Shoot and root single cell sequencing reveals tissue- and daytime-specific transcriptome profiles.

Although several large-scale single-cell RNA sequencing (scRNAseq) studies addressing the root of Arabidopsis (Arabidopsis thaliana) have been published, there is still need for a de novo reference map for both root and especially above-ground cell types. As the plants' transcriptome substantially changes throughout the day, shaped by the circadian clock, we performed scRNAseq on both Arabidopsis root and above-ground tissues at defined times of the day. For the root scRNAseq analysis, we used tissue-specific reporter lines grown on plates and harvested at the end of the day (ED). In addition, we submitted above-ground tissues from plants grown on soil at ED and end of the night to scRNAseq, which allowed us to identify common cell types/markers between root and shoot and uncover transcriptome changes to above-ground tissues depending on the time of the day. The dataset was also exploited beyond the traditional scRNAseq analysis to investigate non-annotated and di-cistronic transcripts. We experimentally confirmed the predicted presence of some of these transcripts and also addressed the potential function of a previously unidentified marker gene for dividing cells. In summary, this work provides insights into the spatial control of gene expression from nearly 70,000 cells of Arabidopsis for below- and whole above-ground tissue at single-cell resolution at defined time points.

Knockdown of the plastid-encoded acetyl-CoA carboxylase gene uncovers functions in metabolism and development.

De novo fatty acid biosynthesis in plants relies on a prokaryotic-type acetyl-CoA carboxylase (ACCase) that resides in the plastid compartment. The enzyme is composed of four subunits, one of which is encoded in the plastid genome, whereas the other three subunits are encoded by nuclear genes. The plastid gene (accD) encodes the ß-carboxyltransferase subunit of ACCase and is essential for cell viability. To facilitate the functional analysis of accD, we pursued a transplastomic knockdown strategy in tobacco (Nicotiana tabacum). By introducing point mutations into the translational start codon of accD, we obtained stable transplastomic lines with altered ACCase activity. Replacement of the standard initiator codon AUG with UUG strongly reduced AccD expression, whereas replacement with GUG had no detectable effects. AccD knockdown mutants displayed reduced ACCase activity, which resulted in changes in the levels of many but not all species of cellular lipids. Limiting fatty acid availability caused a wide range of macroscopic, microscopic, and biochemical phenotypes, including impaired chloroplast division, reduced seed set, and altered storage metabolism. Finally, while the mutants displayed reduced growth under photoautotrophic conditions, they showed exaggerated growth under heterotrophic conditions, thus uncovering an unexpected antagonistic role of AccD activity in autotrophic and heterotrophic growth.

Phytochromes control metabolic flux, and their action at the seedling stage determines adult plant biomass.

Phytochrome photoreceptors are known to regulate plastic growth responses to vegetation shade. However, recent reports also suggest an important role for phytochromes in carbon resource management, metabolism, and growth. Here, we use 13CO2 labelling patterns in multiallele phy mutants to investigate the role of phytochrome in the control of metabolic fluxes. We also combine quantitative data of 13C incorporation into protein and cell wall polymers, gas exchange measurements, and system modelling to investigate why biomass is decreased in adult multiallele phy mutants. Phytochrome influences the synthesis of stress metabolites such as raffinose and proline, and the accumulation of sugars, possibly through regulating vacuolar sugar transport. Remarkably, despite their modified metabolism and vastly altered architecture, growth rates in adult phy mutants resemble those of wild-type plants. Our results point to delayed seedling growth and smaller cotyledon size as the cause of the adult-stage phy mutant biomass defect. Our data signify a role for phytochrome in metabolic stress physiology and carbon partitioning, and illustrate that phytochrome action at the seedling stage sets the trajectory for adult biomass production.

Response of the Circadian Clock and Diel Starch Turnover to One Day of Low Light or Low CO2.

Diel starch turnover responds rapidly to changes in the light regime. We investigated if these responses require changes in the temporal dynamics of the circadian clock. Arabidopsis (Arabidopsis thaliana) was grown in a 12-h photoperiod for 19 d, shifted to three different reduced light levels or to low CO2 for one light period, and returned to growth conditions. The treatments produced widespread changes in clock transcript abundance. However, almost all of the changes were restricted to extreme treatments that led to carbon starvation and were small compared to the magnitude of the circadian oscillation. Changes included repression of EARLY FLOWERNG 4, slower decay of dusk components, and a slight phase delay at the next dawn, possibly due to abrogated Evening Complex function and sustained expression of PHYTOCHROME INTERACTING FACTORs and REVEILLEs during the night. Mobilization of starch in the night occurred in a linear manner and was paced to dawn, both in moderate treatments that did not alter clock transcripts and in extreme treatments that led to severe carbon starvation. We conclude that pacing of starch mobilization to dawn does not require retrograde carbon signaling to the transcriptional clock. On the following day, growth decreased, sugars rose, and starch accumulation was stimulated in low-light-treated plants compared to controls. This adaptive response was marked after moderate treatments and occurred independently of changes in the transcriptional clock. It is probably a time-delayed response to low-C signaling in the preceding 24-h cycle, possibly including changes in PHYTOCHROME INTERACTING FACTOR and REVEILLE expression.

