Twenty-five years ago, a seminal paper demonstrated that warm temperatures increase auxin levels to promote hypocotyl growth in Arabidopsis thaliana. Here we highlight recent advances in auxin-mediated thermomorphogenesis and identify unanswered questions. In the warmth, PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF7 bind the YUCCA 8 gene promoter and, in concert with histone modifications, enhance its expression to increase auxin synthesis in the cotyledons. Once transported to the hypocotyl, auxin promotes cell elongation. The meta-analysis of expression of auxin-related genes in seedlings exposed to temperatures ranging from cold to hot shows complex patterns of response. Changes in auxin only partially account for these responses. The expression of many SMALL AUXIN UP RNA (SAUR) genes reaches a maximum in the warmth, decreasing towards both temperature extremes in correlation with the rate of hypocotyl growth. Warm temperatures enhance primary root growth, the response requires auxin, and the hormone levels increase in the root tip but the impacts on cell division and cell expansion are not clear. A deeper understanding of auxin-mediated temperature control of plant architecture is necessary to face the challenge of global warming.

Every living organism on Earth depends on its interactions with other organisms to live. In the rhizosphere, plants and microorganisms constantly exchange signals and influence each other's behavior. Recent studies have shown that many beneficial rhizosphere microbes can produce specific signaling molecules that affect plant root architecture and may therefore have substantial effects on aboveground growth. This review sorts out these chemical signals and summarizes their mechanisms of action, enhancing our understanding of plant-microbe interactions and providing references for the comprehensive development and utilization of these active components in agricultural production. Finally, we have pointed out future research directions and challenges, such as the searching of microbial signals to induce the primary root development.

Eggplant (Solanum melongena) is a major vegetable crop with great potential for genetic improvement owing to its large and mostly untapped genetic diversity. Eggplant is closely related to over 500 species of Solanum subgenus Leptostemonum, belonging to its primary, secondary, and tertiary genepools, and exhibits a wide range of characteristics, including adaptive traits to climate change, that are useful for eggplant breeding. Germplasm banks worldwide hold more than 19,000 accessions of eggplant and related species, most of which have yet to be evaluated. Nonetheless, eggplant breeding using the cultivated S. melongena genepool has yielded significantly improved varieties. To overcome current breeding challenges and adaptation to climate change, a qualitative leap forward in eggplant breeding is necessary. The initial findings from introgression breeding in eggplant indicate that unleashing the diversity present in eggplant relatives can greatly contribute to a revolution in eggplant breeding. The recent creation of new genetic resources, such as mutant libraries, core collections, recombinant inbred lines (RILs), and sets of introgression lines (ILs) will be another crucial element for an eggplant breeding revolution, which will require the support of new genomics tools and biotechnological developments. The systematic utilization of eggplant genetic resources supported by international initiatives will be critical for a much-needed eggplant breeding revolution to address the challenges posed by climate change.

Genome-wide association study (GWAS) has improved our understanding of complex traits, but challenges exist in distinguishing causation versus association caused by linkage disequilibrium. Instead, the transcriptome-wide association study (TWAS) detects direct associations between expression levels and phenotypic variations, providing an opportunity to better prioritize candidate genes. To assess the feasibility of TWAS, we investigated the association among transcriptomes, genomes, and various traits, including flowering time in Arabidopsis. First, the associated genes formerly known to regulate growth allometry or metabolite production were identified by TWAS. Then, for flowering time, six TWAS-newly identified genes were functionally validated. Analysis of the expression quantitative trait locus (eQTL) further revealed a trans-regulatory hotspot affecting the expression of several TWAS-identified genes. The hotspot covers the FRIGIDA (FRI) gene body, which possesses multiple haplotypes differentially affecting the expression of downstream genes, such as FLOWERING LOCUS C (FLC) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). We also revealed multiple independent paths towards the loss of FRI function in natural accessions. Altogether, this study demonstrates the potential of combining TWAS with eQTL analysis to identify important regulatory modules of the FRI-FLC-SOC1 for quantitative traits in natural populations.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO2. Although the atomic resolution structure and basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording chlorophyll-a fluorescence induction kinetics (ChlF). According to the 'mainstream' model, the rise from the minimum level (Fo) to the maximum (Fm) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the Fv/Fm ratio is equated with the maximum photochemical quantum yield of PSII (Fv=Fm-Fo). However, this model has never been free of controversies. A range of recent experimental data confirmed that the first single-turnover saturating flash (STSF), generating closed state (PSIIC), produces F1<Fm; and uncovered rate-limiting steps - Δτ1/2 half-waiting times - in the multi-STSF induced F1-to-Fm increments, originating from the gradual formation of light-adapted charge-separated state (PSIIL) with significantly increased stability of charges compared to PSIIC elicited by a single STSF. All data show that the interpretation of ChlF must be laid on new grounds. Here we discuss the underlying physical mechanisms and the significance of structural/functional dynamics of PSII - as reflected by ChlF and variations of the novel parameter Δτ1/2.

Although many plant cell types are capable of producing hormones and plant hormones can in most cases act in the same cells in which they are produced, they also act as signaling molecules that coordinate physiological responses between different parts of the plant, indicating that their action is subject to spatial regulation. Numerous publications have reported that all levels of plant hormonal pathways, i.e., metabolism, transport, and perception/signal transduction, can help determine the spatial ranges of hormone action. For example, polar auxin transport or localized auxin biosynthesis contribute to creating a differential hormone accumulation across tissues that is instrumental for specific growth and developmental responses. On the other hand, tissue specificity of cytokinin actions has been proposed to be regulated by mechanisms operating at the signaling stages. Here, we review and discuss current knowledge about the contribution of the three levels mentioned above in providing spatial specificity to plant hormone action. We also explore how new technological developments, such as plant hormone sensors based on FRET or single-cell RNA-seq, can provide an unprecedented level of resolution in defining the spatial domains of plant hormone action and its dynamics.

