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Plants modify their root system architecture (RSA) in response to nitrogen (N) deficiency. The plant steroidal hormone, brassinosteroid (BR), plays important roles in root growth and development. This study demonstrates that optimal levels of exogenous BR impact significant increases in lateral root length and numbers in Arabidopsis seedlings under mild N-deficient conditions as compared to untreated seedlings. The impact of BR on RSA was stronger under mild N deficiency than under N-sufficient conditions. The BR effects on RSA were mimicked in dominant mutants of BZR1 and BES1 ( bzr1-1D and bes1-D ) transcription factors, while the RSA was highly reduced in the BR-insensitive mutant bri1-6 , confirming that BR signaling is essential for the development of RSA under both N-sufficient and N-deficient conditions. Exogenous BR and constitutive activity of BZR1 and BES1 in dominant mutants led to enhanced root meristem, meristematic cell number, and cortical cell length. Under mild N deficiency, bzr1-1D displayed higher fresh and dry shoot weights, chlorophyll content, and N levels in the shoot, as compared to the wild type. These results indicate that BR modulates RSA under both N-sufficient and N-deficient conditions via the transcription factors BES1/BZR1 module and confers tolerance to N deficiency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Al-Mamun, Cazzonelli and Krishna.)

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or β-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The challenges facing tree orchard production in the coming years will be largely driven by changes in the climate affecting the sustainability of farming practices in specific geographical regions. Identifying key traits that enable tree crops to modify their growth to varying environmental conditions and taking advantage of new crop improvement opportunities and technologies will ensure the tree crop industry remains viable and profitable into the future. In this review article we 1) outline climate and sustainability challenges relevant to horticultural tree crop industries, 2) describe key tree crop traits targeted for improvement in agroecosystem productivity and resilience to environmental change, and 3) discuss existing and emerging genomic technologies that provide opportunities for industries to future proof the next generation of orchards., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kerr, Shehnaz, Paudel, Manivannan, Shaw, Johnson, Velasquez, Tanurdžić, Cazzonelli, Varkonyi-Gasic and Prentis.)

High energy costs are a barrier to producing high-quality produce at protected cropping facilities. A potential solution to mitigate high energy costs is film technology, which blocks heat-producing radiation; however, the alteration of the light environment by these films may impact crop yield and quality. Previous studies have assessed the impact of ULR 80 [i.e., light-blocking film (LBF)] on crop yield and photosynthetically active radiation (PAR); however, an assessment of the spectral environment over different seasons is important to understand potential crop impacts through different developmental phases. In this study, two varieties (red and orange) of Capsicum annuum were grown across two crop cycles: one cycle with primary crop growth in the autumn (i.e., autumn experiment [AE]) and the other with primary crop growth in the summer (i.e., summer experiment [SE]). LBF reduced PAR (roof level: 26%-30%, plant canopy level: 8%-25%) and net radiation (36%-66%). LBF also reduced total diffuse PAR (AE: 8%, SE: 15%), but the diffuse fraction of PAR increased by 7% and 9% for AE and SE, respectively, potentially resulting in differential light penetration throughout the canopy across treatments. LBF reduced near-infrared radiation (700 nm-2,500 nm), including far-red (700 nm-780 nm) at mid- and lower-canopy levels. LBF significantly altered light quantity and quality, which determined the amount of time that the crop grew under light-limited (<12 mol m -2 d -1 ) versus sufficient light conditions. In AE, crops were established and grown under light-limited conditions for 57% of the growing season, whereas in SE, crops were established and grown under sufficient light conditions for 66% of the growing season. Overall, LBF significantly reduced the yield in SE for both varieties (red: 29%; orange: 16%), but not in AE. The light changes in different seasons in response to LBF suggest that planting time is crucial for maximizing fruit yield when grown under a film that reduces light quantity. LBF may be unsuitable for year-round production of capsicum, and additional development of LBF is required for the film to be beneficial for saving energy during production and sustaining good crop yields in protected cropping., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maier, Chavan, Klause, Liang, Cazzonelli, Ghannoum, Chen and Tissue.)

Light-blocking films (LBFs) can contribute to significant energy savings for protected cropping via altering light transmitting, such as UVA, photosynthetically active radiation, blue and red spectra affecting photosynthesis, and capsicum yield. Here, we investigated the effects of LBF on orange color capsicum (O06614, Capsicum annuum L.) fruit transcriptome at 35 (mature green) and 65 (mature ripe) days after pollination (DAP) relative to untreated control in a high-technology glasshouse. The results of targeted metabolites showed that LBF significantly promotes the percentage of lutein but decreased the percentage of zeaxanthin and neoxanthin only at 35 DAP. At 35 DAP, fruits were less impacted by LBF treatment (versus control) with a total of 1,192 differentially expressed genes (DEGs) compared with that at 65 DAP with 2,654 DEGs. Response to stress and response to light stimulus in biological process of Gene Ontology were found in 65-DAP fruits under LBF vs. control, and clustering analysis revealed a predominant role of light receptors and phytohormone signaling transduction as well as starch and sucrose metabolism in LBF adaptation. The light-signaling DEGs, UV light receptor UVR8 , transcription factors phytochrome-interacting factor 4 ( PIF4 ), and an E3 ubiquitin ligase ( COP1 ) were significantly downregulated at 65 DAP. Moreover, key DEGs in starch and sucrose metabolism ( SUS , SUC , and INV ), carotenoid synthesis ( PSY2 and BCH1 ), ascorbic acid biosynthesis ( VTC2 , AAO , and GME ), abscisic acid (ABA) signaling ( NCED3 , ABA2 , AO4 , and PYL2/4 ), and phenylpropanoid biosynthesis ( PAL and DFR ) are important for the adaptation of 65-DAP fruits to LBF. Our results provide new candidate genes for improving quality traits of low-light adaptation of capsicum in protected cropping., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 He, Solis, Chavan, Maier, Wang, Liang, Klause, Ghannoum, Cazzonelli, Tissue and Chen.)

