Accumulation Of Cytokinins Research Articles

Tyrosylprotein Sulfotransferase Positively Regulates Symbiotic Nodulation and Root Growth.

Posttranslational tyrosine sulfation of peptides and proteins is catalysed by tyrosylprotein sulfotransferases (TPSTs). In Arabidopsis, tyrosine sulfation is essential for the activities of peptide hormones, such as phytosulfokine (PSK) and root meristem growth factor (RGF). Here, we identified a TPST-encoding gene, MtTPST, from model legume Medicago truncatula. MtTPST expression was detected in all organs, with the highest level in root nodules. A promoter:GUS assay revealed that MtTPST was highly expressed in the root apical meristem, nodule primordium and nodule apical meristem. The loss-of-function mutant mttpst exhibited a stunted phenotype with short roots and reduced nodule number and size. Application of both of the sulfated peptides PSK and RGF3 partially restored the defective root length of mttpst. The reduction in symbiotic nodulation in mttpst was partially recovered by treatment with sulfated PSK peptide. MtTPST-PSK module functions downstream of the Nod factor signalling to promote nodule initiation via regulating accumulation and/or signalling of cytokinin and auxin. Additionally, the small-nodule phenotype of mttpst, which resulted from decreased apical meristematic activity, was partially complemented by sulfated RGF3 treatment. Together, these results demonstrate that MtTPST, through its substrates PSK, RGF3 and other sulfated peptide(s), positively regulates nodule development and root growth.

Cytokinin oxidase2-deficient mutants improve panicle and grain architecture through cytokinin accumulation and enhance drought tolerance in indica rice.

The Osckx2 mutant accumulates cytokinin thereby enhancing panicle branching, grain yield, and drought tolerance, marked by improved survival rate, membrane integrity, and photosynthetic function. Cytokinins (CKs) are multifaceted hormones that regulate growth, development, and stress responses in plants. Cytokinins have been implicated in improved panicle architecture and grain yield; however, they are inactivated by the enzyme cytokinin oxidase (CKX). In this study, we developed a cytokinin oxidase 2 (Osckx2)-deficient mutant using CRISPR/Cas9 gene editing in indica rice and assessed its function under water-deficit and salinity conditions. Loss of OsCKX2 function increased grain number, secondary panicle branching, and overall grain yield through improved cytokinin content in the panicle tissue. Under drought conditions, the Osckx2 mutant conserved more water and demonstrated improved water-saving traits. Through reduced transpiration, Osckx2 mutants showed an improved survival response than the wild type to unset dehydration stress. Further, Osckx2 maintained chloroplast and membrane integrity and showed significantly improved photosynthetic function under drought conditions through enhanced antioxidant protection systems. The OsCKX2 function negatively affects panicle grain number and drought tolerance, with no discernible impact in response to salinity. The finding suggests the utility of the beneficial Osckx2 allele in breeding to develop climate-resilient, high-yielding cultivars for future food security.

Regulatory role of Prunus mume DAM6 on lipid body accumulation and phytohormone metabolism in the dormant vegetative meristem.

Bud dormancy is a crucial process in the annual growth cycle of woody perennials. In Rosaceae fruit tree species, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factor genes regulating bud dormancy have been identified, but their molecular roles in meristematic tissues have not been thoroughly characterized. In this study, molecular and physiological analyses of transgenic apple plants overexpressing the Japanese apricot DAM6 gene (PmDAM6) and Japanese apricot cultivars and F1 individuals with contrasting dormancy characteristics revealed the metabolic pathways controlled by PmDAM6. Our transcriptome analysis and transmission electron microscopy examination demonstrated that PmDAM6 promotes the accumulation of lipid bodies and inhibits cell division in the dormant vegetative meristem by down-regulating the expression of lipid catabolism genes (GDSL ESTERASE/LIPASE and OIL BODY LIPASE) and CYCLIN genes, respectively. Our findings also indicate PmDAM6 promotes abscisic acid (ABA) accumulation and decreases cytokinin (CTK) accumulation in vegetative buds by up-regulating the expression of the ABA biosynthesis gene ARABIDOPSIS ALDEHYDE OXIDASE and the CTK catabolism gene CYTOKININ DEHYDROGENASE, while also down-regulating the expression of the CTK biosynthesis genes ISOPENTENYL TRANSFERASE (IPT) and CYP735A. Additionally, PmDAM6 modulates gibberellin (GA) metabolism by up-regulating GA2-OXIDASE expression and down-regulating GA3-OXIDASE expression. Furthermore, PmDAM6 may also indirectly promote lipid accumulation and restrict cell division by limiting the accumulation of CTK and GA in buds. In conclusion, using our valuable genetic platform, we clarified how PmDAM6 modifies diverse cellular processes, including lipid catabolism, phytohormone (ABA, CTK, and GA) biosynthesis and catabolism, and cell division, in the dormant vegetative meristem.

