Malus Zumi Research Articles

Transcriptome and Metabolome Analysis Reveals Salt-Tolerance Pathways in the Leaves and Roots of ZM-4 (Malus zumi) in the Early Stages of Salt Stress.

The breeding of salt-tolerant rootstock relies heavily on the availability of salt-tolerant Malus germplasm resources. The first step in developing salt-tolerant resources is to learn their molecular and metabolic underpinnings. Hydroponic seedlings of both ZM-4 (salt-tolerant resource) and M9T337 (salt-sensitive rootstock) were treated with a solution of 75 mM salinity. ZM-4's fresh weight increased, then decreased, and then increased again after being treated with NaCl, whereas M9T337's fresh weight continued to decrease. The results of transcriptome and metabolome after 0 h (CK) and 24 h of NaCl treatment showed that the leaves of ZM-4 had a higher content of flavonoids (phloretinm, naringenin-7-O-glucoside, kaempferol-3-O-galactoside, epiafzelechin, etc.) and the genes (CHI, CYP, FLS, LAR, and ANR) related to the flavonoid synthesis pathway showed up-regulation, suggesting a high antioxidant capacity. In addition to the high polyphenol content (L-phenylalanine, 5-O-p-coumaroyl quinic acid) and the high related gene expression (4CLL9 and SAT), the roots of ZM-4 exhibited a high osmotic adjustment ability. Under normal growing conditions, the roots of ZM-4 contained a higher content of some amino acids (L-proline, tran-4-hydroxy-L-prolin, L-glutamine, etc.) and sugars (D-fructose 6-phosphate, D-glucose 6-phosphate, etc.), and the genes (GLT1, BAM7, INV1, etc.) related to these two pathways were highly expressed. Furthermore, some amino acids (S-(methyl) glutathione, N-methyl-trans-4-hydroxy-L-proline, etc.) and sugars (D-sucrose, maltotriose, etc.) increased and genes (ALD1, BCAT1, AMY1.1, etc.) related to the pathways showed up-regulation under salt stress. This research provided theoretical support for the application of breeding salt-tolerant rootstocks by elucidating the molecular and metabolic mechanisms of salt tolerance during the early stages of salt treatment for ZM-4.

Physiological Changes and Transcriptome Analysis of Malus zumi in Response to Salt Stress

Physiological Changes and Transcriptome Analysis of Malus zumi in Response to Salt Stress

Effects of Salt Stress on the Antioxidant Activity and Malondialdehyde, Solution Protein, Proline, and Chlorophyll Contents of Three Malus Species.

Understanding the different physiological responses of Malus species under salt stress in the seedling stages will be useful in breeding salt-tolerant dwarfing apple rootstocks. Seedlings of Malus Zumi (Mats.) Rehd. (M. zumi), Malus sieversii (Led.) Roem. (M. sieversii), and Malus baccata (L.) Borkh. (M. baccata) were treated with solution of 0, 0.20%, 0.40%, and 0.60% salinity. Physiological parameters of their leaves and roots were measured at 0 d, 4 d, 8 d and 12 d after salinity treatments. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA), solution protein (SP), and proline (PRO) initially increased and then decreased. The activities and contents of these parameters were higher in the 0.40% and 0.60% NaCl treatments than in the 0.20% treatment and in the 0% control. M. zumi was the most resistant to salt stress, showing the lowest content of MDA in the leaves and roots, which increased slightly under salt stress. M. baccata had the highest increase in both the content and proportion of MDA. High enzyme activity was shown to play an important role in the salt resistance of M. zumi. Moreover, it can be speculated that there are other substances that also play a major role. We found that osmotic regulation played a key role in response to salt stress for M. baccata even though it was sensitive to salt stress. For M. sieversii, both the osmotic regulation and enzymatic antioxidants were observed to play a major role in mitigating salt stress.

Variability and correlations of traits of the flower, leaf and fruit of Malus zumi

Malus zumi (Matsum.) Rehder is a carrier of powdery mildew resistance genes and also has ornamental traits. However, the variability of its traits is still poorly understood. The aim of the research is establishing the variability and relationship between the 17 traits of the Malus zumi. It was found that the variability of the traits of the flower and fruit is at a very low level, and the traits of the leaf are more variable. There were found strong correlations between the seeds’ weight and its number in the fruit (r=0.83), the diameter and weight of the fruit (r=0.79), and there is a strong inverse relationship between fruit’s weight and anther’s length (r=-0.73). The length of the filament has a strong relationship with the length of the fruit (r=-0.71) and the leaf’s length (r=-0.71), also it is closely related to all leaf traits with a positive relationship. This information is important for the implementation of directed breeding programs, as well as recommendations for use in ornamental horticulture.

