Peanut violaxanthin de-epoxidase alleviates the sensitivity of PSII photoinhibition to heat and high irradiance stress in transgenic tobacco.

KEY MESSAGE: This is the first study on peanut VDE, which led to multiple biochemical and physiological changes to heat and HI stress by improving de-epoxidation of the xanthophylls cycle. A peanut (Arachis hypogaea L.) violaxanthin de-epoxidase gene (AhVDE) was isolated by RT-PCR and RACE methods. The deduced amino acid sequence of AhVDE showed high identities with violaxanthin de-epoxidase of other plant species. The expression of AhVDE was obviously upregulated by 4, 40 °C and high light, NaCl, and abscisic acid. Sense and RNAi transgenic tobaccos were further used to investigate the physiological effects and functional mechanism of AhVDE. Compared with WT, the content of Z, the ratio of (A + Z)/(V + A + Z) and the non-photochemical quenching were higher in sense plants, and lower in the RNAi lines under heat and high irradiance (HI) stress, respectively. Additionally, photoinhibition of photosystem II (PSII) reflected by the maximal photochemical efficiency in WT lines was more severe, and in the RNAi lines was the most severe compared with that in the sense lines. Meanwhile, overexpressing AhVDE also led to multiple biochemical and physiological changes under heat and HI stress. Higher activities of superoxide dismutase and ascorbate peroxidase, lower content of reactive oxygen species and slighter membrane damage were observed in sense lines after heat and HI stress. These results suggested that, peanut VDE can alleviate PSII photoinhibition to heat and HI stress by improving the xanthophyll cycle-dependent energy dissipation.

Calcium contributes to photoprotection and repair of photosystem II in peanut leaves during heat and high irradiance.

In this study, we investigated the effects of exogenous calcium nitrate on photoinhibition and thylakoid protein level in peanut plants under heat (40°C) and high irradiance (HI) (1,200 µmol/m(2) per s) stress. Compared with control seedlings (cultivated in 0 mmol/L Ca(NO3 )2 medium), the maximal photochemical efficiency of photosystem II (PSII) in Ca(2+) -treated plants showed a slight decrease after 5 h stress, accompanied by lower degree of PSII closure (1-qP), higher non-photochemical quenching, and lower level of membrane damage. Ca(2+) inhibitors were used to analyze the varieties of antioxidant enzymes activity and PSII proteins. These results indicated that Ca(2+) could protect the subunits of PSII reaction centers from photoinhibition by reducing the generation of reactive oxygen species. In the presence of both ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid and ascorbic acid (AsA), the net degradation of the damaged D1 protein was faster than that only treated with AsA. Our previous study showed that either the transcriptional or the translational level of calmodulin was obviously higher in Ca(2+) -treated plants. These results suggested that, under heat and HI stress, the Ca(2+) signal transduction pathway can alleviate the photoinhibition through regulating the protein repair process besides an enhanced capacity for scavenging reactive oxygen species.

[Soil Bacterial Community Structure and Function Prediction of Millet/Peanut Intercropping Farmland in the Lower Yellow River].

The objective of this study was to explore the microecological variability in farmland soil fertility in response to millet-peanut intercropping patterns by clarifying the effects of millet-peanut 4:4 intercropping on soil bacterial community structure and its diversity, as well as to provide a reference basis for promoting ecological restoration and arable land quality improvement in the lower Yellow River farmland. The Illumina MiSeq high-throughput sequencing technology and QIIME 2 platform were used to analyze the differences in bacterial community composition and their influencing factors in five soils[sole millet (SM), sole peanut (SP), intercropping millet (IM), intercropping peanut (IP), and millet-peanut intercropping (MP)] and to predict their ecological functions. The results showed that the α-diversity of intercropping soil bacterial communities differed from that of monocropping, though not significantly, whereas the ß-diversity was significantly different (P<0.05). A total of 7081 ASVs were obtained from all soil samples, classified into 34 phyla, 109 orders, 256 class, 396 families, 710 genera, and 1409 species, of which 727 ASVs were shared, accounting for 24.5% to 27.8% in five soil species. The bacterial communities of millet-peanut intercropping and its monocropping soils were similar in phylum composition but varied in relative abundance. All five soils were dominated by the Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, with a relative abundance of 79.40%-81.33%. Soil organic carbon and alkaline nitrogen were the most important factors causing differences in the structures of the five soil bacterial communities at the phylum and genus levels, respectively. The PICRUSt functional prediction revealed that the relative abundance of primary functional metabolism was the largest (78.9%-79.3%), and the relative abundance of secondary functional exogenous biodegradation and metabolism fluctuated the most (CV=3.782%). In terms of the BugBase phenotype, the relative abundance of oxidative stress-tolerant bacteria increased in intercropping millet or peanut soils compared to that in the corresponding monocultures and significantly increased in intercropping millet soils compared to that in sole millet (P<0.05). Oxidative stress-tolerant, Gram-positive, and aerobic phenotypes were highly significantly positively correlated with each other (P<0.01), and all three showed highly significant negative correlations with potential pathogenicity and Gram-negative and anaerobic phenotypes (P<0.01). This showed that millet-peanut intercropping resulted in differences in soil bacterial community diversity, abundance, and metabolic functions and the possibility of reducing the occurrence of potential soil diseases. It can be used to regulate the soil microbiological environment to promote ecological restoration and sustainable development of farmland in the lower Yellow River.

