Endogenous Cytokinin Content Research Articles

Altering endogenous cytokinin content by GmCKX13 as a strategy to develop drought-tolerant plants

Climate-resilient crops are essential to meet the growing food demands of an ever-increasing world population. The phytohormone cytokinins (CKs) have been well established to regulate plant adaptation to drought. Previously, we reported that GmCKX13, encoding a CK oxidase/dehydrogenase in soybean (Glycince max), was differentially expressed under drought. Here, we further characterized its in planta function to assess if GmCKX13 could be a candidate gene to develop drought-tolerant crops. Transgenic Arabidopsis 35S:GmCKX13 plants ectopically and constitutively expressing GmCKX13 using the 35S promoter had an increase in root length, while showing a reduction in shoot height and biomass. CK contents were reduced in 35S:GmCKX13 plants, coupled with the reduced expression of all AtCKX genes and increased expression of all Arabidopsis isopentenyl transferase (AtIPT) genes, except AtIPT2 and AtIPT9 that are responsible for the production of cis-zeatin-type CKs. The 35S:GmCKX13 plants showed improved tolerance to drought and more sensitivity to the treatment with exogenous abscisic acid (ABA). Under the control of the drought-responsive promoter RD29A, the transgenic RD29A:GmCKX13 Arabidopsis plants had a similar phenotype as that of the wild-type (WT) plants under normal conditions. Under dehydration, we found a significant increase in the expression of the transgene coupled with a higher leaf relative water content in RD29A:GmCKX13 plants when compared with WT plants. In consistence with this finding, the RD29A:GmCKX13 plants exhibited higher survival rates and 30 % higher seed yield than the WT plants under drought conditions. Taken together, our results demonstrated that GmCKX13 is an excellent gene for developing drought-tolerant crops by altering endogenous CK levels and ABA responsiveness when being driven by a drought-responsive promoter.

Seed yield as a function of cytokinin-regulated gene expression in wild Kentucky bluegrass (Poa pratensis)

BackgroundKentucky bluegrass (Poa pratensis L.) panicle development is a coordinated process of cell proliferation and differentiation with distinctive phases and architectural changes that are pivotal to determine seed yield. Cytokinin (CK) is a key factor in determining seed yield that might underpin the second “Green Revolution”. However, whether there is a difference between endogenous CK content and seed yields of Kentucky bluegrass, and how CK-related genes are expressed to affect enzyme regulation and downstream seed yield in Kentucky bluegrass remains enigmatic.ResultsIn order to establish a potential link between CK regulation and seed yield, we dissected and characterized the Kentucky bluegrass young panicle, and determined the changes in nutrients, 6 types of endogenous CKs, and 16 genes involved in biosynthesis, activation, inactivation, re-activation and degradation of CKs during young panicle differentiation of Kentucky bluegrass. We found that high seed yield material had more meristems compared to low seed yield material. Additionally, it was found that seed-setting rate (SSR) and lipase activity at the stage of spikelet and floret primordium differentiation (S3), as well as 1000-grain weight (TGW) and zeatin-riboside (ZR) content at the stages of first bract primordium differentiation (S1) and branch primordium differentiation (S2) showed a significantly positive correlation in the two materials. And zeatin, ZR, dihydrozeatin riboside, isopentenyl adenosine and isopentenyl adenosine riboside contents were higher in seed high yield material than those in seed low yield material at S3 stage. Furthermore, the expressions of PpITP3, PpITP5, PpITP8 and PpLOG1 were positively correlated with seed yield, while the expressions of PpCKX2, PpCKX5 and PpCKX7 were negatively correlated with seed yield in Kentucky bluegrass.ConclusionsOverall, our study established a relationship between CK and seed yield in Kentucky bluegrass. Perhaps we can increase SSR and TGW by increasing lipase activity and ZR content. Of course, using modern gene editing techniques to manipulate CK related genes such as PpITP3/5/8, PpLOG1 and PpCKX2/5/7, will be a more direct and effective method in Kentucky bluegrass, which requires further trial validation.

