Pollen Development In Arabidopsis Research Articles

Ectopic expression of a thaumatin-like protein impairs the timely deposition and dissolution of callose during microsporogenesis, leading to microspore death and male sterility in Arabidopsis.

Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis.

PXL1 and SERKs act as receptor–coreceptor complexes for the CLE19 peptide to regulate pollen development

Gametophyte development in angiosperms occurs within diploid sporophytic structures and requires coordinated development; e.g., development of the male gametophyte pollen depends on the surrounding sporophytic tissue, the tapetum. The mechanisms underlying this interaction remain poorly characterized. The peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 19 (CLE19) plays a “braking” role in preventing the harmful overexpression of tapetum transcriptional regulators to ensure normal pollen development in Arabidopsis. However, the CLE19 receptor is unknown. Here, we show that CLE19 interacts directly with the PXY-LIKE1 (PXL1) ectodomain and induces PXL1 phosphorylation. PXL1 is also required for the function of CLE19 in maintaining the tapetal transcriptional regulation of pollen exine genes. Additionally, CLE19 induces the interactions of PXL1 with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors required for pollen development. We propose that PXL1 and SERKs act as receptor and coreceptor, respectively, of the extracellular CLE19 signal, thereby regulating tapetum gene expression and pollen development.

Overexpression of sesame polyketide synthase A leads to abnormal pollen development in Arabidopsis

BackgroundSesame is a great reservoir of bioactive constituents and unique antioxidant components. It is widely used for its nutritional and medicinal value. The expanding demand for sesame seeds is putting pressure on sesame breeders to develop high-yielding varieties. A hybrid breeding strategy based on male sterility is one of the most effective ways to increase the crop yield. To date, little is known about the genes and mechanism underlying sesame male fertility. Therefore, studies are being conducted to identify and functionally characterize key candidate genes involved in sesame pollen development. Polyketide synthases (PKSs) are critical enzymes involved in the biosynthesis of sporopollenin, the primary component of pollen exine. Their in planta functions are being investigated for applications in crop breeding.ResultsIn this study, we cloned the sesame POLYKETIDE SYNTHASE A (SiPKSA) and examined its function in male sterility. SiPKSA was specifically expressed in sesame flower buds, and its expression was significantly higher in sterile sesame anthers than in fertile anthers during the tetrad and microspore development stages. Furthermore, overexpression of SiPKSA in Arabidopsis caused male sterility in transgenic plants. Ultrastructural observation showed that the pollen grains of SiPKSA-overexpressing plants contained few cytoplasmic inclusions and exhibited an abnormal pollen wall structure, with a thicker exine layer compared to the wild type. In agreement with this, the expression of a set of sporopollenin biosynthesis-related genes and the contents of their fatty acids and phenolics were significantly altered in anthers of SiPKSA-overexpressing plants compared with wild type during anther development.ConclusionThese findings highlighted that overexpression of SiPKSA in Arabidopsis might cause male sterility through defective pollen wall formation. Moreover, they suggested that SiPKSA modulates vibrant pollen development via sporopollenin biosynthesis, and a defect in its regulation may induce male sterility. Therefore, genetic manipulation of SiPKSA might promote hybrid breeding in sesame and other crop species.

The N-terminal acetyltransferase Naa50 regulates tapetum degradation and pollen development in Arabidopsis

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9–11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

Tetraketide \u03b1-pyrone reductases in sporopollenin synthesis pathway in Gerbera hybrida: diversification of the minor function

The structurally robust biopolymer sporopollenin is the major constituent of the exine layer of pollen wall and plays a vital role in plant reproductive success. The sporopollenin precursors are synthesized through an ancient polyketide biosynthetic pathway consisting of a series of anther-specific enzymes that are widely present in all land plant lineages. Tetraketide α-pyrone reductase 1 (TKPR1) and TKPR2 are two reductases catalyzing the final reduction of the carbonyl group of the polyketide synthase-synthesized tetraketide intermediates to hydroxylated α-pyrone compounds, important precursors of sporopollenin. In contrast to the functional conservation of many sporopollenin biosynthesis associated genes confirmed in diverse plant species, TKPR2’s role has been addressed only in Arabidopsis, where it plays a minor role in sporopollenin biosynthesis. We identified in gerbera two non-anther-specific orthologues of AtTKPR2, Gerbera reductase 1 (GRED1) and GRED2. Their dramatically expanded expression pattern implies involvement in pathways outside of the sporopollenin pathway. In this study, we show that GRED1 and GRED2 are still involved in sporopollenin biosynthesis with a similar secondary role as AtTKPR2 in Arabidopsis. We further show that this secondary role does not relate to the promoter of the gene, AtTKPR2 cannot rescue pollen development in Arabidopsis even when controlled by the AtTKPR1 promoter. We also identified the gerbera orthologue of AtTKPR1, GTKPR1, and characterized its crucial role in gerbera pollen development. GTKPR1 is the predominant TKPR in gerbera pollen wall formation, in contrast to the minor roles GRED1 and GRED2. GTKPR1 is in fact an excellent target for engineering male-sterile gerbera cultivars in horticultural plant breeding.

