Insights into the Impact of Trans-Zeatin Overproduction-Engineered Sinorhizobium meliloti on Alfalfa (Medicago sativa L.) Tolerance to Drought Stress.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti-induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Insights into drought stress response mechanism of tobacco during seed germination by integrated analysis of transcriptome and metabolome.

Drought stress inhibits seed germination, plant growth and development of tobacco, and seriously affects the yield and quality of tobacco leaves. However, the molecular mechanism underlying tobacco drought stress response remains largely unknown. In this study, integrated analysis of transcriptome and metabolome was performed on the germinated seeds of a cultivated variety K326 and its EMS mutagenic mutant M28 with great drought tolerance. The result showed that drought stress inhibited seed germination of the both varieties, while the germination rate of M28 was faster than that of K326 under drought stress. Besides, the levels of phytohormone ABA, GA19, and zeatin were increased by drought stress in M28. Five vital pathways were identified through integrated transcriptomic and metabolomic analysis, including zeatin biosynthesis, aspartate and glutamate synthesis, phenylamine metabolism, glutathione metabolism, and phenylpropanoid synthesis. Furthermore, 20 key metabolites in the above pathways were selected for further analysis of gene modular-trait relationship, and then four highly correlated modules were found. Then analysis of gene expression network was carried out of Top30 hub gene of these four modules, and 9 key candidate genes were identified, including HSP70s, XTH16s, APX, PHI-1, 14-3-3, SCP, PPO. In conclusion, our study uncovered some key drought-responsive pathways and genes of tobacco during seeds germination, providing new insights into the regulatory mechanisms of tobacco drought stress response.

Changes in Phytohormones and Transcriptomic Reprogramming in Strawberry Leaves under Different Light Qualities.

Strawberry plants require light for growth, but the frequent occurrence of low-light weather in winter can lead to a decrease in the photosynthetic rate (Pn) of strawberry plants. Light-emitting diode (LED) systems could be used to increase Pn. However, the changes in the phytohormones and transcriptomic reprogramming in strawberry leaves under different light qualities are still unclear. In this study, we treated strawberry plants with sunlight, sunlight covered with a 50% sunshade net, no light, blue light (460 nm), red light (660 nm), and a 50% red/50% blue LED light combination for 3 days and 7 days. Our results revealed that the light quality has an effect on the contents of Chl a and Chl b, the minimal fluorescence (F0), and the Pn of strawberry plants. The light quality also affected the contents of abscisic acid (ABA), auxin (IAA), trans-zeatin-riboside (tZ), jasmonic acid (JA), and salicylic acid (SA). RNA sequencing (RNA-seq) revealed that differentially expressed genes (DEGs) are significantly enriched in photosynthesis antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolisms, carotenoid biosynthesis, tryptophan metabolism, phenylalanine metabolism, zeatin biosynthesis, and linolenic acid metabolism. We then selected the key DEGs based on the results of a weighted gene co-expression network analysis (WGCNA) and drew nine metabolic heatmaps and protein-protein interaction networks to map light regulation.

Flavin mononucleotide regulated photochemical isomerization and degradation of zeatin.

cis-Zeatin (cZ), a cytokinin often overlooked compared to trans-zeatin (tZ), can now be controlled in live cells and plants through a new biocompatible reaction. Using flavin photosensitizers, cZ can be isomerized to tZ or degraded, depending on the presence of a reducing reagent. This breakthrough offers a novel approach for regulating plant growth through chemical molecules.

Cytokinin-mediated shoot proliferation and its correlation with phytoremediation effects in Cd-hyperaccumulator ecotype of Sedum alfredii.

