interactions of nanomaterials can be interpreted on a spatial scale: from local interactions in cells to systemic effects on whole plants and on ecosystems. Interpreted on a time scale, the effects of nanomaterials on plants may be immediate or subsequent. At the cellular level, the composition and structure of the cell wall and membranes are modified by nanomaterials, promoting internalization. The effects of nanomaterials on germination and seedling physiology and on the primary and secondary metabolism in the shoot are realized at organ and organism levels. Nanomaterials interact with the beneficial ecological partners of plants. The effects of nanomaterials on plant growth-promoting rhizobacteria and legume-rhizobia symbiosis can be stimulating or inhibitory, depending on the concentration and type of nanomaterial. Nanomaterials exert a negative effect on arbuscular mycorrhiza, and vice versa. Pollinators are exposed to nanomaterials, which may affect plant reproduction. The substances released by the roots influence the availability of nanomaterials in the rhizosphere, and components of plant cells trigger internalization, translocation, and transformation of nanomaterials. Understanding of the multilevel and bidirectional relationship between plants and nanomaterials is of great relevance.

Comment on J Exp Bot. 2023 Jun 27;74(12):3488-3502.

The fusion of male and female gametes is a fundamental process in the perpetuation and diversification of species. During the last 50 years, significant efforts have been made to isolate and characterize sperm cells from flowering plants, and to identify how these cells interact with female gametes to achieve double fertilization. The first techniques and analytical approaches not only provided structural and biochemical characterizations of plant sperm cells but also paved the way for in vitro fertilization studies. Further technological advances then led to unique insights into sperm biology at the transcriptomic, proteomic, and epigenetic level. Starting with a historical overview of sperm cell isolation techniques, we provide examples of how these contributed to create our current knowledge of sperm cell biology, and point out remaining challenges.

PHYTOCHROME INTERACTING FACTORS (PIFs) integrate light and temperature signs to control plant growth and development. However, little is known about PIFs in crop plants such as cotton. Here, we identified 68 PIF proteins and their coding genes from an allotetraploid and three diploid ancestors. Cotton PIFs contain typical ACTIVEPHYA-BINDING (APA) and ACTIVE PHYB-BINDING (APB) motifs by which they bind to phytochrome phyA and phyB, respectively, and have a BASIC HELIX-LOOP-HELIX (bHLH) domain and a nuclear localization sequence necessary for bHLH-type transcription factors. Bioinformatics analysis showed that the promoter of each PIF gene contains multiple cis-acting elements and that the evolution of cotton genomes probably underwent loss, recombination, and tandem replication. Further observations indicated that the sensitivity of cotton PIF expression to high temperature was significantly different from that to low temperature. We found that allotetraploid Gossypium hirsutum PIF4a (GhPIF4a) was induced by high temperature. GhPIF4a promotes flowering in cotton and Arabidopsis and binds to the promoter of GhFT (G. hirsutum FLOWERING LOCUS T), and binding increased with increasing temperature. Our work identifies the evolutionary and structural characteristics and functions of PIF family members in cotton.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

Plant architecture plays a major role in flowering and therefore in crop yield. Attempts to visualize and analyse strawberry plant architecture have been few to date. Here, we developed open-source software combining two- and three-dimensional representations of plant development over time along with statistical methods to explore the variability in spatio-temporal development of plant architecture in cultivated strawberry. We applied this software to six seasonal strawberry varieties whose plants were exhaustively described monthly at the node scale. Results showed that the architectural pattern of the strawberry plant is characterized by a decrease of the module complexity between the zeroth-order module (primary crown) and higher-order modules (lateral branch crowns and extension crowns). Furthermore, for each variety, we could identify traits with a central role in determining yield, such as date of appearance and number of branches. By modeling the spatial organization of axillary meristem fate on the zeroth-order module using a hidden hybrid Markov/semi-Markov mathematical model, we further identified three zones with different probabilities of production of branch crowns, dormant buds, or stolons. This open-source software will be of value to the scientific community and breeders in studying the influence of environmental and genetic cues on strawberry architecture and yield.

