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Main conclusion The combination of image-based phenotyping with in-depth anatomical analysis allows for a thorough investigation of plant physiological plasticity in acclimation, which is driven by environmental conditions and mediated by anatomical traits. Abstract Understanding the ability of plants to respond to fluctuations in environmental conditions is critical to addressing climate change and unlocking the agricultural potential of crops both indoor and in the field. Recent studies have revealed that the degree of eco-physiological acclimation depends on leaf anatomical traits, which show stress-induced alterations during organogenesis. Indeed, it is still a matter of debate whether plant anatomy is the bottleneck for optimal plant physiology or vice versa. Here, we cultivated ‘Salanova’ lettuces in a phenotyping chamber under two different vapor pressure deficits (VPDs; low, high) and watering levels (well-watered, low-watered); then, plants underwent short-term changes in VPD. We aimed to combine high-throughput phenotyping with leaf anatomical analysis to evaluate their capability in detecting the early stress signals in lettuces and to highlight the different degrees of plants’ eco-physiological acclimation to the change in VPD, as influenced by anatomical traits. The results demonstrate that well-watered plants under low VPD developed a morpho-anatomical structure in terms of mesophyll organization, stomatal and vein density, which more efficiently guided the acclimation to sudden changes in environmental conditions and which was not detected by image-based phenotyping alone. Therefore, we emphasized the need to complement high-throughput phenotyping with anatomical trait analysis to unveil crop acclimation mechanisms and predict possible physiological behaviors after sudden environmental fluctuations due to climate changes.

Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (g

Main conclusion The leaf patch clamp pressure probe combined with gas exchange measurements provides a non-invasive approach for measuring leaf aerenchyma pressure and study its physiological role in plants. Abstract The non-invasive leaf patch clamp pressure probe (LPCP) measures the output pressure, Pp, in response to the pressure applied by two magnets clamped to a leaf. In many plant species, it has been observed that the diel pattern of Pp follows the changes in the leaf turgor pressure reversely. The genus Hippeastrum comprises 143 species and many hybrids and cultivars of high economic value within Amaryllidaceae. Their leaves are characterized by the presence of aerenchyma composed of lacunae, running throughout the leaf and composing most of the mesophyll volume. In Hippeastrum, the diel changes of the LPCP output pressure are the reverse of that observed on the air pressure in the leaf aerenchyma, Pa, which depends on the changes in the leaf vapor pressure occurring during photosynthesis. A theoretical model is proposed and confirmed experimentally by LPCP and gas exchange measurements. The output pressure, Pp, in Hippeastrum can be related to the plant water status through the gas exchange processes that occur during photosynthesis. Considering the natural habitats of Hippeastrum species, these results agree with the physiological role of leaf aerenchyma in facilitating gas transport and light scattering in leaves, thus contributing to the photosynthetic efficiency of these plants under adverse environments. A second, but supplemental, interpretation of the LPCP output pressure, Pp, when applied on species in which the aerenchyma constitutes most of the mesophyll volume is presented.

OsAPL positively controls the seedling growth and grain size in rice by targeting the plasma membrane H

Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm

This study suggests that stomatal and leaf structures are highly correlated, and mesophyll cell size is an important anatomical trait determining the coordination between stomatal size and mesophyll porosity. A comprehensive study of the correlations between the structural traits and on their relationships with gas exchange parameters may provide some useful information into leaf development and improvement in efficiencies of photosynthetic CO

Based on transcriptomic analysis of wild-type and mutant tomato plants, ARPC1 was found to be important for trichome formation and development and it plays a key role in terpene synthesis. Trichomes are protruding epidermal cells in plant species. They function as the first defense layer against biotic and abiotic stresses. Despite the essential role of tomato trichomes in defense against herbivores, the understanding of their development is still incomplete. Therefore, the aim of this study was to identify genes involved in trichome formation and morphology and terpene synthesis, using transcriptomic techniques. To achieve this, we examined leaf morphology and compared the expression levels of some putative genes involved in trichome formation between wild-type (WT) and hairless-3 (hl-3) tomato mutant. The hl-3 plants displayed swollen and distorted trichomes and reduced trichome density (type I and IV) and terpene synthesis compared with that of the WT plants. Gene expression analysis showed that Actin-Related Protein Component1 (ARPC1) was expressed more highly in the WT than in the hl-3 mutant, indicating its critical role in trichome morphology and density. Additionally, the expression of MYC1 and several terpene synthase genes (TPS9, 12, 20), which are involved in type VI trichome initiation and terpene synthesis, was lower in the hl-3 mutant than in the WT plants. Moreover, transformation of the hl-3 mutant with WT ARPC1 restored normal trichome structure and density, and terpene synthesis. Structural and amino acid sequence analysis showed that there was a missplicing mutation in the hl-3 mutant, which was responsible for the abnormal trichome structure and density, and impaired terpene synthesis. Overall, the findings of this study demonstrated that ARPC1 is involved in regulating trichome structure and terpene synthesis in tomato.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-β-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

Glandular trichomes of Artemisia argyi H. Lév.Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév.Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography-mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4-6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi.

Main conclusion The oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by ≈5%. Abstract Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 °C, 500 µmol photons m−2 s−1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin–Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin–Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if ≤ 21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of ≥ 62%. Last, we discuss interactions between the OPPP and the cytosolic oxidation–reduction cycle in supplying leaf cytosolic NADPH.

