Reduced Ethylene Production Research Articles

Role of Ethylene Biosynthesis Genes in the Regulation of Salt Stress and Drought Stress Tolerance in Petunia.

Ethylene plays a critical signaling role in the abiotic stress tolerance mechanism. However, the role of ethylene in regulating abiotic stress tolerance in petunia has not been well-investigated, and the underlying molecular mechanism by which ethylene regulates abiotic stress tolerance is still unknown. Therefore, we examined the involvement of ethylene in salt and drought stress tolerance of petunia using the petunia wild type cv. “Merage Rose” and the ethylene biosynthesis genes (PhACO1 and PhACO3)-edited mutants (phaco1 and phaco3). Here, we discovered that editing PhACO1 and PhACO3 reduced ethylene production in the mutants, and mutants were more sensitive to salt and drought stress than the wild type (WT). This was proven by the better outcomes of plant growth and physiological parameters and ion homeostasis in WT over the mutants. Molecular analysis revealed that the expression levels of the genes associated with antioxidant, proline synthesis, ABA synthesis and signaling, and ethylene signaling differed significantly between the WT and mutants, indicating the role of ethylene in the transcriptional regulation of the genes associated with abiotic stress tolerance. This study highlights the involvement of ethylene in abiotic stress adaptation and provides a physiological and molecular understanding of the role of ethylene in abiotic stress response in petunia. Furthermore, the finding alerts researchers to consider the negative effects of ethylene reduction on abiotic stress tolerance when editing the ethylene biosynthesis genes to improve the postharvest quality of horticultural crops.

Effects of 1-methylcyclopropene (1-MCP) treatment on ethanol fermentation of Nanguo pear fruit during ripening.

The aim of this study was to analyze the effects of 1-methylcyclopropene (1-MCP) on ethanol fermentation of Nanguo pears during ripening. Pears were exposed to 1µl/L 1-MCP and stored at 20±2°C. Our data indicated that postharvest application of 1-MCP maintained flesh firmness and reduced ethylene production and respiration rate during storage compared with untreated fruits. 1-MCP treatment delayed the second glucose peak during fruit ripening. The contents of pyruvate and acetyl-CoA were generally reduced by 1-MCP treatment, and at the same time, their peaks were delayed by it during storage compared with controls. The contents of citric acid (CA) and oxaloacetate (OA) were increased by 1-MCP, whereas the contents of acetaldehyde and ethanol were reduced during the whole storage period compared with controls. Activities of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) were reduced by application of 1-MCP during the early stage of storage, and the appearance of their peak activities was also delayed after treatment with 1-MCP. These data showed that postharvest application of 1-MCP could effectively delay the production of the "alcohol taste" of Nanguo pears during ripening. PRACTICAL APPLICATIONS: Nanguo pear is one of the most common cultivars that widely grow in northeast China. It is preferred by many consumers for its unique "alcohol taste", which is mainly produced through ethanol fermentation during ripening. Until now, the mechanisms for regulating ethanol fermentation in ripening Nanguo pears are still unclear. Our data indicated that postharvest application of the ethylene action inhibitor 1-MCP could effectively delay the production of the "alcohol taste" of Nanguo pears during ripening. The data from this study can provide reference data for maintaining the quality of postharvest Nanguo pears.

Transcriptional and post-transcriptional regulation of ethylene biosynthesis by exogenous acetylsalicylic acid in kiwifruit.

Levels of ethylene, implicated in the induction of fruit ripening in a diverse array of plants, are influenced by genetic and environmental factors, such as other plant hormones. Among these, salicylic acid (SA) and its derivative, acetylsalicylic acid (ASA), have been demonstrated to inhibit ethylene biosynthesis in fruit, yet the underlying regulatory mechanisms remain elusive. Here, we showed that treatment with exogenous ASA dramatically reduced ethylene production, as well as activities of ACC synthase (ACS) and ACC oxidase (ACO), in kiwifruit tissues. Comparative transcriptome analysis indicated the differential expression of ethylene biosynthetic genes (AdACS1/2 and AdACO5). A screen of transcription factors indicated that AdERF105L and AdWRKY29 were ASA-responsive regulators of AdACS1/2 and AdACO5, respectively. In addition to these genes, AdACS3 and AdACO3 were abundantly expressed in both ASA-treated and control tissues. AdACS3 protein was phosphorylated and stabilized by AdMPK16, a mitogen-activated protein kinase, while AdACO3 activity was enhanced by AdAP, an aspartic peptidase. Exogenous ASA downregulated AdMPK16 and AdAP, thereby influencing ethylene biosynthesis at a post-transcriptional level. These findings led us to propose a multidimensional system for inhibition of ethylene biosynthesis by ASA, inducing differential expression of some ethylene biosynthesis genes, as well as differential effects on protein activity on other targets.

