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The increment in global temperature reduces crop productivity, which in turn threatens food security. Currently, most of our food supply is produced by plants and the human population is estimated to reach 9 billion by 2050. Gaining insights into how plants navigate heat stress in their reproductive phase is essential for effectively overseeing the future of agricultural productivity. The reproductive success of numerous plant species can be jeopardized by just one exceptionally hot day. While the effects of heat stress on seedlings germination and root development have been extensively investigated, studies on reproduction are limited. The intricate processes of gamete development and fertilization unfold within a brief timeframe, largely concealed within the flower. Nonetheless, heat stress is known to have important effects on reproduction. Considering that heat stress typically affects both male and female reproductive structures concurrently, it remains crucial to identify cultivars with thermotolerance. In such cultivars, ovules and pollen can successfully undergo development despite the challenges posed by heat stress, enabling the completion of the fertilization process and resulting in a robust seed yield. Hereby, we review the current understanding of the molecular mechanisms underlying plant resistance to abiotic heat stress, focusing on the reproductive process in the model systems of Arabidopsis and Oryza sativa.

Key message Overview of the current understanding of PG development, PT growth and the role of AGPs in these processes. The pollen grain (PG) is a complex structure composed of three cells: the vegetative cell which develops into a pollen tube (PT) and two sperm cells that will fuse with the egg cell and central cell, giving rise to the embryo and endosperm, respectively. This resilient gametophyte is constantly subjected to selective pressures, leading to a diverse range of characteristics, with one of its defining features being the pollen cell wall. In this review, we closely examine the developmental stages of PG formation and PT growth, with a specific focus on the dynamic roles of arabinogalactan-proteins (AGPs) throughout these processes. AGPs are initially present in pollen mother cells and persist throughout PT growth. In the early stages, AGPs play a crucial role in primexine anchoring, followed by nexine and intine formation as well as cellulose deposition, thereby providing essential structural support to the PG. As PGs mature, AGPs continue to be essential, as their absence often leads to the collapse of PGs before they reach full maturity. Moreover, the absence of AGPs during PT growth leads to abnormal growth patterns, likely due to disruptions of cellulose, callose, and F-actin deposition, as well as perturbations in calcium ion (Ca 2+ ) signalling. Understanding the intricate interplay between AGPs and PG development sheds light on the underlying mechanisms that drive reproductive success and highlights the indispensable role of AGPs in ensuring the integrity and functionality of PGs.

Summary 483 I. Introduction 483 II. Progress of research on pollen wall development 485 III. The developmental role of the special cell wall 487 IV. Meiosis and the establishment of microspore symmetry 489 V. The origins of the exine during the tetrad stage 490 VI. The free microspore stage to pollen maturation 495 VII. Conclusions 495 Acknowledgements 496 References 496 The outer pollen wall, or exine, is more structurally complex than any other plant cell wall, comprising several distinct layers, each with its own organizational pattern. Since elucidation of the basic events of pollen wall ontogeny using electron microscopy in the 1970s, knowledge of their developmental genetics has increased enormously. However, self-assembly processes that are not under direct genetic control also play an important role in pollen wall patterning. This review integrates ultrastructural and developmental findings with recent models for self-assembly in an attempt to understand the origins of the morphological complexity and diversity that underpin the science of palynology.

Background Pollen development is central for plant reproduction and is assisted by changes of the transcriptome and proteome. At the same time, pollen development and viability is largely sensitive to stress, particularly to elevated temperatures. The transcriptomic and proteomic changes during pollen development and of different stages in response to elevated temperature was targeted to define the underlying molecular principles. Results The analysis of the transcriptome and proteome of Solanum lycopersicum pollen at tetrad, post-meiotic and mature stage before and after heat stress yielded a decline of the transcriptome but an increase of the proteome size throughout pollen development. Comparison of the transcriptome and proteome led to the discovery of two modes defined as direct and delayed translation. Here, genes of distinct functional processes are under the control of direct and delayed translation. The response of pollen to elevated temperature occurs rather at proteome, but not as drastic at the transcriptome level. Heat shock proteins, proteasome subunits, ribosomal proteins and eukaryotic initiation factors are most affected. On the example of heat shock proteins we demonstrate a decoupling of transcript and protein levels as well as a distinct regulation between the developmental stages. Conclusions The transcriptome and proteome of developing pollen undergo drastic changes in composition and quantity. Changes at the proteome level are a result of two modes assigned as direct and delayed translation. The response of pollen to elevated temperature is mainly regulated at the proteome level, whereby proteins related to synthesis and degradation of proteins are most responsive and might play a central role in the heat stress response of pollen.

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 tapetumexpressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

Key messageHexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for mutant analysis in wheat.Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize Male sterile 45 (Ms45) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of Ms45 gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat Ms45 homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of Ms45 gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species. Ms45 based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.

