Physiological and transcriptomic analyses of roots from Malus sieversii under drought stress

Water deficit is one of the main limiting factors for apple growth and production.Root architecture plays an important role in drought tolerance of plants.However,little is known about the molecular basis of root system in apple trees under drought.In this study,we compared root morphology of two widely used rootstocks of apple(R3 and Malus sieversii)under drought.Our results suggested that M.sieversii is more tolerant to drought than R3,since M.sieversii had a higher ratio of root to shoot as well as root hydraulic conductivity under long-term drought conditions.We then performed whole-genome transcriptomic analysis to figure out the molecular basis of root responses in M.sieversii under drought.It was found that genes involved in transcription regulation,signaling or biosynthesis of hormones,and oxidative stress were differentially expressed under drought.Consistent with the gene expression profile,roots of M.sieversii had higher activities of peroxidase(POD)and superoxide dismutase(SOD)under drought,as well as higher content of abscisic acid(ABA)and lower content of auxin.Taken together,our results revealed the physiological and transcriptomic analyses of M.sieversii roots in response to drought.

Tissue distribution and changes in dopamine during development and stress responses in Malus germplasm

Dopamine is a catecholamine and an anti-oxidant which functions in responses to stress and it interacts with plant hormones to mediate plant development.At present,there are few studies on the functions of dopamine in apple.This study developed a method for dopamine determination which was used to analyze dopamine in Malus germplasm,in order to clarify the tissue distribution,developmental changes,diurnal variations,and stress responses in apple trees.First,the proposed method was verified.The linear range of quantification was robust from 0.1 to 20 ng mL^(–1).The instrumental,inter-day precision,and sample repeatability relative standard deviations were 1.024,5.607,and 7.237%,respectively.The spiked recovery was greater than 100%,indicating the feasibility of the method and its suitability for the rapid analysis of dopamine in Malus.Next,the dopamine content was measured in 322 Malus tissues.The results showed that the dopamine level in Malus was low and the average dopamine content in leaf was higher than in peel and flesh.The dopamine had a skewed distribution that deviated to the right in cultivars and wild accessions.Finally,the tissue specificity,developmental changes,diurnal changes,and responses to stress were analyzed.In cultivar ‘Pinova’(Malus domestica),the dopamine concentration was the highest in leaf buds and lowest in flesh.The dopamine contents in leaf and flesh decreased with the growth and development of cultivar ‘Liangxiang’(Malus domestica).The dopamine content of apple leaves was higher after either drought or salinity stress as compared to the control.In this study,a dopamine detection method for apple was established based on HPLC-MS and shown to be a robust approach.This study provides a framework for future research on elucidating the tissue distribution,developmental changes,diurnal variation,and stress responses of dopamine in apple trees.

Genome-wide identification of the radiation sensitivity protein-23(RAD23)family members in apple(Malus×domestica Borkh.)and expression analysis of their stress responsiveness

Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple （Malusxdomestica Borkh.）. We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses. Various members showed a ubiqui- tous pattern of expression in all selected apple parts. Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid （ABA） treatment and water deficiency, suggesting their possible roles in plant stress responses. These results provide essential information about RAD23 genes in apple and will contribute to further functional studies.

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium(MS basal salts, 30 g Lsucrose, 8 g Lagar, 5 mg L6-benzylaminopurine(6-BA), 2 mg Lthidiazuron(TDZ), and 1 mg L1-naphthlcetic acid(NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime(150 mg L) and kanamycin(15 mg L). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

农艺与种业专业学位研究生实践教学模式探讨——以西北农林科技大学园艺学院为例

实践教学是全日制专业学位硕士研究生教学培养的重要环节,实践教学质量水平对实现培养目标、提升教育质量和就业质量及提高实践基地服务研究生教育能力等具有重要意义。为了满足园艺产业发展对高层次技术型专业人才的需求,西北农林科技大学园艺学院通过导师队伍、实践基地服务研究生教育能力管理,以及研究生实践教学内容及考评体系等多方面的探索与建设,形成了符合产业发展且具有西北园艺产业特色的农艺与种业专业学位研究生实践教学管理模式。

反季节杧果花器官结构对胚胎败育的影响

[目的]研究反季节杧果花器官结构对胚胎败育的影响。[方法]观察反季节杧果生产中花器官和胚胎的结构,探索反季节杧果胚胎败育过程中"金煌"品种的细胞学结构变化,研究胚胎败育的细胞学机理。[结果]"金煌"雄蕊退化,花粉囊间发育差异十分显著,花粉囊小的花粉数量少,而花粉囊大的花粉数量多且发育至成熟,仅有少部分花粉囊的花粉退化,花粉生活力为76.9%。雌器官出现子房萎缩变形、胚珠横生现象;胚珠周围的输导组织断裂现象普遍;在胚囊8核期间,在同一或不同胚珠中出现极核细胞消失或者退化,反足细胞个别退化、助细胞个别或者全部退化的现象。[结论]以上因素都会影响胚珠受精,引起授粉受精不良。

苹果酸度相关基因MdPH1的克隆、表达及亚细胞定位分析

以苹果属(Malus)植物沧江海棠(M.ombrophila Hand.-Mazz)的果实为材料,对其发育过程中苹果酸的含量进行测定,并结合转录组测序的方法筛选控制果实酸度的候选基因。结果显示:MdPH1候选基因的编码区包含2829 bp,编码942个氨基酸;基因组序列全长为4269 bp,包含8个外显子和7个内含子。对10份苹果种质资源中PH1基因序列的分析结果表明,该基因序列中存在22个单核苷酸多态性(SNP),其中13个位于内含子区,9个位于外显子区;位于最后一个外显子上SNP(G/A)的变异导致了编码氨基酸从缬氨酸变为异亮氨酸。MdPH1蛋白包含8个跨膜结构域,其中蛋白N端包含3个跨膜结构域,C端包含5个跨膜结构域。系统进化分析结果显示,苹果中的PH家族成员与梨(Pyrus communis L.)中的PH家族成员聚集成一簇。组织特异性表达结果发现,MdPH1基因在苹果果实中的表达量最高,其次是叶、花和根,茎中表达量最低。亚细胞定位分析表明MdPH1蛋白定位于液泡膜上。

外源施加腐殖酸液态膜对苹果光合特性和果实品质的影响

以苹果(Malus domestica Borkh.)品种‘玉华早富’(‘YH’)和‘秦脆’(‘QC’)为材料,设置每15 d、30 d喷施1次腐殖酸液态膜以及非套袋3个处理,通过测定不同处理下苹果叶片的光合特性、果实色差与质地特性、可溶性固形物、可滴定酸及果实中糖酸含量相关基因的相对表达量,分析比较外源施加腐殖酸液态膜对苹果叶片光合指标和果实品质指标的影响。结果显示,与对照相比,喷施腐殖酸液态膜处理可提高苹果叶片的光合特性,改善苹果果实的着色,使果实中可溶性固形物含量增加、可滴定酸含量降低、提高固酸比,且果实中糖含量相关基因MdTST1、MdTST2、MdERDL6-1相对表达量升高,酸含量相关基因MdMa1、MdMa10、MdMDH5、MdWRKY126相对表达量降低。其中每15 d喷施1次腐殖酸液态膜处理作用最显著。研究结果表明,喷施腐殖酸液态膜处理能提高苹果的光合特性,促进树体生长发育,改善果实品质,提高果实风味。

Structure and expression analysis of the sucrose synthase gene family in apple

Sucrose synthases(SUS) are a family of enzymes that play pivotal roles in carbon partitioning, sink strength and plant development. A total of 11 SUS genes have been identified in the genome of Malus domestica(Md SUSs), and phylogenetic analysis revealed that the Md SUS genes were divided into three groups, named as SUS I, SUS II and SUS III, respectively. The SUS I and SUS III groups included four homologs each, whereas the SUS II group contained three homologs. SUS genes in the same group showed similar structural characteristics, such as exon number, size and length distribution. After assessing four different tissues, Md SUS1 s and Md SUS2.1 showed the highest expression in fruit, whereas Md SUS2.2/2.3 and Md SUS3 s exhibit the highest expression in shoot tips. Most Md SUSs showed decreased expression during fruit development, similar to SUS enzyme activity, but both Md SUS2.1 and Md SUS1.4 displayed opposite expression profiles. These results suggest that different Md SUS genes might play distinct roles in the sink-source sugar cycle and sugar utilization in apple sink tissues.

Comprehensive evaluation of tolerance to alkali stress by 17 genotypes of apple rootstocks

Alkaline soils have a great inlfuence on apple production in Northern China. Therefore, comprehensive evaluations of toler-ance to such stress are important when selecting the most suitable apple rootstocks. We used hydroponics culturing to test 17 genotypes of apple rootstocks after treatment with 1：1Na2CO3and NaHCO3. When compared with the normaly grown controls, stressed plants produced fewer new leaves, and had shorter roots and shoots and lower fresh and dry weights after 15 d of exposure to alkaline conditions. Their root/shoot ratios were also reduced, indicating that the roots had been severely damaged. For al stressed rootstocks, electrolyte leakage （EL） and the concentration of malondialdehyde （MDA） increased while levels of chlorophyl decreased. Changes in root activity （up or down）, as wel as the activities of peroxidase （POD）, superoxide dismutase （SOD）, and catalase （CAT） were rootstock-dependent, possibly relfecting their differences in alkali tolerance. Using alkali injury index （AI）, adversity resistance coefifcients （ARC）,cluster analysis, and evaluation of their physiological responses, we classiifed these 17 genotypes into three groups： （1） high tolerance： Hubeihaitang, Wushanbianyehaitang, Laoshanhaitang Ls2, Xiaojinbianyehaitang, and Fupingqiuzi; （2） moderate tolerance： Pingyitiancha, Laoshanhaitang Ls3, Hubeihaitang A1, Deqinhaitang, Balenghaitang, Maoshandingzi, Shandingzi, and Xinjiangyepingguo; or （3） low tolerance： Pingdinghaitang, Hongsanyehaitang, Xiaojinhaitang, and Sanyehaitang. These results wil signiifcantly contribute to the selection of the most suitable materials for rootstocks with desired levels of tolerance to alkali stress.

Transcriptome analysis reveals the effects of alkali stress on root system architecture and endogenous hormones in apple rootstocks

Soil alkalinity is a major factor that restricts the growth of apple roots.To analyze the response of apple roots to alkali stress, the root structure and endogenous hormones of two apple rootstocks, Malus prunifolia (alkali-tolerant) and Malus hupehensis (alkali-sensitive), were compared. To understand alkali tolerance of M. prunifolia at the molecular level, transcriptome analysis was performed. When plants were cultured in alkaline conditions for 15 d, the root growth of M. hupehensis with weak alkali tolerance decreased significantly. Analysis of endogenous hormone levels showed that the concentrations of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in M. hupehensis under alkali stress were lower than those in the control. However, the trend for IAA and ZR in M. prunifolia was the opposite. The concentration of abscisic acid (ABA) in the roots of the two apple rootstocks under alkali stress increased, but the concentration of ABA in the roots of M. prunifolia was higher than that in M. hupehensis. The expression of IAA-related genes ARF5, GH3.6, SAUR36, and SAUR32 and the Cytokinin (CTK)-related gene IPT5 in M. prunifolia was higher than those in the control, but the expression of these genes in M. hupehensis was lower than those in the control. The expression of ABA-related genes CIPK1 and AHK1 increased in the two apple rootstocks under alkali stress, but the expression of CIPK1 and AHK1 in M. prunifolia was higher than in M. hupehensis. These results demonstrated that under alkali stress, the increase of IAA, ZR, and ABA in roots and the increase of the expression of related genes promoted the growth of roots and improved the alkali tolerance of apple rootstocks.

Effects of progressive drought on photosynthesis and partitioning of absorbed light in apple trees

To understand how drought stress affects CO2 assimilation and energy partitioning in apple（Malus domestica Borkh.）, we investigated photosynthesis and photo-protective mechanisms when irrigation was withheld from potted Fuji trees. As the drought progressing, soil relative water content（SRWC） decreased from 87 to 24% in 15 d; this combined the decreasing in leaf relative water content（LRWC）, net photosynthesis rate（P n） and stomatal conductance（G s）. However, the concentrations of chlorophylls（Chl） remained unchanged while P n values were declining. Photochemistry reactions were slightly down-regulated only under severe drought. Rubisco activity was significantly decreased as drought conditions became more severe. The actual efficiency of photosystem II（ΦPSII） was diminished as drought became more intense. Consequently, xanthophyll-regulated dissipation of thermal energy was greatly enhanced. Simultaneously, the ratio of ΦPSII to the quantum yield of carbon metabolism, which is measured under non-photorespiratory conditions, increased in parallel with drought severity. Our results indicate that, under progressive drought stress, the reduction in photosynthesis in apple leaves can be attributed primarily to stomatal limitations and the inhibited capacity for CO2 fixation. Xanthophyll cycle-dependent thermal dissipation and the Mehler reaction are the most important pathways for dispersing excess energy from apple leaves during periods of drought stress.

Genome-wide identification and function analysis of the sucrose phosphate synthase MdSPS gene family in apple

Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

Differences in the Efficiency of Potassium(K) Uptake and Use in Five Apple Rootstock Genotypes

Plants that grow well while accumulating and transporting less potassium（K） perform better than more-sensitive plants when under deficiency conditions, which makes low-K-input and environmentally friendly agriculture possible. We conducted hydroponics and sand culture experiments to evaluate the efficiency of various apple（Malus domestica Borkh） rootstocks in their K uptake and utilization. Five genotypes were selected which are widely used in China- M. hupehensis Rehd, M. prunifolia Borkh, M. robusta Rehd, M. sieversii Roem, and M. rockii Rehd. Plant heights, root and shoot dry weights, and K concentrations were recorded. These genotypes differed markedly in dry weights, absolute and relative K concentrations, absolute and relative K accumulations, and their K efficiency ratio under deficient K conditions. The last parameter, expressed as relative shoot dry weight, was strongly and positively correlated with the other four parameters in each genotype. Therefore, we suggest that this parameter could serve as an index when selecting K-efficient genotypes. In this study, we have determined that M. sieversiiand M. rockii are K-inefficient genotypes; M. prunifolia is K-efficient genotype; M. hupehensis and M. robusta have moderate levels of potassium efficiency.

Effects of light intensity on photosynthesis and photoprotective mechanisms in apple under progressive drought

The effects of light intensity on photosynthesis and photoprotective mechanisms under progressive drought were studied on apple trees (Malus domestica Borkh.) Fuji. The potted trees were exposed to drought stress for 12 days and different light conditions (100, 60 and 25%sunlight). During the progressive drought, the relative water content (RWC) in leaf declined and was faster in ful light than in 60 and 25%sunlight. However, the decrease in the net photosynthetic rate (Pn), stomatal conductance (Gs) and Rubisco activity were slower under 100%sunlight condition than other light conditions. After the 6 days of drought, the maximum PSII quantum yield (Fv/Fm), the capacity of electrons move beyond QA?(1–VJ) and electron move from intersystem to PSI acceptor side (1–VI)/(1–VJ) decreased, with greater decline extent in brighter light. While RWCs were>75%, the variations in different light intensities of Gs and Rubisco activity at identical RWC, suggested the direct effects of light. While the little difference in the state of photosynthetic electron transport chain among tested light intensities indicates the results of faster water loss rate of light. Our results also demonstrated that the enhancement the de-epoxidations of xanthophyl cycle, activities of ascorbate peroxidase (APX) and catalase (CAT) were directly regulated by light intensity. While the higher photorespiration rate (Pr) under stronger light condition was mainly caused by faster water loss rate of light.

Physiological evaluation of nitrogen use efficiency of different apple cultivars under various nitrogen and water supply conditions

Nitrogen(N) deficiency is a common problem for apple(Malus×domestica) production in arid regions of China.However,N utilization efficiency(NUE) of different apple cultivars grown under low N conditions in arid regions has not been evaluated.In this study,NUE was assessed for one-year-old seedlings of six apple cultivars,Golden Delicious,Qinguan,Jonagold,Honeycrisp,Fuji and Pink Lady,grafted onto Malus hupehensis Rehd.rootstocks.Four treatments were used,including control water with control N(CWCN),limited water with control N(LWCN),control water with low N(CWLN) and limited water with low N(LWLN).Our results showed that growth indices such as biomass,plant height and stem diameter,and photosynthetic rate of all cultivars decreased in the order CWCN>CWLN>LWCN>LWLN.When subjected to LWLN treatment,Qinguan showed better growth and photosynthetic characters than other tested cultivars.Additionally,Qinguan and Pink Lady had higher NUE,while Honeycrisp and Jonagold had lower NUE,based on the determination of biomass,photosynthetic parameters,chlorophyll content,the maximal photochemical efficiency of PSII(Fv/Fm),15 N and N contents.

Overexpression of MdMIPS1 enhances drought tolerance and water-use efficiency in apple

Myo-inositol and its derivatives play important roles in the tolerance of higher plants to abiotic stresses, and myo-inositol-1-phosphate synthase(MIPS) is the rate-limiting enzyme in myo-inositol biosynthesis. In this study, we found that increased myo-inositol biosynthesis enhanced drought tolerance in MdMIPS1-overexpressing apple lines under shortterm progressive drought stress. The effect of myo-inositol appeared to be mediated by the increased accumulation of osmoprotectants such as glucose, sucrose, and proline, and by the increased activities of antioxidant enzymes that eliminate reactive oxygen species. Moreover, enhanced water-use efficiency(WUE) was observed in MdMIPS1-overexpressing apple lines under long-term moderate water deficit conditions that mimicked the water availability in the soil of the Loess Plateau. Enhanced WUE may have been associated with the synergistic regulation of osmotic balance and stomatal aperture mediated by increased myo-inositol biosynthesis. Taken together, our findings shed light on the positive effects of MdMIPS1-mediated myo-inositol biosynthesis on drought tolerance and WUE in apple.

Response of carbohydrate metabolism-mediated sink strength to auxin in shoot tips of apple plants

Auxin(indole-3-acetic acid, IAA) has a considerable impact on the regulation of plant carbohydrate levels and growth, but the mechanism by which it regulates sugar levels in plants has received little attention. In this study, we found that exogenous IAA altered fructose(Fru), glucose(Glc), and sucrose(Suc) concentrations in shoot tips mainly by regulating MdSUSY1, MdFRK2, MdHxK1 and MdSDH2 transcript levels. Additionally, we used 5-year-old ’Royal Gala’ apple trees to further verify that these genes play primary roles in regulating sink strength. The results showed that MdSUSY1, MdFRK2, MdHxK1/3 and MdSDH2 might be major contributors to sink strength regulation. Taken together, these results provide new insight into the regulation of the carbohydrate metabolism mechanism, which will be helpful for regulating sink strength and yield.

Factors affecting hydraulic conductivity and methods to measure in plants

The plant hydraulic network faces several challenges under drought stress. Hydraulic conductivity is one of the major indicators of the hydraulic network’s response to drought stress. Here, we review our current understanding of the factors directly affecting hydraulic conductivity and the methods used to measure it.