Root hairs are single-celled tubular structures produced from the epidermis, which play an essential role in water and nutrient uptake from the soil. Therefore, root hair formation and elongation are controlled not only by developmental programs but also by environmental factors, enabling plants to survive under fluctuating conditions. Phytohormones are key signals that link environmental cues to developmental programs; indeed, root hair elongation is known to be controlled by auxin and ethylene. Another phytohormone, cytokinin, also affects root hair growth, while whether cytokinin is actively involved in root hair growth and, if so, how it regulates the signaling pathway governing root hair development have remained unknown. In this study, we show that the two-component system of cytokinin, which involves the B-type response regulators ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12, promotes the elongation process of root hairs. They directly up-regulate ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) encoding a basic helix-loop-helix (bHLH) transcription factor that plays a central role in root hair growth, whereas the ARR1/12-RSL4 pathway does not crosstalk with auxin or ethylene signaling. These results indicate that cytokinin signaling constitutes another input onto the regulatory module governed by RSL4, making it possible to fine-tune root hair growth in changing environments.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo, and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.

Plants respond to vegetative shade with developmental and physiological changes that are collectively known as shade avoidance syndrome (SAS). Although LONG HYPOCOTYL IN FAR-RED 1 (HFR1) is known to be a negative regulator of SAS by forming heterodimers with other basic helix-loop-helix (bHLH) transcription factors to inhibit them, its function in genome-wide transcriptional regulation has not been fully elucidated. Here, we performed RNA-sequencing analyses of Arabidopsis thaliana hfr1-5 mutant and HFR1 overexpression line [HFR1(ΔN)-OE] to comprehensively identify HFR1-regulated genes at different time points of shade treatment. We found that HFR1 mediates the trade-off between shade-induced growth and shade-repressed defence, by regulating the expression of relevant genes in the shade. Genes involved in promoting growth, such as auxin biosynthesis, transport, signalling and response were induced by shade but suppressed by HFR1 under both short and long durations of shade. Likewise, most ethylene-related genes were shade-induced and HFR1-repressed. However, shade suppressed defence-related genes, while HFR1 induced their expression, especially under long durations of shade treatment. We demonstrated that HFR1 confers increased resistance to bacterial infection under shade.

Lack of phosphorus (P) is a major environmental factor affecting rapeseed (Brassica napus. L) root growth and development. For breeding purposes, it is crucial to identify the molecular mechanisms underlying root system architecture traits that confer low-P tolerance in rapeseed. Natural variations in the glycine-rich protein gene BnGRP1 were analysed in the natural population of 400 rapeseed cultivars under low-P stress through genome-wide association study and transcriptome analysis. Based on 11 single nucleotide polymorphism mutations in the BnGRP1 sequence, 10 haplotypes (Hap) were formed. Compared with the other types, the cultivar BnGRP1Hap1 in the panel demonstrated the longest root length and heaviest root weight. BnGRP1Hap1 overexpression in rapeseed led to enhanced low-P tolerance. CRISPR/Cas9-derived BnGRP1Hap4 knockout mutations in rapeseed can lead to sensitivity to low-P stress. Furthermore, BnGRP1Hap1 influences the expression of the phosphate transporter 1 gene (PHT1) associated with P absorption. Overall, the findings of this study highlight new insights into the mechanisms of GRP1 enhancement of low-P tolerance in rapeseed.

High temperature (HT) at flowering hinders pollen shedding, but the mechanisms underlying stress-induced spikelet closure are poorly understood in maize. In this study, yield components, spikelet opening, and lodicule morphology/protein profiling upon HT stress during flowering were examined in two contrasting maize inbred lines, Chang 7-2 and Qi 319. HT induced spikelet closure and reduced pollen shed weight (PSW) and seed set in both lines, but Qi 319 had a 7-fold lower PSW than Chang 7-2, and was thus more susceptible to HT. In Qi 319, a smaller lodicule size reduced the spikelet opening rate and angle, and relatively more vascular bundles hastened lodicule shrinking compared with Chang 7-2. Lodicules were collected for proteomics analysis. In lodicules of HT-stressed plants, proteins involved in stress signals, cell wall, cell constructure, carbohydrate metabolism, and phytohormone signaling were associated with stress tolerance. HT down-regulated the expression of ADP-ribosylation factor GTPase-activating protein domain2, SNAP receptor complex member11, and sterol methyltransferase2 in Qi 319 but not in Chang 7-2, which was in good agreement with the observed changes in protein abundance. Exogenous epibrassinolide increased the spikelet opening angle and extended the duration of spikelet opening. These results suggest that dysfunction of the actin cytoskeleton and membrane remodeling induced by HT probably limits lodicule expansion. In addition, a reduction in the vascular bundles in the lodicules and application of epibrassinolide might confer spikelet tolerance to HT stress.