Transcription factor FveMYB117a inhibits axillary bud outgrowth by regulating cytokinin homeostasis in woodland strawberry.

Shoot branching affects plant architecture. In strawberry (Fragaria L.), short branches (crowns) develop from dormant axillary buds to form inflorescences and flowers. While this developmental transition contributes greatly to perenniality and yield in strawberry, its regulatory mechanism remains unclear and understudied. In the woodland strawberry (Fragaria vesca), we identified and characterized 2 independent mutants showing more crowns. Both mutant alleles reside in FveMYB117a, a R2R3-MYB transcription factor gene highly expressed in shoot apical meristems, axillary buds, and young leaves. Transcriptome analysis revealed that the expression of several cytokinin pathway genes was altered in the fvemyb117a mutant. Consistently, active cytokinins were significantly increased in the axillary buds of the fvemyb117a mutant. Exogenous application of cytokinin enhanced crown outgrowth in the wild type, whereas the cytokinin inhibitors suppressed crown outgrowth in the fvemyb117a mutant. FveMYB117a binds directly to the promoters of the cytokinin homeostasis genes FveIPT2 encoding an isopentenyltransferase and FveCKX1 encoding a cytokinin oxidase to regulate their expression. Conversely, the type-B Arabidopsis response regulators FveARR1 and FveARR2b can directly inhibit the expression of FveMYB117a, indicative of a negative feedback regulation. In conclusion, we identified FveMYB117a as a key repressor of crown outgrowth by inhibiting cytokinin accumulation and provide a mechanistic basis for bud fate transition in an herbaceous perennial plant.

Insights into the Root Sprouts of Toona fargesii in a Natural Forest: From the Morphology, Physiology, and Transcriptome Levels

Toona fargesii (T. fargesii) is a deciduous tree of the Meliaceae family which is utilized for high-value timber. Interestingly, root sprouting is a typical reproductive pattern in T. fargesii. Nevertheless, the genetics underlying this phenomenon are still unclear. Here, three type of roots of T. fargesii were used for histological observation, plant endogenous hormone determination, non-structural carbohydrate (NSC) determination, and sequencing using the Illumina next-generation and PacBio SMRT platforms. Our results indicated that root sprouts originated from cork cambiums. Furthermore, indole-3-acetic acid (IAA), zeatin riboside (ZR), gibberellic acid 3 (GA3), and abscisic acid (ABA) content was significantly increased, while soluble sugar content was significantly decreased in the root sprouts. In addition, transcriptomic analysis suggested a total of 36.19 G of raw data from which 210 differentially expressed genes (DEGs) of were identified in RS vs. SR. Of these, the candidate DEGs were largely enriched in the citrate cycle, gluconeogenesis, starch and sucrose metabolism, and plant hormone signal transduction pathways. We therefore speculated that the accumulation of cytokinin and auxin might be induced by ATP-binding cassette-B 19 (ABCB19) and ABCG14, which were necessary for root sprouting. Additionally, transcription factors SQUAMOSA promoter binding protein-like 18 (SPL18) and NAM, ATAF1/2, and CUC2-14 (NAC14) were found in response to environmental water and epigenetic modification in RS. Overall, this study was to unravel the physiological and transcriptomic levels of the development of root sprouting in T. fargesii.