The phytohormones cytokinins (CKs) are known to regulate apical/auxiliary meristems, control shoot growth and are associated with nutrient uptake and high biomass production. In this study, different cytokinins were tested on Sedum alfredii (S.alfredii) for shoot proliferation and growth performance as well as their correlation with phytoextraction efficiency. Among the tested cytokinins, Zeatin (ZTN) treatments produced the highest number of shoots (5-6 per explant) with 5 and 10 µM ZTN concentrations which are shown as zeatin (ZTN) > kinetin (KTN) > benzylaminopurine (BA) > thidiazuron (TDZ). Maximum biomass production was produced on these media. The maximum biomass (0.14 g) was found in 10 µM ZTN concentration with a 1-fold difference (mean value: 0.02 g) from CK (0.12 g). However, the lowest biomass (0.11 g) was found with 4 µM TDZ, with a 1-fold difference (mean value: 0.02 g) from CK (0.13 g) which suppressed shoot growth. The leaf area and leaf chlorophyll index were significantly increased in all cytokinins except TDZ, and the relation was ZTN > KTN > BA>CK > TDZ. Cadmium accumulation was significantly higher in treatments containing cytokinins as compared to cytokinin-free media. Zeatin at 10 µM concentration was the most effective for high biomass production and correlated with higher cadmium uptake efficiency. The results suggest that cytokinins particularly ZTN, play a crucial role in enhancing both biomass production and cadmium, uptake efficiency in S. alfredii. Therefore, in large-scale phytoremediation initiatives conducted in field conditions, cytokinins can be utilized as growth regulators to enhance biomass production and cadmium extraction efficiency in S.alfredii.

Effect of acupuncture and moxibustion on intestinal flora in the rats with diarrhea-predominant irritable bowel syndrome based on 16S rDNA technique. / 基于16S rDNAæŠ€æœ¯æŽ¢è®¨é’ˆç¸å¯¹è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾æ¨¡åž‹å¤§é¼ è‚ é“èŒç¾¤çš„å½±å“.

OBJECTIVES: To explore the effect of acupuncture and moxibustion on intestinal flora in the rats with diarrhea-predominant irritable bowel syndrome (IBS-D) based on 16S rDNA technique. METHODS: Ten rats were randomized from 58 SPF-grade male SD rats to be the blank group. The remained 48 rats were prepared to be IBS-D models by the modified method of acetic acid enema combined with binding tail-clip stress. Forty successfully-modeled rats were randomly divided into a model group, an acupuncture group, a moxibustion group and a western medication group, with 10 rats in each one. In the acupuncture group, the needle was inserted at bilateral "Zusanli" (ST 36) and remained for 15 min in each rat. In the moxibustion group, the suspending moxibustion was delivered at bilateral "Zusanli" (ST 36) for 15 min. The rats in the western medication group were given pinaverium bromide suspension (10 mL/kg) by intragastric administration. The above interventions were performed once daily for consecutive 14 days. The body mass and the score of fecal trait were compared before and after modeling, as well as after intervention in each group. Fecal water content, diarrhea index and colon transit time (CTT) were measured after modeling and intervention in the rats of each group separately. After intervention, the colonic morphology of rats in each group was observed, and using 16S rDNA technique, the intestinal flora was detected. RESULTS: After modeling, compared with the blank group, the body mass and CTT were reduced (P<0.01); fecal trait scores, fecal water contents and diarrhea index increased (P<0.01) in the other 4 groups. After intervention, the body mass and CTT of the rats decreased (P<0.01), and fecal trait score, fecal water content and diarrhea index increased (P<0.01) in the model group compared with those in the blank group. In the acupuncture group, the moxibustion group and the western medication group, when compared with the model group, the body mass and CTT were elevated (P<0.01), while fecal trait scores, fecal water contents and diarrhea index declined (P<0.01). Compared with the western medication group, fecal water content decreased in the acupuncture group and the moxibustion group (P<0.05), while CTT increased in the acupuncture group (P<0.01), the body mass increased and fecal trait score was dropped in the moxibustion group (P<0.05). The colonic mucosa structure was clear and complete, and there was no obvious inflammatory cell infiltration in the blank group. The mild interstitial edema of intestinal mucosa was presented with the infiltration of few inflammatory cells in the model group. There was the infiltration of few inflammatory cells in the mucosa of the acupuncture group, the moxibustion group and the western medication group. Compared with the blank group, the indexes of Richness, Chao1, ACE and Shannon decreased in the model group (P<0.05). Indexes of Richness, Chao1 and ACE increased in the acupuncture group and the moxibustion group (P<0.05), and the Richness index in the western medication group increased (P<0.05) when compared with those in the model group. The relative abundance of Bacteroidetes, Proteobacteria and Prevotella increased (P<0.05), and that of Firmicutes and Muribaculaceae decreased (P<0.05) in the model group compared with those in the blank group. When compared with the model group, the relative abundance of Bacteroidetes, Proteobacteria and Prevotella was reduced (P<0.05), while that of Firmicutes and Muribaculaceae increased (P<0.05) in the acupuncture group, the moxibustion group and the western medication group; and that of Actinobacteria and Bifidobacterium increased in the acupuncture group and the moxibustion group (P<0.05). Compared with the blank group, the relative abundance of lipopolysaccharide (LPS) biosynthesis was elevated (P<0.05), and that of folate biosynthesis, lipoic acid metabolism, zeatin biosynthesis, ubiquinone and other terpenoid quinone biosynthesis decreased (P<0.05) in the model group. The relative abundance of LPS biosynthesis was dropped (P<0.05), and that of folate biosynthesis, lipoic acid metabolism, zeatin biosynthesis, ubiquinone and other terpenoid quinone biosynthesis increased (P<0.05) in the acupuncture group, the moxibustion group and the western medication group compared with those of the model group. CONCLUSIONS: Either acupuncture or moxibustion can relieve the symptoms of IBS-D and protect intestinal mucosa, which may be associated with regulating the structure of intestinal flora and promoting nutrient metabolism and biosynthesis.