Transcriptomics of manually dissected leaf layers from Medicago truncatula identifies genes with preferential expression in upper and/or lower epidermis. The promoters of these genes confer epidermal-specific expression of transgenes. Improving the quality and quantity of proanthocyanidins (PAs) in forage legumes has potential to improve the nitrogen nutrition of ruminant animals and protect them from the risk of pasture bloat, as well as parasites. However, ectopic constitutive accumulation of PAs in plants by genetic engineering can significantly inhibit growth. We selected the leaf epidermis as a candidate tissue for targeted engineering of PAs or other pathways. To identify gene promoters selectively expressed in epidermal tissues, we performed comparative transcriptomic analyses in the model legume Medicago truncatula, using five tissue samples representing upper epidermis, lower epidermis, whole leaf without upper epidermis, whole leaf without lower epidermis, and whole leaf. We identified 52 transcripts preferentially expressed in upper epidermis, most of which encode genes involved in flavonoid biosynthesis, and 53 transcripts from lower epidermis, with the most enriched category being anatomical structure formation. Promoters of the preferentially expressed genes were cloned from the M. truncatula genome and shown to direct tissue-selective promoter activities in transient assays. Expression of the PA pathway transcription factor TaMYB14 under control of several of the promoters in transgenic alfalfa resulted in only modest MYB14 transcript accumulation and low levels of PA production. Activity of a subset of promoters was confirmed by transcript analysis in field-grown alfalfa plants throughout the growing season, and revealed variable but consistent expression, which was generally highest 3-4 weeks after cutting. We conclude that, although the selected promoters show acceptable tissue-specificity, they may not drive high enough transcription factor expression to activate the PA pathway.

The divergences in propagule mass have been more consistently associated with divergences in seed development duration or fruit pedicel cross-sectional area than with divergences in any other biotic factors. Allometry and Corner's rule became an important theme in evolutionary biology of plant trait structure and function. Being one of the most widely noticed plant traits, propagule (seed and fruit) mass variation mechanism across species is still controversial. Here we examined correlations between propagule mass and seed development duration as well as other traits, such as cross-sectional area of fruit pedicel, life form, fruit type and leaf area over four census years, to test an important life history strategy for propagule biomass allocation. We investigated 491 species, belonging to 91 families and 320 genera, representing 95% of native wild species in Beijing Botanical Garden. The scaling correlations between propagule mass and seed development duration and the other traits were determined using phylogenetic generalized linear models. Results show a significant positive relationship among propagule mass and seed development duration, leaf area and pedicel cross-sectional area for all species and for each life form (except vines) regardless of phylogeny. The variation in seed mass has been more consistently associated with variation in seed development duration than with divergences in any other variable, such as growth form, fruit type, pedicel cross-sectional area and leaf area, whereas variation in fruit weight was found to be more associated with variation in pedicel cross-sectional area than the other. Biotic factors, such as seed development duration, pedicel cross-sectional area, growth form and leaf area, mediate propagule size variation, of which seed development duration or pedicel cross-sectional area is the most important. The results further supported a hypothesis that large-seeded species evolved owing to high photosynthate availability and growth allometry in plant body. A mechanistic mathematical model involving seed development duration was provided to expound propagule mass variations across species.

The difference in leaf color among the three cultivars of A. bettzickiana is due to different chloroplast morphology and chlorophyll-to-anthocyanin ratios. Alternanthera bettzickiana is one of the most important ornamental plants in modern flower beds because of its colorful leaves. The present study examined the mechanism of leaf color formation in A. bettzickiana. Three cultivars of A. bettzickiana (red, green, and mixed red and green) were selected for comprehensive analyses of leaf color formation by examining cellular and subcellular structures and pigment biosynthesis and metabolism. The difference in leaf colors between the three cultivars of A. bettzickiana was due to different chlorophyll-to-anthocyanin ratios. A. bettzickiana 'Green' showed very low expression of CHS, F3H, and DFR, the key genes of the anthocyanin biosynthesis pathway, and a low anthocyanin content but had mature chloroplasts and a green color. A. bettzickiana 'Red' exhibited a low chlorophyll content and deformed chloroplasts but a high cyanidin content and, thus, a red color. A. bettzickiana 'Variegated' presented high anthocyanin and chlorophyll contents and exhibited red and green variegation, indicating a balance between coloration and photosynthetic efficiency. These data provide a good explanation for the coloration of different cultivars of A. bettzickiana and an important reference for better explaining the color formation mechanisms of plant leaves.

The novel C-methyltransferase, MaMT1, could catalyze the conversion of piperidine to 2-methylpiperidine, which may be involved in the methylation step of DNJ biosynthesis in mulberry leaves. Mulberry (Morus alba L.) is a worldwide crop with medicinal, feeding and nutritional value, and 1-deoxynojirimycin ((2R, 3R, 4R, 5S)-2-hydroxymethyl-3, 4, 5-trihydroxypiperidine, DNJ) alkaloid, a potent α-glucosidase inhibitor, is its main active ingredient. Our previous researches clarified the biosynthetic pathway of DNJ from lysine to Δ

The poor-soil-tolerant wild soybean resist phosphorus deficiency by remodeling membrane lipids to reuse phosphorus. The plants synthesize phenolic acids and flavonoids to remove reactive oxygen species and protect membrane stability. Poor soil largely limits plant yields, and the development and utilization of high-quality wild plant resources is an effective approach to resolving this problem. Two ecotypes of wild soybean were used as experimental materials in this experiment. We integrated metabolomics and transcriptomics to determine whether wild soybean (Glycine soja) could resist phosphorus deficiency by remodeling and protecting its membrane system. Under phosphorus-deficient conditions, the plant height and aboveground fresh and dry weight of poor-soil-tolerant wild soybean seedlings were less inhibited than those in common wild soybean. In poor-soil-tolerant wild soybean seedling leaves, the glycerol-3-phosphate content decreased significantly, while caffeic acid, ferulic acid, shikimic acid, phenylalanine, tyrosine, and tryptophan increased significantly. β-Glucosidase and chalcone synthase genes and those that encode SQD2, a crucial enzyme in thiolipid biosynthesis, were specifically up-regulated, whereas the glucosyltransferase UGT74B1 gene was down-regulated. The poor-soil-tolerant wild soybean enhanced glycerolipid metabolism to decompose phospholipids and release phosphorus for reuse to improve resistance to phosphorus deficiency. The plants synthesized thiolipids to replace phospholipids and maintain membrane structure integrity and inhibited glucosinolate biosynthesis to promote phenylpropanoid biosynthesis, leading to the production of phenolic acids and flavonoids that removed reactive oxygen species and protected membrane system stability. The experiments evaluated and provided insight into the innovative utilization of wild soybean germplasm resources.

