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Increasing atmospheric CO 2 concentrations accompanied by abiotic stresses challenge food production worldwide. Elevated CO 2 ( e [CO 2 ]) affects plant water relations via multiple mechanisms involving abscisic acid (ABA). Here, two tomato ( Solanum lycopersicum ) genotypes, Ailsa Craig (AC) and its ABA-deficient mutant ( flacca ), were used to investigate the responses of plant hydraulic conductance to e [CO 2 ] and drought stress. Results showed that e [CO 2 ] decreased transpiration rate ( E ) increased plant water use efficiency only in AC, whereas it increased daily plant water consumption and osmotic adjustment in both genotypes. Compared to growth at ambient [CO 2 ], AC leaf and root hydraulic conductance ( K leaf and K root ) decreased at e [CO 2 ], which coincided with the transcriptional regulations of genes of plasma membrane intrinsic proteins (PIPs) and OPEN STOMATA 1 (OST1), and these effects were attenuated in flacca during soil drying. Severe drought stress could override the effects of e [CO 2 ] on plant water relation characteristics. In both genotypes, drought stress resulted in decreased E , K leaf , and K root accompanied by transcriptional responses of PIPs and OST1. However, under conditions combining e [CO 2 ] and drought, some PIPs were not responsive to drought in AC, indicating that e [CO 2 ] might disturb ABA-mediated drought responses. These results provide some new insights into mechanisms of plant hydraulic response to drought stress in a future CO 2 -enriched environment., 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 © 2021 Li, Fang, Hegelund and Liu.)

Hairy root (HR) cultures are quickly evolving as a fundamental research tool and as a bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4 week period. Nine anthocyanins and 24 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by >20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalization of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants, and provide new insight into the regulation of secondary metabolism in black carrot., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Increasing agricultural losses due to biotic and abiotic stresses caused by climate change challenge food security worldwide. A promising strategy to sustain crop productivity under conditions of limited water availability is the use of plant growth promoting rhizobacteria (PGPR). Here, the effects of spore forming Bacillus licheniformis (FMCH001) on growth and physiology of maize ( Zea mays L. cv. Ronaldinho) under well-watered and drought stressed conditions were investigated. Pot experiments were conducted in the automated high-throughput phenotyping platform PhenoLab and under greenhouse conditions. Results of the PhenoLab experiments showed that plants inoculated with B. licheniformis FMCH001 exhibited increased root dry weight (DW) and plant water use efficiency (WUE) compared to uninoculated plants. In greenhouse experiments, root and shoot DW significantly increased by more than 15% in inoculated plants compared to uninoculated control plants. Also, the WUE increased in FMCH001 plants up to 46% in both well-watered and drought stressed plants. Root and shoot activities of 11 carbohydrate and eight antioxidative enzymes were characterized in response to FMCH001 treatments. This showed a higher antioxidant activity of catalase (CAT) in roots of FMCH001 treated plants compared to uninoculated plants. The higher CAT activity was observed irrespective of the water regime. These findings show that seed coating with Gram positive spore forming B. licheniformis could be used as biostimulants for enhancing plant WUE under both normal and drought stress conditions., (Copyright © 2020 Akhtar, Amby, Hegelund, Fimognari, Großkinsky, Westergaard, Müller, Moelbak, Liu and Roitsch.)

Elevated CO 2 concentration in the air ( e [CO 2 ]) decreases stomatal density (SD) and stomatal conductance ( g s ) where abscisic acid (ABA) may play a role, yet the underlying mechanism remains largely elusive. We investigated the effects of e [CO 2 ] (800 ppm) on leaf gas exchange and water relations of two tomato ( Solanum lycopersicum ) genotypes, Ailsa Craig (WT) and its ABA-deficient mutant ( flacca ). Compared to plants grown at ambient CO 2 (400 ppm), e [CO 2 ] stimulated photosynthetic rate in both genotypes, while depressed the g s only in WT. SD showed a similar response to e [CO 2 ] as g s , although the change was not significant. e [CO 2 ] increased leaf and xylem ABA concentrations and xylem sap pH, where the increases were larger in WT than in flacca . Although leaf water potential was unaffected by CO 2 growth environment, e [CO 2 ] lowered osmotic potential, hence tended to increase turgor pressure particularly for WT. e [CO 2 ] reduced hydraulic conductance of leaf and root in WT but not in flacca , which was associated with downregulation of gene expression of aquaporins. It is concluded that ABA-mediated regulation of g s , SD, and gene expression of aquaporins coordinates the whole-plant hydraulics of tomato grown at different CO 2 environments., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2019.)

