Introducing single cell stereo-sequencing technology to transform the plant transcriptome landscape.

Single cell RNA-sequencing (scRNA-seq) advancements have helped detect transcriptional heterogeneities in biological samples. However, scRNA-seq cannot currently provide high-resolution spatial transcriptome information or identify subcellular organs in biological samples. These limitations have led to the development of spatially enhanced-resolution omics-sequencing (Stereo-seq), which combines spatial information with single cell transcriptomics to address the challenges of scRNA-seq alone. In this review, we discuss the advantages of Stereo-seq technology. We anticipate that the application of such an integrated approach in plant research will advance our understanding of biological process in the plant transcriptomics era. We conclude with an outlook of how such integration will enhance crop improvement.

Improved photosynthetic performance under unilateral weak light conditions in a wide-narrow-row intercropping system is associated with altered sugar transport.

Intercropping improves resource utilization. Under wide-narrow-row maize (Zea mays) intercropping, maize plants are subjected to weak unilateral illumination and exhibit high photosynthetic performance. However, the mechanism regulating photosynthesis under unilateral weak light remains unknown. We investigated the relationship between photosynthesis and sugar metabolism in maize under unilateral weak light. Our results showed that the net photosynthetic rate (Pn) of unshaded leaves increased as the level of shade on the other side increased. On the contrary, the concentration of sucrose and starch and the number of starch granules in the unshaded leaves decreased with increased shading due to the transfer of abundant C into the grains. However, sink loss with ear removal reduced the Pn of unshaded leaves. Intense unilateral shade (40% to 20% normal light), but not mild unilateral shade (60% normal light), reduced grain yield (37.6% to 54.4%, respectively). We further found that in unshaded leaves, Agpsl, Bmy, and Mexl-like expression significantly influenced sucrose and starch metabolism, while Sweet13a and Sut1 expression was crucial for sugar export. In shaded leaves, expression of Sps1, Agpsl, and Sweet13c was crucial for sugar metabolism and export. This study confirmed that unshaded leaves transported photosynthates to the ear, leading to a decrease in sugar concentration. The improvement of photosynthetic performance was associated with altered sugar transport. We propose a narrow-row spacing of 40 cm, which provides appropriate unilateral shade and limits yield reduction.

FLS2-RBOHD module regulates changes in the metabolome of Arabidopsis in response to abiotic stress.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants toward both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 (ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE, PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5, and NITRILASE 3. Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

Cotton proteomics: Dissecting the stress response mechanisms in cotton.

The natural environment of plants comprises a complex set of biotic and abiotic stresses, and plant responses to these stresses are complex as well. Plant proteomics approaches have significantly revealed dynamic changes in plant proteome responses to stress and developmental processes. Thus, we reviewed the recent advances in cotton proteomics research under changing environmental conditions, considering the progress and challenging factors. Finally, we highlight how single-cell proteomics is revolutionizing plant research at the proteomics level. We envision that future cotton proteomics research at the single-cell level will provide a more complete understanding of cotton's response to stresses.

PIN1 regulates epidermal cells development under drought and salt stress using single-cell analysis.

Over the course of evolution, plants have developed plasticity to acclimate to environmental stresses such as drought and salt stress. These plant adaptation measures involve the activation of cascades of molecular networks involved in stress perception, signal transduction and the expression of stress related genes. Here, we investigated the role of the plasma membrane-localized transporter of auxin PINFORMED1 (PIN1) in the regulation of pavement cells (PCs) and guard cells (GCs) development under drought and salt stress conditions. The results showed that drought and salt stress treatment affected the development of PCs and GCs. Further analysis identified the different regulation mechanisms of PIN1 in regulating the developmental patterns of PCs and GCs under drought and salt stress conditions. Drought and salt stress also regulated the expression dynamics of PIN1 in pif1/3/4/5 quadruple mutants. Collectively, we revealed that PIN1 plays a crucial role in regulating plant epidermal cells development under drought and salt stress conditions, thus contributing to developmental rebustness and plasticity.

Research strategies for single-cell transcriptome analysis in plant leaves.

