RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis.

Stomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H2 O2 ) signatures in guard cells are poorly understood. We used the H2 O2 sensor roGFP2-Orp1 and a ROS-specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over-oxidation of roGFP2-Orp1 by a pathogen-associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2-Orp1 oxidation. In contrast, RBOHF was necessary for PAMP-mediated ROS production measured by a fluorescein-based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H2 O2 -mediated stomatal closure at 100 µm while higher H2 O2 concentration up to 1 mm did not promote stomatal closure in wild-type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.

Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses.

Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst dependent on the NADPH oxidase isoform RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). We used the H2O2 biosensor roGFP2-Orp1 and the glutathione redox state biosensor GRX1-roGFP2 targeted to various organelles to reveal unsuspected oxidative events during the pattern-triggered immune response to flagellin (flg22) and after inoculation with Pseudomonas syringae. roGFP2-Orp1 was oxidized in a biphasic manner 1 and 6 h after treatment, with a more intense and faster response in the cytosol compared to chloroplasts, mitochondria, and peroxisomes. Peroxisomal and cytosolic GRX1-roGFP2 were also oxidized in a biphasic manner. Interestingly, our results suggested that bacterial effectors partially suppress the second phase of roGFP2-Orp1 oxidation in the cytosol. Pharmacological and genetic analyses indicated that the pathogen-associated molecular pattern-induced cytosolic oxidation required the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) signaling components involved in the immune response but was largely independent of NADPH oxidases RBOHD and RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and apoplastic peroxidases peroxidase 33 (PRX33) and peroxidase 34 (PRX34). The initial apoplastic oxidative burst measured with luminol was followed by a second oxidation burst, both of which preceded the two waves of cytosolic oxidation. In contrast to the cytosolic oxidation, these bursts were RBOHD-dependent. Our results reveal complex oxidative sources and dynamics during the pattern-triggered immune response, including that cytosolic oxidation is largely independent of the preceding extracellular oxidation events.

The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.

Ascorbate (vitamin C) is one of the most abundant primary metabolites in plants. Its complex chemistry enables it to function as an antioxidant, as a free radical scavenger, and as a reductant for iron and copper. Ascorbate biosynthesis occurs via the mannose/l-galactose pathway in green plants, and the evidence for this pathway being the major route is reviewed. Ascorbate accumulation is leaves is responsive to light, reflecting various roles in photoprotection. GDP-l-galactose phosphorylase (GGP) is the first dedicated step in the pathway and is important in controlling ascorbate synthesis. Its expression is determined by a combination of transcription and translation. Translation is controlled by an upstream open reading frame (uORF) which blocks translation of the main GGP-coding sequence, possibly in an ascorbate-dependent manner. GGP associates with a PAS-LOV protein, inhibiting its activity, and dissociation is induced by blue light. While low ascorbate mutants are susceptible to oxidative stress, they grow nearly normally. In contrast, mutants lacking ascorbate do not grow unless rescued by supplementation. Further research should investigate possible basal functions of ascorbate in severely deficient plants involving prevention of iron overoxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilization during seed development and germination.

Ascorbic acid metabolism and functions.

This Special Issue was assembled to mark the 25th anniversary of the proposal of the d -mannose/ l -galactose (Smirnoff-Wheeler) ascorbate biosynthesis pathway in plants ( Wheeler et al., 1998 ). The issue aims to assess the current state of knowledge and to identify outstanding questions about ascorbate metabolism and functions in plants.

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity.

Comparative experimental evolution reveals species-specific idiosyncrasies in marine phytoplankton adaptation to warming.

A number of experimental studies have demonstrated that phytoplankton can display rapid thermal adaptation in response to warmed environments. While these studies provide insight into the evolutionary responses of single species, they tend to employ different experimental techniques. Consequently, our ability to compare the potential for thermal adaptation across different, ecologically relevant, species remains limited. Here, we address this limitation by conducting simultaneous long-term warming experiments with the same experimental design on clonal isolates of three phylogenetically diverse species of marine phytoplankton; the cyanobacterium Synechococcus sp., the prasinophyte Ostreococcus tauri and the diatom Phaeodoactylum tricornutum. Over the same experimental time period, we observed differing levels of thermal adaptation in response to stressful supra-optimal temperatures. Synechococcus sp. displayed the greatest improvement in fitness (i.e., growth rate) and thermal tolerance (i.e., temperature limits of growth). Ostreococcus tauri was able to improve fitness and thermal tolerance, but to a lesser extent. Finally, Phaeodoactylum tricornutum showed no signs of adaptation. These findings could help us understand how the structure of phytoplankton communities may change in response to warming, and possible biogeochemical implications, as some species show relatively more rapid adaptive shifts in their thermal tolerance.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Chloroplast immunity illuminated.