Brassica napus (oilseed rape) is a major oil crop worldwide. Due to the short domestication period of oilseed rape the genetic variability is limited compared to other crops. Transfer of rol and aux genes from Agrobacterium rhizogenes is used in horticulture to increase genetic variability. In the current study, we explore transformation by A. rhizogenes as a biotechnological approach in breeding for more branched and shorter oilseed rape. In the 2nd generation of transformed oilseed rape, branch numbers increased significantly by 49% from 7.7 ± 0.4 to 11.5 ± 1.9 when comparing rol+/aux+ plants with WT. Simultaneously, the apical height of plants was reduced by 25% from 81.3 ± 1.9 cm to 62.4 ± 6.7 cm in rol+/aux+ plants at the onset of flowering. Reproductive parameters affecting yield as seed size and number were negatively affected in rol+/aux+ plants. Interestingly, oil composition was changed in rol+/aux+ seeds. Oleic acid (ω9) contents were reduced by more than 3% whereas α-linolenic acid (ω6) increased by more than 25% in mature seeds. To obtain shorter and more branched breeding material of oilseed rape we suggest crossing plants with the rol+/aux+ genotype back into the parental breeding line. This could reduce the negative impact of rol+/aux+ on yield., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers are sensitive to even minute concentrations of ethylene., Results: Monitoring flower longevity in three Campanula species revealed C. portenschlagiana (Cp) as ethylene sensitive, C. formanekiana (Cf) with intermediate sensitivity and C. medium (Cm) as ethylene insensitive. We identified key elements in ethylene signal transduction, specifically in Ethylene Response Sensor 2 (ERS2), Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 3- Like 1 and 2 (EIL1 and EIL2) homologous. Transcripts of ERS2, CTR1 and EIL1 were constitutively expressed in all species both throughout flower development and in response to ethylene. In contrast, EIL2 was found only in Cf and Cm. We identified a natural mutation in Cmeil2 causing a frameshift which resulted in difference in expression levels of EIL2, with more than 100-fold change between Cf and Cm in young flowers., Conclusions: This study shows that the naturally occurring 7 bp frameshift discovered in Cmeil2, a key gene in the ethylene signaling pathway, correlates with ethylene insensitivity in flowers. We suggest that transfer of the eil2 mutation to other plant species will provide a novel tool to engineer ethylene insensitive flowers.

Various strategies of plant breeding have been attempted in order to improve the ethylene resistance of flowering ornamental plants. These approaches span from conventional techniques such as simple cross-pollination to new breeding techniques which modify the plants genetically such as precise genome-editing. The main strategies target the ethylene pathway directly; others focus on changing the ethylene pathway indirectly via pathways that are known to be antagonistic to the ethylene pathway, e.g. increasing cytokinin levels. Many of the known elements of the ethylene pathway have been addressed experimentally with the aim of modulating the overall response of the plant to ethylene. Elements of the ethylene pathway that appear particularly promising in this respect include ethylene receptors as ETR1, and transcription factors such as EIN3. Both direct and indirect approaches seem to be successful, nevertheless, although genetic transformation using recombinant DNA has the ability to save much time in the breeding process, they are not readily used by breeders yet. This is primarily due to legislative issues, economic issues, difficulties of implementing this technology in some ornamental plants, as well as how these techniques are publically perceived, particularly in Europe. Recently, newer and more precise genome-editing techniques have become available and they are already being implemented in some crops. New breeding techniques may help change the current situation and pave the way toward a legal and public acceptance if products of these technologies are indistinguishable from plants obtained by conventional techniques.

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10 d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification., (© 2013 John Wiley & Sons Ltd.)

