Cell- and non-cell-autonomous ARF3 coordinates meristem proliferation and organ patterning in Arabidopsis

In cell-cell communication, non-cell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR 3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression is mainly activated at the periphery of the SAM by auxin, where ARF3 cell-autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2) and TARGETS UNDER ETTIN CONTROL3 (TEC3) to determine organ patterning. We also show that ARF3 is translocated into the organizing center, where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity non-cell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.

Cytokinin regulates vegetative phase change in Arabidopsis thaliana through the miR172/TOE1-TOE2 module

During vegetative growth plants pass from a juvenile to an adult phase causing changes in shoot morphology. This vegetative phase change is primarily regulated by the opposite actions of two microRNAs, the inhibitory miR156 and the promoting miR172 as well as their respective target genes, constituting the age pathway. Here we show that the phytohormone cytokinin promotes the juvenile-to-adult phase transition through regulating components of the age pathway. Reduction of cytokinin signalling substantially delayed the transition to the adult stage. tZ-type cytokinin was particularly important as compared to iP- and the inactive cZ-type cytokinin, and root-derived tZ influenced the phase transition significantly. Genetic and transcriptional analyses indicated the requirement of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors and miR172 for cytokinin activity. Two miR172 targets, TARGET OF EAT1 (TOE1) and TOE2 encoding transcriptional repressors were necessary and sufficient to mediate the influence of cytokinin on vegetative phase change. This cytokinin pathway regulating plant aging adds to the complexity of the regulatory network controlling the juvenile-to-adult phase transition and links cytokinin to miRNA action.

The Cytokinin Status of the Epidermis Regulates Aspects of Vegetative and Reproductive Development in Arabidopsis thaliana

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development.

Influence of different concentrations of 6-benzylaminopurine and thidiazuron on the proliferative activity of apple varieties in in vitro culture

An essential role in the process of clonal micropropagation is played by cytokinins, with the help of which apical dominance is removed and the formation of additional shoots and buds is activated. The article presents the study results of microshoots’ proliferative activity of apple varieties on media with 6-benzylaminopurine at a concentration of 1.0 mg/l, 2.0 mg/l and thidiazuron at a concentration of 0.1 mg/l, 0.2 mg/l and 0.3 mg/l. The objects of research were apple varieties of the VNIISPK selection - Bolotovskoe, Imrus, Veteran, Kandil orlovsky, Girlyanda, Priokskoe. The aim of the research was to study the effect of substances with cytokinin activity on the morphogenesis of apple varieties. As a result of the research, it was revealed that both cytokinins and varietal characteristics influence the multiplication factor. The highest multiplication factor was observed when using 6-BAP. Thidiazuron reduced the proliferation rate. When using 6-BAP, Bolotovskoe, Girlyanda and Priokskoe varieties had the highest multiplication factor among the varieties. When using TDZ - varieties Bolotovskoe and Kandil Orlovsky.

Rhizogenesis in clonal apple rootstocks (Malus Mill.) in vitro depending on the influence of abiotic factors

In vitro culture at the rooting stage the aft ereff ect of the use of earlier (during proliferation) growth regulators with cytokinin activity (6-benzylaminopurine and thidiazuron) and the eff ect of lamps of diff erent spectral composition on the percentage of rooting and biometric parameters of the development of the roots of clonal apple rootstocks were studied. The following forms of apple clonal rootstocks were taken as objects of research: 54-118, 57-490, 57-545, ММ 106 and М 26. The research methods used at the stages of in vitro culture studies corresponded to those generally accepted for this section of research. Studies were performed on two variants of explants, one part was cultured at the proliferation stage on media with 6-benzylaminopurine and the other on media with tidiazurone. It was found that explants that were cultivated at the stage of proliferation on media with thidiazuron had a tendency to a subsequent increase in the number of roots (at the stage of rooting) by one microscopic cut, in comparison with cultivation on media with 6-benzylaminopurine, by 1.1-1.3 times, and this regularity was observed in all studied clonal rootstocks of the apple tree. It was noted that when explants were illuminated, explants had a greater percentage of rooting when illuminated with phytolamps in comparison with lighting with lamps with a color temperature of 6500K by 1.1-1.4 times in all studied apple rootstocks, and an increase in the number of roots per one microstalk was observed (for excluding rootstock 57-490), by 1.1-1.9 times. During the research, the specificity of rooting of clonal apple rootstocks was observed under the infl uence of the composition of light and the use of 6-benzylaminopurine and thidiazuron at the stage of proliferation, which was expressed in a higher ability to rhizogenesis of some rootstocks compared to others. The rootstocks MM 106 (83.3-93.9 %) and M26 (87.8-92.7 %) had a greater percentage of rooting, according to the average number of roots per one microcut, rootstocks 57-545 (15.4 pcs.), MM 106 (11.2 pcs.), and M 26 along the average root length (27.0 mm).

