Heat stress at the bicellular stage inhibits sperm cell development and transport into pollen tubes.

For successful double fertilization in flowering plants (angiosperms), pollen tubes deliver 2 nonmotile sperm cells toward female gametes (egg and central cell, respectively). Heatwaves, especially during the reproduction period, threaten male gametophyte (pollen) development, resulting in severe yield losses. Using maize (Zea mays) as a crop and grass model system, we found strong seed set reduction when moderate heat stress was applied for 2 d during the uni- and bicellular stages of pollen development. We show that heat stress accelerates pollen development and impairs pollen germination capabilities when applied at the unicellular stage. Heat stress at the bicellular stage impairs sperm cell development and transport into pollen tubes. To understand the course of the latter defects, we used marker lines and analyzed the transcriptomes of isolated sperm cells. Heat stress affected the expression of genes associated with transcription, RNA processing and translation, DNA replication, and the cell cycle. This included the genes encoding centromeric histone 3 (CENH3) and α-tubulin. Most genes that were misregulated encode proteins involved in the transition from metaphase to anaphase during pollen mitosis II. Heat stress also activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. In summary, misregulation of the identified genes during heat stress at the bicellular stage results in sperm cell development and transport defects ultimately leading to sterility.

Maize stigmas react differently to self- and cross-pollination and fungal invasion.

During sexual reproduction in flowering plants, tip-growing pollen tubes travel from the stigma inside the maternal tissues of the pistil towards ovules. In maize (Zea mays L.), the stigma is highly elongated, forming thread-like strands known as silks. Only compatible pollen tubes successfully penetrate and grow through the transmitting tract of the silk to reach the ovules. Like pollen, fungal spores germinate at the surface of silks and generate tube-like structures (hyphae) penetrating silk tissue. To elucidate commonalities and differences between silk responses to these distinctive invading cells, we compared growth behavior of the various invaders as well as the silk transcriptome after self-pollination, cross-pollination and infection using two different fungi. We report that self-pollination triggers mainly senescence genes, whereas incompatible pollen from Tripsacum dactyloides leads to downregulation of rehydration, microtubule, and cell wall-related genes, explaining the slower pollen tube growth and arrest. Invasion by the ascomycete Fusarium graminearum triggers numerous defense responses including the activation of monolignol biosynthesis and NAC as well as WRKY transcription factor genes, whereas responses to the basidiomycete Ustilago maydis are generally much weaker. We present evidence that incompatible pollination and fungal infection trigger transcriptional reprograming of maize silks cell wall. Pathogen invasion also activates the phytoalexin biosynthesis pathway.

Global identification of LIM genes in response to different heat stress regimes in Lactuca sativa.

BACKGROUND: LIM (Lineage-11 (LIN-11), Insulin-1 (ISL-1), and Mechanotransduction-3 (MEC-3)) genes belong to a family that hold ubiquitous properties contributing to organ, seed, and pollen development as well as developmental and cellular responses to biotic and abiotic stresses. Lettuce (Lactuca sativa) is a highly consumed vegetable crop susceptible heat stress. High temperatures limit lettuce's overall yield, quality and marketability. Lettuce LIM genes have not been identified and their role in response to high temperatures is not known. Aiming to identify potential new targets for thermoresilience, we searched for LIM genes in lettuce and compared them with orthologous of several dicotyledons and monocotyledons plant species. RESULTS: We identified fourteen lettuce LIM genes distributed into eight different subgroups using a genome-wide analysis strategy. Three belonging to DAR (DA means "large" in Chinese) class I, two DAR class II, one in the WLIM1, two in the WLIM2, one in the PLIM1, two in the PLIM2 class, one ßLIM and two δLIMs. No DAR-like were identified in any of the species analyzed including lettuce. Interestingly, unlike other gene families in lettuce which underwent large genome tandem duplications, LIM genes did not increase in number compared to other plant species. The response to heat stress induced a dynamic transcriptional response on LsLIM genes. All heat stress regimes, including night stress, day stress and day and night stress were largely responsible for changes in LIM transcriptional expression. CONCLUSIONS: Our global analysis at the genome level provides a detailed identification of LIM genes in lettuce and other dicotyledonous and monocotyledonous plant species. Gene structure, physical and chemical properties as well as chromosomal location and Cis-regulatory element analysis together with our gene expression analysis under different temperature regimes identified LsWLIM1, LsWLIM2b, LsDAR3 and LsDAR5 as candidate genes that could be used by breeding programs aiming to produce lettuce varieties able to withstand high temperatures.