The uptake and accumulation of silicon (Si) in grass plants play a crucial role in alleviating both biotic and abiotic stresses. Si supplementation has been reported to increase activity of defence-related antioxidant enzyme, which helps to reduce oxidative stress caused by reactive oxygen species (ROS) following herbivore attack. Atmospheric CO 2 levels are known to affect Si accumulation in grasses; reduced CO 2 concentrations increase Si accumulation whereas elevated CO 2 concentrations often decrease Si accumulation. This can potentially affect antioxidant enzyme activity and subsequently insect herbivory, but this remains untested. We examined the effects of Si supplementation and herbivory by Helicoverpa armigera on antioxidant enzyme (catalase, CAT; superoxide dismutase, SOD; and ascorbate peroxidase, APX) activity in tall fescue grass ( Festuca arundinacea ) grown under CO 2 concentrations of 200, 410, and 640 ppm representing reduced, ambient, and elevated CO 2 levels, respectively. We also quantified foliar Si, carbon (C), and nitrogen (N) concentrations and determined how changes in enzymes and elemental chemistry affected H. armigera relative growth rates and plant consumption. Rising CO 2 concentrations increased plant mass and foliar C but decreased foliar N and Si. Si supplementation enhanced APX and SOD activity under the ranging CO 2 regimes. Si accumulation and antioxidant enzyme activity were at their highest level under reduced CO 2 conditions and their lowest level under future levels of CO 2 . The latter corresponded with increased herbivore growth rates and plant consumption, suggesting that some grasses could become more susceptible to herbivory under projected CO 2 conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Biru, Cazzonelli, Elbaum and Johnson.)

The Heirloom Golden tangerine tomato fruit variety is highly nutritious due to accumulation of tetra-cis-lycopene, that has a higher bioavailability and recognised health benefits in treating anti-inflammatory diseases compared to all-trans-lycopene isomers found in red tomatoes. We investigated if photoisomerization of tetra-cis-lycopene occurs in roots of the MicroTom tangerine (tang mic ) tomato and how this affects root to shoot biomass, mycorrhizal colonization, abscisic acid accumulation, and responses to drought. tang mic plants grown in soil under glasshouse conditions displayed a reduction in height, number of flowers, fruit yield, and root length compared to wild-type (WT). Soil inoculation with Rhizophagus irregularis revealed fewer arbuscules and other fungal structures in the endodermal cells of roots in tang mic relative to WT. The roots of tang mic hyperaccumulated acyclic cis-carotenes, while only trace levels of xanthophylls and abscisic acid were detected. In response to a water deficit, leaves from the tang mic plants displayed a rapid decline in maximum quantum yield of photosystem II compared to WT, indicating a defective root to shoot signalling response to drought. The lack of xanthophylls biosynthesis in tang mic roots reduced abscisic acid levels, thereby likely impairing endomycorrhizal colonisation and drought-induced root to shoot signalling., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Smart Glass Film (SGF) is a glasshouse covering material designed to permit 80% transmission of photosynthetically active light and block heat-generating solar energy. SGF can reduce crop water and nutrient consumption and improve glasshouse energy use efficiency yet can reduce crop yield. The effect of SGF on the postharvest shelf life of fruits remains unknown. Two capsicum varieties, Red (Gina) and Orange (O06614), were cultivated within a glasshouse covered in SGF to assess fruit quality and shelf life during the winter season. SGF reduced cuticle thickness in the Red cultivar (5%) and decreased ascorbic acid in both cultivars (9-14%) without altering the overall morphology of the mature fruits. The ratio of total soluble solids (TSSs) to titratable acidity (TA) was significantly higher in Red (29%) and Orange (89%) cultivars grown under SGF. The Red fruits had a thicker cuticle that reduced water loss and extended shelf life when compared to the Orange fruits, yet neither water loss nor firmness were impacted by SGF. Reducing the storage temperature to 2 °C and increasing relative humidity to 90% extended the shelf life in both cultivars without evidence of chilling injury. In summary, SGF had minimal impact on fruit development and postharvest traits and did not compromise the shelf life of mature fruits. SGF provides a promising technology to block heat-generating solar radiation energy without affecting fruit ripening and marketable quality of capsicum fruits grown during the winter season.

A single event of mechanical stimulation is perceived by mechanoreceptors that transduce rapid transient signalling to regulate gene expression. Prolonged mechanical stress for days to weeks culminates in cellular changes that strengthen the plant architecture leading to thigmomorphogenesis. The convergence of multiple signalling pathways regulates mechanically induced tolerance to numerous biotic and abiotic stresses. Emerging evidence showed prolonged mechanical stimulation can modify the baseline level of gene expression in naive tissues, heighten gene expression, and prime disease resistance upon a subsequent pathogen encounter. The phenotypes of thigmomorphogenesis can persist throughout growth without continued stimulation, revealing somatic-stress memory. Epigenetic processes regulate TOUCH gene expression and could program transcriptional memory in differentiating cells to program thigmomorphogenesis. We discuss the early perception, gene regulatory and phytohormone pathways that facilitate thigmomorphogenesis and mechanical stress acclimation in Arabidopsis and other plant species. We provide insights regarding: (1) the regulatory mechanisms induced by single or prolonged events of mechanical stress, (2) how mechanical stress confers transcriptional memory to induce cross-acclimation to future stress, and (3) why thigmomorphogenesis might resemble an epigenetic phenomenon. Deeper knowledge of how prolonged mechanical stimulation programs somatic memory and primes defence acclimation could transform solutions to improve agricultural sustainability in stressful environments., (© 2022 John Wiley & Sons Ltd.)

Background: Some plastid-derived metabolites can control nuclear gene expression, chloroplast biogenesis, and chlorophyll biosynthesis. For example, norflurazon (NFZ) induced inhibition of carotenoid biosynthesis in leaves elicits a protoporphyrin IX (Mg-ProtoIX) retrograde signal that controls chlorophyll biosynthesis and chloroplast development. Carotenoid cleavage products, known as apocarotenoids, also regulate plastid development. The key steps in carotenoid biosynthesis or catabolism that can regulate chlorophyll biosynthesis in leaf tissues remain unclear. Here, we established a foliar pigment-based bioassay using Arabidopsis rosette leaves to investigate plastid signalling processes in young expanding leaves comprising rapidly dividing and expanding cells containing active chloroplast biogenesis., Results: We demonstrate that environmental treatments (extended darkness and cold exposure) as well as chemical (norflurazon; NFZ) inhibition of carotenoid biosynthesis, reduce chlorophyll levels in young, but not older leaves of Arabidopsis. Mutants with disrupted xanthophyll accumulation, apocarotenoid phytohormone biosynthesis (abscisic acid and strigolactone), or enzymatic carotenoid cleavage, did not alter chlorophyll levels in young or old leaves. However, perturbations in acyclic cis-carotene biosynthesis revealed that disruption of CAROTENOID ISOMERASE (CRTISO), but not ZETA-CAROTENE ISOMERASE (Z-ISO) activity, reduced chlorophyll levels in young leaves of Arabidopsis plants. NFZ-induced inhibition of PHYTOENE DESATURASE (PDS) activity caused higher phytoene accumulation in younger crtiso leaves compared to WT indicating a continued substrate supply from the methylerythritol 4-phosphate (MEP) pathway., Conclusion: The Arabidopsis foliar pigment-based bioassay can be used to differentiate signalling events elicited by environmental change, chemical treatment, and/or genetic perturbation, and determine how they control chloroplast biogenesis and chlorophyll biosynthesis. Genetic perturbations that impaired xanthophyll biosynthesis and/or carotenoid catabolism did not affect chlorophyll biosynthesis. The lack of CAROTENOID ISOMERISATION reduced chlorophyll accumulation, but not phytoene biosynthesis in young leaves of Arabidopsis plants growing under a long photoperiod. Findings generated using the newly customised foliar pigment-based bioassay implicate that carotenoid isomerase activity and NFZ-induced inhibition of PDS activity elicit different signalling pathways to control chlorophyll homeostasis in young leaves of Arabidopsis., (© 2022. The Author(s).)