The chloroplast has recently emerged as pivotal to co-ordinating plant defence responses and as a target of plant pathogens. Beyond its central position in oxygenic photosynthesis and primary metabolism - key targets in the complex virulence strategies of diverse pathogens - the chloroplast integrates, decodes and responds to environmental signals. The capacity of chloroplasts to synthesize phytohormones and a diverse range of secondary metabolites, combined with retrograde and reactive oxygen signalling, provides exquisite flexibility to both perceive and respond to biotic stresses. These processes also represent a plethora of opportunities for pathogens to evolve strategies to directly or indirectly target 'chloroplast immunity'. This review covers the contribution of the chloroplast to pathogen associated molecular pattern and effector triggered immunity as well as systemic acquired immunity. We address phytohormone modulation of immunity and surmise how chloroplast-derived reactive oxygen species underpin chloroplast immunity through indirect evidence inferred from genetic modification of core chloroplast components and direct pathogen targeting of the chloroplast. We assess the impact of transcriptional reprogramming of nuclear-encoded chloroplast genes during disease and defence and look at future research challenges.

Spatiotemporal patterns of intracellular Ca2+ signalling govern hypo-osmotic stress resilience in marine diatoms.

Diatoms are globally important phytoplankton that dominate coastal and polar-ice assemblages. These environments exhibit substantial changes in salinity over dynamic spatiotemporal regimes. Rapid sensory systems are vital to mitigate the harmful consequences of osmotic stress. Population-based analyses have suggested that Ca2+ signalling is involved in diatom osmotic sensing. However, mechanistic insight of the role of osmotic Ca2+ signalling is limited. Here, we show that Phaeodactylum Ca2+ elevations are essential for surviving hypo-osmotic shock. Moreover, employing novel single-cell imaging techniques we have characterised real-time Ca2+ signalling responses in single diatom cells to environmental osmotic perturbations. We observe that intracellular spatiotemporal patterns of osmotic-induced Ca2+ elevations encode vital information regarding the nature of the osmotic stimulus. Localised Ca2+ signals evoked by mild or gradual hypo-osmotic shocks are propagated globally from the apical cell tips, enabling fine-tuned cell volume regulation across the whole cell. Finally, we demonstrate that diatoms adopt Ca2+ -independent and dependent mechanisms for osmoregulation. We find that efflux of organic osmolytes occurs in a Ca2+ -independent manner, but this response is insufficient to mitigate cell damage during hypo-osmotic shock. By comparison, Ca2+ -dependent signalling is necessary to prevent cell bursting via precise coordination of K+ transport, and therefore is likely to underpin survival in dynamic osmotic environments.

Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.

Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells. Here, direct incubation of leaf explants from the non-medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High-throughput LC-MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells. A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA-treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin-dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis-(+)-12-oxophytodienoic acid, in synergy, could act as anticancer compounds. Our work suggests a universal role for MeJA-treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti-cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments.

Self-Incompatibility Triggers Irreversible Oxidative Modification of Proteins in Incompatible Pollen.

Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-induced oxidative posttranslational modifications (oxPTMs) can regulate protein structure and function. Here, we have identified and mapped oxPTMs triggered by SI in incompatible pollen. Notably, SI-induced pollen had numerous irreversible oxidative modifications, while untreated pollen had virtually none. Our data provide a valuable analysis of the protein targets of ROS in the context of SI-induction and comprise a benchmark because currently there are few reports of irreversible oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy metabolism are a prominent target of ROS. Oxidative modifications to a phosphomimic form of a pyrophosphatase result in a reduction of its activity. Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI and provide evidence that this modification can affect protein function. We suggest that this reduction in cellular activity could lead to PCD.

Responses of a Newly Evolved Auxotroph of Chlamydomonas to B12 Deprivation.

The corrinoid B12 is synthesized only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions, and we previously demonstrated that experimental evolution of the non-B12-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupts C1 metabolism, causes an accumulation of starch and triacylglycerides, and leads to a decrease in photosynthetic pigments, proteins, and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species, which preceded rapid cell death. Survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to coping with B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing a suitable environment for evolving mechanisms to tolerate B12 limitation better.