Comparative Transcriptome Sequencing and Endogenous Phytohormone Content of Annual Grafted Branches of Zelkova schneideriana and Its Dwarf Variety HenTianGao.

Zelkova schneideriana is a fast-growing tree species endemic to China. Recent surveys and reports have highlighted a continued decline in its natural populations; therefore, it is included in the Red List of Threatened Species by The International Union for Conservation of Nature. A new variety "HenTianGao" (H) has been developed with smaller plant height, slow growth, and lower branching points. In this study, we attempted to understand the differences in plant height of Z. schneideriana (J) and its dwarf variety H. We determined the endogenous hormone content in the annual grafted branches of both J and H. J exhibited higher gibberellic acid (GA)-19 and trans-Zeatin (tZ) levels, whereas H had higher levels of indole-3-acetic acid (IAA) catabolite 2-oxindole-3-acetic acid (OxIAA), IAA-Glu conjugate, and jasmonic acid (JA) (and its conjugate JA-Ile). The transcriptome comparison showed differential regulation of 20,944 genes enriched in growth and development, signaling, and metabolism-related pathways. The results show that the differential phytohormone level (IAA, JA, tZ, and GA) was consistent with the expression of the genes associated with their biosynthesis. The differences in relative OxIAA, IAA-Glu, GA19, trans-Zeatin, JA, and JA-Ile levels were linked to changes in respective signaling-related genes. We also observed significant differences in the expression of cell size, number, proliferation, cell wall biosynthesis, and remodeling-related genes in J and H. The differences in relative endogenous hormone levels, expression of biosynthesis, and signaling genes provide a theoretical basis for understanding the plant height differences in Z. schneideriana.

Modification of Gene Expression of Tomato Plants through Foliar Flavonoid Application in Relation to Enhanced Growth.

The exogenous application of phenolic compounds is increasingly recognized as a valuable strategy for promoting growth and mitigating the adverse effects of abiotic stress. However, the biostimulant effect under optimal conditions has not been thoroughly explored. In this study, we investigated the impact of foliar application of flavonoids, specifically CropBioLife (CBL), on tomato plants grown under controlled conditions. Our study focused on determining growth parameters, such as cell size, and assessing the concentration of hormones. Principal component analysis (PCA) from all physiological variables was determined. Additionally, we utilized high-throughput mRNA-sequencing technology and bioinformatic methodologies to robustly analyze the transcriptomes of tomato leaves regulated by flavonoids. The findings revealed that CBL primarily influenced cell enlargement by 60%, leading to increased growth. Furthermore, CBL-treated plants exhibited higher concentrations of the hormone zeatin, but lower concentrations of IAA (changes of 50%). Moreover, RNA-seq analysis indicated that CBL-treated plants required increased mineral transport and water uptake, as evidenced by gene expression patterns. Genes related to pathways such as fatty acid degradation, phenylpropanoid biosynthesis, and ABC transporters showed regulatory mechanisms governing internal flavonoid biosynthesis, transport, and tissue concentration, ultimately resulting in higher flavonoid concentrations in tomato leaves.