Transcriptional regulation is crucial to control of gene expression. Both spatio-temporal expression patterns and expression levels of genes are determined by the interaction between cis-acting elements and trans-acting factors. Numerous studies have focused on the trans-acting factors that mediate transcriptional regulatory networks. However, cis-acting elements, such as enhancers, silencers, transposons, and natural variations in the genome, are also vital for gene expression regulation and could be utilized by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing to improve crop quality and yield. In this review, we discuss the current understanding of cis-element-mediated transcriptional regulation in major crops, including rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays), as well as the latest advancements in gene editing techniques and their applications in crops to highlight prospective strategies for crop breeding.

Switchgrass, a forage and bioenergy crop, occurs as two main ecotypes with different but overlapping ranges of adaptation. The two ecotypes differ in a range of characteristics, including flowering time. Flowering time determines the duration of vegetative development and therefore biomass accumulation, a key trait in bioenergy crops. No causal variants for flowering time differences between switchgrass ecotypes have, as yet, been identified. In this study, we mapped a robust flowering time quantitative trait locus (QTL) on chromosome 4K in a biparental F2 population and characterized the flowering-associated transcription factor PvHd1, an ortholog of CONSTANS in Arabidopsis and Heading date 1 in rice, as the underlying causal gene. Protein modeling predicted that a serine to glycine substitution at position 35 (p.S35G) in B-Box domain 1 greatly altered the global structure of the PvHd1 protein. The predicted variation in protein compactness was supported in vitro by a 4ºC-shift in denaturation temperature. Overexpressing the PvHd1-p.35S allele in a late-flowering CONSTANS-null Arabidopsis mutant rescued earlier flowering, whereas PvHd1-p.35G had a reduced ability to promote flowering, demonstrating that the structural variation led to functional divergence. Our findings provide us with a tool to manipulate the timing of floral transition in switchgrass cultivars and, potentially, expand their cultivation range.

The phytohormones strigolactones crosstalk with abscisic acid (ABA) in acclimation to osmotic stress, as ascertained in leaves. However, our knowledge about underground tissues is limited, and null in Arabidopsis. Namely, if strigolactones affect ABA transport across plasma membranes has never been addressed. We evaluated the effect of strigolactones on the localisation of ATP BINDING CASSETTE G25 (ABCG25), an ABA exporter in Arabidopsis thaliana. Wild-type, strigolactone-insensitive and -depleted seedlings expressing a GFP:ABCG25 construct were treated with ABA or strigolactones, and GFP was quantified by confocal microscopy in different subcellular compartments of epidermal root cells. We show that strigolactones promote the localisation of an ABA transporter at the plasma membrane by enhancing its endosomal recycling. Genotypes altered in strigolactone synthesis or perception are not impaired in ABCG25 recycling promotion by ABA, which acts downstream or independent of strigolactones in this respect. Additionally, we confirm that osmotic stress decreases strigolactone synthesis in A. thaliana root cells; and that such decrease may support local ABA retention under low water availability, by allowing ABCG25 internalisation. Thus, a new mechanism for ABA homeostasis regulation is proposed in the context of osmotic stress acclimation: the fine tuning by strigolactones of ABCG25 localisation in root cells.

Fruit ripening is a complex and highly regulated process where tomato and strawberry have been the model species classically used for studying climacteric and non-climacteric fleshy fruit ripening types, respectively. Melon has emerged as an alternative ripening model because climacteric and non-climacteric cultivars exist, which makes it possible to dissect the regulation of ripening using a genetic approach. Several QTLs that regulate climacteric fruit ripening have been identified to date, and their combination in both climacteric and non-climacteric genetic backgrounds resulted in lines with different ripening behaviors, demonstrating that the climacteric intensity can be genetically modulated. The review discusses our current knowledge of the physiological changes observed during melon climacteric fruit ripening as ethylene production, fruit abscission, chlorophyll degradation, firmness and aroma, as well as their complex genetic control. From pioneer experiments in which ethylene biosynthesis was silenced, to the recent genetic edition of ripening regulators, current data suggest that the climacteric response is determined by the interaction of several loci under quantitative inheritance. The exploitation of the rich genetic diversity of melon will enable the discovery of additional genes involved in the regulation of the climacteric response, ultimately leading to breeding aromatic melon fruits with extended shelf life.

Phenolic acids are the main active ingredients in Salvia miltiorrhiza, which can be used for the treatment of many diseases, particularly cardiovascular diseases. It is known that salicylic acid (SA) can enhance phenolic acid content, but the molecular mechanism of its regulation is still unclear. The nonexpresser of PR genes 1 (NPR1) plays a positive role in the SA signaling pathway. In this study, we identified a SmNPR1 gene that responds to SA induction and systematically investigated its function. We found that SmNPR1 positively affected phenolic acid biosynthesis. Then, we identified a novel TGA transcription factor, SmTGA2, which interacts with SmNPR1. SmTGA2 positively regulates phenolic acid biosynthesis by directly up-regulating SmCYP98A14 expression. After double-gene transgenic analysis and other biochemical assays, it was found that SmNPR1 and SmTGA2 work synergistically to regulate phenolic acid biosynthesis. In addition, SmNPR4 forms a heterodimer with SmNPR1 to inhibit the function of SmNPR1, and SA can alleviate this effect. Collectively, these findings elucidate the molecular mechanism underlying the regulation of phenolic acid biosynthesis by SmNPR1-SmTGA2/SmNPR4 modules and provide novel insights into the study of the SA signaling pathway regulating plant secondary metabolism.

