R2R3-type Lo MYB21 affects jasmonate-regulated development and dehiscence of anthers in lily(Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers,but their large anthers carry a considerable amount of colored pollen that is dispersed easily.Studying the molecular mechanism of anther development and dehiscence could help solve this problem.LoMYB21,encoding a putative R2R3v-myb avian myeloblastosis viral oncogene homolog(MYB)transcription factor,was identified from oriental lilies(Lilium‘Siberia’).Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther,filament and stigma and had high expression during the late stages of lily anther development.LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana.Suppression of LoMYB21 by virus-induced gene silencing(VIGS)in Lilium‘Siberia’led to anther indehiscence and reduced the expression of genes related to Jasmonate acid(JA)biosynthesis and signal transduction.Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile,accompanied by higher expression of JA biosynthetic and signaling genes.These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis,which affected anther development.Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

ZmMs33 promotes anther elongation via modulating cell elongation regulators,metabolic homeostasis,and cell wall remodeling in maize

Plant cell elongation depends on well-defined gene regulations,adequate nutrients,and timely cell wall modifications.Anther size is positively correlated with the number and viability of pollen grains,while little is known about molecular mechanisms underlying anther cell elongation.Here,we found that properly activated cell elongation regulators at transcriptional levels in loss-of-function ZmMs33 mutant(ms33-6038)anthers failed to promote maize anther elongation.ZmMs33 deficiency disrupted metabolic homeostasis mainly by inhibiting both photosynthesis in anther endothecium and lipid accumulation in anther tapetum.Importantly,ms33-6038 anthers displayed ectopic,premature and excessive secondary cell wall thickening in anther middle layer,which constrained cell elongation structurally and blocked nutrient flows across different anther wall layers.The metabolic disorder was only found in ms33-6038 mutant rather than several representative male-sterility lines at transcriptional and post-translational levels.Collectively,the disordered metabolisms and blocked nutrient flows defeated the activated cell elongation regulators,and finally inhibited anther elongation and growth with a unique‘‘idling effect”in ms33-6038 mutant.

Maize MS2 encodes an ATP-binding cassette transporter that is essential for anther development

The anther cuticle and pollen exine play a critical role in male gametophyte development. The sporopollenin precursors and cuticular lipid monomers are transported to the surface of the microspores and the epidermis by lipid transport proteins(LTPs) and ATP-binding cassette G(ABCG) transporters for the formation of the pollen wall and anther cuticle, respectively. However, the function of ABCG transporters in maize anther development is unclear. Here, we cloned the MS2 gene from the maize male sterile2 mutant using map-based cloning and determined that it encodes an ABCG transporter. MS2 protein was experimentally confirmed to be located on the cell membrane. The quantitative real-time PCR(qRT-PCR)results showed that MS2 was ubiquitously expressed in all vegetative and reproductive tissues, whereas a high transcriptional level of MS2 was observed in anthers, especially at the young microspore stage. Gas chromatography-mass spectrometry(GC–MS) analysis showed decreased accumulation of cutin and wax components in ms2 anthers, indicating that MS2 plays a role in the transport of lipid molecules to anther cuticle and pollen exine. To our knowledge, MS2 is the first reported ABCG transporter gene that participates in anther development in maize.

Identification and Characterization of a LEA-like Gene, CaMF5,Specifically Expressed in the Anthers of Male-fertile Capsicum annuum

To improve the understanding of molecular mechanisms of anther and/or pollen development in Chili pepper, in the present study, fulllength cDNA and DNA sequences of the pollen development-related gene CaMF5 were obtained from the anthers of a Capsicum annuum nuclear male-fertile line. Sequence analysis indicated that the full length of CaMF5 was 747 bp, containing a maximum opening reading frame of 447 bp.Amino acid sequence alignment and phylogenetic analysis revealed that CaMF5 shared approximately 37%–77% homology with a series of uncharacterized or hypothetical proteins and late embryogenesis abundant(LEA) proteins from other plants. However, no LEA structural domain was detected in CaMF5, which indicated that it might be a new type of LEA gene. CaMF5 was only expressed in flower buds at stages 7 and 8 and in open flowers of the male-fertile line, whereas it exhibited no expression in any examined organs of the male-sterile line. In addition, CaMF5 showed the highest transcript abundance in the anthers of the male-fertile line, with no expression being detected in any other examined organs, such as the sepals, petals, pistils, roots, stems, or leaves. Taken together, our results suggest that CaMF5 is an anther-specific gene that might encode a new type of LEA protein related to anther and/or pollen development in C. annuum.

