Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

Hello darkness, my old friend: 3-KETOACYL-COENZYME A SYNTHASE4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis thaliana.

Plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness or extended darkness to a panel of ∼300 Arabidopsis (Arabidopsis thaliana) accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding an enzyme involved in very long chain fatty acid (VLCFA) synthesis, underlies the differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression of KCS4 in yeast and plants proved that KCS4 is a functional enzyme localized in the endoplasmic reticulum with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and leaf wax coverage, puTAG accumulation, and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlates with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation.

Metabolite-mediated adaptation of crops to drought and the acquisition of tolerance.

Drought is one of the major and growing threats to agriculture productivity and food security. Metabolites are involved in the regulation of plant responses to various environmental stresses, including drought stress. The complex drought tolerance can be ascribed to several simple metabolic traits. These traits could then be used for detecting the genetic architecture of drought tolerance. Plant metabolomes show dynamic differences when drought occurs during different developmental stages or upon different levels of drought stress. Here, we reviewed the major and most recent findings regarding the metabolite-mediated plant drought response. Recent progress in the development of drought-tolerant agents is also discussed. We provide an updated schematic overview of metabolome-driven solutions for increasing crop drought tolerance and thereby addressing an impending agricultural challenge.

Genome-wide association studies identify loci controlling specialized seed metabolites in Arabidopsis.

Plants synthesize specialized metabolites to facilitate environmental and ecological interactions. During evolution, plants diversified in their potential to synthesize these metabolites. Quantitative differences in metabolite levels of natural Arabidopsis (Arabidopsis thaliana) accessions can be employed to unravel the genetic basis for metabolic traits using genome-wide association studies (GWAS). Here, we performed metabolic GWAS on seeds of a panel of 315 A. thaliana natural accessions, including the reference genotypes C24 and Col-0, for polar and semi-polar seed metabolites using untargeted ultra-performance liquid chromatography-mass spectrometry. As a complementary approach, we performed quantitative trait locus (QTL) mapping of near-isogenic introgression lines between C24 and Col-0 for specific seed specialized metabolites. Besides common QTL between seeds and leaves, GWAS revealed seed-specific QTL for specialized metabolites, indicating differences in the genetic architecture of seeds and leaves. In seeds, aliphatic methylsulfinylalkyl and methylthioalkyl glucosinolates associated with the ALKENYL HYDROXYALKYL PRODUCING loci (GS-ALK and GS-OHP) on chromosome 4 containing alkenyl hydroxyalkyl producing 2 (AOP2) and 3 (AOP3) or with the GS-ELONG locus on chromosome 5 containing methylthioalkyl malate synthase (MAM1) and MAM3. We detected two unknown sulfur-containing compounds that were also mapped to these loci. In GWAS, some of the annotated flavonoids (kaempferol 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-rhamnoside) were mapped to transparent testa 7 (AT5G07990), encoding a cytochrome P450 75B1 monooxygenase. Three additional mass signals corresponding to quercetin-containing flavonols were mapped to UGT78D2 (AT5G17050). The association of the loci and associating metabolic features were functionally verified in knockdown mutant lines. By performing GWAS and QTL mapping, we were able to leverage variation of natural populations and parental lines to study seed specialized metabolism. The GWAS data set generated here is a high-quality resource that can be investigated in further studies.

Genome-wide association of the metabolic shifts underpinning dark-induced senescence in Arabidopsis.