Genome-wide gene network uncover temporal and spatial changes of genes in auxin homeostasis during fruit development in strawberry (F. × ananassa).

BACKGROUND: The plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle. RESULTS: Genes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Heat Shock Transcription Factor and Heat Shock Proteins (HSF/HSP), APETALA2/Ethylene Responsive Factor (AP2/ERF) and MYB transcription factor groups. CONCLUSIONS: These results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation.

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.

The Arabidopsis HDZIP class II transcription factor ABA INSENSITIVE TO GROWTH 1 functions in leaf development.

Leaf laminar growth and adaxial-abaxial boundary formation are fundamental outcomes of plant development. Boundary and laminar growth coordinate the further patterning and growth of the leaf, directing the differentiation of cell types within the top and bottom domains and promoting initiation of lateral organs along their adaxial or abaxial axis. Leaf adaxial-abaxial polarity specification and laminar outgrowth are regulated by two transcription factors, REVOLUTA (REV) and KANADI (KAN). ABA INSENSITIVE TO GROWTH 1 (ABIG1) encodes a HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) class II transcription factor and is a direct target of the adaxial-abaxial regulators REV and KAN. To investigate the role of ABIG1 in leaf development and in the establishment of polarity, we examined the phenotypes of both gain-of-function and loss-of-function mutants. Through genetic interaction analysis with REV and KAN mutants, we determined that ABIG1 plays a role in leaf laminar growth as well as in adaxial-abaxial polarity establishment. Genetic and physical interaction assays showed that ABIG1 interacts with the transcriptional TOPLESS corepressor. This study provides new evidence that ABIG1, another HD-ZIP II, facilitates growth through the corepressor TOPLESS.

Genome-wide identification of heat shock factors and heat shock proteins in response to UV and high intensity light stress in lettuce.

BACKGROUND: Heat shock factors (Hsfs) and Heat shock proteins (Hsps) belong to an essential group of molecular regulators involved in controlling cellular processes under normal and stress conditions. The role of Hsfs and Hsps is well known in model plant species under diverse stress conditions. While plants Hsfs are vital components of the signal transduction response to maintain cellular homeostasis, Hsps function as chaperones helping to maintain folding of damaged and newly formed proteins during stress conditions. In lettuce (Lactuca sativa), a highly consumed vegetable crop grown in the field and in hydroponic systems, the role of these gene families in response to artificial light is not well characterized. RESULTS: Using a genome-wide analysis approach, we identified 32 Hsfs and 22 small heat shock proteins (LsHsps) in lettuce, some of which do not have orthologs in Arabidopsis, poplar, and rice. LsHsp60s, LsHsp90s, and LsHsp100s are highly conserved among dicot and monocot species. Surprisingly, LsHsp70s have three times more members than Arabidopsis and two times more than rice. Interestingly, the lettuce genome triplication did not contribute to the increased number of LsHsp70s genes. The large number of LsHsp70s was the result of genome tandem duplication. Chromosomal distribution analysis shows larger tandem repeats of LsHsp70s genes in Chr1, Chr7, Chr8, and Chr9. At the transcriptional level, some genes of the LsHsfs, LsHsps, LsHsp60s, and LsHsp70s families were highly responsive to UV and high intensity light stress, in contrast to LsHsp90s and LsHsp100s which did not respond to a light stimulus. CONCLUSIONS: Our genome-wide analysis provides a detailed identification of Hsfs and Hsps in lettuce. Chromosomal location and syntenic region analysis together with our transcriptional analysis under different light conditions provide candidate genes for breeding programs aiming to produce lettuce varieties able to grow healthy under hydroponic systems that use artificial light.

Male Sterility in Maize after Transient Heat Stress during the Tetrad Stage of Pollen Development.

Shifts in the duration and intensity of ambient temperature impair plant development and reproduction, particularly male gametogenesis. Stress exposure causes meiotic defects or premature spore abortion in male reproductive organs, leading to male sterility. However, little is known about the mechanisms underlying stress and male sterility. To elucidate these mechanisms, we imposed a moderate transient heat stress on maize (Zea mays) plants at the tetrad stage of pollen development. After completion of pollen development at optimal conditions, stress responses were assessed in mature pollen. Transient heat stress resulted in reduced starch content, decreased enzymatic activity, and reduced pollen germination, resulting in sterility. A transcriptomic comparison pointed toward misregulation of starch, lipid, and energy biosynthesis-related genes. Metabolomic studies showed an increase of Suc and its monosaccharide components, as well as a reduction in pyruvate. Lipidomic analysis showed increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids. In contrast, the majority of genes involved in developmental processes such as those required for auxin and unfolded protein responses, signaling, and cell wall biosynthesis remained unaltered. It is noteworthy that changes in the regulation of transcriptional and metabolic pathway genes, as well as heat stress proteins, remained altered even though pollen could recover during further development at optimal conditions. In conclusion, our findings demonstrate that a short moderate heat stress during the highly susceptible tetrad stage strongly affects basic metabolic pathways and thus generates germination-defective pollen, ultimately leading to severe yield losses in maize.