Treatment with nitric oxide in controlled atmosphere storage to preserve the quality of ‘Laetitia’ plums

This study aims to evaluate the effect of nitric oxide (NO) treatment under controlled atmosphere (CA) on quality of ‘Laetitia’ plums, mainly on internal browning. The experiment followed a completely randomized design, with five repetitions and a 25-fruit experimental unit. The evaluated treatments were: control (no NO); NO at 2 μL L−1 (low dose) applied every 5 days of storage; NO at 5 μL L−1 (medium dose) applied every 5 days of CA storage; and NO at 10 μL L−1 (high dose) applied only at the beginning of CA storage. The treatments with NO in CA storage did not delay the loss of flesh firmness and compression force of the fruit. Treatments with NO reduced ethylene production and respiratory rates and delayed the skin color evolution, especially when applied at medium dose every 5 days of CA storage and high dose at the beginning of CA storage. The treatment with low dose of NO every 5 days of CA storage was the most effective to reduce the internal browning of ‘Laetitia’ plums.

Nitric oxide supply reduces ethylene production, softening and weight loss in papaya fruit

Nitric oxide application has been seen as a promising technique to extend the postharvest life of various fresh fruits and vegetables. This is the first work involving the spray application of nitric oxide donor S-nitrosoglutathione (GSNO) on Golden papaya. Considering that results are very distinct depending on the type of the nitric oxide donor, the form of application, the concentration used and the species studied, the application must be adapted to each necessity. The aim of this study was to relate the application of GSNO, spray applied at 10, 100, 1,000 ?M, with the physiological, physical-chemical, and biochemical changes of Golden papaya, in the first 72 h of ripening. Control fruit was sprayed with distilled water. GSNO application did not interfere on color and chlorophyll fluorescence of the peel, on soluble solids, titratable acidity, lipid peroxidation, and in the level of S-nitrosothiols. Control fruit and 10-?M GSNO sprayed showed lower respiration. After 72 h of ripening at 25°C, all fruits showed an increase in ethylene biosynthesis, except for those treated with 10 ?M GSNO. Papaya sprayed with 10-?M GSNO showed the highest pulp firmness and 52% less weight loss when compared to control fruit. GSNO was also responsible for increasing the ascorbic acid in papayas, besides showing an increase in total antioxidant activity production. The results indicated that the application of 10 ?M of GSNO by spray can potentially preserve the quality characteristics of Golden papaya, mainly due to the lower ethylene production, the delay in the firmness loss, and the less weight loss.

Effect of chitosan coatings supplemented with chitosan-montmorillonite nanocomposites on postharvest quality of ‘Hom Thong’ banana fruit

The chitosan (CTS) solutions supplemented with chitosan-montmorillonite (CTS-MMT) nanocomposites at various concentrations were prepared for free-standing films by the casting technique. Incorporating 2% CTS-MMT nanocomposites into the free-standing CTS films could improve the water-resistance and oxygen barrier of the film. For the postharvest experiment, CTS and CTS supplemented with CTS-MMT nanocomposite solutions were applied as banana fruit coating by the dipping technique. The CTS supplemented with 2% CTS-MMT showed a significant retarding in peel color change, reduced electrolyte leakage, and MDA content, while CTS coating could maintain fruit firmness and reduce plasma membrane destruction for only the first few days. In addition, the CTS supplemented with 2% CTS-MMT coating could reduce ethylene production and respiration rate of the banana fruit. Overall results suggest that the CTS supplemented with 2% CTS-MMT nanocomposites is a novel coating material for maintaining the postharvest quality of ‘Hom Thong’ banana fruit.

