Early detection of graft incompatibility in apricot (Prunus armeniaca) using in vitro techniques

Graft compatibility has been studied in vitro using callus tissues of apricot ( Prunus armeniaca) and different Prunus rootstocks to form scion/rootstock combinations with different degrees of graft compatibility. In these species, incompatibility is manifested by a breakdown of the trees at the union area that can occur some years after grafting. Here, the possibility of obtaining an early detection method to determine graft incompatibility is explored by callus fusion in vitro. The adhesion of the two callus partners, the development of the cells at the contact surface (cell arrangement, intensity of cell-wall staining), and the presence of lipid and phenolic compounds have been studied during the first 3 weeks after grafting in both compatible and incompatible combinations. Differences were observed at the second and the third week of callus co-culture in most of the characters determined, although these differences were present as early as the first week in the case of phenolic compounds. The behaviour of the grafts grown in vitro was correlated to that of the same combinations in the field, suggesting that callus fusion in vitro could be a possible and reliable method for an early detection of graft incompatibility in different Prunus combinations.     

GRAFT UNION FORMATION IN DOUGLAS-FIR

Greenhouse-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) graft unions were examined between 2 and 84 days after grafting. Room temperature was maintained at 60-70 F throughout the growing season. In most respects grafts of Douglas-fir followed development patterns previously reported for spruce and pine grafts, but specific differences were noted in contributing cell types, time of formation, and mode of healing. The time interval from first occurrence to occurrence in 80% of the grafts is as follows: contact layers, 2 days; callus bridges, 10-14 days; periderm, 10-17 days; cambia, 17-23 days. Callus bridges were generally of secondary phloem or cortex origin. Callus lignification began along cut edges of the union at 14 days and was completed across the entire length of the union by 17 days. Lignified tracheids were continuous across union zones at 35 days. When proper grafting techniques were used, all tissue systems necessary for a successful union were present 35 days after grafting. Poor grafting techniques at times retarded cambium formation for 3 months or more.     

Phenylalanine ammonia-lyase-mediated biosynthesis of 2-hydroxy-4-methoxybenzaldehyde in roots of Hemidesmus indicus

The fragrant rootstocks of Hemidesmus indicus are known to accumulate 2-hydroxy-4-methoxybenzaldehyde (MBALD), yet, the enzymatic route to this hydroxybenzoate is not known. Therefore, root organs of H. indicus hold promises to unravel the biosynthesis related to this phenolic fragrance. Chitosan treatment at 200mg/L concentration to the excised roots effectively increased phenolic accumulation in both the cortex and cork tissues reaching a peak after 24h treatment and decreasing thereafter. The activity of phenylalanine ammonia-lyase (PAL) enzyme in excised roots also increased upon chitosan elicitation, and the maximum specific activity was recorded after 12h of elicitation. Suppression of PAL in vivo by using a specific irreversible inhibitor aminooxyacetic acid (AOAA) resulted in the decrease in MBALD content, indicating its formation via phenylpropanoid pathway.     

Graft establishment between compatible and incompatible Prunus spp

Graft compatibility has been studied in apricot (prunus armeniacaL.) grafted on Prunus cerasifera Ehrh. Two apricot cultivars,one compatible and one incompatible on this rootstock, wereselected for this study. In these species incompatibility isonly manifested by tree breakdown at a late phase of the tree'slife. The process of graft union formation was observed forthe first month following grafting. No differences were foundeither in the process of healing or in its kinetics. Thus, callusproliferation, callus differentiation and vascular connectionsare established in the same way and at the same time in bothcompatible and incompatible grafts. However, clear differencesexist in the level of differentiation of the callus produced.While in compatible grafts, callus quickly differentiates intocambium and vascular tissue, in incompatible grafts this differentiationis not complete and a portion of the tissue evolves into a parenchymatoustissue that coexists with the differentiated vascular tissue. Key words: Graft, Prunus, compatibility     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> as a Model System for Graft Union Development in Homografts and Heterografts

