Rapid and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis>) employing standard binary vectors and <Emphasis Type="Italic">bar</Emphasis> gene as a selectable marker

Key message

A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker.

Abstract

Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7–12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40–50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

     

Identification of loci associated with embryo yield in microspore culture of <Emphasis Type="Italic">Brassica rapa</Emphasis> by segregation distortion analysis

Key message

We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield.

Abstract

Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. ‘Ho Mei’ is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01–A10) was performed using a population of microspore-derived embryos obtained from an F₁ hybrid between ‘CR-Seiga’, a low yield cultivar in microspore-derived embryos, and ‘Ho Mei’. Three regions showing significant segregation distortion with increasing ‘Ho Mei’ alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F₂ population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC₃F₁ population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

     

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Background

Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂).

Results

Using geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H₂O₂. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.

Conclusion

Taken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

     

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings

Key message

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings.

Abstract

Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

     

MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple

Key message

Salt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple.

Abstract

Salinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser³⁹⁶ site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1–MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1–MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

     

Sequence diversification in recessive alleles of two host factor genes suggests adaptive selection for bymovirus resistance in cultivated barley from East Asia

Key message

Two distinct patterns of sequence diversity for the recessive alleles of two host factors HvPDIL5 - 1 and HvEIF4E indicated the adaptive selection for bymovirus resistance in cultivated barley from East Asia.

Abstract

Plant pathogens are constantly challenging plant fitness and driving resistance gene evolution in host species. Little is known about the evolution of sequence diversity in host recessive resistance genes that interact with plant viruses. Here, by combining previously published and newly generated targeted re-sequencing information, we systematically analyzed natural variation in a broad collection of wild (Hordeum spontaneum; Hs) and domesticated barleys (Hordeum vulgare; Hv) using the full-length coding sequence of the two host factor genes, HvPDIL5-1 and HvEIF4E, conferring recessive resistance to the agriculturally important Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV). Interestingly, two types of gene evolution conferred by sequence variation in domesticated barley, but not in wild barley were observed. Whereas resistance-conferring alleles of HvEIF4E exclusively contained non-synonymous amino acid substitutions (including in-frame sequence deletions and insertions), loss-of-function alleles were predominantly responsible for the HvPDIL5-1 conferred bymovirus resistance. A strong correlation between the geographic origin and the frequency of barley accessions carrying resistance-conferring alleles was evident for each of the two host factor genes, indicating adaptive selection for bymovirus resistance in cultivated barley from East Asia.

     

The <Emphasis Type="Italic">Populus trichocarpa PtHSP17.8</Emphasis> involved in heat and salt stress tolerances

Key message

PtHSP17.8 was regulated by various abiotic stresses. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses through maintain ROS homeostasis and cooperate with stress-related genes in Arabidopsis.

Abstract

Small heat shock proteins (sHSPs) play important roles in response to diverse biotic and abiotic stresses, especially in heat tolerance. However, limited information is available on the stress tolerance roles of sHSPs in woody species. To explore the function of sHSPs in poplar, we isolated and characterized PtHSP17.8 from Populus trichocarpa. Phylogenetic analysis and subcellular localization revealed that PtHSP17.8 was a cytosolic class I sHSP. The gene expression profile of PtHSP17.8 in various tissues showed that it was significantly accumulated in stem and root, which was consistent with the GUS expression pattern driven by promoter of PtHSP17.8. The expression of PtHSP17.8 could be induced by various abiotic stresses and significantly activated by heat stress. Overexpression of PtHSP17.8 enhanced the tolerance to heat and salt stresses in Arabidopsis. The seedling survival rate, root length, relative water content, antioxidative enzyme activities, proline, and soluble sugar content were increased in transgenic Arabidopsis under heat and salt stresses, but not in normal condition. The co-expression network of PtHSP17.8 were constructed and demonstrated many stress responsive genes included. The stress-related genes in the co-expression network were up-regulated in the PtHSP17.8 overexpression seedlings. These results suggest that PtHSP17.8 confers heat and salt tolerances in plants.

     

Alternative pathway is involved in the tolerance of highland barley to the low-nitrogen stress by maintaining the cellular redox homeostasis

Key message

Alternative pathway (AP) is involved in the tolerance of highland barley seedlings to the low-nitrogen stress by dissipating excessive reducing equivalents generated by photosynthesis and maintaining the cellular redox homeostasis.