The role of hydrogen water in delaying ripening of banana fruit during postharvest storage

Experiments were conducted to identify the role of hydrogen water (HW) in banana fruit ripening. Banana fruit soaked with 0.8 ppm HW showed longer ripening than control fruit. HW treatment significantly reduced ethylene production and respiratory rate, and inhibited the expressions of ethylene synthesis- and signaling-related genes. Similarly, HW treatment inhibited the down-regulation of chlorophylls binding proteins and delayed the increase of chromaticity a*, b* and L* in banana peel. Furthermore, HW-treated peel exhibited lower expressions of cell wall degradation-related genes and higher levels of fruit firmness, pectin, hemicellulose and lignin. In addition, HW-treated pulp exhibited higher levels of starch, lower level of total soluble solids (TSS) and lower expression of flavor-related genes. Microstructural observation further confirmed that HW treatment delayed the degradations of starch and cell walls. Those results indicated that HW treatment delayed banana ripening via the role of ethylene in relation to degreening, flavor and softening.

Editing of 1-aminocyclopropane-1-carboxylate oxidase genes negatively affects petunia seed germination.

Editing of ACO genes involved in ethylene biosynthesis pathway reduces ethylene production in petunia seeds and inhibits seed germination. Ethylene production in the seeds of Petunia hybrida cv. 'Mirage Rose' was associated with expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes (PhACO1, PhACO3, and PhACO4). Suppression of their expression by ethylene inhibitor silver thiosulphate (STS) significantly reduced ethylene production and inhibited seed germination. When it was combined with ethylene precursor ACC, ethylene production was re-promoted via activation of the genes and higher seed germination was restored. This was confirmed using the mutants editing the genes and WT. In the present study, compared with wild type plants, three different mutants (phaco1, phaco3, and phaco4) showed significantly decreased germination percentages as well as delayed germination time and seedling growth. These reductions were associated with lighter seed weight, lower ACO transcript levels, and lower ethylene production in mutants. Inhibited seed germination owing to reduced ethylene production was further verified by the supplementation of exogenous ACC and gibberellic acid (GA3) to growth medium, which restored high seed germination activity in all mutants via enhanced ethylene production. In this study, we reported a key regulatory role of ethylene in seed germination mechanisms in petunia. Further, we highlighted on need to consider the negative effects of ethylene reduction in seed germination and plant growth when editing genes in the ethylene biosynthesis pathway for the maintenance of postharvest fruit, vegetable, and flower quality.

An ethylene biosynthesis enzyme controls quantitative variation in maize ear length and kernel yield

Maize ear size and kernel number differ among lines, however, little is known about the molecular basis of ear length and its impact on kernel number. Here, we characterize a quantitative trait locus, qEL7, to identify a maize gene controlling ear length, flower number and fertility. qEL7 encodes 1-aminocyclopropane-1- carboxylate oxidase2 (ACO2), a gene that functions in the final step of ethylene biosynthesis and is expressed in specific domains in developing inflorescences. Confirmation of qEL7 by gene editing of ZmACO2 leads to a reduction in ethylene production in developing ears, and promotes meristem and flower development, resulting in a ~13.4% increase in grain yield per ear in hybrids lines. Our findings suggest that ethylene serves as a key signal in inflorescence development, affecting spikelet number, floral fertility, ear length and kernel number, and also provide a tool to improve grain productivity by optimizing ethylene levels in maize or in other cereals.

Development of a New Essential Oil-Based Technology to Maintain Fruit Quality in Tomato

In this study, a new technology was tested for its efficacy in the conservation of tomato fruits. An initial experiment was conducted to determine the effects of eugenol on the main quality parameters of Raf tomato fruits; then, a main experiment was performed to test the effectiveness of the new technology. In both experiments, fruits of the tomato cultivar Raf at the mature green stage were used. The preliminary experiment demonstrated the effectiveness of eugenol in maintaining fruit quality during the postharvest life of the fruit. In the main experiment, the fruits were packed using a sachet filled with a mixture consisting of 10:1:1 clinoptilolite clay, ground clove buds, and activated charcoal. This mixture was evaluated against a commercial ethylene scavenger composed mainly of KMnO4-impregnated sepiolite. Three lots of fruits were used: One batch was considered as the control, where the fruits were not packed but kept in an open box at room temperature. The fruits in the other two batches were packed in 2 L PET containers with lids at a rate of three fruits per container, and a sachet of ground cloves and a commercial scavenger was added inside each container in each batch. The containers were kept at room temperature, and the following main quality parameters were analyzed: ethylene production rate, firmness, color, content of soluble solids, and pigments. The results showed that ground clove buds led to a reduction in ethylene production which was associated with a delay in maturation and could be a good alternative for use in the active fruit packaging of horticultural products.