Diphenylurea-derived cytokinin oxidase/dehydrogenase inhibitors for biotechnology and agriculture

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10–8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

Application of auxin-cytokinin substitute in vitro culture Solanacea crops

Aim. The purpose of our research was to establish the possibility of initiation of potato and tomato culture plants using industrial growth regulators, which are legal for use in Ukraine, which include substances with pronounced auxin-cytokinin activity. Methods. In this work, we used varieties of tomato: Khoriv, Borivsky and Bozhedar, Povin potatos. Work in culture in vitro was performed according to conventional methods. Results. A phytohormone substitute was created in a known nutrient agar medium according to Murasige-Skuga. For the in vitro cultivation of tomato and potato plants, phytohormones were replaced by solutions of Ecostim and Ecostim 1, which exhibited auxin-cytokinin activity. The cost of these substitutes is much lower than that of commercial phytohormones. Conclusions. It is shown that optimal for growth in the MS medium in the callusogenesis of Solanacea cultures in vitro. That variant with the use of cytokinin substitutes Ecostim and Ecostim 1 with the rate of using of 35.0 and 40.0 mg/L. Keywords: modification of MS medium, potatoes, tomatoes, cell culture in vitro.

Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins

The biosynthesis of aromatic cytokinins in planta, unlike isoprenoid cytokinins, is still unknown. To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike active cytokinins-bases, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2′-deoxyribo- and 5′-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2′-deoxyribo- and 5′-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5′-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be “activated” by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid (true hormonal activity) and aromatic (almost no activity) cytokinins indicates a significant difference in the biosynthesis of these compounds.

Auxin and cytokinin coordinate the dormancy and outgrowth of axillary bud in strawberry runner

Background Axillary buds allow the production of either vegetative or reproductive shoots, which display a plastic developmental potential of the plant to suit the prevailing environmental changes. Strawberry represents one of many plant species which displays horizontal above-ground growth of shoot development for asexual reproduction. Two distinct runner growth patterns exist in different strawberry species: one is called sympodial type such as Fragaria vesca, and the other one is called monopodial type such as Fragaria pentaphylla. Despite the runner growth morphology of these strawberry species have been well known, the mechanisms that determine the distinct patterns have rarely been reported. Results In this study, we used Fragaria vesca Hawaii-4 and Fragaria pentaphylla as model species, and captured the initiated dormant bud and non-dormant bud as materials to compare their transcriptome profiles and phytohormone content. Comparisons revealed that relatively higher auxin activity is present in the dormant bud and relatively higher cytokinin activity is in the non-dormant bud. Decapitation and pharmacological experiments on dormant buds showed that the reduction of auxin accumulation triggers the regeneration of vegetative shoots in dormant buds, and exogenous cytokinin application triggers cell fate turnover and generation of reproductive shoots. Conclusion Here, we uncover a mechanism by which auxin and cytokinin coordinate the dormancy and outgrowth of axillary bud in strawberry runner. Our results suggest a contrasting behavior of auxin and cytokinin in control of axillary bud development, facilitating a preliminary understanding of shoot architecture formation in strawberry.