EST sequencing and gene expression profiling of cultivated peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world. However, biotechnological based improvement of peanut is far behind many other crops. It is critical and urgent to establish the biotechnological platform for peanut germplasm innovation. In this study, a peanut seed cDNA library was constructed to establish the biotechnological platform for peanut germplasm innovation. About 17,000 expressed sequence tags (ESTs) were sequenced and used for further investigation. Among which, 12.5% were annotated as metabolic related and 4.6% encoded transcription or post-transcription factors. ESTs encoding storage protein and enzymes related to protein degradation accounted for 28.8% and formed the largest group of the annotated ESTs. ESTs that encoded stress responsive proteins and pathogen-related proteins accounted for 5.6%. ESTs that encoded unknown proteins or showed no hit in the GenBank nr database accounted for 20.1% and 13.9%, respectively. A total number of 5066 EST sequences were selected to make a cDNA microarray. Expression analysis revealed that these sequences showed diverse expression patterns in peanut seeds, leaves, stems, roots, flowers, and gynophores. We also analyzed the gene expression pattern during seed development. Genes that were upregulated (≥twofold) at 15, 25, 35, and 45 days after pegging (DAP) were found and compared with 70 DAP. The potential value of these genes and their promoters in the peanut gene engineering study is discussed.

Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthase (I, II, III), beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.

[Effects of spraying paclobutrazol at different stages on physiological characteristics, yield and quality of peanut]. / 不同时期喷施多效唑对花生生理特性、产量和品质的影响.

To explore the optimum stage of spraying with paclobutrazol (PBZ) for different peanut cultivars under high yield condition, we investigated the effects of spraying PBZ at different stages on chlorophyll content, root activity, protective enzymes, nitrogen and carbon metabolism enzymes of leaves, pod yield and kernel quality of peanut, with Huayu 20 and Huayu 25 as materials. The results showed that spraying PBZ at different stages increased root activity, chlorophyll content, SOD, POD, CAT, SS, SPS, PEPC activities and decreased the MDA content, NR, GS, GDH and GOGAT activities for pod setting stage of two cultivars. These results suggested that the effects would be more obvious at earlier spray time. For HY25, the most significant effects of spraying PBZ on those parameters of pod filling stage could be obtained when the main stem height was 25 cm. For HY20, spraying PBZ when the main stem height was 25 cm decreased the activities of protective enzymes. Earlier spraying time to HY20 would lead to early senescence, lower chlorophyll content, root activity and carbon metabolism enzyme activity. For HY20, the most significant effects of spraying PBZ on those parameters of pod filling stage could be obtained when the main stem height was 30 cm. Our results indicated that PBZ treatments at the optimum stage could improve pod yield and economic coefficient of both cultivars and enhance the fat content and the relative content of oleic acid and the O/L. Under high yield condition, the optimum stage of spraying PBZ was 25 cm height of the main stem for HY25 and 30 cm for HY20.

[Effects of exogenous Ca2+ on growth and development, physiology and yield of peanut under salt stress]. / 盐胁迫下外源Ca2+对花生生长发育、生理及产量的影响.