Полиморфизм признаков цветка и плода яблони Цуми (Malus zumi (Matsum.) Rehder)

The Tsumi apple tree is a valuable crop due to the presence of a gene for resistance to powdery mildew, abundant annual fruiting and a special decorative effect during flowering and fruiting. Its breeding potential has been little studied; therefore, it is necessary to study the morphometric characteristics of a flower and a fruit and establish connections between them. The collection of flowers and fruits of the Tsumi apple tree was carried out in the laboratory of cultivated plants of the Tsytsin Main Botanical Garden in 2021. The coefficients of variation and correlation according to Spearman were determined. It was found that the polymorphism of flower traits is at a very low level (from 4.4% to 14.2%). The number of flowers per inflorescence varies within 15.9%, which corresponds to the average level of polymorphism, as well as in the weight of the fruit (17.3%) and in the weight of seeds (19.5%). Variations in the length of the peduncle, the number of seeds, the length of the fruit, and the diameter of the fruit are at a very low level (6.9-13.6%). A significant number of reliable inverse (negative) correlations were found between signs of moderate strength (0.5 <r <0.6): a strong relationship between fruit weight and anther length (r = -0.73), fruit length and filament length (r = -0.71). A strong positive relationship was found between the following traits: fruit weight and fruit diameter (r = 0.79), seed weight. Keywords: POLYMORPHISM, FLOWER, CRABAPPLES, SORBOMALUS, MALUS ZUMI, TSUMI APPLE TREE, VARIABILITY, MORPHOLOGY

Expression Analysis of AUX/IAA Family Genes in Apple Under Salt Stress.

Members of the auxin/indoleacetic acid (Aux/IAA) gene family in plants are primary auxin-responsive genes that play important roles in many aspects of plant development and in responses to abiotic stress. Recently, 33 Aux/IAA have been identified in the apple genome. The biological responses of MdIAAs to salt stress are still unknown. In this study, Malus zumi, Malus baccata, and Malus × domestica 'Fuji' plantlets were subjected to salt stress by supplementing hydroponic media with NaCl at various concentrations. M. zumi showed the strongest salt resistance, followed by 'Fuji', and M. baccata was the most sensitive to salt stress. Tissue-specific expression profiles of MdIAAs were determined by quantitative real-time polymerase chain reaction. When apple plantlets were subjected to salt stress, most of salt-responsive MdIAAs were up-regulated by 1h, 3h, and 6h in roots, shoot tips, and leaves, respectively. Highly expressed MdIAAs in roots, especially for M. zumi, consisted with the salt tolerance of apple rootstocks. Transgenic apple calli were tolerant to salt stress when over-expressed salt-responsive genes, MdIAA8, -9, and -25. These results provide clues about salt resistance in these three Malus species, which helps apple breeding of salt tolerance by genetic transformation.

Overexpression of MzASMT 1, a Gene From Malus zumi Mats, Enhances Salt Tolerance in Transgenic Tobacco.

Melatonin, widely found in various plants as a new antioxidant, could protect plants from various biotic and/or abiotic stresses, including salt stress. MzASMT 1 (KJ123721), a gene from Malus zumi Mats, is a key enzyme required for melatonin synthesis. However, whether the overexpression of MzASMT 1 could regulate the synthesis of melatonin and improve the salt tolerance in tobacco remains unknown. In this study, the overexpression of MzASMT 1 in tobacco increased the melatonin content, and the transgenic lines owned higher salt tolerance capacity. The transgenic lines overexpressing MzASMT 1 exhibited lower degree of leaf wilting; much more fresh weight; higher plant height; longer root; higher relative water content (RWC) of leaves, stem, and root; and higher chlorophyll content and Fv/Fm, which makes transgenic lines better adapt to salt stress. The transgenic lines also had higher accumulation of proline, lower accumulation of malondialdehyde (MDA), and improved antioxidant systems, which protected plants from cell damage and oxidative stress due to excess reactive oxygen species (ROS) accumulation under salt treatment. The transcription of salt response genes was much more highly activated in transgenic lines than in wild type under salt stress. The above results contributed to the understanding of functions for MzASMT 1 in tobacco under salt stress and provided a new choice for the application of MzASMT 1 in improving plant salt tolerance.