Abstract

Low nitrogen (N) is a major limiting factor for plant growth and crop productivity. In this study, we investigated the roles of the alternative pathway (AP) in the tolerance of two barley seedlings, highland barley (Kunlun12) and barley (Ganpi6), to low-N stress. The results showed that the chlorophyll content and the fresh weight decreased more in Ganpi6 than those in Kunlun12 under low-N stress, suggesting that Kunlun12 has higher tolerance to low-N stress than Ganpi6. AP capacity was markedly induced by low-N stress; and it was higher in Kunlun12 than in Ganpi6. Comparatively, the cytochrome pathway capacity was not affected under all conditions. Western-blot analysis showed that the protein level of the alternative oxidase (AOX) increased under low-N stress in Kunlun12 but not in Ganpi6. Under low-N stress, the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio in Ganpi6 increased more than those in Kunlun12. Furthermore, photosynthetic parameters (Fv/Fm, qP, ETR and Yield) decreased markedly and qN increased, indicating photoinhibition occurred in both barley seedlings, especially in Ganpi6. When AP was inhibited by salicylhydroxamic acid (SHAM), the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio dramatically increased under all conditions, resulting in the marked accumulation of H₂O₂ and malondialdehyde in leaves of both barley seedlings. Meanwhile, the malate–oxaloacetate shuttle activity and the photosynthetic efficiency were further inhibited. Taken together, AP is involved in the tolerance of highland barley seedlings to low-N stress by dissipating excess reducing equivalents and maintaining the cellular redox homeostasis.

     

Root proteomics reveals cucumber 24-epibrassinolide responses under Ca(NO<Subscript>3</Subscript>)<Subscript>2</Subscript> stress

Key message

The application of exogenous 24-epibrassinolide promotes Brassinosteroids intracellular signalling in cucumber, which leads to differentially expressed proteins that participate in different life process to relieve Ca(NO ₃ ) ₂ damage.

Abstract

NO₃ ^? and Ca²⁺ are the main anion and cation of soil secondary salinization during greenhouse cultivation. Brassinosteroids (BRs), steroidal phytohormones, regulate various important physiological and developmental processes and are used against abiotic stress. A two-dimensional electrophoresis gel coupled with MALDI-TOF/TOF MS was performed to investigate the effects of exogenous 24-epibrassinolide (EBL) on proteomic changes in cucumber seedling roots under Ca(NO₃)₂ stress. A total of 80 differentially accumulated protein spots in response to stress and/or exogenous EBL were identified and grouped into different categories of biological processes according to Gene Ontology. Under Ca(NO₃)₂ stress, proteins related to nitrogen metabolism and lignin biosynthesis were induced, while those related to cytoskeleton organization and cell-wall neutral sugar metabolism were inhibited. However, the accumulation of abundant proteins involved in protein modification and degradation, defence mechanisms against antioxidation and detoxification and lignin biosynthesis by exogenous EBL might play important roles in salt tolerance. Real-time quantitative PCR was performed to investigate BR signalling. BR signalling was induced intracellularly under Ca(NO₃)₂ stress. Exogenous EBL can alleviate the root indices, effectively reduce the Ca²⁺ content and increase the K⁺ content in cucumber roots under Ca(NO₃)₂ stress. This study revealed the differentially expressed proteins and BR signalling-associated mRNAs induced by EBL in cucumber seedling roots under Ca(NO₃)₂ stress, providing a better understanding of EBL-induced salt resistance in cucumber seedlings. The mechanism for alleviation provides valuable insight into improving Ca(NO₃)₂ stress tolerance of other horticultural plants.

     

Development of a complete set of wheat–barley group-7 Robertsonian translocation chromosomes conferring an increased content of β-glucan

Key message

A complete set of six compensating Robertsonian translocation chromosomes involving barley chromosome 7H and three chromosomes of hexaploid wheat was produced. Grain β-glucan content increased in lines containing 7HL.

Abstract

Many valuable genes for agronomic performance, disease resistance and increased yield have been transferred from relative species to wheat (Triticum aestivum L.) through whole-arm Robertsonian translocations (RobT). Although of a great value, the sets of available translocations from barley (Hordeum vulgare L.) are limited. Here, we present the production of a complete set of six compensating RobT chromosomes involving barley chromosome 7H and three group-7 chromosomes of wheat. The barley group-7 long-arm RobTs had a higher grain β-glucan content compared to the wheat control. The β-glucan levels varied depending on the temperature and were higher under hot conditions. Implicated in this increase, the barley cellulose synthase-like F6 gene (CslF6) responsible for β-glucan synthesis was physically mapped near the centromere in the long arm of barley chromosome 7H. Likewise, wheat CslF6 homoeologs were mapped near the centromere in the long arms of all group-7 wheat chromosomes. With the set of novel wheat–barley translocations, we demonstrate a valuable increase of β-glucan, along with a resource of genetic stocks that are likely to carry many other important genes from barley into wheat.