A pan-grass transcriptome reveals patterns of cellular divergence in crops.

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits1. Key traits that distinguish these species are mediated by specialized cell types2. Here we compare the transcriptomes of root cells in three grass species-Zea mays, Sorghum bicolor and Setaria viridis. We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others-driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.

Linking genome wide RNA sequencing with physio-biochemical and cytological responses to catalogue key genes and metabolic pathways for alkalinity stress tolerance in lentil (Lens culinaris Medikus).

BACKGROUND: Alkaline soils cause low productivity in crop plants including lentil. Alkalinity adaptation strategies in lentil were revealed when morpho-anatomical and physio-biochemical observations were correlated with transcriptomics analysis in tolerant (PDL-1) and sensitive (L-4076) cultivars at seedling stage. RESULTS: PDL-1 had lesser salt injury and performed better as compared to L-4076. Latter showed severe wilting symptoms and higher accumulation of Na+ and lower K+ in roots and shoots. PDL-1 performed better under high alkalinity stress which can be attributed to its higher mitotic index, more accumulation of K+ in roots and shoots and less aberrantly dividing cells. Also, antioxidant enzyme activities, osmolytes' accumulation, relative water content, membrane stability index and abscisic acid were higher in this cultivar. Differentially expressed genes (DEGs) related to these parameters were upregulated in tolerant genotypes compared to the sensitive one. Significantly up-regulated DEGs were found to be involved in abscisic acid (ABA) signalling and secondary metabolites synthesis. ABA responsive genes viz. dehydrin 1, 9-cis-epoxycarotenoid dioxygenase, ABA-responsive protein 18 and BEL1-like homeodomain protein 1 had log2fold change above 4.0. A total of 12,836 simple sequence repeats and 4,438 single nucleotide polymorphisms were identified which can be utilized in molecular studies. CONCLUSIONS: Phyto-hormones biosynthesis-predominantly through ABA signalling, and secondary metabolism are the most potent pathways for alkalinity stress tolerance in lentil. Cultivar PDL-1 exhibited high tolerance towards alkalinity stress and can be used in breeding programmes for improving lentil production under alkalinity stress conditions.

Identification of climate induced optimal rice yield and vulnerable districts rankings of the Punjab, Pakistan.

The study attracted to insinuate the inhabitant anomalies of the crop yield in the districts of the Punjab where climate variation, inputs utilization, and district exponents are indispensable factors. Impact evaluation of sowing and harvesting dates for rice yield has been analyzed. Suitable sowing and harvesting dates and potential districts for the crop are proposed. Data consisting of 13,617 observations of more than 90 factors encompassing valuable dimensions of the growth of the crops collected through comprehensive surveys conducted by the Agriculture Department of Punjab are formulated to incorporate in this study. The results establish the significant negative repercussions of climate variability while the impacts vary in the districts. The crop yield deteriorates considerably by delaying the sowing and harvesting times. Districts climate-induced vulnerability ranking revealed Layyah, Jhelum, Mianwali, Khanewal and Chinniot, the most vulnerable while Kasur, Gujrat, Mandi Bhauddin, Nankana Sahib and Hafizabad, the least vulnerable districts. Spatial mapping explains the geographical pattern of vulnerabilities and yield/monetary losses. The study ranks districts using climate-induced yield and monetary loss (222.30 thousand metric tons of rice which are equal to 27.79 billion PKR climatic losses in single rice season) and recommends: the formation of district policy to abate the adverse climate impact, utilization of suitable climate variation by adhering proper sowing and harvesting times, setting the prioritized districts facing climate-induced losses for urgent attention and preferable districts for rice crop.

CropPAL for discovering divergence in protein subcellular location in crops to support strategies for molecular crop breeding.

Agriculture faces increasing demand for yield, higher plant-derived protein content and diversity while facing pressure to achieve sustainability. Although the genomes of many of the important crops have been sequenced, the subcellular locations of most of the encoded proteins remain unknown or are only predicted. Protein subcellular location is crucial in determining protein function and accumulation patterns in plants, and is critical for targeted improvements in yield and resilience. Integrating location data from over 800 studies for 12 major crop species into the cropPAL2020 data collection showed that while >80% of proteins in most species are not localised by experimental data, combining species data or integrating predictions can help bridge gaps at similar accuracy. The collation and integration of over 61 505 experimental localisations and more than 6 million predictions showed that the relative sizes of the protein catalogues located in different subcellular compartments are comparable between crops and Arabidopsis. A comprehensive cross-species comparison showed that between 50% and 80% of the subcellulomes are conserved across species and that conservation only depends to some degree on the phylogenetic relationship of the species. Protein subcellular locations in major biosynthesis pathways are more often conserved than in metabolic pathways. Underlying this conservation is a clear potential for subcellular diversity in protein location between species by means of gene duplication and alternative splicing. Our cropPAL data set and search platform (https://crop-pal.org) provide a comprehensive subcellular proteomics resource to drive compartmentation-based approaches for improving yield, protein composition and resilience in future crop varieties.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.