The nuclear lamina in plant cells is composed of plant-specific proteins, including Nuclear Matrix Constituent Proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and helps stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared to cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared to Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Plant architecture imposes a large impact on crop yield. IDEAL PLANT ARCHITECTURE 1 (IPA1), which encodes a SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor, is a target of molecular design for improving grain yield. However, the roles of SPL transcription factors in regulating tomato (Solanum lycopersicum) plant architecture are unclear. Here, we show that the expression of SPL13 is downregulated in the lateral buds of strigolactones (SLs)-deficient ccd mutants and is induced by GR24 (a synthetic analogue of SL). Knockout of SPL13 by CRISPR/Cas9 resulted in higher levels of cytokinins (CKs) and transcripts of CK synthesis gene ISOPENTENYL TRANSFERASES 1 (IPT1) in the stem nodes and more growth of lateral buds. GR24 suppresses CKs synthesis and lateral bud growth in ccd mutants but is not effective in spl13 mutants. Meanwhile, silencing of IPT1 gene inhibited bud growth of spl13 mutants. Interestingly, SLs levels in root extracts and exudates are significantly increased in spl13 mutants. Molecular studies indicated that SPL13 directly represses the transcription of IPT1 and the SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and MORE AXILLARY GROWTH 1 (MAX1). The results demonstrate that SPL13 acts downstream of SL to suppress lateral bud growth by inhibiting CKs synthesis in tomato. Tuning the expression of SPL13 is a potential approach for decreasing the number of lateral shoots in tomato.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, as they do in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens, especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

Phytohormones play a central role in plant development and environmental responses. Auxin is a classical hormone that is required for organ formation, tissue patterning, and defense responses. Auxin pathways have been extensively studied across numerous land plant lineages, including bryophytes and eudicots. In contrast, our understanding of the roles of auxin in maize morphogenesis and immune responses are limited. Here, we will review evidence for auxin-mediated processes in maize and describe promising areas for future research in the auxin field. Several recent transcriptomic and genetic studies have demonstrated that auxin is a key influencer of both vegetative and reproductive development in maize (namely roots, leaves and kernels). Auxin signaling has been implicated in both maize shoot architecture and immune responses through genetic and molecular analyses of the conserved co-repressor RAMOSA ENHANCER LOCUS2. Polar auxin transport is linked to maize drought responses, root growth, shoot formation, and leaf morphogenesis. Notably, maize has been a key system for delineating auxin biosynthetic pathways and offers many opportunities for future investigations on auxin metabolism. In addition, crosstalk between auxin and other phytohormones has been uncovered through gene expression studies and are important for leaf and root development in maize. Collectively these studies point to auxin as a cornerstone for maize biology that could be leveraged for improved crop resilience and yield.

Mitochondria is a semi-autonomous organelle that provides energy for cell activities through oxidative phosphorylation. In this study, we identified a defective kernel 66 (dek66)-mutant maize with defective kernels. We characterized the candidate gene DEK66, encoding a ribosomal assembly factor, locating in mitochondria, and possessing GTPase activity (the GTPase belonged to ribosome biogenesis GTPase A family). In the dek66 mutant, abolishment of mitochondrial structure and function led to the accumulation of reactive oxygen species and promoted programmed cell death in endosperm cells. Furthermore, transcript level of most of the key genes associated with nutrient storage, mitochondrial respiratory chain complex, and mitochondrial ribosomes in the dek66 mutant were significantly altered. Collectively, the results suggested that DEK66 is essential for the development of maize kernels via affecting the mitochondrial function. This study provided a reference to understand the impact of mitochondrial ribosomal assembly factor in maize kernel development.

Parasitic plants invade their host through their invasive organ, the haustoria. This organ connects to the vasculature of the host roots and hijacks water and nutrients. Although plant parasitism has evolved independently in plants, haustoria formation follows a similar mechanism throughout different plant species, highlighting the developmental plasticity of plant tissues. Here, we compare three types of haustoria formed by root and shoot in the plant parasites Striga and Cuscuta. We discuss mechanisms underlying the interactions with their hosts and how different approaches have contributed to major understanding of haustoria formation and host invasion. We also illustrate the role of auxin and cytokinin in controlling this process.

Photorespiration has been considered a 'futile' cycle in C3 plants, necessary to detoxify and recycle the metabolites generated by the oxygenating activity of RUBISCO. However, several reports indicate that this metabolic route plays a fundamental role in plant metabolism and constitutes a very interesting topic of research. Many open questions still remain in regard to photorespiration. One of these questions is how the photorespiratory process is regulated in plants and what factors contribute to this regulation. In this paper, we summarize the progress made on this topic. Here, we review recent advances in the regulation of the photorespiratory pathway with a special focus on the transcriptional and posttranslational regulation of photorespiration and the interconnections of this process with nitrogen and sulfur metabolism. Recent findings on sulfide signaling and protein persulfidation are described.

Diel regulation of protein levels and protein modification had been less studied than transcript rhythms. Here, we compare transcriptome data under light-dark cycles to partial proteome and phosphoproteome data, assayed using shotgun mass-spectrometry, from the alga Ostreococcus tauri, the smallest free-living eukaryote. 10% of quantified proteins but two-thirds of phosphoproteins were rhythmic. Mathematical modelling showed that light-stimulated protein synthesis can account for the observed clustering of protein peaks in the daytime. Prompted by night-peaking and apparently dark-stable proteins, we also tested cultures under prolonged darkness, where the proteome changed less than under the diel cycle. Among the dark-stable proteins were prasinophyte-specific sequences that were also reported to accumulate when O. tauri formed lipid droplets. In the phosphoproteome, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation suggests that a clock-regulated phospho-dawn prepares green cells for daytime functions. Acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase, implicating the clock-related, casein kinases 1 and 2 in phase-specific regulation, alternating with the CMGC protein kinase family. Understanding the dynamic phosphoprotein network should be facilitated by the minimal kinome and proteome of O. tauri. The data are available from ProteomeXchange, with identifiers PXD001734, PXD001735 and PXD002909.

Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia-resistant and light-dependent lesion mimic mutant from an EMS-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, which mapped onto chromosome C04 by bulked segregant analysis (BSA) within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance (SAR), which is consistent with the higher salicylic acid levels in lmm1. Combining of BSA and transcriptome, we identified a significantly up-regulated gene BnaC4.PR2, which encodes β-1,3-glucanase, as the candidate gene. Overexpression of BnaC4.PR2 may induce reactive oxygen species burst to trigger partial cell death and SAR. Our study provides a new genetic resource for S. sclerotiorum resistance as well as fresh insights into disease resistance breeding in B. napus.

Diversification and breeding following domestication and current climate change across the globe are the two most significant events experienced by major crops. Diversification of crops from their wild ancestors has favored dramatic changes in the plant's sensitivity to environment, significantly in transducing light inputs to the circadian clock, which allowed the growth of major crops in the shorter growing season in the Northern hemisphere. Historically, mutants and quantitative trait loci (QTL) mapping have facilitated the identification and cloning of genes underlying major changes of the clock and flowering regulation. Recent studies suggest that thermal plasticity of circadian clock output, not just the core genes that follow temperature compensation, was also under selection during diversification and breeding. Wild alleles that accelerate output rhythmicity could be beneficial for crop resilience. Furthermore, wild alleles with beneficial and flowering-independent effects under stress indicate their possible role in maintaining a balanced source-sink relationship, allowing productivity under climatic change. Because the chloroplast genome also regulates the plasticity of the clock output, mapping populations including cytonuclear interactions should be utilized in an integrated field and clock phenomics framework. We highlight the need to integrate physiological and developmental approaches (Physio-Devo) to gain a better understanding while re-domesticating wild gene alleles into modern cultivars to increase their robustness under abiotic heat and drought stresses.

The ureides allantoin and allantoate serve as nitrogen (N) transport compounds in plants, and more recently, allantoin has been shown to play a role in signaling. In planta, ureide tissue levels are controlled by the activity of enzymes of the purine degradation pathway and by ureide transporters called UPS (ureide permease). Still little is known about the physiological function of UPS proteins in crop plants, and especially in monocotyledon species. Here, we identified 13 TaUPS genes in the wheat (Triticum aestivum L.) genome. Phylogenetic and genome location analyses revealed a close relationship of wheat UPSs to orthologues of grasses and a division into TaUPS1, TaUPS2.1 and TaUPS2.2 groups, each existing of three homeologs, and with a total of four tandem duplications. Expression, localization, and biochemical analyses resolved spatial-temporal expression patterns of TaUPS genes, transporter localization to the plasma membrane, and a role of TaUPS2.1 proteins in cellular import of ureides, and phloem and seed loading. In addition, positive correlations between TaUPS1 and TaUPS2.1 transcripts, and ureide levels were determined. Together the data support TaUPS function in regulating ureide pools in source and sink, and source-to-sink transport. Moreover, comparative studies between wheat cultivars grown at low and high N strengthened a role of TaUPS1 and TaUPS2.1 transporters in efficient N use and in controlling primary metabolism. Co-expression, protein-protein interaction, and haplotype analyses further support TaUPS involvement in N partitioning, N use efficiency, and domestication. Overall, this work provides important knowledge on UPS transporters in grasses and clues for breeding resilient wheat varieties with improved N use efficiency.

Abscisic acid (ABA) is critical in drought tolerance and plant growth. Group A protein type 2C phosphatases (PP2Cs) are negative regulators of ABA signaling and plant adaptation to stress. However, our knowledge about the functions of potato group A PP2Cs is limited. Here, we report that potato group A PP2C StHAB1 is broadly expressed in potato plants and strongly induced by ABA and drought. Suppression of StHAB1 enhanced potato ABA sensitivity and drought tolerance, whereas overexpression of the dominant mutant StHAB1G276D compromised ABA sensitivity and drought tolerance. StHAB1 interacts with almost all ABA receptors and the Snf1-Related Kinase OST1. Suppressing StHAB1 and overexpressing StHAB1G276D alter potato growth morphology; notably, overexpression of StHAB1G276D causes excessive shoot branching in potato. RNA-seq analyses identified that auxin efflux carrier genes, StPIN3, StPIN5, and StPIN8, were upregulated in StHAB1G276D-overexpressed axillary buds. Correspondingly, auxin concentration was reduced in StHAB1G276D-overexpressed axillary buds, consistent with the repressing role of auxin in lateral branch outgrowth. The expression of BRANCHED1s (StBRC1a and StBRC1b) does not change in StHAB1G276D-overexpressed axillary buds, suggesting that overexpression of StHAB1G276D caused axillary bud outgrowth is not due to regulating BRC1 expression. Our findings demonstrate that StHAB1 is vital in potato drought tolerance and shoot branching.