The recent and continuous improvement in single-cell RNA sequencing (scRNA-seq) technology has led to its emergence as an efficient experimental approach in plant research. However, compared with single-cell research in animals and humans, the application of scRNA-seq in plant research is limited by several challenges, including cell separation, cell type annotation, cellular function analysis, and cell-cell communication networks. In addition, the unavailability of corresponding reliable and stable analysis methods and standards has resulted in the relative decentralization of plant single-cell research. Considering these shortcomings, this review summarizes the research progress in plant leaf using scRNA-seq. In addition, it describes the corresponding feasible analytical methods and associated difficulties and problems encountered in the current research. In the end, we provide a speculative overview of the development of plant single-cell transcriptome research in the future.

Single-Cell RNA Sequencing for Plant Research: Insights and Possible Benefits.

In recent years, advances in single-cell RNA sequencing (scRNA-seq) technologies have continued to change our views on biological systems by increasing the spatiotemporal resolution of our analysis to single-cell resolution. Application of scRNA-seq to plants enables the comprehensive characterization of both common and rare cell types and cell states, uncovering new cell types and revealing how cell types relate to each other spatially and developmentally. This review provides an overview of scRNA-seq methodologies, highlights the application of scRNA-seq in plant science, justifies why scRNA-seq is a master player of sequencing, and explains the role of single-cell transcriptomics technologies in environmental stress adaptation, alongside the challenges and prospects of single-cell transcriptomics. Collectively, we put forward a central role of single-cell sequencing in plant research.

Creation of cotton mutant library based on linear electron accelerator radiation mutation.

Cotton (Gossypium spp.) is one of the most important cash crops worldwide. At present, new cotton varieties are mainly produced through conventional cross breeding, which is limited by available germplasm. Although the genome of cotton has been fully sequenced, research on the function of specific genes lags behind due to the lack of sufficient genetic material. Therefore, it is very important to create a cotton mutant library to create new, higher-quality varieties and identify genes associated with the regulation of key traits. Traditional mutagenic strategies, such as physical, chemical, and site-directed mutagenesis, are relatively costly, inefficient, and difficult to perform. In this study, we used a radiation mutation method based on linear electron acceleration to mutate cotton variety 'TM-1', for which a whole-genome sequence has previously been performed, to create a high throughput cotton mutant library. Abundant phenotypic variation was observed in the progeny population for three consecutive generations, including cotton fiber color variation, plant dwarfing, significant improvement of yield traits, and increased sensitivity to Verticillium wilt. These results show that radiation mutagenesis is an effective and feasible method to create plant mutant libraries.

Identification of the Regulators of Epidermis Development under Drought- and Salt-Stressed Conditions by Single-Cell RNA-Seq.

As sessile organisms, plants constantly face challenges from the external environment. In order to meet these challenges and survive, plants have evolved a set of sophisticated adaptation strategies, including changes in leaf morphology and epidermal cell development. These developmental patterns are regulated by both light and hormonal signaling pathways. However, our mechanistic understanding of the role of these signaling pathways in regulating plant response to environmental stress is still very limited. By applying single-cell RNA-Seq, we determined the expression pattern of PHYTOCHROME INTERACTING FACTOR (PIF) 1, PIF3, PIF4, and PIF5 genes in leaf epidermal pavement cells (PCs) and guard cells (GCs). PCs and GCs are very sensitive to environmental stress, and our previous research suggests that these PIFs may be involved in regulating the development of PCs, GCs, and leaf morphology under environmental stress. Growth analysis showed that pif1/3/4/5 quadruple mutant maintained tolerance to drought and salt stress, and the length to width ratio of leaves and petiole length under normal growth conditions were similar to those of wild-type (WT) plants under drought and salt treatment. Analysis of the developmental patterns of PCs and GCs, and whole leaf morphology, further confirmed that these PIFs may be involved in mediating the development of epidermal cells under drought and salt stress, likely by regulating the expression of MUTE and TOO MANY MOUTHS (TMM) genes. These results provide new insights into the molecular mechanism of plant adaptation to adverse growth environments.

Identification of novel regulators required for early development of vein pattern in the cotyledons by single-cell RNA-sequencing.

The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3-day-old cotyledons using single-cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3-day-old cotyledons based on single-cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein.

