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Effects of Different Sugar Sources on Protein Kinase Gene Expression in Grape Plantlets

LIANG GuoPing,LI WenFang,CHEN BaiHong,ZUO CunWu,MA LiJuan,HE HongHong,WAN Peng,AN ZeShan,MAO Juan

Scientia Agricultura Sinica 2019, 52 (7): 1119-1135. DOI: 10.3864/j.issn.0578-1752.2019.07.001

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【Objective】 To explore the effects of different exogenous sugars on the growth and development of grape plantlets and the regulation of protein kinase gene transcription, the candidate genes were tapped in the process of protein phosphorylation by using transcription, which made a foundation for the verification of grape protein kinase-related gene function.【Method】 Sucrose (2%), glucose (2%) and fructose (2%) were added to the basic medium, and the free-sugar treatment was as control, which were named as S20, G20, F20 and CK, respectively. After 37 days of culture, the fresh weight of the leaf-stem and root under different treatments was determined. Transcriptome sequencing of each treated foliages was performed by using Illumina HiSeq ^TM 2000, and a series of protein kinases related genes were screened by integrated bioinformatics analysis, including reference genomic alignment, differentially expressed gene (DEGs) screening, COG (Cluster of Orthologous Groups of proteins) annotation, GO (Gene Ontology) annotation, etc., and the expression characteristic of these genes were further analyzed by qRT-PCR. 【Result】 Compared with CK, ‘Red Globe’ grape plantlets under F20, G20 and S20 treatments exhibited significant differences in the fresh weight of leaf-stem, and the highest was obtained by F20 treatment, while the weight of fresh root under G20 was the highest. The SNP statistics found that the Transition was the main type of mutation, the second was Transversion. The highest number of SNPs that occurred in the Intergenic, and the next was the Upstream. Splice_Site_Donor and Synonymous_Stop events occurred with the least number of genes and equal. A total of 2 633 deferentially expressed genes were obtained in the 4 samples. The Venn diagram showed that there were a total of 180 differential genes under the 3 treatments compared with CK, and these genes were clustered into 3 groups. In the first group, 127 genes were only highly expressed under CK. The 19 genes of the second group were only highly expressed under G20, while the expression patterns of the 34 genes in third group were different under three treatments. The common 180 differential genes were annotated with 26 genes in the COG database to 11 functional categories, and these DEGs were mainly enriched in general functional categories. In the annotation of GO, the common genes were annotated in 14, 22 and 13 functional categories of molecular function, biological process and cellular component, respectively. Seven kinds of protein kinases were screened by this sequencing, including Glucokinase (GK), Mitogen-activated protein kinases (MAPKs), Calcineurin protein kinase (CBL), Protein phosphatase 2 (PP2A), Hexokinase (HXK), Histidine protein kinase (HPK) and Tyrosine kinase (TK), and these different protein kinases genes showed their own expression patterns among different treatments. By qRT-PCR analysis, 17 out of 20 screened genes expression were consistent with the transcriptome sequencing results. 【Conclusion】 Compared with glucose and sucrose, fructose was the best sugar during grape culture process. The sequencing results showed that 180 DEGs all responded to three different sugars. In the COG annotation, these genes were mainly enriched in membrane ester transport and metabolism, the synthesis, transport and decomposition of secondary metabolites and carbohydrates. In the GO databases, the most of common DEGs were annotated in the activities of protein kinases and oxidoreductases. Seven protein kinases were identified, which were selectively in responses to different exogenous sugars in quantity, functional, category and metabolic pathways, and had their own choice of expression specificity.

