Nitrogen Uptake and Distribution in Different Chinese Cabbage Genotypes under Low Nitrogen Stress.

In order to understand the effects of low nitrogen (LN) stress on the growth and development in different genotypes of Chinese cabbage, the L40 genotype with high nitrogen utilization and the L14 genotype with LN utilization were selected as experimental materials. Field experiments and indoor hydroponic methods were used to study the different responses of two Chinese cabbage genotypes to low nitrogen levels. In this study, we also analyzed the genome-wide gene expression profiles of L40 and L14 in response to LN stress by high-throughput RNA sequencing technology. The results reveal that the L40 root system responds better to LN compared with L14. After LN stress, L40 can effectively absorb and transport NO3- and store it in the ground. It is precisely because of this characteristic of the L40 genotype that LN treatment did not have a significant effect on the chlorophyll (Chl) content and net photosynthetic rate (Pn) of the L40 Chinese cabbage compared with the L14 Chinese cabbage. These two different Chinese cabbage genotypes were shown to have differently expressed genes related to nitrate transport, auxin synthesis, and glutamate dehydrogenase synthesis. These genes function in the nitrogen pathway, which are important candidates for understanding the molecular host-response mechanisms to LN stress.

Nitric Oxide Extends the Postharvest Life of Water Bamboo Shoots Partly by Maintaining Mitochondrial Structure and Energy Metabolism.

Harvested water bamboo shoots can be stored for only a few days before they lose weight and become soft. Nitrogen oxide (NO) and modified atmosphere packaging (MAP) have previously been used to prolong horticultural crop storage. In the present study, we analyzed the joint effect of these two methods on extending the postharvest quality of water bamboo shoots. Water bamboo shoots were treated with (1) 30 µL L-1 NO, (2) MAP, and (3) a combination of NO and MAP. The NO treatment delayed the softness and weight loss through maintaining the integrity of the mitochondrial ultrastructure and enhancing the ATP level by activating the expressions and activities of succinic dehydrogenase, malic acid dehydrogenase, and cytochrome oxidase. MAP improved the effect of NO on the mitochondrial energy metabolism. These results indicate that NO and MAP treatments are effective at suppressing the quality deterioration of water bamboo shoots, MAP improves the effect of NO in extending postharvest life, and NO may be the main effective factor in the combination of NO and MAP.

Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.).

Drought tolerance is a complex trait controlled by many metabolic pathways and genes and identifying a solution to increase the resilience of plants to drought stress is one of the grand challenges in plant biology. This study provided compelling evidence of increased drought stress tolerance in two sugar beet genotypes when treated with exogenous putrescine (Put) at the seedling stage. Morpho-physiological and biochemical traits and gene expression were assessed in thirty-day-old sugar beet seedlings subjected to drought stress with or without Put (0.3, 0.6, and 0.9 mM) application. Sugar beet plants exposed to drought stress exhibited a significant decline in growth and development as evidenced by root and shoot growth characteristics, photosynthetic pigments, antioxidant enzyme activities, and gene expression. Drought stress resulted in a sharp increase in hydrogen peroxide (H2O2) (89.4 and 118% in SBT-010 and BSRI Sugar beet 2, respectively) and malondialdehyde (MDA) (35.6 and 27.1% in SBT-010 and BSRI Sugar beet 2, respectively). These changes were strongly linked to growth retardation as evidenced by principal component analysis (PCA) and heatmap clustering. Importantly, Put-sprayed plants suffered from less oxidative stress as indicated by lower H2O2 and MDA accumulation. They better regulated the physiological processes supporting growth, dry matter accumulation, photosynthetic pigmentation and gas exchange, relative water content; modulated biochemical changes including proline, total soluble carbohydrate, total soluble sugar, and ascorbic acid; and enhanced the activities of antioxidant enzymes and gene expression. PCA results strongly suggested that Put conferred drought tolerance mostly by enhancing antioxidant enzymes activities that regulated homeostasis of reactive oxygen species. These findings collectively provide an important illustration of the use of Put in modulating drought tolerance in sugar beet plants.

Methionine-induced regulation of growth, secondary metabolites and oxidative defense system in sunflower (Helianthus annuus L.) plants subjected to water deficit stress.

