Short photoperiod attenuates CO2 fertilization effect on shoot biomass in Arabidopsis thaliana.

The level of carbon dioxide (CO2) in the air can affect several traits in plants. Elevated atmospheric CO2 (eCO2) can enhance photosynthesis and increase plant productivity, including biomass, although there are inconsistencies regarding the effects of eCO2 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 eCO2 enhances plant productivity. This study aimed to investigate the growth response of Arabidopsis thaliana to eCO2 (800 ppm) under short photoperiod (8/16 h, light/dark cycle). Here, we report an attenuated fertilization effect of eCO2 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 eCO2 compared to the ambient CO2 (aCO2, 400 ppm) i.e. control. However, the number of rosette leaves, rosette area, and shoot biomass were similar in mature plants under both CO2 conditions, despite 40% higher photosynthesis in eCO2 exposed plants. The levels of chlorophylls and carotenoids were similar in the fully expanded rosette leaves regardless of the level of CO2. In conclusion, CO2 enrichment moderately increased Arabidopsis shoot biomass at the juvenile stage, whereas the eCO2-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 eCO2 fertilization effect on the shoot biomass in Arabidopsis plants grown under short photoperiod.

Prior exposure of Arabidopsis seedlings to mechanical stress heightens jasmonic acid-mediated defense against necrotrophic pathogens.

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.

Effect of high light on canopy-level photosynthesis and leaf mesophyll ion flux in tomato.

MAIN CONCLUSION: This study highlights the potential link between high light-induced canopy-level photosynthesis and mesophyll cell K+, Cl-, Ca2+, 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 Ca2+ 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 Ca2+ 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.

An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis.

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.

BZR1 and BES1 transcription factors mediate brassinosteroid control over root system architecture in response to nitrogen availability.

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.

Molecular Evolution and Interaction of Membrane Transport and Photoreception in Plants.

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.

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids.

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 ß-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 ß-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in ß-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state ß-carotene amounts to up to 500 µg g-1 dry mass. Furthermore, we identified oxidative ß-carotene degradation products which correlated with pathway activities, yielding ß-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 ß-carotene sequestration by co-localization experiments which revealed that ß-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 ß-carotene levels prior to their application in crops.

In vivo characterization of plant promoter element interaction using synthetic promoters.

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, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing.

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.

Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco.

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.