The invertase gene PWIN1 confers chilling tolerance of rice at the booting stage via mediating pollen development.

Pollen fertility is a primary regulator of grain yield and is highly susceptible to cold and other environmental stress. We revealed the roles of rice cell wall invertase gene PWIN1 in pollen development and chilling tolerance. We uncovered its preferential expression in microspores and bicellular pollen and identified its knock-down and knock-out mutants. pwin1 mutants produced a higher proportion of abnormal pollen than wild-type plants. The contents of sucrose, glucose, and fructose were increased, while ATP content and primary metabolism activity were reduced in the mutant pollen. Furthermore, the loss of function of PWIN1 coincided with an increase in SnRK1 activity and a decrease in TOR activity. Under chilling conditions, pwin1 mutants displayed significantly reduced pollen viability and seed-setting rate, while overexpressing PWIN1 notably increased pollen viability and seed-setting rate as compared with the wild-type, indicating that PWIN1 is essential for rice pollen development and grain yield under cold stress. This study provides insights into the molecular mechanisms underlying rice pollen fertility during chilling stress, and a new module to improve chilling tolerance of rice at the booting stage by molecular design.

Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene.

BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

Exogenous spraying of IAA improved the efficiency of microspore embryogenesis in Wucai (Brassica campestris L.) by affecting the balance of endogenous hormones, energy metabolism, and cell wall degradation.

BACKGROUND: Microspore embryogenesis is an extraordinarily complicated process, comprehensively regulated by a composite network of physiological and molecular factors, among which hormone is one of the most crucial factors. Auxin is required for stress-induced microspore reprogramming, however, the mechanism of its regulation of microspore embryogenesis is still unclear. RESULTS: In this study, we found exogenously spraying 100 mg·L- 1 IAA on the buds of Wucai significantly increased the rate of microspore embryogenesis, and moreover accelerated the process of embryogenesis. Physiological and biochemical tests showed that the contents of amino acids, soluble total sugar, soluble protein, and starch were significantly increased after IAA treatment. Furthermore, exogenously spraying 100 mg·L- 1 IAA significantly enhanced IAA, GA4, and GA9 content, increased catalase (CAT) and malondialdehyde (MDA) activity, and reduced abscisic acid (ABA), MDA and soluble protopectin content, H2O2 and O2·- production rate in the bud with the largest population of late-uninucleate-stage microspores. Transcriptome sequencing was performed on buds respectively treated with 100 mg·L- 1 IAA and fresh water. A total of 2004 DEGs were identified, of which 79 were involved in micropores development, embryonic development and cell wall formation and modification, most of which were upregulated. KEGG and GO analysis revealed that 9.52% of DEGs were enriched in plant hormone synthesis and signal transduction pathways, pentose and glucuronic acid exchange pathways, and oxidative phosphorylation pathways. CONCLUSIONS: These findings indicated that exogenous IAA altered the contents of endogenous hormone content, total soluble sugar, amino acid, starch, soluble protein, MDA and protopectin, the activities of CAT and peroxidase (POD), and the production rate of H2O2 and O2·-. Combined with transcriptome analysis, it was found that most genes related to gibberellin (GA) and Auxin (IAA) synthesis and signal transduction, pectin methylase (PME) and polygalacturonase (PGs) genes and genes related to ATP synthesis and electron transport chain were upregulated, and genes related to ABA synthesis and signal transduction were downregulated. These results indicated that exogenous IAA treatment could change the balance of endogenous hormones, accelerate cell wall degradation, promote ATP synthesis and nutrient accumulation, inhibit ROS accumulation, which ultimately promote microspore embryogenesis.

Brassica napus Haploid and Double Haploid Production and Its Latest Applications.

Rapeseed is one of the most important oil crops in the world. Increasing demand for oil and limited agronomic capabilities of present-day rapeseed result in the need for rapid development of new, superior cultivars. Double haploid (DH) technology is a fast and convenient approach in plant breeding as well as genetic research. Brassica napus is considered a model species for DH production based on microspore embryogenesis; however, the molecular mechanisms underlying microspore reprogramming are still vague. It is known that morphological changes are accompanied by gene and protein expression patterns, alongside carbohydrate and lipid metabolism. Novel, more efficient methods for DH rapeseed production have been reported. This review covers new findings and advances in Brassica napus DH production as well as the latest reports related to agronomically important traits in molecular studies employing the double haploid rapeseed lines.

