Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress

ABSTRACT: BACKGROUND: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew. RESULTS: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles. CONCLUSIONS: The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.     

葡萄属植物叶片显微和超微结构观察

利用光学显微镜和透射电镜,对葡萄属(Vitis L.)15种、1亚种和4栽培品种植物的叶片显微和超微结构进行了观察研究。结果显示:(1)东亚种群中桦叶葡萄的叶片总厚度和各组织厚度最大;北美种群的河岸葡萄和沙地葡葡叶片总厚度、上表皮厚度、栅栏组织厚度、海绵组织厚度和下表皮厚度较大,但叶片组织紧密度最小。(2)各种类间栅栏组织中叶绿体数目均多于海绵组织,且刺葡萄、桦叶葡萄、葛藟葡萄和秋葡萄叶肉细胞中叶绿体数目最多,北红、蘡薁、网脉葡萄和菱叶葡萄叶片中叶绿体数目最少,巨峰和桦叶葡萄叶绿体中含有大量的淀粉粒,桦叶葡萄、网脉葡萄、秋葡萄、葛藟葡萄、刺葡萄和华东葡萄含有较多的嗜锇颗粒。研究表明,中国野生葡萄的许多种类在叶片组织紧密度、叶绿体数量、淀粉粒数量、嗜锇颗粒数量方面均高于北美种群的河岸葡萄和沙地葡萄,具有更高的结构抗性基础。     

葡萄Golden2-like转录因子家族的鉴定及其表达分析

本研究对葡萄（Vitis vinifera L.）的Golden2-like （GLK）转录因子家族进行了全基因组鉴定和表达模式分析,并利用品种‘玫瑰香’（V.vinifera cv.Muscat Hamburg）进一步验证其在低温胁迫下的响应。结果显示,葡萄Golden2-like家族共46个成员,分为5个亚族,同一亚族的保守结构域相似。46个VvGLK分别定位于细胞核、叶绿体、细胞质和过氧化物酶体中,其启动子区域含多种逆境应答顺式作用元件。基因芯片分析结果表明,22个Golden2-like基因在果实发育过程中变化显著。同时,有15、15和9个基因分别响应盐、干旱和低温胁迫。qRT-PCR分析发现26个基因参与低温应答。VvGLK41在所有胁迫处理中均下调表达。     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.     

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK₃-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK₃-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.     

Accumulation of tocopherols and tocotrienols during seed development of grape (Vitis vinifera L. cv. Albert Lavallée)

Tocopherols and tocotrienols are present in mature seeds. Yet, little is known about the physiological role and the metabolism of these compounds during seed development. Here we present data on tocopherol and tocotrienol accumulation during seed development in Vitis vinifera L. cv. Albert Lavallée (Royal). This species was chosen for its ability to synthesize both tocopherols and tocotrienols. It is shown here for the first time that during seed development there are significant differences in localization and accumulation kinetics of tocopherols and tocotrienols. Tocopherols are found homogeneously dispersed throughout all tissues of the seed, in concentrations ranging from 20 to 100 microg tocopherol per g dry weight. Tocopherol levels decrease gradually during seed development. In contrast, tocotrienols are only found in the endosperm of the seeds, accumulating in a sigmoid fashion during the maturation period of seed development. Tocotrienol levels were found to be (54+/-7.4) microg/g dry seed in 90-day-old seeds of V. vinifera L. Furthermore, tocotrienol biosynthesis is demonstrated in these seeds during tocotrienol accumulation and in an endosperm fraction isolated at 75 days after flowering.     

