The DEFECTIVE IN ANTHER DEHISCENCE1 Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in Arabidopsis

The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1–specific manner, and DAD1–green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::β-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.     

The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis

The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1-specific manner, and DAD1-green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::beta-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Epidermal jasmonate perception is sufficient for all aspects of jasmonate‐mediated male fertility in Arabidopsis

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1‐YFP transgene in the coi1‐1 mutant background. As COI1 is an essential component of the JA co‐receptor complex, the null coi1‐1 mutant is male sterile due to lack of JA perception. We show that expression of COI1‐YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA‐dependent non‐autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.     

Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella

Ubiquitylation is one of the cardinal post‐translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image‐based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING‐between‐RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response.     

Heteranthery as a solution to the demand for pollen as food and for pollination – Legitimate flower visitors reject flowers without feeding anthers

• Heteranthery, the presence of feeding and pollinating anthers in the same flower, seems to mediate the evolutionary dilemma for plants to protect their gametes and yet provide food for pollinators. This study aims to elucidate the role of heteranthery in the buzz‐pollinated Senna reniformis.
• The fecundity of pollen from long‐, medium‐ and short‐sized anthers was determined by hand cross‐pollination experiments, and the quantity, size, ornamentation and viability of pollen of different anthers were compared. Rates of flower rejection by bees were measured in anther removal experiments to assess the preferences of flower visitors for feeding or pollinating anthers.
• Large bees, which were the effective pollinators of self‐incompatible S. reniformis, avoided flowers without short feeding anthers, but not those without medium or long anthers. Illegitimate small and medium‐sized bees were unresponsive to anther exclusion experiments. Long anthers deposited pollen on the back and short anthers on the venter of large bees. Pollen from long anthers had higher in vitro viability and higher fruit and seed set after cross‐pollination than pollen from other sized anthers.
• Short anthers produce feeding pollen to effective pollinators and long anthers are related to pollination of S. reniformis. Bee behaviour and size was found to directly influence the role of anthers in the ‘division of labour’. Only large bee pollinators that carry the pollinating pollen from long anthers in ‘safe sites’ associated short anthers with the presence of food. In the absence of these larger bee pollinators, the role of heteranthery in S. reniformis would be strongly compromised and its function would be lost.

     

Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development

The tapetum is a layer of cells covering the inner surface of pollen sac wall. It contributes to anther development by providing enzymes and materials for pollen coat biosynthesis and nutrients for pollen development. At the end of anther development, the tapetum is degenerated, and the anther is dehisced, releasing mature pollen grains. In Arabidopsis, several genes are known to regulate tapetum formation and pollen development. However, little is known about how tapetum degeneration and anther dehiscence are regulated. Here, we show that an activation-tagged mutant of the S HI-R ELATED S EQUENCE 7 (SRS7) gene exhibits disrupted anther dehiscence and abnormal floral organ development in addition to its dwarfed growth with small, curled leaves. In the mutant hypocotyls, cell elongation was reduced, and gibberellic acid sensitivity was diminished. Whereas anther development was normal, its dehiscence was suppressed in the dominant srs7-1D mutant. In wild-type anthers, the tapetum disappeared at anther development stages 11 and 12. In contrast, tapetum degeneration was not completed at these stages, and anther dehiscence was inhibited, causing male sterility in the mutant. The SRS7 gene was expressed mainly in the filaments of flowers, where the DEFECTIVE-IN-ANTHER-DEHISCENCE 1 (DAD1) enzyme catalyzing jasmonic acid (JA) biosynthesis is accumulated immediately before flower opening. The DAD1 gene was induced in the srs7-1D floral buds. In fully open flowers, the SRS7 gene was also expressed in pollen grains. It is therefore possible that the abnormal anther dehiscence and floral development of the srs7-1D mutant would be related with JA.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

Minor Differences in Anther Extrusion Affect Resistance to Fusarium Head Blight in Wheat