Overexpression of an evolutionarily conserved drought-responsive sugarcane gene enhances salinity and drought resilience.

BACKGROUND AND AIMS: Improving drought adaptation is more pressing for crops such as sugarcane, rice, wheat and maize, given the high dependence of these crops on irrigation. One option for enhancing adaptation to water limitation in plants is by transgenic approaches. An increasing number of genes that are associated with mechanisms used by plants to cope with water scarcity have been discovered. Genes encoding proteins with unknown functions comprise a relevant fraction of the genes that are modulated by drought. We characterized a gene in response to environmental stresses to gain insight into the unknown fraction of the sugarcane genome. Scdr2 (Sugarcane drought-responsive 2) encodes a small protein and shares highly conserved sequences within monocots, dicots, algae and fungi. METHODS: Plants overexpressing the Scdr2 sugarcane gene were examined in response to salinity and drought. Measurements of the gas exchange parameters, germination rate, water content, dry mass and oxidative damage were performed. Seeds as well as juvenile plants were used to explore the resilience level of the transgenic plants when compared with wild-type plants. KEY RESULTS: Overexpression of Scdr2 enhanced germination rates in tobacco seeds under drought and salinity conditions. Juvenile transgenic plants overexpressing Scdr2 and subjected to drought and salinity stresses showed higher photosynthesis levels, internal CO2 concentration and stomatal conductance, reduced accumulation of hydrogen peroxide in the leaves, no penalty for photosystem II and faster recovery after submission to both stress conditions. Respiration was not strongly affected by both stresses in the Scdr2 transgenic plants, whereas wild-type plants exhibited increased respiration rates. CONCLUSIONS: Scdr2 is involved in the response mechanism to abiotic stresses. Higher levels of Scdr2 enhanced resilience to salinity and drought, and this protection correlated with reduced oxidative damage. Scdr2 confers, at the physiological level, advantages to climate limitations. Therefore, Scdr2 is a potential target for improving sugarcane resilience to abiotic stress.

Heat stress yields a unique MADS box transcription factor in determining seed size and thermal sensitivity.

Early seed development events are highly sensitive to increased temperature. This high sensitivity to a short-duration temperature spike reduces seed viability and seed size at maturity. The molecular basis of heat stress sensitivity during early seed development is not known. We selected rice (Oryza sativa), a highly heat-sensitive species, to explore this phenomenon. Here, we elucidate the molecular pathways that contribute to the heat sensitivity of a critical developmental window during which the endosperm transitions from syncytium to the cellularization stage in young seeds. A transcriptomic comparison of seeds exposed to moderate (35°C) and severe (39°C) heat stress with control (28°C) seeds identified a set of putative imprinted genes, which were down-regulated under severe heat stress. Several type I MADS box genes specifically expressed during the syncytial stage were differentially regulated under moderate and severe heat stress. The suppression and overaccumulation of these genes are associated with precocious and delayed cellularization under moderate and severe stress, respectively. We show that modulating the expression of OsMADS87, one of the heat-sensitive, imprinted genes associated with syncytial stage endosperm, regulates rice seed size. Transgenic seeds deficient in OsMADS87 exhibit accelerated endosperm cellularization. These seeds also have lower sensitivity to a moderate heat stress in terms of seed size reduction compared with seeds from wild-type plants and plants overexpressing OsMADS87 Our findings suggest that OsMADS87 and several other genes identified in this study could be potential targets for improving the thermal resilience of rice during reproductive development.

Rice fertilization-Independent Endosperm1 regulates seed size under heat stress by controlling early endosperm development.