Endogenous cytokinins in plants of Secale cereale (Poaceae) under the effects of soil drought

Due to ongoing global climate changes and anthropogenic stress, soil drought has emerged as a significant threat, hindering plant growth, development, and resulting in crop losses. While phytohormones play a vital role in the formation of stress resistance mechanisms, cytokinins, in particular, remain poorly understood in cultivated cereals. The objective of our study was to investigate the impact of soil drought on plant growth and the homeostasis of endogenous cytokinins in both the aerial parts and roots of winter rye (Secale cereale) during the initial stages of vegetation. We aimed to elucidate the relationship between growth processes and the balance of these phytohormones. The plants were cultivated in a phytochamber using sand culture, and drought stress was induced by withholding water from nine-day-old plants for a period of eight days. The shoots and roots of 17-day-old plants were collected when dehydrated plants reached the critical wilting point. The content of endogenous cytokinins was analyzed using HPLC-MS. Our findings revealed that the inhibition of shoot growth and root elongation in stressed plants coincided with a reduction in the content of trans-zeatin riboside. This observation suggests that trans-zeatin riboside acts as a growth regulator in winter rye under soil drought conditions. Moreover, we observed an elevation in the levels of trans-zeatin and isopentenyladenine in the shoots and roots of stressed rye plants, indicating the involvement of these hormones in the formation of a "protective anti-stress block." These results highlight the multifunctional activity of cytokinins and demonstrate their role in regulating various components of the water deficit response. Consequently, our study expands our understanding of the role of cytokinins in the development of stress resistance in cereals.

Shoot differentiation from Dendrocalamus brandisii callus and the related physiological roles of sugar and hormones during shoot differentiation.

Only a few calli regeneration systems of bamboos were successfully established, which limited the research on the physiological mechanism of callus differentiation. In this study, we successfully established the callus differentiation systems of Dendrocalamus brandisii (Munro) via seeds. The results showed that the best medium for the callus induction of D. brandisii seeds was basal Murashige and Skoog (1962) (MS) media amended with 5.0mgl-1 2,4-D and 0.5mgl-1 kinetin (KT), and the optimal medium for shoot differentiation was the basal MS media supplemented with 4.0mgl-1 6-benzylaminopurine (6-BA) and 0.5mgl-1 1-Naphthaleneacetic acid (NAA). Callus tissues had apparent polarity in cell arrangement and developed their own meristematic cell layers. Alpha-amylase (α-amylase), starch phosphorylase (STP) and sucrose synthase (SUSY) played a dominant role in carbohydrate degradation in callus during shoot differentiation. The pentose phosphate pathway (PPP) and TCA pathways were up-regulated in the shoot-differentiated calli. The dynamics of 6-BA and KT contents in calli were consistent with their concentrations applied in medium. Indoleacetic acid (IAA) synthesis and the related signal transduction were down-regulated, whereas the endogenous CTK contents were up-regulated by the exogenous cytokinin (CTK) application in shoot-differentiated calli, and their related synthesis, transport and signal transduction pathways were also up-regulated. The down-regulated signal transduction pathways of IAA and abscisic acid (ABA) revealed that they did not play the key role in the shoot differentiation of bamboos. Gibberellins (GAs) also played a role in shoot differentiation based on the down-regulation of DELLA and the up-regulation of PIF4 genes. The overexpression of DbSNRK2 and DbFIF4 genes further confirmed the negative role of ABA and the positive role of GAs in shoot differentiation.

Paclobutrazol And Cytokinin Regulation On Culm Growht Of Black Rice (Oryza sativa L. “Cempo Ireng”)

Rice is a staple food for the majority of Asians; nevertheless, black rice has not been commonly consumed among them. The components of the yield will be determined by the allocation of photosynthate on rice and the tall phenotype of black rice, which is a significant contributing factor to lodging. The growth inhibitor paclobutrazol prevents the manufacture of gibberellin, which results in the tall phenotype of black rice. The endogenous cytokinin content is high at the beginning of the grain filling and then rapidly decreases after that. The purpose of the study was to compare the effects of paclobutrazol and cytokinin on culm height, internode length, culm diameter, lodging resistance, culm parenchyma cell arrangement, and flag leaf structure. Throughout the inquiry, Cempo Ireng black rice cultivar seeds were employed. The seeds were planted in plastic containers that held 10 kg of soil for growth medium and organic fertilizer, and a random block pattern was used for the experiment's design. At 10 weeks following sowing, paclobutrazol was sprayed as a foliar application at concentrations of 0, 50, and 100 ppm. By injecting the kinetin diluted in 0.8 percent agar gel at a rate of 10-5 M in the flag leaf internode lacuna twice, separated by two days, two weeks after anthesis. Eight replications and six combinations were made. DMRT and the significant different were used in the statistical analysis of the mean comparisons (P=0.05). Plant height and internode length were decreased by paclobutrazol (100 ppm), although internode diameter was raised and lodging resistance was induced. The organization of the parenchyma cells in the culm (100 ppm packlobutrazol) changed, and the cells were small and lacked intercellular gaps.