VQ motif-containing proteins play important roles in plant abiotic and biotic stresses. In this study, we cloned the VQ-protein gene TaVQ4-D that is induced by drought stress. Arabidopsis and wheat plants overexpressing TaVQ4-D showed increased tolerance to drought stress. In contrast, wheat lines in which TaVQ4-D expression had been silenced showed decreased drought tolerance. Under drought stress conditions, the contents of superoxide dismutase and proline increased and the content of malondialdehyde decreased in transgenic wheat plants overexpressing TaVQ4-D compared with WT. At the same time, the expression of ROS scavenging-related genes and stress-related genes was up-regulated. However, plants of TaVQ4-D-silenced wheat lines showed decreased activities of antioxidant enzymes and reduced expression of some stress-related and antioxidant-related genes. In addition, the TaVQ4-D protein physically interacts with two mitogen-activated protein kinases (MPK3 and MPK6) and plays a role in plant drought stress as the phosphorylated substrates of MPK3 and MPK6. In summary, the results of our study suggest that TaVQ4-D can positively regulate drought stress tolerance in wheat.

Cereals are a group of grasses cultivated by humans for their grain. It is from these cereal grains that the majority of all human calorie consumption is derived. The production of these grains is the result of the development of a series of hierarchical reproductive structures that form the distinct shoot architecture of the grasses. Being spatiotemporally complex, the coordination of grass shoot development is tightly controlled by a network of genes and signals, including the key phytohormone auxin. Hormonal manipulation has therefore been identified as a promising potential approach to increasing cereal crop yields and therefore ultimately global food security. Recent work translating the substantial body of auxin research from model plants into cereal crop species is revealing the contribution of auxin biosynthesis, transport and signalling to the development of grass shoot architecture. This review discusses this still-maturing knowledge base and examines the possibility that changes in auxin biology could have been a causative agent in the evolution of differences in shoot architecture between key grass species, or could underpin the future selective breeding of cereal crops.

Nitrogen (N) is an essential macronutrient for plants, and N remobilization is key for plant adaptation to N deficiency stress. However, there is a limited understanding of the regulatory mechanisms of N remobilization in Brassica napus. Here, we report a transcription factor, BnaA9.WRKY47, induced by N starvation. At the seedling stage, BnaA9.WRKY47-OE lines displayed earlier senescence of older leaves and preferential growth of juvenile leaves compared to the wild-type 'Westar 10' under N starvation. At the field scale, the seed yield was significantly increased in BnaA9.WRKY47-OE lines compared to 'Westar 10' grown under N deficiency conditions and, conversely, reduced in BnaA9.WRKY47-mutants. Biochemical analyses demonstrate that BnaA9.WRKY47 directly activates BnaC7.SGR1 to accelerate older leaf senescence. In line with leaf senescence, the amino acid concentration was elevated, and the N distribution was reduced in the older leaves of OE lines. This result is because BnaA9.WRKY47 activates the amino acid permease BnaA9.AAP1 and the nitrate transporter BnaA2.NRT1.7. Therefore, the expression of BnaA9.WRKY47 efficiently facilitates N remobilization from older to younger leaves or seeds. Taken together, the results of this study demonstrate that BnaA9.WRKY47 upregulates the expression of BnaC7.SGR1, BnaA2.NRT1.7 and BnaA9AAP1 thus promoting the remobilization of N in B. napus under N starvation.

The surrounding gene networks of Nod factor receptors that govern the symbiotic process remain largely unexplored. In the present study, we identified 13 novel GmNFR1α-associated proteins by Y2H screening, and a potential interacting protein GmBI-1α was described. GmBI-1α had the highst positive correlation with GmNFR1α in the co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α- GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the Endoplasmic Reticulum (ER) and Plasma Membrane (PM). Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference (RNAi) reduced the nodule number. Besides, the nodules in GmBI-1α-OX plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-OX transgenic soybean, the elevated GmBI-1α level also promoted plant growth and suppressed the expression of defense signaling-related genes. IT analysis of GmBI-1α-OX showed that GmBI-1α promoted rhizobial infection. Collectively, our current findings supported a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

Nucleotide-binding leucine-rich repeat receptors (NLRs) recognize pathogen effectors to mediate plant disease resistance often involving host cell death. Effectors escape NLR recognition through polymorphisms, allowing the pathogen to proliferate on previously resistant host plants. The powdery mildew effector AVRA13-1 is recognized by the barley NLR MLA13 and activates host cell death. We demonstrate here that a virulent form of AVRA13, called AVRA13-V2, escapes MLA13 recognition by substituting a serine for a leucine residue at the C-terminus. Counterintuitively, this substitution in AVRA13-V2 resulted in an enhanced MLA13 association and prevented the detection of AVRA13-1 by MLA13. Therefore, AVRA13-V2 is a dominant-negative form of AVRA13 and has likely contributed to the breakdown of Mla13 resistance. Despite this dominant-negative activity, AVRA13-V2 failed to suppress host cell death mediated by the MLA13 auto-active MHD variant. Neither AVRA13-1 nor AVRA13-V2 interacted with the MLA13 auto-active variant, implying that the binding moiety in MLA13 that mediates association with AVRA13-1 is altered after receptor activation. We also show that mutations in the MLA13 coiled-coil domain, which were thought to impair Ca 2+ channel activity and NLR function, instead resulted in MLA13 auto-active cell death. The data constitute an important step to define intermediate receptor conformations during NLR activation.

The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss-of-function of BTSL1 and BTSL2, which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana. Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots as the wild type, but suppressed the accumulation of excess Fe in roots. RNA-seq analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake (IRT1, FRO2, NAS) and in Zn storage (MTP3, ZIF1). Surprisingly, mutant shoots did not show the transcriptional Fe-deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe-deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn-Fe interactions in plants.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the 'design-build-test-learn' cycle, which is relatively slow in plant research. Despite their potential, challenges remain in expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new 'age of synthetic biology'.