Dark-induced senescence provokes profound metabolic shifts to recycle nutrients and to guarantee plant survival. To date, research on these processes has largely focused on characterizing mutants deficient in individual pathways. Here, we adopted a time-resolved genome-wide association-based approach to characterize dark-induced senescence by evaluating the photochemical efficiency and content of primary and lipid metabolites at the beginning, or after 3 or 6 days in darkness. We discovered six patterns of metabolic shifts and identified 215 associations with 81 candidate genes being involved in this process. Among these associations, we validated the roles of four genes associated with glycine, galactinol, threonine, and ornithine levels. We also demonstrated the function of threonine and galactinol catabolism during dark-induced senescence. Intriguingly, we determined that the association between tyrosine contents and TYROSINE AMINOTRANSFERASE 1 influences enzyme activity of the encoded protein and transcriptional activity of the gene under normal and dark conditions, respectively. Moreover, the single-nucleotide polymorphisms affecting the expression of THREONINE ALDOLASE 1 and the amino acid transporter gene AVT1B, respectively, only underlie the variation in threonine and glycine levels in the dark. Taken together, these results allow us to present a very detailed model of the metabolic aspects of dark-induced senescence, as well as the process itself.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Autophagy in maternal tissues contributes to Arabidopsis seed development.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development, including autophagy, which facilitates cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy affects nutrient remobilization from mother plants and functions in the embryo. However, it is impossible to distinguish between the contribution of autophagy in the source (i.e. the mother plant) and the sink tissue (i.e. the embryo) when using autophagy knockout (atg mutant) plants. To address this, we employed an approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between wild type and atg mutant Arabidopsis (Arabidopsis thaliana) plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from altered seed coat development. Our study emphasizes the importance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It also sheds light on the tissue-specific functions of autophagy, offering potential for research into the underlying mechanisms governing seed development and crop yield.

The Alteration of Tomato Chloroplast Vesiculation Positively Affects Whole-Plant Source-Sink Relations and Fruit Metabolism under Stress Conditions.

Changes in climate conditions can negatively affect the productivity of crop plants. They can induce chloroplast degradation (senescence), which leads to decreased source capacity, as well as decreased whole-plant carbon/nitrogen assimilation and allocation. The importance, contribution and mechanisms of action regulating source-tissue capacity under stress conditions in tomato (Solanum lycopersicum) are not well understood. We hypothesized that delaying chloroplast degradation by altering the activity of the tomato chloroplast vesiculation (CV) under stress would lead to more efficient use of carbon and nitrogen and to higher yields. Tomato CV is upregulated under stress conditions. Specific induction of CV in leaves at the fruit development stage resulted in stress-induced senescence and negatively affected fruit yield, without any positive effects on fruit quality. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/CAS9) knockout CV plants, generated using a near-isogenic tomato line with enhanced sink capacity, exhibited stress tolerance at both the vegetative and the reproductive stages, leading to enhanced fruit quantity, quality and harvest index. Detailed metabolic and transcriptomic network analysis of sink tissue revealed that the l-glutamine and l-arginine biosynthesis pathways are associated with stress-response conditions and also identified putative novel genes involved in tomato fruit quality under stress. Our results are the first to demonstrate the feasibility of delayed stress-induced senescence as a stress-tolerance trait in a fleshy fruit crop, to highlight the involvement of the CV pathway in the regulation of source strength under stress and to identify genes and metabolic pathways involved in increased tomato sink capacity under stress conditions.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Starch deficiency in tomato causes transcriptional reprogramming that modulates fruit development, metabolism, and stress responses.

Tomato (Solanum lycopersicum) fruit 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 that 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.

The genetics underlying metabolic signatures in a brown rice diversity panel and their vital role in human nutrition.

Brown rice (Oryza sativa) possesses various nutritionally dense bioactive phytochemicals exhibiting a wide range of antioxidant, anti-cancer, and anti-diabetic properties known to promote various human health benefits. However, despite the wide claims made about the importance of brown rice for human nutrition the underlying metabolic diversity has not been systematically explored. Non-targeted metabolite profiling of developing and mature seeds of a diverse genetic panel of 320 rice cultivars allowed quantification of 117 metabolites. The metabolite genome-wide association study (mGWAS) detected genetic variants influencing diverse metabolic targets in developing and mature seeds. We further interlinked genetic variants on chromosome 7 (6.06-6.43 Mb region) with complex epistatic genetic interactions impacting multi-dimensional nutritional targets, including complex carbohydrate starch quality, the glycemic index, antioxidant catechin, and rice grain color. Through this nutrigenomics approach rare gene bank accessions possessing genetic variants in bHLH and IPT5 genes were identified through haplotype enrichment. These variants were associated with a low glycemic index, higher catechin levels, elevated total flavonoid contents, and heightened antioxidant activity in the whole grain with elevated anti-cancer properties being confirmed in cancer cell lines. This multi-disciplinary nutrigenomics approach thus allowed us to discover the genetic basis of human health-conferring diversity in the metabolome of brown rice.