We identified a typical rice premature senescence leaf mutant 86 (psl86) and exhibited the first global ubiquitination data during rice leaf senescence. Premature leaf senescence affects the yield and quality of rice, causing irreparable agricultural economic losses. In this study, we reported a rice premature senescence leaf mutant 86 (psl86) in the population lines of rice (Oryza sativa) japonica cultivar 'Yunyin' (YY) mutagenized using ethyl methane sulfonate (EMS) treatment. Immunoblotting analysis revealed that a higher ubiquitination level in the psl86 mutant compared with YY. Thus, we performed the proteome and ubiquitylome analyses to identify the differential abundance proteins and ubiquitinated proteins (sites) related to leaf senescence. Among 885 quantified lysine ubiquitination (Kub) sites in 492 proteins, 116 sites in 94 proteins were classified as up-regulated targets and seven sites in six proteins were classified as down-regulated targets at a threshold of 1.5. Proteins with up-regulated Kub sites were mainly enriched in the carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis and the pentose phosphate pathway. Notably, 14 up-regulated Kub sites in 11 proteins were enriched in the carbon fixation in photosynthetic organism pathway, and seven proteins (rbcL, PGK, GAPA, FBA5, ALDP, CFBP1 and GGAT) were down-regulated, indicating this pathway is tightly regulated by ubiquitination during leaf senescence. To our knowledge, we present the first global data on ubiquitination during rice leaf senescence.

Main conclusion Higher acclimated freezing tolerance improved winter survival, but reduced reproductive fitness of Arabidopsis thaliana accessions under field and controlled conditions. Abstract Low temperature is one of the most important abiotic factors influencing plant fitness and geographical distribution. In addition, cold stress is known to influence crop yield and is therefore of great economic importance. Increased freezing tolerance can be acquired by the process of cold acclimation, but this may be associated with a fitness cost. To assess the influence of cold stress on the fitness of plants, long-term field trials over 5 years were performed with six natural accessions of Arabidopsis thaliana ranging from very tolerant to very sensitive to freezing. Fitness parameters, as seed yield and 1000 seed mass, were measured and correlation analyses with temperature and freezing tolerance data performed. The results were compared with fitness parameters from controlled chamber experiments over 3 years with application of cold priming and triggering conditions. Winter survival and seed yield per plant were positively correlated with temperature in field experiments. In addition, winter survival and 1000 seed mass were correlated with the cold-acclimated freezing tolerance of the selected Arabidopsis accessions. The results provide strong evidence for a trade-off between higher freezing tolerance and reproductive fitness in A. thaliana, which might have ecological impacts in the context of global warming.

Main conclusion In accordance with a key role of WHIRLY1 in light-acclimation mechanisms, typical features of acclimation to high light, including photosynthesis and leaf morphology, are compromised in WHIRLY1 deficient plants. Abstract Acclimation to the environment requires efficient communication between chloroplasts and the nucleus. Previous studies indicated that the plastid-nucleus located WHIRLY1 protein is required for the communication between plastids and the nucleus in situations of high light exposure. To investigate the consequences of WHIRLY1 deficiency on the light acclimation of photosynthesis and leaf anatomy, transgenic barley plants with an RNAi-mediated knockdown of HvWHIRLY1 were compared to wild-type plants when growing at low and high irradiance. While wild-type plants showed the typical light acclimation responses, i.e. higher photosynthetic capacity and thicker leaves, the WHIRLY1 deficient plants were not able to respond to differences in irradiance. The results revealed a systemic role of WHIRLY1 in light acclimation by coordinating responses at the level of the chloroplast and the level of leaf morphology.

Main conclusion Elms, which have received insect eggs as a ‘warning’ of larval herbivory, enhance their anti-herbivore defences by accumulating salicylic acid and amplifying phenylpropanoid-related transcriptional and metabolic responses to hatching larvae. Abstract Plant responses to insect eggs can result in intensified defences against hatching larvae. In annual plants, this egg-mediated effect is known to be associated with changes in leaf phenylpropanoid levels. However, little is known about how trees—long-living, perennial plants—improve their egg-mediated, anti-herbivore defences. The role of phytohormones and the phenylpropanoid pathway in egg-primed anti-herbivore defences of a tree species has until now been left unexplored. Using targeted and untargeted metabolome analyses we studied how the phenylpropanoid pathway of Ulmus minor responds to egg-laying by the elm leaf beetle and subsequent larval feeding. We found that when compared to untreated leaves, kaempferol and quercetin concentrations increased in feeding-damaged leaves with prior egg deposition, but not in feeding-damaged leaves without eggs. PCR analyses revealed that prior insect egg deposition intensified feeding-induced expression of phenylalanine ammonia lyase (PAL), encoding the gateway enzyme of the phenylpropanoid pathway. Salicylic acid (SA) concentrations were higher in egg-treated, feeding-damaged leaves than in egg-free, feeding-damaged leaves, but SA levels did not increase in response to egg deposition alone—in contrast to observations made of Arabidopsis thaliana. Our results indicate that prior egg deposition induces a SA-mediated response in elms to feeding damage. Furthermore, egg deposition boosts phenylpropanoid biosynthesis in subsequently feeding-damaged leaves by enhanced PAL expression, which results in the accumulation of phenylpropanoid derivatives. As such, the elm tree shows similar, yet distinct, responses to insect eggs and larval feeding as the annual model plant A. thaliana.