Physiological and Transcriptomic Analysis Provides Insights into Low Nitrogen Stress in Foxtail Millet (Setaria italica L.).

Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop that is well adapted to nutrient-poor soils. However, our understanding of how different LN-tolerant foxtail millet varieties adapt to long-term low nitrogen (LN) stress at the physiological and molecular levels remains limited. In this study, two foxtail millet varieties with contrasting LN tolerance properties were investigated through analyses of physiological parameters and transcriptomics. The physiological results indicate that JG20 (high tolerance to LN) exhibited superior biomass accumulation both in its shoots and roots, and higher nitrogen content, soluble sugar concentration, soluble protein concentration, zeatin concentration in shoot, and lower soluble sugar and soluble protein concentration in its roots compared to JG22 (sensitive to LN) under LN, this indicated that the LN-tolerant foxtail millet variety can allocate more functional substance to its shoots to sustain aboveground growth and maintain high root activity by utilizing low soluble sugar and protein under LN conditions. In the transcriptomics analysis, JG20 exhibited a greater number of differentially expressed genes (DEGs) compared to JG22 in both its shoots and roots in response to LN stress. These LN-responsive genes were enriched in glycolysis metabolism, photosynthesis, hormone metabolism, and nitrogen metabolism. Furthermore, in the shoots, the glutamine synthetase gene SiGS5, chlorophyll apoprotein of photosystem II gene SiPsbQ, ATP synthase subunit gene Sib, zeatin synthesis genes SiAHP1, and aldose 1-epimerase gene SiAEP, and, in the roots, the high-affinity nitrate transporter genes SiNRT2.3, SiNRT2.4, glutamate synthase gene SiGOGAT2, fructose-bisphosphate aldolase gene SiFBA5, were important genes involved in the LN tolerance of the foxtail millet variety. Hence, our study implies that the identified genes and metabolic pathways contribute valuable insights into the mechanisms underlying LN tolerance in foxtail millet.

Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways.

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC-MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-ß-D-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Wheat cis-zeatin-O-glucosyltransferase cZOGT1 interacts with the Ca2+-dependent lipid binding protein TaZIP to regulate senescence.

Premature senescence is an important factor affecting wheat yield and quality. Wheat yield can be increased by delaying senescence and prolonging the effective photosynthetic time. Previously, we found that the cis-zeatin-O-glucosyltransferase (cZOGT1) gene plays an important role in the stay-green wheat phenotype. In this study, cZOGT1-overexpressing lines exhibited a delayed senescence phenotype, despite a significant reduction in the total cytokinin content. Further, we found that cZOGT1 interacted with the Ca2+-dependent lipid binding protein TaZIP (cZOGT1-interacting protein), and that a high level of cZOGT1 expression led to the suppression of TaZIP expression, which in turn, reduced abscisic acid (ABA) content. The synergistic reduction in cytokinins and ABA levels eventually caused the stay-green phenotype in cZOGT1-overexpressing lines. This study provides a new theoretical basis to explain the mechanism underlying the wheat stay-green phenotype and provides a genetic resource for wheat molecular-design breeding.

Comparative physicochemical, hormonal, transcriptomic and proteomic analyses provide new insights into the formation mechanism of two chemotypes of Pogostemon cablin.