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins believed to play a role in cytosolic zinc (Zn) and copper (Cu) homeostasis. However, evidence for the functional properties of MTs has been hampered by methodological problems in the isolation and characterization of the proteins. Here, we document that barley (Hordeum vulgare) MT3 and MT4 proteins exist in planta and that they differ in tissue localization as well as in metal coordination chemistry. Combined transcriptional and histological analyses showed temporal and spatial correlations between transcript levels and protein abundance during grain development. MT3 was present in tissues of both maternal and filial origin throughout grain filling. In contrast, MT4 was confined to the embryo and aleurone layer, where it appeared during tissue specialization and remained until maturity. Using state-of-the-art speciation analysis by size-exclusion chromatography inductively coupled plasma mass spectrometry and electrospray ionization time-of-flight mass spectrometry on recombinant MT3 and MT4, their specificity and capacity for metal ion binding were quantified, showing a strong preferential Zn binding relative to Cu and cadmium (Cd) in MT4, which was not the case for MT3. When complementary DNAs from barley MTs were expressed in Cu- or Cd-sensitive yeast mutants, MT3 provided a much stronger complementation than did MT4. We conclude that MT3 may play a housekeeping role in metal homeostasis, while MT4 may function in Zn storage in developing and mature grains. The localization of MT4 and its discrimination against Cd make it an ideal candidate for future biofortification strategies directed toward increasing food and feed Zn concentrations.

Transmembrane nine (TM9) proteins are localized in the secretory pathway of eukaryotic cells and are involved in cell adhesion and phagocytosis. The mechanism by which TM9 proteins operate is, however, not well understood. Here we have utilized elemental profiling by inductively coupled plasma mass spectrometry (ICP-MS) to further investigate the physiological function of TM9 proteins. Cellular copper contents in Saccharomyces cerevisiae varied depending on the presence of TM9 homologues from both yeast and Arabidopsis thaliana. A yeast tmn1-3 triple mutant lacking all three yeast endogenous TMNs showed altered metal homeostasis with a reduction in the cellular Cu contents to 25% of that in the wild-type. Conversely, when TMN1 was overexpressed in yeast, cellular Cu concentrations were more than doubled. Both Tmn1p-GFP and Tmn2p-GFP fusion proteins localized to the tonoplast. Yeast vacuolar biogenesis was not affected by the lack or presence of TM9 proteins neither in the tmn1-3 triple mutant nor in TM9 overexpressing strains. Heterologous expression in yeast of AtTMN7, a TM9 homologue from Arabidopsis, affected Cu homeostasis similar to the overexpression of TMN1. In Arabidopsis, the two TM9 homologues AtTMN1 and AtTMN7 were ubiquitously expressed. AtTMN7 promoter constructs driving the expression of GFP showed elevated expression of AtTMN7 in the root elongation zone. It is concluded that TM9 homologues from S. cerevisiae and A. thaliana have the ability to affect the intracellular Cu balance. Tmn1p and Tmn2p operate from the yeast vacuolar membrane without influencing vacuolar biogenesis. A new physiological function of the TM9 family coupled to vacuolar Cu homeostasis is proposed., (Copyright © Physiologia Plantarum 2010.)

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1-3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.

Perennial ryegrass (Lolium perenne L.) is the most important turf and forage grass species of the temperate regions. It requires substantial input of nitrogen fertilizer for optimum yield. Improved nitrogen use efficiency (NUE) is therefore one of the main breeding targets. However, limited knowledge is currently available on the genes controlling NUE in perennial ryegrass. The aim of the present study was to isolate genes involved in ammonium transport and assimilation. In silico screening of a Lolium EST-library using known sequences of tonoplast intrinsic proteins (TIPs) and cytosolic glutamine synthetase (GS1) revealed a number of homologous sequences. Using these sequences, primers were designed to obtain the full-length sequences by RACE-PCR. Three TIP genes (LpTIP1;1, LpTIP1;2 and LpTIP2;1) and two GS genes (LpGS1a and LpGS1b) were isolated. Characterization in S. cerevisiae confirmed a function in ammonium transport for LpTIP1;1 and LpTIP2;1 and in synthesis of glutamine for LpGS1a and LpGS1b. Cytoimmunochemical studies showed that GS protein was present in the chloroplasts and cytosol of leaf cells, while TIP1 proteins localized to the tonoplast. At the expression level, Lolium GS1 genes responded to N starvation and re-supply in a manner consistent with functions in primary N assimilation and N remobilization. Similarly, the expression of LpTIPs complied with a role in vacuolar ammonium storage. Together, the reported results provide new understanding of the genetic basis for N assimilation and storage in ryegrass.