MAIN CONCLUSION After tobacco topping, changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the NtARF genes, thus causing changes in K+ content. Tobacco (Nicotiana tabacum) is a valuable industrial and commercial crop, and the leaf is its primary product. Topping (removing apical buds) is a common agronomic practice that significantly improves the yield of tobacco leaves. Potassium (K+) plays an important physiological role in tobacco growth and leaf traits, including combustibility, aroma, and safety in cigarette products, and its levels are significantly decreased after topping. Here, to present global spatial-temporal gene expression profiles and gene regulatory networks of the core elements of K+ uptake, leaves and roots from topped and untopped plants at short- and long-term time points after topping were sampled for transcriptome analysis. We found that the wounding response was initiated in leaves in the early stages after topping. Then, in the long term, processes related to metabolism and transcription regulation, as well as ion binding and transport, were altered. The expression profiles showed that core elements of K+ uptake and xylem loading were drastically suppressed in roots after topping. Finally, transient expression experiments confirmed that changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the tobacco auxin response factor (NtARF) genes, thus causing changes in the K+ content. These results suggest that some ARFs could be selected as targets to enhance the expressions of K+ uptake transporters, leading to increment of K+ contents and improvement of leaf quality in tobacco breeding.

Main conclusion Time-dependent contact angle measurements of pure water on barley leaf surfaces allow quantifying the kinetics of surfactant diffusion into the leaf. Abstract Barley leaf surfaces were sprayed with three different aqueous concentrations (0.1, 1.0 and 10%) of a monodisperse (tetraethylene glycol monododecyl ether) and a polydisperse alcohol ethoxylate (BrijL4). After 10 min, the surfactant solutions on the leaf surfaces were dry leading to surfactant coverages of 1, 10 and 63 µg cm−2, respectively. The highest surfactant coverage (63 µg cm−2) affected leaf physiology (photosynthesis and water loss) rapidly and irreversibly and leaves were dying within 2–6 h. These effects on leaf physiology did not occur with the lower surfactant coverages (1 and 10 µg cm−2). Directly after spraying of 0.1 and 1.0% surfactant solution and complete drying (10 min), leaf surfaces were fully wettable for pure water and contact angles were 0°. Within 60 min (0.1% surfactant) and 6 h (1.0% surfactant), leaf surfaces were non-wettable again and contact angles of pure water were identical to control leaves. Scanning electron microscopy investigations directly performed after surfactant spraying and drying indicated that leaf surface wax crystallites were partially or fully covered by surfactants. Wax platelets with unaltered microstructure were fully visible again within 2 to 6 h after treatment with 0.1% surfactant solutions. Gas chromatographic analysis showed that surfactant amounts on leaf surfaces continuously disappeared over time. Our results indicate that surfactants, applied at realistic coverages between 1 and 10 µg cm−2 to barley leaf surfaces, leading to total wetting (contact angles of 0°) of leaf surfaces, are rapidly taken up by the leaves. As a consequence, leaf surface non-wettability is fully reappearing. An irreversible damage of the leaf surface fine structure leading to enhanced wetting and increased foliar transpiration seems highly unlikely at low surfactant coverages of 1 µg cm−2.

A new molecular mechanism of tetrahydrofolate deformylase involved in the salt response presumably affects mitochondrial and chloroplast function by regulating energy metabolism and accumulation of reactive oxygen species. High salinity severely restrains plant growth and development, consequently leading to a reduction in grain yield. It is therefore critical to identify the components involved in plant salt resistance. In our previous study, we identified a rice leaf early-senescence mutant hpa1, which encodes a formyl tetrahydrofolate deformylase (Xiong et al. in Sci China Life Sci 64(5):720-738, 2021). Here, we report that HPA1 also plays a role in the salt response. To explore the molecular mechanism of HPA1 in salt resistance, we attempted to identify the differentially expressed proteins between wild type and hpa1 mutant for salinity treatment using an iTRAQ-based comparative protein quantification approach. A total of 4598 proteins were identified, of which 279 were significantly altered, including 177 up- and 102 down-regulated proteins. A functional analysis suggested that the 279 differentially expressed proteins are involved mainly in the regulation of oxidative phosphorylation, phenylpropanoid biosynthesis, photosynthesis, posttranslational modifications, protein turnover and energy metabolism. Moreover, a deficiency in HPA1 impaired chlorophyll metabolism and photosynthesis in chloroplasts and affected the electron flow of the electron transport chain in mitochondria. These changes led to abnormal energy metabolism and accumulation of reactive oxygen species, which may affect the permeability and integrity of cell membranes, leading to cell death. In addition, the results were verified by transcriptional or physiological experiments. Our results provide an insight into a new molecular mechanism of the tetrahydrofolate cycle protein formyl tetrahydrofolate deformylase, which is involved in the salt response, presumably by affecting mitochondrial and chloroplast function regulating energy metabolism and accumulation of reactive oxygen species under salt stress.