Melatonin Relieves Ozone Stress in Grape Leaves by Inhibiting Ethylene Biosynthesis.

Ozone (O3) stress severely affects the normal growth of grape (Vitis vinifera L.) leaves. Melatonin (MT) plays a significant role in plant response to various abiotic stresses, but its role in O3 stress and related mechanisms are poorly understood. In order to understand the mechanism of MT in alleviate O3 stress in grape leaves, we perform a transcriptome analyses of grapes leaves under O3 stress with or without MT treatment. Transcriptome analysis showed that the processes of ethylene biosynthesis and signaling were clearly changed in “Cabernet Sauvignon” grapes under O3 and MT treatment. O3 stress induced the expression of genes related to ethylene biosynthesis and signal transduction, while MT treatment significantly inhibited the ethylene response mediated by O3 stress. Further experiments showed that both MT and aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis) enhanced the photosynthetic and antioxidant capacities of grape leaves under O3 stress, while ethephon inhibited those capacities. The combined treatment effect of MT and ethylene inhibitor was similar to that of MT alone. Exogenous MT reduced ethylene production in grape leaves under O3 stress, while ethephon and ethylene inhibitors had little effect on the MT content of grape leaves after O3 stress. However, overexpression of VvACO2 (1-aminocyclopropane-1-carboxylate oxidase2) in grape leaves endogenously induced ethylene accumulation and aggravated O3 stress. Overexpression of the MT synthesis gene VvASMT1 (acetylserotonin methyltransferase1) in tobacco (Nicotiana tabacum L.) alleviated O3 stress and reduced ethylene biosynthesis after O3 stress. In summary, MT can alleviate O3 stress in grape leaves by inhibiting ethylene biosynthesis.

Ethylene-regulated leaf lifespan explains divergent responses of plant productivity to warming among three hydrologically different growing seasons.

Leaf senescence is known to be regulated by the plant hormone ethylene, but how leaf lifespan responds to global environmental change and links to ecosystem-level responses remains largely unexplored. Here we investigated the effects of climate warming and nitrogen addition on plant functional traits, plant hormone ethylene and net primary production in a 13-year field experiment in a desert steppe. Across the last 3years of the experiment (2016-2018), plant productivity increased under warming only in 2016, when there was above normal precipitation, but consistently increased with nitrogen addition. Warming enhanced net photosynthesis, leaf nitrogen and ethylene production and reduced leaf lifespan in 2016 (a wet year), but not in 2017 (a drought year); the effect of warming in 2018 (a year with normal precipitation) was opposite to 2016, likely due to the below-normal precipitation in the mid-growing season in 2018. Nitrogen addition led to increases in leaf nitrogen, ethylene production and net photosynthesis, and declines in leaf lifespan in 2016 and 2018, but not in 2017. The ethylene-regulated lifespan was further evidenced by the addition of CoCl2 (an ethylene biosynthesis inhibitor) that reduced ethylene production and prolonged lifespan. Structural equation modeling showed that leaf lifespan had a negative effect on plant productivity, both directly and indirectly via its negative effect on net photosynthesis, across all 3years. Our results demonstrate the divergent responses of leaf lifespan and, in turn, plant productivity to warming under inter-annual and intra-annual precipitation variation, thus linking plant hormone production, functional traits and ecosystem functioning in the face of global environmental change.