When interacting with the environment, plant roots integrate sensory information over space and time in order to respond appropriately under non-uniform conditions. The complexity and dynamic properties of soil across spatial and temporal scales pose a significant technical challenge for research into the mechanisms that drive metabolism, growth, and development in roots, as well as on inter-organismal networks in the rhizosphere. Synthetic environments, combining microscopic access and manipulation capabilities with soil-like heterogeneity, are needed to elucidate the intriguing antagonism that characterizes subsurface ecosystems. Microdevices have provided opportunities for innovative approaches to observe, analyse, and manipulate plant roots and advanced our understanding of their development, physiology, and interactions with the environment. Initially conceived as perfusion platforms for root cultivation under hydroponic conditions, microdevice design has, in recent years, increasingly shifted to better reflect the complex growth conditions in soil. Heterogeneous micro-environments have been created through co-cultivation with microbes, laminar flow-based local stimulation, and physical obstacles and constraints. As such, structured microdevices provide an experimental entry point into the complex network behaviour of soil communities.

Gene targeting can be used to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant gene targeting. Several studies have recently demonstrated improvements in gene targeting efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified gene-targeting-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated gene targeting in plants and discuss potential efficiency improvements. Increasing the efficiency of gene targeting technology will help pave the way for increased crop yields and food safety in environmentally friendly agriculture.

Plant morphology and anatomy strongly influence agricultural yield. Crop domestication has strived for desirable growth and developmental traits, such as larger and more fruits and semi-dwarf architecture. Genetic engineering has accelerated rational, purpose-driven engineering of plant development, but it can be unpredictable. Developmental pathways are complex and riddled with environmental and hormonal inputs, as well as feedback and feedforward interactions, which occur at specific times and places in a growing multicellular organism. Rational modification of plant development would probably benefit from precision engineering based on synthetic biology approaches. This review outlines recently developed synthetic biology technologies for plant systems and highlights their potential for engineering plant growth and development. Streamlined and high-capacity genetic construction methods (Golden Gate DNA Assembly frameworks and toolkits) allow fast and variation-series cloning of multigene transgene constructs. This, together with a suite of gene regulation tools (e.g. cell type-specific promoters, logic gates, and multiplex regulation systems), is starting to enable developmental pathway engineering with predictable outcomes in model plant and crop species.

The fascination produced by the possibility of engineering plants with augmented capabilities has accompanied plant biotechnology since its origins. This prospect has become even more relevant in present times under the pressure imposed by climate change and population growth. Today's plant biotechnologists approach this challenge with the tools of synthetic biology, which facilitate the assembly of synthetic gene circuits (SGCs) from their modular components. Transcriptional SGCs take environmental or endogenous inputs and operate them using transcriptional signals in ways that do not necessarily occur in nature, generating new physiological outputs. Many genetic components have been developed over the years that can be employed in the design and construction of plant SGCs. This review aims to provide an updated view of the components available, proposing a general scheme that facilitates the classification of circuit components in sensor, processor, and actuator modules. Following this analogy, we review the latest advances in the design of SGCs and discuss the main challenges ahead.

Nitrogen is an essential macronutrient for plant growth and development, and its availability to some extent is regulated by drought stress. Calcium-dependent protein kinases (CPKs) are a unique family of Ca 2+ sensors with diverse functions in nitrogen uptake and drought tolerance signaling pathways. However, how CPKs involve in the crosstalk between drought stress and nitrogen transportation remains largely unknown. Here we identify the drought tolerant function of Arabidopsis CPK6 under high nitrogen condition. CPK6 expression is induced by the treatments of ABA and drought. The mutant cpk6 is insensitive to the ABA treatment and low nitrogen, but sensitive to drought only under high nitrogen condition. CPK6 interacts with NRT1.1 protein and phosphorylates the Thr447 residue, which then represses the NO3  - transporting activity of Arabidopsis under HN and drought stress. Taken together, we showed the evidences that CPK6 regulates Arabidopsis drought tolerance through changing NRT1.1 phosphorylation states, and that enriches the knowledge of nitrogen uptake in plants during drought stress.

Microalgae hold enormous potential to provide a safe and sustainable source of high-value compounds, acting as carbon-fixing biofactories that could help to mitigate rapidly progressing climate change. Bioengineering microalgal strains will be key to optimizing and modifying their metabolic outputs, and to render them competitive with established industrial biotechnology hosts, such as bacteria or yeast. To achieve this, precise and tuneable control over transgene expression will be essential, which would require the development and rational design of synthetic promoters as a key strategy. Among green microalgae, Chlamydomonas reinhardtii represents the reference species for bioengineering and synthetic biology; however, the repertoire of functional synthetic promoters for this species, and for microalgae generally, is limited in comparison to other commercial chassis, emphasizing the need to expand the current microalgal gene expression toolbox. Here, we discuss state-of-the-art promoter analyses, and highlight areas of research required to advance synthetic promoter development in C. reinhardtii. In particular, we exemplify high-throughput studies performed in other model systems that could be applicable to microalgae, and propose novel approaches to interrogating algal promoters. We lastly outline the major limitations hindering microalgal promoter development, while providing novel suggestions and perspectives for how to overcome them.