Patchouli (Pogostemon cablin) is an aromatic plant, and its oil has diverse applications in medicine, food, and cosmetics. Patchouli alcohol is the principal bioactive constituent of its volatile oil. In China, patchouli is typically categorized into two types: patchoulol-type (PA-type) and pogostone-type (PO-type). The study evaluated physiological and biochemical indicators, phytohormone metabolites and conducted transcriptome and proteome analyses on both two chemotypes. The PA-type exhibited higher levels of chlorophyll a, b, and carotenoids than the PO-type. In total, 35 phytohormone metabolites representing cytokinin, abscisic acid, gibberellin, jasmonic acid, and their derivatives were identified using UPLC-MS/MS, 10 of which displayed significant differences, mainly belong to cytokinins and jasmonates. Transcriptome analysis identified 4,799 differentially expressed genes (DEGs), while proteome analysis identified 150 differentially expressed proteins (DEPs). Regarding the transcriptome results, the DEGs of the PO-type showed significant downregulation in the pathways of photosynthesis, photosynthesis-antenna protein, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, sesquiterpene and triterpenoid biosynthesis, and starch and sucrose metabolism, but upregulation in the pathway of zeatin synthesis. A combination of transcriptome and proteome analyses revealed that the DEGs and DEPs of lipoxygenase (LOX2), ß-glucosidase, and patchouli synthase (PTS) were collectively downregulated, while the DEGs and DEPs of Zeatin O-xylosyltransferase (ZOX1) and α-amylase (AMY) were jointly upregulated in the PO-type compared to the PA-type. Differential levels of phytohormones, variations in photosynthetic efficiency, and differential expression of genes in the sesquiterpene synthesis pathway may account for the morphological and major active component differences between the two chemotypes of patchouli. The findings of this study offer novel perspectives on the underlying mechanisms contributing to the formation of the two patchouli chemotypes.

Hormonal crosstalk controls cell death induced by kinetin in roots of Vicia faba ssp. minor seedlings.

Studies of vitality/mortality of cortex cells, as well as of the concentrations of ethylene (ETH), gibberellins (GAs), indolic compounds/auxins (ICs/AUXs) and cytokinins (CKs), were undertaken to explain the hormonal background of kinetin (Kin)-regulated cell death (RCD), which is induced in the cortex of the apical parts of roots of faba bean (Vicia faba ssp. minor) seedlings. Quantification was carried out with fluorescence microscopy, ETH sensors, spectrophotometry and ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS). The results indicated that Kin was metabolized to the transport form, i.e., kinetin-9-glucoside (Kin9G) and kinetin riboside (KinR). KinR was then converted to cis-zeatin (cZ) in apical parts of roots with meristems, to cis-zeatin riboside (cZR) in apical parts of roots without meristems and finally to cis-zeatin riboside 5'-monophosphate (cZR5'MP), which is indicated to be a ligand of cytokinin-dependent receptors inducing CD. The process may be enhanced by an increase in the amount of dihydrozeatin riboside (DHZR) as a byproduct of the pathway of zeatin metabolism. It seems that crosstalk of ETH, ICs/AUXs, GAs and CKs with the cZR5'MP, the cis-zeatin-dependent pathway, but not the trans-zeatin-dependent pathway, is responsible for Kin-RCD, indicating that the process is very specific and offers a useful model for studies of CD hallmarks in plants.

Effects of plant age on antioxidant activity and endogenous hormones in Alpine Elymus sibiricus of the Tibetan Plateau.

Elymus sibiricus L. is a perennial forage species that has potential to serve as a forage source in livestock grazing systems. However, E. sibiricus has been shown to have a rapid and substantial reduction of aboveground biomass and seed yield after 3 or 4 years and an accelerated aging process. To determine possible aging mechanisms, we planted E. sibiricus seeds in triplicate blocks in 2012, 2015, and 2016, respectively, and harvested samples of leaves and roots at the jointing and heading stages in 2018 and 2019 to determine oxidative indices and endogenous hormones. The fresh aboveground biomass of 4- and 5-year old plants declined by 34.2% and 52.4% respectively compared with 3-year old plants, and the seed yield declined by 12.7% and 34.1%, respectively. The water content in leaves was 51.7%, 43.3%, and 35.6%, and net photosynthesis was 7.73, 6.35, and 2.08 µmol/m2·s in 3-, 4-, and 5-year old plants, respectively. The superoxide anion radical generation rate in leaves and roots did not show any aging pattern. There was a non-significant increase in malondialdehyde concentration with plant age, particularly in leaves and roots at the heading stage in 2019. The superoxide dismutase activity showed a declining trend with age of plant roots at the jointing stage in both 2018 and 2019. The peroxidase activity declined with plant age in both leaves and roots, for example, and the catalase activity in roots 4- and 7-year old plants declined by 13.8% and 0.85%, respectively, compared to 3-year old plants at the heading stage in 2018. Therefore, the reduced capacity of the antioxidant system may lead to oxidative stress during plant aging process. Overall, the concentrations of plant hormones, auxin (IAA), gibberellin (GA), zeatin (ZT), and abscisic acid (ABA) were significantly lower in roots than in leaves. The IAA concentration in leaves and roots exhibited different patterns with plant age. The ZT concentrations in leaves of 3-year old plants was 2.39- and 2.62-fold of those in 4- and 7-year old plants, respectively at the jointing stage, and in roots, the concentration declined with plant age. The changes in the GA concentration with plant age varied between the physiological stages and between years. The ABA concentrations appeared to increase with plant age, particularly in leaves. In conclusion, the aging process of E. sibiricus was apparently associated with an increase in oxidative stress, a decrease of ZT and an increase of ABA, particularly in roots. These findings highlight the effects of plant age on the antioxidant and endogenous hormone activity of E. sibiricus. However, these plant age-related trends showed variations between plant physiological stages and between harvest years that needs to be researched in the future to develop strategies to manage this forage species.