Transcriptomics integrated with metabolomics reveals the effect of cold stress on rice microspores.

BACKGROUND: Microspore culture is one of the important biotechnological tools in plant breeding. The induction of microspore embryogenesis is a critical factor that affects the yield of microspore-derived embryo productions. Cold treatment has been reported to reprogram the gametophytic pathway in various plant species. However, the exact mechanism(s) underlying the effect of cold pre-treatment of floral buds on the efficiency of ME is still not clear. RESULTS: In this study, the effects of cold stress on the microspore totipotency of rice cultivar Zhonghua 11 were investigated. Our results revealed that a 10-day cold treatment is necessary for microspore embryogenesis initiation. During this period, the survival rate of microspores increased and reached a peak at 7 days post treatment (dpt), before decreasing at 10 dpt. RNA-seq analysis showed that the number of DEGs increased from 3 dpt to 10 dpt, with more downregulated DEGs than upregulated ones at the same time point. GO enrichment analysis showed a shift from 'Response to abiotic stimulus' at 3 dpt to 'Metabolic process' at 7 and 10 dpt, with the most significant category in the cellular component being 'cell wall'. KEGG analysis of the pathways revealed changes during cold treatment. Mass spectrometry was used to evaluate the variations in metabolites at 10 dpt compared to 0 dpt, with more downregulated DEMs being determined in both GC-MS and LC-MS modes. These DEMs were classified into 11 categories, Most of the DEMs belonged to 'lipids and lipid-like molecules'. KEGG analysis of DEMs indicates pathways related to amino acid and nucleotide metabolism being upregulated and those related to carbohydrate metabolism being downregulated. An integration analysis of transcriptomics and metabolomics showed that most pathways belonged to 'Amino acid metabolism' and 'Carbohydrate metabolism'. Four DEMs were identified in the interaction network, with stearidonic acid involving in the most correlations, suggesting the potential role in microspore totipotency. CONCLUSIONS: Our findings exhibited the molecular events occurring during stress-induced rice microspore. Pathways related to 'Amino acid metabolism' and 'Carbohydrate metabolism' may play important roles in rice microspore totipotency. Stearidonic acid was identified, which may participate in the initiation of microspore embryogenesis.

Au-courant and novel technologies for efficient doubled haploid development in barley (Hordeum vulgare L.).

Bounteous modern and innovative biotechnological tools have resulted in progressive development in the barley breeding program. Doubled haploids developed (homozygous lines) in a single generation is significant. Since the first discovery of haploid plants in 1920 and, in particular, after discovering in vitro androgenesis in 1964 by Guha and Maheshwari, the doubled haploidy techniques have been progressively developed and constantly improved. It has shortened the cultivar development time and has been extensively used in: genetic studies, gene mapping, marker/trait association, and QTL studies. In barley, the haploid occurrence developed gradually from being a sporadic and random process (spontaneous) to haploid development by in vivo method of modified pollination or by in vitro culture of immature male or female gametophytes. Although significant improvement in DH induction protocols has been made, challenges still exist for improvement in areas such as: low efficiency, albinism, genotypic specificity etc. Here, the paper focuses on: haploidization via different in vitro, in vivo techniques, the recent advances technologies like centromere-mediated haploidization, hap induction gene, and Doubled haploid CRISPR. The au-courant work of different researchers in barley using these technologies is reviewed. Studies on different factors affecting haploid induction and work on genome doubling of barley haploids to produce DH lines via spontaneous and induced technologies has also been highlighted.

Transcriptomic and Metabolomic Analyses Reveal the Importance of Lipid Metabolism and Photosynthesis Regulation in High Salinity Tolerance in Barley (Hordeum vulgare L.) Leaves Derived from Mutagenesis Combined with Microspore Culture.

