Genome-Wide Identification and Comparison of Cysteine Proteases in the Pollen Coat and Other Tissues in Maize

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis-elements were identified in the upstream sequence of 39 CPs, especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and cotton (Gossypium hirsutum), together with 39 maize CPs, were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize.

Gametophyte genome activation occurs at pollen mitosis I in maize

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase (1-3). Pollen actively transcribes its haploid genome (4), providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

Genotoxic and Antitumor Activity of Pollen Grains against Prostate Cancer Cell Line

Since the use of engineered antioxidants and antitumor is under investigation, inferable from its likely poisonousness, scientists have deflected their thoughtfulness regarding the quest for characteristic sources to meet the human medication and diet requests. Therefore the study aimed to evaluate the antitumor and antioxidant activities of maize pollen grains against the Prostate Cancer Cell (Pc3) line. Maize pollen grains were collected by Bee through a pollen trap, and then subjected for flavonoids and alkaloids analysis by HPLC method. an in vitro assays, were used to test the antitumor properties, against Pc3 cells. Furthermore, its antioxidant potential was also evaluated by DPPH. The detected flavonoids were identified to be quercetin, luteolin kaempferol, rutin, apigenin and naringin and the alkaloids were quinolone, hydroxyindolenine and conofoline. The antitumor efficacy of pollen grains extract increased with concentration and reached to 94.92 % that similar to the toxicity % of adriamycin at 1000 µg/mL, however, the IC50 (339.81 µg) of pollen grains extract was highest than IC50 (58.07 µg) of adriamycin. At 500 μg/mL of pollen grains extract, morphological changes of Pc3 were recorded. These changes deformed more at 1000 μg/mL. DPPH scavenging activity was found to be 92.26 % at 1280 µg/mL of pollen grains extracted with IC50 425.4 µg/mL compared with IC50 (13.9 µg/mL) of the ascorbic acid. DNA fragmentation and quantitative RT-PCR examinations of Bax and Bcl-2 genes demonstrated that pollen grains extract induced cellular apoptosis of Pc3 cells. This study concluded that the maize pollen grains may applied as natural safe source for inhibit Pc3 Cells proliferation as well as applied as antioxidant.

Bootstrap simulations for evaluating the model estimation of the extent of cross-pollination in maize at the field-scale level

With the recent advent of genetic engineering, numerous genetically modified (GM) crops have been developed, and field planting has been initiated. In open-environment cultivation, the cross-pollination (CP) of GM crops with wild relatives, conventional crops, and organic crops can occur. This exchange of genetic material results in the gene flow phenomenon. Consequently, studies of gene flow among GM crops have primarily focused on the extent of CP between the pollen source plot and the adjacent recipient field. In the present study, Black Pearl Waxy Corn (a variety of purple glutinous maize) was used to simulate a GM-maize pollen source. The pollen recipient was Tainan No. 23 Corn (a variety of white glutinous maize). The CP rate (%) was calculated according to the xenia effect on kernel color. We assessed the suitability of common empirical models of pollen-mediated gene flow (PMGF) for GM maize, and the field border (FB) effect of the model was considered for small-scale farming systems in Asia. Field-scale data were used to construct an optimal model for maize PMGF in the maize-producing areas of Chiayi County, southern Taiwan (R.O.C). Moreover, each model was verified through simulation and by using the 95% percentile bootstrap confidence interval length. According to the results, a model incorporating both the distance from the source and the FB can have optimal fitting and predictive abilities.

Pollen grains are suitable alternative food for rearing the commercially used predatory mite Neoseiulus cucumeris (Acari: Phytoseiidae)

The generalist predatory mite, Neoseiulus cucumeris (Oudemans) is one of the most effective natural enemies on many crops in indoor cultivations. As supply of alternative food such as pollen is generally thought to enhance the reproductive and biological control performance of generalist predatory mites, the life table parameters of the predatory mite, N. cucumeris were determined in laboratory at 25±1°C, 60±5% RH, and a photoperiod of 16:8 (L: D) hours when fed on seven different pollen (almond, bitter orange, maize, date palm, castor bean, sunflower, and bee-collected). Our results indicated that feeding the predator on castor bean, almond, date palm, and sunflower pollen led to better performance in terms of higher oviposition (38.18, 33.53, 32.87, and 29.48 eggs/female, respectively), intrinsic rate of increase (0.146, 0.129, 0.152, and 0.123 day-1, respectively) and net reproductive rate (19.55, 12.58, 14.93, and 14.06 eggs/individual, respectively), and shorter development time (7.08, 7.73, 7.19, and 9.30 days, respectively). Bitter orange and bee-collected pollen were not suitable diets for both development and reproduction of N. cucumeris. The shortest (7.08 days) and longest (16.09 days) development times were observed on castor bean and bee-collected pollen, respectively. Adult longevity varied from 6.88 days on maize to 60.4 days on castor bean pollen. The highest fecundity was obtained on almond, followed by date palm, and castor bean pollen, while feeding on maize pollen did not lead to oviposition. Overall, we found that castor bean along with date palm, almond, and sunflower pollen are suitable diets for large scale rearing of N. cucumeris for subsequent use in biological control of pests in greenhouses.