Nano-silver controls transcriptional regulation of ethylene- and senescence-associated genes during senescence in cut carnations

Flower senescence in three carnation cultivars (‘Koba Kabana,’ ‘Purple Sky,’ and ‘Lufa’) was associated with increased ethylene production in floral tissues (petals and gynoecia). Treatment with silver nanoparticles (NAg) significantly reduced ethylene production in petals and gynoecia and significantly delayed petal senescence compared with that in control (untreated) flowers, as indicated by higher relative fresh weight. Gene expression analyses performed on petals and gynoecia from initial flower opening through senescence indicated that the expression levels of ethylene biosynthesis genes (ACS1 and ACO1) and a petal senescence-related gene (CP1) were significantly higher in control flowers than in NAg-treated flowers. In contrast, the expression level of the petal senescence inhibitor gene CPi was more upregulated in NAg-treated flowers than in control flowers. The expression levels of EIL1/2, ERS2, and EBF1 were significantly higher in control flowers, in which ethylene was highly produced, than in NAg-treated flowers, whereas the expression levels of CTR1 and ETR1 were lower. This indicates that EIL1/2, ERS2, and EBF1 positively regulate and CTR1 and ETR1 negatively regulate the ethylene signal transduction pathway in carnations. In addition, NAg can inhibit the expression of these positive regulator genes in carnations. Therefore, this study suggests that NAg is helpful in significantly repressing ethylene production and delaying petal senescence in carnations via the suppression of ethylene biosynthesis genes, a petal senescence-related gene, and genes positively regulating ethylene signaling in petals and gynoecia.

Melatonin Treatment of Apricot Trees Leads to Maintenance of Fruit Quality Attributes during Storage at Chilling and Non-Chilling Temperatures

The effects of preharvest melatonin treatment on apricot crop yield and fruit quality properties at harvest and during storage have not yet been investigated. Apricot trees, of the ‘Colorado’ and ‘Mikado’ cultivars, were sprayed with 0.1 mM melatonin at three key points of fruit development. Fruit were harvested at commercial ripening stage and yield was higher in melatonin treated trees than in the controls. Fruit were stored at 1 and 8 °C for 21 and 28 days, respectively. Samples were taken weekly and left at 20 °C for 1 day. Weight losses, as well as reduction in firmness and acidity, were delayed in fruits from melatonin treated trees, showing an effect of treatment on delaying the postharvest ripening process, which was attributed to a reduced ethylene production in both cultivars and at both storage temperatures. In addition, chilling injury symptoms were observed in apricots stored at 1 °C, which were reduced by preharvest melatonin treatment. Moreover, apricot from melatonin-treated fruit retained higher total phenolic content than the controls after 14 days of storage, although the phenolic profile was not affected by treatment. Thus, melatonin could be a useful tool for practical purposes to improve apricot crop yield and maintain fruit quality properties during storage.

Brassinosteroids suppress ethylene-induced fruitlet abscission through LcBZR1/2-mediated transcriptional repression of LcACS1/4 and LcACO2/3 in litchi

Abscission in plants is tightly controlled by multiple phytohormones and the expression of various genes. However, whether the plant hormone brassinosteroids (BRs) are involved in this process is largely unknown. Here, we found that exogenous application of BRs reduced the ethylene-induced fruitlet abscission of litchi due to lower ethylene (ET) production and suppressed the expression of the ethylene biosynthetic genes LcACS1/4 and LcACO2/3 in the fruitlet abscission zone (FAZ). Two genes that encode the BR core signaling components brassinazole resistant (BZR) proteins, namely, LcBZR1 and LcBZR2, were characterized. LcBZR1/2 were localized to the nucleus and acted as transcription repressors. Interestingly, the LcBZR1/2 transcript levels were not changed during ET-induced fruitlet abscission, while their expression levels were significantly increased after BR application. Moreover, gel shift and transient expression assays indicated that LcBZR1/2 could suppress the transcription of LcACS1/4 and LcACO2/3 by specifically binding to their promoters. Importantly, ectopic expression of LcBZR1/2 in Arabidopsis significantly delayed floral organ abscission and suppressed ethylene biosynthesis. Collectively, our results suggest that BRs suppress ET-induced fruitlet abscission through LcBZR1/2-controlled expression of genes related to ethylene biosynthesis in litchi. In addition, similar results were observed in the Arabidopsis gain-of-function mutant bzr1-1D, which showed delayed floral organ abscission in parallel with lower expression of ACS/ACO genes and reduced ethylene production, suggesting that the mechanism of BZR-controlled organ abscission via regulation of ethylene biosynthesis might be conserved in Arabidopsis.