Evolutionary temperature compensation of carbon fixation in marine phytoplankton.

The efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short-term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long-term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short-term increases in temperature. Long-term experimental evolution under high temperature reversed the short-term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

MAIN CONCLUSION: Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Hydrogen peroxide metabolism and functions in plants.

Contents Summary 1197 I. Introduction 1198 II. Measurement and imaging of H2 O2 1198 III. H2 O2 and O2·- toxicity 1199 IV. Production of H2 O2 : enzymes and subcellular locations 1200 V. H2 O2 transport 1205 VI. Control of H2 O2 concentration: how and where? 1205 VII. Metabolic functions of H2 O2 1207 VIII. H2 O2 signalling 1207 IX. Where next? 1209 Acknowledgements 1209 References 1209 SUMMARY: Hydrogen peroxide (H2 O2 ) is produced, via superoxide and superoxide dismutase, by electron transport in chloroplasts and mitochondria, plasma membrane NADPH oxidases, peroxisomal oxidases, type III peroxidases and other apoplastic oxidases. Intracellular transport is facilitated by aquaporins and H2 O2 is removed by catalase, peroxiredoxin, glutathione peroxidase-like enzymes and ascorbate peroxidase, all of which have cell compartment-specific isoforms. Apoplastic H2 O2 influences cell expansion, development and defence by its involvement in type III peroxidase-mediated polymer cross-linking, lignification and, possibly, cell expansion via H2 O2 -derived hydroxyl radicals. Excess H2 O2 triggers chloroplast and peroxisome autophagy and programmed cell death. The role of H2 O2 in signalling, for example during acclimation to stress and pathogen defence, has received much attention, but the signal transduction mechanisms are poorly defined. H2 O2 oxidizes specific cysteine residues of target proteins to the sulfenic acid form and, similar to other organisms, this modification could initiate thiol-based redox relays and modify target enzymes, receptor kinases and transcription factors. Quantification of the sources and sinks of H2 O2 is being improved by the spatial and temporal resolution of genetically encoded H2 O2 sensors, such as HyPer and roGFP2-Orp1. These H2 O2 sensors, combined with the detection of specific proteins modified by H2 O2 , will allow a deeper understanding of its signalling roles.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Quantitative proteomics of a B12 -dependent alga grown in coculture with bacteria reveals metabolic tradeoffs required for mutualism.

The unicellular green alga Lobomonas rostrata requires an external supply of vitamin B12 (cobalamin) for growth, which it can obtain in stable laboratory cultures from the soil bacterium Mesorhizobium loti in exchange for photosynthate. We investigated changes in protein expression in the alga that allow it to engage in this mutualism. We used quantitative isobaric tagging (iTRAQ) proteomics to determine the L. rostrata proteome grown axenically with B12 supplementation or in coculture with M. loti. Data are available via ProteomeXchange (PXD005046). Using the related Chlamydomonas reinhardtii as a reference genome, 588 algal proteins could be identified. Enzymes of amino acid biosynthesis were higher in coculture than in axenic culture, and this was reflected in increased amounts of total cellular protein and several free amino acids. A number of heat shock proteins were also elevated. Conversely, photosynthetic proteins and those of chloroplast protein synthesis were significantly lower in L. rostrata cells in coculture. These observations were confirmed by measurement of electron transfer rates in cells grown under the two conditions. The results indicate that, despite the stability of the mutualism, L. rostrata experiences stress in coculture with M. loti, and must adjust its metabolism accordingly.

Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year.

Seasonal behavior is important for fitness in temperate environments but it is unclear how progeny gain their initial seasonal entrainment. Plants use temperature signals to measure time of year, and changes to life histories are therefore an important consequence of climate change. Here we show that in Arabidopsis the current and prior temperature experience of the mother plant is used to control germination of progeny seeds, via the activation of the florigen Flowering Locus T (FT) in fruit tissues. We demonstrate that maternal past and current temperature experience are transduced to the FT locus in silique phloem. In turn, FT controls seed dormancy through inhibition of proanthocyanidin synthesis in fruits, resulting in altered seed coat tannin content. Our data reveal that maternal temperature history is integrated through FT in the fruit to generate a metabolic signal that entrains the behavior of progeny seeds according to time of year.