Multipartite viral vectors provide a simple, inexpensive and effective biotechnological tool to transiently manipulate (i.e. reduce or increase) gene expression in planta and characterise the function of genetic traits. The development of virus-induced gene regulation (VIGR) systems usually involve the targeted silencing or overexpression of genes involved in pigment biosynthesis or degradation in plastids, thereby providing rapid visual assessment of success in establishing RNA- or DNA-based VIGR systems in planta. Carotenoids pigments provide plant tissues with an array of yellow, orange, and pinkish-red colours. VIGR-induced transient manipulation of carotenoid-related gene expression has advanced our understanding of carotenoid biosynthesis, regulation, accumulation and degradation, as well as plastid signalling processes. In this review, we describe mechanisms of VIGR, the importance of carotenoids as visual markers of technology development, and knowledge gained through manipulating carotenogenesis in model plants as well as horticultural crops not always amenable to transgenic approaches. We outline how VIGR can be utilised in plants to fast-track the characterisation of gene function(s), accelerate fruit tree breeding programs, edit genomes, and biofortify plant products enriched in carotenoid micronutrients for horticultural innovation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

The pathways regulated in ectomycorrhizal (EcM) plant hosts during the establishment of symbiosis are not as well understood when compared to the functional stages of this mutualistic interaction. Our study used the EcM host Eucalyptus grandis to elucidate symbiosis-regulated pathways across the three phases of this interaction. Using a combination of RNA sequencing and metabolomics we studied both stage-specific and core responses of E. grandis during colonization by Pisolithus microcarpus. Using exogenous manipulation of the abscisic acid (ABA), we studied the role of this pathway during symbiosis establishment. Despite the mutualistic nature of this symbiosis, a large number of disease signalling TIR-NBS-LRR genes were induced. The transcriptional regulation in E. grandis was found to be dynamic across colonization with a small core of genes consistently regulated at all stages. Genes associated to the carotenoid/ABA pathway were found within this core and ABA concentrations increased during fungal integration into the root. Supplementation of ABA led to improved accommodation of P. microcarpus into E. grandis roots. The carotenoid pathway is a core response of an EcM host to its symbiont and highlights the need to understand the role of the stress hormone ABA in controlling host-EcM fungal interactions., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Reverse-phase high-performance liquid chromatography (HPLC) is a preferred method used to identify and quantify carotenoids. Here, we describe a straightforward, reliable, and cost-effective protocol to purify and develop individual carotenoid standards for absolute quantification of carotenoids, including selected cis-trans (geometric) isomers. Analytical techniques to extract, purify and collect individual carotenoids using an HPLC system equipped with a Diode Array Detector (DAD) and fraction collector are described. Carotenoids were separated and identified by their characteristic ultraviolet-visible (UV-Vis) absorption spectra and individually isolated based on their retention times using a C30 column. This chapter outlines how to prepare standard calibration curves using known quantities of purified and/or commercially available carotenoids. A series of molar extinction and slope coefficients for phytoene, phytofluene, ζ-carotene, neurosporene, pro-lycopene, all trans-lycopene, lutein, β-carotene, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, capsanthin, capsorubin and β-cryptoxanthin are defined to enable absolute quantification of their abundance in plant, animal, and bacterial tissues. Different approaches for reporting carotenoid abundance by absolute concentration, relative composition, and/or using ratios of different pigments are provided as a convenient resource for carotenoid researchers., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Silicon (Si) can adversely affect insect herbivores, particularly in plants that evolved the ability to accumulate large quantities of Si. Very rapid herbivore-induced accumulation of Si has recently been demonstrated, but the level of protection against herbivory this affords plants remains unknown. Brachypodium distachyon, a model Si hyperaccumulating grass, was exposed to the chewing herbivore, Helicoverpa armigera, and grown under three conditions: supplied Si over 34 d (+Si), not supplied Si (-Si), or supplied Si once herbivory began (-Si → +Si). We evaluated the effectiveness of each Si treatment at reducing herbivore performance and measured Si-based defenses and phenolics (another form of defense often reduced by Si). Although Si concentrations remained lower, within 72 h of exposure to Si, -Si → +Si plants were as resistant to herbivory as +Si plants. Both +Si and -Si → +Si treatments reduced herbivore damage and growth, and increased mandible wear compared to -Si. After 6 h, herbivory increased filled Si cell density in -Si → +Si plants, and within 24 h, -Si → +Si plants reached similar filled Si cell densities to +Si plants, although decreased phenolics only occurred in +Si plants. We demonstrate that plants with short-term Si exposure can rapidly accumulate Si-based antiherbivore defenses as effectively as plants with long-term exposure., (© 2021 by the Ecological Society of America.)