FLS2-RBOHD-PIF4 Module Regulates Plant Response to Drought and Salt Stress.

As sessile organisms, plants are constantly challenged by several environmental stresses. Different kinds of stress often occur simultaneously, leading to the accumulation of reactive oxygen species (ROS) produced by respiratory burst oxidase homolog (RBOHD) and calcium fluctuation in cells. Extensive studies have revealed that flagellin sensitive 2 (FLS2) can sense the infection by pathogenic microorganisms and activate cellular immune response by regulating intracellular ROS and calcium signals, which can also be activated during plant response to abiotic stress. However, little is known about the roles of FLS2 and RBOHD in regulating abiotic stress. In this study, we found that although the fls2 mutant showed tolerance, the double mutant rbohd rbohf displayed hypersensitivity to abiotic stress, similar to its performance in response to immune stress. An analysis of the transcriptome of the fls2 mutant and rbohd rbohf double mutant revealed that phytochrome interacting factor 4 (PIF4) acted downstream of FLS2 and RBOHD to respond to the abiotic stress. Further analysis showed that both FLS2 and RBOHD regulated the response of plants to drought and salt stress by regulating the expression of PIF4. These findings revealed an FLS2-RBOHD-PIF4 module in regulating plant response to biotic and abiotic stresses.

COE2 Is Required for the Root Foraging Response to Nitrogen Limitation.

There are numerous exchanges of signals and materials between leaves and roots, including nitrogen, which is one of the essential nutrients for plant growth and development. In this study we identified and characterized the Chlorophyll A/B-Binding Protein (CAB) (named coe2 for CAB overexpression 2) mutant, which is defective in the development of chloroplasts and roots under normal growth conditions. The phenotype of coe2 is caused by a mutation in the Nitric Oxide Associated (NOA1) gene that is implicated in a wide range of chloroplast functions including the regulation of metabolism and signaling of nitric oxide (NO). A transcriptome analysis reveals that expression of genes involved in metabolism and lateral root development are strongly altered in coe2 seedlings compared with WT. COE2 is expressed in hypocotyls, roots, root hairs, and root caps. Both the accumulation of NO and the growth of lateral roots are enhanced in WT but not in coe2 under nitrogen limitation. These new findings suggest that COE2-dependent signaling not only coordinates gene expression but also promotes chloroplast development and function by modulating root development and absorption of nitrogen compounds.

Gravity Reduced Nitrogen Uptake via the Regulation of Brace Unilateral Root Growth in Maize Intercropping.

Water, nutrient, light, and interspecific facilitation regulation of soil physicochemical properties and root morphology modulate nitrogen (N) uptake in cereal and legume intercropping systems. However, maize root morphological plasticity and N uptake capability response to gravity in the intercropping system remains to be determined. In this study, maize was grown under 20 cm (I20), 40 cm (I40), and 60 cm (I60) of narrow row spacing in an intercropping system (maize-soybean strip relay intercropping) and equal row spacing of monoculture (M) in a 2-year field experiment. As a supplementary for the field experiment, maize root barrier and plant inclination experiments were conducted. Plant inclination, brace root morphology, N uptake, indole-3-acetic acid (IAA) level, IAA synthesis genes, and grain yield were assessed. The result showed that the plant inclination increased with decreasing narrow row spacing in intercropping system. Also, the brace unilateral root growth ratio (BURR) increased with increasing plant inclination in intercropping treatments. The plant inclination experiment showed the BURR achieved 94% after inclination at 45°. BURR tended to be positively correlated (p = 0.00) with plant inclination. Thus, gravity (plant inclination) causes brace unilateral root growth. The IAA concentration of stem nodes in the wide row increased with increasing plant inclination, while the IAA accumulation decreased in the narrow row. The Zmvt2 and ZM2G141383 genes (associated with IAA biosynthesis) were highly expressed in a wide row. There was a strong correlation (p = 0.03) between the IAA concentration of wide row and the BURR. Therefore, gravity regulates the IAA level, which affects BURR. In addition, the brace root number, volume, and surface area were decreased when BURR was increased. Subsequently, the leaf N, cob N, and kernel N accumulation were reduced. These organs N and grain yield in I60 were not significantly different as compared to the control treatment. The excessive brace unilateral root growth was not conducive to N uptake and increased yield. Our results suggest that gravity is essential in regulating root morphology plasticity by regulating IAA levels and decreasing N uptake capacity. Furthermore, these results indicate that plant inclination can regulate root phenotype and N uptake of maize and by adjusting the spacing of narrow maize row, we can improve the N uptake and yield of the maize-soybean strip relay-intercropping system.