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest β-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.

Arabidopsis Flowers Unlocked the Mechanism of Jasmonate Signaling.

The Arabidopsis male-sterile phenotype has been a wonderful model for jasmonate action in plants. It has allowed us to identify transcription factors that control gene expression during stamen and pollen maturation and provided for the discovery of the JAZ repressor proteins and the mechanism of jasmonate signaling. More recently, it has revealed intriguing details of the spatial localization of jasmonate synthesis and perception in stamen tissues. The extensive and thoughtful application of protein–protein interaction assays to identify JAZ-interacting partners has led to a much richer appreciation of the mechanisms by which jasmonate integrates with the actions of other hormones to regulate plant growth and physiological responses. This integration is strikingly evident in stamen and pollen development in Arabidopsis, which requires the actions of many hormones. Just as importantly, it is now evident that jasmonate has very different actions during flower development and reproduction in other plant species. This integration and diversity of action indicates that many exciting discoveries remain to be made in this area of jasmonate hormone signaling and response.

Tapetal Expression of BnaC.MAGL8.a Causes Male Sterility in Arabidopsis.

Monoacylglycerol lipase (MAGL) hydrolyzes monoacylglycerol, producing free fatty acid and glycerol. Although this enzyme has been shown to play important roles in mammal, its potential function in plants remains poorly understood. In a survey of the MAGL genes in Brassica napus, we found tapetal expression of BnaC.MAGL8.a, a homolog of AtMAGL8, results in male sterility in Arabidopsis thaliana. Retarded tapetal PCD and defective pollen wall were observed in the transgenic plants. The tapetal cells became vacuolated at stage 9, and then degenerated at stage 11. Most microspores degenerated with the tapetal cells, and only few pollen grains with an irregular-shaped exine layer were produced in the transgenic plants. Transcriptome analysis identified 398 differentially expressed genes. Most of them are involved in pollen development and stress response. ABORTED MICROSPORES and its downstream pollen wall biosynthesis genes were down-regulated, but genes related with reactive oxygen species homeostasis and jasmonates signaling were up-regulated in the transgenic plants. These results suggest that expression of BnaC.MAGL8.a in tapetum invokes stress response and impairs pollen development. The apparent phenotypic similarity between atgpat1 mutant and BnA9::BnaC.MAGL8.a transgenic plants lead us to propose a role for monoacylglycerol (MAG) in pollen development in Arabidopsis. Our study provides insights on not only the biological function of plant MAGL genes but also the role of MAG in pollen development.

PRX9 and PRX40 Are Extensin Peroxidases Essential for Maintaining Tapetum and Microspore Cell Wall Integrity during Arabidopsis Anther Development.

Pollen and microspore development are essential steps in the life cycle of all land plants that generate male gametes. Within flowering plants, pollen development occurs inside of the anther. Here, we report the identification of two class III peroxidase-encoding genes, PEROXIDASE9 (PRX9) and PRX40, that are genetically redundant and essential for proper anther and pollen development in Arabidopsis (Arabidopsis thaliana). Arabidopsis double mutants devoid of functional PRX9 and PRX40 are male sterile. The mutant anthers display swollen, hypertrophic tapetal cells and pollen grains, suggesting disrupted cell wall integrity. These phenotypes lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 likely cross-link extensins to contribute to tapetal cell wall integrity during anther development. This work suggests that PRX9 and PRX40 encode Arabidopsis extensin peroxidases and highlights the importance of extensin cross-linking during pollen development.

Transcriptomics of Arabidopsis sperm cells at single-cell resolution.

