Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

Root-specific reduction of cytokinin perception enhances shoot growth with an alteration of trans-zeatin distribution in Arabidopsis thaliana

Compelling evidence demonstrates that root-derived cytokinins (CKs) contribute to shoot growth via long-distance transport; therefore, we hypothesized that an increase in root-derived CKs enhances shoot growth. To demonstrate this, we grafted Arabidopsis Col-0 (WT) scion onto rootstock originated from WT or a double-knockout line of CK receptors AHK2 and AHK3 (ahk23) because the knockout line over accumulates CKs in the body due to feedback homeostasis regulation. The grafted plants (scion/rootstock: WT/WT and WT/ahk23) were grown in vermiculite pots or solid media under high and low nitrogen regimes for vegetative growth and biochemical analysis. The root-specific deficiency of AHK2 and AHK3 increased root concentrations of trans-zeatin (tZ)-type and N6-(Δ2-isopentenyl) adenine (iP)-type CKs, induced CK biosynthesis genes, and repressed CK degradation genes in the root. Shoot growth, shoot concentrations of tZ-type CKs, and shoot expression of CK-inducible marker genes were consistently larger in the WT/ahk23 plants than in the WT/WT plants. Moreover, the root-specific deficiency of AHK2 and AHK3 enhanced shoot growth in the WT scion more strongly than in the ahk23 scion. Given that tZ-type CKs are predominantly produced from iP-type CKs in the root and xylem-mobile, it is concluded that the root-specific reduction of CK perception would enhance shoot growth by increasing the amount of root-derived tZ-type CKs and their perception by the shoot. This study will present a novel approach to improve plant growth and productivity.

Effects on Plant Hormone levels at Different Growth Stages in Diverse Grape Organs with Root Restriction

Endogenous plant hormones play important roles in germination, blossom, senescence, abscission of plants by a series of signal transduction and molecular regulation. The purpose of this research was to investigate the influence of root restriction (RR) cultivation on plant hormones variation tendency at different growth stages in diverse organs or tissues, ‘Muscat Hamburg’ (Vitis ‘Muscat of Alexandria’ × Vitis ‘Trollinger’) grapevine was used as test material. High Performance Liquid Chromatography (HPLC) was used to quantify hormone levels, aiming to investigate the influence of root restriction on the formation and transportation of plant hormones. The results revealed that RR treatment increased abscisic acid, salicylic acid, zeatin riboside, N6-(delta 2-isopentenyl)-adenine nucleoside concentrations, while reduced auxin, 3-indolepropionic acid, 3-indolebutyric acid, gibberellin A3, zeatin, N6-(delta 2-Isopentenyl)-adenine, kinetin, jasmonic acid and methyl jasmonate concentrations. To sum up, our results suggested that RR treatment could initiate stress responses via up-regulating abscisic acid and salicylic acid contents while down-regulating auxin and kinetin contents, resulting in the changes of fruit appearance and improvement of berry quality.

Coupling proteomics and metabolomics for the unsupervised identification of protein–metabolite interactions in Chaetomium thermophilum

Protein–metabolite interactions play an important role in the cell’s metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.

Tissue-Specific Hormonal Variations in Grapes of Irrigated and Non-irrigated Grapevines (Vitis vinifera cv. “Merlot”) Growing Under Mediterranean Field Conditions