This article intends to summarize all the up-to-date information on coffee leaves, rendering it to be used as a potential agri-food resource in the growing functional foods and pharma industries. Coffee leaves have been processed for herbal tea and ethno-medicine since centuries in the parts of the world where coffee is grown traditionally. Currently, interest in the valorisation of coffee leaves for its application in the food industry is proliferating and the research related to it is scanty and, therefore, worthwhile to congregate. The current review compromises the botanical description, chemical composition, bio-actives and ethnomedicinal properties of coffee leaves. It encompasses the existing pharmacological studies on coffee leaves including the anti-oxidant, anti-inflammatory and anti-obesity activities to pave path for future research. Furthermore, applications and patents associated with coffee leaves in different fields such as therapeutic agents, beverages, packaging material, tobacco substitute etc. have been summarized. The investigation reveals that, despite of many patents on coffee leaves only few products could reach the worldwide market; also in spite of coffee leaves having a rich ethno-medicinal use the study on its pharmacological activities are scarce which creates a huge scope to carry out in-vitro and in-vivo research on its various bio-activities. Future insights reflecting the supplementary research regarding the sensory attributes, changes in phytochemical composition, flavour development and product formulations which is vital are also discussed. In conclusion, this review addresses the breach and specifies the requirements to convert the existing knowledge into commercialized food products with functional properties. Thus, coffee leaves being a copious resource of bio-actives serve as a potential agri-food resource and a promising future in the emerging functional food and nutraceutical industry.

Silicon enhances photosynthetic efficiency, biomass, and lignification of root structures possibly limiting antimony translocation and mitigating phytotoxicity in giant reed plants. Antimony (Sb) is a non-essential metalloid causing toxic effects in plants. Silicon has been reported to impart tolerance against biotic and abiotic stress in plants. Fast-growing plant, giant reed (Arundo donax L.) is a promising energy crop, can be a suitable plant for phytoremediation. However, information regarding the tolerance capacity with respect to Sb toxicity and potential of Si to mitigate the Sb phytotoxicity in giant reed are very scarce. Rhizomes of giant reed were grown for ten weeks in hydroponics exposed to Sb, Si, and their combination wherein treatment without Sb/Si served as control. Effect of these treatments on rate of net photosynthesis and photosynthetic pigments, phytoextraction ability of Sb, Si and Sb uptake, plant biomass, and lignification and suberization of roots along with localization of Sb and Si were analysed. We found that Si considerably improved the growth and biomass of giant reed under Sb toxicity. Antimony reduced the photosynthesis and decreased the content of photosynthetic pigments, which was completely alleviated by Si. Silicon amendment to Sb treated plants enhanced root lignification. Silicon enhanced lignification of root structures probably restricted the Sb translocation. However, co-localization of Sb with Si has not been observed neither at the shoot nor at the root levels. Similarly, Sb was also not detected in leaf phytoliths. These findings suggest that Si treatment promotes overall plant growth by improving photosynthetic parameters and decreasing Sb translocation from root to shoot in giant reed by improving root lignification.

The possible molecular mechanisms regulating sorghum callus regeneration were revealed by RNA-sequencing. Plant callus regeneration has been widely applied in agricultural improvement. Recently, callus regeneration has been successfully applied in the genetic transformation of sorghum by using immature sorghum embryos as explants. However, the mechanism underlying callus regeneration in sorghum is still largely unknown. Here, we describe three types of callus (Callus I-III) with different redifferentiation abilities undergoing distinct induction from immature embryos of the Hiro-1 variety. Compared with nonembryonic Callus III, Callus I produced only some identifiable roots, and embryonic Callus II was sufficient to regenerate whole plants. Genome-wide transcriptome profiles were generated to reveal the underlying mechanisms. The numbers of differentially expressed genes for the three types of callus varied from 5906 to 8029. In accordance with the diverse regeneration abilities observed for different types of callus and leaf tissues, the principal component analysis revealed that the gene expression patterns of Callus I and Callus II were different from those of Callus III and leaves regenerated from Callus II. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, pharmacological treatment, and substance content determinations revealed that plant ribosomes, lignin metabolic processes, and metabolism of starch and sucrose were significantly enriched, suggesting that these factors are associated with callus regeneration. These results helped elucidate the molecular regulation of three types of callus with different regeneration abilities in sorghum.

Rice genotypes with larger stomata maintain higher nocturnal stomatal conductance, thus having lower nocturnal leaf temperature via transpirational cooling. Incomplete night stomatal closure has been widely observed, but the mechanisms and functions of nocturnal stomatal conductance (gs,n) are not fully understood. Stomatal anatomy, leaf morphology, gs,n and nocturnal leaf temperature (Tleaf,n) were measured in 30 Oryza genotypes. Nocturnal leaf conductance (gn) showed a significant circadian rhythm; it gradually increased by 58% from 20:30 to 04:30. Contrary to cuticular conductance (gcut), gs,n was highly correlated with gn. Moreover, gs,n accounted for 76% of gn. Tleaf,n was significantly lower than the air temperature, and was negatively correlated with both gs,n and nocturnal transpiration rate (En). gs,n was positively correlated with stomatal size, intervein distance between major veins (IVDmajor), leaf thickness (LT), individual leaf area (LA), and leaf width (LW). It was also found negatively correlated with stomatal density. Reversely, Tleaf,n was negatively correlated with stomatal size, IVDmajor, intervein distance between minor veins, LA and LW. Tleaf,n presented a positive correlation with stomatal density. This study highlights the importance of stomatal anatomy and leaf morphology on regulating gs,n and Tleaf,n. The underlying mechanisms to the determinants of gs,n and the physiological and ecological functions of the Tleaf,n regulation on rice growth and production were carefully discussed.