Cytokinins act synergistically with heat acclimation to enhance rice thermotolerance affecting hormonal dynamics, gene expression and volatile emission.

Heat stress is a frequent environmental constraint. Phytohormones can significantly affect plant thermotolerance. This study compares the effects of exogenous cytokinin meta-topolin-9-(tetrahydropyran-2-yl)purine (mT9THP) on rice (Oryza sativa) under control conditions, after acclimation by moderate temperature (A; 37 °C, 2h), heat stress (HS; 45 °C, 6h) and their combination (AHS). mT9THP is a stable cytokinin derivative that releases active meta-topolin gradually, preventing the rapid deactivation reported after exogenous cytokinin application. Under control conditions, mT9THP negatively affected jasmonic acid in leaves and abscisic and salicylic acids in crowns (meristematic tissue crucial for tillering). Exogenous cytokinin stimulated the emission of volatile organic compounds (VOC), especially 2,3-butanediol. Acclimation upregulated trans-zeatin, expression of stress- and hormone-related genes, and VOC emission. The combination of acclimation and mT9THP promoted the expression of stress markers and antioxidant enzymes and moderately increased VOC emission, including 2-ethylhexyl salicylate or furanones. AHS and HS responses shared some common features, namely, increase of ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), cis-zeatin and cytokinin methylthio derivatives, as well as the expression of heat shock proteins, alternative oxidases, and superoxide dismutases. AHS specifically induced jasmonic acid and auxin indole-3-acetic acid levels, diacylglycerolipids with fewer double bonds, and VOC emissions [e.g., acetamide, lipoxygenase (LOX)-derived volatiles]. Under direct HS, exogenous cytokinin mimicked some positive acclimation effects. The combination of mT9THP and AHS had the strongest thermo-protective effect, including a strong stimulation of VOC emissions (including LOX-derived ones). These results demonstrate for the first time the crucial contribution of volatiles to the beneficial effects of cytokinin and AHS on rice thermotolerance.

The trans-zeatin-type side-chain modification of cytokinins controls rice growth.

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-(Δ2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in the model dicot plant Arabidopsis (Arabidopsis thaliana) have demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the importance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles other than Arabidopsis, such as rice (Oryza sativa), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant cyp735a3 cyp735a4 demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and CK and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that tZ-type CKs control the growth of both roots and shoots in response to internal and environmental cues in rice.

Overexpression of the Wheat TaPsb28 Gene Enhances Drought Tolerance in Transgenic Arabidopsis.