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The Accumulation of Monoterpenes and the Expression of Its Biosynthesis Related Genes in ‘Aishen Meigui’ Grape Berries Cultivated in Different Trellis Systems During Ripening Stage

WANG HuiLing,WANG XiaoYue,YAN AiLing,SUN Lei,ZHANG GuoJun,REN JianCheng,XU HaiYing

Scientia Agricultura Sinica 2019, 52 (7): 1136-1149. DOI: 10.3864/j.issn.0578-1752.2019.07.002

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【Objective】 The effects of two trellis systems on the synthesis of monoterpenes and the expression of its biosynthesis related genes during the maturity of grape berries were studied, in order to reveal the relationship between gene transcription and monoterpenoids accumulation and provide a theoretical basis for trellis selection in production and improvement of aroma quality of grape fruits. 【Method】 The grape berries of Aishen Meigui cultivated in the T and V trellis were used as materials. The berry samples were collected from the beginning of the color change period until the fruit ripened completely. Total soluble solid and titratable acid content in fruits were detected. The changes of monoterpene components and contents in fruits were determined by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (SPEME-GC-MS) in two consecutive years. Additionally, the expression of key enzyme genes in monoterpene biosynthesis pathway including 1-deoxy-D-xylulose 5-phosphate synthase genes (DXS1 and DXS3), 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (DXR), isopentenyl pyrophosphate reductase gene (HDR), linalool synthase gene (Liner syn) and terpineol synthase gene (Terp syn) were analyzed by real-time fluorescence quantitative analysis (qPCR).【Result】During grape berries maturity, the total soluble solids content increased gradually, while titratable acid content decreased gradually. The total soluble solids content of the Aishen Meigui grape cultivated in T trellis was significantly higher than that of grape cultivated in V trellis at ripening stage, and there was no significant difference in titratable acid content. 27 and 28 monoterpene compounds were detected in the grape berries of Aishen Meigui in 2016 and 2017, respectively. The main monoterpene components changed during berry maturity and were different in grape berries cultivated in T and V trellis. As the results shown, linalool, limonene, α-Terpineol, β-cis-Ocimene and geraniol were the main monoterpenes in grape berries cultivated in T trellis, while abundant linalool, α-Terpineol, limonene, nerol oxide and β-cis-Ocimene were detected in the berries cultivated in V trellis, and the content of linalool was the highest among them. At the maturity stage, total monoterpene content of T trellis grape berries reached 108.18 μg?L ^-1, which was 1.9 times higher than that of V trellis berries in 2016. While in 2017, total monoterpene content of T trellis grape berries reached 403.24 μg?L ^-1, which was 1.5 times higher than that of V trellis berries. The content of most monoterpene compounds in T trellis fruits were significantly higher than that in V trellis fruits at maturity. During grape berry development, the changes of monoterpenes accumulation in grape berries cultivated in both two trellis systems exhibited two patterns. Most of the monoterpenes including linalool, geraniol, nerol and α-Terpineol followed the first pattern: the content of monoterpenes reached the peak at maturity. But differences were observed between trellises. In the T trellis fruits, the content of compounds, such as (E,Z)-Allo-Ocimene, β-cis-Ocimene, limonene and α-terpineol, firstly decreased, and then increased sharply at 57 days after flowering, followed by a reduction during the later part of ripening stage (76 days after flowering). However, in V trellis berries, the content of these compounds increased gradually during fruit ripening, peaked at 48 days after flowering, and then decreased to the lowest level. The expression of monoterpene synthesis pathway genes (DXS1, DXS3, DXR, DHR, Liner syn and Terp syn) increased with berry ripening, of them, the changes of DXS3, HDR, Liner syn and Terp syn expression were corresponding well to the total monoterpenes accumulation patterns in grape berries cultivated in distinct trellis systems during ripening. The expression of each gene in T-trellis grape berries was significantly higher than that in V-trellis berries, which was consistent with the accumulation pattern of different monoterpenes. 【Conclusion】More accumulation of monoterpenes were detected in Aishen Meigui grape berries cultivated in T trellis. The synthesis of monoterpenes was highly correlated with the expression level of several key genes in the synthesis pathway.

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Effects of Two Trellis Systems on Viticultural Characteristics and Fruit Quality of Three Table Grape Cultivars

WANG XiaoYue,ZHANG GuoJun,SUN Lei,ZHAO Yin,YAN AiLing,WANG HuiLing,REN JianCheng,XU HaiYing

Scientia Agricultura Sinica 2019, 52 (7): 1150-1163. DOI: 10.3864/j.issn.0578-1752.2019.07.003