Stomatal density suppressor PagSDD1 is a “generalist” gene that promotes plant growth and improves water use efficiency

The serine protease SDD1 regulates stomatal density, but its potential impact on plant vegetative growth is unclear. Our study reveals a substantial upregulation of SDD1 in triploid poplar apical buds and leaves, suggesting its possible role in their growth regulation. We cloned PagSDD1 from poplar 84 K (Populus alba × P. glandulosa) and found that overexpression in poplar, soybean, and lettuce led to decreased leaf stomatal density. Furthermore, PagSDD1 represses PagEPF1, PagEPF2, PagEPFL9, PagSPCH, PagMUTE, and PagFAMA expression. In contrast, PagSDD1 promotes the expression of its receptors, PagTMM and PagERECTA. PagSDD1-OE poplars showed stronger drought tolerance than wild-type poplars. Simultaneously, PagSDD1-OE poplar, soybean, and lettuce had vegetative growth advantages. RNA sequencing revealed a significant upregulation of genes PagLHCB2.1 and PagGRF5, correlating positively with photosynthetic rate, and PagCYCA3;4 and PagEXPA8 linked to cell division and differentiation in PagSDD1-OE poplars. This increase promoted leaf photosynthesis, boosted auxin and cytokinin accumulation, and enhanced vegetative growth. SDD1 overexpression can increase the biomass of poplar, soybean, and lettuce by approximately 70, 176, and 155 %, respectively, and increase the water use efficiency of poplar leaves by over 52 %, which is of great value for the molecular design and breeding of plants with growth and water-saving target traits.

Peculiarities of soybean-rhizobial systems subject to different levels of water supply fol-lowing treatment with succinic acid and epibrassinolide

All around the world, one of the leading – according to area of cultivated fields – oleic crops is soybean, which has a high demand for moisture. Given the significance of this crop and negative impact of drought on its yield, integrated research of the influence of insufficient water supply on the intensity of physiological-biochemical processes in those plants is necessary for identifying and understanding the drought-tolerance mechanisms of soybean, as well as symbiotic systems created with its participation, and also for search for ways to adapt it to this stressor. Therefore, our objective was determining the specifics of formation and functioning of the symbiotic systems of soybean and Bradyrhizobium japonicum, following treatment with succinic acid (0.01 g/L) and 24-epibrassinolide (0.00001 g/L), subject to different levels of watering. Our studies revealed that pre-sowing treatment of the seeds with a solution of 24-epibrassinolide with their subsequent inoculation with B. japonicum Т21-2 resulted in the most pronounced stimulation of formation and functioning of the symbiotic systems of soybean in the optimal growing conditions. At the same time, during water shortage, the intensity of nitrogen fixation was the highest in the plants grown from seeds that had been successively treated with the acid and the inoculant. We confirmed that water deficit led to significant increase in the overall content of phytohormones of cytokinin nature in the soybean root nodules, depending on the way the seeds were treated. However, the largest pool of cytokinins was seen in the plants that had been treated with succinic acid against the background of both optimal and insufficient water supply. Treatment of the seeds with 24-epibrassinolide caused significant excess of content of zeatin riboside over the content of zeatin during the flowering stage, whereas in the stage of pods formation it led to an opposite effect – excess of zeatin over zeatin riboside. Fourteen days-long water deficit decreased the content of chlorophylls in the leaves and grain productivity of the plants of all variants of the experiment. The use of growth regulators managed to alleviate the negative impact of stress and protect the pigment complex from ruination. Treatment of the seeds with solutions of succinic acid and 24-epibrassinolide provided the growth of soybean grain productivity regardless on water-supply level. The most efficient was 24-epibrassinolide. Therefore, use of 24-epibrassinolide for pre-sowing treatment of the soybean seeds provided formation of effective symbiotic systems with high nitrogen-fixing activity and caused a number of specific changes in the pattern of accumulation of free and complex forms of cytokinins in the root nodules of those plants. At the same time, the treatment provided the highest concentration of photosynthesis pigments in the soybean leaves, and as a result produced the greatest increase in grain productivity of plants of all the variants, regardless of levels of water supply. In turn, use of succinic acid produced the highest level of nitrogen-fixing activity in the case of the lowest number of root nodules in the conditions of insufficient water supply, and also caused significant accumulation of cytokinins in the nodules, compared with other studied variants against the background of both optimal and insufficient water supply. Therefore, it did result in increase in soybean grain productivity, but this was lower than in the plants treated with 24-epibrassinolide.