Autophagy is required for lipid homeostasis during dark-induced senescence.

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

The significance of WRKY45 transcription factor in metabolic adjustments during dark-induced leaf senescence.

Plants are constantly exposed to environmental changes that affect their performance. Metabolic adjustments are crucial to controlling energy homoeostasis and plant survival, particularly during stress. Under carbon starvation, coordinated reprogramming is initiated to adjust metabolic processes, which culminate in premature senescence. Notwithstanding, the regulatory networks that modulate transcriptional control during low energy remain poorly understood. Here, we show that the WRKY45 transcription factor is highly induced during both developmental and dark-induced senescence. The overexpression of Arabidopsis WRKY45 resulted in an early senescence phenotype characterized by a reduction of maximum photochemical efficiency of photosystem II and chlorophyll levels in the later stages of darkness. The detailed metabolic characterization showed significant changes in amino acids coupled with the accumulation of organic acids in WRKY45 overexpression lines during dark-induced senescence. Furthermore, the markedly upregulation of alternative oxidase (AOX1a, AOX1d) and electron transfer flavoprotein/ubiquinone oxidoreductase (ETFQO) genes suggested that WRKY45 is associated with a dysregulation of mitochondrial signalling and the activation of alternative respiration rather than amino acids catabolism regulation. Collectively our results provided evidence that WRKY45 is involved in the plant metabolic reprogramming following carbon starvation and highlight the potential role of WRKY45 in the modulation of mitochondrial signalling pathways.

Transcriptomic and Metabolomic Analysis of a Pseudomonas-Resistant versus a Susceptible Arabidopsis Accession.

Accessions of one plant species may show significantly different levels of susceptibility to stresses. The Arabidopsis thaliana accessions Col-0 and C24 differ significantly in their resistance to the pathogen Pseudomonas syringae pv. tomato (Pst). To help unravel the underlying mechanisms contributing to this naturally occurring variance in resistance to Pst, we analyzed changes in transcripts and compounds from primary and secondary metabolism of Col-0 and C24 at different time points after infection with Pst. Our results show that the differences in the resistance of Col-0 and C24 mainly involve mechanisms of salicylic-acid-dependent systemic acquired resistance, while responses of jasmonic-acid-dependent mechanisms are shared between the two accessions. In addition, arginine metabolism and differential activity of the biosynthesis pathways of aliphatic glucosinolates and indole glucosinolates may also contribute to the resistance. Thus, this study highlights the difference in the defense response strategies utilized by different genotypes.

Reduced Endocannabinoid Tone in Saliva of Chronic Orofacial Pain Patients.

BACKGROUND: the endocannabinoid system (ECS) participates in many physiological and pathological processes including pain generation, modulation, and sensation. Its involvement in chronic orofacial pain (OFP) in general, and the reflection of its involvement in OFP in salivary endocannabinoid (eCBs) levels in particular, has not been examined. OBJECTIVES: to evaluate the association between salivary (eCBs) levels and chronic OFP. METHODS: salivary levels of 2 eCBs, anandamide (AEA), 2-arachidonoylglycerol (2-AG), 2 endocannabinoid-like compoundsN-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), and their endogenous precursor and breakdown product, arachidonic acid (AA), were analyzed using liquid chromatography/tandem mass spectrometry in 83 chronic OFP patients and 43 pain-free controls. The chronic OFP patients were divided according to diagnosis into musculoskeletal, neurovascular/migraine, and neuropathic pain types. RESULTS: chronic OFP patients had lower levels of OEA (p = 0.02) and 2-AG (p = 0.01). Analyzing specific pain types revealed lower levels of AEA and OEA in the neurovascular group (p = 0.04, 0.02, respectively), and 2-AG in the neuropathic group compared to controls (p = 0.05). No significant differences were found between the musculoskeletal pain group and controls. Higher pain intensity was accompanied by lower levels of AA (p = 0.028), in neuropathic group. CONCLUSIONS: lower levels of eCBs were found in the saliva of chronic OFP patients compared to controls, specifically those with neurovascular/migraine, and neuropathic pain. The detection of changes in salivary endocannabinoids levels related to OFP adds a new dimension to our understanding of OFP mechanisms, and may have diagnostic as well as therapeutic implications for pain.