Agricultural practices in grapevines management include water restrictions due to its positive effect on wine quality, especially when applied at fruit ripening. Although the effects of water stress in some groups of phytohormones have already been described in leaves and whole grapes, information regarding tissue-specific variations in hormones during ripening in grapes is scarce. Field-grown grapevines from the cv. “Merlot” were subjected to two differential water supplies, including only rainfed, non-irrigated vines (T0) and vines additionally irrigated with 25Lweek−1 vine−1 (T1). Tissue-specific variations in the hormonal profiling of grapes [including changes in the contents of abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid (ACC), the auxin indole-3-acetic acid, gibberellins 1, 3, 4, and 7 (GA1, GA3, GA4, and GA7), the cytokinins trans-zeatin, and 2-isopentenyl adenine, including as well their respective ribosylated forms] were periodically evaluated from veraison to harvest. The hormonal profiling in leaves was also measured at the beginning and end of the season for comparison. Results showed that grape growth dynamics were transiently affected by the differences in water regimes, the increased water supply leading to an accelerated growth, slightly reduced accumulation of sugars, and transiently lowered pH, although grape quality did not differ between treatments at harvest. Hormonal profiling of whole berries did not reveal any difference in the endogenous contents of phytohormones between treatments, except for a transient decrease in GA4 contents in T1 compared to T0 vines, which was not confirmed at the tissular level. Hormonal profiling at the tissue level highlighted a differential accumulation of phytohormones during ripening in berry tissues, with pulps being particularly poor in ABA, JA, and SA contents, seeds particularly accumulating ACC, gibberellins, and zeatin-type cytokinins, and the skin being particularly rich in auxin and active cytokinins. Changes in water supply led to very small and transient changes in the endogenous contents of phytohormones in the seeds, pulp, and skin of berries, the most remarkable variations being observed in cytokinin contents, which increased earlier [between 5 and 12days after veraison (DAV)] but later kept more constant in the skin from T1 compared to T0 vines and were also 3-fold higher at 40 DAV in seeds of T1 compared to T0 vines. It is concluded that small changes in water supply can trigger hormonal-driven physiological adjustments at the tissular level affecting the evolution of fruit growth and quality throughout grape berry ripening.

Improvement of Tillering and Grain Yield by Application of Cytokinin Derivatives in Wheat and Barley

Three cytokinin derivatives (CKd) designated as RR-G, RR-O, and RR-V applied by foliar spraying at tillering, and one compound previously described as a cytokinin antagonist (CKa) designated as RR-P applied as a seed coating were tested in winter wheat and spring barley in field trial experiments. The aim of the study was to examine the influence of the compounds that were tested on the number of productive tillers, grain yield, and endogenous CK content. With the exception of the compound RR-V, the measured parameters clearly showed the stimulatory effects of CKd on tillering and grain yield in spring barley and winter wheat. The RR-V showed a stimulatory effect on the number of productive tillers and yield in spring barley, but not in winter wheat. Although in winter wheat CKa stimulated both the number of productive tillers and the grain yield, there was an inhibitory effect in terms of the number of productive tillers observed in spring barley. The results of the endogenous cytokinin analysis suggested, among others, the importance of the role of isopentenyl-adenine types of cytokinins in the tillering of spring barley. In conclusion, the cytokinin derivative compounds with an agonistic or antagonistic role showed strong potential for application in the future development of plant growth regulators.

Overproduction of ABA in rootstocks alleviates salinity stress in tomato shoots

To determine whether root-supplied ABA alleviates saline stress, tomato (Solanum lycopersicum L. cv. Sugar Drop) was grafted onto two independent lines overexpressing the SlNCED1 (9-cis-epoxycarotenoid dioxygenase) gene (NCED OE) and wild type rootstocks. After 200 days of salinity irrigation (EC = 3.5 dS m-1), plants with NCED OE rootstocks had 30% higher fruit yield, but root biomass and lateral root development was reduced. Although NCED OE rootstocks upregulated ABA-signalling (AREB, ATHB12), ethylene-related (ACCs, ERFs), aquaporin (PIPs) and stress-related (TAS14, KIN, LEA) genes, downregulation of PYL ABA receptors and signalling components (WRKYs), ethylene synthesis (ACOs) and auxin responsive factors occurred. Elevated SlNCED1 expression enhanced ABA levels in reproductive tissue while ABA catabolites accumulated in leaf and xylem sap suggesting homeostatic mechanisms. NCED OE also reduced xylem cytokinin transport to the shoot and stimulated foliar 2-isopentenyl adenine (iP) accumulation and phloem transport. Moreover, increased xylem gibberellin GA3 levels in growing fruit trusses was associated with enhanced reproductive growth. Improved photosynthesis without changes in stomatal conductance was consistent with hormone-mediated alteration of leaf growth and mesophyll structure, which combined with lower assimilate requirement in the roots and systemic changes in hormone balances could explain enhanced vigour, reproductive growth and yield under saline stress.