Effects of Endogenous Cytokinin on Physicochemical Properties of Superior and Inferior Grain Starch in Rice

AbstractDifferent grain positions on the rice panicle have asynchronous flowering phenomenon. The difference between superior and inferior grains not only affects the yield, but also restricts the quality of rice. In this study, the cytokinin‐related transgenic lines VIN3‐LIKE 2 (OsVIL2)‐OX (increased endogenous cytokinin content), Oryza sativa A‐type response regulator (OsRR1)‐OX, and Early heading date 1 (Ehd1)‐OX (B‐type response regulator) are used to analyze nutrient accumulation and starch physicochemical properties of superior and inferior grains from the physiological level. The research conclusions are as follows: The overexpression transgenic plants of OsVIL2, OsRR1, and Ehd1 result in an increase of average starch granule size and solubility, but decrease in apparent amylose content of superior and inferior grains, which further reduce the surface order of superior and inferior grains, and increase the relative crystallinity of superior grains. Increased cytokinin content possibly accelerates the accumulation of carbohydrates in superior and inferior grains by promoting the expression of OsRR1. In addition, Ehd1 might be the main gene that affects the production of inferior grains, and Ehd1 is also involved in the regulation of the surface order structure and crystallinity of starch from superior and inferior grains by cytokinin.

MdIPT8, an isopentenyl transferase enzyme, enhances the resistance of apple to Colletotrichum gloeosporioides infection

• Cytokinin enhances resistance to anthracnose in apple. • MdIPT8 responses C. gloeosporioides infection. • Overexpression of MdIPT8 increases apple resistance to C. gloeosporioides infection. Apple ( Malus domestica ) is a delicious fruit of high economic value. However, numerous destructive fungi affect apples, greatly limiting their production. Many studies have shown that salicylic acid and jasmonic acid improve plant resistance to fungal diseases, but the potential mechanisms of cytokinin action and fungal diseases remain largely unreported. The IPT gene family encodes the proteins required for cytokinin biosynthesis and plays a central role in plant biological progresses. In our previous study, we found that the disease resistance in autotetraploid apple was much better than ‘Hanfu’ diploid and analyzed the transcriptome data between ‘Hanfu’ diploid and autotetraploid apple. We found that the MdIPT8 gene was significantly differentially expressed. Therefore, we focused on gene family functions in cytokinin biosynthesis and biotic stress responses. We identified ten IPT genes in the ‘Hanfu’ whole genome that are unevenly distributed across the five apple chromosomes. Phylogenetic analysis indicated that this gene family belonged to two groups. Transcriptional analysis of the ten IPT genes indicated that MdIPT8 could be induced under Colletotrichum gloeosporioides infection in apple leaves. The MdIPT8 protein, when fused to the green fluorescent protein, identified the chloroplast as the site of protein localization. Finally, we confirmed that overexpression of MdIPT8 increased the resistance of C. gloeosporioides infection. Taken together, we have identified that the endogenous cytokinin content of ‘Hanfu’ leaves increased during C. gloeosporioides infection and the application of exogenous cytokinin improved the resistance of apple leaves to C. gloeosporioides and found that MdIPT8 can increase apple anthracnose resistance by improving the biosynthesis of cytokinin.