Multiple Amino Acids Inhibit Postharvest Senescence of Broccoli

The function of free amino acids in protein synthesis, as a source of energy and unique roles in catabolism have been well studied in plant development but their function in postharvest fruit and vegetables has received little attention. This study evaluated 11 amino acids—arginine, alanine, aspartic acid, glutamic acid, glycine, ornithine, phenylalanine, serine, tyrosine, tryptophan and valine—on the development of senescence of broccoli. Broccoli florets were dipped in 5 mM solution of amino acids, then stored at 10 °C in air containing 0.1 µL L−1 ethylene. Senescence was assessed by green life, ethylene production, respiration rate and ion leakage. Green life was increased by all the amino acids except valine. Similarly, ethylene production and ion leakage were decreased by all the amino acids except valine, while respiration rate was reduced by all amino acids. It is speculated that the early reduction in ethylene production could be the mechanism by which the amino acids delayed senescence. The beneficial effect of naturally occurring amino acids in inhibiting senescence has potential commercial relevance, as the amino acids have Generally Recognised As Safe (GRAS) status which should assist gain regulatory approval, and gain acceptance by consumers wary of synthetic chemicals on foods.

Polyamine oxidase (PAO)–mediated polyamine catabolism plays potential roles in peach (Prunus persica L.) fruit development and ripening

Recently, there is an increased interest in the function of polyamine (PA) catabolism during fruit ripening, but little is known about its role during the ripening of peach (Prunus persica L.). In this study, the contents of both free and conjugated PAs markedly decreased as the peach fruit ripened. However, RNA-seq analysis showed that the transcript levels of PA synthesis–related genes increased, suggesting that the decreasing PA content during peach ripening was associated with PA catabolism. To better understanding the function of PA catabolism in peach ripening, genes encoding potential polyamine oxidases (PAOs), which are involved in PA catabolism, were identified across the peach genome. Four putative PAO genes (PpePAO1-PpePAO4) were identified in peach using the recently released genome database. The mRNA level of PpePAO1 was significantly increased during peach fruit ripening. Detection of free PAs in tobacco leaves transiently over-expressing PpePAO1 suggested that this gene is probably involved in terminal catabolism of PA. Treatment with the PAO inhibitor guazatine significantly reduced ethylene production and flesh softening of peach fruit, decreased the expression levels of fruit ripening-related genes, and significantly decreased the expression level of PpePAO1 and PAO activity, while PA contents were dramatically higher compared to control treatments. Our results suggests that PpePAO1-mediated PA catabolism is associated with decreased PA content during peach fruit ripening. These data provide valuable knowledge for better understanding the roles of PA catabolism in peach development and ripening.

The mechanism for brassinosteroids suppressing climacteric fruit ripening.

The plant hormone ethylene is important for the ripening of climacteric fruit, such as pear (Pyrus ussuriensis), and the brassinosteroid (BR) class of phytohormones affects ethylene biosynthesis during ripening via an unknown molecular mechanism. Here, we observed that exogenous BR treatment suppressed ethylene production and delayed fruit ripening, whereas treatment with a BR biosynthesis inhibitor promoted ethylene production and accelerated fruit ripening in pear, suggesting BR is a ripening suppressor. The expression of the transcription factor BRASSINAZOLE-RESISTANT 1PuBZR1 was enhanced by BR treatment during pear fruit ripening. PuBZR1 interacted with PuACO1, which converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, and suppressed its activity. BR-activated PuBZR1 bound to the promoters of PuACO1 and of PuACS1a, which encodes ACC synthase, and directly suppressed their transcription. Moreover, PuBZR1 suppressed the expression of transcription factor PuERF2 by binding its promoter, and PuERF2 bound to the promoters of PuACO1 and PuACS1a. We concluded that PuBZR1 indirectly suppresses the transcription of PuACO1 and PuACS1a through its regulation of PuERF2. Ethylene production and expression profiles of corresponding apple (Malus domestica) homologs showed similar changes following epibrassinolide treatment. Together, these results suggest that BR-activated BZR1 suppresses ACO1 activity and the expression of ACO1 and ACS1, thereby reducing ethylene production and suppressing fruit ripening. This likely represents a conserved mechanism by which BR suppresses ethylene biosynthesis during climacteric fruit ripening.