Chloroplastic biosynthesis of melatonin and its involvement in protection of plants from salt stress

Within the chloroplasts reactive oxygen species (ROS) are generated during photosynthesis and stressful conditions. Excessive ROS damages chloroplasts and reduces photosynthesis if not properly detoxified. In this current study, we document that chloroplasts produce melatonin, a recently-discovered plant antioxidant molecule. When N-acetylserotonin, a substrate for melatonin synthesis, was fed to purified chloroplasts, they produced melatonin in a dose-response manner. To further confirm this function of chloroplasts, the terminal enzyme for melatonin synthesis, N-acetylserotonin-O-methyltransferase (ASMT), was cloned from apple rootstock, Malus zumi. The in vivo fluorescence observations and Western blots confirmed MzASMT9 was localized in the chloroplasts. A study of enzyme kinetics revealed that the Km and Vmax of the purified recombinant MzASMT9 protein for melatonin synthesis were 500 μM and 12 pmol/min·mg protein, respectively. Arabidopsis ectopically-expressing MzASMT9 possessed improved melatonin level. Importantly, the MzASMT9 gene was found to be upregulated by high light intensity and salt stress. Increased melatonin due to the highly-expressed MzASMT9 resulted in Arabidopsis lines with enhanced salt tolerance than wild type plants, as indicated by reduced ROS, lowered lipid peroxidation and enhanced photosynthesis. These findings have agricultural applications for the genetic enhancement of melatonin-enriched plants for increasing crop production under a variety of unfavorable environmental conditions.

Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis.

The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with Vmax values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis.

MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation, Altered the Drought and Salt Tolerance of Transgenic Arabidopsis.

BackgroundPlants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant.ResultsThe MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K+/Na+ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment.ConclusionThe MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance.

Overexpression of MzASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants.

Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-O-methyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E.coli converted its substrates to melatonin with an activity of approximately 5.5pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2-4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.

Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na+/H+ Antiport Activity of Three Malus species with Different Salt Tolerance

Salt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol (PEG) has only osmotic stress. This study aimed to compare the different effects on the activity of H+-ATPase, proton pump and Na+/H+ antiport in Malus seedlings between osmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malus baccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activity of H+-ATPase, proton pump and Na+/H+ antiport of plasmolemma and tonoplast in Malus seedlings were obviously increased under salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H+-ATPase, proton pump and Na+/H+ antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher than those in salt-sensitive one. Higher Na+/H+ antiport activity of plasmolemma and tonoplast in salt-tolerant species could help to extrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H+-ATPase, proton pump and Na+/H+ antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmotic PEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H+-ATPase, proton pump and Na+/H+ antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization in roots and is therefore more salt tolerant.

A genome-wide expression profile of salt-responsive genes in the apple rootstock Malus zumi.

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees.

Isolation and preliminary function analysis of a Na + /H + antiporter gene from Malus zumi

A full-length cDNA Na+/H+ antiporter gene (MzNHX1) was isolated from Malus zumi according to the homologous Na+/H+ antiporter gene region in plants. Sequence analysis indicated that the cDNA was 2062 bp in length, including an open reading frame (ORF) of 1629 bp, which encoded a predicted polypeptide of 542 amino acids. The MzNHX1 protein shared high identity with other reported plant vacuolar Na+/H+ antiporters. Southern blot analysis detected multiple copies of MzNHX1 in the M. zumi genome. Northern blot analysis showed negligible expression of the gene in roots, but expression was detected in stems and leaves. To test the function of MzNHX1, we expressed the gene in the saltsensitive AXT3 yeast mutant. No differences in yeast cell growth were detected given the presence or absence of MzNHX1 on a NaCl free medium. However, on a 70 mM NaCl medium, growth in the control transformant was noticeably suppressed, and yeast overexpression of MzNHX1 showed increased population growth rates. These results indicated that the MzNHX1 protein increased AXT3 salt tolerance. Alignments of the deduced Na+/H+ antiporter amino acid sequence of different plants from NCBI revealed that MzNHX1 shared high identity (>86%) with vacuolar Na+/H+ antiporters, including RhNHX1 from rose, cNHX1 from citrus, and AtNHX1 from Arabidopsis thaliana. However, MzNHX1 shared very low identity ( thaliana. These results indicated that MzNHX1 was localized to the vacuolar membrane.