Optimum water availability at different growth stages is one the major prerequisites of best growth and yield production of plants. Exogenous application of plant growth regulators considered effective for normal functioning of plants under water-deficit conditions. A study was conducted to examine the influence of exogenously applied L-methionine on sunflower (Helianthus annuus L.) plants grown under water-deficit conditions. Twenty-five-day old seedlings of four sunflower cultivars, FH331, FH572, FH652 and FH623 were exposed to control (100% F.C.) and drought stress (60% F.C.) conditions. After 30-day of drought stress, L-methionine (Met; 20 mg/L) was applied as a foliar spray to control and drought stressed plants. Water deficit stress significantly reduced shoot fresh and dry weights shoot and root lengths, and chlorophyll a content in all four cultivars. While a significant increase was observed due to water deficiency in relative membrane permeability (RMP), malondialdehyde (MDA), total soluble proteins (TSP), total soluble sugars (TSS), ascorbic acid (AsA) and activity of peroxidase (POD). Although, exogenously applied Met was effective in decreasing RMP, MDA and H2O2 contents, it increased the shoot fresh weight, shoot length, chlorophyll a, chlorophyll a/b ratio, proline contents and the activities of SOD, POD and CAT enzymes in all four cultivars under water deficit stress. No change in AsA and total phenolics was observed due to foliar-applied Met under water stress conditions. Of all sunflower cultivars, cv. FH-572 was the highest and cv. FH-652 the lowest of all four cultivars in shoot fresh and dry weights as well as shoot length under drought stress conditions. Overall, foliar applied L-methionine was effective in improving the drought stress tolerance of sunflower plants that was found to be positively associated with Met induced improved growth attributes and reduced RMP, MDA and H2O2 contents under water deficit conditions.

Impact of Phytomediated Zinc Oxide Nanoparticles on Growth and Oxidative Stress Response of In Vitro Raised Shoots of Ochradenus arabicus.

Biogenic nanoparticles have potential roles in the growth and development of plants and animals as they are ecofriendly and free of chemical contaminants. In this study, we assessed the effects of phytomediated zinc oxide nanoparticles (ZnONPs) on shoot growth, biochemical markers, and antioxidant system response in Ochradenus arabicus, which is a medicinal plant. The shoot length and fresh and dry weights were found to be higher in groups with 5 and 10 mg/L ZnONPs than in the control. At high concentrations of ZnONPs (50, 100, and 300 mg/L), biomass was decreased in a concentration-dependent manner. The shoot number was observed to be highest at 50 mg/L among all applied concentrations of ZnONPs. The levels of the stress markers proline and TBARS were found to be higher in shoots treated with 100 and 300 mg/L ZnONPs than in the control as well as NP-treated shoots. The levels of antioxidant enzymes were significantly increased at high concentrations of nanoparticles compared with the control. Thus, synthesized phytomediated ZnONPs from shoots of O. arabicus and their application to the same organ of O. arabicus in vitro were found to be effective as a low concentration of nanoparticles promoted shoot growth, resulting in high biomass accumulation. Thus, using green nanotechnology, such endemic plants could be conserved in vitro and multiple shoots could be produced by reducing the phytohormone concentration for multiple uses, such as the production of potential secondary metabolites.

Short-term changes related to autotetraploidy in essential oil composition of Eucalyptus benthamii Maiden & Cambage and its applications in different bioassays.

Some forest trees have been polyploidized to improve their traits and to supply new germplasms for breeding programs. As trees have a long juvenile stage, the early characterization of the chromosome set doubling effects is crucial for previous selection. Thus, we aimed to characterize the chemical variability of essential oils from diploid and autotetraploid germplasms (autotetraploid A and B) of Eucalyptus benthamii, as well as to evaluate their larvicidal and allelopathic effects. Autotetraploid A showed a higher essential oil yield than diploid and autotetraploid B, which did not differ quantitatively. Aromadendrene, viridiflorol and α-pinene were the major compounds in the diploid essential oil. In contrast, compounds were present in autotetraploids, such as 1,8-cineole, limonene, α-terpineol, and α-terpinyl-acetate. Essential oils from the diploid at 50-200 ppm were twice as larvicidal than those from autotetraploids against Aedes aegypti larvae. Considering the phytotoxicity bioassays using Lactuca sativa, essential oils from both ploidy levels affected root growth. Moreover, the essential oils inhibited shoot growth at all concentrations tested (187.5; 375; 750; 1500; and 3000 ppm). Autotetraploid A and B had the same effect on shoot growth as glyphosate. The essential oils had no cytogenotoxic effect on root meristematic cells of L. sativa, whereas phytotoxic potential was identified mainly in shoot growth. This work demonstrated a dramatic change in secondary metabolism (terpene composition) related to an increase in the ploidy level in Eucalyptus germplasms. In addition, we report the novelty of the chemical composition of essential oils among germplasms and their potential use as larvicidal and post-emergence weed control agents.