Huayu 22, one of the peanut (Arachis hypogaea) cultivars, was used as material in this study. Peanuts, which grew under normal conditions and 100 mmol·L-1 NaCl stress, were treated with 0, 6, 12 mmol·L-1 Ca(NO3)2 respectively to elucidate the effects of exogenous calcium on peanut salt tolerance. The effects of different Ca2+ concentrations on the physiological indices and yield of peanut during the whole growth period under salt stress were investigated in potted plants, with the aim to provide theoretical basis for the growth and production of peanut in saline soil. The results showed that, under salt stress, the activities of superoxide dismutase (SOD), catalase (CAT), and chlorophyll content increased whereas the MDA content and electrolytes decreased when treated with different concentrations of exogenous calcium. Calcium also improved root activity, biomass, improved agronomic traits, and finally increased peanut yield. Among all the exogenous calcium treatments, the effect of 12 mmol·L-1 Ca2+ treatment was the most significant. These results indicated that exogenous calcium could alleviate the salt stress on peanut plants and enhance the yield of pods by enhancing the scavenging ability of active oxygen, maintaining the stability and integrity of cell membrane.

[Effects of two phenolic acids on root zone soil nutrients, soil enzyme activities and pod yield of peanut]. / ä¸¤ç§é šé ¸ç±»ç‰©è´¨å¯¹èŠ±ç”Ÿæ ¹éƒ¨åœŸå£¤å »åˆ†ã€é ¶æ´»æ€§å’Œäº§é‡çš„å½±å“.

In order to investigate the relationship between the accumulation of phenolic acids in peanut continuous cropping soil and the continuous cropping obstacle of peanut, the effects of p-hydroxy benzoic acid and cinnamic acid on peanut root zone soil nutrients, soil enzyme activities and yield of peanut were studied by pot experiment at three stages of peanut, i.e. the pegging stage of peanut (45 days after seedling), the early podding (75 days after seedling) and the end of podding (105 days after seedling) stages. The results showed that the peanut root zone soil nutrients and enzyme activities changed obviously under the two phenolic acids treatment, especially at the pegging stage of peanut. The soil alkali-hydrolyzable nitrogen, available phosphorus, available potassium, and soil enzyme activities (urease, sucrose, neutral phosphatase) were decreased significantly. At the early and end of podding stages of peanut, the effects of the two phenolic acids on peanut root zone soil nutrients and soil enzyme activities were under a weakening trend. The allelopathy of cinnamic acid was stronger than that of p-hydroxy benzoic acid at the same initial content. The pod yield per pot was reduced by 45.9% and 52.8%, while the pod number of per plant was reduced by 46.2% and 48.9% at higher concentration (80 mg·kg-1 dry soil) of p-hydroxy benzoic acid and cinnamic acid treatments, respectively.

[Effects of single-seed sowing on canopy microenvironment, photosynthetic characteristics and pod yield of peanut (Arachis hypogaca)].

The large-seed peanut cultivar of Huayu 22 was used to study the differences of canopy microenvironment, photosynthetic characteristics, and pod yield at three single-seed sowing densities, i.e., 225000 (S1), 195000 (S2) and 165000 (S3) holes per hectare, in field experiments. The results showed that the canopy light transmittance, canopy air temperature and canopy CO2concentration all increased at these three single-seed sowing densities compared with those of double-seed sowing pattern (150000 holes per hectare), while the canopy humidity decreased. It seemed that single-seed sowing was helpful to improve microenvironment and the growth of peanut, especially at late growth stage. Meanwhile, the photosynthetic pigment contents and the net photosynthetic rate of peanut under single-seed sowing, especially in S2 and S3, were remarkably higher than those under traditional double-seed sowing. S2 had the optimum population size with an equal distribution of individuals, which reduced the contradiction between individuals and population, optimized the canopy microenvironment, enhanced the photosynthetic characteristics, and increased the synthesis and accumulation of photosynthetic products to maximize the yield production of peanut.

Isolation and functional characterisation of CDPKs gene from Arachis hypogaea under salt stress.

One of salt-induced calcium-dependent protein kinases (CDPKs) gene was isolated from Arachis hypogeae L. by RACE method. The cDNA full length was 2241bp deposited in GenBank (number KF437909), designated as AhCDPK. The coding region sequence of AhCDPK was 1629bp and encoded a protein of 542 amino acids. The molecular weight and the theoretical isoelectric point of AhCDPK was 60.96kDa and 5.61 respectively. Amino acid sequence analysis indicated that AhCDPK has highest similarity and homology with Glycine max L. In addition, the AhCDPK amino acids were predicted to encode a hydrophilic protein which localised in the endoplasmic reticulum. AhCDPK seemed to transcript in all peanut organs, and had the highest expression in seeds. The expression of AhCDPK could be strongly induced by both Ca2+ and NaCl. When exposed to salt stress, overexpressing AhCDPK in tobacco could alleviate PSII photoinhibition by improving physiological states, such as reducing the accumulation of reactive oxygen species (ROS), improving the activity of antioxidant defence system enzymes and improving the accumulation of osmotic regulation substance. These results showed that AhCDPK has the same functions as that of G. max, and it could play an important role for peanut to resist salt stress.