GUN4 Affects the Circadian Clock and Seedlings Adaptation to Changing Light Conditions.

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 µmol photons m-2s-1) compared to normal light (100 µmol photons m-2s-1), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect.

Heterogeneous Light Conditions Reduce the Assimilate Translocation Towards Maize Ears.

The border row crop in strip intercropped maize is often exposed to heterogeneous light conditions, resulting in increased photosynthesis and yield decreased. Previous studies have focused on photosynthetic productivity, whereas carbon allocation could also be one of the major causes of decreased yield. However, carbon distribution remains unclear in partially shaded conditions. In the present study, we applied heterogeneous light conditions (T), and one side of plants was shaded (T-30%), keeping the other side fully exposed to light (T-100%), as compared to control plants that were exposed entirely to full-light (CK). Dry weight, carbon assimilation, 13C abundance, and transport tissue structure were analyzed to clarify the carbon distribution in partial shading of plants. T caused a marked decline in dry weight and harvest index (HI), whereas dry weight in unshaded and shaded leaves did not differ. Net photosynthesis rate (Pn), the activity of sucrose phosphate synthase enzymes (SPS), and sucrose concentration increased in unshaded leaves. Appropriately, 5.7% of the 13C from unshaded leaves was transferred to shaded leaves. Furthermore, plasmodesma density in the unshaded (T-100%) and shaded (T-30%) leaves in T was not significantly different but was lower than that of CK. Similarly, the vascular bundle total area of T was decreased. 13C transfer from unshaded leaves to ear in T was decreased by 18.0% compared with that in CK. Moreover, 13C and sucrose concentration of stem in T were higher than those in CK. Our results suggested that, under heterogeneous light, shaded leaves as a sink imported the carbohydrates from the unshaded leaves. Ear and shaded leaf competed for carbohydrates, and were not conducive to tissue structure of sucrose transport, resulting in a decrease in the carbon proportion in the ear, harvest index, and ear weight.

Gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature interaction.

Soybean is an important oilseed crop grown globally. However, two examples of environmental stresses that drastically regulate soybean growth are low light and high-temperature. Emerging evidence suggests a possible interconnection between these two environmental stimuli. Low light and high-temperature as individual factors have been reported to regulate plant hypocotyl elongation. However, their interactive signal effect on soybean growth and development remains largely unclear. Here, we report that gibberellins (GAs) and auxin are required for soybean hypocotyl elongation under low light and high-temperature interaction. Our analysis indicated that low light and high-temperature interaction enhanced the regulation of soybean hypocotyl elongation and that the endogenous GA3 , GA7 , indole-3-acetic acid (IAA), and indole-3-pyruvate (IPA) contents significantly increased. Again, analysis of the effect of exogenous phytohormones and biosynthesis inhibitors treatments showed that exogenous GA, IAA, and paclobutrazol (PAC), 2, 3, 5,-triiodobenzoic acid (TIBA) treatments significantly regulated soybean seedlings growth under low light and high-temperature interaction. Further qRT-PCR analysis showed that the expression level of GA biosynthesis pathway genes (GmGA3ox1, GmGA3ox2 and GmGA3) and auxin biosynthesis pathway genes (GmYUCCA3, GmYUCCA5 and GmYUCCA7) significantly increased under (i) low light and high-temperature interaction and (ii) exogenous GA and IAA treatments. Altogether, these observations support the hypothesis that gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature stress interaction.

Evidence that melatonin promotes soybean seedlings growth from low-temperature stress by mediating plant mineral elements and genes involved in the antioxidant pathway.