Freeze-thaw-induced embolism is a key limiting factor for perennial plants in frost-exposed environments. Gas bubbles are formed during freezing and expand during thawing resulting in xylem embolism. However, when water freezes, its volume increases by 9%, generating local pressures, which can affect the formation of bubbles. To characterize local dynamic of pressure-tension and physical state of the sap during freeze-thaw cycles, we simultaneously used ultrasonic acoustic emissions analysis and synchrotron-based high resolution computed tomography on the diffuse-porous species Betula pendula. Visualization of individual air-filled vessels was performed to measure freeze-thaw induced embolism after successive freeze-thaw cycles down to -10°C or -20°C during the leafy and the leafless periods. We also measured the distribution of gas bubbles in frozen xylem of Betula pendula, and made additional continuous monitoring of embolism spreading during one freeze-thaw cycle using a dedicated cooling system that allowed X-ray scanning during freezing and thawing. Experiments confirmed that ultrasonic emissions occurred after the onset of ice formation, together with bubble formation, whereas the development of embolism took place after thawing in all cases. The pictures of frozen tissues indicated that the positive pressure induced by the volumetric increase of ice can provoke inward flow from the cell wall toward the lumen of the vessels. We found no evidence that wider vessels within a tissue were more prone to embolism although the occurrence of gas bubbles in larger conduits would make them prone to earlier embolism. These results highlight the need to monitor local pressure as well as ice and air distribution during xylem freezing to understand the mechanism leading to frost-induced embolism.

Integration of reactive oxygen species (ROS)-mediated signal transduction pathways via redox sensors and the thiol-dependent signalling network is of increasing interest in cell biology for their implications in plant growth and productivity. Redox regulation is an important point of control in the protein structure, interactions, cellular location and function, thioredoxins (TRXs) and glutaredoxins (GRXs) being key players in the maintenance of cellular redox homeostasis. The crosstalk between second messengers, ROS, thiol redox signalling and redox homeostasis-related genes, controls almost every aspect of plant development and stress response. We review the emerging roles of TRXs and GRXs in redox-regulated processes interacting with other cell signalling systems such as organellar retrograde communication and gene expression, especially in plants during their development and under stressful environments. This approach will throw light on the specific role of these proteins as redox signalling components, and their importance in different developmental processes during abiotic stress.

Senescence is the final stage of plant growth and development and is a highly regulated process at the molecular, cellular, and organismal levels. When triggered by age, hormonal, or environmental cues, plants actively adjust metabolism and gene expressions to execute the progression of senescence. Proper regulation of senescence is vital for the reallocation of nutrients to sink organs to ensure reproductive success and adaptations to stresses. Identification and characterization of hallmarks of leaf senescence are of great importance for understanding the molecular regulatory mechanisms of plant senescence and breeding future crops with more desirable senescence traits. Tremendous progress has been made in elucidating the genetic network underpinning the metabolic and cellular changes in leaf senescence. In this review, we focus on three hallmarks of leaf senescence, chlorophyll and chloroplast degradation, loss of proteostasis, and activation of senescence-associated genes (SAGs), and discuss recent findings of the molecular players and the cross-talks of senescence pathways.

The circadian clock system acts as an endogenous timing reference that coordinates many metabolic and physiological processes in plants. Previous studies have shown that the application of osmotic stress delays circadian rhythms via 3'-Phospho-Adenosine 5'-Phosphate (PAP), a retrograde signalling metabolite that is produced in response to redox stress within organelles. PAP accumulation leads to the inhibition of EXORIBONUCLEASEs (XRNs), which are responsible for RNA degradation. Interestingly, we are now able to demonstrate that post-transcriptional processing is crucial for the circadian response to osmotic stress. Our data show that osmotic stress increases the stability of specific circadian RNAs, suggesting that RNA metabolism plays a vital role in circadian clock coordination during drought. Inactivation of XRN4 is sufficient to extend circadian rhythms as part of this response, with PRR7 and LWD1 identified as transcripts that are post-transcriptionally regulated to delay circadian progression.

Fruit ripening involves numerous physiological, structural and metabolic changes that result in the formation of edible fruits. This process is controlled at different molecular levels, with essential roles for phytohormones, transcription factors and epigenetic modifications. Fleshy fruits are classified as either climacteric or non-climacteric species. Climacteric fruits are characterized by a burst in respiration and ethylene production at the onset of ripening, while regulation of non-climacteric fruit ripening has been commonly attributed to abscisic acid (ABA). However, there is controversy as to whether mechanisms regulating fruit ripening are shared between non-climacteric species, and to what extent other hormones contribute alongside ABA. In this review, we summarize classic and recent studies on the accumulation profile and role of ABA and other important hormones in the regulation of non-climacteric fruit development and ripening, as well as their cross-talk, paying special attention to the two main non-climacteric plant models, strawberry and grape. We highlight both the common and different roles of these regulators in these two crops, and discuss the importance of the transcriptional and environmental regulation of fruit ripening, as well as the need to optimize genetic transformation methodologies to facilitate gene functional analyses.

Plant use different receptors to detect potential pathogens: membrane-anchored pattern-recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI), and intracellular nucleotide-binding leucine-rich repeats proteins (NLRs) proteins activated by detection of pathogen-derived effectors, triggering effector-triggered immunity (ETI). The interconnections between PTI and ETI responses are increasingly reported. Elevated NLR levels may cause autoimmunity with symptoms ranging from fitness-cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide microRNAs (miRNAs) with the unique ability to trigger secondary short interfering RNA (siRNA) production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR-target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize the current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22-nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

For many years, research has been carried out to understand the mechanism of auxin action, its biosynthesis, catabolism, perception, and transport. One central interest is understanding the auxin-dependent gene expression regulation mechanism involving AUXIN RESPONSE FACTOR (ARF) transcription factors and their repressors, the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins. Numerous studies have been focused on the MONOPTEROS (MP)/ARF5, an activator of auxin-dependent gene expression with a crucial impact on plant development. This review paper summarizes over thirty years of research on MP/ARF5. We indicate the available analytical tools to study MP/ARF5 and point out the known mechanism of MP/ARF5-dependent regulation of gene expression during various developmental processes, i.e., embryogenesis, leaf formation, vascularization, and shoot and root meristem formation. However, many questions remain about the auxin-dose-dependent regulation of gene transcription by MP/ARF5 and its isoforms in plant cells, the composition of the MP/ARF5 protein complex, and finally, the list of genes under its direct control. In addition, the information on post-translational modifications of MP/ARF5 protein is marginal, and knowledge about their consequences on MP/ARF5 function is limited. Moreover, the epigenetic factors and other regulators that act upstream of MP/ARF5 are poorly understood. Their identification will be a challenge in the coming years.