Petal size is controlled by the MYB73/TPL/HDA19-miR159-CKX6 module regulating cytokinin catabolism in Rosa hybrida

The size of plant lateral organs is determined by well-coordinated cell proliferation and cell expansion. Here, we report that miR159, an evolutionarily conserved microRNA, plays an essential role in regulating cell division in rose (Rosa hybrida) petals by modulating cytokinin catabolism. We uncover that Cytokinin Oxidase/Dehydrogenase6 (CKX6) is a target of miR159 in petals. Knocking down miR159 levels results in the accumulation of CKX6 transcripts and earlier cytokinin clearance, leading to a shortened cell division period and smaller petals. Conversely, knocking down CKX6 causes cytokinin accumulation and a prolonged developmental cell division period, mimicking the effects of exogenous cytokinin application. MYB73, a R2R3-type MYB transcription repressor, recruits a co-repressor (TOPLESS) and a histone deacetylase (HDA19) to form a suppression complex, which regulates MIR159 expression by modulating histone H3 lysine 9 acetylation levels at the MIR159 promoter. Our work sheds light on mechanisms for ensuring the correct timing of the exit from the cell division phase and thus organ size regulation by controlling cytokinin catabolism.

Enhancing wheat regeneration and genetic transformation through overexpression of TaLAX1

In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.

Genetic robustness control of auxin output in priming organ initiation

Auxin signaling is essential for organ initiation in plants. How genetic robustness controls auxin output during organ initiation is largely unknown. Here, we identified DORNROSCHEN-LIKE (DRNL) as a target of MONOPTEROS (MP) that plays essential roles in organ initiation. We demonstrate that MP physically interacts with DRNL to inhibit cytokinin accumulation by directly activating ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 and CYTOKININ OXIDASE 6. DRN, the paralogous gene of DRNL, acts redundantly with DRNL but is not coexpressed with DRNL in the organ founder cells in which DRNL is expressed. We demonstrate that DRNL directly inhibits DRN expression in the peripheral zone, whereas DRN transcripts are ectopically activated in drnl mutants and fully restore the functional deficiency of drnl in organ initiation. Our results provide a mechanistic framework for the robust control of auxin signaling in organ initiation through paralogous gene-triggered spatial gene compensation effects.

Girdling promotes tomato fruit enlargement by enhancing fruit sink strength and triggering cytokinin accumulation.

Girdling is a horticultural technique that enhances fruit size by allocating more carbohydrates to fruits, yet its underlying mechanisms are not fully understood. In this study, girdling was applied to the main stems of tomato plants 14 days after anthesis. Following girdling, there was a significant increase in fruit volume, dry weight, and starch accumulation. Interestingly, although sucrose transport to the fruit increased, the fruit's sucrose concentration decreased. Girdling also led to an increase in the activities of enzymes involved in sucrose hydrolysis and AGPase, and to an upregulation in the expression of key genes related to sugar transport and utilization. Moreover, the assay of carboxyfluorescein (CF) signal in detached fruit indicated that girdled fruits exhibited a greater ability to take up carbohydrates. These results indicate that girdling improves sucrose unloading and sugar utilization in fruit, thereby enhancing fruit sink strength. In addition, girdling induced cytokinin (CK) accumulation, promoted cell division in the fruit, and upregulated the expression of genes related to CK synthesis and activation. Furthermore, the results of a sucrose injection experiment suggested that increased sucrose import induced CK accumulation in the fruit. This study sheds light on the mechanisms by which girdling promotes fruit enlargement and provides novel insights into the interaction between sugar import and CK accumulation.

HY5 functions as a systemic signal by integrating BRC1-dependent hormone signaling in tomato bud outgrowth

Light plays an important role in determining plant architecture, which greatly influences crop yield. However, the precise mechanisms by which light signaling regulates bud outgrowth remain to be identified. Here, we show that light regulates bud outgrowth via both HY5 and brassinosteroid (BR)-dependent pathways in tomato. Inactivation of the red-light photoreceptor PHYB, or deficiencies in PHYB or the blue-light photoreceptor CRY1a, inhibits bud outgrowth and leads to decreased accumulation of HY5 protein and increased transcript level of BRANCHED1 (BRC1), a central integrator of branching signals. HY5, functioning as a mobile systemic signal from leaves, promotes bud outgrowth by directly suppressing BRC1 transcript and activating the transcript of BR biosynthesis genes within the lateral buds in tomato. Furthermore, BRC1 prevents the accumulation of cytokinin (CK) and gibberellin (GA) by directly inhibiting the transcript of CK synthesis gene LOG4, while increasing the transcript levels of CK and GA degradation genes (CKX7, GA2ox4, and GA2ox5), leading to an arrest of bud outgrowth. Moreover, bud outgrowth occurs predominantly in the day due to the suppression of BRC1 transcript by HY5. These findings demonstrate that light-inducible HY5 acts as a systemic signaling factor in fine-tuning the bud outgrowth of tomato.