The relationship of fructan to plant growth regulators is clearly more complicated than it looks and is likely related to differences between fructan molecules in size and structure as well as localization. Fructans are a complex group of carbohydrates composed mainly of fructose units linked to a sucrose molecule. Fructans are present in plants as heterogeneous mixtures with diverse molecular structures and mass, different polymerization degrees, and linkage types between fructosyl residues. Like sucrose, they are frequently stored in leaves and other organs, acting as carbohydrate reserves. Fructans are synthesized in the cell vacuole by fructosyltransferase enzymes and catabolized by fructan exohydrolase enzymes. Several publications have shown that fructan metabolism varies with the stage of plant development and in response to the environment. Recent studies have shown a correlation between plant growth regulators (PGR), fructan metabolism, and tolerance to drought and cold. PGR are compounds that profoundly influence the growth and differentiation of plant cells, tissues, and organs. They play a fundamental role in regulating plant responses to developmental and environmental signals. In this review, we summarize the most up-to-date knowledge on the metabolism of fructans and their crosstalk with PGR signaling pathways. We identify areas that require more research to complete our understanding of the role of fructans in plants.

Suppression of the maize phytoglobin ZmPgb1.1 enhances tolerance against Clavibacter nebraskensis by promoting hypersensitive response mechanisms mediated by ethylene and reactive oxygen species. Suppression of the maize phytoglobin, ZmPgb1.1, reduced lesion size and disease severity in leaves following inoculation with Clavibacter nebraskensis, the causal agent of Goss's bacterial wilt disease of corn. These effects were associated with an increase of the transcriptional levels of ethylene biosynthetic and responsive genes, which resulted in the accumulation of reactive oxygen species (ROS) and TUNEL-positive nuclei in the proximity of the inoculation site. An in vitro system, in which maize cells were treated with induced xylem sap, was employed to define the cause-effect relationship of these events. Phytoglobins (Pgbs) are hemoglobins able to scavenge nitric oxide (NO). Suppression of ZmPgb1.1 elevated the level of NO in cells exposed to the induced xylem sap causing a rise in the transcript levels of ethylene biosynthesis and response genes, as well as ethylene. Accumulation of ethylene in the same cells was sufficient to elevate the amount of reactive oxygen species (ROS), through the activation of the respiratory burst oxidase homologs (Rboh) genes, and trigger programmed cell death (PCD). The sequence of these events was demonstrated by manipulating the content of NO and ethylene in culture through pharmacological treatments. Collectively, our results illustrated that suppression of ZmPgb1.1 evokes tolerance against C. nebraskensis culminating in the execution of PCD, a key step of the hypersensitive response.

The roles of microRNA-mediated epigenetic regulation were highlighted in the bud dormancy-activity cycle, implying that certain differentially expressed miRNAs play crucial roles in apical bud burst, such as csn-miR319c/TCP2. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by targeting mRNA transcripts for cleavage or directing translational inhibition. To investigate whether miRNAs regulate bud dormancy-activation transition in tea plant, which largely affects the yield and price of tea products and adaptability of tea trees, we constructed small RNA libraries from three different periods of bud dormancy-burst transition. Through sequencing analysis, 262 conserved and 83 novel miRNAs were identified, including 118 differentially expressed miRNAs. Quantitative RT-PCR results for randomly selected miRNAs exhibited that our comprehensive analysis is highly reliable and accurate. The content of caffeine increased continuously from the endodormancy bud to flushing bud, and differentially expressed miRNAs coupling with their targets associated with bud burst were identified. Remarkably, csn-miR319c was downregulated significantly from the quiescent bud to burst bud, while its target gene CsnTCP2 (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR 2) displayed opposite expression patterns. Co-transformation experiment in tobacco demonstrated that csn-miR319c can significantly suppress the functions of CsnTCP2. This study on miRNAs and the recognition of target genes could provide new insights into the molecular mechanism of the bud dormancy-activation transition in tea plant.

The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.

Primitive wheat follows an opposite metabolic law from modern wheat with regard to leaf biomass/reproductive growth vs above-ground biomass that is under the regulation of non-hydraulic root signals and that influences resource acquisition and utilization. Non-hydraulic root signals (nHRS) are so far affirmed as a unique positive response to drying soil in wheat, and may imply huge differences in energy metabolism and source-sink relationships between primitive and modern wheat species. Using a pot-culture split-root technique to induce nHRS, four primitive wheat genotypes (two diploids and two tetraploids) and four modern wheat ones (released from different breeding decades) were compared to address the above issue. The nHRS was continuously induced in drying soil, ensuring the operation of energy metabolism under the influence of nHRS. We found that primitive wheat followed an opposite size-dependent allometric pattern (logy = αlogx + logβ) in comparison with modern wheat. The relationships between ear biomass (y-axis) vs above-ground biomass (x-axis), and between reproductive biomass (y-axis) and vegetative (x-axis) biomass fell into a typical allometric pattern in primitive wheat (α 1), and the nHRS significantly increased α (P 0.01). However, in modern wheat, they turned to be in an isometric pattern (α ≈ 1). Regardless of nHRS, either leaf (i.e., metabolic rate) or stem biomass generally exhibited an isometric relationship with above-ground biomass in primitive wheat (α ≈ 1), while in modern wheat they fell into an allometric pattern (α 1). Allometric scaling of specific leaf area (SLA) or biomass density showed superior capabilities of resource acquisition and utilization in modern wheat over primitive ones. We therefore proposed a generalized model to reveal how modern wheat possesses the pronounced population yield advantage over primitive wheat, and its implications on wheat domestication.