Psb28 is a soluble protein in the photosystem II (PSII) complex, but its role in the drought stress response of wheat remains unclear. Here, we functionally characterized the TaPsb28 gene, which positively regulates drought tolerance in wheat. When the full-length 546-bp TaPsb28 cDNA was transferred into Arabidopsis thaliana, it was located in the guard cell chloroplast around the stroma. Overexpression of TaPsb28 conferred drought tolerance, as exhibited by the increases in the survival rate. Transgenic plants maintained lower MDA content and higher chlorophyll content by inducing chlorophyll synthase (ChlG) gene transcription. The content of abscisic acid (ABA) and zeatin increased significantly in wild-type (WT) plants under drought stress, and the transcriptional expression levels of RD22, dihydroflavonol 4-reductase (DFR) and anthocyanin reductase (ANR) genes were induced, thus enhancing the contents of endogenous cyanidin, delphinidin, and proanthocyanidins. However, in transgenic plants, although anthocyanins were further aggregated, the ABA increase was inhibited, zeatin was restored to the control level under drought stress, and stomatal closure was promoted. These findings indicate ABA and zeatin have opposite synergistic effects in the process of drought tolerance caused by TaPsb28 because only after the effect of zeatin is alleviated can ABA better play its role in promoting anthocyanin accumulation and stomatal closure, thus enhancing the drought tolerance of transgenic plants. The results suggest that overexpression of TaPsb28 exerts a positive role in the drought response by influencing the functional metabolism of endogenous hormones. The understanding acquired through the research laid a foundation for further in-depth investigation of the function of TaPsb28 in drought resistance in wheat, especially its relationship with anthocyanidin accumulation.

Trans-Zeatin induce regulation the biosynthesis of 2-acetyl-1-pyrroline in fragrant rice (Oryza sativa L.) seedlings.

BACKGROUND: In plants, cytokinin is activated into trans-zeatin to fight abiotic stresses. However, the mechanism of the effect of trans-zeatin on 2-acetyl-1-pyrroline (2-AP) biosynthesis in fragrant rice has yet to be studied. The present study was conducted to explore the effects of exogenous trans-zeatin on enzymes activities, genes expression, and precursors involved in 2-AP biosynthesis and 2-AP contents as well as the seedling quality of a fragrant rice cultivar viz., Meixiangzhan2. Four concentrations of trans-zeatin solutions at 20, 40, and 80 µmol L- 1 (ZT1, ZT2, and ZT3) were sprayed onto rice seedlings. RESULTS: Compared to the control, trans-zeatin treatments showed significantly higher 2-AP contents of fragrant rice seedlings. Increased plant height and stem width were observed due to trans-zeatin treatments. The trans-zeatin application increased 1-pyrroline, methylglyoxal, proline, and P5C contents, enhanced P5CS and OAT activities, and reduced glutamic acid contents. In addition, expressions of ProDH, P5CS2, and DAO4 were comparatively higher under trans-zeatin treatments than CK in fragrant rice seedlings. CONCLUSIONS: Overall, up-regulation of P5C, 1-pyrroline, and proline and down-regulation of glutamic acid under appropriate trans-zeatin concentrations (20 and 40 µmol L- 1) resulted in enhanced 2-AP biosynthesis in fragrant rice seedlings and 20-40 µmol L- 1 was considered as the suggested concentrations of trans-zeatin application in fragrant rice seedling.

Zeatin: The 60th anniversary of its identification.

While various labs had shown cell division-inducing activity in a variety of plant extracts for over a decade, the identification of zeatin (Z) in 1964, the first known naturally occurring cytokinin, belongs to Letham and co-workers. Using extracts from maize (Zea mays), they were the first to obtain crystals of pure Z and in sufficient quantity for structural determination by MS, NMR, chromatography, and mixed melting-point analysis. This group also crystallized Z-9-riboside (ZR) from coconut (Cocos nucifera) milk. However, their chemical contributions go well beyond the identification of Z and ZR and include two unambiguous syntheses of trans-Z (to establish stereochemistry), the synthesis of 3H-cytokinins that facilitated metabolic studies, and the synthesis of deuterated internal standards for accurate mass spectral quantification. Letham and associates also unequivocally identified Z nucleotide, the 7-and 9-glucoside conjugates of Z, and the O-glucosides of Z, ZR, dihydro Z (DHZ) and DHZR as endogenous compounds and as metabolites of exogenous Z. Their contributions to the role of cytokinins in plant physiology and development were also substantial, especially the role of cytokinins moving in the xylem. These biological advances are described and briefly related to the genetic/molecular biological contributions of others that established that plants have an absolute requirement for cytokinin.

Dynamics of endogenous levels and subcellular localization of ABA and cytokinins during pollen germination in spruce and tobacco.

We used the enzyme-linked immunosorbent assay (ELISA) to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.