Preliminary Study on Anther in vitro Culture of Lily“Prato”

The effects of the microspore developmental stage,hormones and culture condition on anther in vitro culture of lily(Lilium spp.) were discussed.The results showed that when the flower buds were about 23-26 mm long,the microspores were at the uninucleate stage which was suitable for culture and the culture under the darkness would promote the callus induction of anther.The induction frequency could reach 42.5% in the optimized medium which was MS+[6-BA(0.5)+KT(2.0)+2,4-D(1.0)] mg·L-1.The rate of callus differentiation could reach 31.57% in the optimized medium which was MS+ NAA(1.5,2.0) mg·L-1.

Effect of Maltose Concentration on Plant Regeneration of Anther Culture with Different Genotypes in Rice (<i>Oryza sativa </i>L.)

This study describes the impact of different concentrations of maltose on plant regeneration of anther culture for five genotypes of rice (Oryza sativa). N6 medium was used for calli induction, while N6 medium supplemented with different concentrations of maltose, 2.0 mg/L NAA and 0.5 mg/L kinase was used for plant regeneration. The result showed that during the initial stages of calli induction the anther cultures had varying rates of calli formation among genotypes, with the best frequency being observed for Dreami2/CaMsrB2-8-DH-1 with a calli frequency of 27.8%. Different genotypes of rice cultured in regeneration media showed varying plantlet regeneration on media supplemented with different concentrations of maltose, with low concentrations (0.04 g/L) leading to low frequency regeneration plantlet but high green plant production. Indeed, when Dreami2/CaMsrB2-8-DH-2 and Dreami2/CaMsrB2-8-DH-5 were cultivated under these conditions, 100% green plants were observed. Another genotype also showed a small rate of albino frequency in response to the lowest concentration of maltose, while increased maltose concentrations resulted in increased rates of albino plants. Overall, the results of this study should facilitate establishment of an efficient plant regeneration system from anther culture in rice.

Cytological Observation of Pollen Abortion of Lycium barbarum Haploids from Anther Culture

To reveal the sterility characteristics of Lycium barbarum haploids, cytological observations were made on the anthers of Ningqi No.1 and its haploids obtained from anther culture. The results showed that there were no significant differences in anther development between Ningqi No.1 and its haploids at the stage of pollen mother cell, and tetrads were formed successfully after the meiosis stage. The tetrads of Ningqi No.1 could release microspores. At the same time, the tapetal cells can provide nutrition for the development of the microspores, which eventually developed into mature pollen grains. Although the haploids could also release microspores at the tetrad stage, the tapetal cells degraded in advance, which made the released microspores unable to develop into mature pollen grains, resulting in pollen abortion of haploids.

Isolation and Characterization the ADP-ribosylation Factor(ARF) Gene from Cotton Anther

ADP-ribosylation factor(ARF)is considered asthe family of small GTP binding proteins,withmolecular mass around 21KD.It has beenknown that ARF plays important roles in cells toregulate membrane traffic and structure andintracellular signal transduction system in yeastand mammalian.Although ARFs have

Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in <i>Paris polyphylla</i>var. Yunnanensis