Extensins are hydroxyproline-rich glycoproteins (HRGPs) and generally play a structural role in cell wall integrity. In this study, we determined a novel role of tomato (Solanum lycopersicum) senescence-associated extensin1 (SAE1) in leaf senescence. Both gain-of-function and loss-of-function analyses suggest that SAE1 plays a positive role in leaf senescence in tomato. Transgenic tomato plants overexpressing the SAE1 gene (SAE1-OX) exhibited premature leaf senescence and enhanced dark-induced senescence, whereas SAE1 knock-out (SAE1-KO) plants displayed delayed development-dependent or dark-induced leaf senescence. Heterologous overexpression of SAE1 in Arabidopsis also led to premature leaf senescence and enhanced dark-induced senescence. In addition, the SAE1 protein interacted with the tomato ubiquitin ligase SlSINA4 and SlSINA4 promoted SAE1 degradation in a ligase-dependent manner when co-expressed in Nicotiana benthamiana leaves, suggesting SlSINA4 controls SAE1 protein levels via the ubiquitin-proteasome pathway (UPS). Consistently, introduction of the SlSINA4 overexpression construct into the SAE1-OX tomato completely eliminated accumulation of SAE1 protein and suppressed the phenotypes conferred by overexpression of SAE1. Taken together, our data suggest that the tomato extensin SAE1plays a positive role in leaf senescence and is regulated by the ubiquitin ligase SlSINA4.

Pepper (Capsicum annuum) employs distinct defence responses against Ralstonia solanacearum infection (RSI); however, the mechanisms by which pepper activates these defence responses in a context-dependent manner is unclear. Here we study pepper plants defence response to RSI under room temperature-high humidity (RSRT, 28 °C / 90%) and high temperature-high humidity (RSHT, 37 °C / 90%) conditions, and non-infected plants under high temperature-high humidity (HTHH, 42 °C / 90%) stress. Herein, we found that the MADS-box transcription factor CaAGL8 was up-regulated by HTHH stress and RSRT or RSHT, and its silencing significantly reduced pepper thermotolerance and susceptibility to infection under both room and high temperature-high humidity (RSRT and RSHT). This was coupled with down-regulation of CaSTH2 and CaDEF1 upon RSRT, down-regulation of CaMgst3 and CaPRP1 upon RSHT, and down-regulation of CaHSP24 upon HTHH. In contrast, the ectopic overexpression of CaAGL8 significantly increased the resistance of Nicotiana benthamiana plants to RSRT, RSHT, and HTHH. In addition, CaAGL8 was found to interact with CaSWC4, which acted as a positive regulator of the pepper response to RSRT, RSHT, and HTHH. Silencing of either CaAGL8 or CaSWC4 blocked the hypersensitive response (HR) cell death and context-dependent up-regulation of defence-related genes triggered by the other. Importantly, enrichment of H4K5Ac, H3K9Ac, H3K4me3, and H3K9me2 on the tested defence-related genes was context- and gene-specifically regulated through synergistic interaction between CaSWC4 and CaAGL8. Our results indicate that pepper employs CaAGL8 to modulate chromatin remodelling by interacting with CaSWC4, thereby activating defence responses to RSRT, RSHT, and HTHH.

Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, we described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid between N. tabacum and Physochlaina orientalis, and re-analyzed the somatic hybrids that we previously generated. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them (~80%) were asymmetrical, consistent with the break-induced replication pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. Break-induced replication is the main pathway of interparental recombination in somatic hybrid mitochondria. Findings of this study are relevant to mitogenome editing assays and to mechanistic aspects of DNA integration following mitochondrial DNA horizontal transfer events.

The corn leaf aphid (Rhopalosiphum maidis) is a major maize pest that frequently causes substantial yield losses. Exploring the genetic basis of resistance to aphids is important for improving maize yield and quality. Here, we used a maize recombinant inbred line population derived from two parents with different susceptibility to aphids, B73 (susceptible) and Abe2 (resistant), and performed quantitative trait locus (QTL) mapping using aphid resistance scores as an indicator. We mapped a stable QTL, qRTA6, to chromosome 6 using data from 2 years of field trials, which explained 40.12-55.17% of the phenotypic variation. To further investigate the mechanism of aphid resistance in Abe2, we constructed transcriptome and metabolome libraries from Abe2 and B73 leaves with or without aphid infestation at different time points. Integrating QTL mapping and transcriptome data revealed three aphid resistance candidate genes (Zm00001d035736, Zm00001d035751, and Zm00001d035767) associated with the hypersensitive response, the jasmonic acid pathway, and protein ubiquitination. Integrated transcriptomic and metabolomic analysis revealed that the differentially expressed genes and metabolites were enriched in flavonoid biosynthesis. These findings extend our understanding of the molecular mechanisms controlling aphid resistance in maize, and the QTL and candidate genes are valuable resources for increasing this resistance.