Rice transcription factor OsDOF18 enlarges the starch granule size by cytokinin

Nitrogen is an essential element for rice biomass and starch quality. However, the molecular mechanism affecting starch characteristic by nitrogen remains poorly defined. Previously, we noted that Oryza sativa DNA BINDING WITH ONE FINGER 18 (OsDOF18) mediated ammonium transport by ammonium transporters. Here, we found that OsDOF18 induced nitrogen accumulation in seed. By analyzing the osdof18 mutant and OsDOF18 overexpression transgenic seed, OsDOF18 inhibited small size, granule formation of starch, but induced large size starch granules and apparent amylose content. By transcriptome analysis and qRT-PCR in OsDOF18 mutant plants, OsDOF18 affected the transcription level of cytokinin metabolism-related genes. We further found that the content of cytokinin- zeatin and trans-zeatin-riboside was reduced in the osdof18 mutant. By analyzing the Cytokinin-related transgenic lines-VIN3-LIKE 2(OsVIL2)-OX (increased endogenous cytokinin content), Oryza sativa A-type response regulator (OsRR1)-OX (cytokinin signaling pathway Type A response factor) and Early heading date 1 (Ehd1)-OX (cytokinin signaling pathway Type B response factor), we found that cytokinin content and signaling enhanced lines had more large size granules, but less small size granules, which is similar with the OsDOF18 overexpression lines. These results demonstrated that OsDOF18 affected starch granule size by cytokinin at a physiological and genetic level, and provides new insight that OsDOF18 coordinated with carbon and nitrogen metabolism in rice development.

Effect of low-temperature stress on the growth of plants of Secale cereale (Poaceae) and endogenous cytokinin content in roots and shoots

Phytohormones play a key role in the regulation of plant acclimation to low temperature. To elucidate the role of cytokinins in rye plant response to chilling, we studied the dynamics of these hormones in shoots and roots under short-term and prolonged cold stress. The 7-day-old plants were exposed to cold stress (2 °C) for 2 h (alarm phase of response) or for 6 h for two days (acclimation phase of response). Endogenous content of cytokinins was analyzed by HPLC-MS method. Low temperature had a differential effect on the content of individual cytokinins and their localization in rye plants. During the short-term stress, a decrease in the content of active cytokinins (trans-zeatin and trans-zeatin riboside) in the roots and an increase in the shoots were shown. Prolonged low-temperature stress declined the amount of cytokinins except trans-zeatin riboside, which was detected in both roots and shoots. Significant rise in trans-zeatin riboside content in roots and shoots in this period evidenced an important role of this cytokinin during cold acclimation of rye plants.

Physiological and transcription analyses reveal regulatory pathways of 6-benzylaminopurine delaying leaf senescence and maintaining quality in postharvest Chinese flowering cabbage

The regulatory role of cytokinins (CTKs) in leaf senescence has been documented in different species, including Chinese flowering cabbage. However, its physiological and molecular basis relatively remains unknown. In this study, exogenous application of a CTK analogue 6-benzylaminopurine (6-BA) significantly retarded leaf senescence of Chinese flowering cabbage, with less chlorophyll degradation and lower accumulation of reactive oxygen species (ROS) and malondialdehyde compared with the control group. Meanwhile, higher levels of soluble sugars and proteins, flavonoids, cellulose, amino acids, total phenols, procanthocyanins, and vitamin C were retained in 6-BA-treated leaves. 6-BA treatment also prevented the decline in endogenous CTK content and the increase in ethylene, abscisic acid, and jasmonic acid contents. Moreover, the comparative transcriptome analysis revealed that a total of 21,895 differently expressed genes (DEGs) were identified from four comparisons of 6-BA treatment versus the control during senescence. Further analysis showed that most of the identified DEGs were enriched in ROS, respiratory metabolism, and phytohormone pathways, and a total of 50 classes of transcription factors that were possibly involved in modulating these DEGs were obtained. The transcriptional levels of 18 DEGs were verified by Quantitative real-time PCR (qRT-PCR), which confirmed the accuracy of the transcriptomic data. Overall, these findings and data provide a comprehensive view of physiological and molecular events concerning with the CTK-mediated leaf senescence and -maintained quality in economical leafy vegetables.

ВЛИЯНИЕ ИНДУКЦИИ СИНТЕЗА ЦИТОКИНИНОВ НА ФЛОЭМНЫЙ ТРАНСПОРТ АУКСИНА У IPT ТРАНСГЕННЫХ РАСТЕНИЙ ТАБАКА

As a model for the changes in the content of endogenous cytokinins, ipt-transgenic tobacco plants (Nicotiana tabacum L. cv. Petit Havana SR-1) were used. The total heating of plants caused the accumulation of cytokinins in shoots and a decrease in the delivery of auxins with phloem flow. The effect of cytokinins on the transport of auxins through the phloem is discussed, as well as limitations for the use of ipt transgenic tobacco plants as a model for studying the interaction of hormones.