Homografting of Arabidopsis thaliana scions on stocks of A. thaliana and heterografting on other species were used to study the compatibility and the ontogeny of graft union formation. Highly compatible homografting with scions of young leafy inflorescence stems was obtained on stocks of inflorescence stems growing from large 3-month-old A. thaliana plants. Histologic analysis revealed four developmental stages of graft union formation in Arabidopsis homografting: (1) development of a necrotic layer, (2) callus proliferation in the grafted scion, (3) differentiation of new vascular tissues within the scion, and (4) a full vascular graft union formation between the scion and the stock. Vascular connections were formed within the callus bridge between rootstocks and scions 15 days after grafting. Heterografts of Arabidopsis on two members of Brassicaceae, cabbage (Brassica) and radish (Raphanus), showed partial incompatible interaction with a lower level of vascular differentiation. Arabidopsis grafting on tomato (Solanaceae) rootstock showed complete incompatibility and limited noncontinuous differentiation of new vascular tissues that did not cross the scion/stock boundary. Although lacking scion/stock vascular connections, Arabidopsis scions grafted onto tomato rootstock flowered and produced seeds. This may indicate some nonvascular functional connections between the two plants, probably of parenchyma cells, further emphasizing the usefulness of Arabidopsis as a model plant for studying various levels of the complicated scion/stock relationships expressed in grafting biology. Experiments with dye transport in the xylem showed that although in general there was an agreement between the histologic study and dye transport, in Arabidopsis homografts water transport frequency was lower than functional and histologic compatability. We conclude that homografting and heterografting of Arabidopsis inflorescence stems is a convenient and reproducible method for studying the fundamental cellular genetic and molecular aspects of grafting biology.     

Spectral composition of irradiation regulates the cell growth and flavonoids biosynthesis in callus cultures of Saussurea medusa Maxim

Cell growth, flavonoids biosynthesis and L-phenylalanine ammonia-lyase (PAL) activity were studied in callus cultures of Saussurea medusa Maxim. under different types of spectral radiance. After 21 days, red light significantly improved the callus growth, but inhibited the biosynthesis of flavonoids in callus cultures. However, blue light was found to enhance the biosynthesis of flavonoids, although callus growth under this spectrum was comparable with that under white and other coloured spectra, such as green and yellow. The accumulation of flavonoids in callus cultures was related to the PAL activity, which was found to be stimulated by the spectral composition of irradiation.     

Phenylalanine ammonia-lyase,chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree

Summary During the first growth phase of walnut (Juglans sp.) stump shoots, the concentrations of the two major phenolic compounds are not correlated with an increasing rate of shoot growth. The concentration of hydrojuglone glucoside (naphtoquinone) decreases as shoot growth rate increases, whereas the concentration of myricitrin (flavonol) remains constant. In contrast, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity is proportional to the growth rate of shoots. Rejuvenation, which induces a higher growth rate and vegetative propagation ability, results in an increase of both PAL and chalcone synthase (CHS, EC 2.3.1.74) activities and hydrojuglone glucoside/myricitrin ratio. Moreover, physiological ageing is characterized by an accelerated functioning of polyphenolic metabolism. Fluctuations in PAL activity are associated with changes in shoot growth rate and with rejuvenation, but PAL does not directly control the accumulation of flavonoid compounds during rejuvenation. On the contrary, mathematical correlation of CHS activity and flavonoid accumulation during annual shoot growth of both adult and rejuvenated trees, indicates that CHS is the rate-limiting enzyme of the pathway.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Effect of Ultraviolet (UV-B) Radiation on the Formation and Localization of Phenolic Compounds in Tea Plant Callus Cultures

The effect of ultraviolet (UV-B) radiation on the accumulation and tissue localization of phenolic compounds in two strains of callus cultures of tea plant (Camellia sinensisL.) were investigated. The strains differed in their morphological and physiological characteristics and biosynthetic capacity. UV-B radiation hampered culture growth, decreased the size of callus-forming cells and promoted the accumulation of soluble and, to a lesser extent, polymeric forms of phenolic compounds, such as lignin. This accumulation was accompanied by an increase in the phenolic compound deposition in cell walls and intercellular space and by deposition of a lignin-like material on the surface of callus cultures. The strain characterized by an increased formation of phenolic compounds was more resistant to UV-B radiation as compared to that with lower phenolic productivity.     

Molecular cloning and functional characterization of a phenylalanine ammonia-lyase from liverwort Plagiochasma appendiculatum

Liverworts are rich in phenolic compounds, including flavonoids and the distinctive type of bisbibenzyls. The biosynthesis of both types of compounds is believed to involve the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is thought to be the key enzyme in the biosynthesis of bisbibenzyls and flavonoids in liverworts. In this study, PAL (designated as PaPAL) was cloned and characterized from both the cDNA and genomic DNA of the liverwort Plagiochasma appendiculatum. The full-length cDNA sequence contains 2,202 bp and is predicted to encode a protein with 733 amino acids. Sequence alignment showed that PaPAL’s predicted amino acid sequence shares more than 70 % identity with PAL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited high PAL activity, catalyzing the conversion of l-phenylalanine to trans-cinnamic acid. However, the enzyme exhibited lower activity in catalyzing the formation of p-coumaric acid from l-tyrosine. Additionally, when the thallus of P. appendiculatum was treated with abiotic stress inducers methyl jasmonate and abscisic acid, PaPAL expression was enhanced, thereby augmenting bisbibenzyl formation. These results suggest that PaPAL plays a key role in the early steps of bisbibenzyl biosynthesis and that abiotic stress can stimulate the expression of PaPAL, resulting in the accumulation of bisbibenzyls in the plant.     