Ethylene receptor is crucial for PCD and aerenchyma formation in Typha angustifolia leaves. Not only does it receive and deliver the ethylene signal, but it probably can determine the cell fate during aerenchyma morphogenesis, which is due to the receptor expression quantity. Aquatic plant oxygen delivery relies on aerenchyma, which is formed by a programmed cell death (PCD) procedure. However, cells in the outer edge of the aerenchyma (palisade cells and septum cells) remain intact, and the mechanism is unclear. Here, we offer a hypothesis: cells that have a higher content of ethylene receptors do not undergo PCD. In this study, we investigated the leaf aerenchyma of the aquatic plant Typha angustifolia. Ethephon and pyrazinamide (PZA, an inhibitor of ACC oxidase) were used to confirm that ethylene is an essential hormone for PCD of leaf aerenchyma cells in T. angustifolia. That the ethylene receptor was an indispensable factor in this PCD was confirmed by 1-MCP (an inhibitor of the ethylene receptor) treatment. Although PCD can be avoided by blocking the ethylene receptor, excessive ethylene receptors also protect cells from PCD. TaETR1, TaETR2 and TaEIN4 in the T. angustifolia leaf were detected by immunofluorescence (IF) using polyclonal antibodies. The result showed that the content of ethylene receptors in PCD-unsusceptible cells was 4-14 times higher than that one in PCD-susceptible cells, suggesting that PCD-susceptible cells undergo the PCD programme, while PCD-unsusceptible cells do not due to the content difference in the ethylene receptor in different cells. A higher level of ethylene receptor content makes the cells insensitive to ethylene, thereby avoiding cell death and degradation.

The present study is the first to integrate physiological and proteomic data providing information on Fe, Mn and Zn deficiency-responsive mechanisms of potato plants in vitro. Micronutrient deficiency is an important limiting factor for potato production that causes substantial tuber yield and quality losses. To under the underlying molecular mechanisms of potato in response to Fe, Mn and Zn deficiency, a comparative proteomic approach was applied. Leaf proteome change of in vitro-propagated potato plantlets subjected to a range of Fe-deficiency treatments (20, 10 and 0 μM Na-Fe-EDTA), Mn-deficiency treatments (1 and 0 μM MnCl2·4H2O) and Zn-deficiency treatment (0 μM ZnCl2) using two-dimensional gel electrophoresis was analyzed. Quantitative image analysis showed a total of 146, 55 and 42 protein spots under Fe, Mn and Zn deficiency with their abundance significantly altered (P

Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids. Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.

Morphological, physiological, and gene expression analyses showed that Os4BGlu10, Os6BGlu24, and Os9BGlu33 played specific roles in seed germination, root elongation, and drought tolerance of rice, with various relations with indole-3-acetic acid (IAA) and abscisic acid (ABA) signaling. β-Glucosidases (BGlus) belong to glycoside hydrolase family 1 and have many functions in plants. In this study, we investigated the function of three BGlus in seed germination, drought tolerance, and root elongation using the loss-of-function mutants bglu10, bglu24, and bglu33. These mutants germinated slightly later under normal conditions and had significantly longer roots than the wild type. In the presence of ABA, bglu10 and bglu24 exhibited a higher germination inhibition percentage, whereas bglu33 had a lower germination inhibition percentage, compared to the wild type. All of the mutants exhibited less drought tolerance, with the survival rates significantly lower than that of the wild type, which was also confirmed by a decrease in relative leaf water content and Fv/Fm ratio after drought treatment. The root length of bglu10 did not respond to IAA, whereas that of bglu24 responded to a high (0.25 µM) concentration of IAA, and that of bglu33 to a low (0.05 µM) concentration of IAA. The root length of bglu10 and bglu24 did not respond to ABA, whereas that of bglu33 increased significantly in response to a high (0.05 µM) concentration of ABA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that expression of Os4BGlu10 was up-regulated by polyethylene glycol (PEG), whereas that of Os6BGlu24 was up-regulated by 0.25 µM IAA, and Os9BGlu33 was up-regulated by PEG, IAA, and ABA. Taken together, we demonstrate that Os4BGlu10, Os6BGlu24, and Os9BGlu33 play specific roles in seed germination, root elongation, and drought tolerance with various relation with IAA and ABA signaling.

Glufosinate is primarily toxic to plants due to a massive light-dependent generation of reactive oxygen species rather than ammonia accumulation or carbon assimilation inhibition. Glutamine synthetase (GS) plays a key role in plant nitrogen metabolism and photorespiration. Glufosinate (C5H12NO4P) targets GS and causes catastrophic consequences leading to rapid plant cell death, and the causes for phytoxicity have been attributed to ammonia accumulation and carbon assimilation restriction. This study aimed to examine the biochemical and physiological consequences of GS inhibition to identify the actual cause for rapid phytotoxicity. Monocotyledonous and dicotyledonous species with different forms of carbon assimilation (C3 versus C4) were selected as model plants. Glufosinate sensitivity was proportional to the uptake of herbicide between species. Herbicide uptake also correlated with the level of GS inhibition and ammonia accumulation in planta even with all species having the same levels of enzyme sensitivity in vitro. Depletion of both glutamine and glutamate occurred in glufosinate-treated leaves; however, amino acid starvation would be expected to cause a slow plant response. Ammonia accumulation in response to GS inhibition, often reported as the driver of glufosinate phytotoxicity, occurred in all species, but did not correlate with either reductions in carbon assimilation or cell death. This is supported by the fact that plants can accumulate high levels of ammonia but show low inhibition of carbon assimilation and absence of phytotoxicity. Glufosinate-treated plants showed a massive light-dependent generation of reactive oxygen species, followed by malondialdehyde accumulation. Consequently, we propose that glufosinate is toxic to plants not because of ammonia accumulation nor carbon assimilation inhibition, but the production of reactive oxygen species driving the catastrophic lipid peroxidation of the cell membranes and rapid cell death.