Background and AimsThe arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated.MethodsReal-time reverse transcription–PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing.Key ResultsThe BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues.ConclusionsThe phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix.

Background Seedless grapes are in high demand for fresh and dry fruit consumption. Seedlessness in grapes ( Vitis vinifera L.) is triggered by two different mechanisms: stenospermocarpy and parthenocarpy. However, the key regulators of seed development and their targets in grapes are not well characterized. The present study used the seeded grape hybrid ARI 516 and its seedless mutant to understand the molecular mechanisms controlling the seedless phenotype in grapes. Results Gametogenesis studies demonstrated that the seedless mutant exhibits pollen sterility due to abnormal pollen morphology, significantly low viability, and a complete lack of germination ability. The macrogametophyte in the seedless mutant was significantly smaller than in ARI 516. Transcriptomic comparisons were performed during three developmental stages, including pre-flowering stage E-L 15, anthesis stage E-L 23, and berry formation E-L 31, to study altered developmental processes in the seedless mutant and ARI 516. Genes downregulated in the seedless mutant were enriched in the male gametophyte development-related pathways, which may cause pollen sterility. The RNAseq results were validated by qRT-PCR. Genome sequence data was also used to identify induced mutations in the seedless mutant, which revealed three homozygous and 25 heterozygous InDels in the genes related to male gametophyte development. RNAseq and genome sequencing data collectively indicate parthenocarpic seedless phenotype due to aberrant developmental and physiological processes involved in pollen formation, maturation and germination in the seedless mutant of ARI 516. Conclusion The study showed the downregulation of transcription factors and their target genes involved in cell division, gibberellin biosynthesis and signalling, cell wall development, organization, and pollen germination. This study represents a comprehensive attempt to identify putative candidate genes associated with parthenocarpic pollen sterility in grapes using genomic approaches.

Background Artemisia vulgaris L. (Asteraceae family), a wind-pollinated perennial weed, is a significant source of allergenic pollen, responsible for respiratory allergies in late summer. Six allergenic proteins—Art v 1, Art v 2, Art v 3, Art v 4, Art v 5, and Art v 6—have been identified in A. vulgaris pollen. However, knowledge regarding significant scientific questions, such as where, when, and in what quantities these proteins are expressed, remains limited. Results This study fills these gaps by determining the expression profiles of all six genes encoding allergenic proteins in mugwort pollen. The real-time PCR method was used to analyze the level of allergen expression at three stages of pollen development: microsporocytes before meiosis (stage I), tetrads after meiosis (stage II), and enclosed mature pollen (stage IIIa), as well as isolated mature pollen grains (stage IIIb). The results showed that the expression levels of the most immunogenic allergens, Art v 1 and Art v 3, are extremely high at stage IIIa but very low at stage IIIb, suggesting their production occurs in mature inflorescence tissues. The expression levels of these two major allergens are significantly higher than those of other minor allergens in Artemisia pollen. Art v 2 is expressed in both pollen grains and anther tissues, whereas Art v 5 and Art v 6 are transcribed only in mature pollen, with no noticeable expression in earlier stages of pollen development. Art v 4 expression begins at the tetrad stage and reaches its highest levels in mature pollen grains, where it surpasses the expression level of all other allergens. Conclusions Our study provides new insights into allergen expression in A. vulgaris pollen, highlighting significant quantitative and developmental differences. These findings may help explain why some proteins are more likely to cause pollen allergies than others.

Background The myeloblastosis (MYB) gene family plays crucial roles in the development of anthers and the establishment of pollen morphology during plant growth. However, little is known about the role of MYB transcription factors in pollen development in alfalfa ( Medicago sativa L.). Results In this study, we identified 161 MsMYBs in the alfalfa genome, including 34 1R-MYBs, 123 R2R3-MYBs, 3 3R-MYBs, and 1 4R-MYBs (categorized by the number of repeats). These were classified into six subfamilies based on the phylogenetic analysis, conserved structural domains, and gene structures. All MsMYBs were predicted to be hydrophilic and localized in the cell nucleus. The promoter regions contained three classes of cis-regulatory elements related to pollen development, as well as a variable set of functionally diverse elements, including hormone responsiveness, growth and development, and stress responsiveness elements. A transcriptome and qRT-PCR analysis revealed 12 MsMYBs with anther-specific expression and exhibited distinct expression patterns. Some MsMYBs showed a close phylogenetic relationship with Arabidopsis MYBs related to pollen development, such as MsMYB49 and MsMYB100 , were found to be localized in the nucleus upon subcellular localization analysis. This genetic proximity suggests a potential role for these MsMYBs in the developmental processes of pollen. Conclusions This study provides a comprehensive understanding of MsMYBs in alfalfa and elucidates their potential roles and expression patterns in pollen development.