Diazotrophic Bacteria Pantoea dispersa and Enterobacter asburiae Promote Sugarcane Growth by Inducing Nitrogen Uptake and Defense-Related Gene Expression.

Sugarcane is a major crop in tropical and subtropical regions of the world. In China, the application of large amounts of nitrogen (N) fertilizer to boost sugarcane yield is commonplace, but it causes substantial environmental damages, particularly soil, and water pollution. Certain rhizosphere microbes are known to be beneficial for sugarcane production, but much of the sugarcane rhizosphere microflora remains unknown. We have isolated several sugarcane rhizosphere bacteria, and 27 of them were examined for N-fixation, plant growth promotion, and antifungal activity. 16S rRNA gene sequencing was used to identify these strains. Among the isolates, several strains were found to have a relatively high activity of nitrogenase and ACC deaminase, the enzyme that reduces ethylene production in plants. These strains were found to possess nifH and acdS genes associated with N-fixation and ethylene production, respectively. Two of these strains, Pantoea dispersa-AA7 and Enterobacter asburiae-BY4 showed maximum plant growth promotion (PGP) and nitrogenase activity, and thus they were selected for detailed analysis. The results show that they colonize different sugarcane tissues, use various growth substrates (carbon and nitrogen), and tolerate various stress conditions (pH and osmotic stress). The positive effect of AA7 and BY4 strains on nifH and stress-related gene (SuCAT, SuSOD, SuPAL, SuCHI, and SuGLU) expression and the induction of defense-related processes in two sugarcane varieties, GT11 and GXB9, showed their potential for stress amelioration and PGP. Both bacterial strains increased several sugarcane physiological parameters. i.e., plant height, shoot weight, root weight, leaf area, chlorophyll content, and photosynthesis, in plants grown under greenhouse conditions. The ability of rhizobacteria on N-fixing in sugarcane was also confirmed by a 15N isotope-dilution study, and the estimate indicates a contribution of 21–35% of plant nitrogen by rhizobacterial biological N fixation (BNF). This is the first report of sugarcane growth promotion by N-fixing rhizobacteria P. dispersa and E. asburiae strains. Both strains could be used as biofertilizer for sugarcane to minimize nitrogen fertilizer use and better disease management.

Enhancement of the flower longevity of petunia by CRISPR/Cas9-mediated targeted editing of ethylene biosynthesis genes

The transcriptional activation of genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (PhACO3 and PhACO4) during petunia flower senescence has been reported. However, no studies have elaborately investigated their specific roles in ethylene production and flower longevity using genetic manipulation. Hence, we used the CRISPR/Cas9 system to edit the genes (PhACO3 and/or PhACO4) involved in ethylene production and flower longevity in petunia cv. Mirage Rose. The use of the CRISPR/Cas9 system with a sgRNA, which was designed from exon 2 of PhACO3, allows for the specific editing of the genes PhACO3 and/or PhACO4 with high mutation frequency, consequently producing different types of zygotes. The PhACO3 and PhACO4-edited lines 8 and 9 showed remarkably reduced ethylene production (approximately 2.8- to 3.0-fold in corollas and 1.5-fold in pistils) during flowering and extended flower longevity (approximately 9.5 d), while the PhACO3-edited bi-allelic and PhACO4-edited homozygous T0 mutant lines (14 and 23) showed enhanced flower longevity (approximately 8.0 d) compared with 6.0 d for the WT line. This was associated with reduction of PhACO4 protein levels in PhACO4-edited lines, which was confirmed using Western blot analysis and Image J software. Moreover, there was no undesirable editing effect on the PhACO1 gene. The transmission of the edited alleles to the T1 generation was also observed, and ethylene production and flower longevity were identical to those of the T0 mutant lines. Taken together, this study demonstrated not only the single and combined role of PhACO3 and PhACO4 in ethylene production in petunia flowers but also reports improvements in flower longevity by editing of the aforementioned genes using the CRISPR/Cas9 system. Therefore, our study can pave the way for the editing of homologous genes in other ornamental plants using the CRISPR/Cas9 system with a common sgRNA, thus allowing for a time- and cost-effective approach to advancing plant biology and the floricultural industry.