Phosphate (Pi) Starvation Up-Regulated GmCSN5A/B Participates in Anthocyanin Synthesis in Soybean (Glycine max) Dependent on Pi Availability.

Phosphorus (P) is an essential macronutrient for plant growth and development. Among adaptive strategies of plants to P deficiency, increased anthocyanin accumulation is widely observed in plants, which is tightly regulated by a set of genes at transcription levels. However, it remains unclear whether other key regulators might control anthocyanin synthesis through protein modification under P-deficient conditions. In the study, phosphate (Pi) starvation led to anthocyanin accumulations in soybean (Glycine max) leaves, accompanied with increased transcripts of a group of genes involved in anthocyanin synthesis. Meanwhile, transcripts of GmCSN5A/B, two members of the COP9 signalosome subunit 5 (CSN5) family, were up-regulated in both young and old soybean leaves by Pi starvation. Furthermore, overexpressing GmCSN5A and GmCSN5B in Arabidopsis thaliana significantly resulted in anthocyanin accumulations in shoots, accompanied with increased transcripts of gene functions in anthocyanin synthesis including AtPAL, AtCHS, AtF3H, AtF3'H, AtDFR, AtANS, and AtUF3GT only under P-deficient conditions. Taken together, these results strongly suggest that P deficiency leads to increased anthocyanin synthesis through enhancing expression levels of genes involved in anthocyanin synthesis, which could be regulated by GmCSN5A and GmCSN5B.

The Combined Effect of ZnO and CeO2 Nanoparticles on Pisum sativum L.: A Photosynthesis and Nutrients Uptake Study.

Cerium oxide nanoparticles (CeO2 NPs) and zinc oxide nanoparticles (ZnO NPs) are emerging pollutants that are likely to occur in the contemporary environment. So far, their combined effects on terrestrial plants have not been thoroughly investigated. Obviously, this subject is a challenge for modern ecotoxicology. In this study, Pisum sativum L. plants were exposed to either CeO2 NPs or ZnO NPs alone, or mixtures of these nano-oxides (at two concentrations: 100 and 200 mg/L). The plants were cultivated in hydroponic system for twelve days. The combined effect of NPs was proved by 1D ANOVA augmented by Tukey's post hoc test at p = 0.95. It affected all major plant growth and photosynthesis parameters. Additionally, HR-CS AAS and ICP-OES were used to determine concentrations of Cu, Mn, Fe, Mg, Ca, K, Zn, and Ce in roots and shoots. Treatment of the pea plants with the NPs, either alone or in combination affected the homeostasis of these metals in the plants. CeO2 NPs stimulated the photosynthesis rate, while ZnO NPs prompted stomatal and biochemical limitations. In the mixed ZnO and CeO2 treatments, the latter effects were decreased by CeO2 NPs. These results indicate that free radicals scavenging properties of CeO2 NPs mitigate the toxicity symptoms induced in the plants by ZnO NPs.

Short-term effects of cadmium on leaf growth and nutrient transport in rice plants.

Consumption of rice grains contaminated with high concentrations of cadmium (Cd) can cause serious long-term health problems. Moreover, even low Cd concentrations present in the soil can result in the abatement of plant performance, leading to lower grain yield. Studies examining the molecular basis of plant defense against Cd-induced oxidative stress could pave the way in creating superior rice varieties that display an optimal antioxidative defense system to cope with Cd toxicity. In this study, we showed that after one day of Cd exposure, hydroponically grown rice plants exhibited adverse shoot biomass and leaf growth effects. Cadmium accumulates especially in the roots and the leaf meristematic region, leading to a disturbance of manganese homeostasis in both the roots and leaves. The leaf growth zone showed an increased amount of lipid peroxidation indicating that Cd exposure disturbed the oxidative balance. We propose that an increased expression of genes related to the glutathione metabolism such as glutathione synthetase 2, glutathione reductase and phytochelatin synthase 2, rather than genes encoding for antioxidant enzymes, is important in combating early Cd toxicity within the leaves of rice plants. Furthermore, the upregulation of two RESPIRATORY BURST OXIDASE HOMOLOG genes together with a Cd concentration-dependent increase of abscisic acid might cause stomatal closure or cell wall modification, potentially leading to the observed leaf growth reduction. Whereas abscisic acid was also elevated at long term exposure, a decrease of the growth hormone auxin might further contribute to growth inhibition and concomitantly, an increase in salicylic acid might stimulate the activity of antioxidative enzymes after a longer period of Cd exposure. In conclusion, a clear interplay between phytohormones and the oxidative challenge affect plant growth and acclimation during exposure to Cd stress.

