The Arabidopsis U–box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells,and its mutation leads to conditional male sterility

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male‐sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U–box/ARM repeat‐containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male‐sterility phenotype caused by the Atpub4 mutation is temperature‐dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4‐mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.     

Tomato SlIDA has a critical role in tomato fertilization by modifying reactive oxygen species homeostasis

Anther development and pollen tube elongation are key steps for pollination and fertilization. The timing and spatial distribution of reactive oxygen species (ROS) and programmed cell death are central to these processes, but the regulatory mechanism of ROS production is not well understood. Inflorescence deficient in abscission (IDA) is implicated in many plant development and responses to environmental stimuli. However, their role in reproductive development is still unknown. We generated tomato knockout lines (CR‐slida) of an IDA homolog (SlIDA), which is expressed in the tapetum, septum and pollen tube, and observed a severe defect in male gametes. Further analysis indicated that there was a programmed cell death defect in the tapetum and septum and a failure of anther dehiscence in the CR‐slida lines, likely related to insufficient ROS signal. Liquid chromatography‐tandem mass spectrometry identified mature SlIDA as a 14‐mer EPIP peptide, which was shown to be secreted, and a complementation experiment showed that application of a synthetic 14‐mer EPIP peptide rescued the CR‐slida defect and enhanced the ROS signal. Moreover, the application of the ROS scavengers diphenyleneiodonium or Mn‐TMPP suppressed peptide function. Collectively, our results revealed that SlIDA plays an essential role in pollen development and pollen tube elongation by modulating ROS homeostasis.     

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7–ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4′,6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified. Received: 14 August 1997 / Accepted: 7 October 1997     

Classic myrosinase‐dependent degradation of indole glucosinolate attenuates fumonisin B1‐induced programmed cell death in Arabidopsis

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1‐induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1‐induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col‐0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β‐thioglucoside glucohydrolase (TGG)‐deficient double mutant tgg1 tgg2, rather than atypical myrosinase‐deficient mutant pen2‐2, is more sensitive to FB1 than Col‐0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG‐dependent IGS hydrolysis is involved in FB1‐induced PCD. Indole‐3‐acetonitrile (IAN) and indole‐3‐carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS‐scavenging ability. Despite the involvement of indole‐3‐acetic acid (IAA) in restricting FB1‐induced PCD, feeding of IAN and I3C attenuated FB1‐induced PCD in the IAA receptor mutant tir1‐1 just as in Col‐0. Taken together, our results indicate that TGG‐catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA‐independent way.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

BAT1, a putative acyltransferase,modulates brassinosteroid levels in Arabidopsis

Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR‐related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full‐length cDNA over‐expressor (FOX) screen. Over‐expression of BAT1 resulted in typical BR‐deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6‐deoxotyphasterol, typhasterol and 6‐deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.