GENETIC CHARACTERIZATION AND MAPPING OF THE MAJOR POWDERY MILDEW RESISTANCE GENE PLBJ FROM MALUS BACCATA JACKII

Powdery mildew, caused by the obligate biotrophic ascomycete fungus Podosphaera leucotricha, is one of the main fungal diseases in commercial apple production. Only a few major resistance sources are currently used in apple breeding programmes to include mildew resistance. Some of them such as Pl1 from Malus robusta and Pl2 from Malus zumi have already been reported to be not fully active against the whole race spectrum of the apple powdery mildew fungus. New mildew resistance sources are needed that could extend the genetic basis for mildew resistance breeding. The mildew resistant Malus bacctata accession no. 419 (M. baccata var. jackii) was selected as crossing parent to produce an apple progeny segregating for resistance. Based on clearly resistant/susceptible individuals, an AFLP-based bulked segregant analysis (BSA) was performed. Several polymorphic AFLP markers were identified. Among them one marker co-segregated with the resistance phenotype. This AFLP fragment was cloned and a specific SCAR primer pair (called PLBJ-SCAR) was developed. By using an SSR-based genome-scan, the Plbj gene was located close to the SSRs CH02a10 and CH02c11 on the apple linkage group LG 10. For a verification of the monogenic inheritance of Plbj, as well as for testing the practical usefulness of the developed SCAR marker, a second population has been raised in 2004 from a cross between ‘Cox Orange’ and M. baccata 419. The correspondence between the mildew resistance phenotype and the PLBJ-SCAR is demonstrated also for this apple family.

Efficient Agrobacterium tumefaciens-mediated transformation of Malus zumi (Matsumura) Rehd using leaf explant regeneration system

Malus zumi is known as an excellent dwarfing apple rootstock occurring in natural or arid/semiarid soil or salina. Gene manipulation of M. zumi through transgenic technology can modify plant feature for further improvement fruit tree production by grafting the scion on a transgenic rootstock. Here, we report the establishment of an efficient, in vitro , shoot regeneration system and Agrobacterium tumefaciens - mediated transformation from the leaf explants for Malus zumi (Matsumura) Rehd. Leaf explants were infected with Agrobacterium strains containing npt II and gus gene. The highest frequency of shoot regeneration was obtained on MS medium containing 500 mg l -1 Lactalbumin hydrolysate, 30 g l -1 fructose, supplemented with 3.0 mg l -1 BA, 0.2 mg l -1 NAA.Using fructose instead of sucrose significantly increases the shoot regeneration and decreases vitrification. This regeneration procedure was incorporated into an Agrobacterium -mediated transformation procedure in M. zumi . Kanamycin was an efficient selective agent for selection. Pre-selection (5 days after co-cultivation) improved the transformation efficiency. The emergence of expected bands by PCR analysis and Southern blot in transgenic plantlets confirmed the transformation of foreign DNA into plant genome.

Development of apple powdery mildew on sources of resistance to Podosphaera leucotricha, exposed to an inoculum virulent against the major resistance gene Pl‐2

AbstractThe major resistance gene Pl‐2 in apple has recently been shown to be overcome by a virulent population of powdery mildew, Podosphaera leucotricha, in genotypes of the fourth generation after introgression of Pl‐2 from crab apple, Malus zumi. Tests of pathogenicity in controlled conditions and observations in an orchard showed that the initial progenitor of Pl‐2, MAL 68/1, was overcome by a virulent inoculum. As a consequence, all selections derived from MAL 68/1 may lose their resistance to powdery mildew, if Pl‐2 is used alone. Sources of resistance other than Pl‐2 remained efficient to the virulent population: M. hupehensis, M. mandshurica, M. robusta, M. sargentii, M. sieboldii, D12, Mildew Immune Selection and ‘White Angel’. The combination of Pl‐2 with quantitative resistance genes resulted in a high level of resistance.

Breakdown of Pl2, a major gene of resistance to apple powdery mildew, in a French experimental orchard

Pl2, a major resistance gene to powdery mildew originating from Malus zumi, is mainly used in apple‐breeding programmes. In an experimental orchard in France, an increase of susceptibility to powdery mildew was observed on apple genotypes carrying Pl2. This increase of susceptibility could not be explained by an effect of the age of the trees, or by an effect of the amount of inoculum on expression of the resistance Pl2. It was demonstrated by tests of pathogenicity in controlled conditions that isolates of Podosphaera leucotricha sampled in this orchard were virulent to Pl2. The appearance of virulent isolates within 6 years after planting apple genotypes selected for the presence of Pl2 resulted in a breakdown of the resistance. Ten years after planting, the percentage of genotypes that were still resistant to powdery mildew varied between 2 and 56%, depending on the progenies. This suggested that additional quantitative resistance genes may influence the durability of the resistance Pl2.