The interplay of specific hormonal profile in fruit parts of sunflower inbred lines with contrasting dormancy levels during germination and dormancy breaking by exogenous application of plant growth regulators

AbstractSunflower (Helianthus annuus L.) seed dormancy at harvest is an undesirable characteristic regarding productive systems. From a commercial point of view, the dormancy generates production and marketing problem with a negative impact on crop yields. To overcome this problem, we studied different treatments, including the application of growth regulators. In addition, as the interaction of various hormones in different sunflower fruit parts is not fully understood regarding regulation of dormancy and germination processes, we also (1) characterized the dormancy level of sunflower seeds of Xi3 and X274 inbred lines; (2) assessed the effect of exogenous growth regulators on dormancy release; (3) evaluated the endogenous content of abscisic acid (ABA), gibberellins (GAs), 12‐oxo‐phytodienoic acid (OPDA), jasmonic acid (JA), salicylic acid (SA), auxins, and cytokinins (CKs) in embryonic axis, cotyledons, pericarp, and seed coat‐endosperm; and (4) analyzed the kinetics of the accumulation of these phytohormones in Xi3 embryonic axis during early imbibition (3 to 12 h) of cypselas treated, or not, with growth regulators effective in the dormancy release. We were able to classify the two sunflower lines studied according to dormancy level: The Xi3 line was dormant and X274 line was non‐dormant at harvest. The results showed that the dormancy of Xi3 seeds is associated with a high level of ABA, OPDA, and JA, while germination of X274 dry seeds at harvest time is related to high levels of GA1. Exogenous treatment with GA3 and Ethephon overcame Xi3 seed dormancy through the changes in endogenous hormonal profiles of the embryonic axis. The exogenous application of GA3 induced JA, SA, and CKs accumulation and to modify the ABA/GA1 ratio, whereas Ethephon treatments mediated the ABA/GA1 ratio with the consequent Xi3 seed germination. Thus, the interplay of different phytohormones during early imbibition time seems to be the responsible for breaking sunflower seed dormancy.

Auxin-independent effects of apical dominance induce changes in phytohormones correlated with bud outgrowth.

The inhibition of shoot branching by the growing shoot tip of plants, termed apical dominance, was originally thought to be mediated by auxin. Recently, the importance of the shoot tip sink strength during apical dominance has re-emerged with recent studies highlighting roles for sugars in promoting branching. This raises many unanswered questions on the relative roles of auxin and sugars in apical dominance. Here we show that auxin depletion after decapitation is not always the initial trigger of rapid cytokinin (CK) increases in buds that are instead correlated with enhanced sugars. Auxin may also act through strigolactones (SLs) which have been shown to suppress branching after decapitation, but here we show that SLs do not have a significant effect on initial bud outgrowth after decapitation. We report here that when sucrose or CK is abundant, SLs are less inhibitory during the bud release stage compared to during later stages and that SL treatment rapidly inhibits CK accumulation in pea (Pisum sativum) axillary buds of intact plants. After initial bud release, we find an important role of gibberellin (GA) in promoting sustained bud growth downstream of auxin. We are, therefore, able to suggest a model of apical dominance that integrates auxin, sucrose, SLs, CKs, and GAs and describes differences in signalling across stages of bud release to sustained growth.

WOX11 and CRL1 act synergistically to promote crown root development by maintaining cytokinin homeostasis in rice.

Crown root (CR) morphogenesis is critical for normal growth and nutrition absorption in cereals. In rice, WUSCHEL-RELATED HOMEOBOX11 (WOX11) and CROWN ROOTLESS1 (CRL1) play vital roles in controlling CR development. Despite their importance, whether and how the two regulators coordinate CR formation remains unclear. Electrophoretic mobility shift assays, transient expression, and chromatin immunoprecipitation qPCR suggested that WOX11 and CRL1 directly bind to OsCKX4 to regulate its expression during CR development. CRL1 enhances OsCKX4 activation through direct interaction with WOX11 at root emergence and elongation stages. Genetic dissection showed that the wox11/crl1 double mutant exhibits a more severe root phenotype. OsCKX4 knockout plants generated by CRISPR/Cas9 exhibited fewer CRs and higher cytokinin levels in the root meristem. Increased expression of OsCKX4 could partially complement the CR phenotypes of both crl1 and wox11 mutants. Furthermore, cytokinin can promote WOX11 protein accumulation in the root meristem. Together, these findings show that cytokinin accumulation is tightly regulated by the WOX11-CRL1 complex during CR elongation by counteracting the negative regulatory effects of cytokinin on root development. Importantly, these results reveal an intrinsic link between WOX11 protein accumulation and cytokinin to maintain CR growth.