Silicon (Si) has an important role in mitigating diverse biotic and abiotic stresses in plants, mainly via the silicification of plant tissues. Environmental changes such as atmospheric CO 2 concentrations may affect grass Si concentrations which, in turn, can alter herbivore performance. We recently demonstrated that pre-industrial atmospheric CO 2 increased Si accumulation in Brachypodium distachyon grass, yet the patterns of Si deposition in leaves and whether this affects insect herbivore performance remains unknown. Moreover, it is unclear whether CO 2 -driven changes in Si accumulation are linked to changes in gas exchange (e.g. transpiration rates). We therefore investigated how pre-industrial (reduced; rCO 2 , 200 ppm), ambient (aCO 2 , 410 ppm) and elevated (eCO 2 , 640 ppm) CO 2 concentrations, in combination with Si-treatment (Si+ or Si-), affected Si accumulation in B. distachyon and its subsequent effect on the performance of the global insect pest, Helicoverpa armigera. rCO 2 increased Si concentrations by 29% and 36% compared to aCO 2 and eCO 2 respectively. These changes were not related to observed changes in gas exchange under different CO 2 regimes, however. The increased Si accumulation under rCO 2 decreased herbivore relative growth rate (RGR) by 120% relative to eCO 2, whereas rCO 2 caused herbivore RGR to decrease by 26% compared to eCO 2 . Si supplementation also increased the density of macrohairs, silica and prickle cells, which was associated with reduced herbivore performance. There was a negative correlation among macrohair density, silica cell density, prickle cell density and herbivore RGR under rCO 2 suggesting that these changes in leaf surface morphology were linked to reduced performance under this CO 2 regime. To our knowledge, this is the first study to demonstrate that increased Si accumulation under pre-industrial CO 2 reduces insect herbivore performance. Contrastingly, we found reduced Si accumulation under higher CO 2 , which suggests that some grasses may become more susceptible to insect herbivores under projected climate change scenarios., (© 2021 John Wiley & Sons Ltd.)

Optical films that alter light transmittance may reduce energy consumption in high-tech greenhouses, but their impact on crop physiology remains unclear. We compared the stomatal responses of Capsicum plants grown hydroponically under control glass (70% diffuse light) or the smart glass (SG) film ULR-80, which blocked >50% of short-wave radiation and ~9% of photosynthetically active radiation (PAR). SG had no significant effects on steady-state (gs) or maximal (gmax) stomatal conductance. In contrast, SG reduced stomatal pore size and sensitivity to exogenous abscisic acid (ABA), thereby increasing rates of leaf water loss, guard cell K+ and Cl- efflux, and Ca2+ influx. SG induced faster stomatal closing and opening rates on transition between low (100 µmol m-2 s-1) and high PAR (1500 µmol m-2 s-1), which compromised water use efficiency relative to control plants. The fraction of blue light (0% or 10%) did not affect gs in either treatment. Increased expression of stomatal closure and photoreceptor genes in epidermal peels of SG plants is consistent with fast stomatal responses to light changes. In conclusion, stomatal responses of Capsicum to SG were more affected by changes in light intensity than spectral quality, and re-engineering of the SG should maximize PAR transmission, and hence CO2 assimilation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The level of carbon dioxide (CO 2 ) in the air can affect several traits in plants. Elevated atmospheric CO 2 (eCO 2 ) can enhance photosynthesis and increase plant productivity, including biomass, although there are inconsistencies regarding the effects of eCO 2 on the plant growth response. The compounding effects of ambient environmental conditions such as light intensity, photoperiod, water availability, and soil nutrient composition can affect the extent to which eCO 2 enhances plant productivity. This study aimed to investigate the growth response of Arabidopsis thaliana to eCO 2 (800 ppm) under short photoperiod (8/16 h, light/dark cycle). Here, we report an attenuated fertilization effect of eCO 2 on the shoot biomass of Arabidopsis plants grown under short photoperiod. The biomass of two-, three-, and four-week-old Arabidopsis plants was increased by 10%, 15%, and 28%, respectively, under eCO 2 compared to the ambient CO 2 (aCO 2 , 400 ppm) i.e. control. However, the number of rosette leaves, rosette area, and shoot biomass were similar in mature plants under both CO 2 conditions, despite 40% higher photosynthesis in eCO 2 exposed plants. The levels of chlorophylls and carotenoids were similar in the fully expanded rosette leaves regardless of the level of CO 2 . In conclusion, CO 2 enrichment moderately increased Arabidopsis shoot biomass at the juvenile stage, whereas the eCO 2 -induced increment in shoot biomass was not apparent in mature plants. A shorter day-length can limit the source-to-sink resource allocation in a plant in age-dependent manner, hence diminishing the eCO 2 fertilization effect on the shoot biomass in Arabidopsis plants grown under short photoperiod., (© Prof. H.S. Srivastava Foundation for Science and Society 2021.)

Background: Prolonged mechanical stress (MS) causes thigmomorphogenesis, a stress acclimation response associated with increased disease resistance. What remains unclear is if; 1) plants pre-exposed to a short period of repetitive MS can prime defence responses upon subsequent challenge with necrotrophic pathogens, 2) MS mediates plant immunity via jasmonic acid (JA) signalling, and 3) a short period of repetitive MS can cause long-term changes in gene expression resembling a stress-induced memory. To address these points, 10-days old juvenile Arabidopsis seedlings were mechanically stressed for 7-days using a soft brush and subsequently challenged with the necrotrophic pathogens, Alternaria brassicicola, and Botrytis cinerea. Here we assessed how MS impacted structural cell wall appositions, disease symptoms and altered gene expression in response to infection., Results: The MS-treated plants exhibited enhanced cell wall appositions and jasmonic acid (JA) accumulation that correlated with a reduction in disease progression compared to unstressed plants. The expression of genes involved in JA signalling, callose deposition, peroxidase and phytoalexin biosynthesis and reactive oxygen species detoxification were hyper-induced 4-days post-infection in MS-treated plants. The loss-of-function in JA signalling mediated by the JA-insensitive coronatine-insensitive 1 (coi1) mutant impaired the hyper-induction of defense gene expression and promoted pathogen proliferation in MS-treated plants subject to infection. The basal expression level of PATHOGENESIS-RELATED GENE 1 and PLANT DEFENSIN 1.2 defense marker genes were constitutively upregulated in rosette leaves for 5-days post-MS, as well as in naïve cauline leaves that differentiated from the inflorescence meristem well after ceasing MS., Conclusion: This study reveals that exposure of juvenile Arabidopsis plants to a short repetitive period of MS can alter gene expression and prime plant resistance upon subsequent challenge with necrotrophic pathogens via the JA-mediated COI1 signalling pathway. MS may facilitate a stress-induced memory to modulate the plant's response to future stress encounters. These data advance our understanding of how MS primes plant immunity against necrotrophic pathogens and how that could be utilised in sustainable agricultural practices.