Exogenous calcium alleviates photoinhibition of PSII by improving the xanthophyll cycle in peanut (Arachis hypogaea) leaves during heat stress under high irradiance.

Peanut is one of the calciphilous plants. Calcium (Ca) serves as a ubiquitous central hub in a large number of signaling pathways. The effect of exogenous calcium nitrate [Ca(NO3)2] (6 mM) on the dissipation of excess excitation energy in the photosystem II (PSII) antenna, especially on the level of D1 protein and the xanthophyll cycle in peanut plants under heat (40°C) and high irradiance (HI) (1 200 µmol m(-2) s(-1)) stress were investigated. Compared with the control plants [cultivated in 0 mM Ca(NO3)2 medium], the maximal photochemical efficiency of PSII (Fv/Fm) in Ca(2+)-treated plants showed a slighter decrease after 5 h of stress, accompanied by higher non-photochemical quenching (NPQ), higher expression of antioxidative genes and less reactive oxygen species (ROS) accumulation. Meanwhile, higher content of D1 protein and higher ratio of (A+Z)/(V+A+Z) were also detected in Ca(2+)-treated plants under such stress. These results showed that Ca(2+) could help protect the peanut photosynthetic system from severe photoinhibition under heat and HI stress by accelerating the repair of D1 protein and improving the de-epoxidation ratio of the xanthophyll cycle. Furthermore, EGTA (a chelant of Ca ion), LaCl3 (a blocker of Ca(2+) channel in cytoplasmic membrane), and CPZ [a calmodulin (CaM) antagonist] were used to analyze the effects of Ca(2+)/CaM on the variation of (A+Z)/(V+A+Z) (%) and the expression of violaxanthin de-epoxidase (VDE). The results indicated that CaM, an important component of the Ca(2+) signal transduction pathway, mediated the expression of the VDE gene in the presence of Ca to improve the xanthophyll cycle.

Isolation and expression analysis of LEA genes in peanut (Arachis hypogaea L.).

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.

Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance.

A known arabidopsis cDNA clone, the CRT/DRE binding factor 1 (CBF1), was isolated and introduced into tobacco plants. It has been reported that CBF1 is one member of CBF gene family related to low temperature and enhancing low temperature tolerance of plants. In the present work, the transcripts could be detected in the transgenic lines. The photochemical efficiency of PSII (F(v)/F(m)) and the photo-oxidizable P700 in the transgenic lines overexpressing CBF1 were higher than that in the wild type plants during the chilling stress under low irradiance. Similarly, the higher NPQ, higher qf, lower Phi(NF), higher activity of SOD, and lower content of MDA were also detected in the transgenic tobacco lines. Additionally, higher expression levels of Nicotiana tabacum copper/zinc superoxide dismutase (Cu/Zn SOD) were also detected in the transgenic lines. These results suggest that CBF1 protein plays an important role in protection of PSII and PSI during the chilling stress under low irradiance.

Isolation and expression analysis of LEA genes in peanut (Arachis hypogaea L.).

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.

Cloning and analysis of a NBS-LRR disease resistance gene candidate PnAG1 from peanut (Arachis hypogaea L. )

Background: Based on the conserved sequences of a known NBS resistance gene, a pair of degenerate primers was designed to amplify the NBS-LRR resistance gene from peanut using PCR and RACE methods. Results: Analyzing the amino acid sequence by BLAST on NCBI, which was deduced from the 1088bp-long gene named PnAG1-2, showed that it had a certain homology with some resistance proteins, among which Arachis cardenasii resistance protein gene had the highest homology (66 percent). Relative quantification PCR analysis indicated that PnAG1-2 gene expresses more in J11 (an A. flavus-resistant variety) than in JH1012 (an A. flavus-susceptible variety) when the harvest time was coming. Conclusions: In this study, the NBS-LRR resistance sequence was successfully cloned from peanut and prokaryotic expression was done on the gene, which provided a foundation for cultivating anti-A. flavus peanut varieties.

Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.

The cultivated peanut is a valuable source of dietary oil and ranks fi fth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase (I, II, III), β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.