VaAPRT3 Gene is Associated With Sex Determination in Vitis amurensis.

In the past decade, progress has been made in sex determination mechanism in Vitis. However, genes responsible for sexual differentiation and its mechanism in V. amurensis remain unknown. Here, we identify a sex determination candidate gene coding adenine phosphoribosyl transferase 3 (VaAPRT3) in V. amurensis. Cloning and sequencing of the VaAPRT3 gene allowed us to develop a molecular marker able to discriminate female individuals from males or hermaphrodites based on a 22-bp InDel. Gene expression and endogenous cytokinin content analysis revealed that the VaAPRT3 gene is involved in sex determination or, to be precise, in female organ differentiation, through regulating cytokinin metabolism in V. amurensis. This study enlarged the understanding of sex determination mechanism in the genus Vitis, and the sex marker could be used as a helpful tool for sexual identification in breeding programs as well as in investigation and collection of V. amurensis germplasms.

Effect of seed priming with zeatin on Secale cereale L. growth and cytokinins homeostasis under hyperthermia

ABSTRACT The rise in temperature during autumn is one of the most serious negative factors for winter rye (Secale cereale L.), which leads to wilting, plant damage by fungal diseases and yield losses. The aim of the current study was to elucidate the possibility of overcoming the consequences of heat stress through seed priming with zeatin solution. Rye seeds (cv. Boguslavka) were soaked in zeatin (Z) solution (10−6 M) or water for 3 h. Seedlings were grown at a temperature of +16° C in sand culture. The 7-day-old plants were exposed to heat stress (+35° C) for 2 h (alarm phase of response) or for 6 h for 2 days (acclimation phase of response). Then plants recovered for 5 d under initial conditions. Endogenous cytokinins content was analyzed by HPLC-MS method. Hyperthermia had a differential effect on the content of individual cytokinins and their localization in the shoots and roots of rye plants. Pre-sowing treatment of rye seeds with zeatin solution resulted in significant changes in cytokinin status in shoots and roots of young plants. Effect of hyperthermia on cytokinin amount in plants grown from primed seeds was less pronounced compared with the plants grown from untreated seeds. So, cytokinins are considered an important regulator of rye plants’ response and adaptation to hyperthermia. Seed priming with cytokinins could be a relevant tool to improve growth and development of rye.

Plant Growth Regulators INCYDE and TD-K Underperform in Cereal Field Trials.

Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels.

Integration of genome wide association studies and co-expression networks reveal roles of PtoWRKY 42-PtoUGT76C1-1 in trans-zeatin metabolism and cytokinin sensitivity in poplar.

Cytokinins are important for in vitro shoot regeneration in plants. Cytokinin N-glucosides are produced via an irreversible glycosylation pathway, which regulates the endogenous cytokinin content. Although cytokinin N-glucoside pathways have been uncovered in higher plants, no regulator has been identified to date. We performed a metabolome genome-wide association study (mGWAS), weighted gene co-expression network analysis (WGCNA), and expression quantitative trait nucleotide (eQTN) mappings to build a core triple genetic network (mGWAS-gene expression-phenotype) for the trans-zeatin N-glucoside (ZNG) metabolite using data from 435 unrelated Populus tomentosa individuals. Variation of the ZNG level in poplar is attributed to the differential transcription of PtoWRKY42, a member of WRKY multigene family group IIb. Functional analysis revealed that PtoWRKY42 negatively regulated ZNG accumulation by binding directly to the W-box of the UDP-glycosyltransferase 76C 1-1 (PtoUGT761-1) promoter. Also, PtoWRKY42 was strongly induced by leaf senescence, 6-BA, wounding, and salt stress, resulting in a reduced ZNG level. We identified PtoWRKY42, a negative regulator of cytokinin N-glucosides, which contributes to the natural variation in ZNG level and mediates ZNG accumulation by directly modulating the key glycosyltransferase gene PtoUGT76C1-1.

Limitation of Cytokinin Export to the Shoots by Nucleoside Transporter ENT3 and its Linkage with Root Elongation in Arabidopsis.