Grasses are hyper-accumulators of silicon (Si), which they acquire from the soil and deposit in tissues to resist environmental stresses. Given the high metabolic costs of herbivore defensive chemicals and structural constituents (e.g. cellulose), grasses may substitute Si for these components when carbon is limited. Indeed, high Si uptake grasses evolved in the Miocene when atmospheric CO 2 concentration was much lower than present levels. It is, however, unknown how pre-industrial CO 2 concentrations affect Si accumulation in grasses. Using Brachypodium distachyon , we hydroponically manipulated Si-supply (0.0, 0.5, 1, 1.5, 2 mM) and grew plants under Miocene (200 ppm) and Anthropocene levels of CO 2 comprising ambient (410 ppm) and elevated (640 ppm) CO 2 concentrations. We showed that regardless of Si treatments, the Miocene CO 2 levels increased foliar Si concentrations by 47% and 56% relative to plants grown under ambient and elevated CO 2 , respectively. This is owing to higher accumulation overall, but also the reallocation of Si from the roots into the shoots. Our results suggest that grasses may accumulate high Si concentrations in foliage when carbon is less available (i.e. pre-industrial CO 2 levels) but this is likely to decline under future climate change scenarios, potentially leaving grasses more susceptible to environmental stresses.

Main Conclusion: This study highlights the potential link between high light-induced canopy-level photosynthesis and mesophyll cell K + , Cl - , Ca 2+ , and H + homeostasis in tomato. Light is a primary energy source for photosynthesis and a vital regulator of mineral nutrient uptake and distribution in plants. Plants need to optimize photosynthesis and nutrient balance in leaves for performance in fluctuating light conditions that are partially regulated by light-induced ion homeostatsis in the mesophyll cells. It is still elusive whether high light-induced leaf mesophyll ion fluxes affect leaf photosynthesis at different canopy levels in Solanum lycopersicum L. Leaf gas exchange and microelectrode ion flux (MIFE) measurements were employed to study the effects of prolonged light-induced canopy-level leaf physiological responses of tomato plants. High light resulted in a significant lowering in photosynthesis in the fully-exposed top canopy leaves of tomato, but not to mid- or low-canopy leaves. Leaf mesophyll K + effluxes of all canopies were significantly decreased after three weeks of high light treatment. However, high light-induced leaf mesophyll Ca 2+ effluxes were significantly enhanced only in the top and mid canopies. Moreover, we found that photosynthetic parameters were significantly correlated with leaf mesophyll ion fluxes. We thus propose that canopy-level significant Ca 2+ efflux and K + efflux of leaf mesophyll may serve as early indicators for light-induced regulation on photosynthesis. We conclude that light-induced differential photosynthetic performance and ion fluxes in leaves may implicate a requirement of more uniform light irradiance and spectra at different canopy levels of tall greenhouse tomato plants. This can be achieved through new innovative greenhouse lighting technologies and covering materials towards the enhancement of crop photosynthesis and yield.

Environmental factors such as photoperiod, temperature, phytohormones, sugars, and soil nutrients can affect the development of axillary meristems and emergence of shoot branches in plants. We investigated how an extended period of cold exposure to Arabidopsis plants before and after inflorescence meristem differentiation would affect plant growth and shoot branching. The number of rosette leaves and shoot branches increased when wild type (WT) juvenile seedlings, but not adult plants, were subjected to a prolonged cold exposure (10/7°C day/night cycle). As the duration of cold exposure to WT juvenile seedlings increased, so too did the rosette area, number of leaves, and rosette branches revealing an extended period of vegetative growth. The prolonged cold treatment also increased the primary inflorescence stem height and number of cauline branches in WT plants revealing a delay in reproductive development that could be altered by early ( set domain group 8; sdg8 ) and late ( methyltransferase 1; met1 ) flowering mutants. The axillary buds/leaf and rosette branches/leaf ratios declined significantly in WT, yet were enhanced in the loss-of-function of carotenoid cleavage dioxygenase 7 ( ccd7 ) and teosinte branched 1 ( brc1 ) hyper-branched mutants. This indicated that axillary meristem differentiation continued during the cold exposure, which did not directly impact axillary bud formation or shoot branching. We conclude that a prolonged cold exposure to juvenile seedlings prior to inflorescence meristem development extended vegetative growth and delayed the reproductive phase to allow additional leaf primordia and axillary meristems to differentiate that enhanced the number of shoot branches in Arabidopsis .

Heatwaves are becoming more frequent with climate warming and can impact tree growth and reproduction. Eucalyptus parramattensis can cope with an extreme heatwave in the field via transpiratory cooling and enhanced leaf thermal tolerance that protected foliar tissues from photo-inhibition and photo-oxidation during natural midday irradiance. Here, we explored whether changes in foliar carotenoids and/or the xanthophyll cycle state can facilitate leaf acclimation to long-term warming and/or an extreme heatwave event. We found that leaves had similar carotenoid levels when grown for one year under ambient and experimental long-term warming (+ 3 °C) conditions in whole tree chambers. Exposure to a 4-day heatwave (> 43 °C) significantly altered the xanthophyll de-epoxidation state of carotenoids revealing one mechanism by which trees could minimise foliar photo-oxidative damage. The levels of zeaxanthin were significantly higher in both young and old leaves during the heatwave, revealing that violaxanthin de-epoxidation and perhaps de novo zeaxanthin synthesis contributed to enhancement of the xanthophyll cycle state. In a future climate of long-term warming and increased heatwave events, leaves of E. parramattensis will be able to utilise biochemical strategies to alter the xanthophyll cycle state and cope with extreme temperatures under natural solar irradiation., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Prof. H.S. Srivastava Foundation for Science and Society 2020.)

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 ( ccr2 ), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a ζ-carotene isomerase mutant ( ziso-155 ) and ccr2 blocked the biosynthesis of specific cis -carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1 , thereby controlling plastid development., Competing Interests: CC, XH, YA, JR, ND, JL, SM, BP No competing interests declared, (© 2020, Cazzonelli et al.)