Although heat stress reduces seed size in rice (Oryza sativa), little is known about the molecular mechanisms underlying the observed reduction in seed size and yield. To elucidate the mechanistic basis of heat sensitivity and reduced seed size, we imposed a moderate (34°C) and a high (42°C) heat stress treatment on developing rice seeds during the postfertilization stage. Both stress treatments reduced the final seed size. At a cellular level, the moderate heat stress resulted in precocious endosperm cellularization, whereas severe heat-stressed seeds failed to cellularize. Initiation of endosperm cellularization is a critical developmental transition required for normal seed development, and it is controlled by Polycomb Repressive Complex2 (PRC2) in Arabidopsis (Arabidopsis thaliana). We observed that a member of PRC2 called Fertilization-Independent Endosperm1 (OsFIE1) was sensitive to temperature changes, and its expression was negatively correlated with the duration of the syncytial stage during heat stress. Seeds from plants overexpressing OsFIE1 had reduced seed size and exhibited precocious cellularization. The DNA methylation status and a repressive histone modification of OsFIE1 were observed to be temperature sensitive. Our data suggested that the thermal sensitivity of seed enlargement could partly be caused by altered epigenetic regulation of endosperm development during the transition from the syncytial to the cellularized state.

Transcriptome analysis highlights changes in the leaves of maize plants cultivated in acidic soil containing toxic levels of Al(3+).

Soil acidity limits crop yields worldwide and is a common result of aluminum (Al) phytotoxicity, which is known to inhibit root growth. Here, we compared the transcriptome of leaves from maize seedlings grown under control conditions (soil without free Al) and under acidic soil containing toxic levels of Al. This study reports, for the first time, the complex transcriptional changes that occur in the leaves of maize plants grown in acidic soil with phytotoxic levels of Al. Our data indicate that 668 genes were differentially expressed in the leaves of plants grown in acidic soil, which is significantly greater than that observed in our previous work with roots. Genes encoding TCA cycle enzymes were upregulated, although no specific transporter of organic acids was differentially expressed in leaves. We also provide evidence for positive roles for auxin and brassinosteroids in Al tolerance, whereas gibberellin and jasmonate may have negative roles. Our data indicate that plant responses to acidic soil with high Al content are not restricted to the root; tolerance mechanisms are also displayed in the aerial parts of the plant, thus indicating that the entire plant responds to stress.

Cell size differences affect photosynthetic capacity in a Mesoamerican and an Andean genotype of Phaseolus vulgaris L.

The efficiency of CO2 flux in the leaf is hindered by a several structural and biochemical barriers which affect the overall net photosynthesis. However, the dearth of information about the genetic control of these features is limiting our ability for genetic manipulation. We performed a comparative analysis between three-week-old plants of a Mesoamerican and an Andean cultivar of Phaseolus vulgaris at variable light and CO2 levels. The Mesoamerican bean had higher photosynthetic rate, maximum rate of rubisco carboxylase activity and maximum rate of photosynthetic electron transport at light saturation conditions than its Andean counterpart. Leaf anatomy comparison between genotypes showed that the Mesoamerican bean had smaller cell sizes than the Andean bean. Smaller epidermal cells in the Mesoamerican bean resulted in higher stomata density and consequently higher stomatal conductance for water vapor and CO2 than in the Andean bean. Likewise, smaller palisade and spongy mesophyll cells in the Mesoamerican than in the Andean bean increased the cell surface area per unit of volume and consequently increased mesophyll conductance. Finally, smaller cells in the Mesoamerican also increased chlorophyll and protein content per unit of leaf area. In summary, we show that different cell sizes controls the overall net photosynthesis and could be used as a target for genetic manipulation to improve photosynthesis.

Examining preharvest genetic and morphological factors contributing to lettuce (Lactuca sativa L.) shelf-life.

Lettuce is a highly perishable horticultural crop with a relatively short shelf-life that limits its commercial value and contributes to food waste. Postharvest senescence varies with influences of both environmental and genetic factors. From a larger pool of romaine lettuce genotypes, we identified three genotypes with variable shelf lives and evaluated their leaf morphology characteristics and transcriptomic profiles at preharvest to predict postharvest quality. Breeding line 60184 had the shortest shelf-life (SSL), cultivar 'Manatee' had an intermediate shelf-life (ISL), and 'Okeechobee' had the longest shelf-life (LSL). We observed significantly larger leaf lamina thickness and higher stomatal index in the SSL genotypes relative to the LSL cultivar. To identify molecular indicators of shelf-life, we used a transcriptional approach between two of the contrasting genotypes, breeding line 60184 and cultivar 'Okeechobee' at preharvest. We identified 552 upregulated and 315 downregulated differentially expressed genes between the genotypes, from which 27% of them had an Arabidopsis thaliana ortholog previously characterized as senescence associated genes (SAGs). Notably, we identified several SAGs including several related to jasmonate ZIM-domain jasmonic acid signaling, chlorophyll a-b binding, and cell wall modification including pectate lyases and expansins. This study presented an innovative approach for identifying preharvest molecular factors linked to postharvest traits for prolonged shelf.