Epigenetic Landmarks of Leaf Senescence and Crop Improvement.

This review synthesizes knowledge on epigenetic regulation of leaf senescence and discusses the possibility of using this knowledge to improve crop quality. This control level is implemented by different but interacting epigenetic mechanisms, including DNA methylation, covalent histone modifications, and non-covalent chromatin remodeling. The genetic and epigenetic changes may act alone or together and regulate the gene expression, which may result in heritable (stress memory) changes and may lead to crop survival. In the review, the question also arises whether the mitotically stable epigenetic information can be used for crop improvement. The barley crop model for early and late events of dark-induced leaf senescence (DILS), where the point of no return was defined, revealed differences in DNA and RNA modifications active in DILS compared to developmental leaf senescence. This suggests the possibility of a yet-to-be-discovered epigenetic-based switch between cell survival and cell death. Conclusions from the analyzed research contributed to the hypothesis that chromatin-remodeling mechanisms play a role in the control of induced leaf senescence. Understanding this mechanism in crops might provide a tool for further exploitation toward sustainable agriculture: so-called epibreeding.

MULTI-FLORET SPIKELET 2, a MYB Transcription Factor, Determines Spikelet Meristem Fate and Floral Organ Identity in Rice.

An understanding of flower and panicle development is crucial for improving yield and quality in majority of grass crops. In this study, we used mapping-based cloning to identify MULTI-FLORET SPIKELET2 (MFS2), which encodes a MYB transcription factor and regulates flower and spikelet development in rice (Oryza sativa). In the mfs2 mutant, specification of palea identity was severely disturbed and showed degradation or transformation into a lemma-like organ, and the number of all floral organs was increased to varying degrees. Due to the increase in the number of floral organs and development of extra transformed palea/marginal region of the palea-like organs, some mfs2 spikelets had a tendency to produce two florets. These defects implied that the mfs2 mutation caused abnormal specification of palea identity and partial loss of spikelet determination. We confirm that MFS2 is a transcriptional repressor that shows strong repression activity by means of two typical ethylene-responsive element binding factor-associated amphiphilic motifs, one of which locates at the C terminus and is capable of interaction with three rice TOPLESS and TOPLESS-related proteins. The results indicate that MFS2 acts as a repressor that regulates floral organ identities and spikelet meristem determinacy in rice by forming a repression complex with rice TOPLESS and TOPLESS-related proteins.

Pollen-Specific Protein PSP231 Activates Callose Synthesis to Govern Male Gametogenesis and Pollen Germination.

Spatiotemporally regulated callose deposition is an essential, genetically programmed phenomenon that promotes pollen development and functionality. Severe male infertility is associated with deficient callose biosynthesis, highlighting the significance of intact callose deposition in male gametogenesis. The molecular mechanism that regulates the crucial role of callose in production of functional male gametophytes remains completely unexplored. Here, we provide evidence that the gradual upregulation of a previously uncharacterized cotton (Gossypium hirsutum) pollen-specific SKS-like protein (PSP231), specifically at the post pollen-mitosis stage, activates callose biosynthesis to promote pollen maturation. Aberrant PSP231 expression levels caused by either silencing or overexpression resulted in late pollen developmental abnormalities and male infertility phenotypes in a dose-dependent manner, highlighting the importance of fine-tuned PSP231 expression. Mechanistic analyses revealed that PSP231 plays a central role in triggering and fine-tuning the callose synthesis and deposition required for pollen development. Specifically, PSP231 protein sequesters the cellular pool of RNA-binding protein GhRBPL1 to destabilize GhWRKY15 mRNAs, turning off GhWRKY15-mediated transcriptional repression of GhCalS4/GhCalS8 and thus activating callose biosynthesis in pollen. This study showed that PSP231 is a key molecular switch that activates the molecular circuit controlling callose deposition toward pollen maturation and functionality and thereby safeguards agricultural crops against male infertility.

Autophagy in crop plants: what's new beyond Arabidopsis?

Autophagy is a major degradation and recycling pathway in plants. It functions to maintain cellular homeostasis and is induced by environmental cues and developmental stimuli. Over the past decade, the study of autophagy has expanded from model plants to crop species. Many features of the core machinery and physiological functions of autophagy are conserved among diverse organisms. However, several novel functions and regulators of autophagy have been characterized in individual plant species. In light of its critical role in development and stress responses, a better understanding of autophagy in crop plants may eventually lead to beneficial agricultural applications. Here, we review recent progress on understanding autophagy in crops and discuss potential future research directions.