The trans-membrane carrier AtENT3 is known to transport externally supplied cytokinin ribosides and thus promote uptake by cells. However, its role in distributing either exogenous or endogenous cytokinins within the intact plant has not hitherto been reported. To test this, we used ent3-1 mutant Arabidopsis seedlings in which the gene is not expressed due to a T-DNA insertion, and examined the effect on the concentration and distribution of either endogenous cytokinins or exogenous trans-zeatin riboside applied to the roots. In the mutant, accumulation of endogenous cytokinins in the roots was reduced and capacity to deliver externally supplied trans-zeatin riboside to the shoots was increased suggesting involvement of equilibrative nucleoside (ENT) transporter in the control of cytokinin distribution in the plants. Roots of ent3-1 were longer in the mutant in association with their lower cytokinin concentration. We concluded that the ENT3 transporter participates in partitioning endogenous cytokinins between the apoplast and the symplast by facilitating their uptake by root cells thereby limiting cytokinin export to the shoots through the xylem. Dilution of the mineral nutrient solution lowered endogenous cytokinin concentration in the roots of both wild type (WT) and ent3-1 plants accompanied by promotion of root elongation. Nevertheless, cytokinin content was lower, while roots were longer in the ent3-1 mutant than in the WT under either normal or deficient mineral nutrition suggesting a significant role of ENT3 transporter in the control of cytokinin level in the roots and the rate of their elongation.

Effect of Exogenous Gibberellin, Paclobutrazol, Abscisic Acid, and Ethrel Application on Bulblet Development in Lycoris radiata.

Lycoris species have great ornamental and medicinal values; however, their low regeneration efficiency significantly restricts their commercial production. Exogenous hormone application is an effective way to promote bulblet development, but their effect on Lycoris radiata has not been verified to date. In the present study, we examined the effect of different exogenous hormones on bulblet development in L. radiata, and found that gibberellic acid (GA) significantly inhibited, whereas paclobutrazol (PBZ), abscisic acid (ABA), and ethrel promoted bulblet development, especially PBZ, a GA biosynthesis inhibitor. Furthermore, GA reduced endogenous cytokinin (CK) content, as well as the activities of carbohydrate metabolism enzymes, including sucrose synthase (SUS) and glucose-1-phosphate adenylyltransferase (AGPase), by downregulating the expression levels of LrSUS1, LrSUS2, and genes encoding AGPase large and small subunits. This resulted in the decrease in carbohydrate accumulation in the bulblets, thus hindering their development. PBZ had the opposite effect to GA on carbohydrate metabolism; it decreased endogenous GA15 and GA24, thereby promoting bulblet development. ABA promoted endogenous auxin content and the activities of starch synthesis enzymes, especially soluble starch synthase (SSS) and granule-bound SS (GBSS), through the up-regulation of the expression levels of LrSS1, LrSS2, and LrGBSS1 genes, which could also result in the accumulation of carbohydrates in the bulblets and promote their development. In addition, ethrel application partly promoted bulblet development by promoting endogenous CK content. Although the accumulation of carbohydrates and the activity of starch enzymes were increased by ethrel treatment, we hypothesized that the effect of ethrel on regulating carbohydrate metabolism may be indirect. Our results could provide a basis for improving the propagation efficiency of L. radiata for production, as well as propose some directions for future research.

Rice gene, OsCKX2-2, regulates inflorescence and grain size by increasing endogenous cytokinin content

Panicle size is one of the yield determining traits in rice (Oryza sativa L.). In the current study, a rice mutant named cytokinin oxidase/dehydrogenase (Osckx2-2) was developed from the indica cultivar 9311 by ethyl methanesulfonate (EMS) mutagenesis. A comparison of agronomic traits showed that the panicle of the Osckx2-2 mutant was longer with dense branching pattern and higher seed setting rate compared to the wild-type (WT). In addition, the grains in the Osckx2-2 mutant were markedly longer, wider, and heavier compared to the WT. Histological analysis revealed that a higher number of the spikelet cells with bigger size resulted in larger grains in the Osckx2-2 mutant. By map-based cloning, SNP was detected in the 3′-UTR of the OsCKX2-2 gene which encodes cytokinin dehydrogenase and the cleaved amplified polymorphic sequence (CAPS) marker was developed based on the detected SNP. The RT-qPCR analysis displayed a decreased OsCKX2-2 expression in the Osckx2-2 mutant panicles. Meanwhile, the enzymatic activity assay showed the reduced cytokinin dehydrogenase enzyme activity, whereas the enzyme-linked immunosorbent assay (ELISA) revealed remarkably higher endogenous cytokinin content in the Osckx2-2 mutant in contrast to WT. The analysis of grain quality parameters showed that the degree of chalkiness and gel consistency decreased, while the amylose content and alkaline spreading value increased in the Osckx2-2 mutant. The OsCKX2-2 gene and Osckx2-2 mutant would be beneficial in marker-assisted backcrossing to improve yield in rice.