Most plant growth and development processes are regulated one way or another by auxin. The best-studied mechanism by which auxin exerts its regulatory effects is through the nuclear auxin pathway (NAP). In this pathway, AUXIN RESPONSE FACTORs (ARFs) are the transcription factors that ultimately determine which genes become auxin-regulated by binding to specific DNA sequences. ARFs have primarily been studied in Arabidopsis thaliana, but recent studies in other species have revealed family-wide DNA-binding specificities for different ARFs and the minimal functional system of the NAP system, consisting in a duo of competing ARFs of the A and B classes. In this review, we provide an overview of key aspects of ARF DNA-binding such as auxin response elements (TGTCNN) and tandem repeat motifs, and consider how structural biology and in vitro studies help us understand ARF DNA preferences. We also highlight some recent aspects related to the regulation of ARF levels inside a cell, which may alter the DNA-binding profile of ARFs in different tissues. We finally emphasize on the need to study minimal NAP systems to understand fundamental aspects of ARF function, the need to characterize algal ARFs to understand how ARFs evolved, how cutting-edge techniques can increase our understanding of ARFs, and which remaining questions can only be answered by structural biology.

Nematode migration, feeding site formation, withdrawal of plant assimilates, and activation of plant defence responses have a significant impact on plant growth and development. Plants display intraspecific variation in tolerance limits for root-feeding nematodes. Although disease tolerance has been recognised as a distinct trait in biotic interactions of mainly crops, we lack mechanistic insights. Progress is hampered by difficulties in quantification and laborious screening methods. We turned to the model plant Arabidopsis thaliana, since it offers extensive resources to study the molecular and cellular mechanisms underlying nematode-plant interactions. Through imaging of tolerance-related parameters the green canopy area was identified as an accessible and robust measure for assessing damage due to cyst nematode infection. Subsequently, a high-throughput phenotyping platform simultaneously measuring the green canopy area growth of 960 A. thaliana plants was developed. This platform can accurately measure cyst- and root-knot nematode tolerance limits in A. thaliana through classical modelling of tolerance limits. Furthermore, real-time monitoring provided data for a novel view of tolerance, identifying a compensatory growth response. These findings show that our phenotyping platform will enable further studies into a mechanistic understanding of tolerance to below-ground biotic stress.

The plant immune system is constituted by two functionally interdependent branches that provide the plant with an effective defense against microbial pathogens. They can be considered separate since one detects extracellular pathogenassociated molecular patterns by means of receptors on the plant surface, while the other detects pathogen-secreted virulence effectors via intracellular receptors. Plant defense depending on both branches can be effectively suppressed by host-adapted microbial pathogens. In this review we will focus on bacterially driven suppression of the latter, usually known as ETI for Effector-Triggered Immunity and depending on diverse NOD-like receptors, or NLRs. We will examine how some effectors secreted by pathogenic bacteria carrying Type III Secretion Systems can be subject to specific NLR-mediated detection, which can be evaded by the action of additional co-secreted effectors (suppressors), implying that virulence depends on the coordinated action of the whole repertoire of effectors of any given bacteria, and their complex epistatic interactions within the plant. We will consider how, to avoid ETI activation, suppressors can directly alter compromised cosecreted effectors, modify plant defense-associated proteins, or occasionally both. We will also comment on the potential assembly within the plant cell of multi-protein complexes comprising both bacterial effectors and defense protein targets.

Carbon concentrating mechanisms enhance the carboxylase efficiency of the central photosynthetic enzyme rubisco by providing supra-atmospheric concentrations of CO2 in its surrounding. In the C4 photosynthesis pathway, this feat is realised by combinatory changes to leaf biochemistry and anatomy. In contrast to the C4 pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 to 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation between tested plant species. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4 classified taxa indicating a crucial role of anatomical features for CO2 concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of PEPC activities and metabolite composition suggests that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct and fit photosynthesis type.

Roses are significant botanical species with both ornamental and economic value, displaying diverse floral traits, particularly an extensive array of petal colors. The red pigmentation of rose petals is predominantly attributed to anthocyanin accumulation. However, the underlying regulatory mechanism of anthocyanin biosynthesis in roses remains elusive. This study presents a novel light-responsive regulatory module governing anthocyanin biosynthesis in rose petals, which involves the transcription factors RhHY5, RhMYB114a, and RhMYB3b. Under light conditions, RhHY5 represses RhMYB3b expression, and induces RhMYB114a expression that positively regulates anthocyanin biosynthesis in rose petals through the direct activation of anthocyanin structural genes via the MYB114a-bHLH3-WD40 complex. Notably, this function likely involves an interaction and synergy between RhHY5 and the MYB114a-bHLH3-WD40 complex. Additionally, RhMYB3b is activated by RhMYB114a to prevent excessive accumulation of anthocyanin. Conversely, in low light conditions, the degradation of RhHY5 leads to down-regulation of RhMYB114a and up-regulation of RhMYB3b, which in turn inhibits the expression of both RhMYB114a and anthocyanin structural genes. Additionally, RhMYB3b competes with RhMYB114a for binding to RhbHLH3 and the promoters of anthocyanin-related structural genes. Overall, our study uncovers a complex light-mediated regulatory network that governs anthocyanin biosynthesis in the rose, thereby advancing our understanding of the underlying molecular mechanisms of anthocyanin biosynthesis in rose flowers.