Reverse phase high-performance liquid chromatography (HPLC) is the method of choice used in biological, health, and food research to identify, quantify, and profile carotenoid species. The identification and quantification of cis- and/or trans-carotene and xanthophyll isomers in plant tissues can be affected by the method of sample preparation and extraction, as well as the HPLC column chemistry and the solvent gradient. There is a high degree of heterogeneity in existing methods in terms of their ease, efficiency, and accuracy. We describe a simple carotenoid extraction method and two different optimised HPLC methods utilizing C18 or C30 reverse-phase columns. We outline applications, advantages, and disadvantages for using these reverse phase columns to detect xanthophylls and cis-carotenes in wild-type photosynthetic leaves and mutant dark-grown etiolated seedlings, respectively. Resources are provided to profile individual species based upon their spectral properties and retention time, as well as quantify carotenoids by their composition and absolute levels in different plant tissues.

Light is a vital regulator that controls physiological and cellular responses to regulate plant growth, development, yield, and quality. Light is the driving force for electron and ion transport in the thylakoid membrane and other membranes of plant cells. In different plant species and cell types, light activates photoreceptors, thereby modulating plasma membrane transport. Plants maximize their growth and photosynthesis by facilitating the coordinated regulation of ion channels, pumps, and co-transporters across membranes to fine-tune nutrient uptake. The signal-transducing functions associated with membrane transporters, pumps, and channels impart a complex array of mechanisms to regulate plant responses to light. The identification of light responsive membrane transport components and understanding of their potential interaction with photoreceptors will elucidate how light-activated signaling pathways optimize plant growth, production, and nutrition to the prevailing environmental changes. This review summarizes the mechanisms underlying the physiological and molecular regulations of light-induced membrane transport and their potential interaction with photoreceptors in a plant evolutionary and nutrition context. It will shed new light on plant ecological conservation as well as agricultural production and crop quality, bringing potential nutrition and health benefits to humans and animals., (Copyright © 2019 Babla, Cai, Chen, Tissue, Cazzonelli and Chen.)

Plants are subjected to a multitude of stimuli during insect herbivory, resulting in a complex and cumulative defence response. Breaking down the components of herbivory into specific stimuli and identifying the mechanisms of defence associated with them has thus far been challenging. Advances in our understanding of responses to inconspicuous stimuli, such as those induced by microbial symbionts in herbivore secretions and mechanical stimulation caused by insects, have illuminated the intricacies of herbivory. Here, we provide a synthesis of the interacting impacts of herbivory on plants and the consequential complexities associated with uncoupling defence responses. We propose that simulated herbivory should be used to complement true herbivory to decipher the mechanisms of insect herbivore-induced plant defence responses., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Carotenoids are isoprenoid pigments synthesised by plants, algae, photosynthetic bacteria as well as some non-photosynthetic bacteria, fungi and insects. Abundant carotenoids found in nature are synthesised via a linear route from phytoene to lycopene after which the pathway bifurcates into cyclised α- and β-carotenes. Plants evolved additional steps to generate a diversity of cis-carotene intermediates, which can accumulate in fruits or tissues exposed to an extended period of darkness. Enzymatic or oxidative cleavage, light-mediated photoisomerization and histone modifications can affect cis-carotene accumulation. cis-carotene accumulation has been linked to the production of signaling metabolites that feedback and forward to regulate nuclear gene expression. When cis-carotenes accumulate, plastid biogenesis and operational control can become impaired. Carotenoid derived metabolites and phytohormones such as abscisic acid and strigolactones can fine-tune cellular homeostasis. There is a hunt to identify a novel cis-carotene derived apocarotenoid signal and to elucidate the molecular mechanism by which it facilitates communication between the plastid and nucleus. In this review, we describe the biosynthesis and evolution of cis-carotenes and their links to regulatory switches, as well as highlight how cis-carotene derived apocarotenoid signals might control organelle communication, physiological and developmental processes in response to environmental change., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Carotenoids contribute to photosynthesis, photoprotection, phytohormone and apocarotenoid biosynthesis in plants. Carotenoid-derived metabolites control plant growth, development and signalling processes and their accumulation can depend upon changes in the environment. Elevated carbon dioxide (eCO 2 ) often enhances carbon assimilation, early growth patterns and overall plant biomass, and may increase carotenoid accumulation due to higher levels of precursors from isoprenoid biosynthesis. Variable effects of eCO 2 on carotenoid accumulation in leaves have been observed for different plant species. Here, we determined whether the variable response of carotenoids to eCO 2 was potentially a function of leaf age and the impact of eCO 2 on leaf development by growing Arabidopsis in ambient CO 2 (400 ppm) and eCO 2 (800 ppm). eCO 2 increased plant leaf number, rosette area, biomass, seed yield and net photosynthesis. In addition, eCO 2 increased carotenoid content by 10-20% in younger emerging leaves, but not in older mature leaves. Older leaves contained approximately 60% less total carotenoids compared to younger leaves. The age-dependent effect on carotenoid content was observed for cotyledon, juvenile and adult phase leaves. We conclude that younger leaves utilize additional carbon from enhanced photosynthesis in eCO 2 to increase carotenoid content, yet older leaves have less capacity to store additional carbon into carotenoids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis . Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1 -2. PAP also inhibits wild type and abi1 -1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca 2+ ; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.

Thigmomorphogenesis is viewed as being a response process of acclimation to short repetitive bursts of mechanical stimulation or touch. The underlying molecular mechanisms that coordinate changes in how touch signals lead to long-term morphological changes are enigmatic. Touch responsive gene expression is rapid and transient, and no transcription factor or DNA regulatory motif has been reported that could confer a genome wide mechanical stimulus. We report here on a chromatin modifying enzyme, SDG8/ASHH2, which can regulate the expression of many touch responsive genes identified in Arabidopsis. SDG8 is required for the permissive expression of touch induced genes; and the loss of function of sdg8 perturbs the maximum levels of induction on selected touch gene targets. SDG8 is required to maintain permissive H3K4 trimethylation marks surrounding the Arabidopsis touch-inducible gene TOUCH 3 (TCH3), which encodes a calmodulin-like protein (CML12). The gene neighboring was also slightly down regulated, revealing a new target for SDG8 mediated chromatin modification. Finally, sdg8 mutants show perturbed morphological response to wind-agitated mechanical stimuli, implicating an epigenetic memory-forming process in the acclimation response of thigmomorphogenesis.

In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds/clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds/clb5/spc1/pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4., (© 2014 American Society of Plant Biologists. All rights reserved.)