Grain chalkiness is an important index of rice appearance quality and is negatively associated with rice processing and eating quality. However, the genetic mechanism underlying chalkiness formation is largely unknown. To identify the genetic basis of chalkiness, 410 recombinant inbred lines (RILs) derived from two representative indica rice varieties, Shuhui498 (R498) and Yihui3551 (R3551), were used to discover quantitative trait loci (QTLs). The two parental lines and RILs were grown in three locations in China under three controlled fertilizer application levels. Analyses indicated that chalkiness was significantly affected by genotype, the environment, and the interaction between the two, and that heritability was high. Several QTLs were isolated, including the two stable QTLs qCGP6 and qCGP8. Fine mapping and candidate gene verification of qCGP6 showed that Wx may play a key role in chalkiness formation. Chromosomal segment substitution lines (CSSLs) and near-isogenic lines (NILs) carrying the Wxa or Wxin allele produced more chalky grain than the R498 parent. A similar result was also observed in the 3611 background. Notably, the effect of the Wx genotype on rice chalkiness was shown to be dependent on environmental conditions, and Wx alleles exhibited different sensitivities to shading treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), the Wxa promoter region was successfully edited; down-regulating Wx alleviates chalkiness formation in NILR498-Wxa. This study developed a new strategy for synergistic improvement of eating and appearance qualities in rice, and created a novel Wx allele with great potential in breeding applications.

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

In flowering plants, floral induction signals intersect at the shoot apex to modulate meristem determinacy and growth form. Here, we report a single-nucleus RNA sequence analysis of litchi apical buds at different developmental stages. A total of 41 641 nuclei expressing 21 402 genes were analyzed, revealing 35 cell clusters corresponding to 12 broad populations. We identify genes associated with floral transition and propose a model that profiles the key events associated with litchi floral meristem identity by analyzing 567 identified floral meristem cells at single cell resolution. Interestingly, single-nucleus RNA-sequencing data indicated that all putative FT and TFL1 genes were not expressed in bud nuclei, but significant expression was detected in bud samples by RT-PCR. Based on the expression patterns and gene silencing results, we highlight the critical role of LcTFL1-2 in inhibiting flowering and propose that the LcFT1/LcTFL1-2 expression ratio may determine the success of floral transition. In addition, the transport of LcFT1 and LcTFL1-2 mRNA from the leaf to the shoot apical meristem is proposed based on in situ and dot-blot hybridization results. These findings allow a more comprehensive understanding of the molecular events during the litchi floral transition, as well as the identification of new regulators.

In response to changing environmental conditions, plants activate cellular responses to enable them to adapt to these changes. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key insights into how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.

Endoreduplication is the major source of somatic endopolyploidy in higher plants, leading to variation in cell ploidy levels due to iterative rounds of DNA synthesis in the absence of mitosis. Despite its ubiquitous occurrence in many plant organs, tissues and cells, the physiological meaning of endoreduplication is not fully understood, although several roles during plant development have been proposed, mostly related to cell growth, cell differentiation and specialization via transcriptional and metabolic reprogramming. Here we review the recent advances in the knowledge of the molecular mechanisms and cellular characteristics of endoreduplicated cells, and provide an overview of the multi-scale effects of endoreduplication on supporting growth in plant development. Finally, the effects of endoreduplication in fruit development are discussed, since during fruit organogenesis, endoreduplication is highly prominent where it acts as a morphogenetic factor supporting rapid fruit growth as illustrated with case of the fleshy fruit model tomato (Solanum lycopersicum).