Distinct responses of frond and root to increasing nutrient availability in a floating clonal plant.

Current knowledge on responses of aquatic clonal plants to resource availability is largely based on studies manipulating limited resource levels, which may have failed to capture the "big picture" for aquatic clonal plants in response to resource availability. In a greenhouse experiment, we grew the floating clonal plant Spirodela polyrhiza under ten nutrient levels (i.e., 1/64×, 1/32×, 1/16×, 1/8×, 1/4×, 1/2×, 1×, 2×, 4× and 8×full-strength Hoagland solution) and examined their responses in terms of clonal growth, morphology and biomass allocations. The responses of total biomass and number of ramets to nutrient availability were unimodal. A similar pattern was found for frond mass, frond length and frond width, even though area per frond and specific frond area fluctuated greatly in response to nutrient availability. In contrast, the responses of root mass and root length to nutrient availability were U-shaped. Moreover, S. polyrhiza invested more to roots under lower nutrient concentrations. These results suggest that nutrient availability may have distinct influences on roots and fronds of the aquatic clonal plant S. polyrhiza, resulting in a great influence on the whole S. polyrhiza population.

Correlation among Metabolic Changes in Tea Plant Camellia sinensis (L.) Shoots, Green Tea Quality and the Application of Cow Manure to Tea Plantation Soils.

Traditionally, the supplement of organic manure in tea plantations has been a common approach to improving soil fertility and promoting terroir compounds, as manifested by the coordinated increase in yield and quality for the resulting teas. However, information regarding the effect of organic manure in the metabolome of tea plants is still inadequate. The metabolite profiles of tea shoots applied with cow manure, urea or no fertilizer were studied using gas chromatography-mass spectrometry (GC-MS). In total, 73 metabolites were detected, and the modulated metabolites included mainly amino acids, organic acids and fatty acids. In particular, glutamine, quinic acid and proline accumulated more in tea shoots in soils treated with cow manure, but octadecanoic acid, hexadecanoic acid and eicosanoic acid were drastically reduced. Pearson correlation analysis indicated that organic acids and amino acids in tea shoots were the two major metabolite groups among the three treatments. The analysis of metabolic pathways demonstrated that the cow manure treatment significantly changed the enrichment of pathways related to amino acids, sugars and fatty acids. Sensory evaluation showed that the quality of green teas was higher when the plants used to make the tea were grown in soil treated with cow manure rather than urea during spring and late summer. The results indicated that the application of cow manure in soils changed the metabolic characteristics of tea shoots and improved the qualities of the resulting teas.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2-13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.

Effect of Elicitation with (+)-Usnic Acid on Accumulation of Phenolic Acids and Flavonoids in Agitated Microshoots of Eryngium alpinum L.

The present work was aimed at studying the potential of elicitation on the accumulation of phenolic compounds in in vitro shoot cultures of Eryngium alpinum L., a protected plant from the Apiaceae family. The study examined the influence of (+)-usnic acid on the biomass growth as well as on the biosynthesis of the desired flavonoids and phenolic acids in the cultured microshoots. The phenolic compound content was determined by HPLC-DAD. The flavonoid of the highest concentration was isoquercetin, and the phenolic acids of the highest amount were rosmarinic acid, caffeic acid and 3,4-dihydroxyphenylacetic acid, both in the non-elicited and elicited biomass. Isoquercetin accumulation was efficiently increased by a longer elicitation with a lower concentration of lichenic compound (107.17 ± 4.67 mg/100 g DW) or a shorter elicitation with a higher concentration of acid (127.54 ± 11.34 and 108.37 ± 12.1 mg/100 g DW). Rosmarinic acid production generally remained high in all elicited and non-elicited microshoots. The highest content of this acid was recorded at 24 h of elicitation with 3.125 µM usnic acid (512.69 ± 4.89 mg/100 g DW). The process of elicitation with (+)-usnic acid, a well-known lichenic compound with allelopathic nature, may therefore be an effective technique of enhancing phenolic compound accumulation in alpine eryngo microshoot biomass.