Melatonin (MT) regulates several physiological activities in plants. However, information on how MT regulates soybean growth under low-temperature (LT) stress is lacking. To better understand how MT promotes plant growth and development under LT stress, we designed this study to evaluate the role of MT pretreatment on soybean seedlings exposed to LT stress. Our results showed that LT stress increased oxidative damage by increasing reactive oxygen species (ROS) accumulation, which affected the growth and development of soybean seedlings. However, the application of 5 µmol L-1 MT significantly decreased the oxidative damage by increasing plant mineral element concentrations and the transcript abundance of antioxidant related genes, which enhanced the decrease in ROS accumulation. These results collectively suggest the involvement of MT in improving LT stress tolerance of soybean seedlings by mediating plant mineral elements and the expression of genes involved in the antioxidant pathway.

Pre-treatment of salicylic acid enhances resistance of soybean seedlings to Fusarium solani.

KEY MESSAGE: Pre-treatment of soybean seedlings with 200 µM salicylic acid before fungal inoculation significantly alleviated disease resistance in soybean seedlings against Fusarium solani infection. Sudden death syndrome of soybean is largely caused by Fusarium solani (F. solani). Salicylic acid (SA) has been reported to induce resistance in plants against many pathogens. However, the effect of exogenous SA application on F. solani infection of soybean is less reported. This study investigated the effect of foliar application of SA on soybean seedlings before F. solani infection. Seedlings were sprayed with 200 µM SA and inoculated with F. solani after 24 h of last SA application. After 3 days post-inoculation, seedlings treated with 200 µM SA showed significantly fewer disease symptoms with increased endogenous SA level, SA marker genes expression and antioxidant activities in the SA-treated seedlings more than the untreated control seedlings. Furthermore, the decrease in hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels was observed in the SA-treated plants as compared to the untreated plants. Analysis of the effect of SA application on F. solani showed that the mycelia growth of F. solani was not affected by SA treatment. Further investigation in this study revealed a decreased in F. solani biomass content in the SA treated seedlings. Results from the present study show that pre-treatment of 200 µM SA can induce resistance of soybean seedlings against F. solani infection.

Narrow-wide row planting pattern improves the light environment and seed yields of intercrop species in relay intercropping system.

Different planting patterns affect the light interception of intercrops under intercropping conditions. Here we revealed that narrow-wide-row relay-intercropping improves the light interception across maize leaves in wide rows (60cm) and narrow rows (40cm), accelerated the biomass production of intercrop-species and compensated the slight maize yield loss by considerably increasing the soybean yield. In a two-year experiment, maize was planted with soybean in different planting patterns (1M1S, 50:50cm and 2M2S, 40:60cm) of relay-intercropping, both planting patterns were compared with sole cropping of maize (M) and soybean (S). As compared to M and 1M1S, 2M2S increased the total light interception of maize leaves in wide rows (WR) by 27% and 23%, 20% and 10%, 16% and 9% which in turn significantly enhanced the photosynthetic rate of WR maize leaves by 7% and 5%, 12% and 9%, and 19% and 4%, at tasseling, grain-filling and maturity stage of maize, respectively. Similarly, the light transmittance at soybean canopy increased by 218%, 160% and 172% at V2, V5 and R1 stage in 2M2S compared with 1M1S. The improved light environment at soybean canopy in 2M2S considerably enhanced the mean biomass accumulation, and allocation to stem and leaves of soybean by 168%, and 131% and 207%, respectively, while it decreased the mean biomass accumulation, and distribution to stem, leaves and seed of maize by 4%, and 4%, 6% and 5%, respectively than 1M1S. Compared to 1M1S, 2M2S also increased the CR values of soybean (by 157%) but decreased the CR values of maize (by 61%). Overall, under 2M2S, relay-cropped maize and soybean produced 94% and 69% of the sole cropping yield, and the 2M2S achieved LER of 1.7 with net income of 1387.7 US $ ha-1 in 2016 and 1434.4 US $ ha-1 in 2017. Our findings implied that selection of optimum planting pattern (2M2S) may increase the light interception and influence the light distribution between maize and soybean rows under relay-intercropping conditions which will significantly increase the intercrops productivity. Therefore, more attention should be paid to the light environment when considering the sustainability of maize-soybean relay-intercropping via appropriate planting pattern selection.