УЧАСТИЕ ПЕРЕНОСЧИКА ENT3 В РАСПРЕДЕЛЕНИИ ЦИТОКИНИНОВ МЕЖДУ КОРНЕМ И ПОБЕГОМ И В РЕГУЛЯЦИИ РОСТОВОГО ОТВЕТА РАСТЕНИЙ АРАБИДОПСИСА НА ПОВЫШЕННЫЙ УРОВЕНЬ МИНЕРАЛЬНОГО ПИТАНИЯ

The nitrogenous base riboside transporter ENT3 is known for its ability to transport ribosylated cytokinins across the membrane. However, its role in the distribution of cytokinins between plant organs has not yet been studied. For this purpose, we compared the content and distribution of either endogenous cytokinins or the exogenous trans-zeatin riboside introduced into the nutrient solution in mutant ent3-1 plants and the parent genotype Columbia. In the mutant, the accumulation of endogenous cytokinins in the roots was suppressed, and the ability to deliver exogenous trans-zeatin riboside to shoots increased. The roots of ent3-1 were about 15% longer and had a lower cytokinin concentration. A thirtyfold increase in the concentration of macronutrients led to inhibition of root elongation in the original Columbia line, but not in ent3-1 plants. This growth response occured in accordance with the content of cytokinins in the roots: in Columbia plants, cytokinins accumulated in these organs. The increase in the level of hormones in the roots of ent3-1 was to a lesser extent and was not significant. It was concluded that the ENT3 transporter is involved in the distribution of endogenous cytokinins between the apoplast and symplast, facilitating their uptake by root cells, thereby limiting the export of cytokinins to shoots through the xylem, and can play an important adaptive role in changing the level of mineral nutrition.

Forcing vine regrowth to delay ripening and its association to changes in the hormonal balance

The quality and typicality of wines, strongly depends on the management techniques used for grapevine cultivation. Actually, the increment in the average world temperature due to climate change induces not only bigger irrigation necessities, but also earlier grape-ripening processes, which take place in warmer days and shorter nights. Thus, with the aim of delaying grape ripeness of at least two months, a technique has been proposed based on forcing vine regrowth. This technique consists on forcing vine regrowth from the formed latent buds after cutting the green shoots between the second and the third node; lateral shoots, leaves, and primary clusters are also removed. In this study, a forcing treatment was carried out at three different phenological stages (G, I and J). Depending on the phenological stage of vines during the forcing treatment, we wanted to determine the berry ripening delay and to explore how this mechanical pruning interacts with the hormonal balance to modulate bud growth just before shoot decapitation (Control) and later, within the following 7 and 14 days after cutting the green shoots. Forcing treatments carried out at stages G, I and J succeeded to delay ripening 18, 27 and 45 days respectively, as compared to unforced plants. Vine yield was significantly reduced in all treatments as compared to control plants, resulting in a high level of acidity in berries which might be associated with the loss of flowers, a reduction in the fruit set percentage or a combination of both. Endogenous cytokinin (CK) content in control latent buds decreased during the vine vegetative cycle. Contrarily, abscisic acid (ABA) and Jasmonic acid (JA) increased, while minor changes were found in the concentration of gibberellins (GAs), salicylic acid (SA) and the ethylene precursor 1-aminocyclopropane-1-car-boxilic acid (ACC). Moreover, a clear modification of the hormonal balance was found in latent buds 7 and 14 days after forcing regrowth. CK content significantly increased while ABA rapidly decreased after pruning in all treatments. Thus, vine regrowth from the formed latent buds might have been upregulated by CK and promoted by the absence of ABA.