Genome-Wide Association Mapping Reveals That Specific and Pleiotropic Regulatory Mechanisms Fine-Tune Central Metabolism and Growth in Arabidopsis.

Central metabolism is a coordinated network that is regulated at multiple levels by resource availability and by environmental and developmental cues. Its genetic architecture has been investigated by mapping metabolite quantitative trait loci (QTL). A more direct approach is to identify enzyme activity QTL, which distinguishes between cis-QTL in structural genes encoding enzymes and regulatory trans-QTL. Using genome-wide association studies, we mapped QTL for 24 enzyme activities, nine metabolites, three structural components, and biomass in Arabidopsis thaliana We detected strong cis-QTL for five enzyme activities. A cis-QTL for UDP-glucose pyrophosphorylase activity in the UGP1 promoter is maintained through balancing selection. Variation in acid invertase activity reflects multiple evolutionary events in the promoter and coding region of VAC-INVcis-QTL were also detected for ADP-glucose pyrophosphorylase, fumarase, and phosphoglucose isomerase activity. We detected many trans-QTL, including transcription factors, E3 ligases, protein targeting components, and protein kinases, and validated some by knockout analysis. trans-QTL are more frequent but tend to have smaller individual effects than cis-QTL. We detected many colocalized QTL, including a multitrait QTL on chromosome 4 that affects six enzyme activities, three metabolites, protein, and biomass. These traits are coordinately modified by different ACCELERATED CELL DEATH6 alleles, revealing a trade-off between metabolism and defense against biotic stress.

Trehalose 6-phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.).

Trehalose 6-phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2-oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration-dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.

Circadian, Carbon, and Light Control of Expansion Growth and Leaf Movement.

We used Phytotyping4D to investigate the contribution of clock and light signaling to the diurnal regulation of rosette expansion growth and leaf movement in Arabidopsis (Arabidopsis thaliana). Wild-type plants and clock mutants with a short (lhycca1) and long (prr7prr9) period were analyzed in a T24 cycle and in T-cycles that were closer to the mutants' period. Wild types also were analyzed in various photoperiods and after transfer to free-running light or darkness. Rosette expansion and leaf movement exhibited a circadian oscillation, with superimposed transients after dawn and dusk. Diurnal responses were modified in clock mutants. lhycca1 exhibited an inhibition of growth at the end of night and growth rose earlier after dawn, whereas prr7prr9 showed decreased growth for the first part of the light period. Some features were partly rescued by a matching T-cycle, like the inhibition in lhycca1 at the end of the night, indicating that it is due to premature exhaustion of starch. Other features were not rescued, revealing that the clock also regulates expansion growth more directly. Expansion growth was faster at night than in the daytime, whereas published work has shown that the synthesis of cellular components is faster in the day than at nighttime. This temporal uncoupling became larger in short photoperiods and may reflect the differing dependence of expansion and biosynthesis on energy, carbon, and water. While it has been proposed that leaf expansion and movement are causally linked, we did not observe a consistent temporal relationship between expansion and leaf movement.

Response of Arabidopsis primary metabolism and circadian clock to low night temperature in a natural light environment.

Plants are exposed to varying irradiance and temperature within a day and from day to day. We previously investigated metabolism in a temperature-controlled greenhouse at the spring equinox on both a cloudy and a sunny day [daily light integral (DLI) of 7 mol m-2 d-1 and 12 mol m-2 d-1]. Diel metabolite profiles were largely captured in sinusoidal simulations at similar DLIs in controlled-environment chambers, except that amino acids were lower in natural light regimes. We now extend the DLI12 study by investigating metabolism in a natural light regime with variable temperature including cool nights. Starch was not completely turned over, anthocyanins and proline accumulated, and protein content rose. Instead of decreasing, amino acid content rose. Connectivity in central metabolism, which decreased in variable light, was not further weakened by variable temperature. We propose that diel metabolism operates better when light and temperature are co-varying. We also compared transcript abundance of 10 circadian clock genes in this temperature-variable regime with the temperature-controlled natural and sinusoidal light regimes. Despite temperature compensation, peak timing and abundance for dawn- and day-phased genes and GIGANTEA were slightly modified in the variable temperature treatment. This may delay dawn clock activity until the temperature rises enough to support rapid metabolism and photosynthesis.