In plants, continuous formation of lateral roots (LRs) facilitates efficient exploration of the soil environment. Roots can maximize developmental capacity in variable environmental conditions through establishment of sites competent to form LRs. This LR prepattern is established by a periodic oscillation in gene expression near the root tip. The spatial distribution of competent (prebranch) sites results from the interplay between this periodic process and primary root growth; yet, much about this oscillatory process and the formation of prebranch sites remains unknown. We find that disruption of carotenoid biosynthesis results in seedlings with very few LRs. Carotenoids are further required for the output of the LR clock because inhibition of carotenoid synthesis also results in fewer sites competent to form LRs. Genetic analyses and a carotenoid cleavage inhibitor indicate that an apocarotenoid, distinct from abscisic acid or strigolactone, is specifically required for LR formation. Expression of a key carotenoid biosynthesis gene occurs in a spatially specific pattern along the root's axis, suggesting spatial regulation of carotenoid synthesis. These results indicate that developmental prepatterning of LRs requires an uncharacterized carotenoid-derived molecule. We propose that this molecule functions non-cell-autonomously in establishment of the LR prepattern.

Cellular auxin homeostasis controls many aspects of plant growth, organogenesis and development. The existence of intracellular auxin transport mediated by endoplasmic reticulum (ER)-localized PIN5, PIN6 and PIN8 proteins is a relatively recent discovery shaping a new era in understanding auxin-mediated growth processes. Here we summarize the importance of PIN6 in mediating intracellular auxin transport during root formation, leaf vein patterning and nectary production. While, it was previously shown that PIN6 was strongly expressed in rosette leaf cell types important in vein formation, here we demonstrate by use a PIN6 promoter-reporter fusion, that PIN6 is also preferentially expressed in the vasculature of the primary root, cotyledons, cauline leaves, floral stem, sepals and the main transmitting tract of the reproductive silique. The strong, vein- specific reporter gene expression patterns enabled by the PIN6 promoter emphasizes that transcriptional control is likely to be a major regulator of PIN6 protein levels, during vasculature formation, and supports the need for ER-localized PIN proteins in selecting specialized cells for vascular function in land plants.

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin-regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

Background: Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction., Results: Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem., Conclusions: We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues.

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3' half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.

Carotenoids are natural isoprenoid pigments that provide leaves, fruits, vegetables and flowers with distinctive yellow, orange and some reddish colours as well as several aromas in plants. Their bright colours serve as attractants for pollination and seed dispersal. Carotenoids comprise a large family of C40 polyenes and are synthesised by all photosynthetic organisms, aphids, some bacteria and fungi alike. In animals carotenoid derivatives promote health, improve sexual behaviour and are essential for reproduction. As such, carotenoids are commercially important in agriculture, food, health and the cosmetic industries. In plants, carotenoids are essential components required for photosynthesis, photoprotection and the production of carotenoid-derived phytohormones, including ABA and strigolactone. The carotenoid biosynthetic pathway has been extensively studied in a range of organisms providing an almost complete pathway for carotenogenesis. A new wave in carotenoid biology has revealed implications for epigenetic and metabolic feedback control of carotenogenesis. Developmental and environmental signals can regulate carotenoid gene expression thereby affecting carotenoid accumulation. This review highlights mechanisms controlling (1) the first committed step in phytoene biosynthesis, (2) flux through the branch to synthesis of α- and β-carotenes and (3) metabolic feedback signalling within and between the carotenoid, MEP and ABA pathways.

The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5' untranslated region (5'UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5'UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the beta-glucuronidase (GUS) gene was fused to each of the three 5'UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5'UTRs directed higher levels of GUS activity than the PAE 5'UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5'UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5'UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5'UTR.

Carotenoids are a diverse group of colourful pigments naturally found in plants, algae, fungi and bacteria. They play essential roles in development, photosynthesis, root-mycorrhizal interactions and the production of phytohormones, such as abscisic acid and strigolactone. Carotenoid biosynthesis is regulated throughout the life cycle of a plant with dynamic changes in composition matched to prevailing developmental requirements and in response to external environmental stimuli. There are key regulatory nodes in the pathway that control the flux of metabolites into the pathway and alter flux through the pathway. The molecular nature of the mechanisms regulating carotenoid biosynthesis, including evidence for metabolite feedback, transcription and epigenetic control as well as their accumulation, storage and degradation will be the focus of this review., (2010 Elsevier Ltd. All rights reserved.)

Background: In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation., Findings: A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production., Conclusions: Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

Carotenoids are pigments required for photosynthesis, photoprotection and the production of carotenoid-derived hormones such as ABA and strigolactones. The carotenoid biosynthetic pathway bifurcates after lycopene to produce epsilon- and beta-carotenoids and this branch is critical for determining carotenoid composition. Here, we show how the branch point can be regulated by the chromatin-modifying histone methyltransferase, Set Domain Group 8 (SDG8) targeting the carotenoid isomerase (CRTISO). SDG8 is required to maintain permissive expression of CRTISO during seedling development, in leaves, shoot apex, and some floral organs. The CRTISO and SDG8 promoters show overlapping tissue-specific patterns of reporter gene activity. Interestingly, CRTISO showed atypical reporter gene expression in terms of greater variability between different lines compared to the Cauliflower Mosaic Virus 35S promoter (CaMV35s) and epsilonLCY promoters, potentially due to chromosomal position effects. Regulation of the CRTISO promoter was dependent in part upon the presence or absence of SDG8. Knockouts of SDG8 (carotenoid and chloroplast regulation (ccr1)) and CRTISO (ccr2) result in altered carotenoid composition and this could be restored in ccr2 using the CaMV35s or CRTISO promoters. In contrast, varying degrees of GUS expression and carotenoid complementation by CRTISO overexpression using CaMV35S or CRTISO promoters in the ccr1 background demonstrated that both the CRTISO promoter and open reading frame are necessary for SDG8-mediated expression of CRTISO.

Plants utilize sophisticated epigenetic regulatory mechanisms to coordinate changes in gene expression during development and in response to environmental stimuli. Epigenetics refers to the modification of DNA and chromatin associated proteins, which affect gene expression and cell function, without changing the DNA sequence. Such modifications are inherited through mitosis, and in rare instances through meiosis, although it can be reversible and thus regulatory. Epigenetic modifications are controlled by groups of proteins, such as the family of histone lysine methytransferases (HKMTs). The catalytic core known as the SET domain encodes HKMT activity and either promotes or represses gene expression. A large family of SET domain proteins is present in Arabidopsis where there is growing evidence that two classes of these genes are involved in promoting gene expression in a diverse range of developmental processes. This review will focus on the function of these two classes and the processes that they control, highlighting the huge potential this regulatory mechanism has in plants.