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【Objective】The effects of two different trellis systems on viticultural characteristics, fruit quality and disease incidence of three table grape cultivars were studied deeply to provide theoretical basis for the selection of appropriate trellis system for Northern China alluvial plain.【Method】Vitis vinifera L. were used as plant materials, including Ruiducuixia, Ruiduwuheyi and Ruiduhongyu, which were planted with “T” shaped and “V” shaped trellis systems, respectively. Phenological stages, fruiting habits, photosynthetic characteristics, vine structure, labor intensity, fruit quality indexes including cluster weight, berry weight, berry width, berry length, total soluble solids (TSS), titratable acidity (TA), total anthocyanins, flavonoid and proanthocyanidin of three cultivars in both trellis systems were measured.【Result】There was no significant difference in phenological stages or fruiting habits. In most cases, percentage of germination showed higher value with T-trellis, while other fruiting habits showed no significant differences. T-trellis showed considerable advantages in management operations of the vineyard, in terms of the lateral shoot management, topping of primary shoot, fruit branch binding and winter pruning. Both Soil-bury and untying hours in the fall and out-of-soil and tying hours in the spring showed no statistically difference between the two trellis systems. Ruiducuixia showed higher net photosynthetic rate (Pn) with V-trellis in 2017, Ruiduwuheyi showed higher Pn with V-trellis in 2016, in other cases, T-trellis showed significantly higher Pn. Ruiduwuheyi showed higher stomatal conductance with T-trellis for the three years but the other two cultivars showed inconsistent results. Ruiducuixia had a higher transpiration rate with V-trellis; Ruiduxiangyu had the same trend in 2016. In most cases, there was no difference in intercellular CO₂ concentration between the two trellis systems. The shoot width of three cultivars showed trivial difference between the two trellis systems in 2017. However, after the vigor of the vine had been trained to a moderate level, the standard deviation of shoot width of T-trellis had decreased to 2.3, 2.6 and 2.3 for the three cultivars, lower than that of V-trellis (2.7, 2.75 and 3.0), showing higher shoot consistency. Three cultivars showed different results of canopy height and canopy length, while the grapevines grown with V-trellis had larger canopy volume. Leaf area index of Ruiducuixia and Ruiduxiangyu were much higher with V-trellis than T-trellis, while Ruiduwuheyi showed the opposite. The leaf chlorophyll between the two trellis systems showed no statistical difference. As for leaf disease, V-trellis showed much higher disease index and incidence of downy mildew than T-trellis, while no difference was found on powdery mildew. There was no significant difference in cluster weight, single berry weight, vertical diameter, horizontal diameter or total soluble solids. With T-trellis, TSS/TA was in a better range which made the berries taste more balanced and desirable. Ruiducuixia and Ruiduwuheyi showed higher total anthocyanins with T-trellis; all three cultivars had higher level of flavonoid with T-trellis; besides, Ruiduwuheyi showed higher value of proanthocyanidin with T-trellis system, while the other two cultivars showed no significant difference. 【Conclusion】 Based on the three-year study, for soil-bury grape growing areas in Northern China alluvial plain, T-trellis system showed higher photosynthetic use efficiency, less disease incidence, better shoot consistency, higher fruit quality and less labor intensity for vineyard management, manifesting an favorable trellis system to be promoted and practiced in commercial vineyards.

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The Effects of Different Training Systems and Shoot Spacing on the Fruit Quality of ‘Kyoho’ Grape

JI XiaoHao,LIU FengZhi,SHI XiangBin,WANG BaoLiang,LIU PeiPei,WANG HaiBo

Scientia Agricultura Sinica 2019, 52 (7): 1164-1172. DOI: 10.3864/j.issn.0578-1752.2019.07.004