Getting back to nature: a reality check for experiments in controlled environments.

Irradiance from sunlight changes in a sinusoidal manner during the day, with irregular fluctuations due to clouds, and light-dark shifts at dawn and dusk are gradual. Experiments in controlled environments typically expose plants to constant irradiance during the day and abrupt light-dark transitions. To compare the effects on metabolism of sunlight versus artificial light regimes, Arabidopsis thaliana plants were grown in a naturally illuminated greenhouse around the vernal equinox, and in controlled environment chambers with a 12-h photoperiod and either constant or sinusoidal light profiles, using either white fluorescent tubes or light-emitting diodes (LEDs) tuned to a sunlight-like spectrum as the light source. Rosettes were sampled throughout a 24-h diurnal cycle for metabolite analysis. The diurnal metabolite profiles revealed that carbon and nitrogen metabolism differed significantly between sunlight and artificial light conditions. The variability of sunlight within and between days could be a factor underlying these differences. Pairwise comparisons of the artificial light sources (fluorescent versus LED) or the light profiles (constant versus sinusoidal) showed much smaller differences. The data indicate that energy-efficient LED lighting is an acceptable alternative to fluorescent lights, but results obtained from plants grown with either type of artificial lighting might not be representative of natural conditions.

Durum wheat seedling responses to simultaneous high light and salinity involve a fine reconfiguration of amino acids and carbohydrate metabolism.

Durum wheat plants are extremely sensitive to drought and salinity during seedling and early development stages. Their responses to stresses have been extensively studied to provide new metabolic targets and improving the tolerance to adverse environments. Most of these studies have been performed in growth chambers under low light [300-350 µmol m-2 s-1 photosynthetically active radiation (PAR), LL]. However, in nature plants have to face frequent fluctuations of light intensities that often exceed their photosynthetic capacity (900-2000 µmol m-2 s-1 ). In this study we investigated the physiological and metabolic changes potentially involved in osmotic adjustment and antioxidant defense in durum wheat seedlings under high light (HL) and salinity. The combined application of the two stresses decreased the water potential and stomatal conductance without reducing the photosynthetic efficiency of the plants. Glycine betaine (GB) synthesis was inhibited, proline and glutamate content decreased, while γ-aminobutyric acid (GABA), amides and minor amino acids increased. The expression level and enzymatic activities of Δ1-pyrroline-5-carboxylate synthetase, asparagine synthetase and glutamate decarboxylase, as well as other enzymatic activities of nitrogen and carbon metabolism, were analyzed. Antioxidant enzymes and metabolites were also considered. The results showed that the complex interplay seen in durum wheat plants under salinity at LL was simplified: GB and antioxidants did not play a main role. On the contrary, the fine tuning of few specific primary metabolites (GABA, amides, minor amino acids and hexoses) remodeled metabolism and defense processes, playing a key role in the response to simultaneous stresses.

Loss of starch granule initiation has a deleterious effect on the growth of arabidopsis plants due to an accumulation of ADP-glucose.

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.

The transcription factor HSFA7b controls thermomemory at the shoot apical meristem by regulating ethylene biosynthesis and signaling in Arabidopsis.

The shoot apical meristem (SAM) is responsible for overall shoot growth by generating all aboveground structures. Recent research has revealed that the SAM displays an autonomous heat stress (HS) memory of a previous non-lethal HS event. Considering the importance of the SAM for plant growth, it is essential to determine how its thermomemory is mechanistically controlled. Here, we report that HEAT SHOCK TRANSCRIPTION FACTOR A7b (HSFA7b) plays a crucial role in this process in Arabidopsis, as the absence of functional HSFA7b results in the temporal suppression of SAM activity after thermopriming. We found that HSFA7b directly regulates ethylene response at the SAM by binding to the promoter of the key ethylene signaling gene ETHYLENE-INSENSITIVE 3 to establish thermotolerance. Moreover, we demonstrated that HSFA7b regulates the expression of ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-LIKE 1, both of which encode ethylene biosynthesis repressors, thereby ensuring ethylene homeostasis at the SAM. Taken together, these results reveal a crucial and tissue-specific role for HSFA7b in thermomemory at the Arabidopsis SAM.

cDNA cloning and differential expression patterns of ascorbate peroxidase during post-harvest in Brassica rapa L.