Extracts of Emmer Wheatgrass Grown with Distilled Water, Salinity or Selenium Differently Affect Germination and Cytosolic Ca2+ of Maize Pollen

In this work, the biological activity of emmer (Triticum turgidum L. spp. dicoccum (Schrank ex Shubler) Thell.) wheatgrass extracts obtained from grains sprouted with distilled water, or salinity (50 mM) or selenium (45 mg L−1 of Na2SeO3), was tested through an experimental biological model based on the germination and cytosolic Ca2+ homeostasis of maize pollen grains. The effects of thapsigargin (TG) and of four phenolic acids (PAs: ferulic, coumaric, salicylic and 3-HO benzoic) on maize pollen were also tested as controls. Wheatgrass extracts influenced both pollen cytosolic Ca2+ and germination. The Ca2+ agonist activity of emmer wheatgrass was transient, different from that of TG, which caused a depletion of the stored Ca2+ and a permanent alteration of Ca2+ homeostasis. The results obtained with extracts compared to those obtained with pure PAs suggest that PAs in unconjugated forms, which are known to be well represent in emmer wheatgrass, contribute to the biological activity of extracts. The extent of the biological response of emmer wheatgrass extracts was influenced by emmer sprouting conditions (i.e., distilled water, or salinity or selenium). Maize pollen treated with Se-enriched wheatgrass extracts showed a less perturbed cytosolic Ca2+ and a higher germination rate.

Chemical constituents and phytochemical properties of floral maize pollen

Currently, bee-gathered pollen (bee pollen) is commonly used worldwide as a dietary supplement and is recognized for its curative properties. Floral pollen is also important but is less recognized due to a lack of investigation. This study aims to determine the morphological characteristics and nutritional and phytochemical properties of floral maize pollen. Fresh pollen grains harvested from a farm of maize plants are yellow in colour and spheroid in shape. They change to amber and indented prismatic solid shapes when dehydrated. The main composition of floral maize pollen is carbohydrates (44.30±3.73%), followed by moisture (23.38±5.73%), crude proteins (17.16±3.13%), crude fibres (9.56±0.92%), and ash (4.98±0.11%), while the lowest content is observed for crude fats (0.62±0.06%). The predominant mineral is potassium (768.50±11.40 mg 100 g-1), followed by sodium (695.10±9.70 mg 100 g-1), calcium (147.20±12.60 mg 100 g-1), and magnesium (97.30±2.9 mg 100 g-1). The microelements (with average values) consist of iron (49.50±3.30 mg 100 g-1) and zinc (30.00±3.70 mg 100 g-1). Excellent phytochemical properties add value to floral maize pollen. Maize pollen contains a high total phenolic content (TPC) and total flavonoid content (TFC) of 783.02 mg GAE 100 g-1 and 1706.83 mg QE 100 g-1, respectively, and possesses strong antioxidant activity of 10.54 mg mL-1. Maize floral pollen and derived products can serve as future food resources for human consumption and as a source of functional and bioactive compounds in nutraceutical and pharmaceutical industries.

Climatic and anthropogenic influences on vegetation changes during the last 5000 years in a seasonal dry tropical forest at the northern limits of the Neotropics

Seasonal dry tropical forest (STDF) is a widespread vegetation type in western Mexico. Generally, this type of forest develops in semihumid habitats where lacustrine basins are scarce, preventing documentation of the vegetation history and forest dynamics using a paleoecological approach. Here, we present a palynological record from the Santa Maria del Oro crater lake, located within the distribution area of the STDF and the adjacent dry oak forest which gives insight into the changes in diversity and human impact at this tropical site. Pollen data, in combination with geochemical analysis and microcharcoal data from a lacustrine sequence, are used to discuss vegetation change and diversity in the plant assemblages related to drought or anthropogenic activity over the last 5000 years. Our results show three distinct periods of drought in the basin, from 4200 to 3850, 3100 to 2300, 1570 to 1100 and 300 cal year BP, with changes in vegetation composition mainly related to a decrease in taxa diversity during these periods. Based on the presence of maize pollen, two periods of human activity (3790–2160 and 280 cal year BP to present) were detected with an increase in herbaceous pollen used as a surrogate for deforestation. These two disturbance periods, pre-Columbian and postcolonial, occurred during wet conditions in the basin. Our results highlight the correlation between El Niño events, drought and fire with changes in the composition and diversity of STDF.