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【Objective】The aim of this study was to ascertain the effects of different training systems and shoot spacing on the fruit quality of Kyoho grape and provide the theoretical guidance for the canopy management of Kyoho grape. 【Method】 The experimental materials were six-year-old vines of Kyoho grape using Beta grape as rootstock. Two different training systems, including horizontal shoot positioning and Y-shaped training system, were applied. The distance between the shoots was 10 cm, 15 cm and 20 cm controlled by the rope, respectively. Fruit quality indexes, such as berry weight, soluble solids, titratable acid, vitamin C, anthocyanin and aroma, were determined at maturity stage. 【Result】 Berry weight, soluble solids, titratable acid and vitamin C contents were significantly correlated with the training systems, but not significantly correlated with the shoot spacing. The anthocyanin content was significantly correlated with training systems and shoot spacing. The total aroma content was significantly correlated with the training systems, but not significantly correlated with the shoot spacing. Among them, the content of aldehydes, alcohols and terpenes was consistent with the total aroma content, while the content of esters was only correlated with the shoot spacing. The Topsis comprehensive evaluation analysis was conducted for 9 indicators including berry weight, soluble solids, titratable acid, vitamin C, anthocyanin, ester, aldehyde, terpene and alcohol contents, and the result showed that horizontal shoot positioning training system combining with 15 cm shoot spacing scored the highest. 【Conclusion】 These data strongly suggested that horizontal shoot positioning training system combining with 15 cm shoot spacing was the most suitable for Kyoho grape production.

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Effects of Local Root Zone Salinity on Grapevine Injury, Na ⁺ Accumulation and Allocation of Carbon and Nitrogen

SUN Hong,JIANG YiWen,YU Xin,XIANG GuangQing,YAO YuXin

Scientia Agricultura Sinica 2019, 52 (7): 1173-1182. DOI: 10.3864/j.issn.0578-1752.2019.07.005

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【Objective】 Salt stress seriously affects yield and fruit quality of fruit crops. Soil salinity is often heterogeneous in saline fields, and within the different root zones of single plant the salinity of the soil solution might vary widely. This paper was aimed to determine the injury extent of grapevine under the non-uniform salt treatment, and to disclose the corresponding mechanism through the determination of Na ⁺ flux and allocation of carbon and nitrogen in grapevine. 【Method】 Saline environment of vine roots was controlled through split-root system and five treatments with different NaCl concentration (mmol·L ^-1) were set: 0/0, 0/50, 50/50, 0/100, and 100/100. Grapevine injury was evaluated via determining content of chlorophyll and malondialdehyde (MDA) as well as the changes of chlorophyll fluorescence parameters. Na ⁺ transport was analyzed by the determination of Na ⁺ content, Na ⁺ flux and electrical conductivity of culture medium around roots. Nitrogen utilization efficiency and distribution rate of carbon and nitrogen were used to detect the changes of carbon and nitrogen in different tissues under different treatments.【Result】The uniform salt treatment of bilateral roots significantly reduced the content of chlorophyll and enhanced the MDA levels in roots and leaves at 15 and 30 days after treatment. In contrast, salt treatment of local roots alleviated the chlorophyll decrease and the MDA accumulation. Additionally, the determination of chlorophyll fluorescence parameters, such as Fv/Fm and ERT, showed the similar results. Therefore, the roots in the non-saline side could alleviate the grapevine injury in comparison to the uniform salt treatments. All of salt treatments increased Na ⁺ content in roots and leaves to varying extents at 15 days after treatment; particularly, the Na ⁺ content of the roots in the non-saline side was also enhanced; additionally, local root zone salinity significantly decreased the Na ⁺ content in leaves, and local treatment of 100 mmol·L ^-1NaCl significantly reduced the Na ⁺ content in saline side roots, compared to the uniform NaCl treatment. The Na ⁺ efflux was observed in non-treated roots, however, the Na ⁺ flux was reversed to influx in the non-saline side roots under non-uniform salt treatment. Additionally, the electrical conductivity of the culture medium around the roots in the non-saline side was significantly enhanced. Therefore, the Na ⁺ absorbed from the salt-treated side could be transported to the non-saline side roots and thereby expelled out of the roots. Nitrogen utilization efficiency was significantly reduced by the uniform salt treatment and the decline was associated with salt treatment concentration. In contrast, the non-uniform salt treatment alleviated the declines in nitrogen utilization efficiency and particularly, which was significantly enhanced in the non-saline side roots under the 0/100 mmol·L ^-1treatment. The uniform salt treatments and particularly 100 mmol·L ^-1NaCl decreased the distribution rate of nitrogen in roots and leaves and increased the values in the two-year-old shoots, favoring the storage of nitrogen. In contrast, the non-saline side roots alleviated the declines of nitrogen distribution rate in roots and leaves. The uniform salt treatment decreased carbon distribution rate in leaves and roots; in contrast, the non-saline side roots not only alleviated the declines of carbon distribution rate in leaves but also elevated carbon distribution rate in roots. It was noteworthy that 50 and 100 mmol·L ^-1 NaCl treatments imparted different effects on carbon distribution in new shoots and two-year-old shoots, i.e., the uniform and non-uniform treatments of 50 mmol·L ^-1 NaCl enhanced carbon distribution in the two-year-old shoots while the treatments of 100 mmol·L ^-1 NaCl produced the contrary results.【Conclusion】Compared with the uniform salt treatment, NaCl treatment of local roots produced the lesser injury for grapevines. Na ⁺ absorbed from the salt-treated side was transported to the non-treated side, expelled them from the roots, and thereby reduced Na ⁺ accumulation in leaves. The non-saline side roots alleviated the declines in carbon and nitrogen distribution rate of leaves and roots.