Carotenoid pigments are critical for plant survival, and carotenoid composition is tuned to the developmental stage, tissue, and to environmental stimuli. We report the cloning of the CAROTENOID CHLOROPLAST REGULATORY1 (CCR1) gene. The ccr1 mutant has increased shoot branching and altered carotenoid composition, namely, reduced lutein in leaves and accumulation of cis-carotenes in dark-grown seedlings. The CCR1 gene was previously isolated as EARLY FLOWERING IN SHORT DAYS and encodes a histone methyltransferase (SET DOMAIN GROUP 8) that methylates histone H3 on Lys 4 and/or 36 (H3K4 and H3K36). ccr1 plants show reduced trimethyl-H3K4 and increased dimethyl-H3K4 surrounding the CAROTENOID ISOMERASE (CRTISO) translation start site, which correlates with low levels of CRTISO mRNA. Microarrays of ccr1 revealed the downregulation of 85 genes, including CRTISO and genes associated with signaling and development, and upregulation of just 28 genes. The reduction in CRTISO transcript abundance explains the altered carotenoid profile. The changes in shoot branching are additive with more axillary branching mutants, but the altered carotenoid profile may partially affect shoot branching, potentially by perturbed biosynthesis of the carotenoid substrates of strigolactones. These results are consistent with SDG8 regulating shoot meristem activity and carotenoid biosynthesis by modifying the chromatin surrounding key genes, including CRTISO. Thus, the level of lutein, the most abundant carotenoid in higher plants that is critical for photosynthesis and photoprotection, appears to be regulated by a chromatin modifying enzyme in Arabidopsis thaliana.

Short directly-repeated (DR) DNA enhancer elements of plant viral origin were analyzed for their ability, both individually and in combination, to influence in vivo transcription when inserted upstream from a minimal CaMV35S promoter. Synthetic promoters containing multiple copies and/or combinations of DR cassettes were tested for their effect upon reporter gene (luciferase) expression using an Agrobacteria-based leaf-infiltration transient assay and within stably transformed plants (Nicotiana tabacum). Transgenic plants harboring constructs containing different numbers or combinations of DR cassettes were further tested to look for tissue-specific expression patterns and potential promoter response to the infiltration process employed during transient expression. Multimerization of DR elements produced enhancer activity that was in general additive, increasing reporter activity in direct proportion to the number of DR cassettes within the test promoter. In contrast, combinations of different DR cassettes often functioned synergistically, producing reporter enhancement markedly greater then the sum of the combined DR activities. Several of the DR constructs responded to Agrobacteria (lacking T-DNA) infiltration of transgenic leaves by an induction (2 elements) or reduction (1 element) in reporter activity. Combinations of DR cassettes producing the strongest enhancement of reporter activity were used to create two synthetic promoters (SynPro3 and SynPro5) that drive leaf reporter activities at levels comparable to the CaMV35S promoter. Characterization of these synthetic promoters in transformed tobacco showed strong reporter expression at all stages of development and in most tissues. The arrangement of DR elements within SynPro3 and SynPro5 appears to play a role in defining tissue-specificity of expression and/or Agrobacteria-infusion responsiveness.

An in vivo assay system for analyzing transient luciferase expression in tobacco leaves infused with Agrobacterium tumefaciens is described. The system makes use of A. tumefaciens harboring T-DNA vectors containing either an intron-containing firefly (Photinus pyralis) luciferase (EC 1.13.12.7) gene or an intron-containing sea pansy (Renilla reniformis) luciferase (EC 1.13.12.5) gene. Single or mixed Agrobacterium lines were infiltrated into leaf tissues (Nicotiana tabacum or Nicotiana benthamiana) through stomatal openings and leaf disks from infused areas floated on reaction buffers specific to each enzyme. Photons emitted were then measured to determine reporter gene activity. Parameters affecting assay reliability and sensitivity were tested, including: buffer composition; bacterial density; infusion location; reaction kinetics; and environmental factors (light and temperature). The resulting in vivo assay system generates results comparable to those obtained using a commercially available in vitro dual-luciferase(R) reporter gene assay, and reports relative expression levels, as well as induction characteristics, analogous to those obtained using leaf tissue from stably transformed plants harboring the same promoter::gene constructs. Light and temperature were observed to markedly impact transient reporter activities. Co-expression of viral suppressors of post-transcriptional gene silencing (PTGS), HcPro, p19 and AC2, confirms the occurrence of PTGS within infused zones, and provides a convenient mechanism for PTGS analysis. The in vivo transient assay was used to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS.

We report the cloning and characterization in tobacco and Arabidopsis of a Vigna radiata L. (mung bean) promoter that controls the expression of VR-ACS1, an auxin-inducible ACC synthase gene. The VR-ACS1 promoter exhibits a very unusual behavior when studied in plants different from its original host, mung bean. GUS and luciferase in situ assays of transgenic plants containing VR-ACS1 promoter fusions show strong constitutive reporter gene expression throughout tobacco and Arabidopsis development. In vitro quantitative analyses show that transgenic plants harboring VR-ACS1 promoter-reporter constructs have on average 4-6 fold higher protein and activity levels of both reporter genes than plants transformed with comparable CaMV 35S promoter fusions. Similar transcript levels are present in VR-ACS1 and CaMV 35S promoter lines, suggesting that the high levels of gene product observed for the VR-ACS1 promoter are the combined result of transcriptional and translational activation. All tested deletion constructs retaining the core promoter region can drive strong constitutive promoter activity in transgenic plants. This is in contrast to mung bean, where expression of the native VR-ACS1 gene is almost undetectable in plants grown under normal conditions, but is rapidly and highly induced by a variety of stimuli. The constitutive behavior of the VR-ACS1 promoter in heterologous hosts is surprising, suggesting that the control mechanisms active in mung bean are impaired in tobacco and Arabidopsis. The 'aberrant' behavior of the VR-ACS1 promoter is further emphasized by its failure to respond to auxin and cycloheximide in heterologous hosts. VR-ACS1 promoter regulatory mechanisms seem to be different from all previously characterized auxin-inducible promoters.

The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a -46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct-repeat cassette of the element (2x CLE) enhances luciferase activity by 2-fold, independent of the element's orientation, while multiple copies of the cassette (4-12x CLE) increases activity up to 10- to 15-fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-AC2 expression was found to result in a 2-fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter's promoter. These data suggests that the PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.

Background: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection., Results: DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components., Conclusion: Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.