Fusarium head blight (FHB) remains a serious problem due to yield loss and mycotoxin accumulation in wheat production worldwide. We previously reported that the closed‐flowering (no anther extrusion) characteristic was effective for increasing resistance to FHB infection. In this study, we investigated the relationships between the degree of anther extrusion (AE) and FHB damage using double haploid lines (DHLs), derived from F₁ plants from crosses between closed‐flowering and opened‐flowering varieties. These DHLs exhibited various degrees of AE, and the degree of AE was significantly different among DHLs, regardless of the year and environment (pot‐ or field‐grown). FHB severity was the lowest in closed‐flowering DHLs, and DHLs with partially extruded anthers showed significantly higher FHB symptoms than those with closed‐flowering phenotypes. In general, DHLs with partially extruded anthers also had relatively severe FHB symptoms compared with those exhibiting full anther extrusion. FHB severity was significantly correlated with Fusarium‐damaged kernels and deoxynivalenol concentration. The results of this study showed that partially extruded anthers were considered to be a source of FHB infection. The closed‐flowering phenotype improved resistance to FHB infection. Meanwhile, phenotypes with rapid anther extrusion and ejection also could contribute to the avoidance of FHB infection.     

Ethylene regulates the timing of anther dehiscence in tobacco

We investigated the involvement of ethylene signaling in the development of the reproductive structures in tobacco ( Nicotiana tabacum L.) by studying flowers that were insensitive to ethylene. Ethylene-insensitivity was generated either by expression of the mutant etr1-1 ethylene-receptor allele from Arabidopsis thaliana or by treatment with the ethylene-perception inhibitor 1-methylcyclopropene (MCP). Development of ovaries and ovules was unaffected by ethylene-insensitivity. Anther development was also unaffected, but the final event of dehiscence was delayed and was no longer synchronous with flower opening. We showed that in these anthers degeneration of the stomium cells and dehydration were delayed. In addition, we found that MCP-treatment of detached flowers and isolated, almost mature anthers delayed dehiscence whereas ethylene-treatment accelerated dehiscence. This indicated that ethylene has a direct effect on a process that takes place in the anthers just before dehiscence. Because a similar function has been described for jasmonic acid in Arabidopsis, we suggest that ethylene acts similarly to or perhaps even in concurrence with jasmonic acid as a signaling molecule controlling the processes that lead to anther dehiscence in tobacco.     

Antisense inhibition of a nuclear gene,BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment

Male sterility is widely used for the production of hybrid seeds, but the use of genic male sterility is rather limited because of difficulty in maintaining homozygous male sterile plants. Recently, the DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) gene, which encodes a phospholipase A1 involved in the first step of the jasmonic acid (JA) biosynthesis pathway, was isolated from a male sterile Arabidopsis mutant. To utilize this gene in Brassica crops, we characterized the BrDAD1 gene, the putative ortholog of DAD1 in Brassica rapa. Out of 25 plants transformed with an antisense gene constructed from the BrDAD1, 3 plants showed a defect of anther dehiscence at the flower bud opening stage and produced inviable pollen. One of the three showed male sterility only, but the other two showed a delay or a lack of flower opening in addition to male sterility. The male sterile and flower-opening phenotypes were rescued by the application of JA as well as linolenic acid. Furthermore, all these characteristics were inherited to the next generation. The present results demonstrate a novel control system for hybrid seed production by the use of nuclear genes.     

CsSPL functions as an adaptor between HD‐ZIP III and CsWUS transcription factors regulating anther and ovule development in Cucumis sativus (cucumber)

Anther and ovule genesis preconditions crop fertilization and fruit production; however, coordinative regulation of anther and ovule development and underlying molecular pathways remain largely elusive. Here, we found that SPOROCYTELESS (SPL)/NOZZLE (NZZ) expression was nearly abolished in a Cucumis sativus (cucumber) mutant with severely defective anther and ovule development. CsSPL was expressed specifically in the developing anthers and ovules. Knock‐down of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development. Importantly, CsSPL directly interacted with CsWUS (WUSCHEL) in the nucellus and YABBY family genes in integuments, and positively regulated CsWUS expression, meanwhile the HD‐ZIP III gene CsPHB (PHABULOSA), expressed specifically in the nucellus, promoted CsSPL expression by binding to the CsSPL promoter. Thus, CsSPL acts as an adaptor to link CsPHB and CsWUS functioning, underpinning a previously unidentified regulatory pathway orchestrating sex organ development in planta. In addition, auxin accumulation was reduced in the reproductive organs of CsSPL knock‐down plants. Biochemical analyses further showed that CsSPL stimulated the expression of AUXIN RESPONSE FACTOR 3 (CsARF3), and was positively regulated by CsARF13 during reproductive organ development, indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.