Ascorbate is an antioxidant and a cofactor of many dioxygenases in plant and animal cell metabolism. A well-recognized enzyme consuming ascorbate is ascorbate peroxidase (APX), which catalyses the reduction of hydrogen peroxide to water with the simultaneous oxidation of ascorbate with a high specificity. The isolation and characterisation of new Apx cDNAs, could provide new insights about the physiological roles and regulation of these enzymes. In this work chloroplastic (Br-chlApx) and cytosolic (Br-cApx) isoform transcripts were isolated by RT-PCR in Brassica rapa and expression changes were analysed by semi-quantitative RT-PCR performed in different tissues (layer, stalk and florets) at different days (0, 4 and 14 day). The result showed that BrApx isoforms were differentially expressed and the Br-chlApx, in particular in the layer, had the highest expression level and remained unchanged also after 14 day after harvest. In addition, expression changes were compared with total BrAPX activity and the results showed that the activity decreased in all tissues at 14 day after harvest, independently of transcripts. Finally, additional solutes as the substrate of APX ascorbate and its oxidized form, dehydroascorbate, as well as α-tocopherol, the major vitamin E compound that prevents the propagation of lipid peroxidation in thylakoid membranes, were followed. The changes in the BrApx expression, BrAPX activity and metabolites can provide further evidence of the close relationships that exist between antioxidants which compensate for each other and suggest that there are multiple sites of reciprocal control.

Primary carbohydrate metabolism genes participate in heat-stress memory at the shoot apical meristem of Arabidopsis thaliana.

In plants, the shoot apical meristem (SAM) is essential for the growth of aboveground organs. However, little is known about its molecular responses to abiotic stresses. Here, we show that the SAM of Arabidopsis thaliana displays an autonomous heat-stress (HS) memory of a previous non-lethal HS, allowing the SAM to regain growth after exposure to an otherwise lethal HS several days later. Using RNA sequencing, we identified genes participating in establishing the SAM's HS transcriptional memory, including the stem cell (SC) regulators CLAVATA1 (CLV1) and CLV3, HEAT SHOCK PROTEIN 17.6A (HSP17.6A), and the primary carbohydrate metabolism gene FRUCTOSE-BISPHOSPHATE ALDOLASE 6 (FBA6). We demonstrate that sugar availability is essential for survival of plants at high temperature. HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2A) directly regulates the expression of HSP17.6A and FBA6 by binding to the heat-shock elements in their promoters, indicating that HSFA2 is required for transcriptional activation of SAM memory genes. Collectively, these findings indicate that plants have evolved a sophisticated protection mechanism to maintain SCs and, hence, their capacity to re-initiate shoot growth after stress release.

Physiological Profiling of Embryos and Dormant Seeds in Two Arabidopsis Accessions Reveals a Metabolic Switch in Carbon Reserve Accumulation.

In flowering plants, sugars act as carbon sources providing energy for developing embryos and seeds. Although most studies focus on carbon metabolism in whole seeds, knowledge about how particular sugars contribute to the developmental transitions during embryogenesis is scarce. To develop a quantitative understanding of how carbon composition changes during embryo development, and to determine how sugar status contributes to final seed or embryo size, we performed metabolic profiling of hand-dissected embryos at late torpedo and mature stages, and dormant seeds, in two Arabidopsis thaliana accessions with medium [Columbia-0 (Col-0)] and large [Burren-0 (Bur-0)] seed sizes, respectively. Our results show that, in both accessions, metabolite profiles of embryos largely differ from those of dormant seeds. We found that developmental transitions from torpedo to mature embryos, and further to dormant seeds, are associated with major metabolic switches in carbon reserve accumulation. While glucose, sucrose, and starch predominantly accumulated during seed dormancy, fructose levels were strongly elevated in mature embryos. Interestingly, Bur-0 seeds contain larger mature embryos than Col-0 seeds. Fructose and starch were accumulated to significantly higher levels in mature Bur-0 than Col-0 embryos, suggesting that they contribute to the enlarged mature Bur-0 embryos. Furthermore, we found that Bur-0 embryos accumulated a higher level of sucrose compared to hexose sugars and that changes in sucrose metabolism are mediated by sucrose synthase (SUS), with SUS genes acting non-redundantly, and in a tissue-specific manner to utilize sucrose during late embryogenesis.