Expression of KS-type dehydrins is primarily regulated by factors related to organ type and leaf developmental stage during vegetative growth

The expression of a gene, designated as DHN10, was analyzed at the protein level in two Solanum species. The DHN10 protein displays some consensus amino acid sequences of dehydrins, termed K- and S-segments. Unlike most dehydrins, both segments occur only in single copies in the DHN10 sequence and the S-segment is at a C-terminal position. Database searches revealed that KS-type dehydrins constitute a specific subclass distributed in dicotyledons and monocotyledons. In Solanum tuberosum L. plants, a high DHN10 abundance was observed under control conditions, particularly in flowers, stems, tubers and young developing leaves. In other Solanaceae and in barley (Hordeum vulgare L.), the amount of DHN10 was much more elevated in young leaves than in old leaves. DHN10 abundance was investigated in two Solanum species subjected to low temperature or to drought. Under stress conditions, we observed substantially higher protein levels only in mature expanded leaves. These findings clearly indicate that KS-type dehydrins are present at a high level in the absence of stress during vegetative growth and that their expression is primarily regulated by factors related to organ type and to leaf development stage. A potential role for the DHN10 dehydrin during plant development and in tolerance to environmental stress is discussed.Abbreviations DHN10 Dehydrin protein of 10 kDa - His Histidine - KS-type dehydrin Dehydrin containing a single K-segment followed by a single S-segment - LEA Late embryogenesis abundant - NTS Nuclear targeting signal     

Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.     

Root pressure and specific conductivity in temperate lianas: exotic Celastrus orbiculatus (Celastraceae) vs. native Vitis riparia (Vitaceae)

The exotic temperate liana (woody vine) Celastrus orbiculatus has become a weed in Michigan, occurring in many of the same habitats as the native liana Vitis riparia. However, C. orbiculatus frequently develops into extensive monospecific infestations, while V. riparia does not. Freezing-induced embolism may be responsible for limiting liana distribution. Root pressure has been observed in numerous tropical lianas and temperate species of Vitis and has been implicated as vital to the recovery of xylem function in wide vessels following winter freezes. For both of these co-occurring lianas we investigated root pressure and water conductance as possible explanatory factors for their differential spread. According to our hypothesis, C. orbiculatus should have produced greater or more frequent root pressures than V. riparia. However, the reverse proved true, indicating that root pressure is not a prerequisite for weedy proliferation of C. orbiculatus. Additionally, the seasonal patterns of specific conductivity of stem xylem indicate that each species responds differently to environmental constraints. Vitis riparia establishes conductivity early in the growing season, before the leaves emerge, using root pressure to reverse embolism, but loses conductivity with the first freeze in early autumn. Celastrus orbiculatus is slow to establish conductivity, depending on new wood production, but leafs out sooner than V. riparia and maintains green leaves after the first freeze. Vulnerability curves of xylem to cavitation caused by water stress for the two species indicate that they respond similarly to dehydration. These results indicate that root pressures are not responsible for the invasive success of C. orbiculatus and suggest that other factors must be key to its prolific invasion.     

Performance of Hungarian phylloxera strains on Vitis riparia rootstocks

Abstract: Vitis riparia and other rootstocks were used in excised root- and plantlet culture-bioassays to test the performance of Hungarian colonies of phylloxera. Phylloxera colonies from America and Germany were used as comparisons. The Hungarian colonies had a higher level of performance on V. riparia in contrast with the American and German colonies. Plantlet assays found the performance of a Hungarian strain on V. riparia equivalent to that on Vitis vinifera , AXR#1 and 41B root types. In contrast, the American colonies did not survive on V. riparia . Collections of Hungarian gallicole colonies from cultivated and escaped rootstock leaves were assayed using excised roots of the rootstock Teleki 5C. Results indicated that performance of phylloxera collected from feral rootstocks was higher than performance of colonies collected from cultivated rootstock leaves. Lastly, when the attached roots of field-grown V. riparia vines were damaged by girdling, phylloxera performance was much higher than on undamaged roots, suggesting cultural conditions can increase the risk of related vine damage. These laboratory and field results show that V. riparia is capable of supporting phylloxera on its roots. Escaped, feral vines may select for phylloxera with increased virulence on roots. This is of significance in regions such as Hungary where feral rootstock is common.