Heat stress during seed development leads to impaired physiological function and plasticity in seed oil accumulation in Camelina sativa.

Camelina sativa, a member of the Brassicaceae, is a low-cost, renewable oilseed crop that produces seeds up to 40% oil by weight with high potential for use in food, feed, and biofuel applications. Camelina seeds contain high levels of the fatty acids α-linolenic acid (C18:3), linoleic acid (C18:2), oleic acid (C18:1), and gondoic acid (C20:1), which have high nutritional and industrial value. The impact of climate change, especially increased frequency and amplitude of heat waves, poses a serious threat to crop productivity. In this study, we evaluated the effect of elevated temperatures post-anthesis on the developing seeds of C. sativa and performed physiological, morphological, and chemical characterizations at 7, 14, 21, and 28 days post-anthesis (DPA), as well as at maturity. While the seed oil accumulation peaked at 21 DPA under control conditions, reaching 406mg/g dry weight, under heat stress it was only 186mg/g. Physiologically, transpiration rate (E) and internal CO2 concentration (Ci) increased between 2 to 9 days post-stress imposition and overall net photosynthesis was impaired. Seed yield, seed weight, and oil content reduced by 84.5%, 38.5% and 54.1% respectively. We demonstrate that post-anthesis heat stress causes severe yield losses and developmental plasticity in fatty acid accumulation in oilseeds.

Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.

The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.

Unconventional routes to developing insect-resistant crops.

Concerns over widespread use of insecticides and heightened insect pest virulence under climate change continue to fuel the need for environmentally safe and sustainable control strategies. However, to develop such strategies, a better understanding of the molecular basis of plant-pest interactions is still needed. Despite decades of research investigating plant-insect interactions, few examples exist where underlying molecular mechanisms are well characterized, and even rarer are cases where this knowledge has been successfully applied to manage harmful agricultural pests. Consequently, the field appears to be static, urgently needing shifts in approaches to identify novel mechanisms by which insects colonize plants and plants avoid insect pressure. In this perspective, we outline necessary steps for advancing holistic methodologies that capture complex plant-insect molecular interactions. We highlight novel and underexploited approaches in plant-insect interaction research as essential routes to translate knowledge of underlying molecular mechanisms into durable pest control strategies, including embracing microbial partnerships, identifying what makes a plant an unsuitable host, capitalizing on tolerance of insect damage, and learning from cases where crop domestication and agronomic practices enhance pest virulence.

Genome-Wide Identification of Allele-Specific Gene Expression in a Parent-of-Origin Specific Manner.

Upon fertilization, normal endosperm and embryo development require the contribution of both the maternal and paternal genomes. However, certain genes are expressed in a parent-of-origin-dependent manner, an epigenetic phenomenon known as genomic imprinting. Despite the blast of new technologies and the crucial advances of the past decades in the epigenetics field, novel imprinted genes are yet to be discovered and thus key regulators of early seed development. Using rice plant as a model, we describe a method for the identification of imprinted genes based on an RNA-Seq approach, which allows the identification of maternal and paternal gene expression in a parent-of-origin-specific manner.

Analysis of Epigenetic Modifications During Vegetative and Reproductive Development in Cereals Using Chromatin Immunoprecipitation (ChIP).

The study of heritable genetic changes that do not implicate alterations in the DNA sequence-epigenetics-represents one of the most prolific and expanding fields in plant biology during the last two decades. With a focus on DNA methylation and histone modifications, recent advances also reported the identification of epigenetic regulatory mechanisms that control reproductive development in cereal crop plants. One of the most powerful methods to selectively study interactions between epigenetic factors or specific proteins bound to genomic DNA regions is called chromatin immunoprecipitation (ChIP). ChIP can be widely used to determine the presence of particular histones with posttranslational modifications at specific genomic regions or whether and where specific DNA-binding proteins including transcription factors interact with candidate target genes. ChIP is also an exciting tool to study and compare chromatin states under normal and stress conditions. Here, we present a detailed step-by-step ChIP assay to investigate epigenetic chromatin marks during vegetative and reproductive development in cereals. However, the method described here can be used for all plant tissues and plant species.