Transcriptome Analysis Reveals a Comprehensive Virus Resistance Response Mechanism in Pecan Infected by a Novel Badnavirus Pecan Virus.

Pecan leaf-variegated plant, which was infected with a novel badnavirus named pecan mosaic virus (PMV) detected by small RNA deep sequencing, is a vital model plant for studying the molecular mechanism of retaining green or chlorosis of virus-infected leaves. In this report, PMV infection in pecan leaves induced PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PMV infection suppressed the expressions of key genes of fatty acid, oleic acid (C18:1), and very-long-chain fatty acids (VLCFA) biosynthesis, indicating that fatty acids-derived signaling was one of the important defense pathways in response to PMV infection in pecan. PMV infection in pecans enhanced the expressions of pathogenesis-related protein 1 (PR1). However, the transcripts of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) were downregulated, indicating that salicylic acid (SA) biosynthesis was blocked in pecan infected with PMV. Meanwhile, disruption of auxin signaling affected the activation of the jasmonic acid (JA) pathway. Thus, C18:1 and JA signals are involved in response to PMV infection in pecan. In PMV-infected yellow leaves, damaged chloroplast structure and activation of mitogen-activated protein kinase 3 (MPK3) inhibited photosynthesis. Cytokinin and SA biosynthesis was blocked, leading to plants losing immune responses and systemic acquired resistance (SAR). The repression of photosynthesis and the induction of sink metabolism in the infected tissue led to dramatic changes in carbohydrate partitioning. On the contrary, the green leaves of PMV infection in pecan plants had whole cell tissue structure and chloroplast clustering, establishing a strong antiviral immunity system. Cytokinin biosynthesis and signaling transductions were remarkably strengthened, activating plant immune responses. Meanwhile, cytokinin accumulation in green leaves induced partial SA biosynthesis and gained comparatively higher SAR compared to that of yellow leaves. Disturbance of the ribosome biogenesis might enhance the resistance to PMV infection in pecan and lead to leaves staying green.

Genome-wide identification and analysis of the cytokinin oxidase/dehydrogenase (ckx) gene family in finger millet (Eleusine coracana).

Cytokinin dehydrogenase/oxidase (CKX) enzymes play a key role in regulating cytokinin (CK) levels in plants by degrading the excess of this phytohormone. CKX genes have proven an attractive target for genetic engineering, as their silencing boosts cytokinin accumulation in various tissues, thereby contributing to a rapid increase in biomass and overall plant productivity. We previously reported a similar effect in finger millet (Eleusine coracana) somaclonal lines, caused by downregulation of EcCKX1 and EcCKX2. However, the CKX gene family has numerous representatives, especially in allopolyploid crop species, such as E. coracana. To date, the entire CKX gene family of E. coracana and its related species has not been characterized. We offer here, for the first time, a comprehensive genome-wide identification and analysis of a panel of CKX genes in finger millet. The functional genes identified in the E. coracana genome are compared with the previously-identified genes, EcCKX1 and EcCKX2. Exon-intron structural analysis and motif analysis of FAD- and CK-binding domains are performed. The phylogeny of the EcCKX genes suggests that CKX genes are divided into several distinct groups, corresponding to certain isotypes. Finally, the phenotypic effect of EcCKX1 and EcCKX2 in partially silencing the SE7 somaclonal line is investigated, showing that lines deficient in CKX-expression demonstrate increased grain yield and greater bushiness, enhanced biomass accumulation, and a shorter vegetation cycle.

Cytokinins as boron deficiency signals to sustain shoot development in boron-efficient oilseed rape.