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Effects of Different Light Intensity and Temperature on PSII Photochemical Activity in ‘Cabernet Sauvignon’ Grape Leaves Under Ozone Stress

GENG QingWei,XING Hao,ZHAI Heng,JIANG EnShun,DU YuanPeng

Scientia Agricultura Sinica 2019, 52 (7): 1183-1191. DOI: 10.3864/j.issn.0578-1752.2019.07.006

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【Objective】Ozone stress, high light and high temperature usually exist simultaneously in nature, but little is known about the effect of the combination of three stress factors on plants. So the combined effects of different temperature (26, 40℃), light (800, 1 600 μmol?m ^-2?s ^-1) and ozone treatments on the operation of photosynthetic apparatus were investigated to reveal the mechanism of inhibition of photosynthesis, which provided a theoretical basis for reducing ozone injury by regulating the light and temperature. 【Method】 Potted Cabernet Sauvignon grapes were used as materials to be treated with different temperatures (26, 40℃), two kinds of light intensities (800, 1 600 μmol?m ^-2?s ^-1) combined with (120±20) nL?L ^-1 ozone. Chlorophyll a fluorescence parameters were analyzed to investigate the effects of different treatments on the PSII function. 【Result】 40℃ and 1 600 μmol·m ^-2·s ^-1 both significantly decreased the maximal photochemical efficiency (F_v/F_m), linear electron transport rate (ETR), the unit area number of reactive centers (RC/CS_m) and photochemical quenching coefficient (q_P) of leaf under ozone stress. At the same time, the decreased excitation energy partition coefficient (α) of PSI and increased excitation energy partition coefficient (β) of PSII induced a serious imbalance of excitation energy distribution between the two photosystems and caused the damage of photosystem II, and the influence of strong light intensity (1 600 μmol·m ^-2·s ^-1) was more significant. The maximal photochemical efficiency (F_v/F_m) under T2 and T3 treatment decreased 10.3% and 38.8% than that under T1 treatment, F_v/F_m under T4 treatment decreased most for 54.8%. PSII quantum efficiency F_v/F_o under T2, T3 and T4 treatment decreased 30.4%, 69.6% and 80.3% than that of T1 treatment, respectively. The number of reaction centers per unit area (RC/CS_m) under T2, T3 and T4 treatment decreased 30.4%, 69.6% and 80.3% than that of T1 treatment, respectively, and the probability of that a trapped exciton the moves an electron further than Q_A by trapped exciton (Ψo) under T2, T3 and T4 treatment decreased 11.2%, 21.6% and 40.8% than that of T1 treatment, respectively. The effective photochemical efficiency F_v'/F_m' under T2, T3 and T4 treatment decreased 7.9%, 22.1% and 42.3% than that under T1 treatment, respectively. Linear electron transport rate (ETR) of T4 decreased most (decreased 62.5% than that under T1 treatment), and ETR under T2 and T3 treatment decreased 17.6% and 37.5% than that of T1 treatment, respectively. The photochemical quenching coefficient (q_P) under T2, T3 and T4 treatment decreased 10.7%, 19.8% and 39.5% than that of T1 treatment, respectively. The photochemical reactions share under T2, T3 and T4 treatment decreased 18.8%, 38.8% and 62.0% than that of T1 treatment, respectively. While the antennal heat dissipation share under T2, T3 and T4 treatment increased 15.9%, 36.2% and 60.5% than that of T1 treatment, respectively, and the excess light energy share of T2, T3 and T4 treatment increased 12.5%, 19.1% and 25.2% than that of T1 treatment, respectively, the excitation energy partition coefficient (α) of PSI under T2, T3 and T4 treatment decreased 6.6%, 12.8% and 25.1% than that under T1 treatment, respectively, and the excitation energy partition coefficient (β) of PSII under T2, T3 and T4 treatment increased 4.5%, 8.7% and 17.1% than that under T1 treatment, respectively, and the change trend of imbalance of partitioning of excitation energy between PSⅠand PSⅡ(β/α-1) was consistent with the change of the excitation energy partition coefficient, β/α-1 under T2, T3 and T4 increased 37.7%, 78.3% and 187.8% than that under T1 treatment.【Conclusion】Temperature and light stress increased the sensitivity of PSII activity to ozone stress, and the influence of light stress was more significant, while high temperature and high light stress caused serious damage to photosystem II.