We present a detailed protocol for isolation of single sperm cells and transcriptome analysis to study variation in gene expression between sperm cells. Male gametophyte development in flowering plants begins with a microspore mother cell, which upon two consecutive cell divisions forms a mature pollen grain containing a vegetative nucleus and two sperm cells. Pollen development is a highly dynamic process, involving changes at both the transcriptome and epigenome levels of vegetative nuclei and the pair of sperm cells that have their own cytoplasm and nucleus. While the overall transcriptome of Arabidopsis pollen development is well documented, studies at single-cell level, in particular of sperm cells, are still lacking. Such studies would be essential to understand whether and how the two sperm cells are transcriptionally different, in particular once the pollen tube grows through the transmitting tissue of the pistil. Here we describe a detailed protocol for isolation of single sperm cells from growing pollen tubes and analysis of their transcriptome.

Application of rice microspore-preferred promoters to manipulate early pollen development in Arabidopsis: a heterologous system.

Rice microspore-promoters. Based on microarray data analyzed for developing anthers and pollen grains, we identified nine rice microspore-preferred (RMP) genes, designated RMP1 through RMP9. To extend their biotechnological applicability, we then investigated the activity of RMP promoters originating from monocotyledonous rice in a heterologous system of dicotyledonous Arabidopsis. Expression of GUS was significantly induced in transgenic plants from the microspore to the mature pollen stages and was driven by the RMP1, RMP3, RMP4, RMP5, and RMP9 promoters. We found it interesting that, whereas RMP2 and RMP6 directed GUS expression in microspore at the early unicellular and bicellular stages, RMP7 and RMP8 seemed to be expressed at the late tricellular and mature pollen stages. Moreover, GUS was expressed in seven promoters, RMP3 through RMP9, during the seedling stage, in immature leaves, cotyledons, and roots. To confirm microspore-specific expression, we used complementation analysis with an Arabidopsis male-specific gametophytic mutant, sidecar pollen-2 (scp-2), to verify the activity of three promoters. That mutant shows defects in microspore development prior to pollen mitosis I. These results provide strong evidence that the SIDECAR POLLEN gene, driven by RMP promoters, successfully complements the scp-2 mutation, and they strongly suggest that these promoters can potentially be applied for manipulating the expression of target genes at the microspore stage in various species.

MONENSIN SENSITIVITY1 (MON1)/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-Mediated Rab7 Activation Regulates Tapetal Programmed Cell Death and Pollen Development

Programmed cell death (PCD)-triggered degradation of plant tapetum is essential for microspore development and pollen coat formation; however, little is known about the cellular mechanism regulating tapetal PCD Here, we demonstrate that Rab7-mediated vacuolar transport of tapetum degradation-related cysteine proteases is crucial for tapetal PCD and pollen development in Arabidopsis (Arabidopsis thaliana), with the following evidence: (1) The monensin sensitivity1 (mon1) mutants, which are defective in Rab7 activation, showed impaired male fertility due to a combined defect in both tapetum and male gametophyte development. (2) In anthers, MON1 showed preferential high level expression in tapetal cell layers and pollen. (3) The mon1 mutants exhibited delayed tapetum degeneration and tapetal PCD, resulting in abnormal pollen coat formation and decreased male fertility. (4) MON1/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-mediated Rab7 activation was indispensable for vacuolar trafficking of tapetum degradation-related cysteine proteases, supporting that PCD-triggered tapetum degeneration requires Rab7-mediated vacuolar trafficking of these cysteine proteases. (5) MON1 mutations also resulted in defective pollen germination and tube growth. Taken together, tapetal PCD and pollen development require successful MON1/CCZ1-mediated vacuolar transport in Arabidopsis.

Biphasic regulation of the transcription factor ABORTED MICROSPORES (AMS) is essential for tapetum and pollen development in Arabidopsis.

Summary Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis‐associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation.An inducible‐AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild‐type RNA and protein expression.Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS‐regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation.We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum‐expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

IRE1, a component of the unfolded protein response signaling pathway, protects pollen development in Arabidopsis from heat stress.

The unfolded protein response (UPR) is activated by various stresses during vegetative development in Arabidopsis, but is constitutively active in anthers of unstressed plants. To understand the role of the UPR during reproductive development, we analyzed a double mutant, ire1a ire1b. The double mutant knocks out the RNA-splicing arm of the UPR signaling pathway. It is fertile at room temperature but male sterile at modestly elevated temperature (ET). The conditional male sterility in the mutant is a sporophytic trait, and when the double mutant was grown at ET, defects appeared in the structure of the tapetum. As a result, the tapetum in the double mutant failed to properly deposit the pollen coat at ET, which made pollen grains clump and prevented their normal dispersal. IRE1 is a dual protein kinase/ribonuclease involved in the splicing of bZIP60 mRNA, and through complementation analysis of various mutant forms of IRE1b it was demonstrated that the ribonuclease activity of IRE1 was required for protecting male fertility from ET. Itwas also found that overexpression of SEC31A rescued the conditional male sterility in the double mutant. SEC31A is involved in trafficking from the endoplasmic reticulum to Golgi and a major target of the IRE1-mediated UPR signaling in stressed seedlings. Thus, IRE1, a major component of the UPR, plays an important role in protecting pollen development from ET.

Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis.

Combinatorial interactions between LBD10 and LBD27 are essential for male gametophyte development in Arabidopsis

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encodes a unique class of transcription factors that play roles in diverse aspects of lateral organ development in plants. The Arabidopsis LBD gene family comprises 42 members and biological functions of most of the LBD genes are unknown. Our molecular genetic analysis and a variety of functional assays including expression analysis, genetic transmission and pollen viability assays, and pollen development analysis demonstrated that LBD10 co-acts with SIDECAR POLLEN(SCP)/LBD27 to control an early stage of microspore development but also plays a distinct role at later bicellular and tricellular pollen stages and that these 2 LBD genes are essential for Arabidopsis pollen development. We also showed that LBD10 and LBD27 interact with each other to be localized into the nucleus. Our subcellular localization analysis of LBD10 in comparison with LBD27 during pollen development indicated that regulated protein degradation may be involved in determining spatially and temporally distinct and overlapping expression patterns of these LBD transcription factors, contributing to distinct and combinatorial roles of LBD10 and LBD27 in Arabidopsis pollen development.

An endoplasmic reticulum magnesium transporter is essential for pollen development in Arabidopsis

Magnesium is one of the essential macro-elements for plant growth and development, participated in photosynthesis and various metabolic processes. The Mg-transport abilities of the AtMGT (Magnesium Transporter) genes were identified in bacteria or yeast mutant system. In our previous studies, both the AtMGT5 and AtMGT9 were found essential for pollen development in Arabidopsis. Here we report another AtMGT member, AtMGT4, which was localized to the endoplasmic reticulum, was essential for pollen development as well. AtMGT4 expressed notably in pollen grains from bicellular pollen stage to mature pollen stage. A T-DNA insertional mutant of the gene, named mgt4-1, showed pollen abortive phenotype, thus we could not get any homozygous mutant from progenies of self-crossed +/mgt4-1 plants. Meanwhile, nearly half of pollens in AtMGT4-RNAi transgenic lines were sterile, consistent with the phenotype of +/mgt4-1 mutant. Transgenic plants expressing AtMGT4 in the mgt4-1 background could recover the pollen fertility to the wild type. Together, our findings demonstrated that the disruption of AtMGT4 in Arabidopsis could cause a defect of pollen development. The visible pollen abortion appeared at bicellular pollen stage in +/mgt4-1.

Annexin5 plays a vital role in Arabidopsis pollen development via Ca2+-dependent membrane trafficking.

The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca2+ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca2+ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca2+-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca2+-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.

DELLA activity is required for successful pollen development in the Columbia ecotype of Arabidopsis

Excessive gibberellin (GA) signalling, mediated through the DELLA proteins, has a negative impact on plant fertility. Loss of DELLA activity in the monocot rice (Oryza sativa) causes complete male sterility, but not in the dicot model Arabidopsis (Arabidopsis thaliana) ecotype Landsberg erecta (Ler), in which DELLA function has been studied most extensively, leading to the assumption that DELLA activity is not essential for Arabidopsis pollen development. A novel DELLA fertility phenotype was identified in the Columbia (Col-0) ecotype that necessitates re-evaluation of the general conclusions drawn from Ler. Fertility phenotypes were compared between the Col-0 and Ler ecotypes under conditions of chemical and genetic GA overdose, including mutants in both ecotypes lacking the DELLA paralogues REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI). Ler displays a less severe fertility phenotype than Col-0 under GA treatment. Col-0 rga gai mutants, in contrast with the equivalent Ler phenotype, were entirely male sterile, caused by post-meiotic defects in pollen development, which were rescued by the reintroduction of DELLA into either the tapetum or developing pollen. We conclude that DELLA activity is essential for Arabidopsis pollen development. Differences between the fertility responses of Col-0 and Ler might be caused by differences in downstream signalling pathways or altered DELLA expression.

Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.