Barley is the most salt-tolerant cereal crop. However, little attention has been paid to the salt-tolerant doubled haploids of barley derived from mutagenesis combined with isolated microspore culture. In the present study, barley doubled haploid (DH) line 20, which was produced by mutagenesis combined with isolated microspore culture, showed stably and heritably better salt tolerance than the wild type H30 in terms of fresh shoot weight, dry shoot weight, K+/Na+ ratio and photosynthetic characteristics. Transcriptome and metabolome analyses were performed to compare the changes in gene expression and metabolites between DH20 and H30. A total of 462 differentially expressed genes (DEGs) and 152 differentially accumulated metabolites (DAMs) were identified in DH20 compared to H30 under salt stress. Among the DAMs, fatty acids were the most accumulated in DH20 under salt stress. The integration of transcriptome and metabolome analyses revealed that nine key biomarkers, including two metabolites and seven genes, could distinguish DH20 and H30 when exposed to high salt. The pathways of linoleic acid metabolism, alpha-linolenic acid metabolism, glycerolipid metabolism, photosynthesis, and alanine, aspartate and glutamate metabolism were significantly enriched in DH20 with DEGs and DAMs in response to salt stress. These results suggest that DH20 may enhance resilience by promoting lipid metabolism, maintaining energy metabolism and decreasing amino acids metabolism. The study provided novel insights for the rapid generation of homozygous mutant plants by mutagenesis combined with microspore culture technology and also identified candidate genes and metabolites that may enable the mutant plants to cope with salt stress.

Opposite Auxin Dynamics Determine the Gametophytic and Embryogenic Fates of the Microspore.

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore.

Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

DNA methylation dynamics of sperm cell lineage development in tomato.

During the sexual reproduction of higher plants, DNA methylation and transcription are broadly changed to reshape a microspore into two sperm cells (SCs) and a vegetative cell (VC). However, when and how the DNA methylation of SCs is established remains not fully understood. Here we investigate the DNA methylation (5 mC) dynamics of SC lineage and the VC in tomato using whole-genome bisulfite sequencing. We find the asymmetric division of the microspore gives its two daughter cells differential methylome. Compared with the generative cell (GC), the VC is hypomethylated at CG sites while hypermethylated at CHG and CHH sites, with the majority of differentially methylation regions targeted to transposable elements (TEs). SCs have a nearly identical DNA methylome to the GC, suggesting that the methylation landscape in SCs may be pre-established following the asymmetric division or inherited from the GC. The random forest classifier for predicting gene and TE expression shows that methylation within the gene body is a more powerful predictor for gene expression. Among all tested samples, gene and TE expression in the microspore may be more predictable by DNA methylation. Our results depict an intact DNA methylome landscape of SC lineage in higher plants, and reveal that the impact of DNA methylation on transcription is variant in different cell types.

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum- and/or microspore-localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood. In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans-Golgi network/early endosome (TGN/EE)-localized ADAPTOR PROTEIN-1 complex (AP-1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP-1 in pollen wall formation. The deposition of Acyl-CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP-1µ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP-1 subunits at the TGN/EE. Our observations show that AP-1-mediated post-Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.

Effects of genotype and culture conditions on microspore embryogenesis in radish (Raphanus sativus L.).

Radish (Raphanus sativus L.), an important annual or biennial root vegetable crop, is widely cultivated in the world for its high nutritive value. Isolated microspore culture (IMC) is one of the most effective methods for rapid development of homozygous lines. Due to imperfection of the IMC technology system, it is particularly important to establish an efficient IMC system in radish. In this study, the effects of different factors on radish microspore embryogenesis were investigated with 23 genotypes. Buds with the largest population of late-uninucleate-stage microspores were most suitable for embryogenesis, with a ratio of petal length to anther length (P/A) in buds of about 3/4 ~ 1. Cold pretreatment was found to be genotype specific, and the highest microspore-derived embryoid (MDE) yield occurred for treatment of the heat shock of 48 h. In addition, the supplement of 0.75 g/L activated charcoal (AC) could increase the yield of embryoids. It was found that genotypes, bud size, as well as temperature treatments had significant effects on microspore embryogenesis. Furthermore, somatic embryogenesis-related kinase (SERK) genes were profiled by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which indicated that they are involved in the process of MDE formation and plantlet regeneration. The ploidy of microspore-derived plants was identified by chromosome counting and flow cytometry, and the microspore-derived plants were further proved as homozygous plants through expressed sequence tags-simple sequence repeats (EST-SSR) and genetic-SSR markers. The results would facilitate generating the large-scale double haploid (DH) from various genotypes, and promoting further highly efficient genetic improvement in radish. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01312-w.