Vascular tissues serve a dual function in plants providing both physical support as well as controlling the transport of nutrients, water, hormones and other small signaling molecules. Xylem tissues transport water from root to shoot; phloem tissues transfer photosynthates from shoot to root; while divisions of the (pro)cambium increase the number of xylem and phloem cells. Although vascular development constitutes a continuous process from primary growth in the early embryo and meristem regions to secondary growth in the mature plant organs, it can be artificially separated into distinct processes including cell type specification, proliferation, patterning and differentiation. In this review, we focus our attention to how hormonal signals orchestrate the molecular regulation of vascular development in the Arabidopsis thaliana primary root meristem. Although auxin and cytokinin have taken center stage in this aspect since their discovery, other hormones including brassinosteroids, abscisic acid and jasmonic acid are also taking up leading roles during vascular development. All these hormonal cues synergistically or antagonistically participate in the development of vascular tissues, forming a complex hormonal control network.

Tomato fruit (Solanum lycopersicum) store carbon as starch during early development and mobilize it at the onset of ripening. Starch accumulation has been suggested to buffer fluctuations in carbon supply to the fruit under abiotic stress, and contribute to sugar levels in ripe fruit. However, the role of starch accumulation and metabolism during fruit development is still unclear. Here we show that the tomato mutant adpressa (adp) harbors a mutation in a gene encoding the small subunit of ADP-glucose pyrophosphorylase (AGPase), which abolishes starch synthesis. The disruption of starch biosynthesis causes major transcriptional and metabolic remodeling in adp fruit but only minor effects on fruit size and ripening. Changes in gene expression and metabolite profiles indicate that the lack of carbon flow into starch increases levels of soluble sugars during fruit growth, triggers a readjustment of central carbohydrate and lipid metabolism, and activates growth and stress protection pathways. Accordingly, adp fruits are remarkably resistant to blossom-end rot, a common physiological disorder induced by environmental stress. Our results provide insights into the effects of perturbations of carbohydrate metabolism on tomato fruit development, with potential implications for the enhancement of protective mechanisms against abiotic stress in fleshy fruit.

Fruit set is the process by which the ovary develops into a fruit and is an important factor in determining the fruit yield. Fruit set is induced by two hormones, auxin and gibberellin, and the activation of their signaling pathways, partly by suppressing various negative regulators. Many studies have investigated the structural changes and gene networks in the ovary during fruit set, revealing the cytological and molecular mechanisms. In tomato (Solanum lycopersicum), SlIAA9 and SlDELLA/PROCERA act as auxin and gibberellin repressors, respectively, and are important regulators of the activity of transcription factors and downstream gene expression involved in fruit set. Upon pollination, SlIAA9 and SlDELLA are degraded, which subsequently activates downstream cascades and mainly contributes to active cell division and cell elongation, respectively, in ovaries during fruit setting. According to current knowledge, gibberellin appears to be most downstream in fruit set induction, and therefore its role in fruit set has been extensively explored. Furthermore, multi-omics analysis has revealed the detailed dynamics of gene fluctuation and metabolites downstream of gibberellins, highlighting the rapid activation of central carbon metabolism. This review will outline the relevant mechanisms at the molecular and metabolic levels during fruit set, particularly focusing on these mechanisms in tomato.

Fruit quality traits are determined to a large extent by their metabolome. The metabolite content of climacteric fruit changes drastically during ripening and postharvest storage and has been investigated extensively. However, the spatial distribution of metabolites and how it changes in time has received much less attention as fruit are usually considered as homogenous plant organs. Yet, spatio-temporal changes of starch, which is hydrolysed during ripening, has been used for ages as a ripening index. As vascular transport of water, and, hence, convective transport of metabolites slows down in mature fruit and even stalls after detachment, spatio-temporal changes in their concentration are likely affected by diffusive transport of gaseous molecules that act as substrate (O2), inhibitor (CO2) or regulator (ethylene, NO) of the metabolic pathways that are active during climacteric ripening. In this review we discuss such spatio-temporal changes of the metabolome and how they are affected by transport of metabolic gases and gaseous hormones. As there are currently no measurement techniques available to measure the metabolite distribution repeatedly by nondestructive means, we introduce reaction-diffusion models as an in silico tool to compute it. We show how the different components of such a model can be integrated and used to better understand the role of spatio-temporal changes of the metabolome in ripening and postharvest storage of climacteric fruit that is detached from the plant and discuss future research needs.

Root-knot nematodes (RKN) from genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression and auxin is known to be a key player in their development. However, little is known how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their microRNA167 regulators, were identified as robust gene/miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-GUS fusions showed the upregulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.