Optimizing Micropropagation of Apple (Malus × Domestica Borkh) and in Vitro Root Induction By Piriformospora indica

The inoculation of plant species with mycorrhiza fungus Piriformospora indica results in enhancement of growth, increase in yield, and induction of resistance to biotic and abiotic diseases through improvement of the root system. The aim of the present study was to optimize in vitro propagation protocol for three indigenous apples (Malus × domestica) cultivars (ꞌGolbaharꞌ, ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ) and one commercial cultivar (ꞌGolden Deliciousꞌ). Furthermore, the efficiency of P. indica at rooting stage was investigated on three cultivars (ꞌSharbatiꞌ, ꞌSoltani Shabestariꞌ, ꞌGolden Deliciousꞌ). Establishment and proliferation stages were optimized by collecting explants at different seasons and comparing different culture media respectively. Rooting optimization included six treatments containing different concentrations of auxins in the presence or absence of P. indica. Results showed that at the establishment stage, a maximum percent of survival was observed in explants collected in spring. At the proliferation stage, different media had a divergent effects on distinct cultivars. Although all cultivars reacted favourable to micropropagation in MS (Murashige & Skoog 1962) basal medium, the presence or absence of cytokinin 2ip (N6-(2-Isopentenyl) adenine) in the culture media showed significant and incremental improvements in growth indices. In all cultivars highest rooting percent, root length, root thickness, and the number of roots/explant was observed in MS media containing auxins for three weeks followed by a treatment of MS medium containing P. indica for another three weeks. Plantlets treated with P. indica, grow stronger and healthier at the acclimation stage compared to the ones that excluded P. indica.

Differences in xylem development between Dutch and Japanese tomato (Solanum lycopersicum) correlate with cytokinin levels in hypocotyls

Background and Aims Dutch tomato cultivars tend to have a greater yield than Japanese cultivars even if they are grown under the same conditions. Factors contributing to the increased yield of the Dutch cultivars were a greater light use efficiency and greater leaf photosynthetic rate. On the other hand, the relationship between tomato yields and anatomical traits is still unclear. The aim of this study is to identify the anatomical traits related to the difference in yield between Dutch and Japanese cultivars. Methods Anatomical properties were compared during different growth stages of Dutch and Japanese tomatoes. Hormone profiles and related gene expression in hypocotyls of Dutch and Japanese cultivars were compared in the hypocotyls of 3- and 4-week-old plants. Key results Dutch cultivars have a more developed secondary xylem than Japanese cultivars, which would allow for greater transport of water, mineral nutrients and phytohormones to the shoots. The areas and ratios of the xylem in the hypocotyls of 3- to 6-week-old plants were larger in the Dutch cultivars. In reciprocal grafts of the Japanese and Dutch cultivars, xylem development at the scion and rootstock depended on the scion cultivar, suggesting that some factors in the scion are responsible for the difference in xylem development. The cytokinin content, especially the level of N6-(Δ 2-isopentenyl) adenine (iP)-type cytokinin, was higher in the Dutch cultivars. This result was supported by the greater expression of Sl-IPT3 (a cytokinin biosynthesis gene) and Sl-RR16/17 (a cytokinin-responsive gene) in the Dutch cultivars. Conclusions These results suggest that iP-type cytokinins, which are locally synthesized in the hypocotyl, contribute to xylem development. The greater xylem development in Dutch cultivars might contribute to the high yield of the tomato.

Identification of the profile of endogenous cytokinin-like compounds during different plant growth stages and their effects on flower bud abscission in pistachio (Pistacia vera L.)

Alternate bearing is a significant economic problem that affects fruit producers, consumers and the economy of a country. Concentrations of cytokinin (CK)-like compounds in different tissues of pistachio trees (Pistacia vera ‘Uzun’) were measured to elucidate the relationship between endogenous CKs and flower bud abscission (alternate bearing). Identification and quantification of CK-like compounds of the Z, DHZ and iP types from different physiological stages and tissues were performed using high-performance liquid chromatography–electrospray ionisation–tandem mass spectrometry (HPLCESI-MS/MS) techniques. Five to seven different CK-like compounds were detected, respectively. Isopentenyl adenine riboside (iPR) and trans-zeatin-O-glucoside (t-ZOG) were the main isoprenoid CKs, as the dominant conjugates in all the analysed organs of pistachio trees. The leaves and shoots of ‘On’-year trees had significantly higher CK contents than those of ‘Off’-year trees on all the sampling dates. Interestingly, the relatively high CK activity was recorded before flower bud abscission in the fractions corresponding to iPR and t-ZOG, while this activity dropped considerably during the flower bud abscission period (55 DAFB). Consequently, the strong negative correlation between CK concentration and bud abscission could be the first indication of the role of CK-like compounds in this unique phenomenon in the ‘Uzun’ pistachio cultivar. It was concluded that CKs could have an important role in the alternate bearing in pistachio.