Quantitative trait loci (QTL) for low temperature tolerance at the young microspore stage in rice (Oryza sativa L.) in Australian breeding material.

Low temperatures at the young microspore stage (YMS) decreases spikelet fertility and is a major limiting factor to rice production in temperate Australia. Low temperature tolerance is a difficult trait to phenotype, hence there is a strong desire for the identification of quantitative trait loci (QTL) for their use in marker-assisted selection (MAS). Association mapping was used in several breeding populations with a known source of low temperature tolerance, Norin PL8, to identify QTL for low temperature tolerance. A novel QTL for spikelet fertility was identified on chromosome 6, qYMCT6.1, in which the Australian variety, Kyeema, was the donor for increased fertility. Additional five genomics regions were identified that co-located with previously reported QTL, two of which have been previously cloned. Additionally, for the first time a QTL for spikelet fertility qYMCT10.1, has been shown to co-locate with the number of dehisced anthers qYMCTF10.1 which increases the shedding of pollen from the anthers. This study revealed one new QTL for low temperature tolerance at YMS in temperate japonica germplasm and identified an additional five previously reported. These QTL will be utilised for MAS in the Australian rice breeding program and may have merit for temperate breeding programs globally.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage '01-88' under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Defective Microspore Development 1 is required for microspore cell integrity and pollen wall formation in rice.

Highly coordinated pollen wall patterning is essential for male reproductive development. Here, we report the identification of Defective Microspore Development 1 (DMD1), which encodes a nuclear-localized protein possessing transactivation activity. DMD1 is preferentially expressed in the tapetum and microspores during post-meiotic development. Mutations in DMD1 cause a male-sterile phenotype with impaired microspore cell integrity. The mutants display abnormal callose degradation, accompanied by inhibited primexine thickening in the newly released microspores. Several genes associated with callose degradation and primexine formation are downregulated in dmd1 anthers. In addition, irregular Ubisch body morphology and discontinuous endexine occur, and the baculum is completely absent in dmd1. DMD1 interacts with Tapetum Degeneration Retardation (TDR), a basic helix-loop-helix transcription factor required for exine formation. Taken together, our results suggest that DMD1 is responsible for microspore cell integrity, primexine formation and exine pattern formation during Oryza sativa (rice) microspore development.

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture.

BACKGROUND: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. RESULTS: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, 'Jersey' and 'Mercada' that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. 'Mercada' that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating 'Mercada' embryos contained a low number of plastome copies whose replication was not always completed. Contrary to 'Mercada', cv. 'Jersey' that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in 'Jersey' regenerants. CONCLUSIONS: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection.

BACKGROUND: The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. RESULTS: In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. CONCLUSIONS: Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

FLA14 is required for pollen development and preventing premature pollen germination under high humidity in Arabidopsis.

BACKGROUND: As an important subfamily of arabinogalactan proteins (AGPs), fasciclin-like AGPs (FLAs) contribute to various aspects of growth, development and adaptation, yet their function remains largely elusive. Despite the diversity of FLAs, only two members, Arabidopsis FLA3 and rice MTR1, are reported to be involved in sexual reproduction. In this study, another Arabidopsis FLA-encoding gene, FLA14, was identified, and its role was investigated. RESULTS: Arabidopsis FLA14 was found to be a pollen grain-specific gene. Expression results from fusion with green fluorescent protein showed that FLA14 was localized along the cell membrane and in Hechtian strands. A loss-of-function mutant of FLA14 showed no discernible defects during male gametogenesis, but precocious pollen germination occurred inside the mature anthers under high moisture conditions. Overexpression of FLA14 caused 39.2% abnormal pollen grains with a shrunken and withered appearance, leading to largely reduced fertility with short mature siliques and lower seed set. Cytological and ultramicroscopic observation showed that ectopic expression of FLA14 caused disruption at the uninucleate stage, resulting in either collapsed pollen with absent intine or pollen of normal appearance but with a thickened intine. CONCLUSIONS: Taken together, our data suggest a role for FLA14 in pollen development and preventing premature pollen germination inside the anthers under high relative humidity in Arabidopsis.