Paris polyphylla var. yunnanensis (Franch.), one of the best-known medicinal plants in China, has a dehiscent anther which physiologically work in pollination, however, the dehiscent anther always closes in response to darkness every day, and watering or raining every time. To explore this frequently closing and its unkown physiology, next-generation sequencing was performed, and the transcriptome was de novo assembled. RNA-sequencing was carried out in 15 samples including seven openning samples, four closed samples owing to darkness or watering, and tissue samples (leaf, petal, calyx, and stigma) were used for control. We obtained 72.75 GB data, assembled into 79,815 unigenes. Differentially expressed unigenes (DEGs) between opened and closed anther samples were 6231 and the DEGs between anther and control samples were 2831. Comparation between the two DEGs by KEGG enrichment showed that “plant hormone signal transduction” pathway is the most significant pathway for DEGs from closing anther vs. opening anther, and expression model of DEGs in the pathway might elicit change in germination and seed dormancy. Further examination of the action of the signal pathway on physiology showed “chromatin binding” function was prominent in “DNA binding” function of annotated DEGs between opened and closed anthers, of the 215 “chromatin binding” unigenes, 120 were involved in epigenetic silencing, and 50 of the epigenetic unigenes were directly related to germination or seed dormancy, strongly correlating anther closing to epigenetic modification and seed dormancy. These results were verified that at least three auxins involved in seed dormancy showed same expression patterns occurred in abnormal closing anther and seed embryo in Paris polyphylla var. yunnanensis. In conclusion, the information from transcriptome point out that frequent abnormal closing of dehiscent anthers possibly transfer the impact on seed dormancy, and epigenetic modification happened in closing may be the cause.

Anther Culture and Plant Regeneration of Tetraploid Purple Coneflower (Echinacea purpurea L.)

Anthersisolated from tetraploid purple coneflowerplants were cultured in vitro. The highest callus induction rate was obtained when the medium was consisted of N6 basal elements, 4% sucrose, 0.5 mg?L?1 BA, and 0.10 mg?L?1 NAA. Various morphogenesis such as globular, heart-shape, torpedo-shapeand final state embryos as well asvarious texture calluses around were observed. Out of 110 plantlets regenerated, 104 were confirmed as diploid and the rest were as tetraploid. Plants of one diploid offspring strain presented aspecialcharacter in pot: unlike the original tetraploid plants, it grown tubular, bisexual ray florets. The results obtained in the present studies indicated that although the tetraploid purple coneflower plants produced only diploid microspores, the recovery of some useful mutants through in vitro anther cultures might be reasonably expected.

Changes of Oxidative Stress and Soluble Sugar in Anthers Involve in Rice Pollen Abortion Under Drought Stress

Two rice maintaining lines with different drought tolerances, viz., Jin 23B (tolerant) and Zhenshan 97B (sensitive), were used to study the oxidative stress and soluble sugar in rice anthers and pollen viability under drought stress during flowering stage. Higher antioxidant enzyme activities and lower malonaldehyde (MDA) content in rice anthers were observed in Jin 23B than in Zhenshan 97B under drought stress. Further, a great increase in the content of soluble sugar in rice anthers of Jin 23B was observed across the whole drought exposure, while Zhenshan 97B showed significant decrease in soluble sugar during 9-12 d after drought stress (DADS). Accordingly, a marked decline of pollen fertility and activity, pollen numbers in an anther and pollen numbers on a stigma was observed in Zhenshan 97B, whereas little difference was found in Jin 23B. Thus, we suggest that pollen abortion caused by drought stress may be related with the reciprocity between oxidative stress and soluble sugar content in rice anthers.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ‘double T-DNA’ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene (hpt) in one T-DNA region and three target genes (hLF, SB401, RZ10) in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Effect of ^(137)Cs Gamma Rays to Panicles on Rice Anther Culture

Panicles of an indica rice line TM7-5 were subjected to radiation with 137Cs gamma rays at 0 (control), 5, 10, 15 and 20 Gy respectively, and then its anthers were cultured. There were slight differences among the treatments in peak emerging time of callus initiation, from 38 to 44 days after inoculation (DAI) as well as the frequency of callus initiation (2.3-3.5%). About two thirds calli were induced before 44 DAI, and calli derived beyond 60 DAI lost the regeneration ability. Green plant regeneration frequency was significantly stimulated from two- to three-fold by irradiation of the 137Cs gamma rays compared with the control, and the maximum was 22.81% (15 Gy). The culture ability based on callus initiation and green plantlet regeneration was 0.19% for the control while it was over 0.45% for all the irradiated treatments, and the maximum was 0.59% for 15 Gy treatment. The advantages of panicle irradiation before anther culture and the potential application in rice anther culture, especially for recalcitrant indica rice, were discussed.