Boron (B) deficiency is a highly prominent nutrient disorder. While B-efficient accessions have recently been identified in the highly B-demanding crop oilseed rape, it remained unclear which physiological processes underlie B efficiency and which signaling pathways trigger an efficient B-deficiency response. Here, we compared, under three different B supply conditions, two Brassica napus accessions with contrasting B efficiency. Shoot biomass formation, B distribution patterns and metabolic dynamics of different phytohormone species were studied using a combination of mass spectrometry-based analyses and physiological measurements. Our results show that the B-efficient accession CR2267 does not differ from the B-inefficient accession CR2262 in terms of B accumulation and subcellular B-partitioning, although it displays no morphological B-deficiency symptoms under severe B-deficient conditions. Investigating phytohormone metabolism revealed a strong accumulation of cytokinins in CR2267 at a developmental stage when striking B-dependent differences in biomass and organ formation emerge in the two B. napus accessions. In contrast, elevated levels of the stress hormone abscisic acid as well as bioactive auxins, representing functional antagonists of cytokinins in shoots, were detected only in CR2262. Our results indicate that superior B efficiency in CR2267 relies on a higher B utilization efficiency that builds on an earlier and higher cytokinin biosynthesis required for the maintenance of the shoot meristem activity and proper leaf development. We further conclude that an elevated abundance of cytokinins is not a consequence of better plant growth but rather a presumption for better plant growth under low-B conditions.

Cytokinins regulate rice lamina joint development and leaf angle.

Leaf angle is determined by lamina joint inclination and is an important agronomic trait that determines plant architecture, photosynthetic efficiency, and crop yield. Cytokinins (CKs) are phytohormones involved in shaping rice (Oryza sativa L.) architecture, but their role in leaf angle remains unknown. Here, we report that CK accumulation mediated by rice CK OXIDASE/DEHYDROGENASE3 (OsCKX3) controls lamina joint development and negatively regulates leaf angle. Phenotypic analysis showed that rice osckx3 mutants had smaller leaf angles, while the overexpression lines (OsCKX3-OE) had larger leaf angles. Histological sections indicated that the leaf inclination changes in the osckx3 and OsCKX3-OE lines resulted from asymmetric proliferation of the cells and vascular bundles in the lamina joint. Reverse transcription quantitative PCR, promoter-fused β-glucuronidase expression, and subcellular localization assays indicated that OsCKX3 was highly expressed in the lamina joint, and OsCKX3-GFP fusion protein localized to the endoplasmic reticulum. The enzyme assays using recombinant protein OsCKX3 revealed that OsCKX3 prefers trans-zeatin (tZ) and isopentenyladenine (iP). Consistently, tZ and iP levels increased in the osckx3 mutants but decreased in the OsCKX3 overexpression lines. Interestingly, agronomic trait analysis of the rice grown in the paddy field indicated that osckx3 displayed a smaller leaf angle and enhanced primary branch number, grain size, 1,000-grain weight, and flag leaf size. Collectively, our results revealed that enhancing CK levels in the lamina joint by disrupting OsCKX3 negatively regulates leaf angle, highlighting that the CK pathway can be engineered to reduce leaf angle in rice and possibly in other cereals.

Leaf cytokinin accumulation promotes potato growth in mixed nitrogen supply by coordination of nitrogen and carbon metabolism

The source and sink balance determines crop growth, which is largely modulated by nitrogen (N) supplies. The use of mixed ammonium and nitrate as N supply can improve plant growth, however mechanisms involving the coordination of carbon and N metabolism are not well understood. Here, we investigated potato plants responding to N forms and confirmed that, compared with sole nitrate supply, mixed N (75 %/25 % nitrate/ammonium) enhanced leaf area, photosynthetic activity and N metabolism and accordingly resulted in outgrowth of stolons and shoot axillary buds. Cytokinin transportation in xylem sap and local cytokinin synthesis in leaves were up-regulated in mixed-N-treated potato plants relative to sole nitrate provision; and exogenous application of 6-benzylaminopurine in addition to sole nitrate restored leaf area, photosynthetic capacity and N content in leaves to the similar as those under mixed-N treatment. Partial defoliation, an effective method to enhance the sink strength, induced more cytokinin content in leaflets under two treatments relative to their respective controls and ultimately resulted in larger photosynthesis capacity and leaf area. These results suggest that mixed-N-enhanced plant growth through the coordination of carbon and N metabolism largely depends on the signal molecule cytokinin modulated by N supplies.