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 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.

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.

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.

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 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.

Cover crops, hormones and herbicides: Priming an integrated weed management strategy.

Herbicide weed resistance has been a major issue of conventional global row crop agriculture for decades. Still current strategies and novel technologies available to address weed resistance are mainly herbicide-based. Thus, there is a need for innovative means of integrated weed management strategies. Our approach proposed herein integrates cover crops, plant hormones and pre-emergence (PRE) herbicides as part of weed management programs. Plant hormones such as gibberellic acid (GA3) and abscisic acid (ABA) have the potential to induce seed germination and seed dormancy, respectively. Prior to crop emergence, plant hormones are tank mixed with PRE herbicides and sprayed to cover crop residue. Two strategies are proposed (1) PRE herbicides + GA3 and (2) PRE herbicide + ABA. The hormones provide different results; GA3 is likely to stimulate a more uniform weed seed germination, thus enhancing efficacy of PRE herbicides. Conversely, ABA could promote weed seed dormancy, reducing selection pressure and weed infestations until crop canopy closure. Much research is needed to understand the impact of hormones on weed and crop species, optimize products and rates, and compatibility of hormones with herbicides and cover crops. If successful, this approach could open a new opportunity for agricultural business, enhance farming sustainability by reducing dependence on herbicides and minimizing agronomic, economic and environmental issues related to weed resistance.

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.

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.

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.

Transient Heat Stress During Early Seed Development Primes Germination and Seedling Establishment in Rice.

Rice yield is highly sensitive to increased temperature. Given the trend of increasing global temperatures, this sensitivity to higher temperatures poses a challenge for achieving global food security. Early seed development in rice is highly sensitive to unfavorable environmental conditions. Heat stress (HS) during this stage decreases seed size and fertility, thus reducing yield. Here, we explore the transgenerational phenotypic consequences of HS during early seed development on seed viability, germination, and establishment. To elucidate the impact of HS on the developmental events in post-zygotic rice seeds, we imposed moderate (35°C) and severe (39°C) HS treatments initiated 1 day after fertilization and maintained for 24, 48, or 72 h. The transient HS treatments altered the initiation of endosperm (ED) cellularization, seed size and/or the duration of spikelet ripening. Notably, seeds exposed to 24 and 48 h moderate HS exhibited higher germination rate compared to seeds derived from plants grown under control or severe HS. A short-term HS resulted in altered expression of Gibberellin (GA) and ABA biosynthesis genes during early seed development, and GA and ABA levels and starch content at maturity. The increased germination rate after 24 of moderate HS could be due to altered ABA sensitivity and/or increased starch level. Our findings on the impact of transient HS on hormone homeostasis provide an experimental framework to elucidate the underlying molecular and metabolic pathways.

Epigenetic responses to abiotic stresses during reproductive development in cereals.

KEY MESSAGE: Overview of current understanding of epigenetic alterations after abiotic stresses during reproductive development in cereals. Abiotic stresses, including heat, drought, cold, flooding, and salinity, negatively impact crop productivity. Various stages during reproductive development are especially sensitive to environmental stresses, which may lead to complete sterility and severe yield losses. Plants exhibit diverse responses to ameliorate stress damage. Changes in DNA methylation, histone modification as well as regulation of small RNA and long noncoding RNA pathways have been shown to represent key modulators in plant stress responses. During reproductive development in cereals, various protein complexes controlling histone and DNA methylation have been identified, revealing conserved and novel mechanisms regulating abiotic stress responses in cereals and other plant species. New findings highlight the role of transposable elements during stress periods. Here, we review our current understanding of epigenetic stress responses during male and female gametophyte formation (germline development), fertilization, early seed devolvement, and seed maturation in cereals. An integrative model of epigenetic responses during reproductive development in cereals is proposed, emphasizing the role of DNA methylation and histone modifications during abiotic stresses.

Tracking maize pollen development by the Leaf Collar Method.

KEY MESSAGE: An easy and highly reproducible nondestructive method named the Leaf Collar Method is described to identify and characterize the different stages of pollen development in maize. In plants, many cellular events such as meiosis, asymmetric cell division, cell cycle regulation, cell fate determination, nucleus movement, vacuole formation, chromatin condensation and epigenetic modifications take place during pollen development. In maize, pollen development occurs in tassels that are confined within the internal stalk of the plant. Hence, identification of the different pollen developmental stages as a tool to investigate above biological processes is impossible without dissecting the entire plant. Therefore, an efficient and reproducible method is necessary to isolate homogeneous cell populations at individual stages throughout pollen development without destroying the plant. Here, we describe a method to identify the various stages of pollen development in maize. Using the Leaf Collar Method in the maize inbreed line B73, we have determined the duration of each stage from pollen mother cells before meiosis to mature tricellular pollen. Anther and tassel size as well as percentage of pollen stages were correlated with vegetative stages, which are easily recognized. The identification of stage-specific genes indicates the reproducibility of the method. In summary, we present an easy and highly reproducible nondestructive method to identify and characterize the different stages of pollen development in maize. This method now opens the way for many subsequent physiological, morphological and molecular analyses to study, for instance, transcriptomics, metabolomics, DNA methylation and chromatin patterns during normal and stressful conditions throughout pollen development in one of the economically most important grass species.

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 Ovate Family Protein 2 (OFP2) alters hormonal homeostasis and vasculature development.

OFP (Ovate Family Protein) is a transcription factor family found only in plants. In dicots, OFPs control fruit shape and secondary cell wall biosynthesis. OFPs are also thought to function through interactions with KNOX and BELL transcription factors. Here, we have functionally characterized OsOFP2, a member of the OFP subgroup associated with regulating fruit shape. OsOFP2 was found to localize to the nucleus and to the cytosol. A putative nuclear export signal was identified within the OVATE domain and was required for the localization of OsOFP2 to distinct cytosolic spots. Rice plants overexpressing OsOFP2 were reduced in height and exhibited altered leaf morphology, seed shape, and positioning of vascular bundles in stems. Transcriptome analysis indicated disruptions of genes associated with vasculature development, lignin biosynthesis, and hormone homeostasis. Reduced expression of the gibberellin biosynthesis gene GA 20-oxidase 7 coincided with lower gibberellin content in OsOFP2 overexpression lines. Also, we found that OsOFP2 was expressed in plant vasculature and determined that putative vascular development KNOX and BELL proteins interact with OsOFP2. KNOX and BELL genes are known to suppress gibberellin biosynthesis through GA20ox gene regulation and can restrict lignin biosynthesis. We propose that OsOFP2 could modulate KNOX-BELL function to control diverse aspects of development including vasculature development.