Effects of Antimicrotuble Herbicide APM on anther Culture in Rice

Significant differences in the effects of aminoprophosmethyl(APM) on ather culture were observed among 6 Australian cultivars in rice.APM had improved anther culture efficienncy in 5 cultivars,but decreased the induce rate of callus in one.

Anther Culture of Chinese Radish(Raphanus sativus L. var. Longinnatus Bailey):Response of Different Genotypes to the Embryogenesis and the Traits of Regenerated Plantlets

[Objective] The aims were to ① conduct anther culture of Chinese radish varieties; ② observe the development of embryos from anther culture; ③ study the response of different genotypes to embryogenesis in anther culture; ④ observe the morphology of regenerated plantlets; ⑤ analyze the ploidy level of regenerated plantlets arising from the anther culture process. [Method]Anthers of 15 genotypes with diverse genetic backgrounds of Chinese radish have been cultured in vitro and induced to undergo embryogenesis and plant formation. [Result] Of 15 genotypes evaluated,four produced embryos. The genotype was the main factor to influence the embryogenesis. The morphology and the ploidy of the regenerated plantlets were observed,and the anther-derived plantlets included a mix of haploids,diploids and hexaploids. Of the plants that regenerated from anther embryos 60% were diploid. [Conclusion] The plantlets had the high ability to double spontaneously.

Pollen-embryogenesis and chromosomal variability in anther culture of Brassica hirta Moench (Sinapis alba L)

The anther cultures of Brassica hirta underwent pollenembryogenesis and callusing,which showed a wide range of chromosome numbers varying from 9 (n=12) to a highly polyploid.For embryogenesis,pretreatment of floral buds in 0.4 M sucrose solution for 72 hrs at 4℃ was superior to freshly cultured anthers.Culture temperature of 30℃ for 14 days before maintenance of cultures at 25℃ was significantly beneficial for embryo yield in comparison to cultures continuously incubated at 25℃.Dark treatment during culture was more effective for pollen-embryo yield.

Anther Dehiscence Disturbed by High Temperature and Water Stress Presented in Os DIR Gene Expression in Rice(Oryza sativa L.)

In future climates, rice could more frequently be subjected to simultaneous high temperature(HT) and water stress(WS) during sensitive developmental stages such as flowering. In this study, two rice genotypes were exposed to HT, WS and combined high temperature and water stress(WS + HT) during flowering to quantify their response through anther dehiscence. Gene expression profiles of 15 selected Os DIRs revealed differences among stresses and between varieties. The targeted gene Os DIR-08, which was considered to be a HT stress candidate gene, was decreasingly expressed from the 1st d to the 4th d under HT stress in Nagina 22(N22) while increased in Moroberekan. Varies of the expression of Os DIR genes in stresses intuitively reflects on the lignin-staining at the anther dehisced sites, which implied a negative relationship between the lignin biosynthesis and Os DIRs' expression. Anther dehiscence disturbed by HT and WS + HT stress showed a negative cumulative effect in HT sensitive variety Moroberekan but not in N22. Higher level of anther dehiscence in N22 under HT and WS + HT stress indicated its true tolerance of HT and to WS + HT during anthesis. The differentially expressed of Os DIR(s) under various managed stresses caused the difference of the lignin-staining at the anther dehisced site in N22, and thus transformed anther dehiscence correspondingly might be one of the main reasons for the tolerance.

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development

The tapetum,a crucial innermost layer encompassing male reproductive cells within the anther wall,plays a pivotal role in normal pollen development.The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1,a gatekeeper TF in the tapetum.Nevertheless,the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown.In this study,we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089,probably through their K259 site.Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089.Intriguingly,elevated CCoAOMT1 expression resulted in defective pollen development,mirroring the phenotype observed in bhlh010 bhlh089 mutants.Moreover,our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1.Importantly,transgenic plants with the probHLH089::bHLH089^(K259A)construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants.Collectively,these findings emphasize the need to maintain balanced TF homeostasis for male fertility.They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.