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Effects of 1-MCP and Na₂S₂O₅ Composite Preservative on Postharvest Physiology and Storage Quality of Red Globe Grapes

CHEN Hao,ZHANG RunGuang,FU LuYing,ZHANG YouLin

Scientia Agricultura Sinica 2019, 52 (7): 1192-1204. DOI: 10.3864/j.issn.0578-1752.2019.07.007

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【Objective】 Red Globe grapes easily appears dehydration, threshing, decay and dry stem during storage. The objective of this study was to provide theoretical basis and technical parameters for prolonging the storage period of Red Globe grapes by studying the effects of 1-MCP and Na₂S₂O₅ on the postharvest physiology and storage quality.【Method】 Shaanxi Heyang Red Globe grape was selected as the test material and sealed with 0.04 mm perforated polyethylene (PE) film bag. The Red Globe grapes were stored under the condition of low temperature (-1±0.5)℃and relative humidity of 90%-95%. The Red Globe grapes were treated with no preservative agent as the control. And then, fruit respiration intensity, weight loss rate, titratable acid content, fruit hardness, malondialdehyde content, soluble solid content, fruit cell membrane relative permeability, protopectin, soluble pectin, reducing sugar content, antioxidant enzyme activities of superoxide dismutase (SOD), polyphenol oxidase (PPO), polygalacturonase (PG), peroxidase (POD) and catalase (CAT) were used as measurement indexes to study the effects of respiratory types of Red Globe grapes and 1-MCP, Na₂S₂O₅ and 1-MCP+Na₂S₂O₅ three preservative treatments on the postharvest physiological and related physicochemical indexes of Red Globe grapes after harvest and during storage. Commercial fruit percentage were calculated during storage, including sensory characters such as color, aroma, taste and shape of Red Globe grapes, which were comprehensively evaluated when stored for 180 days.【Result】 The spike-stalk of Red Globe grapes was respiratory climacteric, and ear and fruit grain were non-respiratory climacteric type. Three kinds of preservatives had different effects on the quality of Red Globe grapes. 1-MCP and Na₂S₂O₅ combined treatment could inhibit activities of PPO, POD, SOD and PG, enhance the activity of CAT, delay the increase of malondialdehyde, soluble pectin content, fruit cell membrane relative permeability and weight loss rate, slow down fruit hardness, protopectin and respiration intensity, prevent Red Globe grapes decay and deterioration effectively, and maintain the contents of soluble solid, titratable acid and reducing sugar. Sensory evaluation at 180 days after storage showed that the indexes under 1-MCP and Na₂S₂O₅ combined treatment of Red Globe grapes were higher than that under other treatments, the commercial fruit rate under this treatment was 90.4%, and the sensory evaluation was 90.2 points.【Conclusion】 Postharvest Red Globe grapes were treated with 3 mg·kg ^-1 FW 1-MCP and 4 g·kg ^-1 FW Na₂S₂O₅, which could slow down the change rate of fruit softening, protopectin and respiratory intensity, maintain high content of soluble solids, titratable acid and reducing sugar, and keep good quality of fruits.

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