Glycinebetaine: a versatile protectant to improve rice performance against aluminium stress by regulating aluminium uptake and translocation.

KEY MESSAGE: Glycinebetaine alleviates the detrimental effects of aluminium stress by regulating aluminium uptake and translocation, maintaining PSII activity, and activating the oxidative defence, thereby maintaining the growth and development of rice. Aluminium (Al) toxicity is one of the primary growth-limiting factors that limits plant growth and crop productivity in acidic soils. Rice (Oryza sativa L.) plants are susceptible to Al stress and do not naturally accumulate glycinebetaine (GB), one of the most effective protectants. Therefore, the objective of this study was to investigate whether exogenous GB can ameliorate the detrimental effects of Al stress on rice plants. Our results showed that the growth, development and biomass of rice were clearly inhibited under Al stress. However, exogenous GB application increased rice shoot growth and photosynthetic pigments contents, maintained photosystem II (PSII) activity, and activated the antioxidant defence system under Al stress. More importantly, GB may mediate the expression of Al uptake- and translocation-related genes, including OsALS1, OsNrat1, OsSTAR1 and OsSTAR2, and the galacturonic acid contents in rice roots under Al stress. Therefore, our findings highlight exogenous GB application is a valid approach to effectively combat Al toxicity by regulating physiological and biochemical processes in crops.

Epibrassinolide improves the growth performance of Sedum lineare upon Zn stress through boosting antioxidative capacities.

As an essential element, zinc (Zn) can improve or inhibit the growth of plants depending on its concentrations. In this study, the effects of 24-Epibrassinolide (EBR), one well-known steroid phytohormone regulating plant growth and alleviating abiotic stress damage, on morphological parameters and antioxidant capacities of Sedum lineare were investigated under different Zn doses. Compared to plants only exposed to Zn, simultaneously foliar application of 0.75 µM EBR significantly improved multiple morphological characteristics and such growth-improving effects were more significant at high Zn concentrations. At a detrimental 800 µM Zn, EBR benefitted plant growth most prominently, as shown by that the stem length, fresh weight and internode length were increased by 111%, 85% and 157%, respectively; than Zn solely treated plants. EBR spray also enhanced both the activities of antioxidant enzymes such as peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and the contents of antioxidative agents including ascorbic acid (AsA) and glutathione (GSH), which in turn decreased the accumulation of reactive oxygen species (ROS) and alleviated the lipid peroxidation in plants. Thus, by demonstrating that EBR could help S. lineare resist high-zinc stress through strengthening the antioxidant system, this work provided a new idea for expanding the planting range of Crassulaceae plants in heavy metal contaminated soil for phytoremediation purpose in the future.

Overexpression of nicotinamidase 3 (NIC3) gene and the exogenous application of nicotinic acid (NA) enhance drought tolerance and increase biomass in Arabidopsis.

KEY MESSAGE: Overexpressing Nicotinamidase 3 gene, and the exogenous application of its metabolite nicotinic acid (NA), enhance drought stress tolerance and increase biomass in Arabidopsis thaliana. With progressive global climatic changes, plant productivity is threatened severely by drought stress. Deciphering the molecular mechanisms regarding genes responsible for balancing plant growth and stress amelioration could imply multiple possibilities for future sustainable goals. Nicotinamide adenine dinucleotide (NAD) biosynthesis and recycling/ distribution is a crucial feature for plant growth. The current study focuses on the functional characterization of nicotinamidase 3 (NIC3) gene, which is involved in the biochemical conversion of nicotinamide (NAM) to nicotinic acid (NA) in the salvage pathway of NAD biosynthesis. Our data show that overexpression of NIC3 gene enhances drought stress tolerance and increases plant growth. NIC3-OX plants accumulated more NA as compared to WT plants. Moreover, the upregulation of several genes related to plant growth/stress tolerance indicates that regulating the NAD salvage pathway could significantly enhance plant growth and drought stress tolerance. The exogenous application of nicotinic acid (NA) showed a similar phenotype as the effect of overexpressing NIC3 gene. In short, we contemplated the role of NIC3 gene and NA application in drought stress tolerance and plant growth. Our results would be helpful in engineering plants with enhanced drought stress tolerance and increased growth potential.