Primary and secondary metabolism of winter wheat under cold hardening and treatment with antioxidants

The content of saccharides and phenolic compounds (including flavonoids and lignin) and the activity of L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) were determined in leaves and tillering nodes of winter wheat Triticum aestivum L.) cultivars Inna and Moskovskaya 39, differing in the level of frost resistance. These parameters were determined in three groups of plants--control, hardened, and treated with synthetic antioxidants (ambiol and amerol 2000). Cold hardening increased accumulation of primary and secondary metabolites in tissues but decreased the enzyme activity. Treatment with antioxidants also increased the content of saccharides and phenolic compounds (primarily flavonoids) and PAL activity. These changes were more pronounced in cultivar Inna, which is less frost resistant than Moskovskaya 39.     

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants (Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.     

Response of winter wheat to cold: production of phenolic compounds and L-phenylalanine ammonia-lyase activity

The formation of soluble and polymeric (lignin) phenolic compounds, activity of L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and content of free L-phenylalanine during cold hardening of winter wheat plants (Triticum aestivum L.) were studied. Cold treatment increased accumulation of soluble phenolic compounds in leaves while not affecting the content of lignin. The opposite was observed in tillering nodes. The activity of PAL was lower than in control plants in both tissues, and the content of free L-phenylalanine in tissues increased.     

嫁接植株形成过程中接合部组织学和生长素含量的变化

本文应用植物激素间接酶联免疫技术(ELISA),第一次定量检测了嫁接植株形成过程中生长素(IAA)的动态变化。结果表明：嫁接植株发育的前期,亲和性与非亲和性组合其IAA含量的变化相似。在后期,不亲和组合IAA含量急骤减少,而亲和性的组合在第八天即维管束桥分化形成的这一天,可见到IAA高峰值的出现。     

A Study of the graft union in in vitro micrografted apple

Light microscopy was used to study graft union formation in in vitro micrografts of tissue cultured apple (Malus domestica. Borkh). Micrografts were constructed using horizontal incisions to form the grafting surface, and placing the cut ends of rootstock and scion into sterile silicon tubing to permit graft formation to occur.The outer morphological and histological development was similar for different stock-scion micrograft combinations but graft union formation was slower in heterografts than in autografts. Initial leaf expansion at the scion shoot apex occurred in all micrografted plantlets within 1–4 days and was not indicative of graft success. Progressive scion growth and development could be used as an indication of graft success by ten to fourteen days after grafting and probably was related to establisment of cell to cell contact at the graft interface. Microscopy showed initiation of callus proliferation in the vascular cumbium and the pith ten days after grafting. Differentiation was observed subsequently and this was reflected in scion development. Longitudinally orientated cambial cells began to differentiate between twenty and forty days after grafting, and formed a bridge between the vascular cylinders of scion and rootstock. The scions at this stage had as many as eight newly expanded leaves and micrografts were strong enough to permit silicon sleeve removal without damage. Continuity of new vascular elements in rootstock and scion was established around forty days. New vascular elements curved slightly in towards the pith to form a c shaped bridge across the graft union. Vascular development continued until it reached completion after six months.     

Graft Formation in Kalanchoe blossfeldiana

Three phases of cohesion between the stock and scion are observableduring the formation of compatible autografts in Kalanchoe blossfeldiana.The first phase of cohesion: (a) lasts four to five days, (b)is correlated with an accumulation of dictyosomes along thegraft interface and with callus proliferation in the stock andscion, and (c) is characterized by a tensile strength of approximately5 g breaking weight (BW) mm^–2 graft area (GA) by 5 d aftergrafting. The second phase of cohesion lasts from days 5–20after grafting and is correlated with (a) an interdigitationof callus cells at the graft interface, (b) the differentiationof vascular tissue across the graft interface, and (c) a 20-foldincrease in the tensile strength of the graft union to approximately100 g BW mm^–2 GA by 20 d after grafting. This cohesivestrength is comparable to that of an intact, non-grafted stem.The third phase of cohesion is characterized by a levellingoff of the increase in tensile strength of the graft union withtime at approximately 125 g BW mm^–2 GA. The results ofthis study are discussed relative to other structural studiesof and proposed mechanisms for graft development.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase