Comparative Metabolic Profiling of Two Contrasting Date Palm Genotypes Under Salinity

Since specific metabolites may be associated with salinity tolerance, this study aimed to decipher the salinity tolerance mechanism in date palm based on the information encoded by the metabolomic profiles of the salt-tolerant “Umsila” and salt-susceptible “Zabad” cultivars when grown under salinity conditions. Changes in the metabolomic profiles of the leaf and root tissues were determined using hydrophilic interaction liquid chromatography (HILIC) and reverse-phase liquid chromatography (RPLC) mass spectrometry. The global untargeted metabolomic analysis showed the presence of 4878 metabolites accumulated in leaf and root tissues of the date palm seedlings. Principal component analysis (PCA) revealed the presence of unique groups of metabolites for each treatment and tissue type. Pathway analysis showed the involvement of some of these metabolites in the biosynthesis of several types of membranous lipids and glycolipids molecules such as 18:0-lyso-phosphatidylethanolamine (lysoPE), and also the synthesis of cell-wall components such as 16-hydroxy hexadecanoic acid, which is an intermediate metabolite in cutin, suberin, and wax biosynthesis. Moreover, antioxidant flavonoids such as (+)-catechin and epicatechin, vitamins such as B₉, phytohormone-associated compounds such as dihydrozeatin-9-N-glucoside-O-glucoside, and osmolytes such as the sulfonic amino acid taurine were all significantly (p?≤?0.05, FWER?≤?0.05) altered in response to salinity in both cultivars. These results indicate that salinity tolerance in date palm involved a multi-dimensional mechanism, which includes the modification of the cell-wall and cellular membranes, the production of oxygen species scavengers, the adjustment of cellular osmotic pressure, and probably the alteration of the hormonal balance. The intracultivar metabolomic profile comparison strategy performed in this study represents an approach that may pave the road toward the identification of salinity tolerance mechanisms in date palm based on the final protein products.

     

Differences in Grain Ultrastructure,Phytochemical and Proteomic Profiles between the Two Contrasting Grain Cd-Accumulation Barley Genotypes

To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars.     

Comparative Metabolite Profiling of Two Rice Genotypes with Contrasting Salt Stress Tolerance at the Seedling Stage

Background

Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level.

Methods

Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated.

Results

We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na⁺ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment.

Conclusions

Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.     

Grain Yield in Two Contrasting Varieties of Spring Wheat

To investigate factors determining grain yield in spring wheattwo contrasting varieties of high (Jufy I) and low (Peko) yieldingcapacity were grown in field and glasshouse experiments. In a field trial Jufy I yielded approximately 14 per cent moregrain than Peko on both control and nitrogen-treated plots.In another field trial employing small plots, although for bothvarieties doubling the seed-rate did not affect grain yield,the two varieties differed in their response to the increasein density. In Peko the yield level remained unaffected becausedoubling the seed-rate halved the number of fertile tillersper plant, whereas for Jufy I in addition to a shift in tillernumber per plant there were changes in the number of grainsper ear. In both varieties the weight per seed remained constant. When the two varieties were grown alone or as a 1: 1 mixtureat the same seed-rate the grain yields were similar. In themixture, Peko, compared with Jufy I, produced a greater numberof fertile tillers and consequently a greater contribution tothe total grain yield. In glasshouse experiments the grain yield per tiller and theorder of leaf area duration per tiller for the period from earemergence to grain ripening were both higher for Jufy I. Onthe other hand, there was no varietal difference for the netassimilation rate estimated during the vegetative phase, butexcluding the roots. Additional nitrogen primarily stimulated tiller production inboth varieties. Shading either the upper or lower parts of the plant demonstratedthat the ear and flag leaf and its sheath are important sourcesof assimilates for grain formation, whereas the lower partsare relatively unimportant in this respect. There were indicationsthat the response between varieties differed.     

Alleviation of Negative Effects of Water Stress in Two Contrasting Wheat Genotypes by Calcium and Abscisic Acid

The individual and interactive role of calcium and abscisic acid (ABA) in amelioration of water stress simulated by polyethylene glycol (PEG) 6000 was investigated in two contrasting wheat genotypes. PEG solution (osmotic potential –1.5 MPa) was applied to 10-d-old seedlings growing under controlled conditions and changes in photosynthetic rate, activities of ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase, water potential and stomatal conductance were observed in the presence of 0.1 mM ABA, 5 mM calcium chloride, 1 mM verapamil (Ca²⁺ channel blocker), and 1 mM fluridone (inhibitor of ABA biosynthesis). ABA and calcium chloride ameliorated the effects of water stress and the combination of the two was more effective. The two genotypes varied for their sensitivity to ABA and Ca²⁺ under stress. As was evident from application of their inhibitors, ABA caused more alleviation in C 306 (drought tolerant) while HD 2380 (drought susceptible) was more sensitive to Ca²⁺.     

Differential Drought Responses in F-box Gene Expression and Grain Yield Between Two Rice Groups with Contrasting Drought Tolerance

Understanding the molecular mechanisms regulating rice’s response to drought stress is important for cereal crop development. We investigated the physiological and gene expression responses of three drought-tolerant and two drought-sensitive unrelated rice cultivars at the reproductive stage. Leaf water loss and leaf rolling tests distinguished these two groups from each other. Both 7 and 14 days of drought stress affected most of the tested agronomic traits including grain yield, and the effects were stronger in the drought-sensitive cultivars. Fourteen days of drought stress severely reduced grain yield in the sensitive cultivars. Expression levels of 50 genes previously published were examined in panicles. Of these, 25 genes were expressed in panicles and could be classified into 6 groups. LOC_Os02g51350, a Kelch domain-containing F-box gene, was selected for further studies including expression analysis in panicles during the 14-day drought stress, domain analysis, and analysis of cis-acting elements in the promoters. All three drought-resistant cultivars possessed the F-box domain, which was absent in the two drought-sensitive cultivars. In addition, drought-related ABRE and DRE/CRT cis-acting elements were more abundant in Os02g51350 promoters of cultivars with good grain yield under drought stress than in promoters of cultivars with severe yield reduction. Our results suggest that the F-box version of Os02g51350 is important for maintenance of grain yield under drought.     

Transcriptional Profiling of Gene Expression Changes in a PACE-Transfected CHO DUKX Cell Line Secreting High Levels of rhBMP-2

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.     

Comparison of the Drought Stress Responses of Tolerant and Sensitive Wheat Cultivars During Grain Filling: Changes in Flag Leaf Photosynthetic Activity,ABA Levels,and Grain Yield

Water status parameters, flag leaf photosynthetic activity, abscisic acid (ABA) levels, grain yield, and storage protein contents were investigated in two drought-tolerant (Triticum aestivum L. cv. MV Emese and cv. Plainsman V) and two drought-sensitive (cvs. GK élet and Cappelle Desprez) wheat genotypes subjected to soil water deficit during grain filling to characterize physiological traits related to yield. The leaf water potential decreased earlier and at a higher rate in the sensitive than in the tolerant cultivars. The net CO₂ assimilation rate (P _N) in flag leaves during water deficit did not display a strict correlation with the drought sensitivity of the genotypes. The photosynthetic activity terminated earliest in the tolerant cv. Emese, and the senescence of flag leaves lasted 7 days longer in the sensitive Cappelle Desprez. Soil drought did not induce characteristic differences between sensitive and tolerant cultivars in chlorophyll a fluorescence parameters of flag leaves during post-anthesis. Changes in the effective quantum yield of PSII (Φ_PSII) and the photochemical quenching (qP) depended on the genotypes and not on the sensitivity of cultivars. In contrast, the levels of ABA in the kernels displayed typical fluctuations in the tolerant and in the sensitive cultivars. Tolerant genotypes exhibited an early maximum in the grain ABA content during drought and the sensitive cultivars maintained high ABA levels in the later stages of grain filling. In contrast with other genotypes, the grain number per ear did not decrease in Plainsman and the gliadin/glutenin ratio was higher than in the control in Emese during drought stress. A possible causal relationship between high ABA levels in the kernels during late stages of grain filling and a decreased grain yield was found in the sensitive cultivars during drought stress.     

Effect of Source-to-sink Ratio on Partitioning of Dry Matter and14C-photoassimilates in Wheat during Grain Filling

Increasingly, wheat (Triticum aestivumL.) is being grown intropical environments, but there is inadequate information aboutthe physiological processes limiting yield. In this investigation,the source:sink ratio was manipulated to examine the performanceof source-sink interactions after anthesis and the factor(s)limiting grain filling in tropical conditions. Plants of threewheat cultivars, Cuba C-204, Candeias and IAC-60, were artificiallymodified to give different source:sink ratios. The treatmentswere: I, Control; II, all spikelets on one side of the spikeremoved; III, all spikelets removed except the four centralspikelets of the spike; and IV, flag leaf blade removed. Thedistribution of dry matter between kernels and stem internodeswas analysed at harvest in all three cultivars. Partitioningof¹⁴C-photoassimilates was measured on three occasions afteranthesis in the cultivar Cuba C-204. Modifications of source:sinkratio led to different patterns of allocation of dry matterbetween cultivars and sowing dates. The reduction in sink sizein treatment II produced no significant change in the mass pergrain in the January sowing, but this was enhanced in two cultivarsin the November sowing. In treatment III, both mass per grainand translocation of¹⁴C-photoassimilates declined, apparentlydue to feedback inhibition of photosynthesis. The participationof stem reserves in grain filling and the existence of genotypicdifferences in response to availability of photoassimilateswere corroborated. The pattern of partitioning of dry matterobserved in plants in this investigation suggests a source limitation,particularly during the November sowing. This pattern differedmarkedly from that in other studies, most of which have beenmade in temperate areas.Copyright 1999 Annals of Botany Company Photoassimilates, sink, source, partitioning, grain filling, wheat.     

Accumulation and Conversion of Sugars by Developing Wheat Grains : VI. Gradients Along the Transport Pathway from the Peduncle to the Endosperm Cavity during Grain Filling

Gradients along the transport pathway from the peduncle to the endosperm cavity were examined during grain filling in wheat. Sieve tube exudate was collected from severed aphid stylets established on the peduncle and rachis and on the vascular bundles in the creases of grains. Phloem exudate could also be collected from broken grain pedicels, and by puncturing the vascular bundle in the grain crease with a needle. Stylets on excised grains persisted exuding, indicating that grain sieve tubes are capable of loading solutes. There was little, if any, discernible gradient in osmolality or solute composition (sucrose, total amino acids) of sieve tube contents along the phloem pathway from the peduncle to the rachis or along the rachis itself. Neither was a gradient detected in osmolality along the sieve tube pathway from the rachis through the rachilla and grain stalk to the crease. Demonstrable solute gradients occurred only across those tissues of the grain crease between the crease sieve tubes and the endosperm cavity, a distance of just 1 millimeter. However, while the sucrose concentration in the sieve tubes was almost tenfold that in the endosperm cavity sap, total amino acids were only threefold higher, and the potassium concentrations of the two were equal. Our observations strongly implicate the movement of assimilates from the sieve tubes and across the crease tissues as important control points in grain filling.     

大小粒型藜麦籽粒表型、灌浆特性及淀粉合成酶活性的差异分析

【目的】籽粒大小是影响藜麦产量、商品性和加工特性的重要因素,考察灌浆期大小粒型藜麦籽粒表型、灌浆特性和淀粉合成酶活性的差异,为大粒型藜麦品种的选育提供理论指导。【方法】选择千粒重大于5.0 g和小于3.0 g的藜麦材料各两份,在青海省农林科学院种质资源创新试验基地进行田间试验,比较自灌浆期始7 d、14 d、21 d和28 d籽粒表型、灌浆特性和淀粉合成酶活性等在大小粒型藜麦间的差异。【结果】（1）大小粒型藜麦籽粒面积、周长、直径、粒长、粒宽表型性状随着生育时期均极显著增大,且粒型间存在显著差异,并以籽粒面积和周长差异最大,大粒型藜麦显著高于小粒型藜麦9.12%~11.54%和21.49~23.92%。（2）灌浆期间大粒型藜麦百粒干重始终显著高于同期小粒型藜麦,平均增幅在21.23%～31.04%;大小粒型藜麦灌浆速率随生育期均先上升后下降,均符合“慢-快-慢”的变化规律,但达到峰值时间和峰高明显不同,大粒型峰值出现早而高,小粒型则低而迟。（3）淀粉分支酶（SBE）、蔗糖合成酶（SS）、可溶性淀粉合成酶（SSS）和ADPG焦磷酸化酶（AGP）在大小粒型藜麦籽粒灌浆期呈现不同的变化趋势,SBE和SS活性表现为小粒型藜麦强于大粒型藜麦,而SSS和AGP活性则表现为大粒型藜麦强于小粒型藜麦。【结论】藜麦籽粒灌浆期间4种淀粉合成酶活性的差异,致使淀粉合成积累量和灌浆速率峰值的不同,进而形成籽粒表型性状的差异,而SSS和AGPase是影响藜麦籽粒大小形成的关键酶。     

A Microarray-Based Comparative Analysis of Gene Expression Profiles During Grain Development in Transgenic and Wild Type Wheat

Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.     

The Fate of the Dry Matter, Carbohydrates and 14C Lost from the Leaves and Stems of Wheat during Grain Filling

In a field study with six winter wheat genotypes losses of drymatter from the stems between 30 June and maturity averaged172 g m^–2 (range 82–236), there being significantdifferences in loss between genotypes. Respiration from thestems during the same period was estimated to amount to 106g m^–2 (range 104–225). The amount of dry mattermobilized from the stems, calculated by difference, was estimatedas 66 g m^–2. The loss of ethanol- and water-soluble carbohydratefrom the stems (170 g m^–2; range 124–215) was verysimilar to the dry weight loss. Carbon-14 labelling was used to trace the time course and theamount of the movement of assimilates from the vegetative organsto the grain. Only 14•3 per cent (range 10•3–21•0)of the products of photosynthesis over the period 21 May-20June were relocated to the grains. This relocation amountedto an average of 7 per cent (range 5•7–11•4)of the final grain weight. It was estimated that during the18 days following anthesis on 20 June photosynthesis contributed48 per cent (range 39–55) of the final grain dry weight.Of this, about half was translocated to the grain within 10days of initial assimilation. The remainder appeared to be storedtemporarily in the stems and leaves and translocated to thegrains during the period 17–29 July. In general, relocationof dry matter from the vegetative organs to the grains, assessedby carbon-14 labelling, was greatest in those genotypes (Hobbitand Sportsman) which lost most dry weight from the stems andleaves.