Humic Acid Improves Zn Fertilization in Oxisols Successively Cultivated with Maize-Brachiaria.

Zinc (Zn) is an essential micronutrient for plant growth, and Zn deficiency is a global issue, especially in tropical soils. This study aimed to investigate the effects of humic acid (HA) and the Zn addition (Zn sulfate + HA) on the growth of maize and brachiaria in two contrasting Oxisols. The potential complexation of Zn sulfate by HA was evaluated by Fourier-transform infrared (FTIR) spectroscopy analysis. Zinc content and its availability in solution and the shoot and root biomass of maize and brachiaria were determined. FTIR spectroscopy revealed the complexation of Zn sulfate by HA through its S and C functional groups. In both Oxisols, solution Zn increased due to the combined use of Zn and HA. In a soil type-dependent manner, maize biomass and Zn in its shoots were affected only by the exclusive use of Zn fertilization. In the Yellow Oxisol, brachiaria growth and Zn accumulated in its shoot were positively affected by the combined use of Zn fertilization with HA. In the Oxisol with lower organic matter content, HA can assure adequate supplying of residual Zn, while increasing growth of brachiaria cultivated in sequence to maize.

Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album.

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Synergistic Effects of Melatonin and Gamma-Aminobutyric Acid on Protection of Photosynthesis System in Response to Multiple Abiotic Stressors.

GABA (gamma-aminobutyric acid) and melatonin are endogenous compounds that enhance plant responses to abiotic stresses. The response of Vicia faba to different stressors (salinity (NaCl), poly ethylene glycol (PEG), and sulfur dioxide (SO2)) was studied after priming with sole application of GABA and melatonin or their co-application (GABA + melatonin). Both melatonin and GABA and their co-application increased leaf area, number of flowers, shoot dry and fresh weight, and total biomass. Plants treated with GABA, melatonin, and GABA + melatonin developed larger stomata with wider aperture compared to the stomata of control plants. The functionality of the photosynthetic system was improved in primed plants. To investigate the photosynthetic functionality in details, the leaf samples of primed plants were exposed to different stressors, including SO2, PEG, and NaCl. The maximum quantum yield of photosystem II (PS II) was higher in the leaf samples of primed plants, while the non-photochemical quenching (NPQ) of primed plants was decreased when leaf samples were exposed to the stressors. Correlation analysis showed the association of initial PIabs with post-stress FV/FM and NPQ. Stressors attenuated the association of initial PIabs with both FV/FM and NPQ, while priming plants with GABA, melatonin, or GABA + melatonin minimized the effect of stressors by attenuating these correlations. In conclusion, priming plants with both GABA and melatonin improved growth and photosynthetic performance of Vicia faba and mitigated the effects of abiotic stressors on the photosynthetic performance.

Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant.

KEY MESSAGE: We have defined the conditions for citrus transformations using glyphosate as selection agent. This protocol results in high transformation rate and low incidence of chimeric shoots. Glyphosate, the most widely used herbicide in the world, specifically inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an essential enzyme of the shikimate pathway. Various laboratory-generated or naturally evolved glyphosate-resistant EPSPS variants have been used to produce glyphosate-tolerant transgenic crops, enabling highly effective weed control in agriculture. In this study, we explored the potential of using a citrus EPSPS variant that mimics the previously reported Eleusine indica glyphosate-resistant TIPS (T102I + P106S) mutant for selection of transgenic citrus plants in the presence of glyphosate. We found that glyphosate did not suppress bud formation on 'Duncan' grapefruit seedling explants, but inhibited non-transgenic bud outgrowth to produce shoots in a concentration-dependent manner. At certain concentrations, glyphosate had dramatic effect on the transformation rate and the percentage of non-chimeric transgenic shoots in this newly developed selection system. Specifically, at 0, 10, 20, and 50 µM of glyphosate, the citrus TIPS EPSPS-based selection resulted in transformation rates of 4.02, 5.04, 14.46, and 40.78%, respectively, and 6.41, 23.96, 42.94, and 40.17% of non-chimeric transgenic shoots, respectively. These results indicate that the citrus TIPS EPSPS-glyphosate selection system is highly efficient and can be used as an alternative to antibiotic-based selection methods in citrus genetic transformation. Furthermore, the selection conditions defined in this study are expected to greatly facilitate the production of genetically modified, market-friendly citrus plants, such as cisgenic and intragenic plants.