Model-assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways.

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom-up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint-based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.

The Acetate Pathway Supports Flavonoid and Lipid Biosynthesis in Arabidopsis.

The phenylpropanoid pathway of flavonoid biosynthesis has been the subject of considerable research attention. By contrast, the proposed polyketide pathway, also known as the acetate pathway, which provides malonyl-CoA moieties for the C2 elongation reaction catalyzed by chalcone synthase, is less well studied. Here, we identified four genes as candidates for involvement in the supply of cytosolic malonyl-CoA from the catabolism of acyl-CoA, based on coexpression analysis with other flavonoid-related genes. Two of these genes, ACC and KAT5, have been previously characterized with respect to their involvement in lipid metabolism, but no information concerning their relationship to flavonoid biosynthesis is available. To assess the occurrence and importance of the acetate pathway, we characterized the metabolomes of two mutant or transgenic Arabidopsis lines for each of the four enzymes of this putative pathway using a hierarchical approach covering primary and secondary metabolites as well as lipids. Intriguingly, not only flavonoid content but also glucosinolate content was altered in lines deficient in the acetate pathway, as were levels of lipids and most primary metabolites. We discuss these data in the context of our current understanding of flavonoids and lipid metabolism as well as with regard to improving human nutrition.

Interorganelle Communication: Peroxisomal MALATE DEHYDROGENASE2 Connects Lipid Catabolism to Photosynthesis through Redox Coupling in Chlamydomonas.

Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here, we show that knocking out the peroxisome-located MALATE DEHYDROGENASE2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, mdh2 showed increased photosystem II yield and photosynthetic CO2 fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H2O2 in mdh2 Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H2O2-producing peroxisomal ACYL-COA OXIDASE2 and on the effects of H2O2 supplementation, we propose that peroxisome-derived H2O2 acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle- and H2O2-based redox signaling.

When vegetation indicates reproduction: The affinity between leaf morphology and flowering commitment in the lily meristem.

At the reproductive stage, lily plants bear two morphological types of mature leaves, one at the lower and one at the upper part of the stem. At the vegetative stage, all the leaves are similar to each other and to the reproductive plant's lower leaves. This heterophylly has not yet been explored. In this study, we show that it is not a result of the plant's age but rather an outcome of floral induction. The induction appears as an on-going process, during which the meristem still produces leaves but progressively becomes committed to reproduction. This intermediate period lasts until the ultimate switch to flower primordia occurs. The leaves produced during floral induction, termed here as "inductive," appear at the upper part of the stem. Besides their typical higher stomata density, these leaves have a poly-layered palisade mesophyll, whose cells exhibit a unique morphology and contain more chlorophyll than leaves of vegetative plants. These leaves display higher carbon assimilation, soluble sugar production, and chloroplast-lipid accumulation. Accordingly, genes associated with stomata, chloroplast, and photosynthesis are upregulated in these leaves. Our results were obtained when floral induction was achieved either by vernalization or photoperiod signals, ruling out a mere environmental effect. We suggest that lily plants prepare themselves for the high-energy-demanding bloom by producing leaves with enhanced photosynthetic capacity, leading to an increase in soluble sugars. These novel findings introduce an adjacent affinity between photosynthesis and flowering and provide a nondestructive tool for identifying the plant's developmental stage-vegetative or reproductive.