Nuclear Proteome: Isolation of Intact Nuclei, Extraction of Nuclear Proteins, and 2-DE Analysis.

Proteome profiling aims to unravel the mystery of biological complexity encoded by the genome. The successful proteome profiling largely depends upon analytical approaches because single-step proteome characterization of eukaryotic cells is difficult due to the large number of proteins expressed and their complex physiochemical properties. Organellar proteomics helps in identifying a refined set of proteins by pinpointing certain activities to specific organelles, thereby increasing our knowledge of cellular processes. The reliability of a plant organelle proteome is intimately dependent on the purity of the organelle preparation. Methodological improvements in sample handling, organelle fractionation, and protein extraction are therefore crucial to plant subcellular proteomics. The nuclear proteins are organized into complex regulatory networks and perform varied cellular functions. Therefore, characterization of the nuclear proteome is an important step toward accumulating knowledge about regulation of gene expression and function. In this chapter, we present methods for the isolation of nuclei, purification of nuclear proteins, and proteome profiling that have been adapted for proteomic characterization of economically important crop species, such as chickpea.

Redox regulation of a guard cell SNF1-related protein kinase in Brassica napus, an oilseed crop.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn2+). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H2O2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H2O2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

Meiosis, unreduced gametes, and parthenogenesis: implications for engineering clonal seed formation in crops.

KEY MESSAGE: Meiosis and unreduced gametes. Sexual flowering plants produce meiotically derived cells that give rise to the male and female haploid gametophytic phase. In the ovule, usually a single precursor (the megaspore mother cell) undergoes meiosis to form four haploid megaspores; however, numerous mutants result in the formation of unreduced gametes, sometimes showing female specificity, a phenomenon reminiscent of the initiation of gametophytic apomixis. Here, we review the developmental events that occur during female meiosis and megasporogenesis at the light of current possibilities to engineer unreduced gamete formation. We also provide an overview of the current understanding of mechanisms leading to parthenogenesis and discuss some of the conceptual implications for attempting the induction of clonal seed production in cultivated plants.

A faster Rubisco with potential to increase photosynthesis in crops.

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial ß-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the ß-carboxysome shell proteins.

Salt resistant crop plants.

Soil salinity is a major constraint to agriculture. To improve salinity tolerance of crops, various traits can be incorporated, including ion exclusion, osmotic tolerance and tissue tolerance. We review the roles of a range of genes involved in salt tolerance traits. Different tissues and cells are adapted for specific and often diverse function, so it is important to express the genes in specific cell-types and to pyramid a range of traits. Modern biotechnology (marker-assisted selection or genetic engineering) needs to be increasingly used to introduce the correct combination of genes into elite crop cultivars. Importantly, the effects of introduced genes need to be evaluated in the field to determine their effect on salinity tolerance and yield improvement.

How do filamentous pathogens deliver effector proteins into plant cells?

Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens.

Effects of high temperature after pollination on physicochemical properties of waxy maize flour during grain development.

BACKGROUND: Waxy maize is grown in South China, where high temperatures frequently prevail. The effect of high-temperature stress on grain development of waxy maize is not known. RESULTS: High temperature decreased the grain fresh weight and volume, and lowered the grain dry weight and water content after 22 days after pollination (DAP). Plants exposed to high temperature had low starch content, and high protein and soluble sugar contents at maturity. Starch iodine binding capacity and granule size were increased by heat stress at all grain-filling stages. The former parameter decreased, while the latter parameter increased gradually with grain development. High temperature increased the peak and breakdown viscosity before 30 DAP, but the value decreased at maturity. Pasting and gelatinization temperatures at different stages were increased by heat stress and gradually decreased with grain development under both high-temperature and control conditions. Gelatinization enthalpy increased initially but decreased after peaking at 22 DAP under both control and heat stress conditions. High temperature decreased gelatinization enthalpy after 10 DAP. Retrogradation percentage value increased with high temperature throughout grain development. CONCLUSION: High temperature after pollination changes the dynamics of grain filling of waxy maize, which may underlie the observed changes in its pasting and thermal properties.

Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 µM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.

Sustainable agriculture: possible trajectories from mutualistic symbiosis and plant neodomestication.

Food demand will increase concomitantly with human population. Food production therefore needs to be high enough and, at the same time, minimize damage to the environment. This equation cannot be solved with current strategies. Based on recent findings, new trajectories for agriculture and plant breeding which take into account the belowground compartment and evolution of mutualistic strategy, are proposed in this opinion article. In this context, we argue that plant breeders have the opportunity to make use of native arbuscular mycorrhizal (AM) symbiosis in an innovative ecologically intensive agriculture.