This study shows that NgRBP suppresses both local and systemic RNA silencing induced by sense- or double-stranded RNA, and the RNA binding activity is essential for its function. To counteract host defence, many plant viruses encode viral suppressors of RNA silencing targeting various stages of RNA silencing. There is increasing evidence that the plants also encode endogenous suppressors of RNA silencing (ESR) to regulate this pathway. In this study, using Agrobacterium infiltration assays, we characterized NgRBP, a glycine-rich RNA-binding protein from Nicotiana glutinosa, as an ESR. Our results indicated that NgRBP suppressed both local and systemic RNA silencing induced by sense- or double-stranded RNA. We also demonstrated that NgRBP could promote Potato Virus X (PVX) infection in N. benthamiana. NgRBP knockdown by virus-induced gene silencing enhanced PVX and Cucumber mosaic virus resistance in N. glutinosa. RNA immunoprecipitation and electrophoretic mobility shift assays showed that NgRBP bound to GFP mRNA, dsRNA rather than siRNA. These findings provide the evidence that NgRBP acts as an ESR and the RNA affinity of NgRBP plays the key role in its ESR activity. NgRBP responds to multiple signals such as ABA, MeJA, SA, and Tobacco mosaic virus infection. Therefore, it could participate in the regulation of gene expression under specific conditions.

Ficus species have adapted to diverse environments and pests by developing physical or chemical protection strategies. Physical defences are based on the accumulation of minerals such as calcium oxalate crystals, amorphous calcium carbonates and silica that lead to tougher plants. Additional cellular structures such as non-glandular trichomes or laticifer cells make the leaves rougher or sticky upon injury. Ficus have also established structures that are able to produce specialized metabolites (alkaloids, terpenoids, and phenolics) or proteins (proteases, protease inhibitors, oxidases, and chitinases) that are toxic to predators. All these defence mechanisms are distributed throughout the plant and can differ depending on the genotype, the stage of development or the environment. In this review, we present an overview of these strategies and discuss how these complementary mechanisms enable effective and flexible adaptation to numerous hostile environments.

In hybrids of Arabidopsis, cotyledons influence the amount and proportion of hybrid vigor in total plant growth. We found Arabidopsis cotyledons are essential for plant growth and in some hybrids for hybrid vigor. In hybrids between C24 and Landsberg erecta (Ler), biomass vigor (heterosis) occurs in the first few days after sowing (DAS), with hybrid cotyledons being larger than those of their parents. C24xLer hybrids are ahead of their parents in activating photosynthesis and auxin pathway genes in cotyledons at 3-4 DAS. "Earliness" is also present in newly emerged C24xLer hybrid leaves. We showed cotyledon removal at 4 DAS caused significant biomass reduction in later growth in hybrids and parental lines. The biomass decrease caused by cotyledon removal can be partially rescued by exogenous sucrose or auxin with different genotypes responding to sucrose and/or auxin differently. Cotyledon removal has different effects on heterosis in different hybrids. After cotyledon removal, in C24xLer hybrids, both growth and heterosis were reduced in similar proportions, but the level of hybrid vigor was reduced as a proportion of growth in C24xColumbia (Col) and ColxLer hybrids. The removal of cotyledons at 4 DAS markedly decreased the level of growth and eliminated the heterotic phenotype of Wassilewskija (Ws)/Ler hybrids. In mutant Ws/Ler hybrids which had a reduced level of photosynthesis in the cotyledons, there was a reduction in plant growth and loss of heterosis. The variation in contribution of cotyledons to heterosis in different hybrids indicates there are multiple pathways to achieve heterotic phenotypes.

Arabidopsis Mediator subunits 2, 14, 15a, 16, and 25 are required for papillae development on the trichome cell wall surface. Arabidopsis leaf hairs exhibit raised protrusions, termed papillae, on their cell wall surfaces. Here, we show that the glassy hair mutant, glh2, exhibits trichomes with an approximate 11-fold decrease in papillae density on their surfaces in comparison to wild type. This phenotype was found to be the result of mutations in Arabidopsis Mediator subunit 16. MED16 is localized to the nucleus of trichomes, consistent with Mediator's role in transcription. The expression patterns of the trichome development reporters, ETR2pro::GUS and GL2pro::GUS, as well as GL2 transcript levels were not altered in the glh2 mutant. Screening of available T-DNA insertion lines in other subunits of the Mediator tail module revealed glassy trichome phenotypes in med2, med14, and med15a mutants. The data suggest that the Mediator complex is required for expression of genes involved in trichome papillae development.

Main Conclusion The drought conditions and the application of ABA reduce the photosynthetic activity, and the processes related to the transpiration of Dracocephalum moldavica L. At the same time, the plant increases the production of phenolic compounds and essential oil as a response to stress conditions. Abstract In the semi-arid regions, drought stress is the most important environmental limitations for crop production. Abscisic acid (ABA) plays a crucial role in the reactions of plants towards environmental stress such as drought. Field experiments for two consecutive years in 2016 and 2017 were conducted to evaluate the effect of three watering regimes (well-watered, moderate and severe drought) and five exogenous ABA concentrations (0, 5, 10, 20 and 40 μM) on growth, photosynthesis, total phenolic and essential oil content of Dracocephalum moldavica L. Without ABA application, the highest photosynthetic rate (6.1 μmol CO2 m−2 s−1) was obtained under well-watered condition and, moderate and severe drought stress decreased photosynthesis rate by 26.39% and 34.43%, respectively. Some growth parameters such as stem height, leaf area, leaf dry weight and biological yield were also reduced by drought stress. ABA application showed a decreasing trend in photosynthesis rate and mentioned plant growth parameters under all moisture regimes. The highest seed yield (1243.56 kg ha−1) was obtained under well-watered condition without ABA application. Increasing ABA concentration decreased seed yield in all moisture regimes. The highest total phenolic content (8.9 mg g−1 FW) and essential oil yield (20.58 kg ha−1) were obtained from 20 and 5 μM ABA concentration, respectively, under moderate drought stress.

Main conclusion Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions. Abstract Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.